Studies on the Manufacture &
Spray Drying of Liposomes and
Membrane Lipids
Der Naturwissenschaftlichen Fakultät der
Friedrich-Alexander-Universität Erlangen-Nürnberg
zur Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von
Julia Staudenecker
aus Aalen
Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der
Friedrich‐Alexander-Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 22. April 2016
Vorsitzender des Promotionsorgans: Prof. Dr. Jörn Wilms
Gutachter: Prof. Dr. Geoffrey Lee
PD Dr. Henning Gieseler
Für meine Eltern und Christian
Parts of this thesis have already been presented:
Julia Staudenecker and Stefan Seyferth (2014):
“Effect of liposome concentration on spray dried powders.” 9thWorld Meeting on Phar-
maceutics, Biopharmaceutics and Pharmaceutical Technology, Lisbon, Portugal (Poster)
Acknowledgements
The research work presented in this thesis has been performed between December 2011
and November 2015 at the Division of Pharmaceutics, Friedrich-Alexander University of
Erlangen-Nürnberg, Germany.
Prof. Dr. Geoffrey Lee is gratefully acknowledged for offering me the opportunity to work
in the Division of Pharmaceutics, serving as my doctoral advisor and for refereeing this
thesis.
Many thanks go to Dr. Stefan Seyferth for being my supervisor and for choosing this inter-
esting topic. You had always time for discussing current problems and for developing new
ideas for this work. I take the friendly cooperation during the last years not for granted.
Many thanks for that!
PD Dr. Henning Gieseler is gratefully acknowledged for co-refereeing this thesis.
Many thanks go to the Lehrstuhl für Feststoff- und Grenzflächenverfahrenstechnik for giv-
ing me the opportunity to perform all Zetasizer measurements in their laboratory.
I give thanks to the staff of the Cauerstrasse for the support and help with everyday business
and problems. Petra Neubarth, thank you for your coordination and help with the student´s
practical courses, the support in administrative issues and for delivering hundreds of pack-
ets to me. Luise Schedl is thanked for taking numberless SEM pictures and for being my
lab-mate during the first months. Christiane Blaha, thank you for the fast supply with lipids
and other materials. Many thanks go to Josef Hubert for fixing everything in no time espe-
cially during the student´s practical courses.
I would like to thank my former colleagues Dr. Susanne Rutzinger, Dr. Elke Lorenzen,
Dr. Anne Mundstock, Dr. Felix Wolf, Dr. Sabine Ullrich, Dr. Ulrike Stange, Dr. Matthias
Erber and Dr. Joachim Schäfer at the Division of Pharmaceutics for the friendly welcome,
the assistance in initial training to all the laboratory stuff, the funny coffee breaks and the
exciting table soccer games. Many thanks go to Natalie Keil, Alexander Grebner, Jens
Holtappels, Claudia Kunz, Zixin Huang, Melinda Rupp, Veronika Braig and Alexander
Ullrich for the good cooperation and friendly atmosphere not only in the student´s practical
courses and for sharing ups and downs of everyday life in laboratory. Sandra Großberger,
thank you for your company to all Weiterbildungen and all the fun we had through the
years. Special thanks go to my lab-mates Dr. Anders Kunst and Christina Rödel. Anders,
thank you for the funny and easygoing three years in our Olympia-lab. Christina, thanks
for the last months with all the chitchat and good luck with your work.
Finally, I would like to thank my parents for their continuous support through the years.
Christian, thank you for always being there for me. I am so lucky to have you.
I
Table of Contents
TABLE OF CONTENTS ....................................................................................... I
LIST OF ABBREVIATIONS ................................................................................ V
1 INTRODUCTION .......................................................................................... 1
2 LIPOSOMES ................................................................................................ 5
2.1 Classification........................................................................................................ 5
2.2 Preparation ........................................................................................................... 7
2.2.1 Preparation Techniques .............................................................................. 7
2.2.2 Preparation of Liposomes with a Defined Size .......................................... 9
2.2.3 Drug Loading ........................................................................................... 10
2.3 Liposome Composition ...................................................................................... 11
2.3.1 Phospholipids ........................................................................................... 11
2.3.2 Cholesterol ............................................................................................... 13
2.4 Conventional Liposomes ................................................................................... 14
2.5 Long-circulating Liposomes .............................................................................. 15
2.6 Liposomes in Gene Delivery ............................................................................. 17
2.7 Liposomes as Carriers of Proteins and Peptides ................................................ 18
2.7.1 Vaccines ................................................................................................... 18
2.7.2 Further encapsulated Proteins and Peptides ............................................. 18
2.8 Liposome Formulations ..................................................................................... 19
2.8.1 Stability of Liposomes ............................................................................. 19
2.8.2 Stabilization of Liposomes via Drying .................................................... 21
2.8.3 Freeze Drying of Liposomes .................................................................... 23
2.8.4 Overview of approved Liposome Formulations ...................................... 24
II
3 SPRAY DRYING ........................................................................................ 27
3.1 Introduction to Spray Drying ............................................................................. 27
3.2 Atomization ....................................................................................................... 29
3.3 Drying ................................................................................................................ 35
3.4 Particle Size and Morphology ............................................................................ 38
3.5 Powder Separation ............................................................................................. 41
3.6 Spray Drying of Liposomes ............................................................................... 44
4 MATERIALS AND METHODS ................................................................... 47
4.1 Materials ............................................................................................................ 47
4.1.1 Lipids ....................................................................................................... 47
4.1.2 Encapsulated Substances ......................................................................... 49
4.1.3 Excipients and Reagents .......................................................................... 51
4.1.4 Other Materials ........................................................................................ 53
4.2 Methods ............................................................................................................. 54
4.2.1 Liposome Preparation .............................................................................. 54
4.2.2 Spray Drying ............................................................................................ 55
4.2.3 Nozzle Types ........................................................................................... 56
4.2.4 Differential Scanning Calorimetry ........................................................... 57
4.2.5 Dynamic Light Scattering ........................................................................ 58
4.2.6 Karl-Fischer Titration .............................................................................. 58
4.2.7 Laser Diffraction ...................................................................................... 58
4.2.8 Wide-Angle-X-Ray-Diffraction (WAXD)............................................... 59
4.2.9 Scanning Electron Microscopy ................................................................ 59
4.2.10 Levitation ............................................................................................. 59
4.2.11 Encapsulation Efficiency – Insulin ...................................................... 61
4.2.12 Size Exclusion Chromatography – Insulin........................................... 62
III
4.2.13 HPLC – Lipid Recovery....................................................................... 63
4.2.14 Calcein Encapsulation and Membrane Integrity .................................. 64
4.2.15 Freeze Drying ....................................................................................... 65
4.2.16 Maximum Bubble Pressure Tensiometry ............................................. 66
4.2.17 Viscosity Measurements ...................................................................... 66
4.2.18 Stability ................................................................................................ 66
5 RESULTS AND DISCUSSION ................................................................... 67
5.1 Preparation of Liposomes .................................................................................. 67
5.1.1 Liposome Dispersions after Extrusion ..................................................... 67
5.1.2 Phase Transition of Lipids and Liposomes .............................................. 71
5.2 Characterization of Spray Dried Liposomes ...................................................... 73
5.2.1 Dynamic Light Scattering ........................................................................ 73
5.2.2 Atomization of Liposomes ....................................................................... 78
5.2.3 Lipid Recovery ......................................................................................... 80
5.2.4 Calorimetric Study on SD Liposomes ..................................................... 82
5.3 Characterization of Spray Dried Powders.......................................................... 85
5.3.1 Particle Yield ........................................................................................... 85
5.3.2 Particle Size and Morphology .................................................................. 86
5.3.3 Residual Moisture and Glass Transition .................................................. 92
5.3.4 Comparison between Spray Drying and Freeze Drying .......................... 99
5.3.5 Levitation Experiments .......................................................................... 101
5.4 Spray Drying of Liposomes using various Excipients ..................................... 111
5.4.1 Sugars and Sugar Alcohols .................................................................... 111
5.4.2 Polysorbate 80 ........................................................................................ 120
5.5 Stealth Liposomes ............................................................................................ 124
5.5.1 Influence of PEG on Liposome Properties ............................................ 124
IV
5.5.2 Spray Drying of Stealth Liposomes ....................................................... 127
5.6 Experiments with a Fluorescent Marker .......................................................... 131
5.6.1 Encapsulation Efficiency ....................................................................... 131
5.6.2 Studies on the Membrane Integrity ........................................................ 136
5.7 Liposomes with encapsulated Insulin .............................................................. 142
5.7.1 Characterization of Insulin and Liposomes after Spray Drying ............ 142
5.7.2 Characterization of Spray Dried Powders.............................................. 150
5.8 Stability of SD Liposome Formulations .......................................................... 156
6 CONCLUSION .......................................................................................... 165
7 ZUSAMMENFASSUNG ............................................................................ 171
8 REFERENCES ......................................................................................... 177
9 CURRICULUM VITAE .............................................................................. 195
V
List of Abbreviations
Capital letters
D droplet diameter
Dav average droplet diameter
Db diffusivity of vapour in boundary layer
Dc droplet diameter at critical point
Dpowder powder diameter
Dv diffusion coefficient
Fin fluorescence after addition of Co2+
Ftot initial fluorescence
Ftotq fluorescence after addition of Co2+ and Triton X
Fultrasound fluorescence after SD, FD or atomization
Kd thermal conductivity
M molecular weight
Ma mass flow air
Ml mass flow liquid
P∞ partial vapour pressure at outside plane of the stagnant boundary
layer
PS partial vapour pressure at droplet surface
Q volume flow rate
R gas constant
Tc collapse temperature
Tchamber air temperature at the end of the drying chamber
Tg glass transition temperature
Tg´ glass transition temperature of the maximally freeze-concentrated so-
lution
Tinlet air inlet temperature
Tm phase transition temperature
Toutlet air outlet temperature
Z0 vortex length
VI
Small letters
d50 median value of a volume based size distribution
f frequency
mconc mass of insulin in the concentrate
mfilt mass of insulin in the filtrate
mstart initial mass of insulin in the dispersion
pKa acid dissociation constant
r droplet radius
r1 dilution factor
r2 dilution factor
Greek letters
µair air viscosity
λ wavelength
λev latent heat of evaporation
νin inlet velocity
νrel relative velocity
ρ density
ρa spray gas density
ρdroplet density of spray solution
ρpowder density of SD particle
ρs density of solids
σ surface tension
Expressions
BCA bicinchoninic acid
BSA bovine serum albumin
CCD charged coupled device
CF carboxyfluorescein
DLS dynamic light scattering
DLVO Derjaguin, Landau, Verwey, Overbeek
VII
DPI dry powder inhaler
DSC differential scanning calorimetry
EDTA ethylenediaminetetraacetic acid
EE encapsulation efficiency
EPR enhanced permeability and retention effect
FD freeze drying
GM1 monoganglioside
HDL high density lipoprotein
HEPA high-efficiency particulate arrestance
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HMWP high molecular weight protein
HPLC high pressure liquid chromatography
IR infrared
LDL low density lipoprotein
LUV large unilamellar vesicle
MLV multilamellar vesicle
MPS mononuclear phagocyte system
MVV multivesicular vesicle
NIBS non-invasive back scatter
NMWL nominal molecular weight limit
OLV oligolamellar vesicle
PDI polydispersity index
PEG poly-(ethylene glycol)
Ph.Eur. Pharmacopoea Europaea
PMMA polymethyl methacrylate
PUFA polyunsaturated fatty acids
REV reverse phase evaporation vesicles
RH relative humidity
RI refractive index
SD spray drying
SEC size exclusion chromatography
SEM scanning electron microscopy
SOD superoxide dismutase
SUV small unilamellar vesicle
VIII
TEM transmission electron microscopy
TF two fluid
TFN two fluid nozzle
Tris tromethamine
UHPLC ultra high performance liquid chromatography
US ultrasonic
USN ultrasonic nozzle
USP United States Pharmacopeia
UV ultraviolet
VIS visible
WAXD wide-angle-x-ray-diffraction
XRD x-ray diffraction
Lipids
18:1 PEG 2000 PE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-
ethylene glycol)-2000] (18:1)
DHAB dihexadecyldimethylammoniumbromide
DLPC 1,2-didodecanoyl-sn-glycero-3-phosphocholine (12:0)
DMPC 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (14:0)
DMPG 1,2-ditetradecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (14:0)
DOPC 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (18:1)
DOPE 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (18:1)
DOPS 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-L-serine (18:1)
DOTAP 1,2-dioleoyl-3-trimethylammonium-propane (18:1)
DPPC 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (16:0)
DPPG 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (16:0)
DSPC 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (18:0)
DSPE-PEG-2000 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly-
ethylene glycol)-2000] (18:0)
DSPE-PEG-5000 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(pol-
yethylene glycol)-5000] (18:0)
Egg-PG L-α-phosphatidylglycerol (egg, chicken)
Egg-SM N-hexadecanoyl-D-erythro-sphingosylphosphorylcholine (egg,
chicken)
HSPC L-α-phosphatidylcholine, hydrogenated (soy)
IX
PA phosphatidyl acid
PC phosphatidylcholine
PE phosphatidylethanolamine
PI phosphatidylinositol
POPC 1-hexadecanoyl-2--(9Z-octadecenoyl)-sn-glycero-3-phosphocholine
(16:0/18:1)
PS phosphatidylserine
1 INTRODUCTION
The discovery of liposomes more than 50 years ago marked the beginning of a success
story. Today, one application field of liposomes is the use as drug carrier, but the small
vesicles can be found in several other parts of our life as well. Liposomes consist of one or
more phospholipid bilayers and are able to encapsulate hydrophilic substances in their
aqueous core as well as lipophilic components in their bilayer. The spherical, self-closed
vesicles have a diameter of 50 nm up to 5 µm (Immordino et al., 2006). In the early years,
they were used as a membrane model, but their potential as a carrier for different substances
was identified soon. Besides the pharmaceutical industry, the food industry (F. Gibbs,
1999), cosmetics (Meybeck, 1992) and diagnostics (Phillips, 1999) use the small lipid ves-
icles for the transport of various substances.
Liposomes as drug carriers feature several strengths. On the one hand, they can reduce the
toxicity and therefore side effects of several drugs, for example amphotericin (Tiphine et
al., 1999). On the other hand, they are an instrument for drug targeting, which is a key
component particularly in the field of cancer treatment (Torchilin, 2005).
However, the handling of liposomes bears some difficulties. Liposomes tend to aggregate,
the encapsulated drug may vanish due to drug leakage and the phospholipids themselves
are susceptible to undergo oxidation or hydrolysis (Crommelin et al., 1994). These prob-
lems especially occur when liposomes are stored under their original conditions – sus-
pended in an aqueous buffer. The transfer of these liquid formulations to more stable solid
formulations is an approach to overcome the stability problems mentioned. Several publi-
cations have shown that lyophilization is a proven tool to stabilize liposomes by embedding
them into a sugar matrix (Chen et al., 2010).
Besides lyophilization, which is a time-consuming and therefore expensive process, spray
drying is another option for the transfer of a liquid formulation to the preferred solid for-
mulation. Although spray drying is a very fast process applying higher temperatures for the
2 INTRODUCTION
drying, a lot of publications reveal that it is possible to spray dry sensitive biopharmaceu-
ticals, for example vaccines (Ohtake et al., 2010). Goldbach et al. (1993a) and Hauser and
Strauss (1987) were the first to report about the potential to stabilize liposomes through
spray drying. The reconstituted liposomes showed nearly the same size and size distribution
compared to the initial liposome dispersion.
Spray drying in general is a process, which is widely used, for example in food and agri-
culture industries. In pharmaceutics, the production of inhalable powders for drug delivery
is one application. The lungs are a site of action for local therapy but also a possible entry
route for systemic therapy, not only for small molecules but also for peptides and proteins
(Balducci et al., 2014). Here, dry powder particles with a size between 1 and 5 µm are
required. These powders can be designed and produced in a spray dryer, since this process
is capable of generating particles in this size range. In respect thereof, spray drying is su-
perior to lyophilization. Exubera® was the first spray dried dry powder formulation of a
protein, insulin, which was designed for the application via a dry powder inhaler (White et
al., 2005).
Liposomes are also used for the treatment of lung diseases. As Aerosols, they are applied
for the delivery of phospholipids to the alveolar surface for the treatment of neonatal res-
piratory distress syndrome (Labiris and Dolovich, 2003). If active ingredients, for example
antibiotics or antiasthma drugs for the local therapy of lung diseases, are encapsulated into
the lipid vesicles and delivered to the lungs, they show sustained release properties and
decreased systemic effects (Conley et al., 1997).
Bringing the two technologies mentioned together, dry powder formulations of liposomes
prepared by a spray drying process can be a promising concept for the treatment of respir-
atory diseases as well as for the systemic delivery of active ingredients. Chougule et al.
(2008) spray dried dapsone, amiloride and tacrolimus, all of them encapsulated in lipo-
somes and spray dried to obtain a dry powder suitable for pulmonary delivery.
Besides these approaches for a pulmonary delivered liposomal formulation, Lo et al. (2004)
demonstrated that encapsulation of a protein, superoxide dismutase, into liposomes fol-
lowed by a spray drying step is feasible, too. Aside from the above mentioned early studies
of Goldbach et al. (1993a) and Hauser and Strauss (1987), Wessman et al. (2010) published
INTRODUCTION 3
a study of the structural effects of spray drying and freeze drying on liposomes. They re-
ported that liposomes are slightly smaller after drying and rehydration and found some fu-
sions into larger liposomes. They referred this to an osmotic stress, which occurs during
the drying process. However, taken as a whole, there is not much research data about the
spray drying of liposomes.
The aim of this work was to improve the understanding of a liposomal spray drying process,
in doing so the focus was not only on the liposome itself, but also on the powders and the
impact of embedded liposomes on powder properties. In this work, different types of lipo-
somes were manufactured, spray dried and investigated with respect to specific character-
istics of both the individual liposome formulation and the spray drying process conditions.
For this purpose, different nozzle types and varying lipid concentrations were examined. In
addition, the drying behavior of the liposomal formulations was analyzed through acoustic
levitation in order to monitor the rapid spray drying process in slow motion. The idea was
to subject many different liposomal formulations to the spray drying process. Cationic lip-
osomes, stealth liposomes, liposomes with an encapsulated test protein, insulin, or lipo-
somes with an entrapped fluorescence marker, calcein, were processed. The latter was used
for studies on the encapsulation efficiency and membrane integrity during spray drying.
2 LIPOSOMES
2.1 Classification
Liposomes are spherical, self-closed structures formed by one or several concentric phos-
pholipid bilayers (Torchilin, 2005). They enclose a part of the surrounding aqueous solvent.
Lipid vesicles were first made and described by Bangham and Horne (1964). Figure 2-1
shows a liposome consisting of one bilayer. The polar head groups of the phospholipids are
directed towards the aqueous phase whereas the two hydrophobic chains build the inner
structure of the bilayer. In addition, Figure 2-1 demonstrates the two possible ways of en-
capsulating a substance into a liposome. Hydrophobic components are encapsulated be-
tween the hydrophobic chains of the bilayer whereas hydrophilic substances are located in
the aqueous core of the liposome. Figure 2-2 shows the different types of liposomes, clas-
sified based on size and number of bilayers. Multilamellar vesicles (MLV) are larger than
0.5 µm and oligolamellar vesicles (OLV) have a size range of 0.1 – 1 µm. Unilamellar
vesicles are distinguished between small unilamellar vesicles (SUV; 20 – 100 nm) and large
unilamellar vesicles (LUV; > 100 nm). Furthermore, there are multivesicular vesicles
(MVV), with several small vesicles enclosed by one bilayer (Storm and Crommelin, 1998).
Figure 2-1: Schematic draw of a liposome with possible drug encapsulation (Gubernator, 2011).
6 LIPOSOMES
Figure 2-2: (a) multilamellar vesicle (MLV); (b) large unilamellar vesicle (LUV); (c) small
unilamellar vesicle (SUV); (d) multivesicular vesicle (MVV).
As a result of the increasing research in the field of liposomes, it is important to keep in
mind that this is a very simple classification approach with no statement of possible appli-
cations or surface modifications. The following list gives another possible categorization
of liposomes into different subtypes (Torchilin, 2005):
Conventional liposomes
Long-circulation liposomes (Stealth liposomes)
Liposomes for gene delivery
Liposomes with a modified surface for drug targeting (immunoliposomes, for ex-
ample antibody-mediated liposome targeting)
pH sensitive liposomes
A more detailed explanation of some of the mentioned subtypes is given in the chapters
2.4, 2.5 and 2.6. Combinations of the mentioned subtypes are also possible.
LIPOSOMES 7
2.2 Preparation
2.2.1 Preparation Techniques
The most common method for preparing liposomes is the round bottom flask method (film
method, hand shaken method), which was introduced by Bangham et al. (1965). A weighed
amount of phospholipids or a mixture of phospholipids is dissolved in an organic solvent
in a round bottom flask. The organic solvent is removed under reduced pressure by a rotary
evaporator. Afterwards an aqueous buffer is added in order to rehydrate the thin lipid film.
The temperature should be kept above the phase transition temperature (Tm; see 2.3.1) of
the phospholipids. The resulting multilamellar vesicles (MLV) are heterogeneous with re-
spect to their size and lamellarity.
Another method of producing liposomes is the detergent removal. Mixed micelles of phos-
pholipids and detergent in aqueous solution are prepared. Afterwards the detergent is re-
moved and liposomes are formed. Different subtypes of this procedure are explained briefly
in the following: Detergent elimination through dialysis is one possible technique to pro-
duce liposomes with an extreme narrow size distribution (Milsmann et al., 1978, Zumbuehl
and Weder, 1981). Another way to remove the detergent was presented by Brunner et al.
(1976). They add sodium cholate to a milky dispersion of lecithin in aqueous solution.
Small mixed micelles are formed and the detergent is removed through a size exclusion
chromatography with a Sephadex G-50 column. The resulting vesicles have a radius of
150 Å. Schurtenberger et al. (1984) controlled the liposome size through a defined and
rapid dilution of the mixed micellar solutions followed by dialysis.
Ethanol injection is a technique, which was first described by Batzri and Korn (1973). Li-
pids are dissolved in ethanol and the solution is rapidly injected into an aqueous buffer. The
procedure is rapid, reproducible and chemical degradation of the lipids is avoided. How-
ever, there are some limitations to this technique. The final dispersion of unilamellar lipo-
somes contains ethanol, which has to be removed. Additionally, the solubility of phospho-
lipids in ethanol and the maximum volume of ethanol (7.5 % v/v), which can be introduced
into the buffer, are limiting factors (Pons et al., 1993). As a result, the final liposome sus-
pension is dilute. A study of the factors of influence (Kremer et al., 1977) shows that the
8 LIPOSOMES
concentration of the lipids in alcohol is the crucial parameter in controlling the final lipo-
some size. The effect of injection rate, rate of stirring and size of the reaction vessel is
marginal.
The ether infusion technique is another procedure of preparing liposomes through injection
of an organic phospholipid solution into an aqueous solution of the material to be encapsu-
lated. The aqueous solution is warmed above the boiling point of ether in order to vaporize
the ether immediately. This method was first described by Deamer and Bangham (1976).
In a further study, Deamer (1978) pointed out that the technique requires only a minimal
time input, including a filtration step to remove multilamellar vesicles and aggregates and
a gel filtration step to remove residual ether.
Reverse-phase evaporation vesicles (REV) are described in a study of Szoka and
Papahadjopoulos (1978). Phospholipids are dissolved in an organic solvent before adding
an aqueous buffer. Subsequently the two phases are mixed by a bath-type sonicator and the
organic solvent is removed under reduced pressure. The resulting vesicles show a high en-
capsulation efficiency.
The methods mentioned above can be categorized as classical methods. The following tech-
niques are more recent developments, which aim at bigger batch sizes or a tailored vesicle
size for particular applications. Jahn et al. (2007) introduced the microfluidic channel
method. A stream of lipids dissolved in alcohol is focused between two aqueous streams in
order to form liposomes at the liquid interfaces. The heating method was published by
Mozafari et al. (2002). The technique has the advantage of not using any organic solvents
or detergents. Lipids are hydrated in an aqueous solution containing 3 % (v/v) glycerol
before heating the solution to 120 °C. Castor (1998) presented an injection and decompres-
sion method, which works with dense gases. This method includes, for example, the mixing
of lipids, a hydrophobic drug and a supercritical fluid followed by an injection into an
aqueous medium to form liposomes (Frederiksen et al., 1997). Otake et al. (2001) intro-
duced a supercritical reverse phase evaporation process, which is related to the decompres-
sion method of Castor (1998). Lipid, supercritical carbon dioxide and ethanol are stirred
and the aqueous phase is added under subsequent pressure reduction. Another technique
for the preparation of small amounts of liposomes is the dual asymmetric centrifugation
LIPOSOMES 9
(Massing et al., 2008). Further methods, e.g. for the selective production of large unilamel-
lar vesicles (LUV), are listed in the review of Szoka Jr and Papahadjopoulos (1980).
2.2.2 Preparation of Liposomes with a Defined Size
Some of the methods described in the previous chapter have the ability to produce small
unilamellar vesicles (SUV), for example the detergent removal technique or the microflu-
idic channel method, whereas the commonly used round bottom flask method results in
multilamellar vesicles (MLVs). However, various applications require liposomes of a de-
fined size, uniform appearance and narrow size distribution.
A simple approach to reduce liposome size is the vortexing of the rehydrated lipid film.
Another mechanical method of reducing liposome size is the sonication under an inert gas
atmosphere. Two different devices can be used. Probe sonication (Huang, 1969) is one
option, but has an essential disadvantage: Metal particles from the probe tip, which end up
in the liposome suspension during sonication, have to be removed by centrifugation after-
wards (Hamilton et al., 1980). An alternative is the use of a bath type sonicator (Woodbury
et al., 2006).
In a study of Brandl et al. (1990) small unilamellar vesicles with a narrow size distribution
and a size of 25 – 50 nm were produced through a high-pressure homogenizer. Barnadas-
Rodrıguez and Sabés (2001) characterized the factors which affect the liposome size when
using a high-pressure homogenizer. They found that the pressure, the number of cycles and
the bulk ionic strength have an impact on the resulting vesicles.
The French press extrusion is another technique of generating liposomes of a defined size.
Hamilton et al. (1980) used a French pressure cell to extrude liposomes at
20 000 psi through a small orifice. After only a single pass, 70 % of the liposomes built a
homogenous population.
In the laboratory scale, there are two frequently used techniques: the above mentioned son-
ication techniques and filter extrusion through polycarbonate membranes with defined pore
sizes. In this discontinuous process the liposome suspension is extruded several times
through a membrane with a defined pore size. Olson et al. (1979) used polycarbonate mem-
branes of different pore sizes to diminish liposomes starting with a 1.0 µm membrane and
going down to a 0.2 µm membrane. The resulting vesicles had a size of 0.27 µm. In order
10 LIPOSOMES
to perform a smooth extrusion it is fundamental to adjust the temperature of the liposome
dispersion above the phase transition temperature (Tm; see 2.3.1) of the lipids. Otherwise
the membrane clogs and finally breaks. Berger et al. (2001) reported that extrusion through
big filter pores, e.g. 800 nm, results in vesicles smaller than the pore size, whereas small
pore sizes, e.g. 100 nm, results in liposomes, which are somewhat bigger than the pore size.
This effect can be explained by the elastic behavior of liposomes. Another determining
factor is the number of extrusions. With increasing extrusion steps through the same mem-
brane the size distribution becomes more uniform (Cullis, 1986). A disadvantage of classi-
cal extrusion procedures is the limited batch size, but there are many approaches to circum-
vent this problem. One of these approaches is the proposal of Sachse (1995). He used high
pressures and a series of staggered membranes with decreasing pore sizes. Wiggenhorn et
al. (2010) presented a single-pass process using a porous device for the extrusion of the
crude liposome dispersion, which was followed downstream by a nozzle. Subsequently,
droplets were dried through spray drying or spray freeze drying.
2.2.3 Drug Loading
There are two different procedures for the encapsulation of active ingredients into lipo-
somes, passive trapping and active trapping. Passive trapping is an easy approach, which is
based on the ability of liposomes to capture a certain aqueous volume during vesicle for-
mation (Mayer et al., 1986). Large unilamellar vesicles (LUV) and multilamellar vesicles
(MLV) are able to encapsulate more bioactive agents than small unilamellar vesicles
(SUV), since they have a larger internal volume. An increase in phospholipid concentration
leads to an increased number of vesicles formed per volume unit. As a result, the encapsu-
lation efficiency increases.
However, this method has two main disadvantages. First, the encapsulation efficiencies are
low and second, rapid drug leakage may occur especially when working with hydrophilic
drugs. Mayer et al. (1985) presented the freezing-and-thawing technique, which includes
several freezing and thawing steps of multilamellar liposome suspensions (MLVs) in order
to obtain higher percentages of capture. The increasing encapsulation efficiency achieved
with this method can be explained by an increasing distance between the bilayers after the
treatment and hence a bigger liposome volume and better drug penetration.
LIPOSOMES 11
Active trapping methods are based on the fact that lipophilic, non-charged molecules can
easily penetrate the lipid bilayer and charged, ionized molecules are not able to pass the
membrane. The equilibrium between the non-charged penetrating form and the charged
form is defined by the pKa of the molecule and the surrounding pH value. If, for example,
a penetrating, non-charged weak base reaches an inner liposome compartment with a low
pH, it is possible to trap the molecule, because it is ionized in the inner compartment and
is no longer able to cross the bilayer (Gubernator, 2011). This phenomena is the basis of
many procedures for active loading of drugs into liposomes (Bally et al., 1988, Goldbach
et al., 1993b).
The remote loading procedure or so called pH gradient method was described by Dos
Santos et al. (2004) for doxorubicin, an anticancer drug. A lipid film was rehydrated by a
citrate buffer (pH 4.0) before the multilamellar liposomes were extruded through a poly-
carbonate membrane in order to obtain the desired size. Subsequently, the liposomes were
passed through a Sephadex column equilibrated with HEPES (pH 7.4) to exchange the ex-
ternal buffer. After elution the liposomes had a pH gradient > 3.0. Finally, the drug and
ethanol, which in this case was used for an enhanced penetration, was added to load the
liposomes. More than 90 % of the added doxorubicin was encapsulated after 2 hours.
2.3 Liposome Composition
2.3.1 Phospholipids
A variety of lipids is available for the preparation of liposomes. A study of Grazia Calvagno
et al. (2007) showed that the vesicle size, size distribution, zeta potential as well as drug
release, loading capacity and intracellular uptake are influenced by the lipid composition
and preparation method. Although phospholipids are the most commonly used lipids, other
amphiphile molecules such as sphingomyelin, lysophophatides or single chain amphiphiles
may also be possible candidates for liposome formulations. Phospholipids possess a hydro-
philic head group and two fatty acid tails, which are hydrophobic. They consist of glycerol,
which is esterified with two fatty acids building the hydrophobic part and in the third posi-
tion with phosphoric acid. The second acidic group of the phosphoric acid is usually also
esterified with a short chain alcohol. Figure 2-4 gives the chemical structure and possible
head groups. In addition to varying head groups, the fatty acid chains differ in length and
12 LIPOSOMES
degree of unsaturation (Braun-Falco, 1992). When phospholipids or mixtures of phospho-
lipids are hydrated in excess water, they form at least two different lamellar phases. The
first one is the gel phase, which occurs at lower temperatures and in which the molecules
are packed in a quasi-crystalline two-dimensional lattice (Blume, 1991). The second lamel-
lar phase is the fluid liquid-crystalline phase, in which lipid molecules are able to diffuse
rapidly in the plane of the lipid bilayer. In addition, they are able to perform fast rotational
diffusion and trans-gauche isomerization of the fatty acyl chains. The characteristic tem-
perature, at which the gel to liquid-crystalline transition occurs, is called the phase transi-
tion temperature (Tm). With increasing chain lengths the phase transition temperature in-
creases. Unsaturated or branched fatty acid chains in phospholipids decrease the phase tran-
sition temperature. In addition, the head groups also have an impact on Tm. Phophatidylacid
Figure 2-3: Head groups of phospholipids. The following groups may be linked to the phospha-
tidyl moiety: PA = acid; PI = inositol; PS = serine; PE = ethanolamine; PC = choline (taken from
Henry et al. (2012)).
LIPOSOMES 13
and phosphatidylethanolamine head groups are able to form intermolecular hydrogen bonds
and thus have higher phase transition temperatures. In the liquid-crystalline state, which is
the relevant state for almost all biological membranes, the permeability of the bilayer is
increased (Braun-Falco, 1992). Permeability maxima occur in the vicinity of Tm (Kanehisa
and Tsong, 1978).
2.3.2 Cholesterol
Cholesterol is a component of eukaryotic membranes and is used in many liposome formu-
lations. When integrated in a membrane, the hydroxyl group is positioned beside the car-
boxyl groups of the ester linkages in the phospholipids and therefore alters the membrane
properties. The rigid steroid structure creates space between the fatty acid chains of the
phospholipids, while reducing their freedom of motion (New, 1989). The insertion of cho-
lesterol into a membrane, which is arranged in the gel phase, leads to a more fluid mem-
brane, while the incorporation into a liquid-crystalline bilayer results in a condensed mem-
brane with a decreased fluidity.
Cholesterol incorporation has only a marginal effect on the phase transition temperature,
but a major effect on the heat of transition. With increasing cholesterol concentrations the
transition enthalpy decreases. A membrane consisting of linear saturated phosphatidylcho-
lines showed phase transitions until a level of 50 mol% cholesterol is reached (McMullen
et al., 1993).
Figure 2-4: Chemical structure of cholesterol (Caslab, 2015). The hydroxyl group is oriented to-
wards the aqueous surface when embedded in a membrane.
14 LIPOSOMES
2.4 Conventional Liposomes
In contrast to the long-circulating liposomes or the liposomes for gene delivery, the “con-
ventional liposomes” have no further surface modification. Neutrally or negatively charged
molecules and frequently cholesterol are used for their formulation. Regarding the ap-
proved and emerging liposome formulations of the last years (Immordino et al., 2006), their
application as a vehicle for anticancer drugs is the main focus in liposome research. Lipo-
somes can passively accumulate inside tissues, especially solid tumors, due to the fact that
blood capillaries, which supply the tumor, have a discontinuous endothelium. This effect
is called passive targeting. Liposomes take advantage of the missing tight junctions and
consequently are able to accumulate inside the concerned tissue. The discontinuous endo-
thelium is only one factor, which contributes to the enhanced permeability and retention
effect (EPR) of macromolecules and lipophilic particles found in solid tumors (Maeda et
al., 2001).
However, a discontinuous endothelium is not unique to solid tumors, but is also found in
organs, which belong to the mononuclear phagocyte system (MPS). Among these organs
are the liver, spleen and bone marrow. The MPS rapidly captures intravenously adminis-
tered liposomes and removes them from blood circulation, which leads to a short half-life
(Scherphof et al., 1985). In addition, the rapid clearance by the MPS limits the time for
accumulation in the desired tissues. The rapid liposome uptake by the MPS can be used for
the therapy of macrophage-based infections, for example leishmaniasis (Alving et al.,
1978). The liposome clearance of the MPS is preceded by an opsonization of the liposome
surface or by an initiated membrane lysis by the complement system. These two processes
are required for the liposome recognition by the MPS. Another half-time decreasing event
is the interaction with high (HDL) and low density (LDL) lipoproteins. Several studies
revealed that the physicochemical properties of liposomes affect the clearance and therefore
the half-time. Cholesterol incorporation was shown to reduce the transfer of phospholipids
to HDL (Damen et al., 1981). Harashima et al. (1994) demonstrated that the opsonization
is size dependent, with small vesicles being less opsonized than larger liposomes. Besides
cholesterol incorporation and vesicle size, the surface charge is a contributing factor. Neg-
atively charged liposomes activated the complement via the classical pathway, whereas
positively charged vesicles used the alternative pathway (Chonn et al., 1991). In addition,
LIPOSOMES 15
the study revealed that saturated fatty acid chains increase liposome stability in blood.
There are several successful drug formulations, which are based on the improvements men-
tioned in avoiding MPS uptake. These formulations are described more detailed in 2.8.4.
2.5 Long-circulating Liposomes
The above mentioned rapid uptake of liposomes by the MPS motivated the research to
study liposomes with a modified surface in order to avoid MPS clearance and increase half-
life of lipid vesicles. First studies were performed with monoganglioside (GM1), which
was incorporated on the liposome surface in order to mimic the erythrocyte membrane.
GM1 liposomes showed reduced MPS uptake and hence extended liposome half-life (Allen
et al., 1989). Liposome surface coverage with poly-(ethylene glycol) has been widely stud-
ied over the last years, with PEGylated liposomes showing significantly reduced MPS up-
take (Klibanov et al., 1990). PEG can be easily attached on the liposome surface by incor-
Figure 2-5: Schematic draw of a PEG-grafted liposome with different PEG configurations
(taken from Immordino et al. (2006)). At least 4 mol% PEG-2000-lipid is needed to form a “poly-
mer brush” (Kenworthy et al., 1995a).
16 LIPOSOMES
poration of PEG-lipid conjugates into the bilayer membrane. Hydrophilic PEG chains of
different lengths are commonly linked with ethanolamine carrying phospholipids. Possible
PEG configurations are shown in Figure 2-5. The configuration type depends on the molar
ratio of PEG-lipid conjugates used (Kenworthy et al., 1995a). The desired configuration is
the “polymer brush”, which is the cause of the Stealth behavior. In vivo, PEGylated lipo-
somes (“cryptosomes”) are inaccessible to macromolecules due to their steric hindrance
induced by the PEG-chains (Blume and Cevc, 1993). Thereby PEGylated liposomes do not
undergo opsonization. The work of Needham et al. (1992) showed that there is a strong
repulsive pressure between PEG-covered membranes, which originates mainly from a ste-
ric pressure. On this account, PEGylated liposomes tend to show decreased aggregation.
Moreover, Needham et al. (1992) postulated that the grafted polymer extended about 50 Å
from the liposome surface.
Targeting moieties such as monoclonal antibodies, peptides or receptor ligands can be cou-
pled to the PEG chains in order to create liposomes, which are more selectively accumu-
lated in a certain tissue. After the accumulation of liposomes in a tissue, it is important to
enable the release of the encapsulated drug. For this purpose, pH sensitive liposomes are
under investigation, because pathological tissues (for example tumors) exhibit a lower pH
value (Torchilin et al., 1993). These liposomes are stable under physiological pH and are
able to release their content as soon as they are exposed to lower pH values.
LIPOSOMES 17
2.6 Liposomes in Gene Delivery
Gene delivery is the process of introducing genetic material into cells. This operation is
also called gene therapy, if cells are treated, which suffer from genetic dysfunctions (Stone,
2010). Diseases, for example cystic fibrosis, which are possible candidates for gene ther-
apy, are itemized in a review of Verma and Somia (1997). Nayerossadat et al. (2012) listed
different vector systems for gene delivery, among them viral vectors such as herpes simplex
virus and adeno-associated vectors or non-viral delivery systems such as the so-called gene
gun or the naked DNA method. Viral vectors are the most frequently used delivery systems,
as viruses can be easily modified in order to create relatively safe vectors for gene delivery.
Therefore, essential genes for the replication are removed and replaced by the therapeutic
gene (Gardlík et al., 2005).
Positively charged liposomes are used as non-viral vectors for gene delivery. They are sim-
ple and quick to formulate, cheaper and not biologically hazardous (Dass, 2002, Torchilin,
2005). Due to their positive charge they naturally form complexes with the negatively
charged DNA, so-called lipoplexes (Verma and Somia, 1997). Lipoplexes consist of a pos-
itively charged lipid and a neutral helper lipid, which is frequently dioleoylphosphatidyl-
ethanolamine (DOPE). The neutral lipid stabilizes the liposome and enables membrane fu-
sion and therefore the release of the DNA (Felgner et al., 1994, Zuidam and Barenholz,
1998). Frequently used positively charged lipids are DOTMA (1,2-di-O-octadecenyl-3-tri-
methylammonium propane, Lipofectin®), DOTAP (1,2-dioleoyl-3-trimethylammonium-
propane) and DC-Chol (3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hy-
drochloride). Lipoplexes enter cells via endocytosis followed by the release of the en-
trapped DNA and the uptake into the nucleus (Cao et al., 2000). A disadvantage of lipo-
plexes is the toxicity, which is dependent on the lipid species, the charge ratios and the dose
administrated (Dass et al., 2002).
18 LIPOSOMES
2.7 Liposomes as Carriers of Proteins and Peptides
2.7.1 Vaccines
Liposomes are used as carriers for antigens, which are usually peptides or proteins. Anti-
gens can be entrapped in the aqueous liposome core or attached to the liposome surface.
As already reported in the 70s, liposomes are immunological adjuvants, which are able to
elicit higher antibody levels compared to the free antigens (Allison and Gregoriadis, 1974).
Watson et al. (2012) listed some liposome or lipid based vaccines, which are approved for
human use or in clinical trials. This review also emphasizes the influence of liposome com-
position and therefore liposome charge, membrane fluidity and antigen attachment on the
resulting immune response. As described in chapter 2.6, positively charged liposomes can
be used as a transfection reagent. They are also appropriate co-adjuvants for antigens, be-
cause high densities of positive charges are warning signals for cells and trigger several
pathways of immune response (Lonez et al., 2012).
Virosomes are liposomes, which are decorated with viral proteins or viral envelopes and
accordingly acquire viral functions such as fusion, receptor-binding and tissue tropism
(Kaneda, 2000). Virosomes are used both for DNA delivery and for vaccines.
2.7.2 Further encapsulated Proteins and Peptides
Liposome-encapsulated enzymes have the potential to be delivered directly to their site of
action while reducing side effects or protecting the encapsulated enzyme from degradation.
One example of this approach is described by Gaspar et al. (1996). They encapsulated L-
asparaginase, which is used for the removal of L-asparagine from the blood in cancer treat-
ments of tumors, whose cells are not able to synthesize L-asparagine by themselves. How-
ever, the usual treatment with L-asparaginase is limited by allergic side reactions. Liposo-
mal L-asparaginase showed a decreased toxicity and increased mean residence times, when
using small liposomes for the encapsulation. Further studies with liposomal enzymes were
performed with superoxide dismutase (SOD; Stanimirovic et al. (1994)) and tissue plas-
minogen activator (Heeremans et al., 1995). SOD containing liposomes were spray dried
(Lo et al., 2004). For further information refer to chapter 3.4.
LIPOSOMES 19
Extensive research was done on liposomal insulin. As already described in chapter 2.4, the
liver, which is one site of action for insulin, is a natural target of liposomes. In addition,
liposomal insulin shows an increased half-life and an enhanced oral bioavailability. The
latter promotes research with the aim of an orally available insulin drug. Therefore, lipo-
somes were coated with mucin or PEG in order to protect the encapsulated insulin from
degradation under the aggressive conditions of the gastro-intestinal tract and to enhance the
absorption of insulin (Iwanaga et al., 1997). One of many other ideas was to use deformable
vesicles for the buccal delivery of insulin (Yang et al., 2002). Although the results of the
mentioned studies are encouraging, the oral delivery of insulin remains a challenge as there
are still too many factors affecting the bioavailability (Torchilin, 2005).
2.8 Liposome Formulations
2.8.1 Stability of Liposomes
Liposome stability is a key parameter in the development of liposomal formulations. In
general, possible instabilities concerning liposomes are distinguished between chemical
and physical degradations. An overview is given in Table 2-1.
Oxidation of phospholipids is mainly related to unsaturated phospholipids, while polyun-
saturated fatty acids (PUFA) are the most susceptible to undergo peroxidation. The free
radical chain mechanism of phospholipid oxidation starts with the abstraction of a hydrogen
atom and is caused by the exposure to electromagnetic radiation or the contamination with
transition metal ions. If oxygen is present, the process can continue with a formation of
hydroperoxides, which may trigger the fission of fatty acid chains (New, 1989). If it is
necessary to use unsaturated phospholipids, oxidation can be minimized by the use of high
Chemical degradation Physical instabilities
Oxidation of phospholipids Size changes
Hydrolysis of phospholipids Size distribution changes
Aggregation and fusion
Drug leakage
Table 2-1: Overview of possible stability problems.
20 LIPOSOMES
quality raw materials, the exclusion from oxygen and light during storage, the storage at
low temperatures (Grit and Crommelin, 1993) and the addition of α-tocopherol (Hunt and
Tsang, 1981), butyl hydroxyl toluene (Agarwal et al., 2001) or EDTA, which is used for
the chelation of catalyzing heavy metals (Thoma and Jocham, 1992). Hydrolysis of phos-
pholipids mainly concerns the ester bond between the fatty acid and the glycerol group in
the phospholipid molecule (see Figure 2-3) and results in the formation of lyso-phospho-
lipids and free fatty acids. The rate of hydrolysis is strongly pH-dependent and is signifi-
cantly increased at high temperatures. The selection of an appropriate buffer and a low
buffer concentration can increase stability (Grit et al., 1989, Grit and Crommelin, 1993).
Physical instabilities listed in Table 2-1 are interconnected, because, for example, size or
size distribution changes are often related to the fusion of liposomes. All physical instabil-
ities can be promoted by previously proceeding chemical degradations. The fusion of lipo-
some bilayers especially occurs when the bilayer is uncharged and not PEGylated. Charged
phospholipids, such as the positively charged DOTAP or the negatively charged phospha-
tidic acid (PA), phosphatidylglycerol (PG) or phosphatiydylserine (PS), can be incorpo-
rated in the membrane to overcome this problem (New, 1989). Crommelin (1984) showed
that an increased charge density on the liposome surface increased the zeta potential values.
In contrast, higher ionic strengths decreased the zeta potential. In this study, the liposome
stability was compared with the DLVO theory (Derjaguin-Landau-Verwey-Overbeek),
which serves as a model for the prediction of aggregation behavior of colloidal systems.
The results were to a large extend in accordance with the DLVO calculations with higher
zeta potentials leading to more stable systems.
As described earlier (chapter 2.3.1), fluid-crystalline membranes show a higher permeabil-
ity and therefore a higher drug leakage in comparison to membranes, which are in the gel
phase. This behavior was confirmed in a study of Crommelin and van Bommel (1984). The
addition of cholesterol to fluid-crystalline membranes reduced the leakage of the marker
substance carboxyfluorescein, while the addition to the gel phase brought no significant
improvements.
LIPOSOMES 21
2.8.2 Stabilization of Liposomes via Drying
Stability problems of liposomes are often related to the presence of water. In addition, the
drugs encapsulated are frequently expensive and challenging molecules such as proteins
and peptides, which need to be protected from water themselves. Here, the removal of water
might be advantageous resulting in an improved stability. Moreover, solid formulations
usually require no “cold chain” during distribution and have an increased shelf life, which
reduces product costs.
There are a few drying technologies available, with freeze drying being the most commonly
used method. Further technologies are spray drying, spray freeze drying and the supercriti-
cal fluid technology (Ingvarsson et al., 2011). Spray drying of liposomes is specified in
chapter 3.4.
Without the addition of further excipients, liposomes aggregate and fuse during drying.
After rehydration the formulation has not much in common with the original formulation
regarding drug encapsulation or vesicle size. Hence it is important to add excipients, which
are able to protect liposome membranes and their integrity during drying and rehydration.
The most common and efficient excipients are disaccharides such as trehalose or sucrose
(Leslie et al., 1995). Their effect on the lipid bilayer during drying is shown in Figure 2-6.
The water replacement theory is the first theory to describe the stabilizing effect of disac-
charides on membrane bilayers (Crowe et al., 1996a). In the solid state, sugars replace water
molecules and their hydrogen bonds. The phase transition temperature (Tm) usually in-
creases during drying due to missing water molecules and increased van der Waals inter-
actions. However, if sugars are added, Tm is not affected or even decreases during dehydra-
tion. Sugar molecules enlarge the distance between the phospholipid head groups (cf. Fig-
ure 2-6) and weaken the van der Waals interactions (Ohtake et al., 2005). This effect is
particularly important for liposome formulations with a low Tm, because these formulations
undergo phase transitions during drying and rehydration without stabilizing excipients
(Crowe et al., 1985). The pass through Tm has been identified as one of the most destabi-
lizing effects regarding drug retention (Crowe et al., 1986).
The vitrification theory deals with the formation of a highly viscous, concentrated solution
of carbohydrates, which finally builds a viscous glassy matrix surrounding the liposomes
(Koster et al., 1994). This glassy state provides a low mobility, which prevents liposomes
22 LIPOSOMES
from aggregation and fusion or conformational changes. The temperature, where the highly
viscous, glass-like structure changes to a state of decreased viscosity, is called the glass
transition temperature (Tg). This temperature is dependent on the sugar used (Wolkers et
al., 2004) and on the residual moisture content. Tg is important for the definition of storage
conditions, which should be well below Tg. For example, a study of Sun et al. (1996)
showed that the half-life of marker retention could be prolonged by storing the freeze-dried
formulation of liposomes and sucrose below the Tg.
Figure 2-6: Schematic draw of the stabilization mechanisms of sugars (e.g. trehalose). Dehydra-
tion without excipients results in decreased head group spacing and drug leakage (taken from
Ingvarsson et al. (2011)).
LIPOSOMES 23
2.8.3 Freeze Drying of Liposomes
Lyophilization is a technique, which is widely used for the stabilization of biopharmaceu-
ticals such as monoclonal antibodies or vaccines. Freeze drying is a multistage process,
which usually includes a freezing step, a primary drying step applying vacuum below
1 mbar and low temperatures and the secondary drying. All three steps influence the final
product and therefore have an impact on the liposome stability during drying. In addition,
factors like vial type, filling volume, selection of excipients, lyoprotectant concentration
(Crowe et al., 1985), vesicle size (Crowe and Crowe, 1988) etc. contribute to the product
quality.
Generally, the freezing rate during the freezing step influences the ice crystal size. Slow
freezing results in large, less uniform ice crystals, whereas quick freezing leads to small ice
crystals. Big ice crystals have the advantage of a faster sublimation during primary drying,
whereas finer ice crystals produce highly ordered structures and are less detrimental to the
membrane integrity (Chen et al., 2010). In a study of van Winden et al. (1997) the impact
of slow and quick freezing was investigated. They pointed out that a successful drug reten-
tion during freeze drying is mainly dependent on the lipid bilayer composition. Addition-
ally, a slow freezing rate is beneficial. One explanation was the enhanced membrane re-
covery from deformations during slow freezing. These deformations are caused by osmotic
pressures, which are produced by freeze-concentration (Siow et al., 2007).
During primary drying, the product temperature needs to be kept below the collapse tem-
perature (Tc). A compromise between an economical process time and a stable product
needs to be found (Tang and Pikal, 2004). The primary and secondary drying phases have
a minor effect on liposome integrity (Ingvarsson et al., 2011). However, these phases are
important for the achievement of a low residual moisture content, which affects the Tg of
the formulation and therefore the long term stability of liposomes (van Winden and
Crommelin, 1997). The last-mentioned authors freeze dried liposomes with the lyoprotect-
ants lactose, sucrose, trehalose and maltose. The long term stability data revealed that sam-
ples with a moisture content below 1 % were stable upon storage at temperatures up to
30 °C.
24 LIPOSOMES
2.8.4 Overview of approved Liposome Formulations
Product name Drug Drug form Lipid
composition Excipients
Abelcet Amphotericin B Suspension DMPC, DMPG Sodium chloride, water
for injection
Ambisome Amphotericin B Powder,
freeze dried
HSPC, DSPG,
cholesterol
Alpha tocopherol, su-
crose, disodium succinate
hexahydrate, sodium hy-
droxide, hydrochloric
acid
Amphotec Amphotericin B Powder,
freeze dried
Cholesteryl sul-
fate
Tromethamine, lactose
monohydrate, EDTA, hy-
drochloric acid
DaunoXome Daunorubicin Emulsion DSPC, choles-
terol
Citric acid, sucrose, gly-
cine, calcium chloride,
water for injection
DepoCyt Cytarabine Suspension
DOPC, DPPG,
cholesterol, trio-
lein
Sodium chloride, water
for injection
DepoDur Morphine sulfate Suspension
DOPC, choles-
terol, DPPG, tri-
olein
Sodium chloride, tri-
caprylin
Doxil/Caelyx/
Lipo-Dox Doxorubicin Suspension
HSPC, choles-
terol, PEG-2000-
DSPE
Ammonium sulphate, su-
crose, histidine, hydro-
chloric acid, sodium hy-
droxide, water for injec-
tion
Epaxal Inactivated hepa-
titis A virus Suspension Lecithin, cephalin
Sodium chloride, water
for injection
Inflexal V
Inactivated he-
maglutinine of
influenza virus
strains A and B
Suspension Lecithin
Sodium chloride, diso-
dium phosphate dehy-
drate, potassium dihydro-
gen phosphate, water for
injection
Marqibo Vincristine
Suspension,
prepared by
Marqibo Kit
on site
Egg SM, choles-
terol
Sodium citrate, mannitol,
citric acid, sodium phos-
phate, sodium chloride
Myocet Doxorubicin
Suspension,
prepared on
site
PC, cholesterol
Lactose, citric acid, so-
dium hydroxide, sodium
carbonate, water for in-
jection
Visudyne Verteporfin Powder,
freeze dried
Egg PG, DMPC,
ascorbyl palmi-
tate
Lactose monohydrate,
butylated hydroxytoluene
Mepact Mifamurtid Powder,
freeze dried POPC, DOPS –
Table 2-2: Overview of approved liposomal drugs (Rote Liste (2014); Chang and Yeh (2012)).
This list is not exhaustive.
LIPOSOMES 25
In 1995, the first liposome based drug, Doxil®, entered the market (Fan and Zhang, 2013).
Today, there are about 13 liposomal drugs on the market and there is a pipeline of promising
liposomal drugs, which are in clinical trials (Chang and Yeh, 2012). Table 2-2 gives an
overview of approved liposomal drugs and their lipid compositions as well as the whole
formulations. Several liposomal drugs are exemplified in the following section:
Amphotericin B is an antifungal drug and is used for the treatment of invasive
fungal infections. However, treatment is limited due to a dose-dependent ne-
phrotoxicity. Nephrotoxicity of liposomal amphotericin is significantly lowered and
makes the drug more effective. An explanation is the preferential binding to high
density lipoproteins, which promotes the uptake in the reticuloendothelial system.
Thereby the active ingredient concentration in the kidneys is lowered (Walsh et al.,
2001).
Like the majority of liposomal drugs, doxorubicin is an anticancer drug.
Doxil®/Caelyx® contains long-circulating liposomes (chapter 2.5), which use the
principle of passive targeting (chapter 2.4). The concentration of free doxorubicin
in the plasma is lowered. Hence the (cardio)toxicity is reduced (O’brien et al.,
2004).
Verteporfin (Visudyne) is a drug for the treatment of age-related macular degen-
eration. The hydrophobic drug tends to self-aggregate, which is disadvantageous
for the bioavailability. Therefore, it is encapsulated in liposomes (Chang and Yeh,
2012).
Some of the formulations are freeze-dried with lactose or sucrose being the lyoprotectant.
Further excipients are buffer salts and substances for the pH adjustment. Lyophilized pow-
ders must be dissolved in water for injection or physiological saline before administration.
Myocet® and Marqibo® are prepared on site, which means that empty liposomes, the active
ingredient and a buffer are delivered in separate vials and have to be mixed under heating
prior to administration.
3 SPRAY DRYING
3.1 Introduction to Spray Drying
Spray drying is the transformation of a given fluid feedstock into a dried particulate form
by spraying the feed into a hot drying medium. The technique of spray drying is used in
many industries, for example for the production of instant coffee or soups in powdered
form. Spray dried agrochemicals, clay and mineral ores, which demand high-tonnage out-
puts in the continuous spray drying process, are further examples (Masters, 2002).
One advantage compared to other drying technologies is the potential to design the resulting
product in terms of the particle size, shape, density and moisture content (Chow et al.,
2007). This fact is taken into account in pharmaceutics, as it offers a way to produce very
small particles, which are, for example, suitable for the pulmonary delivery of drugs
(Malcolmson and Embleton, 1998).
Figure 3-1 shows a typical setup of a basic spray dryer. The liquid feedstock can be a solu-
tion, suspension, emulsion or fluid paste and has to be pumpable. The first step in spray
drying is the atomization of the liquid feedstock through a nozzle followed by the spray-
gas-mixing in the drying chamber. The droplet size decreases with proceeding evaporation
until the droplets finally turn into particles. These are further dried until they are separated
from the drying gas using a cyclone or a bag-filter (Masters, 2002).
The pictured spray dryer operates in a co-current mode, which implies the same flow di-
rection for the atomized droplets and the drying gas. Further setups are the counter-current
mode and the mixed-flow mode (Cal and Sollohub, 2010). A co-current mode is beneficial
for heat-sensitive substances, because the atomized droplets encounter the hottest drying
gas, whereas already dried particles are exposed to the chilled and humidified gas. Conse-
quently, heat-sensitive materials are cooled due to increased evaporation rates at the begin-
ning of the drying process. This features the co-current design as a relatively mild process
28 SPRAY DRYING
(Broadhead et al., 1992). In contrast, the counter-current mode is more economic and there-
fore used for the large-scale production of heat-resistant substances (Masters, 2002).
Depending on the solvent to be removed, there are spray dryers, which operate in an open
cycle mode or closed cycle mode. The customary design is open cycle, because an aqueous
feedstock is used more frequently. It is possible to remove organic solvents with a closed
cycle mode, which is equipped with a condenser for the collection of removed solvent
(Masters, 2002, Masters, 1999). In this case, the spray dryer is driven with an inert gas, for
example nitrogen, which circulates through the system (Crisp et al., 1991). This closed
setup is also suitable for the drying of oxygen-sensitive materials and hazardous substances
(Masters, 2002).
Figure 3-1: Setup of a basic spray dryer (Masters, 2002).
SPRAY DRYING 29
3.2 Atomization
Figure 3-2: Schematic presentation of the liquid breakup during atomization, exemplified ac-
cording to Hede et al. (2008).
Atomization is the disintegration of a liquid jet or sheet by the kinetic energy of the liquid
itself, by the exposure to high-velocity air or gas or by the application of mechanical energy
through a rotating or vibrating device (Lefebvre, 1988). The mechanism of the liquid
breakup into droplets is shown in Figure 3-2. The atomization device prepares the liquid
feed as a thin film, which breaks up into ligaments. These ligaments finally disintegrate and
form drops, which have preferably a narrow size distribution (Lefebvre, 1988, Masters,
2002). Viscosity and surface tension influence the atomization process.
Different devices are available for the atomization process:
Rotary atomizers
Pressure nozzles
Pneumatic nozzles
Ultrasonic nozzles
Table 3-1 gives suitable mean droplet sizes, which are achieved under normal operating
conditions using the different nozzle types.
30 SPRAY DRYING
Nozzle type Mean droplet size [µm]
Rotary atomizer 20 – 200
Pressure nozzle 50 – 400
Pneumatic nozzle 5 – 75
Sonic nozzle 10 – 50
Table 3-1: Mean droplet sizes of different atomization devices according to Masters (2002).
In a rotary atomizer, the liquid feedstock is supplied to a horizontally spinning wheel or
disk and the liquid film is catapulted to the edge of the disk by centrifugal forces, where it
is atomized into droplets. The particle size can be controlled by the adjustment of the rota-
tional speed of the disk. Rotary atomizers are suitable for the large-scale production as it is
possible to atomize up to 200000 l/h (Schiffter, 2012). However, the centrifugal accelera-
tion of the droplets results in a broad spray pattern and high wall depositions, which makes
this devices not applicable to bench-top spray dryers. Their drying chambers are usually
too small (Huang et al., 2006).
Pressure nozzles, also known as one-fluid nozzles or hydraulic nozzles, work on the prin-
ciple of pressure energy conversion into kinetic energy. The built-in swirl chamber provides
a rotational, air-cored motion of the liquid, which results in a hollow-cone spray pattern
(Datta and Som, 2000, Masters, 2002). In contrast to rotary atomizers, pressure nozzles
exhibit the risk of clogging or fast abrasion and are not able to atomize high-viscosity feeds
(Cal and Sollohub, 2010).
Pneumatic nozzles operate through the atomization of a liquid feed in a stream of com-
pressed gas. Frictional forces disintegrate the liquid into droplets. Pneumatic nozzles are
also known as two fluid nozzles, which arises from the fact that two streams are existent –
a gas stream and the liquid feed stream. The gas stream is pressurized to 1.5 – 10 bar
(Schiffter, 2012). Two basic types of pneumatic nozzles can be distinguished (Figure 3-3):
Nozzles with the mixing zone inside the nozzle (left) and outside the nozzle (right). Internal
mixing has the advantage of a more efficient energy transfer, whereas external mixing per-
mits an independent control of both liquid and air streams (Masters, 2002, Hede et al.,
2008). A benefit of pneumatic nozzles is their flexibility regarding droplet size, type of
feedstock and spray pattern, which is defined through the orifice type (Hede et al., 2008).
SPRAY DRYING 31
Droplet size achieved by pneumatic atomizers is predictable according to Kim and Marshall
(1971):
𝐷 =𝑎
(𝑣𝑟𝑒𝑙2 𝜌𝑎)𝛼
+ 𝑏 (𝑀𝑎
𝑀𝑙)
𝛽
Equation 3-1
The droplet size (𝐷) is dependent on the relative velocity (𝑣𝑟𝑒𝑙) between air and liquid, the
spray gas density (𝜌𝑎) and the mass flow ratio of air to liquid (𝑀𝑎/ 𝑀𝑙). α and β are func-
tions of nozzle design, a and b of both nozzle design and liquid properties.
Drawbacks of pneumatic nozzles are the huge amounts of compressed gas needed (Cal and
Sollohub, 2010), which is connected with high costs and a reduction of the drying efficiency
due to the cold gas stream in the drying chamber (Masters, 2002).
Three- and four-fluid nozzles provide the opportunity of combining two separate gas
streams with one or two separate liquid streams. These nozzle types are used either to at-
omize the liquid into very fine droplets or to combine two different solvent streams (e.g.
one organic and one aqueous). The necessity of a common solvent can be omitted, which
is helpful especially for the formulation of water-insoluble drugs. The latter setup, for ex-
ample, is used for the production of nanoparticle-containing microparticles (Mizoe et al.,
2007).
Figure 3-3: Schematic drawing of pneumatic nozzles (taken from Hede et al. (2008)).
32 SPRAY DRYING
A schematic drawing of an ultrasonic nozzle is given in Figure 3-4. A high-frequency elec-
tric signal is applied to the active electrode, which is placed between piezoelectric trans-
ducers. The electric signal triggers vibrations, which are transferred to the titanium nozzle
tip. The nozzle tip boosts the vibrations and forces the liquid feed to atomize (Cal and
Sollohub, 2010, Sono-Tek, 2010). The nozzle frequency determines the droplet size. Vis-
cosity and surface tension have a minor impact on the resulting droplet size compared to
the other nozzle types (Peskin and Raco, 1963, Sono-Tek, 2010). The following equation
(Lang, 1962) gives a correlation between the frequency (𝑓) and the droplet size (𝐷). Higher
frequencies result in smaller droplets:
𝐷 = 0.34 (8 𝜋 𝜎
𝜌 𝑓2)
1/3
Equation 3-2
𝜎 is the surface tension and 𝜌 the liquid density. However, it is important to note that this
simple correlation is applicable only if the liquid flow rate and the liquid viscosity have no
influence on droplet size at all. Further correlations, which consider this fact, are itemized
in a publication of Dalmoro et al. (2013b).
Figure 3-4: Schematic drawing of an ultrasonic nozzle (Sono-Tek, 2010).
SPRAY DRYING 33
As shown in Figure 3-4, ultrasonic nozzles produce a so-called “soft spray”, which means
that the emerging spray has a low velocity. This fact is advantageous regarding wall depo-
sitions in spray drying. The key benefit of ultrasonic nozzles is the narrow droplet size
distribution and the resulting narrow particle size distribution (Topp and Eisenklam, 1972).
The reproducibility is superior to pneumatic atomizers owing to the ability for self-cleaning
and associated non-clogging (Sono-Tek, 2010). In addition, process variables can be con-
trolled independently.
Regarding atomization in general, liquids and dissolved substances suffer from mechanical
energy inputs or the contact with a high-velocity air or gas stream during atomization. This
is of great importance for proteins, peptides and other sensitive substances, which might be
damaged during the atomization process. Shear stress is one risk factor and is known to
alter enzyme kinetics or to cause inactivation, especially in connection with an air interface
(Charm and Wong, 1981).
Ultrasonic nebulization might be damaging to proteins due to the power input, cavitation
effects and the heat production in the nozzle (Niven et al., 1995, Avvaru et al., 2006). Free
radicals might also occur due to ultrasonic vibrations (Bittner and Kissel, 1999). Cavitation
is the appearance of vapour bubbles within the liquid bulk (Franc and Michel, 2006). The
collapse of these bubbles leads to locally high temperatures, pressure and the irradiation of
acoustic shock waves (Sponer, 1990). Vonhoff (2010) examined the nozzle-induced cavi-
tation through the quantification of free radicals generated by cavitation. The nozzle with
the highest frequency (120 kHz) created most free radicals. Increasing power inputs also
led to an increasing number of free radicals. In addition, temperatures measured at the noz-
zle tips were up to 80 °C.
Ultrasound is also applied for the size reduction of liposomes and for the preparation of
small unilamellar liposomes (chapter 2.2.2). Typically, bath sonicators operate at a fre-
quency of 40 kHz and a power rating of 50 W resulting in a power intensity of 1 – 5 W/cm2
at the transducer face (Capote and de Castro, 2007). Power densities achieved with a probe
tip can be up to several hundred watt per square centimeter (Capote and de Castro, 2007).
In contrast, an ultrasonic nozzle, which operates at 25 kHz and a power input of 10 W has
a relatively low power intensity of 0.725 W/cm2 (Dalmoro et al., 2013a). Considering the
residence time of liposomes in the relevant device, which is typically 5 – 10 min (Avanti,
34 SPRAY DRYING
2015) in a bath sonicator or next to a probe tip and ~ 6 s at the nozzle tip (supposing a flow
rate of 1 ml/min and a volume of 100 µl surrounding the nozzle tip), cavitation effects
during atomization through ultrasonic nozzles are low compared to the treatment with bath
sonicators or probe sonicators. However, power density is not the only parameter influenc-
ing cavitation. Capote and de Castro (2007) itemized further parameters such as tempera-
ture, frequency, surface tension, solvent viscosity and solvent vapour pressure.
SPRAY DRYING 35
3.3 Drying
As soon as the spray-gas mixing takes place, evaporation of the volatiles starts.
Figure 3-5 presents the drying process regarding a single droplet. At the beginning, when
droplets enter the drying chamber, they possess the temperature of the liquid feedstock (A).
Next, their surface is heated up to the wet bulb temperature (B), where the surface of the
droplet maintains 100 % relative humidity. These saturated surface conditions are main-
tained by diffusion and capillary flow of moisture from within the droplet to the surface.
The droplet keeps the wet bulb temperature and shrinks due to evaporation (C). The solu-
tion concentrates until there is no more unbound, free surface water. Consequently a crust
forms, when the critical point is reached (D). Further drying is marked by the increasing
crust and therefore decreasing evaporation rates until the end of drying (E) is achieved
(Masters, 2002, Farid, 2003).
Two simultaneous processes are required for a sufficient evaporation: First, the heat trans-
fer from the surrounding drying gas into the droplet, and second, the transfer of vaporized
moisture from the droplet surface through the boundary layer into the surrounding air. The
difference between the vapour pressure at the saturated droplet surface (𝑃𝑤𝑏) and the partial
pressure of vapour in surrounding air (𝑝𝑤) is the driving force of evaporation. Therefore,
Figure 3-5: Different stages of drying according to Farid (2003).
36 SPRAY DRYING
temperature and humidity of the drying air are important parameters. The relative velocity
between droplet and air is another contributing factor. However, droplet velocity and air
velocity can be considered to be equal during the main part of the droplet stay in the drying
chamber. This results in a relative velocity near zero and – according to the boundary-layer
theory – to the following assumption (Masters, 1979):
The evaporation rate 𝑑𝑊/𝑑𝑡 in terms of mass transfer can be expressed
𝑑𝑊
𝑑𝑡= 2𝜋𝐷𝑣𝐷(𝑃𝑤𝑏 − 𝑝𝑤)
Equation 3-3
where 𝐷𝑣 is the diffusion coefficient and 𝐷 the droplet diameter. The presence of dissolved
solids lowers the vapour pressure at the droplet surface and the driving force for mass trans-
fer, respectively.
Figure 3-6 shows the plot of the moisture content versus the drying time, which can be
divided up into two phases. The first phase, the so-called “constant rate”, is characterized
by a constant moisture evaporation under saturated surface conditions. During this drying
step the majority of moisture is removed.
𝑑𝑊
𝑑𝑡=
2𝜋𝐾𝑑𝐷𝑎𝑣∆𝑇
𝜆
Equation 3-4
The evaporation rate 𝑑𝑊/ 𝑑𝑡 in terms of heat transfer during the constant rate is dependent
on the average droplet diameter (𝐷𝑎𝑣), the thermal conductivity (𝐾𝑑), the temperature dif-
ference ∆𝑇 between the droplet surface temperature and the surrounding air and the latent
heat of evaporation (𝜆) (Masters, 1979).
The second phase, the “falling rate”, starts with the first presence of a solid phase at the
critical point. The average rate of evaporation can be expressed
𝑑𝑊
𝑑𝑡=
−12𝐾𝑑∆𝑇
𝜆 𝐷𝑐2𝜌𝑠
Equation 3-5
SPRAY DRYING 37
Figure 3-6: Spray drying kinetics. Modified after Schiffter (2012).
where 𝐷𝑐 is the droplet diameter at the critical point and 𝜌𝑠 the density of solids. The rate
of evaporation eases during the “falling rate”, as the resistance to mass transfer increases
(Masters, 1979). The particles have a solid shell, which gets thicker and encloses a liquid
core (Figure 3-5; D). The heat transfer rate rises above the rate of mass transfer. From now
on, the volume and shape of the particle is unchanged (Kastner et al., 2000). The tempera-
ture of the particle increases and finally reaches the dry bulb temperature of the air (Maa
and Hsu, 1997). This is marked as “sensible heat” in Figure 3-5 due to the possible heat
stress. If the heat exceeds the boiling point of the residual inner liquid, vapour forms and
the pressure inside the particle increases. The nature of the solid shell is of great importance
for the remaining mass transfer and the resulting particle morphology (see chapter 3.4).
Porous crusts enable a higher vapour flow and therefore higher evaporation during the sec-
ond drying step than impervious films on the particle surface. The end of evaporation is
reached, when the moisture content of the particle is in equilibrium with the surrounding
air. However, powders are usually removed from the system before this equilibrium is
achieved (Masters, 1979). Particles leaving the drying chamber possess the temperature
Toutlet of the drying system (Maa and Hsu, 1997).
38 SPRAY DRYING
3.4 Particle Size and Morphology
In general, particle size and morphology is highly specific to the given product, but also to
the course of drying (Sloth et al., 2009). Particles can be spherical or non-spherical, hollow,
solid or collapsed and their surface can possess craters or cracks as well as the surface can
be smooth or rough. These characteristics influence the flowability and aerodynamic be-
havior (Telko and Hickey, 2005) of the spray dried product and must be taken into account
in order to design a suitable spray drying process.
The particle size is highly dependent on the initial droplet size after atomization. The fol-
lowing equation (Dobry, 2015) gives a relationship between the droplet size (𝐷𝑑𝑟𝑜𝑝𝑙𝑒𝑡) and
the resulting powder size (𝐷𝑝𝑜𝑤𝑑𝑒𝑟):
𝐷𝑝𝑜𝑤𝑑𝑒𝑟 = 𝐷𝑑𝑟𝑜𝑝𝑙𝑒𝑡 × √𝑠𝑜𝑙𝑖𝑑𝑠 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛 ×𝜌𝑑𝑟𝑜𝑝𝑙𝑒𝑡
𝜌𝑝𝑜𝑤𝑑𝑒𝑟
𝟑
Equation 3-6
𝜌𝑑𝑟𝑜𝑝𝑙𝑒𝑡 is the density of the spray solution and 𝜌𝑝𝑜𝑤𝑑𝑒𝑟 the density of an individual spray-
dried particle. However, the prediction of the particle size based on the droplet size is often
more complex (Dobry, 2015). Elversson et al. (2003) found an almost linear relationship
between droplet and particle size. The following list gives an overview of parameters in-
fluencing the droplet size.
Nozzle related parameters, which lead to an increased droplet size:
Larger nozzle orifice diameter (Elversson et al., 2003)
Lower atomizing airflow (Elversson et al., 2003)
Increased feed rate (Anish et al., 2014)
Formulation related parameters, which lead to an increased droplet size:
Increased viscosity of the feed (Anish et al., 2014)
Increased feed concentration (Elversson et al., 2003)
Lower solubility of the carbohydrate (Elversson and Millqvist-Fureby, 2005)
SPRAY DRYING 39
Higher surface tension (Masters, 1979, Dalmoro et al., 2013b)
Figure 3-7 gives a schematic summary of possible particle formation sequences during
spray drying. Phase 1 displays the moment, when the atomized droplet gets in contact with
the hot air. Phase 2 reflects the formation of a skin. The properties of the skin control the
following final particle formation in Phase 3.
Masters (2002) emphasizes the individual drying history of each particle. Every drop-
let/particle is exposed to slightly varying process conditions such as temperature, humidity,
air velocity or the mechanical impact through wall depositions.
Walton and Mumford (1999) reported that high drying temperatures led to an almost im-
mediate skin formation, which was followed by an internal bubble nucleation. As a result
of the increasing internal pressure particles may rupture or collapse (Masters, 1979).
Figure 3-7: Overview of particle shapes formed during the spray drying process (Masters,
2002). 1 = solid, spherical; 2 = shriveled, misshapen; 3 = hollow, spherical; 4 = cenospherical;
5 = disintegrated.
40 SPRAY DRYING
Walton (2000) used a single droplet drying technique for the investigation of the drying
behavior and resulting particle morphology of different materials. He classified spray dried
particles in the following groups:
Skin-forming: Particles consist of a continuous non-liquid phase, which is made up
of a polymeric or sub-microcrystalline material.
Crystalline: Particles consist of individual crystal nuclei, which are interconnected
by a continuous microcrystalline phase.
Agglomerate: Particles are composed of grains. These grains are bound together by
sub-micron dust (i.e. a binder or materials with a diameter less than 1 µm).
Vehring (2008) published further details about the particle formation. The first group of
materials is characterized by a fast diffusional motion of the solutes. This results in particles
with a particle density close to the true density of the dry raw material. He took solid sac-
charide particles as an example. Proteins and polymers belong to the second group. Here,
a shell formation takes place, which triggers the formation of different morphologies.
Besides the outer appearance of the spray dried powder, the inner distribution of the active
ingredient has to be taken into account. This is especially important for peptides and pro-
teins, which tend to accumulate at the particle surface. This behavior was shown for the
enzyme lactate dehydrogenase by Adler and Lee (1999). They found a 10 times higher
surface concentration than expected for a homogenous distribution. Surfactants are able to
counteract by occupying the surface themselves more rapidly after atomization (Führling,
2004).
SPRAY DRYING 41
3.5 Powder Separation
Particle separation is aimed at a collection of 100 %, at its best. The exhaust air in an open
cycle mode spray dryer should be free from particles in order to avoid air pollution and
environmental problems. High particle yields are especially desirable when working with
expensive products.
Several devices and setups are available for the collection of spray dried powders. First,
particles can be collected in a vessel at the base of the drying chamber (Figure 3-1). After-
wards the exhaust air has to be cleaned from the fine particle fraction. This so-called “sec-
ondary dried product discharge” takes place in a cyclone, a bag filter or an electrostatic
precipitator. In another setup the dried particles leave the drying chamber together with the
exhaust air and the separation takes place in a cyclone. Depending on the product type, a
secondary particulate collection equipment such as a bag filter or wet collectors can be
installed downstream (Masters, 2002).
The most common particulate collection equipment, a cyclone, is pictured in Figure 3-8.
Air and particles reach the cyclone and are set in a fast rotational motion by tangential
Figure 3-8: Schematic drawing of a cyclone with the inner and the outer vortex. Taken from
GEA (2015).
42 SPRAY DRYING
centrifugal forces. This motion is intensified by the conical shape of the cyclone. Particles
are separated out at the cyclone walls and leave the base of the cyclone towards a collection
vessel. Clean air quits the cyclone through the top performing spirals (Cal and Sollohub,
2010, Masters, 2002). The cyclone design is a key parameter for the improvement of the
particle yield (Maury et al., 2005). In addition, cyclone walls can be coated in order to
improve the recovery of sticky powders (Lee, 2002).
Another aspect concerning particle separation was reported by Schaefer and Lee (2015).
They found an inactivation of catalase during the residence time in the collection vessel of
a cyclone. They suggested to minimize process times for sensitive products.
Cyclone performance can be defined by two size parameters. First, the critical particle di-
ameter and second, the cut size (Masters, 1979). The critical parameter is defined by the
particle size, which is completely removed. The cut size is defined as the size for which
50 % collection efficiency is obtained. Hence, the latter parameter is more suitable for re-
presentation of the obtainable separation. Figure 3-9 shows grade-efficiency curves, which
are generated by operating a cyclone with different uniform powders of a defined diameter.
Figure 3-9: Theoretical and actual grad-efficiency of a cyclone (Masters, 1979).
SPRAY DRYING 43
According to Shaw (2003) the cut-off size (𝑑𝟓𝟎) is expressed as
𝑑𝟓𝟎 = 𝐾 × √9 𝜇𝑎𝑖𝑟 𝑄
𝜋 𝜌 𝑍0 𝑣𝑖𝑛2
Equation 3-7
where 𝐾 is a correction factor for the cyclone design, 𝜇𝑎𝑖𝑟 the air viscosity, 𝑄 the volume
flow rate through the cyclone, 𝜌 the particle density, 𝑍0 the vortex length and 𝑣𝑖𝑛 the cy-
clone inlet velocity.
44 SPRAY DRYING
3.6 Spray Drying of Liposomes
Table 3-2 gives an overview of publications discussing spray dried liposomes and relevant
parameters of the particular processes. Hauser and Strauss (1987) were the first to report
about the possibility to spray dry liposomes. They attached great importance to the stabi-
lizing agent sucrose, because an experiment without this excipient induced the aggregation
and fusion of vesicles. On the contrary, spray drying had only a little effect on liposome
size and size distribution when the vesicles were embedded in a sucrose matrix. 90 % of
the encapsulated markers, K3Fe(CN)6 and raffinose, could be recovered after spray drying
and rehydration of the powder.
Goldbach et al. published two studies about spray dried liposomes with the purpose to pro-
duce particles suitable for pulmonary delivery. Atomization was performed by a rotary at-
omizer and a pneumatic nozzle and resulted in mean particle sizes of 3.5 µm and 7.1 µm.
Neither the liposome size was affected significantly by the process nor the phospholipids
were significantly oxidized or hydrolyzed.
Spray drying had no effect on the encapsulation of the lipophilic substance α-tocopherol.
In contrast to the results of Hauser and Strauss (1987), they found 65 – 80 % release of the
encapsulated ingredient atropine after spray drying and rehydration of the powder.
Lo et al. (2004) tested different lipids and sugars in order to find an appropriate liposomal
formulation of the enzyme superoxide dismutase. DPPC, which is a natural lung surfactant,
and sucrose showed satisfying results and protected superoxide dismutase effectively from
degradation and activity loss. In addition, this formulation showed the best aerosol powder
performance with particle sizes of about 3 µm. A surface analysis of spray dried powders
revealed that there is an excess of DPPC on the particle surface keeping superoxide dis-
mutase away from the surface.
Chougule et al. presented three studies aiming at the production of aerodynamically light
particles for pulmonary delivery. They tested the spray dried powders using in vitro and in
vivo studies and showed i.e. a prolonged (16 h) drug release for liposomal, spray dried
SPRAY DRYING 45
Author Lipid Nozzle type Tinlet / Toutlet
[°C] Excipients Drug
Liposome
size [nm]
Goldbach et
al. (1993a) SPC
Rotary atom-
izer 110 / 75 – 80 Lactose – ~ 200
Goldbach et al. (1993b)
SPC / HSPC / Chol
Rotary atom-
izer; two fluid nozzle
110 – 140 / 75 – 85
Lactose Atropine;
α-tocopherol ~ 200
Hauser and Strauss
(1987)
POPC /
DOPS ? 140 Sucrose
K3FE(CN)6; raffinose
(labled)
18 – 70
Lo et al.
(2004)
DPPC;
DMPC;
DSPG; DPPG
Two fluid
nozzle
Ø 0.5 mm
168 / 122
Lactose; su-
crose; treha-
lose
Superoxide
dismutase 150 – 200
Chougule et
al. (2008)
DPPC /
Chol
Two fluid
nozzle Ø 0.7 mm
100 / 60 – 65
Lactose; su-
crose; hydro-
lyzed gelatin; leucine
Dapsone 137 ± 15
Chougule et al. (2007)
HSPC / Chol
Two fluid
nozzle Ø 0.7 mm
110 / 60 – 65
Lactose; su-
crose; treha-lose; leucine
Tacrolimus 140
Chougule et
al. (2006)
HSPC /
Chol
Two fluid nozzle
Ø 0.7 mm
120 / 65 – 70 Lactose, su-
crose, manni-
tol, glycine
Amiloride
HCl 198 ± 15
Wessman et
al. (2010)
DSPC /
Chol; DSPC /
Chol /
DSPE-PEG
Two fluid
nozzle 170 / 75 – 81 Lactose – 158 – 190
Wiggenhorn
(2007)
DOTAP /
DOPC
Two fluid
nozzle
Ø 0.7 or 0.5 mm
80 – 220 /
40 – 120 Trehalose Paclitaxel ~ 130
Karadag et al. (2013)
Lecithin;
coated with chitosan
Two fluid
nozzle Ø 1.5 mm
160 / 90 Maltodextrin – ~ 400 – 500
Charnvanich
et al. (2010) HPC / Chol
Two fluid
nozzle
Ø 0.7 mm
120 / 74 – 84 Mannitol Lysozyme ~ 140 – 200
Kim (2001) Egg PC Ultrasonic
nebulizer 90 / 40 – 45 – Amphotericin
192 – 505
(after rehy-
dration)
Skalko-
Basnet et al.
(2000)
Lecithin ? 90 – 120 Mannitol Metronidazol,
verapamil
300 (after re-
hydration)
Table 3-2: Publications discussing the spray drying of liposomes. “/“: A mixture of substances
was used. “;“: The substances were used in separate experiments.
46 SPRAY DRYING
dapsone. A drug residence time of up to 24 h within the lungs for liposomal tacrolimus was
reported. Additionally, they collected data about the long-term stability of liposomal tacro-
limus embedded in a trehalose matrix. After six months at 40 °C / 75 % RH liposome size
increased, the fine particle fraction decreased and drug retention eased to 85 %.
Wessman et al. (2010) examined the structural effects caused by spray drying and freeze
drying of PEGylated and non-PEGylated liposomes and bilayer disks. Cryo-TEM of ex-
truded liposomes showed that PEGylated vesicles were completely unilamellar and non-
PEGylated liposomes had a small portion of bi- and multilamellar structures. The mean
size of PEG-free liposomes after spray drying performed a slight shift towards smaller radii
including some bi-and multilamellar structures. However, spray dried PEGylated lipo-
somes showed clearly larger structures after the drying and rehydration steps. The authors
explained the structural effects through the osmotic stress during dehydration. In a further
experiment, they added lactose to liposomes, which were originally prepared in water and
observed the same changes induced by the osmotic effect. The mean aggregate size de-
creased and liposomes collapsed or rearranged into liposomes with thick walls consisting
of two or several bilayers.
Wiggenhorn (2007) spray dried cationic liposomes with and without the antineoplastic drug
paclitaxel. He showed that liposomes were well retained after spray drying using different
drying conditions. The protective effect of trehalose was especially important at high inlet
temperatures.
Charnvanich et al. (2010) investigated the influence of cholesterol on the properties of spray
dried liposomes and the encapsulation efficiency of lysozyme. Cholesterol improved the
encapsulation and reduced the diameter of the reconstituted liposomes.
Karadag et al. (2013) developed a stable, spray dried formulation of chitosan coated lipo-
somes and the spray drying excipient maltodextrin.
The two last mentioned publications in Table 3-2 deal with the spray drying of organic
solutions of lipid and drug. The rehydration of the spray dried product results in the for-
mation of liposomes with the encapsulated active ingredient.
4 MATERIALS AND METHODS
4.1 Materials
4.1.1 Lipids
DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) is a cationic lipid. It
is a widely known transfection reagent (see chapter 2.6) and is usually used together with
a neutral helper lipid. It consists of a monocationic trimethylammonium head group and
two unsaturated hydrocarbon chains as the lipophilic part of the molecule (Figure 4-1). The
phase transition temperature is quite low, since the molecule possesses two unsaturated
hydrocarbon chains. Regelin et al. (2000) stated a Tm < 5 °C. Platscher and Hedinger (2007)
specified a value of -17.5 °C for the phase transition temperature of the racemate.
Figure 4-1: Chemical structure of DOTAP (Avanti, 2015).
DOPC (1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine) is a neutral lipid. It has a
zwitterionic structure and owns, like DOTAP, two unsaturated hydrocarbon chains, derived
from oleic acid. The phase transition temperature of fully hydrated DOPC is -16.5 °C
(Ulrich et al., 1994). Most experiments in this work were performed using a mixture of
DOTAP and DOPC in the molar ratio 1:1. The same mixture is used for a formulation of
paclitaxel, EndoTAG 1, which is currently in clinical trial phase 2 (Chang and Yeh, 2012).
Paclitaxel is a lipophilic drug, which is encapsulated in the liposome membrane. Liposomes
48 MATERIALS AND METHODS
Figure 4-2: Chemical structure of DOPC (Avanti, 2015).
are a suitable formulation of this antineoplastic drug, because of its extreme low solubility.
The second advantage of this formulation is the targeting of negatively charged proliferat-
ing or activated cells in the tumor blood vessels (Schmitt-Sody et al., 2003). The
EndoTAG 1 formulation was examined in the spray drying experiments of Wiggenhorn
(2007).
18:1 PEG 2000 PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (pro-
pylene glycol)-2000; ammonium salt) is the PEGylated component used for the production
of PEGylated liposomes. It was incorporated up to a concentration of 10 mol% and con-
tributes a negative charge to the membrane composition. The PEG chain attached to the
liposome surface has a length of approximately 50 Å (Needham et al., 1992).
Figure 4-3: Chemical structure of the PEGylated lipid used in this work (Avanti, 2015).
MATERIALS AND METHODS 49
4.1.2 Encapsulated Substances
Insulin is a peptide hormone consisting of two chains of amino acids, which are linked
together by two disulphide bonds. In addition, Chain A has an intramolecular disulphide
bridge. Manufactured in the beta cells of the islets of Langerhans, it is important for the
regulation of the blood glucose level. It triggers for example the transport of glucose into
Molecular Mass 5808 Da
Isoelectric point 5.3 (Conway-Jacobs and Lewin, 1971)
Solubility soluble at 10 mg/ml in pH 2.0 – 2.5
Table 4-1: Characteristics of insulin (Sigma Aldrich, 2015b).
cells and the conversion of glucose into glycogen (Sleigh, 1998). It is used for the treatment
of diabetes type 1 and advanced diabetes type 2. The primary structure is pictured in Figure
4-4. The black marked amino acids designate components, which are invariant among spe-
cies of insulin (e.g. porcine or bovine insulin). Regarding the quaternary structure, the in-
sulin dimer is the relevant species in pharmaceutical concentrations at neutral pH in the
absence of zinc ions. Higher concentrations or the presence of zinc triggers the formation
of hexamers. The monomer exists predominantly at very low concentrations or at pH < 2.0
(Brange and Langkjœr, 1993, Olson et al., 1979).
Figure 4-4: Primary structure of human insulin (Brange and Langkjœr, 1993).
50 MATERIALS AND METHODS
Figure 4-5: Chemical structure of calcein (Sigma Aldrich, 2015a).
Calcein is a fluorescent marker molecule, which is often used for the determination of
encapsulation efficiency (Oku et al., 1982) as well as for the measurement of liposome
leakage (Allen and Cleland, 1980). Characteristics of the molecule are summarized in
Table 4-2. Calcein has, like carboxyfluorescein (CF), the ability to self-quench. At low
concentrations, the fluorescence is proportional to the marker concentration. However, at
high marker concentrations fluorescence drops off because of self-quenching. This phe-
nomenon is used for liposome release studies. The marker is encapsulated into liposomes
at high concentrations, followed by the separation from free, not encapsulated marker mol-
ecules. If there is no leakage, nearly no fluorescence is detectable due to self-quenching.
As soon as the marker leaks out of the vesicles, fluorescence is measurable (Weinstein et
al., 1977).
Molecular Mass 622.5 g/mol
Solubility 50 mg/ml (1 M NaOH)
Excitation λ 470 nm
Emission λ 509 nm (in PBS)
Table 4-2: Characteristics of calcein (Sigma Aldrich, 2015a).
MATERIALS AND METHODS 51
4.1.3 Excipients and Reagents
Table 4-3 gives an overview of the substances used for the preparation of liposomes and
the analyses performed in this work. Double distilled water prepared in an all-glass appa-
ratus was used for the preparation of aqueous solutions. Water was filtered through 0.2 µm
filters, for HPLC mobile phases 0.1 µm, prior to use (Sartorius RC, Sartorius Stedim Bio-
tech GmbH, Goettingen, Germany). All solutions were prepared freshly and stored in the
fridge at 4 °C, if necessary.
Excipient Product No. / Lot No. Supplied by
Liposome Preparation
DOPC
-/556600-2120156-01
various
850375P/181PC-300
Lipoid GmbH, Germany
Merck Eprova AG, Swizer-
land
Avanti Polar Lipids, USA
DOTAP
-/593500-2110001-01
various
Lipoid GmbH, Germany
Merck Eprova AG, Swizer-
land
18:1 PE PEG 2000 880130P/181PEG2PE-45 Avanti Polar Lipids, USA
Methanol 7342.1/various Carl Roth, Germany
Chloroform 733.1/810831 Carl Roth, Germany
Encapsulated Substances
Insulin, From Bovine Pan-
creas I5500/016K1250 Sigma-Aldrich, Germany
Calcein C0875-5G/SLBM2007V Sigma-Aldrich, Germany
Spray Drying Excipients
D-(+)-Trehalose dihydrate T9531/various Sigma-Aldrich, Germany
α-Lactose monohydrate L3625/102K0107 Sigma-Aldrich, Germany
D-(+)-Sucrose 4621.1/494222942 Carl Roth, Germany
D-Mannitol M8429/031M00443V Sigma-Aldrich, Germany
Maltodextrin 419680/MKBJ4272V Sigma-Aldrich, Germany
Polysorbate 80 226953-0001/11D27-N01 Fagron, Germany
52 MATERIALS AND METHODS
Excipient Product No. / Lot No. Supplied by
Laser Diffraction
Miglyol®812 3274/various Sigma-Aldrich, Germany
Sorbitane trioleate 3459/various Sigma-Aldrich, Germany
Karl-Fischer Titration
Hydranal Coulomat AG
Oven 34739/various Sigma-Aldrich, Germany
Molecular Sieve 3Å 8482.2/various Carl Roth, Germany
Methanol 7342.1/various Carl Roth, Germany
Nitrogen gas - Linde, Germany
SEC – Insulin
Acetonitrile 8825.2/various Carl Roth, Germany
Acetic acid A6283/various Sigma-Aldrich, Germany
L-Arginine A5006/MKBN8002V Sigma-Aldrich, Germany
TritonTM X-100 93426/BCBH2984V Sigma-Aldrich, Germany
HCl (0.1 M) 35335/SZBD0040V Sigma-Aldrich, Germany
HPLC – Lipids
Methanol 7342.1/various Carl Roth, Germany
Encapsulation Efficiency – Insulin
BCA Solution 030B/122911 G-Biosciences, USA
Copper Solution 274C/122911 G-Biosciences, USA
TritonTM X-100 93426/BCBH2984V Sigma-Aldrich, Germany
Encapsulation Efficiency and Membrane Integrity – Calcein
Cobaltchloride 232696/BCBN3189V Sigma-Aldrich, Germany
HEPES H4034/SLBK4457V Sigma-Aldrich, Germany
NaOH (1 M) 35256/SZBE2580V Sigma-Aldrich, Germany
MATERIALS AND METHODS 53
Excipient Product No. / Lot No. Supplied by
TritonTM X-100 93426/BCBH2984V Sigma-Aldrich, Germany
Table 4-3: Substances and reagents used in this work.
4.1.4 Other Materials
Material Product No. / Lot No. Supplied by
PC Membranes 0.1 µm 610005/various Avanti Polar Lipids, USA
PC Membranes 0.4 µm 610007/various Avanti Polar Lipids, USA
10 mm Filter Supports 610014/various Avanti Polar Lipids, USA
0.1 µm and
0.2 µm RC membrane fil-
ter
15458--5------N
18407--47------N Sartorius AG, Germany
Amicon Ultra-4 Centrifu-
gal Filter Units (100000
NMWL)
UFC900396 Merck Millipore, Germany
Al-crucibles ME27331 Mettler Toledo, USA
Table 4-4: Materials used in this work.
54 MATERIALS AND METHODS
4.2 Methods
4.2.1 Liposome Preparation
Lipids were weighed and dissolved in about 8 ml of a mixture of chloroform/methanol
(2:1). Liposomes were prepared by the round bottom flask method according to the follow-
ing protocol. Organic solvents were removed in a rotary evaporator (Heidolph VV 2000,
Heidolph Instruments GmbH, Schwabach, Germany) equipped with a water bath (Heidolph
WB 2000, Heidolph Instruments GmbH), which was adjusted to 40 °C. The round bottom
flask rotation was regulated to 120 rpm. The vacuum (Vacuum pump Vario PC 3001, Vac-
uubrand GmbH, Wertheim, Germany) was set to 300 mbar for 2 min followed by 150 mbar
for another 4 min in order to remove the solvents. A lipid film formed on the internal wall
of the round bottom flask. The catchpots were emptied and vacuum was set to 10 mbar for
at least 1 hour in order to dry the lipid film completely. Lipid films were rehydrated with
an aqueous solution for 20 min in the rotating flask keeping the water bath at 40 °C.
Insulin solutions for lipid film rehydration were prepared by dissolving 20.0 mg insulin and
1.0 g trehalose in water pH 2.0 and filling up to 10.0 ml in a volumetric flask. The solution
was used for the rehydration of the lipid film.
A stock solution of calcein was prepared by dissolving calcein in HEPES buffer (50 mM,
pH 7.4) to a concentration of 0.05 mg/ml. For SD experiments, 100 mg/ml trehalose was
dissolved additionally. The stock solution was used for the rehydration of the lipid film.
Figure 4-6: Mini-Extruder. The polycarbonate membrane and filter supports are placed between
two teflon internal membrane supports. 1.0 ml Hamilton syringes are connected to the extruder
outer casting.
MATERIALS AND METHODS 55
The crude liposome dispersion was removed from the flask and filled into Sarstedt tubes
(Sarstedt AG, Nürnbrecht, Germany). Extrusion of liposomes was performed using a Mini-
Extruder (Figure 4-6, Avanti Polar Lipids, Alabaster, USA). Liposomes were extruded 11
times through a 0.4 µm polycarbonate membrane, before extrusion through a 0.1 µm poly-
carbonate membrane for 11 times. The polycarbonate membranes were positioned between
filter supports.
4.2.2 Spray Drying
The extruded liposome dispersions were spray dried using a laboratory scale spray dryer
(model 4M8, ProCepT, Zelzate, Belgium). The spray dryer operated in a co-current mode
and was equipped with an elongated drying chamber of approximately 2.1 meters. Inlet air
is filtered by means of a HEPA filter. The T-piece (Figure 4-7, B) at the end of the drying
chamber was connected to the cyclone (left) and to compressed air supply (right) to opti-
mize airflow through the cyclone. Critical process parameters, such as the Tinlet, Toutlet, the
temperature at the end of the drying chamber (Tchamber) and the inlet air flow were
Figure 4-7: Setup of the spray dryer used in this work (A). Picture B shows the T-piece at the
end of the drying chamber and picture C shows the top of the drying chamber with an inserted
nozzle (here: 25 kHz ultrasonic nozzle).
A B
C
56 MATERIALS AND METHODS
continuously recorded. If not stated otherwise the inlet temperature was 150 °C, the inlet
air flow 0.3 m3/min and compressed air supply 0.2 m3/min. The liquid feed rate was
1 ml/min or 0.5 ml/min. Typical temperatures in the spray dryer were a Tchamber of
70 – 80 °C and a Toutlet of 45 – 55 °C. Liquid feeds were pumped to the nozzle by means of
a syringe pump (Lambda VIT-FIT, Brno, Czech Republic) or a peristaltic pump (Ismatec,
Wertheim, Germany). Spray dried samples were removed quickly after the atomization of
the liquid feed from the collection vessel and filled in Sarstedt tubes.
4.2.3 Nozzle Types
Atomization was performed by the following nozzles:
25 kHz ultrasonic nozzle (Sono-Tek Corp., Milton, USA) connected to a broadband
generator (Sono-Tek Corp), which delivers the high frequency electrical energy.
Output power was set to 2.0 W.
60 kHz ultrasonic nozzle (Sono-Tek Corp) connected to the above mentioned
broadband generator. Output power was set to 5.0 W.
Standard two fluid nozzle, which was delivered with the ProCepT spray dryer. At-
omization air pressure was 2 bar and a nozzle orifice with a diameter of 1.0 mm was
used for all experiments.
Figure 4-8: Nozzle types used in this work. From left to right: Ultrasonic nozzle 25 kHz, ultra-
sonic nozzle 60 kHz, two fluid nozzle.
MATERIALS AND METHODS 57
4.2.4 Differential Scanning Calorimetry
DSC measurements were performed by a Mettler-Toledo DSC 822e (Giessen, Germany),
which is equipped with a liquid nitrogen cooler. The measuring cell is purged and dried
through nitrogen (40 ml/min and 120 ml/min, respectively). Differential scanning calorim-
etry was used both for the determination of Tg of powders and for the determination of
phase transition temperatures of lipids. Approximately 10.0 mg of spray dried powders or
10 µl of liquid samples were filled in 40 µl aluminium crucibles, which were hermetically
sealed. Different procedures were used for the various sample types:
Pure lipids in the non-hydrated and hydrated state (Tm): Samples were cooled and
kept at -30 °C for 10 min before heating with a rate of 7 °C/min up to 50 °C.
Liposomes dispersed in a trehalose solution: Samples were cooled and kept at
-50 °C for 10 min before heating with a rate of 7 °C/min up to 20 °C (Tg´ and Tm
detectable).
Spray dried powders containing trehalose (Tg): Samples were cooled and kept at
10 °C for 10 min before heating with a rate of 7 °C/min up to 90 °C. Samples were
cooled down again to 10 °C and heated up to 120 °C. The midpoint of the glass
transition in the second heating run was used for the determination of Tg in order to
avoid interference from enthalpy relaxation occurring in the first heating run.
Spray dried powders containing trehalose (Tm of liposomes): Samples were cooled
and kept at -110 °C for 10 min before heating with a rate of 7 °C/min up to 25 °C.
Spray dried powders containing various excipients: Heating and cooling procedure
were adapted in order to detect glass transition temperatures or melting points of
the examined powders.
DSC scans were analyzed using the STARe software (Mettler-Tolede, Giessen, Germany).
58 MATERIALS AND METHODS
4.2.5 Dynamic Light Scattering
A Zetasizer Nano ZS (Malvern Instruments, UK) was used for the characterization of lip-
osomes. Liposome dispersions were analyzed before the spray drying process and after
rehydration of the powders. Therefore, SD powder was weighed and dissolved in an appro-
priate volume of water. Liposome size (z-average), size distribution (polydispersity index,
PDI) and zeta potential were determined. Depending on the lipid concentration, samples
were diluted between 1:10 and 1:20 with an aqueous solution of the excipient used in the
spray drying experiment. This was usually a 10 % (w/v) solution of trehalose. Measure-
ments of size and size distribution were performed in standard disposable PS cuvettes
(Brand GmbH, Wertheim, Germany) and the zeta potential was measured in disposable
folded capillary cells (DTS 1070, Malvern). The Zetasizer operated with a measurement
angle of 173° backscatter (non-invasive backscatter technique, NIBS), a measurement tem-
perature of 25 °C and was set to the automatic measurement duration. A refractive index of
1.348 and a viscosity of 1.3 mPas were assumed.
4.2.6 Karl-Fischer Titration
Residual moisture of spray dried samples was measured using Karl-Fischer Titration in a
Metrohm 832 KF Coulometer with a 831 KF Thermoprep oven (Metrohm, Filderstadt,
Germany). The temperature of the oven was set to 110 °C and the nitrogen flow to
70 ml/min. 30 – 40 mg of spray dried powders were filled into vials and sealed with caps.
Three vials were sealed without content and taken for the blank measurements. The vials
were placed in the oven and a needle with two capillaries was inserted through the stopper.
Samples were purged with nitrogen (first capillary) and the water vapour leaves the vial
together with nitrogen towards the reaction vessel (second capillary). Measurements were
started at drifts lower than 5.0 µg/min and stopped automatically when the amount of de-
tected water fell under 10 µg/min. Residual moisture was indicated as percentage of the
powder mass used for the measurement.
4.2.7 Laser Diffraction
A Mastersizer 2000 (Malvern Instruments, UK) connected to a Hydro 2000S wet sample
dispersion unit was used for the determination of particle sizes of spray dried products.
Samples (~ 50 mg) were dispersed thoroughly in a mixture of Miglyol®812 and 1 % (v/v)
MATERIALS AND METHODS 59
Sorbitane trioleate (Span®) before transferring to the dispersion unit, which was also filled
with Miglyol/Span (RI: 1.45). The purpose of sorbitane trioleate was to assure optimal wet-
ting of the powders. The stirrer was set to 1750 rpm and sample was added until obscuration
was in range (~ 8 %). The analysis model “general purpose (spherical)” was chosen and 5
measurement cycles for each measurement run were performed. The d50 value of the vol-
ume based size distribution was usually used for result evaluation.
4.2.8 Wide-Angle-X-Ray-Diffraction (WAXD)
A Philips X´Pert X Ray diffractometer (PANanalytical, Almelo, The Netherlands) was used
for the examination of the physical state of spray dried powders. Acceleration voltage was
40 kV and the anode current was 40 mA. Measurements were performed at ambient tem-
perature under nitrogen atmosphere. Powders were filled in stainless steel sample holders
having an indentation of 2.0 mm. The surface was smoothened using a microscope slide.
Samples were measured in the range of 2θ = 0.5° to 40° (step size 0.02°, time per step 1 s)
with a wavelength of 0.1542 nm.
4.2.9 Scanning Electron Microscopy
Spray dried powders were imaged by a Scanning Electron Microscope (Amray 180T, Bed-
ford, Massachusetts). Samples were fixed onto an aluminium sample stub (Model G301,
Plano) and sputtered with gold for ca. 1.5 min at 5 kV and 20 mA in a sputter unit (Hummer
JR Technics, Munich, Germany).
4.2.10 Levitation
The drying behavior of small droplets of liposome dispersions was observed in an ultra-
sonic levitator (tec5 AG, Oberursel, Germany). Schiffter and Lee (2007a) and Wulsten and
Lee (2008) gave detailed descriptions of the setup, calibration and mode of operation of the
levitation system. In few words, 2 µl droplets were inserted into an acoustical field using a
5 µl Hamilton syringe (Hamilton, Bonaduz, Swizerland). The drying conditions in the
acrylic chamber were 40 °C and < 1 % RH, which was adjusted by a controlled evaporation
mixer (Bronkhorst High-Tech B.V., Ruurlo, The Netherlands). A Schott KL 1500 cold-
light source (Schott AG, Mainz, Germany) provided an optimal lighting of the acrylic
chamber. Droplet drying was observed by an optical charge coupled device camera (CCD-
60 MATERIALS AND METHODS
camera, JAI AG, Copenhagen, Denmark) together with a Nikon Micro 60-mm diaphragm
(Nikon GmbH, Duesseldorf, Germany). An infrared camera (InfraTec GmbH, Dresden,
Germany) was used for the measurement of the droplet surface temperature. The infrared
camera was placed behind an IR transparent germanium window.
Figure 4-9: Setup of the levitation system. The arrow marks the inner acrylic glass chamber with
the ultrasonic levitator. Behind the inner acrylic glass chamber is a germanium window, which is
placed between the droplets and the infrared camera.
Data from the CCD camera was processed using the program Image Pro Plus Software 4.51
(Media Cybernetics, Bethesda, USA). Pictures were taken every 5 s. Pictures taken by the
infrared camera were exploited using the IRBIS professional software (InfraTec GmbH).
The minimal surface temperature was plotted against the time representing the droplet/par-
ticle surface temperature. Data of the optical camera was analyzed with the objective to
calculate an evaporation coefficient, to plot r(t)2/r(0)2, the surface temperature and the as-
pect ratio versus the time (Lorenzen and Lee, 2012). r(t) is the radius of a surface-equivalent
sphere at any given point in time.
controlled evaporation mixer
cold light source
infrared camera
outer acrylic glass chamber
CCD camera
MATERIALS AND METHODS 61
4.2.11 Encapsulation Efficiency – Insulin
Free insulin was separated from encapsulated insulin by means of Amicon Ultra-4 Centrif-
ugal Filter Units (100 000 NMWL, Merck Millipore, Darmstadt, Germany). Liposomes
with encapsulated insulin were not able to pass the regenerated cellulose membrane,
whereas free insulin passes the membrane without hindrance. Every filter unit was weighed
before use, thereby the filter and the collection tube was balanced separately. 1.0 ml of
sample was filled into the centrifugal filter units. Centrifugation took place in a Sigma 3-
16PK centrifuge (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany), which
was cooled to 4 °C. Spin conditions were 20 min at 2000 g followed by two washing steps
with 0.2 ml trehalose solution (10 % w/v) with subsequent centrifugation for 10 min at
2000 g. Filter units and collection tubes were weighed after centrifugation. The volume of
liquid in the filter (now called “concentrate”) and the collection tube (now called “filtrate”)
was calculated assuming a density of 1.03 mg/ml. Both fractions were filled into Eppendorf
tubes for further analysis.
Insulin concentration in the concentrate and in the filtrate was measured using a standard
BCA assay (Bicinchoninic Acid, G-Biosciences, USA). BCA assays are highly sensitive
protein assays and are based on two reactions. First, peptide bonds reduce Cu2+ ions in a
temperature dependent reaction to Cu+. The amount of reduced copper is proportional to
the amount of protein present in the solution. Second, each Cu+ ion chelates with two BCA
molecules and forms a purple product, which absorbs at a wavelength of 562 nm. UV ab-
sorption is linear in the range of 0.02 – 2 mg/ml protein (G-Biosciences, 2013).
A solution of insulin (0.5 mg/ml) was used for the preparation of a calibration line in the
range of 0 – 500 µg/ml.
Samples were diluted 1:6 (initial liposome dispersion, concentrate) or 1:3 (filtrate) with
1 % Triton X (w/v) prior to measurement. Triton X was used for liposome destruction.
The working solution was freshly prepared by mixing 50 parts of BCA solution with 1 part
copper solution.
62 MATERIALS AND METHODS
50 µl of each diluted sample was pipetted in an Eppendorf tube. 1.0 ml of working solution
was added and vortexed. The assay was incubated at 18 °C (air-conditioned room) for 2 h.
Absorbance of each sample was measured using an UV-Vis spectrophotometer (Genesys
10 UV/Vis, Thermo Fisher Scientific, Waltham, USA) at 562 nm.
Insulin concentration was calculated using the calibration line. Subsequently, the absolute
amount of insulin (in µg) of each sample was estimated using the above calculated volumes
of concentrate and filtrate. Then, encapsulation efficiency (𝐸𝐸) and overall recovery of the
method is given as:
The mass of insulin in the concentrate (𝑚𝑐𝑜𝑛𝑐) and in the filtrate (𝑚𝑓𝑖𝑙𝑡) are calculated in
µg. The amount of insulin in the concentrate corresponds with the amount of encapsulated
insulin, whereas the amount of free insulin is represented by the filtrate, respectively. The
recovery rate was determined for verification purposes by comparing the sum of insulin
found in the centrifugal filter unit with the amount of insulin in the initial liposome disper-
sion (𝑚𝑠𝑡𝑎𝑟𝑡). Recovery rates were between 90 and 110 % for all experiments.
4.2.12 Size Exclusion Chromatography – Insulin
Aggregates of insulin were determined by size exclusion chromatography (SEC) according
to a method described in the USP and in the Ph.Eur. The HPLC system consisted of a series
200 lc pump (Perkin Elmer, Waltham, USA) equipped with a VWR L7614 degasser (Rad-
nor, USA), an Advanced LC Sample Processor ISS 200 (Perkin Elmer), a Diode Array
Detector 235C (Perkin Elmer) and a HPLC column chiller/heater (model C030, Torrey
Pines Scientific, Carlsbad, USA).
The mobile phase was composed of arginine solution (1 mg/ml), acetonitrile and glacial
acetic acid in the ratio of 65:20:15. The detector was set to 275 nm, the flow rate to
0.5 ml/min and the injection volume was 30 µl. A Waters insulin HMWP SEC column
𝐸𝐸 [%] = 𝑚𝑐𝑜𝑛𝑐 [μg]
(𝑚𝑐𝑜𝑛𝑐 + 𝑚𝑓𝑖𝑙𝑡)[μg] × 100% Equation 4-1
𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦 [%] = (𝑚𝑐𝑜𝑛𝑐 + 𝑚𝑓𝑖𝑙𝑡)[μg]
𝑚𝑠𝑡𝑎𝑟𝑡[μg] × 100% Equation 4-2
MATERIALS AND METHODS 63
(Waters Corporation, Milford, USA) was placed in the column chiller/heater, which was
set to 20 °C. 250 µl of the sample were mixed with 750 µl mobile phase. This solution was
filled into HPLC vials.
Data was processed using TotalChrom Chromatography Data Systems (Perkin Elmer).
Evaluation of results was conducted according to the USP / Ph.Eur: The areas of peak re-
sponse were measured disregarding any peaks having retention times greater than that of
the insulin monomer.
Then, the percentage of high molecular weight proteins is given as:
𝐻𝑀𝑊𝑃 [%] = 𝑆𝑟𝐻
(𝑆𝑟𝐻 + 𝑟𝑀)× 100%
Equation 4-3
𝑆𝑟𝐻 is the sum of the area for all peaks having retention times less than that of the insulin
monomer and 𝑟𝑀 is the peak area of insulin monomer. The upper limit of HMWP defined
by the USP and Ph.Eur is 1 %. The areas under the curve were calculated as a mean value
of at least 3 injections.
4.2.13 HPLC – Lipid Recovery
The HPLC system used for this analysis consisted of a Flexar UHPLC Autosampler (Perkin
Elmer, Waltham, USA), a Flexar Solvent Manager (Perkin Elmer), a Flexar UHPLC Pump
(Perkin Elmer) and a Flexar UV/VIS UHPLC Detector (Perkin Elmer), which was set to
205 nm. A Brownlee Analytical C18 UHPLC column (1.9 µm, 100 x 2.1 µm, Perkin Elmer)
was connected to the HPLC system. Methanol was used as mobile phase and samples were
diluted 1:6 with methanol prior to injection. The flow rate was 0.3 ml/min and an injection
volume of 5 µl was used.
Evaluation of the results was carried out by the comparison of the area under the curve with
a calibration line, which was injected and measured before and after the sample runs.
64 MATERIALS AND METHODS
4.2.14 Calcein Encapsulation and Membrane Integrity
Liposomes containing encapsulated calcein were used both for the determination of encap-
sulation efficiency and the membrane integrity during atomization, spray drying and freeze
drying. For both methods calcein loaded liposomes were prepared according to the protocol
described in chapter 4.2.1.
The method for the determination of encapsulated marker was presented and described in
detail by Oku et al. (1982). Briefly, it is based on the quenching of free, not encapsulated
calcein with Co2+ ions. The remaining fluorescence corresponds with the percentage of
encapsulated fluorescent dye.
Fluorescence measurements were performed with a Perkin Elmer LS 55 Luminescence
Spectrometer using an excitation wavelength of 490 nm and an emission wavelength of
525 nm. Slit widths were 15 and 20 nm and a 1 % attenuator was used for the detection of
emitted fluorescence.
The procedure for the determination of encapsulation efficiency was as follows:
30 µl of liposome dispersion was diluted to 1.5 ml with HEPES buffer (50 mM, pH 7.4).
The fluorescence was measured (𝐹𝑡𝑜𝑡) using Makro PMMA disposable cuvettes (VWR
International GmbH, Darmstadt, Germany). After the addition of 30 µl CoCl2 (1 mg/ml)
fluorescence was measured again (𝐹𝑖𝑛). 30 µl of Triton X (15 % w/v) were added and the
fluorescence was measured for the last time (𝐹𝑡𝑜𝑡𝑞).
Then, encapsulation efficiency is given as:
𝐸𝐸 [%] = [(𝐹𝑖𝑛 × 𝑟1) − (𝐹𝑡𝑜𝑡𝑞 × 𝑟2)]
[𝐹𝑡𝑜𝑡 − (𝐹𝑡𝑜𝑡𝑞 × 𝑟2)]× 100%
Equation 4-4
𝑟1 and 𝑟2 are dilution factors.
The membrane integrity was determined after the application of stress factors, such as at-
omization, spray drying or freeze drying. The method was adopted from Huang and
MacDonald (2004): 20 µl of CoCl2 (10 mg/ml) were added to 1.0 ml of liposome dispersion
and fluorescence was measured (𝐹𝑖𝑛). Then this mixture was atomized, spray dried or
MATERIALS AND METHODS 65
freeze dried. If powders were generated, they were rehydrated and fluorescence was meas-
ured again (𝐹𝑢𝑙𝑡𝑟𝑎𝑠𝑜𝑢𝑛𝑑). After the addition of Triton X (15 % w/v) the remaining fluores-
cence (𝐹𝑡𝑜𝑡𝑞) was determined. The percentage of initially encapsulated marker, which got
in contact with the surrounding medium, is called “release” and is given as
𝑟𝑒𝑙𝑒𝑎𝑠𝑒 [%] = [𝐹𝑖𝑛 − 𝐹𝑢𝑙𝑡𝑟𝑎𝑠𝑜𝑢𝑛𝑑]
[𝐹𝑖𝑛 − (𝐹𝑡𝑜𝑡𝑞 × 𝑟2)]× 100% Equation 4-5
where 𝑟2 is a dilution factor.
4.2.15 Freeze Drying
Freeze drying experiments were performed using a pilot freeze dryer VirTis™ Genesis (SP
Industries, Warminster, USA). 0.5 ml of liposome dispersions were filled in 2.0 ml vials
and sealed with rubber stoppers (Helvoet Pharma, Hellevoetsluis, The Netherlands). After
freeze drying the vials were immediately closed with Bromobutyl stoppers and sealed with
aluminium caps until analysis. The freeze drying protocol is pictured in Figure 4-10.
Figure 4-10: Freeze drying cycle used in this work.
66 MATERIALS AND METHODS
4.2.16 Maximum Bubble Pressure Tensiometry
The dynamic surface tension of liposome dispersions was measured using a maximum bub-
ble pressure tensiometer MPT2 (Lauda Dr. R. Wobser GmbH, Lauda-Königshofen, Ger-
many). A detailed explanation of the measuring principle is given elsewhere (Führling,
2004). The bubble pressure tensiometer is able to detect surface tensions as a function of
the surface age in the range of 1 – 1000 ms, which is beneficial for the examination of fast
processes such as spray drying. Measuring conditions were a stepwise (8 %) decreasing
flow rate (150 mm3/s – 5 mm3/s), a tempered sample (25 °C) and a constant immersion
depth of the capillary. That was achieved by using the same sample volume (1.5 ml) for
each measurement. For evaluation, the surface tension was plotted against surface life time.
4.2.17 Viscosity Measurements
The dynamic viscosity of liposome dispersions was determined using a capillary viscome-
ter (Ubbelohde) at 23 °C.
4.2.18 Stability
Freshly prepared spray dried samples were filled into Sarstedt tubes and stored at -80 °C
(HERAfreeze, Thermo Fisher Scientific, Waltham, USA), 4 °C (refrigerator, Liebherr, Ger-
many), 25 °C (conditioning cabinet, Heraeus, Hanau, Germany) and 40 °C (conditioning
cabinet CO2-Auto-Zero, Heraeus, Hanau, Germany). The glass transition temperature
(DSC) and liposome properties, such as size, PDI and zeta potential (Zetasizer) were meas-
ured at the beginning of the long-term stability experiment. Samples were retested after 2
weeks, 1 month, 3 months and 6 months with respect to Tg and liposome size, PDI and zeta
potential.
5 RESULTS AND DISCUSSION
5.1 Preparation of Liposomes
The first step in the manufacturing process of spray dried powders containing liposomes
was the preparation of liposome dispersions. After the rehydration of the lipid film the
crude liposomes were extruded in order to get preferably unilamellar vesicles with a size
in the range of 100 nm and a narrow size distribution. A uniform vesicle diameter simplifies
monitoring of changes in size or size distribution induced by liposome fusion or aggrega-
tion. The size was chosen to meet the criteria for an intravenous application. In theory,
liposomes in this size range are able to accumulate in concerned (e.g. tumor) tissues.
5.1.1 Liposome Dispersions after Extrusion
The effect of the first (0.4 µm membrane) and second (0.1 µm membrane) extrusion
through polycarbonate membranes was investigated using liposome dispersions with in-
creasing lipid contents. Sample compositions are given in Table 5-1. Figure 5-1 shows size
distributions of crude and extruded liposomes. Pictures were taken after each extrusion step
(Figure 5-2). Figure 5-3 finally presents liposome properties as a function of the lipid con-
Lipid content [mM] DOTAP/DOPC
concentration [mg/ml]
Trehalose concentration
[mg/ml]
0 0 / 0 100
2 0.70 / 0.79 100
10 3.49 / 3.93 100
20 6.99 / 7.86 100
30 10.48 / 11.79 100
40 13.97 / 15.72 100
60 20.96 / 23.59 100
Table 5-1: Overview of sample compositions used in this section of the work. DOTAP and
DOPC were used in a molar ratio of 1:1.
68 RESULTS AND DISCUSSION
Figure 5-1: Size distributions of a liposome sample (10 mM lipid) before and after one or two
extrusion steps determined using DLS. The z-average diameter is the intensity-weighted mean di-
ameter derived from the cumulants analysis.
tent. Crude liposome dispersions were cloudy (Figure 5-2, left) and possessed a z-average
of 397.9 nm with a broad, polydisperse size distribution (PDI: 0.606). The first extrusion
step through a 0.4 µm membrane results in a significant size reduction to a z-average of
136.3 nm and a PDI of 0.219. The polydispersity index is a dimensionless number, which
represents the broadness of a size distribution. A value of 0 is obtained for a completely
monodisperse distribution, whereas 1 stands for an entirely heterodisperse distribution.
Figure 5-2: Photo of liposome dispersions. Left: crude liposomes. Center: after extrusion
through 0.4 µm membrane. Right: after extrusion through 0.1 µm membrane.
RESULTS AND DISCUSSION 69
PDI values smaller than 0.2 are supposed to represent monodisperse liposomes (Gibis et
al., 2013, Knudsen et al., 2012). Finally, the second extrusion resulted in a clear liquid
(Figure 5-2, right) with the characteristic opalescence of colloidal dispersions. A further
decrease of the z-average to 110.2 nm and to a PDI of 0.165 was observed. This minor
influence of the second extrusion step on the liposome size was most likely the result of a
complete destruction of large multilamellar vesicles during the first extrusion.
Figure 5-1 shows that the extrusion through a 0.4 µm membrane resulted in vesicles, which
were considerably smaller than the pores of the used membrane. However, the outcome of
the final extrusion step was a vesicle size, which was slightly larger in diameter than the
membrane pores. This behavior was also reported by Lesieur et al. (1991) and Berger et al.
(2001). Both found larger liposome diameters compared to the pore size when using
0.2 µm or smaller polycarbonate membranes. They attributed this behavior to the elastic
deformation of liposome spheres to ellipsoid shape during extrusion.
Figure 5-3 pictures the influence of increasing lipid contents on the extrusion results.
Higher lipid concentrations showed an increased viscosity, which was noticeable during
Figure 5-3: Liposome size, PDI (n = 6) and viscosity (n = 3) of extruded liposomes (0.4 µm
membrane followed by 0.1 µm membrane) with increasing lipid content.
70 RESULTS AND DISCUSSION
extrusion of high concentrated liposome dispersions. Filter membranes were often cracked
and the resistance to extrusion increased drastically. Hence it was not possible to prepare
high concentrated liposome dispersions of the same quality. Liposome size was acceptable
up to 40 mM, whereas the PDI already increased starting from a concentration of 20 mM.
Satisfying liposome dispersions with a lipid concentration of 60 mM could not be prepared
using the described method.
Higher lipid concentrations result in an increased liposome population and an increased
liposome density (Torchilin and Weissig, 2003). Thus, interactions between liposomes such
as fusion or aggregation might be promoted.
The overall average of the zeta potential was 65.8 mV with a weak tendency towards higher
values for higher lipid concentrations (refer to chapter 5.2.1). The high zeta potential values
were attributed to the positive charge of DOTAP. Ciani et al. (2004) found slightly lower
zeta potentials of 48 ± 5 mV for DOTAP/DOPE liposomes in Tris/HCl buffer (pH 7.4) and
Wiggenhorn (2007) a value of 50 mV for DOTAP/DOPC liposomes. High zeta potentials
are beneficial for stable formulations, owing to repulsion of the equally charged particles
(Heurtault et al., 2003).
RESULTS AND DISCUSSION 71
5.1.2 Phase Transition of Lipids and Liposomes
Phase transition temperatures of lipids and liposomes prepared thereof were examined us-
ing differential scanning calorimetry. Measurements were performed in order to determine
the type of the lamellar phase of suspended liposomes used in this work and to compare the
values to literature. Figure 5-4 summarizes the results. The curves “DOPC” and “DOTAP”
were obtained by scanning the pure, solid lipids. Hydrated lipid samples were prepared by
filling lipid and water in the molar ratio 1:21 in aluminium crucibles. This ratio was found
to represent a fully hydrated state (Ulrich et al., 1994). Finally, liposomes composed of
DOTAP and DOPC in the molar ratio 1:1 were measured.
The Tm of hydrated DOPC was in accordance with the value found in literature
(-16.5 °C, cf. chapter 4.1.1). However, the slightly higher Tm of DOTAP (-12 °C) conflicted
with the value (-17.5 °C) proposed by Platscher and Hedinger (2007), but was conform
with the statement of Regelin et al. (2000). The phase transition temperatures of non-hy-
drated lipids were higher than those of the hydrated samples. This effect was reported
Figure 5-4: Thermograms of lipids and liposomes used in this work. Note that the event at 0 °C
in the hydrated or liposomal samples is the melting point of water.
72 RESULTS AND DISCUSSION
for example by Ohtake et al. (2005) in conjunction with DPPC. In the hydrated state, water
molecules are associated with the polar head-groups of phospholipids decreasing the pack-
ing density and lowering the van-der-Waals interactions. In the dried state, the van-der-
Waals interactions increase due to missing water molecules. Consequently the spacing be-
tween phospholipid molecules decreases and the Tm of lipids in the dried state increases.
The Tm of hydrated lipids is lower, respectively (Crowe et al., 1998).
Liposomes composed of DOTAP and DOPC had a phase transition temperature of
-8.01 ± 0.07 °C, which was higher than those of the individual lipids. Consequently, sus-
pended liposomes at room temperature were in the more fluid liquid-crystalline state. The
phase transition temperature of the liposomes was in accordance with the thermogram of
an identical liposome formulation presented by Hirsch-Lerner and Barenholz (1999).
However, the measurement of the phase transition temperature of liposomes was only pos-
sible with lipid concentrations ≥ 30 mM. Otherwise, the phase transition event was over-
lapped by the endothermic melting peak of water.
Mixtures of phospholipids, for example DMPC and DPPC, usually possess a phase transi-
tion temperature between the Tm of the individual lipids. If the difference in chain length is
less than two carbon atoms, ideal mixing of phases occurs (Mabrey and Sturtevant, 1976).
This applies to the mixture of DOTAP and DOPC. However, the phase transition tempera-
ture arose to a value higher than the Tm of the individual phospholipids. This phenomenon
is attributed to the rearrangement of head groups, if positively charged phospholipids are
involved (Jurkiewicz et al., 2006). Ryhänen et al. (2005) found an increase in the phase
transition temperature of the mixture DHAB and DMPC compared to the individual lipids.
They concluded that positively charged head groups pair with the negatively charged phos-
phate of the phosphatidylcholine moiety resulting in a reorientation of the phosphatidyl-
choline head group. Altogether, a decreased head group hydration level due to charge neu-
tralization (Hirsch-Lerner and Barenholz, 1999) and an augmented chain-chain interaction
are responsible for the increased phase transition temperature (Ryhänen et al., 2005).
RESULTS AND DISCUSSION 73
5.2 Characterization of Spray Dried Liposomes
The objective of this section was to investigate the influence of spray drying and rehydra-
tion on liposomes. The attention was turned on the impact of different nozzle types: A
25 kHz ultrasonic nozzle, a two fluid nozzle and a 60 kHz ultrasonic nozzle. In addition,
experiments were performed using the 25 kHz ultrasonic nozzle and a reduced flow rate of
the liquid feed (0.5 ml/min). Table 5-1 gives an overview of sample compositions.
Spray dried powders were dissolved in an appropriate amount of water in order to get re-
hydrated samples with the initial (lipid) concentration.
5.2.1 Dynamic Light Scattering
Figure 5-5 – Figure 5-8 present Zetasizer results of vesicles before spray drying and after
spray drying and rehydration of the powder.
Figure 5-5: Size and size distributions before and after the spray drying step in dependence of
lipid contents using the 25 kHz ultrasonic nozzle and a feed rate of 1 ml/min (n = 3).
74 RESULTS AND DISCUSSION
Figure 5-6: Size and size distributions before and after the spray drying step in dependence of
lipid contents using a two fluid nozzle, an atomization pressure of 2 bar and a feed rate of
1 ml/min (n = 3).
Figure 5-7: Size and size distributions before and after the spray drying step in dependence of
lipid contents using the 60 kHz ultrasonic nozzle and a feed rate of 1 ml/min (n = 3).
RESULTS AND DISCUSSION 75
Figure 5-8: Size and size distributions before and after the spray drying step in dependence of
lipid contents using the 25 kHz ultrasonic nozzle and a reduced feed rate of 0.5 ml/min (n = 3).
First of all, size measurements revealed that the chosen liposome formulation was suitable
for spray drying. Changes in size or size distribution were marginal and the rehydrated
liposomes showed nearly the same properties regarding size and size distribution. However,
slight changes could be observed: Figure 5-5 shows results of DLS measurements of re-
constituted liposomes, which were spray dried using the 25 kHz ultrasonic nozzle. They
showed a decrease in diameter (p-value ≤ 0.05 for the 2 mM and 10 mM concentration)
after the rehydration if the lipid concentration was low. Concentrations ≥ 20 mM did not
show this effect. The polydispersity index was almost equal after spray drying and rehy-
dration. As described in chapter 5.1.1, liposome size and PDI of dispersions with 60 mM
lipid did not meet the quality characteristics of low concentrated dispersions. Spray drying
using the 25 kHz ultrasonic nozzle and rehydration slightly improved these characteristics,
but a consistent production of spray dried 60 mM liposomes was not possible. Figure 5-6
presents results of liposomes processed with the two fluid nozzle. Regarding the liposome
size, a diminishing effect was not observed. In contrast, a slight increase in the vesicle
diameter was found for the 30 mM and 40 mM concentration. The two fluid nozzle was
able to downsize the diameter of the highest concentrated liposomes (60 mM) to 130 nm
76 RESULTS AND DISCUSSION
and a PDI of 0.25. In general, no difference between the size distributions (PDI) of the
25 kHz ultrasonic nozzle and the two fluid nozzle was noticeable. Figure 5-7 summarizes
results obtained with the 60 kHz ultrasonic nozzle. Despite the higher frequency a similar
behavior was observed compared to the 25 kHz nozzle: Dispersions with a lipid content of
10 mM featured smaller vesicles after spray drying and reconstitution (p-value ≤ 0.05,
10 mM concentration), whereas the two higher lipid contents resulted in equally sized or
even slightly larger liposomes. Finally, Figure 5-8 shows liposome properties after spray
drying with the 25 kHz ultrasonic nozzle and a reduced flow rate of 0.5 ml/min. Previously
described observations were confirmed: The size of low concentrated liposomes (p-value
≤ 0.05, 10 mM concentration) was reduced after spray drying and rehydration using an
ultrasonic nozzle. The upper limit was 20 mM. Liposome concentrations above this value
did not show this effect as well as liposomes atomized through the two fluid nozzle. In
addition, initially broad size distributions (here: 40 mM) or bigger liposome diameters were
corrected through the spray drying process irrespective of the nozzle type used.
Figure 5-9: Zeta potentials of liposomes before and after (striped bars) spray drying. Left side
(white bars): ultrasonic nozzle 25 kHz; right side (grey bars): two fluid nozzle (n = 3).
RESULTS AND DISCUSSION 77
Figure 5-10: Zeta potentials (n = 3) of liposomes before and after (striped bars) spray drying.
Left side (white bars): ultrasonic nozzle 60 kHz; right side (grey bars): ultrasonic nozzle 25 kHz
with a reduced liquid feed rate (0.5 ml/min).
In general, dynamic light scattering results obtained in these experiments were in good
agreement with the results reported in literature: Hauser and Strauss (1987) referred little
effect of spray drying on the average vesicle size and size distribution. Goldbach et al.
(1993a) and Wessman et al. (2010) reported slight shifts towards smaller radii (197 ± 46
nm before spray drying and 188 ± 36 nm after spray drying). However, they used two fluid
nozzles, which induced no changes in vesicle size in this work. Wiggenhorn (2007) also
found no effects on liposome size and polydispersity index after spray drying and rehydra-
tion. Refer to Table 3-2 for detailed information about the process conditions of the men-
tioned publications.
Figure 5-9 and Figure 5-10 show the results of zeta potential measurements. Figure 5-9
reveals a slight tendency towards increasing zeta potentials for increased lipid contents.
Zeta potentials after spray drying were in the same range irrespective of the nozzle type
used. Figure 5-10 presents the results for the 60 kHz ultrasonic nozzle and the 25 kHz
nozzle conducted with the reduced flow rate. Liposomes processed with the 60 kHz nozzle
78 RESULTS AND DISCUSSION
generally showed a similar behavior compared to the 25 kHz nozzle and the two fluid noz-
zle. However, no tendency of liposomes processed with the 25 kHz nozzle and a reduced
flow rate towards higher zeta potentials for increased lipid contents could be observed.
It was remarkable, that zeta potentials increased after rehydration. In order to assure a com-
plete disintegration, it was necessary to shake samples during rehydration. The increased
liquid/air interface could promote the dissolution of CO2 in the samples. This was likely to
be the reason of a lowered pH value.
This assumption was proved by pH measurements, which yielded in a value of 4.6 of the
starting liposome dispersion and a slightly lower value of 4.5 after rehydration.
In a further experiment, the pH value was adjusted to 3.0 or 2.0 prior to the zeta potential
measurement. Thereby zeta potentials increased remarkably up to 75 or 90 mV, respec-
tively. Hence, it was supposed that the disintegration process and the connected lowering
of the pH value were responsible for the increased zeta potentials after rehydration.
5.2.2 Atomization of Liposomes
In order to evaluate whether changes in liposome size occurred during atomization or dur-
ing drying, liposome dispersions where atomized through the nozzles without the subse-
quent drying step. The spray was collected directly after the nozzle tip in Sarstedt tubes. A
flow rate of 1 ml/min was used and nozzles were operated with the same adjustments as
they were within the spray dryer.
The vesicle size of low concentrated liposome dispersion (Figure 5-11, A) was reduced by
approximately 7 % when spraying with the 25 kHz ultrasonic nozzle (p-value ≤ 0.05) and
by 4 % when using the 60 kHz ultrasonic nozzle (p-value ≤ 0.05). This corresponded to the
results obtained with the spray dried liposomes. However, size reduction was more distinct:
Liposome size was reduced by approximately 13 % after spray drying and reconstitution
using the 25 kHz ultrasonic nozzle and by 9 % using the 60 kHz nozzle, respectively. The
two fluid nozzle and all sprayed samples of the high concentrated liposome dispersion (Fig-
ure 5-11, B) showed no changes in size after spraying. PDI of all samples was < 0.2 and no
effects on the zeta potentials were observed.
RESULTS AND DISCUSSION 79
Although size reductions were less distinct after atomization, the results of this experiment
confirmed data obtained with the spray dried samples. Therefore, changes in liposome size
after spray drying could be – to the most part – attributed to the atomization process. The
ultrasonic nozzles caused a size reduction of liposomes. Hence, the effects of ultrasonic
vibration and cavitation seemed to outweigh the shear stress, which appears during atomi-
zation in two fluid nozzles. The comparatively short residence time at the nozzle tip pre-
vented a further size reduction (cf. chapter 3.2).
A B
Figure 5-11: Bar chart A shows the percentage of liposome size after SD compared to the ves-
icle size before spraying (100 %) of a 10 mM lipid concentration. Bar chart B shows results of
the experiment using a 30 mM lipid concentration (n = 3).
80 RESULTS AND DISCUSSION
5.2.3 Lipid Recovery
The recovery of DOPC before and after the spray drying process was measured using a
HPLC method. First, the lipid concentration was determined after the preparation steps.
93.7 ± 5.6 % of the calculated and weighed amount was found in the samples. A loss of
lipid was likely to occur during the removal of the crude liposome dispersion from the
round bottom flask. In addition, lipids could get lost during extrusion.
Figure 5-12 shows lipid recoveries after spray drying and rehydration. Up to a lipid con-
centration of 40 mM, the lipid recovery was approximately 100 %. However, a lipid loss
was detected with the highest lipid concentration used. Here, the lipid to sugar ratio was
1:4.9. In all other samples, the liposomes were embedded in a high amount of sugar mole-
cules. It was supposed that lipids got physically lost through e.g. wall deposition in the
process chamber or chemically through oxidation or hydrolysis, if high liposome concen-
trations were used. The HPLC method enabled the detection of double bonds in the unsatu-
rated hydrocarbon chains. Oxidations products without double bonds were not detected.
Figure 5-13 shows a typical chromatogram.
Figure 5-12: Lipid recovery in rehydrated, spray dried powders containing liposomes. The val-
ues specified in the diagram refer to the concentration of DOPC in the liquid feed before spray
drying (100 %) (n = 3).
RESULTS AND DISCUSSION 81
Taken as a whole, the results – with the exception of the 60 mM samples – were in good
agreement with the publications of Goldbach et al. (1993a) and Wiggenhorn (2007).
Goldbach et al. (1993a) found that lipids were not significantly hydrolyzed or oxidized
during spray drying. Wiggenhorn (2007) found slight decreases up to 3 % in the lipid con-
tent of a 10 mM liposome formulation when using high drying temperatures up to a Tinlet
of 220 °C and a low trehalose content of 5 % (w/v). The experiments presented were per-
formed using a Tinlet of 150 °C and the twofold amount of trehalose in the liquid feed
(10 % w/v).
Figure 5-13: Typical chromatogram of a reconstituted sample. DOPC has a retention time of
6.5 min. The signals after 1 min are the injection peak and DOTAP, which was not retained under
the described conditions.
82 RESULTS AND DISCUSSION
5.2.4 Calorimetric Study on SD Liposomes
Figure 5-14 shows representative thermograms of a freshly prepared liposome dispersion
and the equivalent spray dried and rehydrated sample. The thermograms are almost identi-
cal, which causes the assumption that liposome membranes remain unaffected during spray
drying. Besides the phase transition temperature, the glass transition temperature of the
maximally freeze-concentrated solution, Tg´, was determined. Table 5-2 summarizes the
results. As described earlier, the measurement of the phase transition temperature was pos-
sible only for high lipid concentrations. No influence of the nozzle type on the Tm or Tg´
could be determined and spray dried samples had only a marginal lower Tm than the initial
dispersions. Tg´ values of liposome dispersions were slightly lower than the value deter-
mined of a trehalose solution (10 % w/v), -29.88 °C. The Tg´ values were in good accord-
ance with literature (Her and Nail, 1994).
Figure 5-15 shows thermograms of spray dried powders. The phase transition temperature
of liposomes in the dried, dehydrated state was detectable. Several publications deal with
Figure 5-14: Thermogram of a liposome dispersion (40 mM) before spray drying and after the
rehydration of the spray dried powder. The magnifications show Tg´ on the left side and Tm on the
right side.
RESULTS AND DISCUSSION 83
Sample Tm [°C] Tg´[°C]
initial liposome dispersion
(30 / 40 / 60 mM lipid) -8.01 ± 0.07 -30.22 ± 0.48
after spray drying using the
25 kHz ultrasonic nozzle
(30 / 40 / 60 mM lipid)
-8.38 ± 0.21 -30.52 ± 0.57
after spray drying using the
two fluid nozzle
(30 / 40 / 60 mM lipid)
-8.23 ± 0.21 -30.46 ± 1.03
Table 5-2: Mean values (n = 3) of phase transition temperatures and glass transition tempera-
tures of the maximally freeze-concentrated solution of liposome dispersions with high lipid con-
tents (30, 40 and 60 mM).
the effect of trehalose or other sugars on the Tm in the dried state (Koster et al., 1994, Ohtake
et al., 2005). Sugars are able to prevent the increase in fluid-to-gel phase transition temper-
ature, which usually occurs during dehydration of phospholipid bilayers. This behavior is
traced back to hydrogen bonding between sugar molecules and polar residues of phospho-
lipid molecules (Crowe et al., 1998). Besides these direct molecular interactions the vitri-
fication of sugars was proposed to be necessary for the stabilization of lipid bilayers (Koster
et al., 1994).
Figure 5-15: Phase transition temperatures of spray dried liposomes in the dehydrated state. Li-
pid concentration in the liquid feed was 40 mM, if not stated otherwise. The lipid to sugar ratio
was 1:7.3, respectively.
84 RESULTS AND DISCUSSION
A special case occurs, if the glass transition temperature, Tg, is greater than Tm. This case
was existent in the samples presented in Figure 5-15. Then, according to Koster et al.
(1994), the Tm is not only kept constant in the dried state but is lowered by approximately
20 °C compared to the Tm of the fully hydrated state. A further publication of Koster et al.
(2000) examined the transition temperatures as a function of hydration of DOPC, which
was a component of liposomes used in this work. Using a trehalose lipid ratio of 2:1, they
found a Tm of DOPC in the dried state of -76.0 °C. This huge depression of more than
50 °C in Tm was also found in the spray dried samples of this work, although a mixture of
DOPC and DOTAP in the molar ratio of 1:1 was used. The mean value was -86.65 ± 0.67
°C. Figure 5-15 shows that neither the nozzle types had an influence on the Tm nor the lipid
concentration. As already observed for the phase transitions in the hydrated state, Tm was
only detectable in samples with high (> 30 mM) lipid concentrations.
In summary, results of DLS and DSC measurements indicated that liposomes were well
protected during spray drying in a wide range of lipid concentrations. The lipid to sugar
molar ratio was 1:146 (2 mM) up to 1:4.9 (60 mM). A limiting factor was the extrusion
process, which was nearly impossible at high lipid concentrations (60 mM). DSC thermo-
grams showed that liposomes were liquid-crystalline at each point in time, both in the fully
hydrated (Tm ~ -8 °C) and in the dried state (Tm ~ -87 °C). The disadvantageous pass (cf.
chapter 2.8.2) through Tm was avoided by the use of the excipient trehalose.
Regarding the influence of the nozzle type, the three tested atomizers had no or only a slight
influence on the liposome size and size distribution. The ultrasonic nozzles triggered a
slight reduction of the liposome size after spray drying and reconstitution, if the lipid con-
centration was ≤ 20 mM. This phenomenon was attributed to the atomizing step through
measurements of the liposome size directly after atomization without drying. Liposomes
spray dried with the two fluid nozzle did not show any changes in size after rehydration of
the powder. Size distributions remained unchanged.
RESULTS AND DISCUSSION 85
5.3 Characterization of Spray Dried Powders
The following section deals with the analysis of spray dried powders containing liposomes.
Particular attention should be paid (a) to the influence of liposomes on the powder proper-
ties and (b) to characteristics, which result from the use of different nozzle types. Sample
compositions are given in Table 5-1.
5.3.1 Particle Yield
Figure 5-16 depicts the powder yields obtained after the spray drying of liposome disper-
sions. Despite the standard deviations, a tendency was noticeable. With increasing lipid
contents the deposition of powder in the cyclone and the T-piece decreased, which led to a
higher powder yield. In addition, powders appeared less electrostatically charged and there
seemed to be a decreased fraction of fine particles. Standard deviations were caused by
fluctuating wall depositions, depositions of powder and water droplets in the T-piece of the
spray dryer and the manual removal of powders from the collection vessel.
Figure 5-16: Powder yield as a function of lipid content and nozzle type (n = 3).
86 RESULTS AND DISCUSSION
The 25 kHz ultrasonic nozzle produced best results irrespective of the flow rate used. The
two fluid nozzle produced finer powders, which could explain the lower particle yields.
With respect to the particle size, the 60 kHz ultrasonic nozzle should perform better than
the two fluid nozzle. However, with increasing spraying times, some bigger droplets
formed at the nozzle tip and impeded an optimal droplet drying resulting in the worst
particle yield.
5.3.2 Particle Size and Morphology
Figure 5-17 displays the mean particle sizes (d50) produced using different nozzles and
increasing lipid concentrations. The 25 kHz ultrasonic nozzle turned out particle sizes of
approximately 23 µm. The flow rate had no significant influence on the particle size. The
second ultrasonic nozzle, 60 kHz, had a main output of particles in the range of 15 – 20 µm
in diameter and the two fluid nozzle around 7 µm in diameter. The lipid content had no or
only little effect on the d50 values of the volume based size distributions. A minor trend
towards increasing particle sizes with increasing lipid content could be observed.
Figure 5-17: d50 values of the volume based size distributions (n = 3).
RESULTS AND DISCUSSION 87
Nozzle type d10 (10 mM) [µm] d10 (40 mM) [µm]
US nozzle 25 kHz 3.03 ± 0.01 4.27 ± 0.26
TF nozzle 2.12 ± 0.07 2.39 ± 0.16
US nozzle 60 kHz 2.16 ± 0.16 2.99 ± 0.09
US nozzle 25 kHz, 50 %
flow 3.11 ± 0.10 4.17 ± 0.19
Table 5-3: d10 values of the volume based size distribution (n = 3). Comparison between samples
of a low lipid content (10 mM) and samples of a high lipid content (40 mM).
However, if the fine particles were taken into account (Table 5-3) the observations made in
the cyclone were confirmed. Increasing lipid concentrations led to an increased d10 value
irrespective of the nozzle used. This could be attributed to the high viscosity of the high
concentrated liposome samples (cf. Figure 5-3). Increasing viscosities enlarge the droplet
size after atomization (Anish et al., 2014) and therefore the particle size. Another reason
might be the higher overall solid content, which triggers the faster formation of a solid crust
during drying (Elversson and Millqvist-Fureby, 2005).
Figure 5-18: Volume based size distributions of spray dried trehalose powders containing
10 mM lipid in the liquid feed.
88 RESULTS AND DISCUSSION
Figure 5-18 shows size distributions of powders produced with the three nozzle types used
in this work. Size distribution curves of the ultrasonic nozzles were similar, with a slight
shift of the 60 kHz nozzle towards smaller particle diameters. Both nozzles produced a fine
particle fraction, which was reflected by the d10 values presented in Table 5-3. The two
fluid nozzle had a broad particle size distribution in the range of 1 – 30 µm.
SEM pictures of particles generated by the 25 kHz ultrasonic nozzle are presented in Figure
5-19. Round particles were formed and the size distribution was quite narrow. However,
some finer particles or fragments of larger particles were visible in pictures a and b. Pure
trehalose particles and particles containing 2 mM lipid had a rough, frayed surface. With
increasing lipid concentrations the surface got smoother and particles had the shape of golf
balls. A further increase in lipid content partly gave completely smooth surfaces.
Powders atomized using the two fluid nozzle are presented in Figure 5-20. First of all, the
broad size distribution was noticeable. Particles up to 20 µm in diameter were identifiable,
but also many fragments and very fine particles were visible. Larger particles showed the
golf ball structure. However, no differences in surface appearance between low and high
lipid concentrations were perceivable.
Similar observations could be made of the powders depicted in Figure 5-21. Pictures a-c
show powders produced with the 60 kHz ultrasonic nozzle. Both the low and the high lip-
osome concentration resulted in particles with a smooth surface. The biggest particles had
a size comparable to that produced by the 25 kHz ultrasonic nozzle (Figure 5-19). This was
in correspondence with the size distributions shown in Figure 5-18, where the upper limit
of both ultrasonic nozzles was in the same region.
Particles, which were atomized by the 25 kHz nozzle with a reduced flow rate, were similar
to the powders obtained with the higher flow rate (cf. Figure 5-19). However, differences
between high and low lipid concentrations were hardly observable and pure trehalose par-
ticles did not show the described roughness.
Regarding the results of laser diffraction and SEM, liposome containing powders were su-
perior compared to pure trehalose particles. They had a lower fine particle fraction, a higher
particle yield and a smoother surface. A possible explanation of the surface smoothening
was the enrichment of phospholipids or complete liposomes at the particle surface. Further
investigations on this question were made using an ultrasonic levitator (cf. 5.3.5). A partial
RESULTS AND DISCUSSION 89
a) pure trehalose, 1000x b) trehalose + 2 mM lipid, 1000x
c) trehalose + 10 mM lipid, 1000x d) trehalose + 20 mM lipid, 1000x
e) trehalose + 30 mM lipid, 1000x f) trehalose + 60 mM lipid, 1000x
Figure 5-19: SEM pictures of spray dried particles of trehalose (10 % m/v in the liquid feed)
containing different amounts of lipid. The pictured powders were atomized using the 25 kHz ul-
trasonic nozzle and a flow rate of 1.0 ml/min. All pictures were taken using the same magnifica-
tion.
melting of the lipid/sugar mixture at the particle surface might be another cause of this
phenomenon. Regarding the roughness at low lipid concentrations, no unequivocal expla-
nation could be found for the different surface appearance of powders produced with the
20µm 20µm
20µm 20µm
20µm 20µm
90 RESULTS AND DISCUSSION
a) pure trehalose, 2000x b) trehalose + 2 mM lipid, 1000x
c) trehalose + 10 mM lipid, 1000x d) trehalose + 60 mM lipid, 1000x
Figure 5-20: SEM pictures of spray dried particles of trehalose (10% w/v in the liquid feed)
containing different amounts of lipid. The pictured powders were atomized using a two fluid noz-
zle with an orifice of 1.0 mm and an atomizing air pressure of 2 bar. Note the different magnifica-
tions of the pictures.
25 kHz ultrasonic nozzle and powders produced with the 60 kHz ultrasonic nozzle or the
reduced flow rate. Perhaps differences in the drying time, residual moisture or low Tg could
be an explanation (cf. chapter 5.3.3.).
20µm
20µm 20µm
20µm
RESULTS AND DISCUSSION 91
a) pure trehalose, 1000x b) trehalose + 10 mM lipid, 1000x
c) trehalose + 40 mM lipid, 1000x d) pure trehalose, 500x
e) trehalose + 10 mM lipid, 1000x f) trehalose + 60 mM lipid, 500x
Figure 5-21: SEM pictures of spray dried particles. Powders pictured in a-c were atomized by
the 60 kHz ultrasonic nozzle. Pictures d-f show powders produced with the 25 kHz ultrasonic
nozzle and a reduced flow rate of 0.5 ml/min. Note the different magnifications of the pictures.
20µm 20µm
20µm
20µm 50µm
50µm
92 RESULTS AND DISCUSSION
5.3.3 Residual Moisture and Glass Transition
Residual moisture contents of spray dried powders varied from almost 6 % to under 2 %
(Figure 5-22). Both the nozzle type used and the lipid content had a strong influence on the
moisture content of spray dried powders. With increasing lipid contents moisture decreased
irrespective of the nozzle type used. The higher ratio of lipophilic substances in the feed
might reduce the amount of solids in the powder, which is able to contain moisture (Hansen
et al., 2004). The differences in residual moisture found between the nozzle types could be
attributed to the different particle sizes generated. Large particles with their longer diffusion
paths gave an increased moisture content. This was especially noticeable for powders at-
omized by the 25 kHz ultrasonic nozzle. A reduction of the feed rate to 0.5 ml/min was
successful in the diminishment of residual moisture. Regarding the corresponding data
points, moisture was reduced by approximately 1 %. Although particles atomized by the
two fluid nozzle had the smallest mean diameter, they exhibited high residual moisture
contents.
This was likely a result of scattered large particles, which could be found in these powders
(Figure 5-20). Another aspect might be the soft spray produced by ultrasonic atomization
Figure 5-22: Residual moisture contents of powders as a function of the lipid content (n = 3).
RESULTS AND DISCUSSION 93
as it might improve the residence time of each droplet/particle in the process chamber and
therefore decrease residual moisture by more extensive drying. In addition, the two fluid
nozzle introduces a cold gas stream into the drying chamber (Masters, 2002).
Figure 5-23 shows glass transition temperatures of spray dried products. The appropriate
thermograms are located on the following pages. Amorphous trehalose with 0 % residual
moisture has a comparably high glass transition temperature of 115 – 117 °C (Surana et al.,
2004, Miller et al., 1997). This temperature marks the transition of a glassy state to a rubber-
like state. The glassy state can be characterized as “kinetically frozen” with a high viscosity,
whereas the rubbery state above Tg features a lower viscosity and increased molecular mo-
tions (Hancock and Zografi, 1997). Water acts as plasticizer, which decreases the glass
transition temperature and increases the matrix mobility (Lai et al., 1999). The plasticizing
effect of water was noticeable in the glass transition temperatures of spray dried powders.
Samples having the lowest residual moisture contents had the highest glass transition tem-
peratures around 70 °C (25 kHz nozzle, 50 % flow and 60 kHz nozzle). Compared to the
results of Adler and Lee (1999), the Tg values found as a function of residual water content
were quite low. This might be an effect of the embedded lipids. Powders spray dried using
the 25 kHz ultrasonic nozzle or the two fluid nozzle had a remarkably lower Tg, if the lipid
content was low and the residual moisture high, respectively. These values, which were
rarely above 40 °C, are likely to become critical with respect to the (long term) stability of
these products.
Sun et al. (1996) published a study about the direct relationship between the glass transition
temperature of sugar glasses and the leakage of carboxyfluorescein from liposomes embed-
ded in sucrose. If liposomes were stored below the glass transition temperature, marker
retention was high and the liposome size was stable. Storing the samples above the glass
transition temperature resulted in a significantly increased leakage and an increased lipo-
some size due to fusion. The correlation between the glassy-to-rubbery transition and the
stability of embedded substances in not only crucial for liposomes, but also for small mol-
ecules and peptides or proteins (Yoshioka and Aso, 2007). Regarding the storage conditions
of pharmaceutical products, it is desirable to produce spray dried powders with a sufficient
high glass transition temperature.
94 RESULTS AND DISCUSSION
Figure 5-23: Glass transition temperatures of spray dried powders (n = 3).
Figure 5-24 – Figure 5-27 show representative thermograms for each formulation and noz-
zle type used. Typically, the second heating scans are depicted and used for the evaluation
in order to avoid interference from enthalpy relaxations. Enthalpy relaxations occurred dur-
ing the first heating steps, especially at low lipid concentrations or rather high residual
moisture contents. Regarding the second heating scans, heat capacity changes decreased
with increasing lipid concentrations.
High lipid concentrations resulted in a second transition, which occurred only in the first
heating scan and is marked with an arrow. These thermal events at 50 °C were limited to
lipid to sugar ratios higher than 1:10 (> 30 mM), but were found irrespective of the nozzle
type used. They were put down to the glassy-to-rubbery transition of a lipid-rich trehalose
phase around or inside the liposomes. A higher lipid concentration is connected to a higher
number of liposomes per volume and hence a larger inner volume (Torchilin and Weissig,
2003). This amount of encapsulated water was probably less easily removable during dry-
ing compared to the water in the surrounding matrix. As a result, an additional, lower Tg
appeared, although the formulation had a lower moisture content. In the second heating
scan, these transitions had vanished without changing the actual Tg. Another aspect might
be the sensitivity of the DSC measurement not being high enough for the detection of these
RESULTS AND DISCUSSION 95
Figure 5-24: Heating scans of spray dried powders atomized using the 25 kHz ultrasonic nozzle.
The lipid contents increase from top to bottom. Thermal events occurring only in samples with
high lipid to sugar ratios are labeled with an arrow. If not stated otherwise, pictured thermograms
were recorded during the second heating scan.
Figure 5-25: Heating scans of spray dried powders atomized using the two fluid nozzle. The li-
pid contents increase from top to bottom. Thermal events occurring only in samples with high li-
pid to sugar ratios are labeled with an arrow. If not stated otherwise, pictured thermograms were
recorded during the second heating scan.
96 RESULTS AND DISCUSSION
Figure 5-26: Heating scans of spray dried powders atomized using the 60 kHz ultrasonic nozzle.
The lipid contents increase from top to bottom. Thermal events occurring only in samples with
high lipid to sugar ratios are labeled with an arrow. If not stated otherwise, pictured thermograms
were recorded during the second heating scan.
Figure 5-27: Heating scans of spray dried powders atomized using the 25 kHz ultrasonic nozzle
and a reduced flow rate of 0.5 ml/min. The lipid contents increase from top to bottom. Thermal
events occurring only in samples with high lipid to sugar ratios are labeled with an arrow. If not
stated otherwise, pictured thermograms were recorded during the second heating scan.
RESULTS AND DISCUSSION 97
phase transitions in formulations with lower lipid contents.
Exothermic events at ~ 110 °C were peaks of crystallization, which were also detectable,
if samples were scanned only once, without cooling and a second heating run. Pronounced
endothermic melting peaks at ~ 95 °C were recorded especially in samples spray dried with
the 25 kHz ultrasonic nozzle at low lipid concentrations. A review of all DSC measure-
ments performed revealed that these melting peaks occurred especially at residual moisture
contents higher than 3.5 %. The melting point of crystalline trehalose dihydrate was re-
ported to be between 95 – 100 °C (Meurant, 1963). Hence, it was suggested that crystalli-
zation occurred to some extent in powders with high moisture contents. This could be dis-
advantageous regarding liposome stability. Therefore, a further examination of the powders
was performed using XRD.
Figure 5-28 shows results of the WAXD measurements of spray dried samples. Crystalline
trehalose dihydrate was used as a reference and showed a typical pattern of crystalline sub-
stances. Spray dried trehalose (marked as 0 mM) and spray dried samples with a lipid con-
centration of 2 mM represent powders with a high amount of residual water. These samples
showed the melting peak in their DSC thermograms at ~ 95 °C. The 40 mM sample repre-
sents powders with a low residual moisture content. Neither the two samples with the high
residual water content nor the 40 mM sample showed any signs of crystallinity. The typical
halo of amorphous materials was recorded. Consequently, the melting peaks in the DSC
thermograms were artifacts of the DSC measurements. A further experiment was per-
formed to prove this assumption:
Pure spray dried trehalose was stored at 40 °C or 80 °C previous to the XRD measurement.
The sample stored at 40 °C showed a slight increase in crystalline patterns whereas the
sample stored at 80 °C showed considerably a partly crystalline structure (Figure 5-29).
In summary, the melting peaks found in the samples with high residual moisture contents
originated from the first heating scan, where a partial crystallization was induced. The slight
exothermic signal at ~ 70 °C, which was likely to be a peak of cold crystallization, proved
the assumption. In brief, the spray dried formulations examined in this part of the work
were amorphous after spray drying. Spray dried powders with a residual moisture content
above 3.5 % exhibited a tendency to crystallize during storage at elevated temperatures
(please refer also to chapter 5.8 “Stability of SD Liposome Formulations”).
98 RESULTS AND DISCUSSION
Figure 5-28: WAXD of crystalline trehalose dihydrate and samples spray dried using the
25 kHz ultrasonic nozzle.
Figure 5-29: WAXD of crystalline trehalose dihydrate and spray dried trehalose, which was di-
rectly measured after spray drying (red), stored at 40 °C (pink) or 80° C (green) prior to the meas-
urement.
RESULTS AND DISCUSSION 99
5.3.4 Comparison between Spray Drying and Freeze Drying
The experiment described in the following section was performed for the purpose of a com-
parative study of the two drying methods. A batch of liposome dispersion containing
10 mM lipid and 100 mg/ml trehalose (refer to Table 5-1 for the exact sample composition)
was freeze dried (FD) using the freeze drying cycle depicted in chapter 4.2.15. The FD cake
was reconstituted with an appropriate volume of water to get the initial lipid concentration.
Results of the subsequent Zetasizer measurements are summarized in Table 5-4. Liposome
size and size distributions were in the same range as those achieved with the spray drying
process. Both drying methods resulted in stable liposomes. Considerable differences be-
tween the freeze dried cake and the spray dried powders were obtained in terms of the
residual moisture content and the connected glass transition temperature. The lyophilisate
had a residual moisture content < 1% and Tg > 80 °C, which is advantageous regarding the
long term stability and storage conditions required for the product. A residual moisture
content below 1 % was identified as crucial with respect to the long-term storage of freeze
dried liposomes (van Winden and Crommelin, 1997). SEM pictures of the lyophilisate (Fig-
ure 5-30) show an acceptable cake with the typical honeycomb structure.
Both spray drying and freeze drying showed comparable results in terms of the liposome
protection during drying. The chosen liposome formulation seemed to be suitable for
freeze drying, too. Regarding the residual moisture content and Tg, the expensive and time-
Liposome and powder properties Spray drying Freeze drying
Before drying
Liposome size [nm] 113.1 ± 7.2 105.3 ± 0.4
PDI < 0.2 0.167 ± 0.01
Zeta potential [mV] 60 – 75 62.6 ± 4.5
After drying and re-
constitution
Liposome size [nm] 100 – 108* 102.3 ± 0.3
PDI 0.17 – 0.21* 0.105 ± 0.02
Zeta potential [mV] 64 – 88* 72.4 ± 5.8
Residual moisture [%] 3.5 – 4.3* 0.92 ± 0.13
Glass transition temperature [°C] 46.8 – 71.1* 83.5 ± 6.7
Table 5-4: Comparison between liposome and powder properties after spray drying and freeze
drying of a liposome dispersion with a lipid content of 10 mM. * depending on the nozzle type
used for atomization.
100 RESULTS AND DISCUSSION
a) view from top, 250x b) fragment of FD cake, 15x
c) fragment of FD cake, 250x d) view from outside wall, 500x
Figure 5-30: SEM pictures of freeze dried liposome dispersions containing 10 mM lipid and
100 mg/ml trehalose. Please note the different magnifications.
consuming lyophilization offered advantages, while the equivalent spray dried products
suffer from high residual moisture contents. Several strategies of decreasing the residual
moisture were pointed out in the previous chapters, such as increasing the lipid content or
reducing the flow rate of the liquid feed. Although the residual moisture might be challeng-
ing, the final product of the spray drying process is a powder, which can be designed for
the intended application, for example in a DPI (Vehring, 2008).
2mm
100µm 50µm
100µm
RESULTS AND DISCUSSION 101
5.3.5 Levitation Experiments
Samples, which were spray dried as described in the previous chapters, were examined
using levitation. A small volume of each dispersion (~ 1 ml) was prepared in the same
manner as liposome dispersions used for the spray drying experiments. Sample composi-
tions are given in Table 5-5.
Figure 5-31 shows a typical evaluation of the levitation process. After the insertion of a
droplet (2 µl) into the acoustic field, the surface temperature was measured by an infrared
camera and recorded as a function of the time (continuous line). The broken line describes
the aspect ratio, which is the ratio between the horizontal diameter and the vertical diameter
of a droplet at each time point. The axial levitation force is greater than the radial one,
which provokes a flattening of the droplets as soon as they are inserted into the acoustic
field (Trinh and Hsu, 1986). This results in aspect ratios > 1.
Finally, the dashed/dotted line represents the decrease in volume of a droplet during drying.
The ratio of the radius of a surface equivalent sphere at each time point to the radius of the
surface equivalent sphere at the starting point (0 s) is plotted against the time, starting with
a value of 1. The calculation of the radius of a surface equivalent sphere is described by
Lorenzen and Lee (2012).
The slope of the r(t)2/r(0)2 curve during the first drying phase was used for the calculation
of the evaporation coefficient according to the d2 law (Frohn and Roth, 2000). A spherical
droplet of pure solvent and an evaporation into a boundary layer of still air and constant
Lipid content [mM] DOTAP/DOPC
concentration [mg/ml]
trehalose concentration
[mg/ml]
0 0 / 0 100
10 3.49 / 3.93 100
15 5.24 / 5.90 100
20 6.99 / 7.86 100
30 10.48 / 11.79 100
40 13.97 / 15.72 100
60 20.96 / 23.59 100
Table 5-5: Overview of sample compositions. DOTAP and DOPC were used in the molar ratio
1:1. Sample compositions were in accordance with the samples used for the spray drying experi-
ments. The 15 mM concentration was added additionally.
102 RESULTS AND DISCUSSION
Figure 5-31: Evaluation of a levitation experiment, exemplified with a lipid concentration of
10 mM.
temperature was supposed:
𝑟(𝑡)2
𝑟(0)2= 1 − {
2𝐷𝑏𝑀
𝜌𝑅(
𝑃𝑆
𝑇𝑆−
𝑃∞
𝑇∞)}
𝑡
𝑟(0)2
Equation 5-1
𝑟(0) is the initial droplet radius and 𝑟(𝑡) is the droplet radius at a point in time. 𝐷𝑏 is the
diffusivity of vapour in the droplet surrounding boundary layer. 𝑀 is the molecular weight
of the solvent (water) and 𝜌 its density. 𝑅 is the gas constant. 𝑃 is the partial vapour pressure
at the droplet surface (S) and at the outside plane of the stagnant boundary layer (∞). The
same applies to the temperature 𝑇. Radii of the surface equivalent sphere were used as
initial and momentary droplet radii. Evaporation coefficients of different liposome formu-
lations are presented in Figure 5-33.
RESULTS AND DISCUSSION 103
Figure 5-31 shows that the evaporation rate decreased drastically at the critical point, be-
cause a solid crust had formed. Diffusion and evaporation of water decelerated in the sec-
ond drying stage.
The end of the constant rate period appeared as an increase in droplet/particle surface tem-
perature as soon as a solid crust had formed. The point of inflection was used for the deter-
mination of the critical point. Figure 5-31 shows that both methods were in good agreement
regarding the critical point. In addition, the aspect ratio changed in the vicinity of the critical
point and increased during the falling rate period. This behavior was also reported by
Schiffter and Lee (2007b) for solutions of trehalose and trehalose particles, respectively. In
this work, this behavior was confirmed not only for trehalose droplets/particles but also for
the liposome containing trehalose droplets/particles (Figure 5-32). Although a solid crust
was formed at the critical point, particles were still deformable in the falling rate period.
This effect was more distinct for particles with high lipid concentrations (40 and 60 mM,
Figure 5-32). Another remarkable effect was observable directly after the insertion of the
droplet into the acoustic field. A significant increase in flattening of the droplet was noticed
visually during the measurement with samples containing liposomes.
Figure 5-32: Aspect ratios as a function of time for different liposome formulations.
104 RESULTS AND DISCUSSION
The calculation of aspect ratios of liposome containing formulations confirmed this obser-
vation (Figure 5-32). The increased flattening was supposed to be a result of a decreased
surface tension of liposome containing samples compared to the pure trehalose solution.
Figure 5-33 shows evaporation coefficients and the time period, which is needed to reach
the critical point determined as the point of inflection of the surface temperature. Both pa-
rameters can be used for the evaluation of the drying behavior of trehalose solutions con-
taining liposomes. The evaporation coefficient increased with increasing lipid contents,
which assumed a faster evaporation during the constant rate. Up to a concentration of
30 mM, the constant rate phase was abbreviated and the critical point emerged earlier. Both
parameters pointed to an improved drying behavior with increasing lipid concentrations.
Partially, this was likely to be a result of the higher solid content. Less water needed to be
removed, which shortened the time to reach the critical point (Schiffter and Lee, 2007b).
In addition, the faster evaporation and the abbreviated constant rate was attributed to the
influence of lipophilic substances on the water binding capabilities of the drying particles.
This assumption has also been made for the residual moisture content of spray dried pow-
ders. The same behavior was assumed for the levitated droplets and particles: The presence
Figure 5-33: Evaporation rate and the time needed to reach the critical point as a function of the
lipid content (n = 4).
RESULTS AND DISCUSSION 105
a) pure trehalose, 100x b) pure trehalose, 1000x
c) trehalose + 20 mM lipid, 100x d) trehalose + 20 mM lipid, 500x
e) trehalose + 60 mM lipid, 100x f) trehalose + 60mM lipid, 500x
Figure 5-34: Levitated particles consisting of trehalose (10 % w/v in the liquid feed) and differ-
ent lipid concentrations. The dried particles were removed from the acoustic field using a small
dip net. Please mind the different magnifications.
of increasing amounts of lipophilic solids decreased the overall hydrophilic interactions,
thus reducing water binding capacity. However, the two highest lipid concentrations (40
and 60 mM) did not show any improvements regarding the drying rate. This was attributed
200µm
200µm
200µm
50µm
50µm
50µm
106 RESULTS AND DISCUSSION
to the formation of a highly lipophilic barrier at the droplet´s surface before or in the vicin-
ity of the critical point. This barrier might have decelerated evaporation at the droplet´s
surface.
Figure 5-34 shows levitated trehalose particles containing different liposome concentra-
tions. Their surfaces were smooth and trehalose particles as well as particles containing
both trehalose and liposomes could be classified as “skin-forming” according to the defini-
tion of Walton (2000). The particle containing the highest liposome concentration (60 mM,
Figure 5-33, e) was clearly flattened, which was in good agreement with the aspect ratio
measured.
In the following section, levitation experiments were carried out without trehalose in order
to determine the drying behavior of liposome dispersions in detail. Sample compositions
were in agreement with Table 5-5. However, no trehalose was used.
Figure 5-35 shows aspect ratios of liquids with different liposome concentrations. Lipo-
some dispersions were flattened to aspect ratios > 1.8 after the insertion into the acoustic
field irrespective of the lipid content, whereas the aspect ratio of pure water was 1.3 after
Figure 5-35: Aspect ratios of levitated droplets containing DOTAP and DOPC.
RESULTS AND DISCUSSION 107
Figure 5-36: Evaporation coefficients of liposome dispersions without the addition of trehalose.
The triangles represents the time, which is needed to reach the critical point (n = 4).
the insertion of the droplet. Due to the extremely low solid content, the monitoring of par-
ticles after the critical point was difficult. Most particles started jumping in the acoustic
field. The signal of pure water droplets ended after an evaporation time of 260 s, because
evaporation was complete.
Figure 5-36 summarizes the evaluation of levitation experiments with liposome disper-
sions. Differences regarding evaporation coefficients were hardly verifiable due to the
standard deviations. The low solid content complicated the exact monitoring of all param-
eters. However, the course of the evaporation coefficients and the course of the time needed
to reach the critical point were similar comparing the results obtained for trehalose contain-
ing samples. Evaporation coefficients were higher compared to the trehalose containing
droplets, which was in accordance with results published by Schiffter and Lee (2007b):
Lower solid contents increase the partial vapour pressure of water at the droplet surface.
The time needed to reach the critical point increased, because more solvent had to be re-
moved.
Figure 5-37 shows SEM pictures of particles simply consisting of DOTAP and DOPC. The
surface was uneven exhibiting several dents.
108 RESULTS AND DISCUSSION
a) 200x b) 500x
Figure 5-37: SEM pictures of pure lipid particles dried in the acoustic field. Please note the dif-
ferent magnifications.
Several experiments and results raised the question, if liposomes or phospholipid molecules
are able to enrich in the droplet surface during spray drying or levitation. The smoothening
effect observed in spray dried particles as well as the flattening effect, which occurred after
the insertion of a droplet into the acoustic field during levitation, caused this suggestion.
Therefore, the surface tension of a liposome dispersion (20 mM) was determined using a
maximum bubble pressure tensiometer. This method for the determination of the surface
tension is able to measure the dynamic surface tension after an interface lifetime ≥ 1 ms.
This is beneficial for the examination of fast processes like spray drying. Droplet drying in
the process chamber of a spray dryer is limited to a time lasting milliseconds to a few sec-
onds. Hence, it was of great interest to determine whether liposomes are able to accumulate
at the surface during these few milliseconds or not.
Figure 5-38 shows the results of the measurements performed with the maximum bubble
pressure tensiometer. The dynamic surface tension (y-axis) is plotted against the surface
age (x-axis). Drying in the spray dryer or the levitator was performed at elevated tempera-
tures. Subsequently the measurements of the surface tension were also executed at different
temperatures. A lipid concentration of 20 mM was used. However, the dispersion was pre-
pared without trehalose, because trehalose interfered the measurements. It was supposed
that crystallization of trehalose in the capillary took place.
200µm 50µm
RESULTS AND DISCUSSION 109
Figure 5-38: Dynamic surface tension of a 20 mM liposome dispersion at different temperatures
compared to pure water.
The black symbols depicts the values obtained for pure water. Here, no changes over the
measured time range were visible, because there were no molecules, which could accumu-
late at the surface. The values measured at different temperatures were in good agreement
with literature (Vargaftik et al., 1983).
Regarding the liposome dispersion, surface tension was lowered of about 1 mN/m com-
pared to water tempered to the relevant temperature. A slight dependence on the surface
age was observable irrespective of the temperature used. However, the effect on the surface
tension was smaller than expected. Nevertheless, the flattening effect during levitation and
the smoothening of the surface could be explained through the slight decrease in surface
tension induced through the presence of liposomes or phospholipids.
Pinazo et al. (2002) measured the dynamic surface tension of DLPC and DPPC liposome
dispersions. They used a pulsating bubble surfactometer and measured the dynamic surface
tension up to 105 s. At least 10 s were needed to record the first drop in the surface tension.
After 100 s they found a dynamic surface tension of 25 mN/m. However, these time spans
are not relevant for the fast spray drying process. Since changes in the surface tension were
observed after 10 s at the earliest, there is no discrepancy to the results found in this work.
110 RESULTS AND DISCUSSION
Summarizing the experiments performed for the characterization of spray dried powders
containing liposomes, the results revealed an influence of liposomes on the residual mois-
ture, the glass transition temperature, the drying behavior observed in a levitator, the parti-
cle yield and the fine particle fraction. Increasing lipid contents caused better product prop-
erties with respect to Tg or the residual moisture. However, at high liposome concentrations
the drying behavior observed in the levitation experiments declined and a second glass
transition at ~ 50 °C occurred in the spray dried product, which might be disadvantageous
regarding the product stability.
The influence of the nozzle type could be summarized as follows: For the spray drying
conditions chosen and a flow rate of 1.0 ml/min the 60 kHz ultrasonic nozzle showed the
best results, with respect to the product properties, the particle size and powder morphol-
ogy. Product properties of powders spray dried with the 25 kHz ultrasonic nozzle could be
improved by reducing the flow rate to 0.5 ml/min. Although the particle size of powders
atomized by the two fluid nozzle was low, powder properties such as residual moisture or
the glass transition temperature were unconvincingly. In addition, the size distribution of
powders generated by the two fluid nozzle was broad.
RESULTS AND DISCUSSION 111
5.4 Spray Drying of Liposomes using various Excipients
The aim of the experiments presented in the following part of this work was to investigate
whether the findings of trehalose based liposomal formulations can be applied to other
spray drying excipients. Lactose, sucrose, maltodextrin and mannitol were tested in the first
section. In the second section, a surfactant, Polysorbate 80, was added.
5.4.1 Sugars and Sugar Alcohols
A solution (10 % w/v) of each excipient was spray dried without liposomes, followed by
the same experiment with an additional lipid content of 10 mM (see Table 5-6). Preparation
of liposomes was performed according to the method described in chapter 4.2.1. The 25
kHz ultrasonic nozzle with a flow rate of 0.5 ml/min was used for atomization. The reduced
flow rate was chosen, since product properties such as the residual moisture content were
superior to powders produced with a flow rate of 1.0 ml/min.
Figure 5-39 shows the results of the Zetasizer measurements. First of all, size and size
distribution of liposomes in the liquid feed before spray drying revealed that the preparation
worked well. Size and size distribution were unaffected by the excipient dissolved in the
rehydration medium. Liposomes had a size of 100 – 120 nm and the PDI was ≤ 0.2 indi-
cating a monodisperse size distribution. However, excipients could be divided into two
groups after spray drying and reconstitution: Liposomes spray dried with lactose or sucrose
showed the previously described slight decrease in diameter (p-values ≤ 0.05), which was
characteristic for the ultrasonic nozzles. Their PDI was unaffected. On the contrary, man-
Excipient Excipient
concentration [mg/ml] Lipid content [mM]
Lactose 100 0
Lactose 100 10
Sucrose 100 0
Sucrose 100 10
Maltodextrin 100 0
Maltodextrin 100 10
Mannitol 100 0
Mannitol 100 10
Table 5-6: Sample compositions. A lipid content of 10 mM corresponds with a DOTAP and
DOPC concentration of 3.49 mg/ml and 3.93 mg/ml, respectively.
112 RESULTS AND DISCUSSION
Figure 5-39: Liposome size and size distribution in the liquid feed (white bars) and after the re-
hydration of the spray dried powders (striped bars) as a function of the excipient used. Liposome
size and PDI of trehalose containing samples are used as a reference. Note that the left y-axis is
broken (n = 3).
nitol and maltodextrin containing samples were cloudy after disintegration of the SD pow-
ders. Consequently, Zetasizer measurements revealed a huge increase in liposome size and
PDI, indicating liposome fusion and damage during spray drying and rehydration. Espe-
cially maltodextrin seemed to have no stabilizing effect on liposomes during spray drying.
It was no surprise that lactose or sucrose were able to prevent fusion during spray drying
and rehydration, since they were used in previous successful studies on the stabilization of
liposomes during spray drying (Lo et al., 2004, Goldbach et al., 1993b, Hauser and Strauss,
1987).
There are poor data records about the spray drying of liposomes using mannitol or malto-
dextrin and the published experiments were performed with strongly different formulations
(Karadag et al., 2013, Charnvanich et al., 2010). However, there is some data about freeze
drying, which confirms the presented results: In terms of mannitol, Stark et al. (2010) pub-
lished a study about the freeze drying of liposomes and the influence of different sugars
and sugar alcohols on the liposome stability after freeze drying. Compared to other sugars,
RESULTS AND DISCUSSION 113
they found a slight but significant increase in liposome size when using mannitol. Hence,
they stated only a slight protection of mannitol. Huang et al. (2001) found an increase in
liposome diameter from 70 nm before freezing to 500 nm after thawing and characterized
mannitol as a poor cryoprotectant.
Maltodextrin is a mixture of glucose oligomers, which is often used for the encapsulation
of food ingredients (Gharsallaoui et al., 2007). Maltodextrin used in this work had a dex-
trose equivalent of 13.0 – 17.0. Ozaki and Hayashi (1997) evaluated the influence of the
glucoside number on the suitability as a cryoprotectant for the lyophilization of liposomes.
Short chain maltodextrins such as maltose were found to be useful cryoprotectants, whereas
larger glucoside numbers (used in the present work) were not suitable due to their poor
hydrogen bonding to phospholipids.
Figure 5-40 shows zeta potentials measured in the different liposome formulations. Sucrose
and maltodextrin containing samples showed an increase in liposome zeta potential after
spray drying and rehydration similar to that reported for trehalose formulations. Lactose
containing formulations showed a lower zeta potential of ~ 50 mV both for the initial lipo-
Figure 5-40: Zeta potentials of liposome dispersions before and after spray drying and rehydra-
tion as a function of the excipient used (n = 3).
114 RESULTS AND DISCUSSION
some dispersion and for the rehydrated sample. A value of 50 mV is high enough to provide
a stable formulation (Heurtault et al., 2003).
Table 5-7 summarizes powder properties of the spray dried samples. In general, particle
yields varied between 40 and 83 %. Liposome containing dispersions could be spray dried
with a higher powder yield compared to the pure sugar solutions, which was in good agree-
ment with the results found for the trehalose based formulations. The comparatively low
lipid content of 10 mM led to no measurable increase in the d10 value of the volume based
size distribution. Mannitol containing powders were an exception. Here, an increase from
1.3 to 3.1 µm of the d10 value occurred. D50 values fluctuated between 21 and 27 µm, but
no influence of the sugar type or the presence of liposomes was observed.
Figure 5-41 shows spray dried powders of pure lactose (a) and sucrose (c). An influence of
liposomes on the surface morphology was not identifiable (b, d). All powders had a smooth
surface. Lactose particles partly showed the golf ball structure, which was found for treha-
lose particles, too. Sucrose powders had a perfectly round and smooth structure.
In contrast, the appearance of maltodextrin and mannitol powders was wrinkled and the
minority of powder particles had a round shape (Figure 5-42). Maltodextrin powders in-
cluded many bowl shaped particles. The surface was smooth but partly wrinkled (a). The
Sample Particle yield [%] Particle size d50 [µm] Particle size d10 [µm]
Lactose 10 % 40 27.3 ± 1.7 3.1 ± 0.2
10 mM lipid +
lactose 10 % 75 21.3 ± 0.1 3.0 ± 0.3
Sucrose 10 % 44 23.8 ± 5.3 2.9 ± 0.6
10 mM lipid +
sucrose 10 % 76 23.2 ± 1.8 3.1 ± 0.1
Maltodextrin 10 % 83 27.0 ± 0.5 4.9 ± 0.5
10 mM lipid +
maltodextrin 10 % 72 24.1 ± 1.4 3.5 ± 0.5
Mannitol 10 % 43 23.7 ± 0.1 1.3 ± 0.3
10 mM lipid +
mannitol 10 % 80 24.1 ± 1.4 3.1 ± 0.3
Table 5-7: Powder yield, d50 and d10 values of the volume based size distribution (n = 3).
RESULTS AND DISCUSSION 115
a) pure lactose, 1000x b) lactose + 10 mM lipid, 1000x
c) pure sucrose, 1000x d) sucrose + 10 mM lipid, 1000x
Figure 5-41: SEM pictures of spray dried powders. Powders were atomized using a 25 kHz ul-
trasonic nozzle and a flow rate of 0.5 ml/min. The concentration of sugar in the liquid feed was
10 % (w/v).
presence of liposomes (b) seemed to increase the extent of particle collapse. Mannitol pow-
ders had a dimpled and rough surface (c). No significant differences could be observed
when liposomes were part of the formulation (d). Both powders composed of mannitol
included a lot of fragments, which was affirmed by the Mastersizer measurements (Table
5-7).
Residual moisture contents of spray dried powders are given in Figure 5-43. Pure lactose
and pure maltodextrin powders had high residual moisture contents compared to the spray
dried trehalose. In terms of the water content, both powders benefited from the presence of
approximately 8 mg/ml lipid, which caused a decrease in the residual moisture content to a
value comparable to trehalose formulations. Glass transition temperatures (Figure 5-44 and
Figure 5-45) of amorphous samples showed the previously reported shift to higher values,
if liposomes were present. Figure 5-46 and Figure 5-47 present XRD graphs, which confirm
20µm
20µm 20µm
20µm
116 RESULTS AND DISCUSSION
a) pure maltodextrin, 1000x b) maltodextrin + 10 mM lipid, 1000x
c) pure mannitol, 1000x d) mannitol + 10 mM lipid, 1000x
Figure 5-42: SEM pictures of spray dried powders. Powders were atomized using a 25 kHz ul-
trasonic nozzle and a flow rate of 0.5 ml/min. The concentration of sugar or sugar alcohol in the
liquid feed was 10 % (w/v).
the amorphous structure of all spray dried powders except both mannitol containing prod-
ucts. The glass transitions of both lactose powders were in the same range as the Tg of
trehalose powders. The Tg of amorphous lactose without any water was found to be 101°C
(Roos and Karel, 1990). Accordingly, water acted as a softener and reduced the glass tran-
sitions to 67.7 and 71.2 °C. The Tg was followed by the crystallization of the product and
an endothermic melting peak. Regarding the results of sucrose containing powders, the
exceptional position of trehalose could be clarified. Sucrose samples had low residual mois-
ture contents of ≤ 2.5 %, but also low glass transition temperatures of ≤ 50 °C, which might
be disadvantageous in respect of product stability. On the contrary, trehalose had higher
glass transitions although the moisture content was > 3 %. Even at 0 % residual moisture
the Tg of sucrose is approximately 50 °C lower than the Tg of trehalose (Crowe et al., 1998).
20µm 20µm
20µm 20µm
RESULTS AND DISCUSSION 117
Figure 5-43: Residual moisture contents of spray dried powders (n = 3).
Regarding the three excipients, which were able to protect liposomes during spray drying,
trehalose and lactose seemed to have the best properties. Mobley and Schreier (1994) found
a similar influence of trehalose and lactose on the Tm of liposomes in the dried state. They
concluded that both have a good capability in terms of liposome stabilization. However, it
has to be taken into account that lactose, in contrast to trehalose, is a reducing sugar. This
might be challenging especially in the development of complex formulations.
Spray dried maltodextrin had an amorphous structure and a glass transition temperature of
108.1 and 116.9 °C, respectively. These values were in good agreement with the values
proposed by Potes and Roos (2011). Mannitol is a crystalline spray drying or freeze drying
excipient (Naini et al., 1998), which featured an extremely low residual moisture content
of less than 0.5 %. The melting endothermic peak occurred between 160 and 170 °C. How-
ever, neither maltodextrin nor mannitol were suitable excipients for the stabilization of lip-
osomes during spray drying.
118 RESULTS AND DISCUSSION
Figure 5-44: Thermograms and glass transition temperatures of spray dried powders of lactose
and sucrose with or without the addition of liposomes. Trehalose curves are pictured for compari-
son.
Figure 5-45: Heating scans of maltodextrin and mannitol containing powders. The glass transi-
tion temperatures of maltodextrin are specified.
RESULTS AND DISCUSSION 119
Figure 5-46: WAXD of spray dried lactose and sucrose powders with or without liposomes. All
samples show the characteristic amorphous halo and the presence of liposomes had no measurable
influence on the product morphology.
Figure 5-47: WAXD of spray dried maltodextrin and mannitol with or without liposomes.
Maltodextrin samples show the amorphous halo, while mannitol samples are clearly crystalline.
The presence of liposomes has no measurable influence on the powder morphology.
120 RESULTS AND DISCUSSION
5.4.2 Polysorbate 80
Polysorbate 80, also known as Tween® 80, is an often used additive in formulations of
peptides and proteins. Denaturation of proteins is strongly correlated to the adsorption of
the protein to an interface, which is the ice-water interface during freeze drying and the
liquid-air interface in spray drying. Surfactants have shown to occupy the interface them-
selves and prevent the protein denaturation in freeze drying (Chang et al., 1996) and spray
drying (Adler et al., 2000).
Hence, the influence of Polysorbate 80 on liposomes and spray dried powders thereof was
investigated. Sample compositions are given in Table 5-8. Polysorbate 80 was dissolved
together with trehalose in the rehydration medium. After the preparation of the three for-
mulations listed, size and size distribution of liposomes were measured (Figure 5-48). For-
mulations with a Polysorbate 80 concentration of 1 mM and 10 mM were spray dried using
the 25 kHz ultrasonic nozzle (flow rate: 1.0 ml/min).
Figure 5-48 displays the changes in liposome size with increasing surfactant concentra-
tions. The final liposome size decreased to 105 nm, while the size distribution remained
constant. The effect on the liposome size was most distinct after the addition of 2 mM
surfactant. A further increasing surfactant concentration of 10 mM resulted in a nearly con-
stant liposome size compared to a surfactant concentration of 2 mM. Smaller liposome
diameters were found for the crude liposomes (~ 250 nm) and the liposomes extruded
through 0.4 µm membranes (~ 120 nm) beforehand. This effect was described by Tasi et
al. (2003). They dissolved the surfactant together with egg-PC and cholesterol in a chloro-
form/methanol mixture. Despite the different formulation and preparation procedure they
found also a decrease in the initial liposome size. This was attributed to a steric repulsion
among the surfactants, which are located on the liposome surface. The curvature increases
due to the steric repulsions on the outer leaflet and results in smaller vesicle radii. In addi-
tion, Tasi et al. (2003) found that Polysorbate 80 molecules might not insert deeply enough
Lipid content [mM] Trehalose concentration
[mg/ml]
Polysorbate 80
concentration [mM]
10 100 1 (= 1.3 mg/ml)
10 100 2 (= 2.6 mg/ml)
10 100 10 (= 13 mg/ml)
Table 5-8: Sample compositions of the surfactant containing experiments.
RESULTS AND DISCUSSION 121
Figure 5-48: Liposome size after extrusion through 0.4 µm or 0.1 µm polycarbonate membranes
and size distribution of the final liposome dispersion (n = 3).
into the lipid bilayer to significantly change phase transition temperatures of liposomes.
Regarding the spray dried product, the appearance of particle fragments in spray dried pow-
ders with a surfactant concentration of 10 mM was remarkable (Figure 5-49, b). Particles
had the golf ball structure, which was already reported for the formulations without surfac-
tant. The diameter of the volume based size distribution (d50) decreased from 25.3 µm for
powders containing 1 mM surfactant to 22.0 µm for powders containing the 10fold con-
centration of surfactant. Lower surface tensions trigger the atomization into smaller drop-
lets (Masters, 1979). The decrease in powder size went along with a lowered residual mois-
ture content of 2.6 % and 3.8 % for the 10 mM Polysorbate 80 concentration and the 1 mM
concentration, respectively. Although the decrease in particle size was less distinct, a de-
crease of the residual moisture of more than 1 % compared to the surfactant free powder
was observed.
Reconstituted liposomes showed the characteristic decrease in size, which was described
earlier for the ultrasonic nozzles. The resuspended liposomes had a size of 100 nm (10 mM
Polysorbate 80) and 93 nm (1 mM Polysorbate 80).
122 RESULTS AND DISCUSSION
a) 1 mM Polysorbate 80, 1000x b) 10 mM Polysorbate 80, 1000x
Figure 5-49: SEM pictures of spray dried trehalose powders containing liposomes (10 mM) and
different amounts of the surfactant Polysorbate 80.
The dynamic surface tension of formulations listed in Table 5-8 were examined using the
maximum bubble pressure tensiometer. The measurements were supplemented by the cor-
responding liposome free formulations. The aim of this experiment was to determine the
extent of the surfactant attachment to the liposome surface and the resulting influence of
the surfactant on the dynamic surface tension. The results are presented in Figure 5-50.
Dynamic interfacial tensions determined for the liposome free formulations were in agree-
Figure 5-50: Dynamic surface tension of different Polysorbate 80 concentrations with or with-
out liposomes. The lipid content of liposome containing solutions was 10 mM.
20µm 20µm
RESULTS AND DISCUSSION 123
ment with the values recorded by Führling (2004). The highest Polysorbate 80 concentra-
tion (10 mM) started with an interfacial tension of 62 mN/m at 1 ms and decreased over the
measured time span to an interfacial tension of 48 mN/m. The liposome containing formu-
lation followed this course having interfacial tensions slightly higher than the liposome free
solution. It was supposed that the excess of free, not attached Polysorbate 80 was high
enough to lower the interfacial tension to nearly the same extent as the liposome free solu-
tion. In contrast, the 2 mM concentration curve of the liposome-free sample strongly di-
verged from the liposome containing dispersion. This was attributed to an almost complete
attachment of the surfactant molecules to the liposome surfaces. Hence, there was only little
excess Polysorbate, which could occupy the air/liquid interface. The solution with the low-
est Polysorbate 80 concentration resulted in a course between 70 mN/m (1 ms) and
60 mN/m at 1 s. The corresponding liposome dispersion started at 65 mN/m and ended at
62 mN/m. Hence, liposomes with attached surfactant were still able to lower the interfacial
tension, since the values were 7 – 10 mN/m lower than the surface tension of pure water,
72 mN/m. However, their time-dependent occupation of the air/liquid interface proceeded
in a different way from the pure solution of surfactant.
Summing up, an influence of the surfactant Polysorbate 80 both on liposomes and liposome
containing powders was found. According to the maximum bubble pressure tensiometer
measurements, 1 – 2 mM of the surfactant were located at the liposome surface and influ-
enced the liposome size. Higher surfactant concentrations did not further decrease the lip-
osome size and the excess surfactant molecules lower the surface tension and therefore
trigger for example the atomization into smaller droplets during spray drying. Although the
reported changes in liposome size, particle size and moisture might be beneficial especially
in terms of protein or peptide containing formulations, it has to be taken into account that
the permeability or the encapsulation efficiency of liposomes might be influenced nega-
tively.
124 RESULTS AND DISCUSSION
5.5 Stealth Liposomes
Stealth liposomes consist of a mixture of “standard” phospholipids with PEGylated phos-
pholipids. In PEGylated lipids, the phospholipids are coupled to monomethoxy pro-
pylenglycol via an amide linkage. Incorporated into liposomal bilayers, the PEG chains
form a steric barrier around the liposomes improving their half-life in vivo (chapter 2.5).
The objective of the following section was to evaluate, if it is possible to spray dry stealth
liposomes and to estimate the influence on the spray dried powder.
5.5.1 Influence of PEG on Liposome Properties
First of all, the influence of the incorporation of PEGylated lipids on the previously used
liposome composition was determined. The PEGylated lipid was dissolved together with
DOTAP and DOPC in chloroform/methanol before the standard preparation procedure was
performed.
Table 5-9 lists the components of the formulations, which were prepared for the experi-
ments. A similar liposome composition with a PEG-lipid content of 5 mol% and the
DOTAP/DOPC mixture was used in a publication of Campbell et al. (2002). However, they
used the saturated version of the PEGylated lipid. After the extrusion through 0.1 µm pol-
ycarbonate membranes, they found a vesicle mean diameter of 150 ± 40 nm. Figure 5-51
shows the results found in this work. PEGylated liposomes had a size of ~ 115 nm irrespec-
tive of the ratio of PEGylated lipid used. 5 mol% of PEGylated lipids are sufficient to ar-
range the PEG chains in the “brush” regime, which means that the distance between graft-
ing sites is small enough to enable the lateral overlapping of adjacent chains (Kenworthy
et al., 1995a). Lower concentrations of the PEGylated lipid would trigger the formation of
Lipid content [mM] PEGylated lipid
[mol%]
Liposome composition:
DOTAP/DOPC/18:1 PEG
2000 PE [mg/ml]
trehalose concentra-
tion [mg/ml]
10 5 3.32 / 3.73 / 1.40 100
10 15 2.97 / 3.34 / 4.20 100
40 5 13.28 / 14.92 / 5.60 100
40 15 11.88 / 13.36 / 16.80 100
Table 5-9: Sample compositions.
RESULTS AND DISCUSSION 125
Figure 5-51: Liposome size, distribution and zeta potential as a function of the amount of
PEGylated lipid in the liposome composition after extrusion through 0.4 and 0.1 µm polycar-
bonate membranes (n = 3). Overall lipid content: 10 mM.
a “mushroom” regime. Refer to Figure 2-5 in chapter 2.5 for a draft of the different config-
urations. A molecular mass of 2000 of the PEG moiety and the arrangement in the “brush”
regime results in a chain length of approximately 50 Å (Needham et al., 1992) and a calcu-
lated increase in the vesicle diameter of 10 nm, respectively. However, no difference in
vesicle size between the conventional liposomes and the stealth liposomes was observed
(Figure 5-51). Yoshida et al. (1999) even found a decrease in liposome diameter with in-
creasing amounts of PEG-lipid. They attributed this to the steric repulsion of PEG chains
exposed from the outer leaflet of the bilayer membrane. However, there are also PEG chains
attached to the inner leaflet decreasing the vesicle curvature. They considered the effect
from the outer leaflet to be predominant in their formulation. Here, an equilibrium of both
effects was supposed.
Regarding the size distribution, the PDI slightly decreased to ~ 0.1 for the formulation with
the highest PEG-lipid content. This was likely to be a result of the steric barrier between
liposomes preventing aggregation and fusion of liposomes. The zeta potential decreased
from ~ 65 mV (0 mol% PEG-lipid) to 45 mV (15 mol% PEG-lipid) as the PEGylated lipid
carries a negative charge. An equal effect was reported by Gjetting et al. (2010).
126 RESULTS AND DISCUSSION
The influence of the PEG-lipid on the phase transition temperature of the liposome formu-
lation was investigated using the two high-concentrated (40 mM) samples listed in Table
5-9. Thermograms of liposome dispersions with increasing molar ratios of the PEG-lipid
are presented in Figure 5-52. Neither the phase transition temperature of the lipid mixture
nor the glass transition temperature of the maximally freeze-concentrated solution, Tg´, was
affected through the PEG-lipid. However, an exact analysis of the endothermic phase tran-
sition was not possible due to the overlapping melting peak of water. Advanced investiga-
tions of the influence of PEGylated lipids on the phase transition temperature were per-
formed by Hashizaki et al. (2000). They found a broadening of the peak and a peak shoulder
for molar ratios up to 14.5 mol% PEG-lipid using liposomes composed of DPPC and PEG-
DSPE. Kenworthy et al. (1995b) used DSPC and several PEGylated lipids to determine the
effect of PEG-lipids on the Tm. They reported a peak broadening and an increase in the
transition temperature up to 10 °C for the incorporation of > 10 mol% PEG-2000 lipid. A
slight broadening was also recognizable in the present measurements, but phase transitions
Figure 5-52: Thermograms of liposome dispersions with increasing molar ratios of PEG 2000
PE. DOTAP and DOPC in the molar ratio 1:1 constituted the remaining part of the liposome com-
position. Tm was -8 °C and Tg´ was -30 °C.
RESULTS AND DISCUSSION 127
remained constant. However, phospholipid mixtures used in this work consisted of the un-
saturated and partially positively charged lipids DOTAP and DOPC in contrast to those
used by Kenworthy et al. (1995b).
5.5.2 Spray Drying of Stealth Liposomes
A larger batch of the liposome dispersion containing 5 mol% PEG-lipid and a total lipid
content of 10 mM (Table 5-9) was prepared in order to perform spray drying experiments.
Dispersions were spray dried using the standard conditions and either the 25 kHz ultrasonic
nozzle or the two fluid nozzle for atomization.
Figure 5-53 summarizes the results of the DLS measurements. Regarding the vesicle size,
no differences were found between the PEGylated liposomes and the conventional lipo-
somes. As already described in the previous chapter, the PEG-chains had no influence on
the liposome size as the steric repulsions increased the curvature. Atomization through the
ultrasonic nozzle triggered a size reduction (p-value ≤ 0.05), which was in agreement with
Figure 5-53: PEGylated liposomes (striped bars, black symbols) compared to conventional lipo-
somes (white bars, white symbols) before and after spray drying using different nozzles and re-
constitution (n = 3).
128 RESULTS AND DISCUSSION
the conventional liposomes. PDI values before and after the spray drying process were
slightly smaller than those of the standard liposomes. Zeta potentials of PEGylated lipo-
somes before spray drying were smaller compared to the PEG-free liposomes, which was
explained by the negative charge of the PEG-lipid. However, no increase in the zeta poten-
tial after reconstitution of the spray dried powders was found as it was observed with the
conventional liposomes. The higher zeta potentials after rehydration of the conventional
liposomes were attributed to a lower pH (cf. chapter 5.2.1). It was hard to find an explana-
tion for the different behavior of the PEGylated liposomes. Perhaps the PEG-coating made
them less sensitive to fluctuating pH values.
Figure 5-54 shows SEM pictures of the spray dried powders. The powder generated by the
ultrasonic nozzle had an equal appearance compared to the corresponding PEG-free for-
mulation (Figure 5-19, c). However, the powder sprayed with the two fluid nozzle differed
distinctly from the round particles of the PEG-free powder (Figure 5-20). Irregularly shaped
fragments and particles with holes were noticeable. The d50 values of the volume based size
distributions were 26.3 µm for the ultrasonic nozzle and 11.0 µm for the two fluid nozzle.
Both values were slightly higher compared to the PEG-free powders (cf. chapter 5.3.2).
Figure 5-55 presents the results of DSC and Karl-Fischer measurements. Powders contain-
ing stealth liposomes had a higher glass transition temperature and higher residual moisture
contents. Although the differences were minor, formulations with PEGylated liposomes
a) US nozzle 25 kHz, 1000x b) two fluid nozzle, 1000x
Figure 5-54: SEM pictures of spray dried powders containing trehalose (10 % w/v in the liquid
feed) and PEGylated liposomes (total lipid concentration in the liquid feed: 10 mM).
20µm 20µm
RESULTS AND DISCUSSION 129
seemed to have the ability to provide high glass transition temperatures coupled with high
residual moisture contents. The plasticizing effect of water did not appear to the same ex-
tend as with the standard liposome formulation – a behavior that was supposed to be a result
of the hydrophilic PEG chains, which were able to bind water without increasing the mois-
ture content of the surrounding trehalose matrix.
In summary, the addition of a PEGylated lipid to the established liposome composition
mainly had an influence on the liposome zeta potential. At a common molar ratio of
5 mol%, no changes in size or size distribution occurred before or after the spray drying
process. In contrast to the results of Wessman et al. (2010), who used DSPC and DSPE-
PEG5000 and the excipient lactose, DLS measurements revealed a monodisperse distribu-
tion of PEGylated vesicles without aggregates after spray drying and reconstitution. It was
supposed that the high zeta potential and the use of trehalose as stabilizing excipient were
the crucial advantage over the liposomes investigated in the publication of Wessman et al.
(2010).
Figure 5-55: Glass transition temperature and residual moisture content of spray dried powders.
The chart uses striped bars and black symbols to indicate powders containing PEGylated lipo-
somes, whereas white bars and symbols represent the standard formulation (n = 3).
130 RESULTS AND DISCUSSION
Regarding the powder properties, some changes were observed compared to the standard
formulation especially when using the two fluid nozzle. The presence of PEG chains
seemed to increase the residual moisture content of the spray dried product while preserving
a comparably high glass transition temperature. Spray drying of PEGylated liposomes was
feasible with powders being only slightly different from PEG-free formulations and narrow
distributed, stable liposomes after spray drying and rehydration.
RESULTS AND DISCUSSION 131
5.6 Experiments with a Fluorescent Marker
Experiments and results presented in the following chapter were performed with the fluo-
rescent dye calcein, which was encapsulated in liposomes. In the first section, the encapsu-
lation efficiency (EE) was determined, both after the preparation of liposome dispersions
and after spray drying and reconstitution. Studies on the membrane integrity are presented
in the second section. Here, the marker leakage during drying was measured by means of
fluorescence quenching of the leaking calcein. In addition to the spray drying experiments,
a batch of liposome dispersion was freeze dried in order to compare the two drying meth-
ods.
5.6.1 Encapsulation Efficiency
The key advantage of the method presented by Oku et al. (1982) is the needlessness of a
separation of the non-encapsulated marker from the liposome dispersion. Separation of the
marker is time-consuming, expensive and difficult to apply to larger batches, as the meth-
ods of choice are dialysis, gel filtration or centrifugation (Dipali et al., 1996). However,
these procedures are necessary for the measurement of the trapped volume/marker accord-
ing to the common method described in chapter 4.1.1.
The approach of Oku et al. (1982) involves the quenching of free fluorescent dye with
cobalt. Calcein possesses two chelating groups. At least one group has to be occupied by a
cobalt ion for the fluorescence to be quenched. Fluorescence is measured before and after
the addition of a cobalt solution. Co2+ ions are not able to pass the lipid bilayer. Hence, the
remaining fluorescence after the addition of cobalt corresponds to the percentage of en-
trapped marker.
Lipid content
[mM]
DOTAP/DOPC
concentration
[mg/ml]
Trehalose
concentration
[mg/ml]
Calcein
concentration
[mg/ml]
HEPES
concentration
[mM]
10 0.33 / 7.48 0 0.05 50
10 0.33 / 7.48 50 0.05 50
10 0.33 / 7.48 100 0.05 50
20 0.66 / 14.96 100 0.05 50
Table 5-10: Sample compositions.
132 RESULTS AND DISCUSSION
Figure 5-56: Calibration curve of calcein. The specified concentration is the actual concentra-
tion of calcein in the cuvette. The addition of cobalt to a calcein solution decreases the fluores-
cence to < 2.
Table 5-10 lists the sample compositions used for the marker experiments. First of all, a
buffer salt was added to guarantee a constant pH (7.4) during the experiments. Calcein
fluorescence was reported to be highly pH dependent (Markuszewski, 1976). Second, the
molar ratio of DOTAP and DOPC was changed from 1:1 to 1:20. The reasons were as
follows: It was essential that the marker fluorescence was not constrained by liposomes or
any other component. However, the positively charged liposomes (DOTAP/DOPC 1:1) in-
terfered with the negatively charged calcein and reduced the fluorescence to less than 50 %
compared to the liposome-free calcein solution of the same concentration. This effect was
self-evident regarding the optical appearance (Figure 5-57) of a dispersion consisting of
calcein and liposomes made up of DOTAP/DOPC in the molar ratio 1:1 (left) or 1:20
(right). The 1:20 mixture looked like the pure solution of calcein. Figure 5-56 displays the
calibration line of calcein. Samples (0.05 mg/ml calcein) were diluted 1:50 prior to the
measurement, resulting in a calcein concentration of 1 µg/ml in the cuvette. Liposome dis-
persions with a DOTAP/DOPC molar ratio of 1:20, which had been rehydrated with a
0.05 mg/ml calcein solution, showed a fluorescence of approximately 750 after dilution
RESULTS AND DISCUSSION 133
Figure 5-57: Optical appearance of calcein-loaded liposomes. Left hand side: “standard” lipo-
some composition. Right hand side: liposomes with a reduced content of the positively charged
lipid DOTAP.
corresponding with an actual calcein concentration of 0.048 mg/ml. This revealed that
> 95 % of the fluorescence dye was unaffected by liposomes composed of a mixture of
DOTAP and DOPC in the molar ratio 1:20. However, as the negatively charged calcein
could bind to positively charged liposomes, encapsulation efficiency could not be put on a
level with the trapped volume. This would only be the case if no interactions between the
marker molecule and the liposomes occur. Despite this limitation, it was stated that meas-
urements of the encapsulation efficiency or the membrane integrity are suitable applications
of this method as the fluorescence quenching by cobalt ions is unaffected by the presence
of positively charged liposomes (Oku et al., 1982).
The modified formulation and the presence of buffer or fluorescent dye did not alter lipo-
some properties such as size and size distribution (Figure 5-64). Due to the decreased
amount of positively charged lipid the liposome zeta potential decreased to a value of
35 mV.
Figure 5-58 displays encapsulation efficiencies (EE) and illustrates the influence of extru-
sion, trehalose content and lipid content on the amount of trapped marker. First of all, the
extrusion of crude liposomes through polycarbonate membranes did not change the encap-
sulation efficiency. Large (multilamellar) vesicles in the crude liposome dispersion were
likely to have a high internal volume. After the extrusion, the amount of liposomes in-
creases since the breakup of MLV produced many small liposomes equalizing the missing
multilamellar liposomes. In addition, the surface area of liposomes increased after extrusion
134 RESULTS AND DISCUSSION
Figure 5-58: Percentage of entrapped marker molecule as a function of the trehalose content (x-
axis), extrusion (non-extruded: clear bars, extruded: striped bars) and lipid content (10 mM: grey
bars, 20 mM: white bars) (n = 3).
offering more “binding sites” for the negatively charged calcein. These effects might ex-
plain the discrepancy to the results presented by Berger et al. (2001) and Schneider et al.
(1995), who found a slight decrease in the encapsulation efficiencies of neutral liposomes
after extrusion.
Encapsulation efficiency significantly decreased with increasing sugar concentrations. Af-
ter the preparation of liposomes without trehalose, an EE of 37 % was measured. The same
formulation with trehalose had an EE of 10 %. Oku et al. (1982) reported approximately
5 % trapped calcein using a lipid concentration of 10 mM and a neutral liposome compo-
sition. The comparably high encapsulation efficiencies found for the DOTAP/DOPC for-
mulation in this work were ascribed to the positive charge of the liposomes.
Regarding the influence of the trehalose content, a further experiment was performed: Tre-
halose was dissolved in a crude liposome dispersion and in an extruded liposome disper-
sion. Usually, trehalose was already present in the rehydration medium. The addition of
trehalose was assumed to decrease the EE, but the EE rather remained at approximately
33 %. Hence, trehalose lowered the encapsulation efficiency only during the rehydration of
the lipid film. If it was added afterwards, no or only a slight effect was observed.
RESULTS AND DISCUSSION 135
It was hard to find a possible explanation of this behavior. Perhaps trehalose prevented a
more distinct “binding” of the negatively charged marker molecule to the positively
charged liposome membrane or the absence of trehalose offered more available volume for
calcein.
A twofold amount of lipid in the dispersion resulted in an approximately twofold higher
encapsulation efficiency. This behavior was attributed to the higher number of vesicles per
volume thus increasing the entrapped volume and the encapsulation efficiency (Schneider
et al., 1995).
Figure 5-59 displays variations in the encapsulation efficiency after atomization using three
different nozzles, spray drying using these nozzles and freeze drying. The encapsulation
efficiency of the individual batch before atomization, spray drying or freeze drying was set
as 100 %. The actual EE was, in conformance with Figure 5-58, approximately 10 %. At-
omization or drying and reconstitution of the calcein loaded liposomes altered the EE only
to a very low extent. With the exception of the EE after atomization using a two fluid noz-
zle, all values were between 90 and 110 %. Accordingly, neither the entrapped volume nor
Figure 5-59: Variations in the encapsulation efficiency after atomization, spray drying and
freeze drying. Experiments were performed using the standard conditions described in chapter
4.2.2 and a flow rate of 1.0 ml/min (n = 3).
136 RESULTS AND DISCUSSION
the fluorescent dye seemed to be affected by atomization, spray drying or freeze drying.
This result was in good agreement with the previously reported results of the marker-free
liposomes and suggested that liposomes were completely intact after atomization, drying
and reconstitution.
5.6.2 Studies on the Membrane Integrity
The presented results of an unchanged amount of encapsulated marker after drying and
reconstitution raised the question whether an exchange between free and entrapped calcein
molecules took place during atomization, spray drying or freeze drying. The previously
described method was suitable only for the determination of the entrapped marker before
and after the spray drying step, but not for the determination of a release or exchange of the
entrapped marker molecules during drying and rehydration of the powder. Therefore, a
modified version of the method presented by Oku et al. (1982) was used. Liposomes were
prepared in exactly the same way as for the measurement of the EE. However, a solution
of cobalt chloride was added before atomization, spray drying or freeze drying experiments
were performed. The remaining fluorescence of the entrapped fluorescent dye was meas-
ured before and after atomization, spray drying or freeze drying. If calcein molecules left
the vesicle or Co2+ ions entered the liposomes during atomization or drying, calcein got in
contact with Co2+ ions and the fluorescence was quenched. Hence, the release was defined
as the percentage of initially entrapped calcein, which got in contact with Co2+ during at-
A B
Figure 5-60: A: time-dependent release of entrapped calcein. B: Marker release during extru-
sion. Extrusion 1 was performed with a liposome dispersion containing 10 mM lipid,
extrusion 2 with a liposome dispersion containing 20 mM lipid (n = 3).
RESULTS AND DISCUSSION 137
omization or drying, to the initial fluorescence. Please refer to chapter 4.2.14 for the calcu-
lation. Huang and MacDonald (2004) used the described method for the determination of
the marker release from acoustically active liposomes.
First of all, it was important to evaluate whether a time-dependent release of calcein took
place (Figure 5-60, A). Lipid bilayers were reported to be impermeable to Co2+ ions (Oku
et al., 1982). A cobalt chloride solution was added to a liposome dispersion and the remain-
ing fluorescence was measured at 0, 10, 30 and 60 min. The values ranged between -3 %
and +3 %, which was attributed to a measuring inaccuracy. Hence, no time-dependent re-
lease was detected during one hour, which was the time span needed to perform atomization
and spray drying experiments.
Figure 5-60 (B) shows the results of another experiment, which was performed in order to
test the plausibility of the method. Co2+ was added to a crude liposome dispersion. This
dispersion was extruded and the fluorescence was measured before and after extrusion.
Extrusion through polycarbonate membranes breaks up larger liposomes. Subsequently, the
encapsulated calcein got in contact with Co2+ ions, which quenched the fluorescence. A
Figure 5-61: Release after atomization through different nozzles. The blank value describes the
release occurring in the tube leading to the nozzle (n = 3).
138 RESULTS AND DISCUSSION
release of > 70 % confirmed this assumption. 70 % of the encapsulated marker was initially
entrapped in liposomes, which were > 0.1 µm in diameter and was quenched after the break-
up of the liposomes. Small liposomes with a diameter < 0.1 µm included approximately 30
% of the fluorescent dye and seemed not to be affected by the extrusion process.
MacDonald et al. (1991) used a slightly different approach: They added calcein to the ex-
ternal phase before extrusion and measured the uptake into liposomes during extrusion.
They also attributed the uptake to the rupture of MLVs during extrusion.
The membrane integrity, expressed as release of calcein, during atomization is presented
in Figure 5-61. The liposome dispersions were collected after atomization below the nozzle
tip and before atomization at the transition tube/nozzle (blank value). This blank value de-
scribed the release, which occurred during pumping through the tube. A release of up to
10 % was measured, which was attributed to the mechanical stress exerted on the liposome
dispersion by the peristaltic pump. Atomization using both ultrasonic nozzles resulted in a
release of 10 – 16 % irrespective of the lipid concentration. The release after droplet gen-
eration using a two fluid nozzle was 20 % for the 10 mM lipid concentration and 32 %
when spraying the dispersion containing the twofold amount of lipid. The comparably high
Figure 5-62: Release after spray drying and reconstitution using different nozzles (SD) and after
freeze drying (FD) and reconstitution of the lyophilisate (n = 3).
RESULTS AND DISCUSSION 139
release during atomization in a two fluid nozzle was attributed to the shear stress in the
nozzle. However, the result was in contrast to the liposome size after atomization. Here,
the ultrasonic nozzles had a great influence, whereas liposomes sprayed by the two fluid
nozzle kept their size (cf. chapter 5.2.1).
Figure 5-62 finally shows the release during spray drying and freeze drying. The nozzle
type had no influence on the membrane permeability. 80 % of the encapsulated fluorescent
dye got in contact with Co2+ during spray drying and rehydration. Freeze drying and recon-
stitution of the lyophilisate triggered a release of 98 %. Compared to atomization, a huge
impact on the release was found after spray drying and freeze drying. Possible stress factors
during spray drying were the temperature and osmotic pressure (Wessman et al., 2010).
The effect of heat on the membrane integrity was examined in a further experiment. Cal-
cein-loaded liposomes were mixed with cobalt chloride and filled in Eppendorf tubes,
which were exposed to 40 °C and 70 °C for 20 min using a water bath. Heating the sample
up to 40 °C resulted in a release of 10 %, whereas the exposure to 70 °C resulted in a release
of 80 %. Spray drying was performed at a Tinlet of 150 °C and a Tchamber of 70-80 °C at the
end of the process chamber. Therefore, droplets/particles were heated up to Tchamber. Subse-
quently, it was concluded that the main part of the release was provoked by the heat stress
and only to a minor extent by the atomization process. This could be explained through the
A B
Figure 5-63: A: Release after heating the liposome dispersions (n = 3). B: SD powders contain-
ing trehalose, liposomes and calcein (left) and SD powders containing trehalose, liposomes, cal-
cein and CoCl2 (right).
140 RESULTS AND DISCUSSION
increased membrane permeability with increasing temperatures (De Gier et al., 1968). In
addition, liposomes used in this work had a phase transition temperature below 0 °C and
thus were in the more permeable liquid-crystalline configuration (Braun-Falco, 1992) dur-
ing the whole process. Figure 5-63 shows spray dried powders containing calcein-loaded
liposomes (B, left) and calcein-loaded liposomes mixed with cobalt chloride (B, right). The
pink coloration was a result of the cobalt chloride hexahydrate formation and vanished as
soon as the powder got in contact with water.
Very high marker leakage was found for the freeze dried liposomes (Figure 5-62). Although
there was no thermal stress, they gave the highest values of release. Here, the pass through
Tm during freezing (Hays et al., 2001), mechanical stress caused by ice crystal formation
(Chen et al., 2010) and osmotic stress provoked by freeze concentration (Siow et al., 2007)
might be possible triggers of the decreased membrane integrity during freeze drying.
Figure 5-64 summarizes the results of Zetasizer measurements during the described exper-
iments. Neither the buffer, the modified formulation, calcein nor the presence of Co2+ al-
tered the size or size distribution of liposomes. They showed the characteristic decrease in
size when using the ultrasonic nozzles for atomization. Freeze drying and reconstitution
Figure 5-64: Properties of calcein-loaded liposomes before processing and after atomization,
spray drying or freeze drying. The striped bars indicate liposome dispersions, where a solution of
cobalt chloride was added.
RESULTS AND DISCUSSION 141
slightly decreased the liposome size, too. All PDI values were below 0.2 indicating a mon-
odisperse size distribution. The zeta potential was in the range of 25 – 35 mV, which was
attributed to the lower percentage of DOTAP in the formulation and a constant pH value
of 7.4, provided by the HEPES buffer. The zeta potential of freeze dried liposomes in-
creased to 50 mV. It was supposed that the pass through Tm might alter the lipid distribution
in the membrane, which might affect the zeta potential.
In conclusion, experiments using a fluorescent dye showed that liposomes and marker were
intact after spray drying or freeze drying. The encapsulation efficiency, liposome size, size
distribution and the overall calcein content remained constant. These results were in good
agreement with the data presented in chapter 5.2.
Regarding the membrane integrity, the experiments revealed that atomization and even
pumping disturbed the membrane integrity. The decrease in the initial fluorescence of ap-
proximately 80 %, expressed as release, was attributed to the heat stress during spray dry-
ing. Here, 80 % of the initially encapsulated marker got in contact with Co2+ ions during
spray drying. Goldbach et al. (1993b) found a similar release of 65 – 80 % of encapsulated
atropine during spray drying. The release remained that high even if a liposome composi-
tion with a high Tm was used. This result and the results found in the presented experiments
were in contrast to a 90 % (hydrophilic) marker retention after spray drying and reconsti-
tution reported by Hauser and Strauss (1987).
Although freeze drying was the method, which produced the highest calcein leakage in the
presented experiments, freeze drying of liposomes with a more satisfying retention of hy-
drophilic molecules is possible in principle (Crowe et al., 1996b). In addition, liposome
compositions with a higher Tm might be beneficial (van Winden et al., 1997).
The encapsulation and retention of water soluble molecules stays challenging (Kreuter,
1994), whereas the entrapment of lipophilic drugs in the membrane bilayer is substantially
more reliable and was reported to be consistent during spray drying (Chougule et al., 2008,
Wiggenhorn, 2007).
142 RESULTS AND DISCUSSION
5.7 Liposomes with encapsulated Insulin
The aim of this chapter was to examine whether it is possible to encapsulate a protein into
liposomes and spray dry these dispersions. The peptide hormone insulin was chosen as a
test substance. Inhaled insulin is efficient and safe (Hollander et al., 2004) and was
launched in 2006 (Exubera®). Pulmonary delivery via liposomes is supposed to decreases
side effects and promote the sustained release of the encapsulated active ingredient (Conley
et al., 1997). Hence, inhaled liposomal insulin (Liu et al., 1993) might be a candidate for
the administration via DPIs, which are prepared, for example, using spray drying. Section
5.7.1 describes the insulin analytics, which were performed in order to characterize insulin
before and after spray drying. A method was developed to determine the encapsulation
efficiency. Insulin aggregation was quantified using size exclusion chromatography (SEC)
according to a method described in the USP and Ph.Eur. SEC results revealed that insulin
formed aggregates in the presence of positively charged liposomes. Therefore, a batch of
DOTAP-free liposomes with encapsulated insulin was prepared and spray dried (Table
5-11).
5.7.1 Characterization of Insulin and Liposomes after Spray Drying
A standard BCA assay (Bicinchoninic Acid) was used for the determination of the overall
insulin content of spray dried and reconstituted powders. In addition, the BCA assay was
used both for the determination of free, non-encapsulated insulin and encapsulated insulin
after separation. Sample compositions are given in Table 5-11. Figure 5-65 shows a typical
calibration curve obtained with the BCA assay. The procedure of the assay as well as the
separation of free insulin from the encapsulated fraction using centrifugal filter units is
described in detail in chapter 4.2.12. Amicon Centrifugal Filter Units with a molecular cut-
Lipid content [mM]
DOTAP/DOPC
concentration
[mg/ml]
Insulin
concentration
[mg/ml]
Trehalose
concentration
[mg/ml]
0 0 2 100
10 3.49 / 3.93 2 100
20 6.99 / 7.86 2 100
10 0 / 7.86 2 100
Table 5-11: Sample compositions.
RESULTS AND DISCUSSION 143
Figure 5-65: Calibration curve of the BCA assay.
off of 100 kDa were chosen for the purpose of assuring 100 % recovery of free insulin
(molecular weight: ~ 6 kDa) in the filtrate. Therefore, an insulin solution (2 mg/ml) was
placed in the sample reservoir. Centrifugation and washing steps were performed and the
recovery in the filtrate and concentrate was measured. 99 % of the insulin were found in
the filtrate, whereas only 1 % was found in the concentrate. The second criterion for the
separation method was the complete retention of liposomes in the concentrate. The pore
size of the membrane was approximately 10 times smaller than the mean diameter of ex-
truded liposomes used in this work and assured the retention of liposomes in the concentrate
(Pall, 2015). After the separation of a liposome containing dispersion, no liposomes were
detected in the filtrate using dynamic light scattering. In conclusion, liposomes were re-
tained in the concentrate and the unhindered pass of insulin through the membrane into the
filtrate was provided.
Encapsulation efficiencies calculated with the described method were compared to the EE
obtained after separation of free insulin from encapsulated insulin using minicolumn cen-
trifugation (Torchilin and Weissig, 2003). Eppendorf cups were packed with Sephadex® G-
50 (Sigma-Aldrich, Germany). The method was not suitable for the separation of liposome
dispersions with a trehalose concentration of 100 mg/ml as the sugar molecules block the
144 RESULTS AND DISCUSSION
Figure 5-66: Encapsulation efficiency of insulin in the liquid feed of different formulations (be-
fore processing) and after spray drying and reconstitution using a 25 kHz ultrasonic nozzle (USN
25 kHz) and a two fluid nozzle (TFN) for atomization as determined using separation in Amicon
Centrifugal Filter Units and subsequent detection using a BCA assay (n = 3).
Figure 5-67: Summary of Zetasizer measurements of samples containing insulin (n = 3).
RESULTS AND DISCUSSION 145
pores, which should retain free insulin. However, the method was used as a reference
method and confirmed the correctness of the separation in Amicon Centrifugal Filter Units.
The measurement of the EE using minicolumn centrifugation of a trehalose-free formula-
tion with a lipid content of 10 mM resulted in an EE of 40.3 ± 6.0 % (n = 8). This value
was in good agreement with the encapsulation efficiencies obtained after the separation in
Amicon Centrifugal Filter Units (Figure 5-66). Increasing the lipid concentration up to
20 mM gave an increase in EE. The same behavior was already reported for the encapsu-
lation of the fluorescent dye calcein (chapter 5.6.1) and was associated with the existence
of an increased number of liposomes. Liposomes composed of DOPC were able to encap-
sulate almost 70 % of the insulin present in the dispersion. This was attributed to the de-
creased aggregation of insulin, which is described in the next section.
Before processing, liposome properties such as liposome size and size distribution were
similar to liposomes not containing insulin. The zeta potential of liposomes composed of
pure DOPC was distinctly lower (30 mV) compared to liposomes containing the positively
charged DOTAP. After spray drying using an ultrasonic nozzle and reconstitution, the lip-
osome size (Figure 5-67) decreased and the size distribution was broader (PDI ~ 0.2).
Hence, the previously reported size reduction triggered by ultrasonic nozzles was also con-
firmed for insulin containing liposomes. Liposomes spray dried with the two fluid nozzle
were larger in diameter and the PDI increased to ~ 0.2 after rehydration. These liposomes
were able to entrap more insulin (Figure 5-66). In contrast, only a slight increase in the
encapsulation efficiency was found when using the ultrasonic nozzle.
Besides the percentage of entrapped insulin, it was of great interest to determine whether
insulin aggregates during the whole process of liposome formation, spray drying and re-
constitution. For this purpose, the method described in the monography of insulin in the
USP and Ph.Eur. for the determination of high molecular weight protein (HMWP) was
implemented. The ratio of the sum of all peaks having retention times less than that of the
insulin monomer to the peak response of the insulin monomer is limited to 1 %.
Figure 5-68 and Figure 5-69 display chromatograms of various formulations of (liposomal)
insulin before and after the spray drying process. Figure 5-68, A shows a chromatogram of
a solution of insulin and trehalose in the absence of liposomes. Calculation of the percent-
age of HMWP revealed that the solution passed the criterion defined by the pharmacopoeia
146 RESULTS AND DISCUSSION
Figure 5-68: SEC chromatograms of a solution of insulin (A), a spray dried and reconstituted
solution of insulin (B), a dispersion containing liposomes (DOTAP/DOPC) and encapsulated in-
sulin (C) and the same dispersion spray dried and reconstituted (D).
D
C
B
A
RESULTS AND DISCUSSION 147
Figure 5-69: SEC chromatograms of a dispersion of liposomes (DOPC) with encapsulated insu-
lin after spray drying and reconstitution (E) and a dispersion of liposomes (DOTAP/DOPC),
which was separated into “filtrate” (F) and the “concentrate” (G) using an Amicon Centrifugal
Filter Unit.
with less than 0.5 % aggregated insulin (see also Figure 5-70). Spray drying and reconsti-
tution of this solution resulted in a slightly increased aggregation (Figure 5-68, B), which
was attributed to the thermal stress and the stress through atomization. The level of aggre-
gation in powders generated by the two fluid nozzle (0.5 %) was superior to the aggregation
found in powders atomized using the ultrasonic nozzle (1.0 %). With approximately 1 %
HMWP, the reconstituted insulin solution did not pass the Ph.Eur. and USP specification.
Chromatograms C and D show dispersions of liposomes, which were prepared by adding
insulin to the rehydration medium of the lipid film. 48 % of the added insulin was entrapped
G
F
E
148 RESULTS AND DISCUSSION
Figure 5-70: Percentage of high molecular weight protein as a function of the formulation and
after spray drying and reconstitution using an ultrasonic nozzle and a two fluid nozzle. Please note
that the y-axis is broken (n = 3).
in liposomes (cf. Figure 5-66). However, SEC analysis revealed that – depending on the
initial lipid content – the liposome dispersion included 12 % or 25 % HMWP. The dupli-
cation of the liposome concentration led to a duplication of the HMWP suggesting a corre-
lation between the presence of liposomes and aggregation of insulin. The shear stress ap-
plied during sample extrusion was ruled out as a reason for the high degree of aggregation:
An extruded insulin solution did not show any aggregates. Spray drying and reconstitution
of liposome dispersions containing insulin did not increase the portion of aggregated insulin
(Figure 5-68, D and Figure 5-70). The total content of insulin in the reconstituted dispersion
determined through the BCA assay remained unchanged (± 3 %). Liposome dispersions
before and after the spray drying and rehydration process were clear with no signs of tur-
bidity. The retention time of insulin aggregates was equal to the retention time of BSA,
suggesting a mean aggregate size of approximately 66.5 kDa.
The encapsulation of insulin in neutral liposomes composed of pure DOPC improved the
aggregation behavior and decreased the percentage of high molecular weight protein in the
initial liposome dispersion to approximately 1 %. However, spray drying and reconstitution
of this formulation increased the amount of HMWP to ~ 2 %. These results indicated that
RESULTS AND DISCUSSION 149
aggregation was limited to the presence of strongly positively charged liposomes. There-
fore, an insulin solution was mixed with a liposome dispersion composed of DOTAP and
DOPC in the molar ratio 1:1. This mixture was analyzed using SEC. No aggregates were
found. Subsequently, the aggregation seemed to be limited to the encapsulation process as
simple mixing of liposomes and insulin did not induce aggregation. To evaluate whether
aggregates were encapsulated or attached to liposomes, a separation in Amicon Centrifugal
Filter Units was performed and the “filtrate” and “concentrate” was examined using SEC.
Figure 5-69 (F and G) shows the chromatograms. Although aggregates would be able to
pass the membrane, no aggregates were found in the “filtrate” indicating that the liposome
dispersion contained no “free” aggregates. On the contrary, aggregates were found in the
“concentrate” suggesting that aggregates were entrapped in liposomes or at least attached
to their surface.
Hence, it was concluded that aggregation took place during the rehydration of a positively
charged lipid film in the round bottom flask and that aggregates were localized in the lipo-
somes or attached to their surface. Sharp et al. (2002) reported that the adsorption of insulin
to a DOTAP-coated surface led to a larger amount of unfolding compared to the bulk solu-
tion of insulin. A negatively charged surface exhibited slower unfolding kinetics.
150 RESULTS AND DISCUSSION
5.7.2 Characterization of Spray Dried Powders
The samples listed in Table 5-11 were spray dried using either the 25 kHz ultrasonic nozzle
or the two fluid nozzle with a flow rate of 1.0 ml/min. Table 5-12 summarizes product
properties such as the powder yield and the d50 value of the volume based size distribution.
There were no major differences between the insulin containing powders and the insulin-
free products (chapter 5.3.1 and 5.3.2). Regarding the powder yield, liposomes triggered a
slight improvement, which was also reported for the insulin-free products. Particles spray
dried using the 25 kHz ultrasonic nozzle had a mean diameter of 20 – 26 µm, whereas the
two fluid nozzle produced powders with a mean size in the range of 5 – 8 µm. Here, lipo-
some containing powders were slightly larger in diameter, which was attributed to an in-
creasing viscosity of the liquid feed. Figure 5-71 shows SEM pictures of various spray dried
powders containing insulin. Some powders were generated from a solution of insulin and
trehalose (a, d), others from a dispersion of liposomes and partly encapsulated insulin and
trehalose (b, c, e, f). All particles had a wrinkled appearance and fragments of the shriveled
particles were visible. Most particles showed craters at their surface. The surface itself was
smooth. Increasing the lipid concentration to 20 mM (c, f) seemed to slightly decrease the
amount of fragments and to enhance the formation of rounded particles.
Composition of the
liquid feed Nozzle
Particle size d50
[µm] Particle yield [%]
2 mg/ml insulin,
100 mg/ml trehalose
25 kHz ultrasonic
nozzle 20.9 ± 0.5 42.9 ± 6.5
two fluid nozzle 5.3 ± 0.3 54.5 ± 9.8
2 mg/ml insulin,
10 mM
DOTAP/DOPC,
100 mg/ml trehalose
25 kHz ultrasonic
nozzle 24.5 ± 0.7 60.1 ± 4.9
two fluid nozzle 6.0 ± 0.1 62.7 ± 12.1
2 mg/ml insulin,
20 mM
DOTAP/DOPC,
100 mg/ml trehalose
25 kHz ultrasonic
nozzle 24.6 ± 0.7 62.8 ± 12.4
two fluid nozzle 6.2 ± 1.0 67.0 ± 5.0
2 mg/ml insulin,
10 mM DOPC,
100 mg/ml trehalose
25 kHz ultrasonic
nozzle 26.2 ± 0.8 65.0 ± 11.0
two fluid nozzle 8.4 ± 1.3 52.9 ± 13.7
Table 5-12: Summary of powder properties (n = 3).
RESULTS AND DISCUSSION 151
a) 10 % trehalose, 2 mg/ml insulin
1000x
b) 10 mM lipid, 10 % trehalose, 2 mg/ml
insulin, 1000x
c) 20 mM lipid, 10 % trehalose, 2 mg/ml
insulin, 1000x
d) 10 % trehalose, 2 mg/ml insulin
1000x
e) 10 mM lipid, 10 % trehalose, 2 mg/ml
insulin, 1000x
f) 20 mM lipid, 10 % trehalose, 2 mg/ml
insulin, 1000x
Figure 5-71: SEM pictures of spray dried powders. The liquid feed was composed of trehalose
(10 % w/v), 0 – 20 mM lipid (DOTAP/DOPC) and 2 mg/ml insulin. Powders of pictures a-c were
atomized using the 25 kHz ultrasonic nozzle and powders of pictures d-f were generated by the
two fluid nozzle.
20µm 20µm
20µm 20µm
20µm 20µm
152 RESULTS AND DISCUSSION
a) 10 mM lipid, 10 % trehalose, 2 mg/ml
insulin, 1000x
b) 10 mM lipid, 10 % trehalose, 2 mg/ml
insulin, 1000x
Figure 5-72: SEM pictures. The spray dried powders were composed of trehalose, DOPC and
insulin. The powder of picture a was atomized using the 25 kHz ultrasonic nozzle and the powder
pictured in b was atomized using the two fluid nozzle.
Figure 5-72 shows SEM pictures of spray dried particles consisting of trehalose and insulin,
which was partly encapsulated in liposomes composed of DOPC instead of a mixture of
DOTAP and DOPC. However, no influence on the particle shape and morphology was
visible with the different phospholipids. Hence, insulin containing powders were in strong
contrast to the insulin-free powders, which had a completely round shape, no craters and
mostly appeared as “golf balls”. The presence of proteins in spray dried powders was re-
ported to alter the morphology towards a more wrinkled appearance (Maa et al., 1997). The
liposomal insulin solution also contained free, non-encapsulated insulin, which – even at
low amounts – triggered the wrinkled appearance. Usage of another phospholipid mixture
did not alter this behavior. Depending on the formulation, dispersions contained between
30 and 50 % free insulin. The lowest amount of free insulin was found in dispersions with
a lipid content of 20 mM. Powders produced thereof tended to be more round shaped com-
pared to powders with a lower lipid concentration. However, liposomes were shown to be
not able to prevent the adsorption of protein to the droplet/air interface during atomization
and therefore have no or only a minor influence on the wrinkled particle appearance (Adler
et al., 2000).
Figure 5-73 displays the residual moisture contents of spray dried powders. Insulin con-
taining powders showed residual moisture contents up to 9.5 %. The use of liposomes de-
creased the residual moisture content considerably. However, powders containing 20 mM
lipid, which were spray dried with the 25 kHz ultrasonic nozzle, still had residual moisture
20µm 20µm
RESULTS AND DISCUSSION 153
contents above 5 %, which is too high regarding the stability of the spray dried product.
The presence of insulin increased the residual moisture content by 2 – 3 % compared to the
equivalent insulin-free formulation. The effect was pronounced using the ultrasonic nozzle.
The glass transition temperature was constant at 41 °C for all powders spray dried using
the 25 kHz ultrasonic nozzle (Figure 5-74). In contrast to the insulin-free powders, the ad-
dition of liposomes did not increase the glass transition temperature. The best powder prop-
erties within these experiments were achieved with a concentration of 20 mM lipid and the
atomization with the two fluid nozzle. Residual moisture contents below 3 % and a glass
transition temperature of 65 °C were measured. The use of liposomes composed of pure
DOPC did not alter the powder properties.
In summary, the presented experiments revealed that the chosen formulation and process
conditions were suitable for the spray drying of insulin containing powders. The results of
the liposome-free SD experiments were in good agreement with a study published by Ståhl
et al. (2002) in terms of the percentage of high molecular weight protein in the spray dried
product. They spray dried insulin without any excipients. However, the reduction of the
high residual moisture contents is crucial with respect to the stability of the spray dried
Figure 5-73: Residual moisture contents of spray dried powders containing 2 mg/ml insulin,
100 mg/ml trehalose and varying amounts of liposomes in the liquid feed. If not stated otherwise,
liposomes were composed of DOTAP and DOPC in the molecular ratio 1:1 (n = 3).
154 RESULTS AND DISCUSSION
Figure 5-74: DSC thermograms of powders containing 2 mg/ml insulin, 100 mg/ml trehalose
and varying amounts of lipid. If not stated otherwise, liposomes were composed of DOTAP and
DOPC in the molecular ratio 1:1. The mean glass transition temperatures ± standard deviations
(n = 3) are specified next to the glass transition.
product (cf. chapter 5.8).
Regarding the encapsulation of insulin into liposomes, aggregation was observed during
the lipid film rehydration. Spray drying and reconstitution did not improve or worsen the
level of aggregation or the encapsulation efficiency of liposomal insulin. A modified for-
mulation with liposomes composed of DOPC revealed that it is possible to prepare and
spray dry insulin-loaded liposomes with a low level of aggregation before and after the
spray drying process.
Only few data are available on the encapsulation (and spray drying) of insulin in liposomes.
Huang and Wang (2006) examined the encapsulation of insulin in liposomes using the con-
ventional film shaking method and the destabilizing/detergent dialyzing method. They
found a better encapsulation efficiency with the second method. The conventional method,
which was similar to the method used in this work, resulted in an EE of 10 %. Liu et al.
(1993) found an entrapment efficiency of 14 % using substantially different lipid composi-
tions compared to the one used in this work. Finally, Park et al. (2011) encapsulated insulin
in positively charged liposomes partly composed of DOTAP. The purpose of this study was
RESULTS AND DISCUSSION 155
to enhance the sustained release delivery of a protein drug for parenteral administration.
Encapsulation efficiency was similar to the results presented in this work. Circular dichro-
ism measurements revealed no changes in the secondary structure of insulin although
DOTAP was part of the liposome composition. These findings are in contrast to the aggre-
gation found in this work. However, zeta potentials strongly differed: The formulation used
in this work had a zeta potential of approximately 70 mV, whereas Park et al. (2011) meas-
ured values < 10 mV. In conclusion, only strongly repulsive surfaces seemed to be respon-
sible for the aggregation of the polycation insulin (Sharp et al., 2002).
156 RESULTS AND DISCUSSION
5.8 Stability of SD Liposome Formulations
Results of a stability test, which was set up for 6 months, are presented in the following
section. Spray dried powders containing liposomes were stored at four different tempera-
tures. All powders had the same composition, but were spray dried using different nozzle
types. Before the manufacturing of the powders took place, preliminary tests were per-
formed in order to define the final sample composition for the stability studies. Several
spray dried powders, which originally had been manufactured in the scope of the experi-
ments presented in chapters 5.1 – 5.3, were examined in terms of liposome properties.
These samples had been stored 3 – 6 months filled in Sarstedt tubes and placed in a desic-
cator at room temperature. Appropriate amounts of these powders were dissolved in water
to get the initial liposome concentration. Subsequently, samples were analyzed using dy-
namic light scattering. Almost all powders analyzed (lipid content: 10 mM) revealed that
fusion or aggregation of liposomes took place during storage since the reconstituted disper-
sion was cloudy, the liposome size was > 130 nm and the PDI > 0.3. Only two powders
showed satisfying results: A powder containing 10 mM lipid, which was spray dried using
the 60 kHz ultrasonic nozzle and a sample containing 40 mM lipid. Both powders had a
low initial moisture content compared to the other products tested. Considering this, a lipid
concentration of 20 mM (Table 5-13) was chosen for the stability tests, as the residual
moisture was remarkably lower compared to the powders containing 10 mM lipid. An im-
proved stability of this formulation was supposed and the extrusion of a large batch of this
formulation was manageable, since the viscosity was only slightly higher than with the
10 mM samples, but considerably lower than that of the 40 or 60 mM samples.
The liposome dispersions were prepared and spray dried using the 25 kHz ultrasonic noz-
zle, the 60 kHz ultrasonic nozzle and the two fluid nozzle with a flow rate of 1.0 ml/min.
In addition, powders were produced using the 25 kHz ultrasonic nozzle and a reduced flow
Lipid content [mM] DOTAP/DOPC
concentration [mg/ml]
Trehalose
concentration
[mg/ml]
20 6.99 / 7.86 100
Table 5-13: Sample composition.
RESULTS AND DISCUSSION 157
A B
C
D
Figure 5-75: Liposome size at different times and under different storage conditions. Please
mind the different scaling of the y-axes.
rate of 0.5 ml/min. Powders spray dried using the 25 kHz nozzle with a reduced flow rate
and the 60 kHz nozzle were supposed to have a lower initial residual moisture content and
therefore a higher glass transition temperature (cf. Figure 5-22 and Figure 5-23). Hence,
one aim of the stability test was to determine the influence of the nozzle type on the stability
of the powder. To examine the influence of common storage conditions, samples were
stored in a refrigerator (2 – 8 °C), a freezer (-80 °C), and in conditioning cabinets adjusted
to 25 °C and 40 °C. The glass transition temperature of the SD powder and liposome prop-
erties such as size, PDI and zeta potential were analyzed at time zero and after 2, 4, 12 and
24 weeks. In general, powders appeared less fluffy after the storage at 40 °C, 25 °C or
2 – 8 °C, whereas powders stored in the freezer maintained their initial appearance.
158 RESULTS AND DISCUSSION
A B
C
D
Figure 5-76: Liposome PDI at different times and under different storage conditions.
Figure 5-75 presents the liposome size after the rehydration of spray dried powders, which
were stored under different conditions. At the beginning, the liposome size of all samples
was between 105 and 125 nm. Aside from minor fluctuations, liposomes, which were stored
at -80 °C maintained their size and all measurements revealed a liposome size in the range
of 105 – 125 nm, even after 6 months of storage (Figure 5-75, A). The same applies to
samples stored in the refrigerator (Figure 5-75, B). Regarding the powders stored at 25 °C
(Figure 5-75, C), after 12 weeks, only one powder contained liposomes > 125 nm, indicat-
ing fusion and aggregation of liposomes. The concerned powder was spray dried using the
two fluid nozzle. The other powders included liposomes, which showed no signs of aggre-
gation or fusion within a time frame of up to 12 weeks. After 24 weeks, all samples stored
at 25 °C revealed that fusion and aggregation of liposomes occurred during storage. Spray
dried liposomes stored at 40 °C were stable up to a storage period of 12 weeks, indicated
RESULTS AND DISCUSSION 159
by a liposome size below 125 nm (Figure 5-75, D) . After 24 months, the reconstitution of
all powders except one revealed that fusion and aggregation of liposomes happened. The
exception was a powder stored at 40 °C, which was spray dried using the 25 kHz ultrasonic
nozzle and a reduced flow rate. In general, powders stored at lower temperatures contained
unchanged and intact liposomes, while the increase in liposome size after 12 or 24 weeks
of storage at elevated temperatures was distinct with z-averages > 140 nm and up to
350 nm.
Figure 5-76 summarizes the size distributions of spray dried liposomes. At t = 0, all PDI
values were < 0.23 indicating a narrow size distributions. The size distributions after the
storage of spray dried liposomes stored at -80 °C and 2 – 8 °C were similar (Figure 5-76,
A, B). A slight increase in the liposome PDI was measured after 2 weeks of storage, but
the PDI values remained below 0.3 at any point of the experiment. Minor fluctuations were
attributed to the measurement inaccuracy. The distribution of liposomes stored at 25 °C
was narrow within a storage period of 4 weeks and remained narrow up to 12 weeks with
one exception. After 6 months, all powders contained broadly distributed liposomes (Figure
5-76, C). Figure 5-76, D shows the size distribution of SD liposomes stored at 40 °C. Up
to a storage period of 12 weeks, aside from a slight increase of the PDI at the time point 2
weeks, all liposomes showed a narrow size distribution. However, PDI values of three sam-
ples distinctly increased after 24 weeks of storage indicating fusion and aggregation. The
results were in good correlation with the liposome size. As long as the liposomes size was
stable, PDI values at least below 0.3 were measured. The sample stored at 25 °C, which
showed an increased size after 12 weeks, also had a heterodisperse size distribution of
~ 0.4. After 24 weeks of storage nearly all powders stored at 25 °C or 40 °C contained
broadly distributed liposome populations, whereas the storage in the freezer or the refrig-
erator resulted in narrow size distributions with a PDI below 0.3.
Zeta potentials of spray dried and reconstituted liposome dispersions at different times are
displayed in Figure 5-77. The measured zeta potentials were between 70 and 85 mV. No
correlation between the stability of the liposome dispersion in terms of size or distribution
and the zeta potential was recognizable. Zeta potentials were constant irrespective of the
size or size distribution measured. Fluctuations were attributed to the measuring method.
160 RESULTS AND DISCUSSION
A B
C
D
Figure 5-77: Liposome zeta potential at different times and under different storage conditions.
Figure 5-78 shows glass transition temperatures of spray dried powders. At time zero, pow-
ders spray dried using the 25 kHz ultrasonic nozzle with a reduced flow rate or the 60 kHz
ultrasonic nozzle had a Tg > 75 °C. In contrast, powders generated by the two fluid nozzle
or the 25 kHz ultrasonic nozzle with the regular flow rate had a glass transition temperature
below 65 °C. These results were in good agreement with the glass transition temperatures
reported in chapter 5.3.3 and emphasized the positive influence of the 60 kHz nozzle or a
decreased flow rate on the moisture content and the Tg, respectively.
The storage at -80 °C (Figure 5-78, A) had a minor influence on the glass transition tem-
perature as the values were almost constant for the time period of storage. Only the Tg of
the sample spray dried using the 60 kHz ultrasonic nozzle decreased distinctly to ~ 65 °C
after 24 weeks of storage. In contrast, powders stored in the refrigerator (Figure 5-78, B)
RESULTS AND DISCUSSION 161
A B
C
D
Figure 5-78: Glass transition temperature of spray dried powders at different times and under
different storage conditions.
showed decreasing glass transition temperatures resulting in a Tg of 45 – 55 °C after 24
weeks of storage. The same applies to powders stored at 25 °C in the conditioning cabinet
(Figure 5-78, C). The Tg decreased to 45 – 55 °C after 12 weeks of storage. However, no
glass transitions were detectable after 24 weeks of storage. The storage at 40 °C (Figure
5-78, D) revealed a different development of the glass transition temperatures. Powders
initially having a low glass transition temperature showed an increased Tg after 4 weeks of
storage. This effect was attributed to a final drying process during the storage at the elevated
temperature. After 24 weeks of storage DSC thermograms revealed the lack of glass tran-
sitions in almost all samples stored at 40 °C.
Especially the storage at 25 °C and 40 °C seemed to approach the initial difference between
the Tg of the powders. The results matched the Zetasizer data well suggesting a correlation
162 RESULTS AND DISCUSSION
Figure 5-79: DSC thermograms of the second heating scan of samples stored at 25 °C. Spray
dried powders were prepared using the 25 kHz ultrasonic nozzle.
Figure 5-80: WAXD of spray dried powders (60 kHz ultrasonic nozzle) after 6 months of stor-
age at different temperatures. Crystalline trehalose dihydrate is depicted as a reference.
RESULTS AND DISCUSSION 163
between the missing glass transition temperature and fusion or aggregation of liposomes.
DSC thermograms with decreasing glass transition temperatures are exemplified in Figure
5-79 presenting the DSC scans of a powder stored at 25 °C. The sample, which had been
stored the longest, did not show a glass transition. Here, a melting peak was visible, whereas
the other powders showed thermal events indicating cold crystallization.
The physical state of powders spray dried with the 60 kHz ultrasonic nozzle was analyzed
using WAXD (Figure 5-80). After 24 weeks of storage, samples stored at -80 °C or
2 – 8 °C clearly showed the amorphous halo, whereas powders stored at 25 °C or 40 °C
were crystalline. Hence, the missing glass transitions in the DSC scans were explained by
the crystalline state of the spray dried powder.
In conclusion, the stability of liposomes embedded in a spray dried trehalose matrix was
highly dependent on the amorphous state of trehalose. Crystallization of the surrounding
matrices triggered fusion or aggregation of liposomes, which was in good agreement with
the results reported in conjunction with the crystalline excipient mannitol. Mannitol was
not able to preserve liposomes during spray drying.
Even low glass transition temperatures of ~ 45 °C after 24 weeks of storage provided the
stability of liposomes. However, the storage temperature of products with such a low Tg is
extremely limited. If powders are stored above their Tg, liposome fusion and leakage is
clearly promoted (Sun et al., 1996).
As soon as the amorphous trehalose matrix crystallized, liposomes were insufficiently sta-
bilized and fused to larger vesicles. An increased z-average and a broader size distribution
were measured, whereas the zeta potential was unaffected.
Regarding the crystallization behavior of trehalose, an elevated temperature (25 or 40 °C)
seemed to be necessary to trigger complete crystallization. Amorphous trehalose is known
to act as a “desiccant”, which is able to partly form crystalline trehalose dihydrate, while
keeping the glass transition temperature of the remaining material high (Crowe et al.,
1996b). Hence, embedded proteins or liposomes can be stabilized even at high humidity.
However, crystallization seemed to be far advanced after 6 months of storage at 40 °C or
25 °C, since the WAXD data revealed a clearly crystalline state and the glass transitions in
the DSC scans disappeared. On closer analysis, the sample stored at 40 °C did not show the
crystalline pattern of trehalose dihydrate, but of crystalline anhydrous trehalose (cf. X-ray
164 RESULTS AND DISCUSSION
diffraction patterns of trehalose depicted in Ohashi et al. (2007)). The pattern of crystalline
trehalose dihydrate was found in samples stored at 25 °C. Moran and Buckton (2007) found
an anhydrous crystalline form arrangement in amorphous trehalose, which was likely to
result in the anhydrous crystalline state of spray dried powders stored at 40 °C in the sta-
bility experiment presented here.
The influence of the nozzle type used for the production of spray dried powders had only a
minor impact on the stability of the embedded liposomes. Depending on the storage condi-
tions, several samples even showed an approximation in terms of Tg, although the initial
glass transition temperatures partially differed more than 20 °C. However, the only stable
sample stored at 40 °C was spray dried using a reduced flow rate. This supported the as-
sumption that low residual moisture contents are a key requirement for the production of
stable spray dried powders. Besides a higher drying temperature or a reduced flow rate an
additional drying step might be appropriate in order to prevent crystallization provoked by
high residual moisture contents.
6 CONCLUSION
In this thesis, the influence of spray drying on liposomes was examined and the question to
what extent liposomes alter the powder properties of the spray dried product was investi-
gated. With respect to literature, especially the latter is insufficiently studied so far. There-
fore, several liposome dispersions were prepared and spray dried in a range of concentra-
tions. In order to analyze liposomes after spray drying, powders were reconstituted with an
appropriate volume of water to get the initial lipid concentration. Spray drying conditions
were constant for all experiments except the nozzle used for atomization. Here, a 25 kHz
and a 60 kHz ultrasonic nozzle and a standard two fluid nozzle were used and their influ-
ence on liposomes and powders was evaluated.
The chosen liposome formulation consisted of the positively charged lipid DOTAP and the
neutral helper lipid DOPC in the molar ratio 1:1 and 100 mg/ml trehalose. After the prepa-
ration in a round bottom flask followed by the extrusion through a 0.1 µm membrane, the
formulations were spray dried. Regarding the liposome size, size distribution or zeta po-
tential, no major changes in the reconstituted product were observed using molar lipid to
sugar ratios between 1:146 (2 mM lipid) and 1:5 (60 mM lipid). However, satisfactory
results of the preceding extrusions were not achieved with the highest liposome concentra-
tion (60 mM), since the high viscosity of the suspension caused numerous ruptures of the
polycarbonate membranes. The phase transition temperature of the rehydrated liposome
dispersion was equal to the Tm of the initial liposome dispersion. Measurements of the lipid
recovery supported this finding. In addition, a calorimetric study of SD trehalose containing
embedded liposomes showed that the phase transition temperature of the phospholipid mix-
ture decreased below -80 °C.
The application of different nozzle types resulted in slight variations of the liposome size
after spray drying and reconstitution of the powder. Both ultrasonic nozzles caused a de-
crease in the vesicle diameter of 9 – 13 %, whereas the liposome size remained constant
when using the two fluid nozzle. The influence of the 25 kHz ultrasonic nozzle was more
166 CONCLUSION
distinct than that of the 60 kHz nozzle. Atomization experiments confirmed these results.
Here, the liposome dispersions were collected below the nozzle tip without heat applica-
tion. The described phenomenon was limited to less concentrated liposome dispersions (<
20 mM), but was not unique to one formulation. Ultrasonic nozzles caused a decrease in
liposome size irrespective of the lipid composition, the entrapment of calcein or insulin, the
incorporation of a PEGylated lipid or the use of different excipients. The size reduction was
attributed to ultrasonic vibration and cavitation effects.
Spray drying of liposomes with different excipients allowed the following conclusions: Su-
crose and lactose were able to stabilize liposomes, whereas mannitol and maltodextrin
failed to stabilize liposomes during spray drying and reconstitution. Sucrose, lactose and
maltodextrin were – like trehalose – in an amorphous state after spray drying. However,
maltodextrin provided insufficient hydrogen bonding to replace water in the dried state.
Regarding the overall properties, trehalose remained the excipient of choice as sucrose had
– despite a low residual moisture content – a low glass transition temperature. Lactose is a
reducing sugar, which might be challenging regarding complex formulations.
The stability of spray dried liposomes was highly dependent on the physical state of the
surrounding trehalose matrix. This was obvious especially during the stability test sched-
uled for a period of 6 months. As long as trehalose was amorphous, liposomes were stable
and DLS measurements revealed no major changes. After six months of storage, fusion and
aggregation of the included liposomes occurred in several samples accompanied by the
crystallization of the concerned powders. Accordingly, an amorphous matrix is a basic re-
quirement of stable liposomes. The lack of stability during spray drying of liposomes using
the crystalline excipient mannitol supported this assumption.
The addition of the surfactant Polysorbate 80, a common excipient used for the stabilization
of proteins during spray drying (Adler and Lee, 1999), to the rehydration medium caused
the attachment of the surfactant to the liposome membrane. Measurements using a maxi-
mum bubble pressure tensiometer showed that a certain quantity of surfactant was attached
to the liposomes, while the excess Polysorbate 80 was able to occupy the liquid/air inter-
face. A slight decrease in the particle size of the spray dried product, which was supposed
to be a result of the lowered surface tension during atomization, went along with a de-
CONCLUSION 167
creased residual moisture content of the powder. The higher number of surfactant mole-
cules at the exterior lipid bilayer and the related increase of steric repulsion increased the
curvature of the vesicle and triggered a size reduction. In the same way, incorporation of
increasing amounts of PEGylated lipid to the standard liposome formulation resulted in an
increased curvature and a constant liposome size, although the PEG chains had a length of
5 nm. Spray drying and rehydration of the powder did not alter the properties of PEGylated
liposomes. Zeta potentials and PDI were slightly smaller compared to the conventional lip-
osomes.
A set of experiments was performed using the fluorescence dye calcein in order to investi-
gate the encapsulation efficiency of liposomes and the membrane integrity during spray
drying. The entrapment of calcein in positively charged liposomes resulted in an encapsu-
lation efficiency of ~ 10 %. If the twofold amount of lipid was used for the liposome prep-
aration, ~ 22 % of calcein was entrapped in liposomes indicating that the number of lipo-
somes had roughly doubled. However, the molar ratio of DOTAP in the lipid composition
had to be reduced from 50 % to 5 % since the positively charged DOTAP interfered with
calcein. The encapsulation efficiency was not affected by the atomization through different
nozzles, spray drying or freeze drying. Hence, it was concluded that the number of lipo-
somes, their size and the distribution of calcein was not notably changed by these processes,
which was in good agreement with the results found in the marker-free experiments. To
evaluate whether there is an exchange between entrapped and free calcein during atomiza-
tion and spray drying, studies on the membrane integrity were performed. Atomization
triggered the decrease in encapsulated marker fluorescence of approximately 20 % and the
complete spray drying process resulted in a decrease of 80 % indicating a poor membrane
integrity during drying. Further experiments showed that the high permeability during dry-
ing in the process chamber could be attributed to the application of heat. Freeze drying of
an equal liposome formulation triggered the decrease in encapsulated marker fluorescence
of almost 100 %.
Since the spray drying of liposomal trapped proteins might be promising, a test protein
– insulin – was added to the rehydration medium and was subsequently encapsulated in
liposomes. The percentage of high molecular weight protein in the dispersion was measured
before and after the spray drying process using SEC. A percentage of 10 – 15 % high mo-
lecular weight protein was found in the liposome dispersions. The experiments revealed
168 CONCLUSION
that insulin aggregation occurred already during the rehydration of the lipid film in the
round bottom flask. The level of aggregation was dependent on the lipid concentration and
was not affected by spray drying and reconstitution. Further experiments showed that these
aggregates were caused by an interaction between the positively charged DOTAP and in-
sulin. Subsequently, a batch of liposomes only composed of the neutral helper lipid DOPC
was prepared. 70 % of insulin was entrapped in these liposomes. The percentage of high
molecular weight protein, which increased from 1 % in the liquid feed to only 2 % in the
reconstituted liposome dispersion, indicated that spray drying of protein-loaded liposomes
is feasible. However, the encapsulation of hydrophilic substances, among them peptides
and proteins, remains challenging. First of all, either the complete encapsulation or the so-
phisticated separation from free, non-encapsulated substances must be ensured. Second, the
hydrophilic molecules must be retained in the vesicles during drying and rehydration. Here,
a lipid mixture with a higher phase transition temperature or the addition of cholesterol
might be promising.
Regarding the powder properties, increasing proportions of lipids improved several im-
portant factors irrespective of the nozzle type used for atomization. A decreasing fine par-
ticle fraction contributed to the higher powder yield of formulations with a higher lipid
concentration. In addition, the particle surface partly became smoother. The most important
factor was the reduction of the residual moisture content and the connected increase of the
glass transition temperature. For example, the residual moisture content of powders gener-
ated by the 25 kHz ultrasonic nozzle decreased from almost 6 % (0 mM lipid) to 2 %
(60 mM lipid). An improved drying behavior – indicated by an increased evaporation co-
efficient in the first drying stage – was also found in the levitation experiments, which were
able to depict the drying process of a droplet in slow motion. Here, the two highest lipid
concentrations resulted in stagnant evaporation coefficients, which was supposed to be a
result of a lipophilic barrier at the droplet´s surface. Hansen et al. (2004) attributed im-
proved drying characteristics of lipophilic mixtures to a limited amount of solids in the
powder with the ability for binding water. In the same way, glass transition temperatures
increased from approximately 45 °C to 75 °C. With respect to the stability of embedded
liposomes, high glass transition temperatures are preferred, since the stability of liposomes
is provided only in powders stored below their glass transition temperature (Sun et al.,
1996). A second glass transition at approximately 50 °C was found in powders with a lipid
CONCLUSION 169
concentration ≥ 30 mM or a lipid to sugar ratio ≥ 1:10, respectively. It was supposed that
the thermal event was the glass transition of a lipid rich phase – probably inside the lipo-
somes – which was insufficiently dried. All spray dried powders were initially amorphous.
If the initial residual moisture content was high (> 3.5 %), the storage at elevated tempera-
tures or heating during a DSC scan triggered the crystallization of trehalose. Similarly, lip-
osome containing powders stored at 25 °C or 40 °C in the framework of the stability test
showed crystallization after 6 months of storage. The comparison of the WAXD data re-
vealed that crystalline anhydrous trehalose was present in the samples stored at 40 °C,
whereas the powder stored at 25 °C formed crystalline trehalose dihydrate.
The influence of the nozzle types used can be summarized as follows: Both ultrasonic noz-
zles produced homogenous powders with narrow size distributions, whereas powders at-
omized using the two fluid nozzle were smaller, broader distributed and had an increased
fine particle fraction. Although particles spray dried using the two fluid nozzle possessed
the smallest mean diameter, residual moisture contents were partly as high as in powders
generated by the 25 kHz ultrasonic nozzle. The combination of the 25 kHz ultrasonic nozzle
with a reduced flow rate of the liquid feed or the use of the 60 kHz nozzle produced best
results regarding the overall powder properties. However, the stability test revealed that
initially different glass transition temperatures approached during storage.
The presented work illustrates the basic impacts of liposomes on spray dried powders.
These findings can facilitate the formulation of liposomes and liposomal drugs, especially
in the context of spray dried powders. Increasing amounts of liposomes in the spray dried
powder resulted in a decreased moisture content, an increased glass transition temperature,
an improved powder yield and a reduced fine particle fraction. Different nozzle types were
presented and their influence on powders and liposomes were demonstrated. The liposome
size was slightly reduced using ultrasonic nozzles for atomization. The amorphous state of
the surrounding matrix was crucial for the stability of liposomes and crystallization must
be avoided. Therefore, the residual moisture content should be as low as possible.
7 ZUSAMMENFASSUNG
Das Thema der vorliegenden Arbeit ist die Sprühtrocknung von Liposomen. Große Teile
der Arbeit befassen sich mit der Stabilität von Liposomen während der Sprühtrocknung,
aber auch während der Lagerung als sprühgetrocknetes Pulver. Ein weiterer Schwerpunkt
liegt auf der Untersuchung des Einflusses von Phospholipiden auf die Eigenschaften sprüh-
getrockneter Pulver. Besonders letzteres ist bislang unzureichend untersucht worden. Lip-
osomendispersionen unterschiedlicher Zusammensetzung und (Lipid-) Konzentration wur-
den hergestellt und sprühgetrocknet. Anschließend wurde ein Teil des Pulvers in Wasser
zur ursprünglichen Konzentration gelöst, um die Liposomen zu untersuchen. Die
Sprühtrocknung erfolgte unter dem Einsatz verschiedener Düsentypen zur Zerstäubung.
Zwei verschiedene Ultraschalldüsen und eine Zweistoffdüse wurden verwendet und deren
Einfluss auf Liposomen und Pulver wurde untersucht. Alle anderen Einstellungen am
Sprühtrockner blieben unverändert.
Das positiv geladene Lipid DOTAP sowie das neutrale Helferlipid DOPC wurden im mo-
laren Verhältnis 1:1 zur Herstellung der Liposomen verwendet. Die Formulierung enthielt
außerdem Trehalose (100 mg/ml). Liposomendispersionen mit Lipid zu Zucker Verhältnis-
sen von 1:146 (2 mM Lipid) bis 1:5 (60 mM Lipid) wurden mit Hilfe der Rundkolbenme-
thode und mittels Extrusion durch eine 0,1 µm-Membran hergestellt und sprühgetrocknet.
Ein Teil des sprühgetrockneten Pulvers wurde für die nachfolgende Analytik wiederaufge-
löst. Mittels dynamischer Lichtstreuung konnten dabei keine größeren Veränderungen bei
sprühgetrockneten Liposomen ausgemacht werden, was dafür sprach, dass die Formulie-
rung grundsätzlich gut zur Sprühtrocknung geeignet war. Allerdings war die Herstellung
der Proben mit dem höchsten eingesetzten Lipidgehalt (60 mM) nicht mehr problemlos
möglich, da die hohe Viskosität der Dispersion die Extrusion behinderte. Nach der
Sprühtrocknung war neben den DLS-Werten Größe, Verteilung und Zetapotenzial auch die
Phasenübergangstemperatur der Lipidmischung sowie deren Wiederfindung unverändert.
Zusätzlich zur Phasenübergangtemperatur der voll hydratisierten Liposomen vor und nach
172 ZUSAMMENFASSUNG
dem Sprühtrocknungsprozess wurde auch die Phasenübergangstemperatur der Liposomen
im getrockneten, in Trehalose eingebetteten Zustand bestimmt. Die DSC-Messungen zeig-
ten hier einen Abfall des Phasenübergangs auf unter -80 °C.
Leicht variierende Liposomengrößen konnten nach der Verwendung unterschiedlicher Dü-
sentypen festgestellt werden. Beide Ultraschalldüsen (25 kHz und 60 kHz) führten zu einer
leichten Verkleinerung der Vesikel nach Trocknung und Wiederauflösen des Pulvers, wäh-
rend gleichbleibend konstante Größen nach dem Zerstäuben mit Hilfe der Zweistoffdüse
gemessen wurden. Die Größenreduktion durch die Ultraschalldüsen betrug 9 – 13 %, wobei
der Effekt bei der Verwendung der 25 kHz Düse ausgeprägter war. Alle in der Arbeit un-
tersuchten Formulierungen, also PEGylierte Liposomen, Liposomen mit verkapseltem In-
sulin oder Calcein und Formulierungen, in denen Trehalose gegen andere Zucker ausge-
tauscht wurde, zeigten dieses Verhalten. Die einzige Voraussetzung für das Auftreten der
leicht kleineren Liposomen nach der Trocknung war eine relativ niedrige Lipidkonzentra-
tion. Die Grenze lag hier bei 20 mM. Liposomendispersionen, die höher konzentriert wa-
ren, zeigten – genau wie alle Proben, die mittels der Zweistoffdüse zerstäubt wurden – nicht
dieses Verhalten. Die Größenreduktion konnte auf den Zerstäubungsprozess zurückgeführt
werden, da Liposomendispersionen, die ohne Hitzeeinwirkung unmittelbar nach der Ultra-
schalldüse aufgefangen wurden, bereits eine Größenreduktion erfahren hatten. Ultraschall-
vibrationen und Kavitationseffekte wurden als Ursache angenommen.
Der Einsatz verschiedener Hilfsstoffe zur Sprühtrocknung erlaubt folgende Rückschlüsse:
Durch den Austausch von Trehalose durch Saccharose oder Lactose konnten Liposomen
ebenso zufriedenstellend stabilisiert werden, während Mannitol und Maltodextrin nicht für
eine Stabilisierung von Liposomen während der Trocknung geeignet erschienen. Nach der
Sprühtrocknung war Maltodextrin genau wie Trehalose, Lactose und Saccharose amorph,
konnte jedoch auf Grund ungenügender Wasserstoffbrückenbindungseigenschaften die Li-
posomen im getrockneten Zustand nicht stabilisieren. Bei Betrachtung aller Produkteigen-
schaften bleibt jedoch Trehalose der Hilfsstoff der Wahl, da Saccharose trotz einer niedri-
gen Restfeuchte sehr niedrige Glasübergangstemperaturen aufwies und Lactose ein redu-
zierender Zucker ist, was die Formulierung allgemein erschwert.
Die Stabilität von sprühgetrockneten Liposomen war stark vom physikalischen Zustand der
umgebenden Trehalose abhängig, was besonders bei einer auf sechs Monate angelegten
ZUSAMMENFASSUNG 173
Stabilitätsuntersuchung deutlich wurde. Solange Trehalose amorph war, waren auch die
Liposomen stabil, was durch DLS Messungen bestätigt wurde. Nach sechs Monaten Lage-
rung waren allerdings einige Proben kristallin und die Zetasizer-Messungen der entspre-
chenden Proben zeigten deutlich eine Fusion und/oder Aggregation der Liposomen. Eine
amorphe Matrix ist folglich eine Grundvoraussetzung für die Stabilität von Liposomen im
getrockneten Zustand. Die fehlende Stabilität der Liposomen bei Verwendung des kristal-
linen Hilfsstoffs Mannitol unterstützt diese Annahme.
Die Zugabe von Polysorbat 80, einem Tensid, das häufig zur Stabilisierung von Proteinen
bei der Sprühtrocknung eingesetzt wird (Adler and Lee, 1999), hatte eine Anheftung der
Polysorbat 80-Moleküle an die Liposomenmembran zur Folge. Mit Hilfe eines Blasen-
drucktensiometers wurde gezeigt, dass eine gewisse Menge an Tensid an die Liposomen-
oberfläche gebunden war, während der Überschuss weiterhin die Grenzfläche zwischen
Flüssigkeit und Luft besetzte. Die durchschnittliche Partikelgröße des sprühgetrockneten
Pulvers war geringfügig kleiner im Vergleich zu einer Polysorbat 80 freien Formulierung,
was auf eine erniedrigte Oberflächenspannung während der Zerstäubung zurückgeführt
wurde. Zudem wurden niedrigere Restfeuchten gemessen. Was die Liposomengröße an-
geht, konnte eine Verkleinerung bei Anwesenheit von Polysorbat 80 gemessen werden.
Durch die sterische Hinderung von Polysorbat 80-Molekülen an der Liposomenoberfläche
wird die Krümmung der Membran vergrößert, was wiederum einen Rückgang der Vesikel-
größe nach sich zieht. Auf gleiche Weise wirkt sich die Zugabe von PEGylierten Lipiden
zur Standardformulierung aus DOTAP und DOPC aus. Der eigentlich zu erwartende Grö-
ßengewinn durch die 5 nm langen PEG-Ketten wurde durch die verstärkte Krümmung auf
Grund sterischer Hinderung der PEG-Ketten egalisiert und die Liposomengröße blieb un-
verändert. PEGylierte Liposomen waren nach der Sprühtrocknung und dem Wiederauflö-
sen des Pulvers unverändert stabil.
Mit Hilfe des Fluoreszenzfarbstoffes Calcein wurde die Verkapselungseffizienz und die
Membranintegrität während der (Sprüh-) Trocknung untersucht. 10 % des Farbstoffs be-
fanden sich nach der Herstellung einer 10 mM Liposomendispersion im Inneren der Vesi-
kel. Bei der doppelten Menge an Lipid pro Volumeneinheit erhöhte sich die Verkapse-
lungseffizienz auf 22 %, was auf eine ungefähre Verdoppelung der Anzahl an Liposomen
rückschließen ließ. Allerdings musste für diese Versuche der Anteil an positivem Lipid von
50 % auf 5 % reduziert werden, da eine Bindung des negativen Calceins an das positive
174 ZUSAMMENFASSUNG
DOTAP erfolgte, was die Messungen störte. Die Verkapselungseffizienz blieb nach dem
Versprühen mit verschiedenen Düsen, der Sprühtrocknung oder der Gefriertrocknung un-
verändert. Es wurde angenommen, dass weder die Liposomen noch Calcein durch diese
Prozesse beeinträchtigt wurden, was gut mit den anderen Ergebnissen übereinstimmte. Um
herauszufinden, ob während dieser Prozesse ein Austausch zwischen verkapseltem und
nicht verkapseltem Calcein stattfindet, wurde die Membranintegrität untersucht. 20 % des
initial verkapselten Calceins kamen während des Zerstäubens in Kontakt mit dem Außen-
medium, während der komplette Sprühtrocknungsprozess incl. Rekonstitution einen Ver-
lust von 80 % zur Folge hatte, was auf eine geringe Membranintegrität während der Trock-
nung rückschließen ließ. Nachfolgende Versuche zeigten, dass der gesteigerte Austausch
zwischen verkapseltem und nicht verkapseltem Material während der Sprühtrocknung auf
die Hitzeeinwirkung zurückzuführen war. Die Gefriertrocknung derselben Formulierung
provozierte einen Austausch von fast 100 %, was allerdings auf andere Einflussfaktoren
zurückzuführen war.
Da eine Anwendung von liposomal verkapselten Proteinen denkbar und vielversprechend
ist, wurde ein Testprotein – Insulin – in die Standardliposomen verkapselt. Allerdings zeig-
ten SEC-Messungen, dass sich bereits bei der Rehydrierung des Lipidfilms im Rundkolben
Insulinaggregate bildeten, die auf eine Wechselwirkung zwischen positiv geladenem Lipid
und Insulin zurückgeführt werden konnten. Die prozentuale Menge an aggregiertem Insulin
war abhängig von der Lipidkonzentration und veränderte sich nicht durch das Sprühtrock-
nen der Formulierung. Verkapselte man Insulin in neutrale Liposomen aus reinem DOPC,
betrug die Verkapselungseffizienz 70 %. Der Anteil an Aggregaten stieg von 1 % in der
Ausgangsdispersion vor dem Sprühtrocknen auf ca. 2 % in der rekonstituierten Liposomen-
dispersion nach der Sprühtrocknung. Im Vergleich zu den DOTAP-haltigen Formulierun-
gen, die 10 – 15 % Verunreinigung in Form von Aggregaten enthielten, zeigten die Versu-
che mit neutralen Liposomen deutlich, dass die Sprühtrocknung von liposomal verkapsel-
tem Insulin grundsätzlich möglich ist. Wie anhand der verwendeten Substanzen Calcein
und Insulin zu sehen ist, bleibt die Formulierung von hydrophilen Substanzen schwierig
und anspruchsvoll. Zum einen muss eine möglichst komplette Verkapselung der Moleküle
in den Liposomen erfolgen oder freie, nicht verkapselte Moleküle müssen aufwändig aus
der Dispersion entfernt werden. Zum anderen dürfen die verkapselten Moleküle durch die
Sprühtrocknung oder Lagerung nicht wieder aus den Liposomen entweichen. Hierfür muss
ZUSAMMENFASSUNG 175
eventuell eine Lipidzusammensetzung mit einer höheren Phasenübergangstemperatur oder
die Zugabe von Cholesterol in Betracht gezogen werden.
Die Pulvereigenschaften der sprühgetrockneten Produkte verbesserten sich teilweise deut-
lich und unabhängig von der verwendeten Düse mit steigenden Lipidgehalt. Die Ausbeute
nahm zu, was zum Teil durch einen sinkenden Feinanteil im Pulver und durch eine glatter
werdende Partikeloberfläche bedingt war. Die schwerwiegendste Verbesserung war jedoch
die geringere Restfeuchte im Produkt und die damit verbundene höhere Glasübergangtem-
peratur. Beispielsweise nahm die Restfeuchte von Pulvern, die mittels der 25 kHz Ultra-
schalldüse hergestellt wurden, von fast 6 % (0 mM Lipid) auf 2 % (60 mM Lipid) ab. Auch
die Messungen am Levitator, mit deren Hilfe das Trocknungsverhalten eines Tropfens „in
Zeitlupe“ mitverfolgt werden kann, zeigten verbesserte Trocknungskoeffizienten und da-
mit eine effizientere Trocknung im ersten Trocknungsabschnitt mit erhöhtem Lipidgehalt.
Mit den höchsten untersuchten Lipidkonzentrationen konnte keine weitere Verbesserung
erzielt werden, was durch die mögliche Bildung einer lipophilen Barriere an der Tropfen-
oberfläche erklärt werden könnte. Hansen et al. (2004) führte das verbesserte Trocknungs-
verhalten mit steigendem Anteil an lipophilen Substanzen auf eine immer begrenztere
Menge an Feststoff zurück, die dazu in der Lage ist, Wasser zu binden. Die Glasübergangs-
temperatur der sprühgetrockneten Pulver stieg von ca. 45 °C auf 75 °C, was mit Blick auf
die Stabilität der eingebetteten Liposomen sehr vorteilhaft ist. Sun et al. (1996) zeigten zum
Beispiel, dass Liposomen in einer Matrix aus Zucker nur unterhalb der Glasübergangstem-
peratur stabil sind. Die in dieser Arbeit hergestellten Pulver wiesen ab einem Lipidgehalt
≥ 30 mM, was einem Lipid/Zucker Verhältnis von ≥ 1:10 entspricht, einen zweiten Glas-
übergang bei ungefähr 50 °C auf. Es wurde vermutet, dass dieser Übergang von einer lip-
idreichen Phase vorzugsweise innerhalb der Liposomen herrührt, die nicht ausreichend ge-
trocknet werden konnte. Alle sprühgetrockneten Pulver, die Trehalose als Hilfsstoff ent-
hielten, waren nach der Entnahme aus dem Auffanggefäße amorph. Jedoch kristallisierten
Proben mit einer hohen Restfeuchte (> 3,5 %) während der Lagerung bei erhöhten Tempe-
raturen oder während des Aufheizens bei einer DSC Messung teilweise aus. Auf gleiche
Weise konnte im Rahmen der Stabilitätsuntersuchung bei Pulvern, die bei 25 °C oder
40 °C gelagert wurden, nach 6 Monaten Kristallisation festgestellt werden. Hier zeigte der
Vergleich der XRD Graphen jedoch, dass nur Proben, die bei 25 °C gelagert wurden, zum
176 ZUSAMMENFASSUNG
Trehalose Dihydrat kristallisiert waren, während Proben bei 40 °C kristalline wasserfreie
Trehalose bildeten.
Der Einfluss verschiedener Düsentypen auf die Pulvereigenschaften kann folgendermaßen
zusammengefasst werden: Beide Ultraschalldüsen produzieren sehr homogene Pulver mit
einer engen Größenverteilung, während die verwendete Zweistoffdüse Pulver mit einem
kleineren Partikeldurchmesser und einer breiteren Größenverteilung generierte. Diese klei-
neren Partikel hatten trotz ihrer geringen Größe eine verhältnismäßig hohe Restfeuchte, die
teilweise so hoch war wie in Pulvern, die mit der 25kHz Ultraschalldüse versprüht wurden.
Betrachtet man alle Pulvereigenschaften erscheint die Kombination einer 25kHz Ultra-
schalldüse mit einer reduzierten Fördergeschwindigkeit oder der Einsatz der 60kHz Ultra-
schalldüse, die eine mittlere Partikelgröße generierte, als sinnvoll. Allerdings haben die
Stabilitätsuntersuchungen auch gezeigt, dass sich Glasübergangstemperaturen, die sich an-
fangs durch den Einsatz der verschiedenen Düsen deutlich unterschieden haben, über den
untersuchten Zeitraum angenähert haben.
Die vorliegende Arbeit untersucht den grundsätzlichen Einfluss von Liposomen auf sprüh-
getrocknete Pulver. Die dabei gefundenen Zusammenhänge können die Formulierung von
Liposomen und darin verkapselten Arzneistoffen vereinfachen, speziell wenn eine Stabili-
sierung durch Sprühtrocknung angestrebt ist. Steigende Lipidkonzentrationen konnten da-
bei die Restfeuchte senken, den Glasübergang erhöhen, die Ausbeute verbessern und den
Feinanteil im Pulver verringern. Verschiedene Düsentypen wurden vorgestellt und deren
Einfluss auf Pulver und Liposomen wurde gezeigt. Ultraschalldüsen verursachten dabei
eine geringfügige Verkleinerung der Liposomengröße. Es wurde außerdem festgestellt,
dass eine amorphe Matrix, die die Liposomen einbettet, überaus wichtig für die Stabilität
der Liposomen ist. Eine Kristallisation muss daher vermieden werden. Aus diesem Grund
sollte die Restfeuchte im Produkt so gering wie möglich sein.
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9 CURRICULUM VITAE
Personal Data
Name Julia Staudenecker
Date of Birth December, 7th 1986 in Aalen, Germany
Education
December 2011 – present Friedrich-Alexander-University Erlangen, Germany
Ph.D. studies in Pharmaceutics under the supervision of
Dr. S. Seyferth and Prof. Dr. G. Lee
November 2011 Passed Licensing Exam as German Pharmacist
(3rd state examination)
May 2011 – October 2011 Hirsch Apotheke Ulm, Germany
Internship at community pharmacy
November 2010 – April 2011 Apotheke des Universitätsklinikums Ulm, Germany
Internship at hospital pharmacy
October 2006 – October 2010 Friedrich-Alexander-University Erlangen, Germany
Studies of Pharmacy
(incl. 1st and 2nd state examination)
June 2006 Abitur, Theodor-Heuss-Gymnasium Aalen, Germany
Further Education
2012 – 2015 Advanced training “Fachapotheker für Pharmazeutische
Technologie”
