Colloids and Surfaces B: Biointerfaces 59 (2007) 184–193
Effects of poly (ethylene glycol) chains conformational transition on theproperties of mixed DMPC/DMPE-PEG thin liquid films and monolayers
Georgi As. Georgiev a, Dipak K. Sarker b,∗, Othman Al-Hanbali b,Georgi D. Georgiev a, Zdravko Lalchev a
a University of Sofia “St. Kliment Ohridski”, Faculty of Biology, Department of Biochemistry, 8 Dragan Tsankov Str., 1164 Sofia, Bulgariab Molecular Mechanisms Group, School of Pharmacy, University of Brighton, Brighton BN2 4GJ, UK
Received 20 January 2007; received in revised form 6 May 2007; accepted 9 May 2007Available online 16 May 2007
bstract
Foam thin liquid films (TLF) and monolayers at the air–water interface formed by DMPC mixed with DMPE-bonded poly (ethylene glycol)sDMPE-PEG550, DMPE-PEG2000 and DMPE-PEG5000) were obtained. The influence of both (i) PEG chain size (evaluated in terms of Mw) andushroom-to-brush conformational transition and (ii) of the liposome/micelle ratio in the film-forming dispersions, on the interfacial properties
f mixed DMPC/DMPE-PEG films was compared.Foam film studies demonstrated that DMPE-PEG addition to foam TLFs caused (i) delayed kinetics of film thinning and black spot expansion
nd (ii) film stabilization. At the mushroom-to-brush transition, due to steric repulsion increased DMPE-PEG films thickness reached 25 nm whileure DMPC films were only 8 nm thick Newton black films. It was possible to differentiate DMPE-PEG2000/5000 from DMPE-PEG550 by the abilityo change foam TLF formation mechanism, which could be of great importance for “stealth” liposome design.
Monolayer studies showed improved formation kinetics and equilibrium surface tension decrease for DMPE-PEG monolayers compared withMPC pure films.SEM observations revealed “smoothing” and “sealing” of the defects in the solid-supported layer surface by DMPE-PEGs adsorption, which
ould explain DMPE-PEGs ability to stabilize TLFs and to decrease monolayer surface tension.All effects in monolayers, foam TLFs and solid-supported layers increased with the increase of PEG Mw and DMPE-PEG concentration.
owever, at the critical DMPE-PEG concentration (where mushroom-to-brush conformational transition occurred) maximal magnitude of theffects was reached, which only slightly changed at further DMPE-PEG content and micelle/liposome ratio increase. 2007 Elsevier B.V. All rights reserved.
Poly (ethylene glycol)s (PEGs) with different moleculareight are membrane active agents commonly used within
he modern pharmaceutical chemistry, medicine, moleculariology and biophysics [1–3]. Widely applied are poly (ethy-
ene glycol)s covalently bonded to phospholipids (so calledhospholipid-bonded PEGs) which possess both—high sur-ace activity (ensured by the phospholipid part anchoring the
∗ Corresponding author at: School of Pharmacy & Biomolecular Sciences, Theniversity of Brighton, Cockcroft Building, rm C705b, Moulsecoomb Scienceampus, Lewes Road, Brighton BN2 4GJ, UK. Tel.: +44 1273 64 2074;
spholipid black films; Phospholipid monolayers; “Stealth” liposomes
olecule to the membrane surface) and good water solubilityprovided by the hydrophilic PEG moiety).
PEG moieties of phospholipid (PL)-bonded PEGs, extendedn “mushroom” (at low PL-PEG surface concentrations) orbrush” (at high PL-PEG concentration) chain conformationrom the membrane plane towards the water solution, form thickydrophilic layer at the membrane surface [4–7]. It was responsi-le for the increased steric disjoining pressure between PL-PEGoated surfaces measured with surface force apparatus technique5]. Thus, the hydrophilic layer creates a steric barrier preventinghe adsorption of lipoproteins and opsonins to the liposome sur-
ace, making the liposomes invisible for the reticulo-endothelialystem (responsible for the uptake of foreign particles out ofhe blood flow). In this way so-called “stealth” liposomes arebtained. They possess increased stability and prolonged life
ime in the blood flow circulation (from hours to days), mak-ng them promising drug vehicles [4,7–9]. Stealth liposomesave been applied as vehicles of known anticancer and anti-ungal drugs (doxorubicin, alphameticin, etc.) and increasedharmacological effect and decreased toxicity of great clinicalnterest are observed [4,10]. It was also found that PL-bondedEGs, pure or mixed (at high molar content) with phospho-
ipids, form “stealth” micelles of decreased size. The “stealth”icelles (instead of liposomes) also can be used as drug vehicles
3,7,11–13]. Recently PL-bonded PEGs were used for obtainingf “stealth” erythrocytes as universal blood substituent [11].
One of the most commonly used phospholipids in mixedormulations with PL-PEGs is dimyristoyl phosphatidylcholineDMPC). DMPC is typical liposome forming phospholipididely applied in biophysics and biopharmaceutics [4,7] exist-
ng in liquid-crystalline phase state at physiological temperature14]. Liposomes composed of phosphatidylcholines and PE-EGs have been used for a decade or more as in vivo drug carriers15–18].
Two model membrane systems preferred for studying theffect of membrane active compounds are phospholipid foamhin liquid films (foam TLFs) and monolayers at the air–waternterface. PL Foam TLFs (foam films) are composed ofwo plane-parallel adsorbed “head-to-head” PL monolayersFig. 1). Thus, PL foam films are structurally analogous to theis-monolayers apposition occurring at close intermembranedhesion and at the onset of membrane fusion [19,20]. Phos-holipid foam TLFs were successfully used as a model systemn biology and medicine [21] to investigate membrane inter-ctions, adhesion and fusion processes, bio-surfactant action atnterfaces, etc. Up to this date there are limited number of studiesf foam films formed by dimyristoyl phosphatidylethanolamineDMPE)-bonded PEGs. Nikolova and Jones [22,23] measuredlm thickness, and gas permeability and Georgiev et al. [24]eported data for film kinetic characteristics and formation prob-bility. Still in these works the relation of PEG conformationmushroom or brush) to foam TLF hydrodynamic behavior andtability was not quantitatively studied. PL monolayers (Fig. 1D)an be regarded as a half of foam TLFs or black lipid membranes21,25] and are common model for studying the interactions
etween phospholipids in the membrane plane.
The aim of the current work was to study the effects of PEGhain size (evaluated in terms of molecular weight, Mw) andushroom-to-brush conformational transition on foam TLFs
mfuh
ig. 1. Schematic representation of TLFs and monolayers at the air/water interfaceewton black film (C). Thick TLF with black spot (A) is also presented. (D) shows p
B: Biointerfaces 59 (2007) 184–193 185
nd monolayers at the air–water interface. Foam films andonolayers were formed by pure DMPC, pure DMPE-bondedEGs (DMPE-PEG550, DMPE-PEG2000 and DMPE-PEG5000)nd their mixtures. The thickness, stability and kinetic behaviorf pure DMPC films were compared with those of DMPE-PEG-ontaining foam TLFs, with different conformation of PEGoiety—mushroom or brush. The conformational transition was
etermined by foam film thickness data, and zeta-potential mea-urements of mixed DMPC/DMPE-PEG liposomes/micellesccording the methodology developed in previous studies9,21,22]. Surface tension measurements of pure DMPC mono-ayers and PEG-containing monolayers were performed. FoamLFs and monolayers formed by pure DMPE-bonded PEGsere also obtained and their properties were compared with
hose of pure DMPC and DMPC/DMPE-PEG mixed films. Inddition to foam film thickness data (giving information abouthe structure of the film core) the morphology of DMPC/DMPE-EG solid-supported layers was registered with scanninglectron microscopy (SEM), for estimation of the mushroom-o-brush transition effect on membrane surface structure. Thelm-forming dispersions were composed, depending on DMPE-EG molar content, of two types of aggregates—liposomes andicelles [12,13]. The ratio of liposome-to-micelles was evalu-
ted by the protocol of Montessano et al. [12] and Belsito etl. [13] and by photon corelation spectroscopy. Thus, the otherurpose of our study was to compare the influence of both PEGhain size and mushroom-to-brush conformational transition tohe effect of liposome/micelle ratio in the dispersions, on theroperties of mixed DMPC/DMPE-PEG films studied.
. Materials and methods
.1. Materials
DMPC, DMPE-bonded PEGs – DMPE-PEG550, DMPE-EG2000 and DMPE-PEG5000 (subscripts denote PEG moietyw) were purchased from “Avanti Polar Lipids”. NaCl was
urchased from “Merck”. Solutions were made with bi-distilledater with conductivity less than 1.10−6 S/cm and surface ten-
ion higher than 72 mN/m. DMPC and DMPE-PEGs (pure or
ixed in desired molar ratio) were initially dissolved in chloro-
orm which was subsequently evaporated under nitrogen streamntil formation of dry film. The dispersions were obtained byydration of the dry film followed by sonication as described in
used in our study. Two types of TLFs are shown: Common black film (B) andhospholipid monolayer, which can be regarded as a half of bilayer film.
1 Surfa
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4,7,12]. All dispersions were formed and used at T = 37 ◦C, pH.8–7.0 and electrolyte concentration Cel = 0.5 M NaCl.
.2. Characterization of the aggregate types in bulkispersions and zeta (�)-potential measurements
Experiments for qualitative determination of the lipo-omes/micelles ratio dependence on PE-PEG molar contentere done using the protocol of Montesano et al. [12] and Belsito
t al. [13] by measuring with SPEKOL-11 the optical densityt 400 nm (OD400) of mixed DMPC/DMPE-PEG dispersions.ccording to the above authors, the initial rapid drop in OD400
PE-bonded PEG mol%) dependence corresponds to a decreasen the lipid aggregates overall size (attributed to disaggregationf the liposomes) and when OD400 becomes very low, the closedilayer vesicles of reduced size convert to micelles.
The process of liposome-to-micelle transformation (quanti-atively evaluated by recording the aggregates hydrodynamicadius) and zeta (�)-potential values were measured with photonorrelation spectroscopy (PCS) and laser Doppler electrophore-is, respectively, using a Zetasizer 3000 system (Malvernnstruments, UK) as described previously [26]. Fourteen PCSeasurements (based on CONTIN analysis) were determined
t a wavelength of 633 nm, scattering angle of 90◦, dispersantiscosity of 0.89 cP and refractive index of 1.35, respectively.or electrophoretic mobility measurements the conditions weres follows: dielectric constant 78.3, current 1.4 mA, mediumefractive index of 1.35, cell field of 29.3 V/cm, viscosity of.89 cP and conductivity of 0.7 mS/cm. All hydrodynamic radiusnd �-potential values presented are averaged from at least fiveeasurements.
.3. Foam thin liquid films (foam TLFs)
Foam TLFs were formed by the method of Exerowa andrugliakov [20] using the modified measuring cell of Lalchev et
l. [21]. A biconcave drop (50 �l volume) of the dispersion waslaced into the cylinder of the measuring cell for 30 min. Afterucking the solution from the drop thick foam TLF was formedFig. 1). Further the film spontaneously thinned and after someharacteristic film thinning time, t0−1 (s), critical thickness waseached (ca. 300 A). Then a black spot (BS), local thinning inhe film, appeared (Fig. 1A) and expanded to fill up the wholerea of the film. The kinetic of this process was measured by BSxpansion time t1−2 (s)—the time from the first BS formationo the moment of its expansion to the whole film area, i.e. tolack film formation. Under different experimental conditionswo types of stable black films is possible to be formed – com-
on black films, CBFs (Fig. 1B) and Newton black films, NBFsFig. 1C).
The film formation was observed with inverted light micro-cope and registered by Olimpus camera (C7070 Wide Zoom).he equivalent water thickness (hw) of foam films was calcu-
ated (with resolution of 0.5 nm) from the data for the intensityf the light reflected from the film measured by the interfero-etric technique as described by [15,27]. The mean value of the
hickness was determined from the data for 5–10 films.
Drsl
ces B: Biointerfaces 59 (2007) 184–193
The probability (W) for formation of stable BFs dependstrongly on surfactant (DMPC and DMPE-PEG) concentration,
[15,18,28], and can be calculated by the equation W = �N/N,here N is the total number of trials (at least 50 for each con-
entration) and �N is the number of trials in which stablelack films are formed (i.e. W varies between 0 and 1). Theependence W(C) is extremely steep which allowed to define ahreshold concentration (Ct)—the minimum surfactant concen-ration at which W = 1 (stable films are always formed) [28]. Its proven that W(C) dependence is sensitive to the compositionf the film-forming dispersion, molecular shape and phase statef the film-forming PLs, pH, electrolyte concentration, appliedressure, etc. [21,29].
.4. Monolayers
Monolayers at the air–water interface (spread and adsorbed)f pure DMPC and of DMPC mixed with DMPE-PEGs wereormed in Langmuir teflon through and the surface tension
(mN/m) was measured by the method of Wilhelmy withccuracy ± 0.5 mN/m, as previously described [29]. Automaticilhelmy tensiometer (Biegler Electronic, Austria) with plat-
num float of size 1 × 1.6 cm was used. Two types Langmuireflon troughs were utilized—with 7 ml subphase volume andrea 706 mm2 (for adsorption monolayers) and with 25 ml sub-hase volume and area 1710 mm2 (for spread monolayers). Fordsorption monolayer studies a Teflon-coated stirring bar dippedn the water subphase was used. Spread monolayers at 200 A2
rea per molecule (approx. 5.10+11 lipid molecules/mm2) wereormed from DMPC/DMPE-PEG mixtures (dissolved in chloro-orm; surfactant concentration in the mixtures was 1000 �g/ml)ith desired molar ratio by careful deposition with Hamiltonicrosyringe of few microlitres sample on the air–water inter-
ace. One hour was given for chloroform evaporation prior tourface tension measurements. The γ (time) and γ(C) depen-ences were recorded. After the sample was applied (injectedn the water subphase for adsorption monolayers or depositedn the interface for spread monolayers) γ decreased to a plateaualue (for up to 40 min for adsorption and up to 10 s for spreadonolayers). It was regarded as equilibrium value only if γ
emained constant for at least 2 h after the plateau value was ini-ially reached. Each measurement was performed at least threeimes with every sample preparation.
.5. Scanning electron microscopy (SEM)
SEM experiments were performed for revealing the morphol-gy of adsorption layer surface. Samples were solid-supportedMPC/DMPE-PEG layers formed on smooth carbon support
Agar Scientic. Ltd., UK). They were located on 1.2 cm alu-inium pin stubs. The layers were formed by adsorption of
urfactant (DMPC and DMPE-PEGs) from dispersion. The sup-ort was covered in the film-forming dispersion (with desired
MPC/DMPE-PEG ratio) for at least 1 h for reaching equilib-
ium adsorption of the surfactant molecules on the hydrophobicurface. The support with the adsorbed surfactant layer was iso-ated and dried according the protocol of Hincha and coworkers
30,31]. The dried samples were then treated with palladium2 nm) and visualized at 5 keV using a Jeol 6310 scanning elec-ron microscope (Jeol, Welwyn Garden City, UK) as describedn [26].
. Results
.1. Evaluation of the ratio of liposomes and micelles in theulk dispersions
The ratio of both aggregate types (liposomes and/or micelles)n DMPC/DMPE-PEGs bulk dispersions was regulated byarying of DMPE-PEG molar content and was determinedxperimentally by measuring the optical density (OD400) of theispersions at 400 nm (Fig. 2 panel A) and by direct measure-ent of the aggregates diameter by PCS (Fig. 2 panel B).The upper region of the plot (Fig. 2A) curved part (at low
olar content) corresponded to dispersions consisting predom-nantly by liposomes [12,13]. With increasing of DMPE-PEGontent OD400 decreased due to the decreased size of the disper-ion forming aggregates transforming from bilayer liposomes toicelles. When the whole dispersion is composed by micelles
he plateau region of minimum OD400 is reached.The process of liposome-to-micelle conversion with increas-
ng of DMPE-PEG content was followed quantitatively by PCSeasurements (Fig. 2 panel B). The average diameter of the
ggregates in the dispersions studied decrease from 500 nmthe size of Large Unilamellar Vesicle) to ca. 100 nm (micel-
ig. 2. OD400 (CDMPE-PEG-n) (panel A) and particle diameter (CDMPE-PEG-n)panel B) dependences on DMPE-PEG mol% in mixed dispersionsMPC/DMPE-PEG. Data labels—DMPE-PEG550 (�), DMPE-PEG2000 (�),MPE-PEG5000 (�). Experiments were done at total lipid (DMPC + DMPE-EG) concentration C = 1000 �g/ml, T = 37 ◦C, Cel = 0.5 M NaCl, pH 6.8–7.0.
3
3t
modbT(P(
Fo(if(N
B: Biointerfaces 59 (2007) 184–193 187
ar size), and the shape of diameter (DMPE-PEG mol%) curvesas practically identical to the shape of OD400(DMPE-PEGol%). This result confirms the suitability of OD measurements
ccording the protocol of Montesano and Belsito, for study-ng the liposome-to-micelle conversion in mixed dispersions ofC with PE-bonded poly(ethylene glycol)s. The diameter of the
iposomes and mixed micelles is also in agreement with thealculations and measurements of Rovira-Bru et al. [32] con-erning mixed dispersions of PC and PE-linked PEGs with PEGize similar to the varieties used in the current study.
It can be seen that the plateau region was reached at lower con-entration for DMPE-PEG2000 (35 mol%) in comparison withMPE-PEG550 and DMPE-PEG5000 (73 mol%). Similar non-
inear dependence of the liposomes-to-micelles transformationn PEG moiety molecular weight was also observed for disper-ions of DPPE-PEGs with DPPC [12,13]. In the current workor most of the experiments with DMPE-PEG containing dis-ersions, three concentrations (CDMPE-PEG) of DMPE-bondedEGs were used—less than 10 mol% DMPE-PEG (where dis-ersions were formed predominantly by liposomes), 20 mol%where mixture of liposomes and micelles existed in the disper-ions) and 80 mol% DMPE-PEG (where DMPC/DMPE-PEGispersions consisted of micelles only).
.2. Foam thin liquid films
.2.1. Detection of the mushroom-to-brush conformationalransition
Two techniques were applied for detection of PEG moietyushroom-to-brush conformational transition—measurement
f foam film thickness and zeta (�)-potential measurements. Theependence of equivalent water thickness (hw) of equilibriumlack films on mol% DMPE-PEG550/2000/5000 is shown in Fig. 3.
he hw thickness of equilibrium DMPC Newtonian black films
8 nm) increased both: (i) with increasing the content of DMPE-EGs and (ii) with increase of PEG moiety molecular weightMw) (hw = 25 nm in presence of DMPE-PEG5000).
ig. 3. Dependence of the equivalent water thickness hw of DMPC black filmsn the DPPE-PEGn content. Data labels—DMPE-PEG550 (�), DMPE-PEG2000
�), DMPE-PEG5000 (�). With eclipse are closed the labels correspond-ng to Ccrit
DMPE−PEG (above which PEG moieties were already transformedrom mushroom-to-brush conformation). Experiments were done at total lipidDMPC + DMPE-PEG) concentration C = 1000 �g/ml, T = 37 ◦C, Cel = 0.5 MaCl, pH 6.8–7.0.
1 Surfaces B: Biointerfaces 59 (2007) 184–193
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Fig. 4. Effect of DMPE-PEG-n on zeta-potential of DMPC large unilamellarvesicles (the starting zeta-potential of the pure DMPC liposomes is −10 mV).Data labels:(�) DMPE-PEG-550; (�) DMPE-PEG-2000; and (�) DMPE-PEG-5 crit
wf
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Film thickness increased gradually with DMPE-bondedEG content until some critical DMPE-PEG concentrationCcrit
DMPE-PEG) is reached where a discontinous jump-like increasef hw was observed, corresponding to PEG conformationalransition from mushroom-to-brush in line with the postu-ates of Nikolova and Jones [23]. Fig. 3 shows that the valuef Ccrit
DMPE-PEG decreased with increasing Mw of PEG moiety9 mol% for DMPE-PEG550, 7 mol% for DMPE-PEG2000 andnly 3 mol% for DMPE-PEG5000) due to the enhanced overlap-ing of poly (ethylene glycol) chains in the film core when theirize is raised [5,6].
The jump-like hw increase at CcritDMPE-PEG was associated
lso with transformation in black film formation mechanismn presence of the longer chain DMPE-PEG2000/5000: from triv-al mechanism of black spot (BS) formation [20] to formationy continuous thinning without BS formation until equilibriumlack, we called, CBF-like films were obtained (see Table 1).hat behaviour can be explained in terms of a strong steric
epulsion disjoining pressure arising between foam TLF sur-aces due to the overlapping of hydrophilic polymer “brushes”n the film liquid core. Such effects were observed for PL linkedEG2000 [5], for DPPE-PEG2000 and DMPC/DPPE-PEG2000ixture [23], three-block co-polymer surfactants [20], etc.The critical concentration of DMPE-PEG at which PEG con-
ormational transition occured was also confirmed by �-potentialeasurements of DMPC/DMPE-PEG dispersions (Fig. 4).The absolute value of �-potential steeply decreased with
MPE-PEG concentration increase from −8 mV to a plateaualue of −2 mV at Ccrit
DMPE-PEG. Our result agrees with theeasurements for PEG-coated liposomes performed by other
uthors [2,33,34] and with the data of Al-Hanbali et al. [26] forolyethylene oxide grafted polystyrene nanoparticles. The alter-tion of �-potential value could be explained with the increase ofhe lateral density of PEG moieties in the membrane plane when
ushroom-to-brush transition occurs, which results in spatialscreening” of the membrane charges, by the poly (ethylene gly-ol) chains lacking of dissociable groups (i.e. of charge carriers)2,33,34].
DDDP
able 1ype, Ct and formation mechanism of black films stabilized by DMPC and DMPC/D
xperiments were done at T = 37 ◦C, Cel = 0.5 M NaCl, pH 6.8–7.0, df = 100 �m. (*ushroom-to-brush conformational transition occurred and foam TLFs transformed
000. With eclipse are closed the labels corresponding to CDMPE−PEG (abovehich PEG moieties were already transformed from mushroom-to-brush con-
ormation). Experiments were done at T = 25 ◦C, Cel = 0.5 M NaCl, pH 6.8–7.0.
.2.2. Kinetic behavior of DMPC/DMPE-PEG foam TLFsThe kinetic behavior of mixed DMPC/DMPE-PEG films
trongly depended on the mol% of DMPE-bonded PEG. Theesults concerning film thinning time (t0−1) and black spotxpansion time (t1−2) of DMPC/DMPE-PEG550 films are shownn Fig. 5A.
It can be seen that t0−1 increased linearly from 30 s (for pureMPC) to 40 s (above 42 mol% DMPE-PEG550 plateau valuef t0−1 was observed). At 9 mol% DMPE-PEG550, the mini-um at which the films became to thin to CBFs (not to NBFs
s for DMPC), t0−1 = 32 s. The value of BS expansion time,1−2 steeply increased from 1 s (for NBFs of pure DMPC) tolateau value of 6 s (at ≥30 mol% DMPE-PEG550) for the mixedlms. At 9 mol% DMPE-PEG550 where the films (and the spots,espectively) became common black t1−2 value was 4 s.
Panels B and C of Fig. 5 represent the dependences of
MPC film thinning time and black spot expansion time onMPE-PEG2000/5000 mol%. For concentrations up to 7 mol%MPE-PEG2000 and up to approximately 3 mol% DMPE-EG5000 typical CBFs were observed with linearly increasing
MPE-PEG mixtures with different PEG chain molecular weight
Ct (mol/l) Black film formation mechanism
2.9 × 10−4 With formation of BS
9.6 × 10−5 With formation of BS2 × 10−4 With formation of BS1.2 × 10−4 With formation of BS9.8 × 10−5 With formation of BS
2.5 × 10−5 Continuous thinning without formation of BS7 × 10−5 Continuous thinning without formation of BS3.2 × 10−5 Continuous thinning without formation of BS2.7 × 10−5 Continuous thinning without formation of BS
4 × 10−6 Continuous thinning without formation of BS1 × 10−5 Continuous thinning without formation of BS7 × 10−6 Continuous thinning without formation of BS5 × 10−6 Continuous thinning without formation of BS
) Denotes the critical DMPE-PEG concentration (CcritDMPE−PEG) at which PEG
dPoatmmand Krugliakov [20]). For revealing the changes occurring in
LFs. Experiments were done at T = 37 ◦C, Cel = 0.5 M NaCl, pH 6.8–7.0, filmiameter df = 200 �m.
0−1 values to ca. 37 s and t1−2 values to around 9 s. Abovemol% DMPE-PEG2000 and 3 mol% DMPE-PEG5000 (after theushroom-to-brush PEG moiety conformational transition) we
bserved that the films started to thin continuously to CBF-likelms without formation of black spot regions (Fig. 5B). In thisase, we recorded the CBF-like formation time (t0−2) on theol% of DMPE-PEGs being increased from 45 s to a plateau
alue of 75 s (at 80 mol% DMPE-PEG2000) and 85 s (at 75 mol%MPE-PEG5000), respectively (Fig. 5C), which was equal to thelm formation times from individual DMPE-PEGs.
Our data show that the kinetics of film thinning and BSxpansion of DMPC/DMPE-PEG foam TLFs was prolongedn a continuous fashion with increasing CDMPE-PEG. This obser-ation correlates with the data for the viscosity increase of filmiquid core due to overlapping of PEG chains [5,6,18]. Despite
he changes occurring in the type and formation mechanism oflack Films at mushroom-to-brush PEG conformational tran-
ition there was no significant change in the slope of kinetic
Dll
s NBF, Fig. 1C); (�) DMPE-PEG550 (the film is CBF, Fig. 1B); (♦) DMPE-EG2000 (the film is CBF-like) and (�) DMPE-PEG5000 (the film is CBF-like).xperiments were done at T = 37 ◦C, Cel = 0.5 M NaCl, pH 6.8–7.0 with filmiameter df = 200 �m.
arameter (t0−1, t1−2 or t0−2) dependences at CcritDMPE−PEG or
ith varying liposome/micelle ratio.
.2.3. Stability of DMPC/DMPE-PEG black filmsThe stability of DMPC/DMPE-PEG films was measured in
erms of probability for stable black film formation (W) on sur-actant concentration (C). W(C) curves of BFs of pure DMPCnd pure DMPE-PEG dispersions are shown in Fig. 6.
The threshold concentrations, Ct, and the formation mecha-isms of films from DMPC/DMPE-PEGs mixed dispersions areisted in Table 1.
It can be clearly seen in Fig. 6 that all DMPE-PEGs examinedormed stable black films at threshold concentration values lowerhan the threshold concentration for DMPC films (2.9 × 10−4 MMPC). For DMPE-PEGs films the value of Ct decreasedith increasing of PEG moiety length and molecular weight
nd the lowest value of Ct was obtained for DMPE-PEG50004 × 10−6 M).
As it was already noted at constant electrolyte concentra-ion (Cel = 0.5 M NaCl) the threshold concentration values andhe formation mechanisms (Table 1) of mixed DMPC/DMPE-EG films depend both on DMPE-PEG content and on the PEGoiety conformation and molecular weight.The Ct of mixed films was lower than that of pure DMPC
lms and higher in comparison with pure DMPE-PEG films.nalogously to DMPE-PEG black films, the lowest Ct value
0.5 × 10−5 M) was obtained for the longest chain PL-bondedEG—for 80 mol% DMPE-PEG5000.
The increased stability of foam TLFs (measured by theecrease of Ct value) suggests that the presence of DMPE-EGs in foam films is responsible not only for the appearancef steric repulsion pressure (resulting in BF thickening) butlso could alter the molecular packing density and the struc-ure of the adsorption layers at the thin film surfaces (since
olecular packing and structure of the adsorption layers are theain determinants of black film stability according Exerowa
MPC/DMPE-PEG adsorption films experiments with mono-ayer surface balance and SEM of solid-supported adsorptionayers were performed.
mstof DMPE-PEGs as in monolayer studies—less than 10 mol%
90 G.As. Georgiev et al. / Colloids and
.2.4. Monolayers of DMPC mixed with DMPE-PEGsThe monolayers examined, adsorbed and spread, were
rom DMPC, DMPE-PEG and mixtures of DMPC/DMPE-EG. DMPE-PEG content in mixed monolayers was less than0 mol% (at Ccrit
DMPE−PEG where mushroom-to-brush confor-ational transition occurs and dispersion is composed mainly
y liposomes), 20 and 80 mol% DMPE-PEG (at which mixediposome/micelles dispersions and dispersions of micelles only,ere formed in the samples, respectively, see Fig. 2). The effects
t 20 and 80 mol% DMPE-PEG on the monolayer equilibriumurface tension were practically identical. Thus, the results for0 mol% DMPE-PEG only are shown below. Equilibrium sur-ace tension was measured as it is well known that its decreases proportional to the increase of molecular packing density inhe membrane plane [35].
The γ(C) dependences of adsorbed and γ(t) dependencesf spread (200 A2 area per molecule) monolayers of DMPC,MPE-PEG and mixed DMPC/DMPE-PEG are represented atig. 7 (panels A and B, respectively).
The comparison of γ(C) dependences (Fig. 7A) showed thathe saturation (γ = 68.5 Mn/m) occurred at the lowest concentra-ion (10−8 M DMPC) for pure DMPC monolayers. The result isn agreement with the data for the low solubility and low critical
icelle concentration of DMPC [4], determining lower degree ofdsorption in comparison with DMPE-PEGs. We also observedncreased surface activity of DMPE-bonded PEGs compared tondividual DMPC and DMPE monolayers (data not shown). ForMPE-PEG monolayers the γ values decreased with increas-
ng PEG moiety Mw—the lowest γ (64 mN/m) was obtainedor DMPE-PEG5000 (at 10−5–10−6 M). As it can be seen thenclusion of DMPE-PEGs in the mixed DMPC/DMPE-PEG
onolayers decreased γ of the saturated DMPC monolayer withhe increase of DMPE-PEG content and PEG moiety Mw. At0 mol% DMPE-PEG the γ(C) dependences of pure DMPE-EG and of the mixed DMPC/DMPE-PEG monolayers werelmost identical.
Comparing γ(t) dependences of spread monolayers (Fig. 7B)t can be seen that DMPC monolayers reached equilibriumurface tension γeq = 58 mN/m after equilibration time (teq) ofs. The presence of DMPE-PEGs in the monolayers resultedoth in improving the kinetics of film spreading and decreas-ng of γeq value (being slight for DMPE-PEG550, and verytrong for DMPE-PEG2000/5000). Both effects increased withMPE-PEG content. At 20 mol% DMPE-PEG the spreadinginetics and �eq were practically the same as for pure DMPE-EG monolayers. It can be seen that at ≥20 mol% DMPE-PEG
he kinetics of spreading was the same for all DMPE-bondedEGs used (teq = 5 s), while γeq value depends on PEG moi-ty Mw and lowest γeq of 42 mN/m for the highest moleculareight DMPE-PEG5000 was reached. These results are in agree-ent with the data for the increase of the effective molecular
rea and lateral steric repulsion force between PL-bonded PEGsith the increase of PEG moiety length and molecular weigh
3,5]. The latter could result in accelerated kinetics of monolayer
preading and uniform molecular packing at the air–water inter-ace, necessary for reaching of low equilibrium surface tension36].
(oc
� and dashed line). Spread monolayers surface density corresponded to 200 Aer molecule. Experiments were done at T = 37 ◦C, Cel = 0.5 M NaCl, pH.8–7.0.
For obtaining morphological information about the changef the structure occurring in the membrane plane when DMPE-EGs were present in brush conformation (at Ccrit
DMPE−PEG andn higher concentrations) in comparison with pure DMPC films,dsorption DMPC/DMPE-PEG films on solid support wereormed and observed with SEM.
.2.5. SEM of adsorption DMPC/DMPE-PEGolid-supported layers
SEM experiments were performed as scanning electronicroscopy is commonly used technique for studying the
urface morphology of adsorption layers formed by nanopar-icles [26]. Experiments were conducted at the same content
at CcritDMPE−PEG), 20 and 80 mol% DMPE-PEG. The images
btained at 20 and 80 mol% DMPE-PEG did not differ signifi-antly and only the results at 20 mol% are shown.
G.As. Georgiev et al. / Colloids and Surfaces B: Biointerfaces 59 (2007) 184–193 191
F The cs
ccffitpral(belmanPnPe“3
Pl(ao
4
opw
[s(iT
ig. 8. SEM images of solid-supported adsorption DMPC/DMPE-PEG layers.amples of pure DMPC, DMPE-PEG550/2000/5000 are also shown.
As it can be seen at Fig. 8 pure DMPC layers surfaceontained numerous micro-heterogeneities and defects, whichould be responsible for the lowest stability of DMPC pureoam TLFs in comparison with the mixed DMPC/DMPE-PEGlms (see Table 1). This observation is in agreement with
he findings of Vassilieff et al. [37] demonstrating incom-lete degradation of PL liposomes and micelles at interfaces,esulting in heterogeneous adsorption layers containing defectsnd vacancies. The presence of DMPE-PEGs in the adsorbedayers reduced the heterogeneity and the amount of defectscavities) in the surface notably. This “smoothing” effectecame stronger with the increase of PEG chain Mw: theffect of DMPE-PEG550 was lowest (which agrees with itsowest effect on foam TLFs and lack of change in BF for-
ation mechanism) and the surface was heterogeneous event ≥20 mol%, while DMPE-PEG2000/5000 were capable to sig-ificantly “smooth” the surface already at Ccrit
DMPE−PEG (whereEG brushes were formed) and at ≥20 mol% practicallyo cavities were observed (the same as for pure DMPE-
EG layers). The most significant visible influence was theffect of the highest molecular weight DMPE-PEG5000 whichsmoothed” the adsorption layer at such a low concentration asmol%.
Dws(
omposition of the film-forming dispersions is noted below each panel. Control
An interesting additional effect was observed for DMPE-EG2000/5000 containing samples—formation of regular mesh-
ike structure. The elemental analysis showed that the stripesthe regions of regular structure) of the mesh are rich of Cnd O atoms, and could represent crystallized long “brushes”f PEG2000/5000 moieties.
. Discussion and concluding remarks
Bulk and interfacial properties of DMPC/DMPE-PEG aque-us dispersions depend on the molar ratio and on the hydrophilicolymer chain size (evaluated in terms of PEG moleculareight).In accordance with the observations of Montessano et al.
12] and Belsito et al. [13] for similar PC/PE-PEG bulk disper-ions a conversion of the dispersion aggregates from liposomesd = 500 nm) to micelles (d ca. 100 nm) was established depend-ng on the molar content and the PEG chain size (Fig. 2).his conversion was completed in the region 35–73 mol%
MPE-PEGn (n = 550, 2000 or 5000 denotes PEG moleculareight). At lower CDMPE-PEG the dispersions consisted of lipo-
omes (<10 mol% DMPE-PEG) or of liposome/micelle mixtures20 mol% DMPE-PEG).
1 Surfa
Ptm
am1(timtTd
bTPohfifeoAgab(prpmttitw
tmCa
sstPitieTtoo
wP(ostdeldssatrsDtTrtrc
shieC
TbvtPltppi
A
bai
R
92 G.As. Georgiev et al. / Colloids and
The interfacial and bulk properties of DMPC/DMPE-EG dispersions depend on another important transition:
he mushroom-to-brush conformational transition of the PEGoiety.In agreement with previous observations of Nikolova
nd Jones (on foam films of DMPC/DPPE-PEG2000) theushroom-to-brush transition occurred at concentrations below
0 mol% DMPE-PEGn. The critical DMPE-PEG concentrationCcrit
DMPE−PEG) corresponding to this conformational transi-ion for the DMPE-PEGs studied was established with twondependent techniques (foam film thickness and �-potential
easurements) in excellent agreement. The critical concentra-ion decreased with increasing PEG chain size (Figs. 3 and 4).he values of Ccrit
DMPE−PEG were in the range where the bulkispersions consisted predominantly of liposomes.
The mushroom-to-brush transition affected also the kineticehavior and the type of the foam TLFs (Figs. 3 and 5,able 1). The most interesting new observation is with DMPE-EG2000/5000: below the critical concentration black films werebtained after formation and expansion of black spots; atigher DMPE-PEG2000/5000 concentrations thicker CBF-likelms were formed by continuous thinning. These changes inoam films thickness, formation mechanism and type can bexplained in terms of the strong steric repulsion arising due to theverlapping of the polymer brushes in the film core [5,20,23].ll the effects observed on foam TLFs and dispersion aggre-ates clearly demonstrated the dramatic influence that even smallmounts of DMPE-PEG can have, if the point of mushroom-to-rush conformational transition is reached. DMPE-PEG effectson foam films type, thickness and formation mechanism) wereroportional to PEG chain size. Their maximal magnitude waseached at the low Ccrit
DMPE−PEG (<10 mol%: i.e. liposomal dis-ersions) and only slightly changed at further CDMPE-PEG oricelle/liposome ratio increase. These observations sustained
he idea for the dominant influence of PEG size and conforma-ion on foam TLFs characteristics. The two parameters of crucialmportance for foam films properties proved to be interrelated ashe critical DMPE-PEG concentration (Ccrit
DMPE−PEG) decreasedith increasing PEG Mw (Figs. 3 and 4).A clear dependence was observed of the threshold concen-
ration (Ct) of DMPC/DMPE-PEG black films on DMPE-PEGolar content and PEG moiety Mw (Fig. 6 and Table 1). Thet values significantly decreased with increasing PEG size (inccordance with the established tendency for Ccrit
DMPE−PEG).Thus, our results demonstrating the determinative role of PEG
ize and mushroom-to-brush transition for the film propertiesupport the hypothesis (proposed also by other authors [36])hat when PEG chains are in brush conformation, mixed PC/PE-EG monolayers with uniform dense molecular packing (which
n turn could increase black film stability) are formed, in whichhe area per molecule only slightly changes at further CDMPE-PEGncrease. This hypothesis was supported both by monolayerxperiments and by SEM of solid-supported adsorption layers.
hese experiments were performed at DMPE-PEG concentra-
ion ≥ CcritDMPE−PEG and no difference was observed in the effects
f 20 and 80 mol% DMPE-PEG. Equilibrium surface tensionf adsorption monolayers decreased (which could be related
ces B: Biointerfaces 59 (2007) 184–193
ith increased molecular packing density) with the increase ofEG moiety Mw in comparison with pure DMPC monolayersFig. 7A). Analogous behaviour was observed for the transientsf the surface tension of the spread monolayers (Fig. 7B). SEMtudies also proved the significance of the mushroom-to-brushransition: the images (Fig. 8) displayed sealing of numerousefects and “smoothing” of DMPC/DMPE-PEG adsorption lay-rs surface at Ccrit
DMPE−PEG (while at DMPE-PEG concentrationower than Ccrit
DMPE−PEG the surface remained heterogeneous –ata not shown). The importance of PEG brush size was demon-trated by the capability of DMPE-PEG2000/5000 to “completelymooth” (i.e. no visible “cavities”) adsorption layer surfacet ≤20 mol% (while for DMPE-PEG550 the surface still con-ained defects at concentration 20 mol% and higher). Thus, theesults of foam TLFs and SEM experiments provide the pos-ibility to differentiate longer chain DMPE-PEG2000/5000 fromMPE-PEG550 by the capability to induce change in BF forma-
ion mechanism (from BS formation to continuous thinning –able 1 and Fig. 5) and to smooth adsorption layer surface. Ouresults are in agreement with the literature data [26] showinghat for formulation of valid pharmaceutical “stealth” drug car-iers grafting of the membrane surface with polyethylene oxidehains with Mw ≥ 2000 is necessary.
In general the inclusion of DMPE-PEGs to DMPC disper-ions results in formation of more stable, densely packed andomogenous films, with the effects of DMPE-PEGs depend-ng mainly on PEG moiety size and conformation. Distinctiveffects of the mushroom-to-brush conformational transition atcritDMPE−PEG were detected with all methods used in this study.he possibility to differentiate DMPE-PEGs with different sizey their capability to change black film formation mechanismia steric repulsion increase, and to measure the resulting BFhickness raise could be of great relevance for selecting PE-EGs in the design of pharmaceutically applicable “stealth”
iposomes and coated nanoparticles. Thus, our study indicateshat phospholipid foam thin liquid films (coupled with phos-holipid monolayers) can be used as a model system for theurposes of pharmaceutical liposome engineering and for thenvestigation of surface interactions of membrane-active agents.
cknowledgements
We gratefully acknowledge the support of this work providedy FEBS Collaborative Experimental Scholarship for Centralnd East Europe and contract TK-Б-1607/06 by Bulgarian Min-stry of Education and Science.
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