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Cite this: DOI: 10.1039/c2sm26178b
www.rsc.org/softmatter PAPER
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Self-assembled gellan-based nanohydrogels as a tool for prednisolonedelivery†
Giorgia D’Arrigo,a Chiara Di Meo,a Elisa Gaucci,b Silvia Chichiarelli,b Tommasina Coviello,a
Donatella Capitani,c Franco Alhaiquea and Pietro Matricardi*a
Received 22nd May 2012, Accepted 6th September 2012
DOI: 10.1039/c2sm26178b
Self-assembled nanohydrogels based on hydrophobically modified polysaccharides have been
extensively studied due to their wide potential applications as drug delivery systems. In the present
study, we developed and characterized self-assembling nanohydrogels based on sonicated gellan gum
chains. Prednisolone (Pred), a poorly water soluble anti-inflammatory drug, was chemically conjugated
to the carboxylic groups of gellan (Ge–Pred) and it was the hydrophobic moiety responsible for the self-
assembling process. Ge–Pred was characterized by proton nuclear magnetic resonance spectra
(1H-NMR) and the cellular cytotoxicity was assessed by the MTS assay. The self-aggregation behavior
in aqueous media of Ge–Pred was evaluated by the pyrene fluorescence technique and the
nanohydrogels (NHs), prepared by bath sonication in water, were analyzed by dynamic light scattering
(DLS) and z-potential. The average size of the nanohydrogels was about 300 nm and their z-potential
values were negative. Our results showed that Ge–Pred NHs are cytocompatible, that the drug is
bioavailable and, consequently, they represent an interesting and innovative carrier for prednisolone.
Introduction
In recent years, significant efforts have been devoted to the
development of amphiphilic polymers consisting of hydrophilic
polysaccharides and hydrophobic moieties because of their
ability to form nanoparticle self-assemblies in aqueous media.
The intermolecular interactions between the hydrophilic and
hydrophobic segments allow the creation of a hydrophilic shell
that faces towards the solvent and an inner hydrophobic core
with minimal interactions with the aqueous medium.1–4 In recent
years, a variety of self-assembled nanoparticles have been
developed for their potential application as drugs and their
ability to increase the therapeutic efficacy of the drugs and to
reduce drug side effects. Numerous examples of nanoparticles
(NPs) are currently used in clinical practice (Doxil and
aDepartment of Drug Chemistry and Technology, Sapienza University ofRome, p.le Aldo Moro 5, 00185 Rome, Italy. E-mail: pietro.matricardi@uniroma1.itbDepartment of Biochemical Sciences ‘‘A. Rossi Fanelli’’, SapienzaUniversity of Rome, p.le Aldo Moro 5, 00185 Rome, ItalycMagnetic Resonance Laboratory Annalaura Segre, Institute of ChemicalMethodologies, CNR, Research Area of Rome, via Salaria Km 29.300,00016 Monterotondo Stazione, Rome, Italy
† Electronic supplementary information (ESI) available: The 400 MHz1H-NMR spectrum of Br–Pred in CDCl3 at 27 �C (Fig. S1). The 400MHz 1H-NMR spectrum of the Br–CH in CDCl3 at 27 �C (Fig. S2).The stability profile of Ge–Pred NHs at 37 �C in DMEM and inDMEM added with 10% of FBS (Fig. S3). The stability profile ofGe–CH NHs at 37 �C in DMEM and in DMEM added with 10% ofFBS (Fig. S4). Assignment of gellan protonic resonances achieved atroom temperature (Table S1). See DOI: 10.1039/c2sm26178b
This journal is ª The Royal Society of Chemistry 2012
Daunoxome) and others are in clinical development (Cyclosert).
Moreover, self-assembled nanoparticles have been proposed as
anticancer drug carriers because they exhibit prolonged systemic
circulation time and can accumulate in the tumor masses (EPR,
Enhanced Permeability and Retention effect). The nanoparticle
surface can then be functionalized to improve targeting to the site
of action of the drug, thereby reducing non-specific distribution.
In particular, surface functionalization can address the drug
release, avoid NP aggregation, and reduce the rapid clearance of
the drug.
As applied to drug delivery systems, natural polysaccharides
have the advantages of their abundance in nature, low cost,
safety, general biocompatibility, biodegradability, and high
stability. Among all natural polysaccharides, gellan is a prom-
ising candidate for biomedical applications because of its pecu-
liar physico-chemical properties and its biocompatibility.5–7
Gellan gum is an anionic polysaccharide produced by Sphingo-
monas elodea with a complex tetrasaccharide repeating unit of
b-D-glucose, b-D-glucuronic acid, b-D-glucose, and a-L-rham-
nose, with a free carboxyl group. Gellan is largely used in the
food industry and biotechnology because it forms transparent
hydrogels that are more resistant to heat and acidic medium as
compared to other polysaccharide hydrogels. Recently, gellan
gum hydrogels showed potential usefulness in the engineering of
cartilaginous tissues due to their viscoelastic properties and lack
of cytotoxicity.8 The polymer solution gelation is due to the
thermally reversible ordered helix-coil transition of the polymer
chains and the formation of junction zones by the stacking of the
macromolecules in the double helix form.9,5 The helix
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aggregation and gel formation are enhanced by cations, with
divalent cations being a stronger promoter of the gelation of
gellan gum than monovalent cations. The ability of gellan gum to
form strong hydrogels in the presence of ions and then to be
transformed into a nanohydrogel offers a promising strategy that
combines the properties of hydrogels with the advantages of
nanotechnology.
Herein, we prepared gellan nanohydrogels (NHs) by self-
assembly of the polymer chains, after an appropriate hydro-
phobic chemical derivatization with cholesterol and prednisolone
moieties. Gellan was ultrasonicated to reduce the molecular
weight, thus obtaining a more reliable and suitable polymer
system for NH formation.10 Prednisolone was chosen as the
hydrophobic moiety for its wide spectrum of activities in cell
trafficking, cell–cell interactions, and cell communication and for
its pronounced anti-inflammatory and immunosuppressive
effects. Prednisolone is a prodrug that is enzymatically activated
to prednisone in the liver. Prednisone then acts as a classical
steroid hormone and diffuses through the cell membrane, inter-
acting with a cytosolic receptor that forms a complex that then
translocates to the nucleus where it directly modulates DNA
transcription of a variety of genes.11 Moreover, over the last two
decades, glucocorticoids have been shown to inhibit solid tumor
growth in experimental animal models.12–15
In the present investigation, the prednisolone moiety, linked to
the polymer chains by means of a short hydrocarbon chain,
simultaneously acts as a pharmacological agent and a promoter
of NH formation whereas cholesterol was chosen as a moiety
able to promote the formation of reference NHs with no phar-
macological activity. This approach allows us to combine the
advantages of nanotechnology, such as high stability, high
carrier capacity, and feasibility of different routes of adminis-
tration with an immobilized form of prednisolone that displays
improved bioavailability, thus increasing its therapeutic effects
and reducing its dosing frequency. In this work, the chemical
derivatization of gellan with prednisolone and cholesterol is
described; the obtained self-assembled NHs were characterized
and the cytotoxicity of the NHs was tested using the MTS assay.
Materials and methods
Materials
Gellan gum tetrabutylammonium salt (GeTBA) was kindly
provided by Fidia Advanced Biopolymers, Abano Terme (PD),
Italy. Cholesterol, prednisolone, 4-bromobutyric acid, N-(3-
dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride
(EDC$HCl), and 4-(dimethylamino)pyridine (DMAP) were
Sigma products. DMEM (Dulbecco’s Minimal Essential
Medium) was from Sigma; the other cell culture reagents were
PAA products. Other chemicals are of analytical grade and were
used without further purification.
Gellan gum controlled depolymerization
Native gellan gum tetrabutylammonium salt (GeTBA) was dis-
solved in distilled water (0.5 g in 100 mL) at 80 �C; 50 mL
aliquots of this solution were sonicated by using a probe type
sonicator (Vibra Cell – VC 750, microtip 6.5 mm), 20 kHz, at
30% of amplitude for 15, 30, 60, 105 and 120 min. The solutions
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were kept cold using an ice bath. To reduce the temperature
increase, a pulsed mode (50% sound–50% silence) was applied.
The solutions were then filtered (0.22 mm, Millipore filter) and
dialyzed against distilled water (Visking tubing, cut-off: 12 000–
14 000) until the conductivity reached a value below 1.2 mS. The
pH of the dialyzed solution was then adjusted to 7, using
TBAOH (0.05 M) and the polymer was recovered by
lyophilization.
Gellan gum characterization
Gel permeation chromatography analysis. The molecular
weight (Mw) and polydispersity index (PDI) of gellan samples,
obtained after sonication, were determined by gel permeation
chromatography (GPC, Model 210 – VARIAN); the polymer
(0.15% w/v) was dissolved in tetramethylammonium chloride
(TMACl, 0.025 M). The elution was then performed on two PL
Aquagel-OH Mixed-H columns (8 mm, 300 � 7.5 mm) using
TMACl (0.025 M) as an eluent, at a flow rate of 1 mL min�1. A
calibration curve based on pullulan (range of Mw 5900–788 000)
in TMACl (0.025 M) was used.6 GPC data were analyzed by
means of Cirrus software.
Intrinsic viscosity measurements. Intrinsic viscosity of GeTBA,
sonicated for 60 min, and of native GeTBA was determined by
dissolving the samples in TMACl (0.025M) (concentration range
0.07% O 0.04% w/v) and using an automated capillary viscom-
eter (Shott-Geraete, diameter 0.54 mm), immersed in a water
bath (LAUDA 0.15 T), equipped with an INSTRUMENT
SCHOTT AVS 370 pump.
NMR analysis. The NMR analysis of sonicated gellan sodium
salt was performed at 27 �C on a Bruker AVANCE AQS 600
spectrometer equipped with a Bruker multinuclear, z-gradient
probe head. In a typical NMR experiment, the polymeric sample
(2 mg) was dissolved in D2O (0.7 mL). In all of the 1H-NMR
spectra, a soft presaturation of the HOD residual signal was
applied. 1H and 13C assignments were obtained using 1H–1H
correlation spectroscopy (COSY), 1H–1H total correlation
spectroscopy (TOCSY), and 1H–13C heteronuclear single
quantum coherence (HSQC) experiments with a z-gradient
coherence selection. All two-dimensional (2D) experiments were
carried out using 1024 data points in the f2 dimension and 512
data points in the f1 dimension; the recycle delay was 1 s. The
TOCSY experiment was performed with a spin-lock duration of
80 ms. The HSQC experiment was performed using a coupling
constant of 150 Hz. The TOCSY and the HSQC experiments
were processed in the phase-sensitive mode (time proportional
phase increment (TPPI)) with 512 � 512 datapoints. 1H and 13C
chemical shifts were reported in ppm with respect to 2,2-
dimethyl-2-silapentane-5-sulfonate (DSS) sodium salt, used as an
internal standard.
Synthesis and characterization of the gellan derivatives
Prednisolone bromo-butyric derivative synthesis. In a typical
synthesis, prednisolone (468 mg, 1.3 mmol) was solubilized in
CH2Cl2 (7 mL); DMAP (79 mg, 0.65 mmol) was added and the
solution was stirred for 15 min. Separately, EDC$HCl (744 mg,
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3.9 mmol) was added to a solution of 4-bromobutyric acid
(648 mg, 3.9 mmol) in CH2Cl2 (7 mL) and the solution was
stirred for 15 min. The two solutions were then mixed and the
reaction was kept for 6 h at room temperature. The reaction was
monitored using TLC on silica gel (eluent cyclo-
hexane : ethylacetate 30 : 70). The final solution was then washed
with NaOH (0.05 M), HCl (0.05 M) and finally three times with
distilled water and then dried over anhydrous Na2SO4. The
solvent was then removed by evaporation and the crude product
was purified on silica column (cyclohexane : ethylacetate
60 : 40). A pure Br–butyric-prednisolone (Br–Pred) product
(366 mg, yield 55%) was obtained. Samples were characterized by1H-NMR (Bruker Avance 400 MHz).
Gellan–prednisolone synthesis. For the preparation of gellan–
prednisolone (Ge–Pred), 60 min sonicated gellan (1 g) was dis-
solved in N-methyl-2-pyrrolidone (NMP) (135 mL); then
Br–Pred (57.4 mg) dissolved in 39 mL of NMP was added, and
the reaction was kept under magnetic stirring for 40 h at 38 �C.An exhaustive dialysis against distilled water (Visking tubing,
cut-off: 12 000–14 000) was then performed and the product
Ge–Pred was finally recovered by lyophilization.
Gellan–prednisolone characterization. The 1H-NMR (600
MHz) of Ge–Pred in the TBA form was obtained by dissolving
the product (2 mg) in 630 mL of DMSO-d6 and 70 mL of D2O, in
order to exchange the hydroxyl hydrogens of the polysaccharide
with deuterium, thus simplifying the obtained spectrum. NMR
spectra were recorded with the above described equipment.
The hydrolysis of Ge–Pred was also performed by an alkaline
treatment of the product (3 mg in 10 mL of H2O) at pH ¼ 13
(NaOH 1 M) and T ¼ 60 �C for 2 h; gellan, prednisolone and
hydroxybutyric derivatives were thus obtained. After this treat-
ment, the solution was left to cool at room temperature (1 h) and
the pH was neutralized with HCl (1 M). The reaction products
were then recovered by lyophilization and prednisolone together
with the butyric-derivative of prednisolone was then extracted
using 2 mL of ethanol. Finally, the suspension was centrifuged at
3000g and the amount of prednisolone and prednisolone butyric-
derivative in the supernatant was quantified by HPLC analysis
(Varian apparatus), using a C18 column (5 mm, 150 mm �4.6 mm) andUV detection at 254 nm; the column temperature was
fixed at 25 �C; isocratic elution H2O : CH3CN 72 : 28 for 9 min,
then a gradient up to 0 : 100 in 10min.Water used for analysis was
always filtered through a 0.45 mm cellulose membrane filter and,
before use, the mobile phase was always de-aerated under vacuum.
Standard solutions of prednisolone prepared for the calibration
curve were treated as described above for Ge–Pred samples.
Gellan–cholesterol (Ge–CH) synthesis and characterization.
Cholesterol (500 mg, 1.3 mmol) and DMAP (79 mg, 0.65 mmol)
were solubilized in CH2Cl2 (7 mL) and the solution was stirred
for 10 min. Separately, EDC$HCl (744 mg, 3.9 mmol) and 4-
bromobutyric acid (648 mg, 3.9 mmol) were solubilized in
CH2Cl2 (7 mL) and the solution was stirred for 10 min. The two
solutions were then mixed and the reaction was kept for 6 h at
room temperature and monitored by TLC on silica gel (eluent
ethylacetate : cyclohexane 2 : 98). The solution was then washed
with NaOH (0.05 M), HCl (0.05 M) and finally three times with
This journal is ª The Royal Society of Chemistry 2012
distilled water and then dried over anhydrous Na2SO4. The
solvent was then removed by rotary evaporation and the crude
product was purified on silica column (ethylacetate : cyclohexane
15 : 85). The yield of the reaction was 70%. The Br–butyric-
cholesterol (Br–CH) product was analyzed by 1H-NMR (400
MHz) spectroscopy. The gellan–cholesterol (Ge–CH) product
was then obtained by dissolving 1 g of 60 min sonicated gellan in
N-methyl-2-pyrrolidone (NMP, 135 mL) and by adding Br–CH
(100 mg) dissolved in NMP (39 mL); the reaction was kept under
magnetic stirring for 40 h at 38 �C. An exhaustive dialysis against
distilled water (Visking tubing, cut-off: 12 000–14 000) was then
performed and the product Ge–CH was finally recovered by
lyophilization.
The 1H-NMR (600 MHz) of Ge–CH in the TBA form was
obtained as previously described for Ge–Pred by dissolving the
product (2 mg) in 630 mL of DMSO-d6 and 70 mL of D2O.
Nanohydrogel preparation and characterization. In a typical
preparation, a dispersion of 5 mg of Ge–Pred (or Ge–CH) in 5
mL of filtered bidistilled water was prepared. The suspension was
then sonicated for 30 min in an ultrasonic bath sonicator (Stra-
sonic-35, Liarre) to form the nanohydrogels (NHs).
The determination of the size, the polydispersity and the z-
potential of the Ge–Pred and Ge–CH NHs were carried out by
means of Dynamic Light Scattering using a Malvern Nano-
ZetaSizer apparatus (Malvern Instruments, Worcestershire,
UK), equipped with a 5 mW HeNe laser (l ¼ 632.8 nm). The
normalized intensity autocorrelation functions were detected at
90� by a logarithmic digital correlator and analyzed by using the
Contin algorithm.
The stability of Ge–Pred and Ge–CH NH suspensions (1
mg mL�1) was followed for 30 days, keeping the samples at room
temperature and measuring the size of the NHs as described
above. The stability of NHs at 1 mg mL�1 in DMEM and
DMEM with added 10% fetal bovine serum (FBS) was assessed
for 24 h at 37 �C.The critical aggregation concentration (CAC) was determined
for Ge–Pred NHs by means of the pyrene fluorescence variation
method.16–19 A pyrene solution (5 � 10�4 M in acetone) was
prepared and filtered at 0.45 mm. Then, an aliquot (25 mL) of this
solution was withdrawn and evaporated under nitrogen flux;
filtered bidistilled water (25 mL) was then added to prepare a 5 �10�7 M aqueous solution of pyrene. Aliquots (2 mL) of the pyrene
solution were added to 2 mL aliquots of Ge–Pred NH suspensions
at concentrations ranging from 1� 10�7 to 0.4 mg mL�1, and left
overnight under magnetic stirring at room temperature. The
fluorescence emission spectra were recorded using a Perkin Elmer
LS50B Luminescence Spectrometer with lexc ¼ 334 nm; the ratio
between the fluorescence intensity at lem ¼ 372 nm (I372) and at
lem ¼ 383 nm (I383) was calculated for each sample and results
were plotted as a function of NP concentration. The CAC was
taken as the point of crossover of the flat region of the graph at a
low concentration of Ge–Pred and the sharp decrease in the value
of I1/I3 at higher concentrations of Ge–Pred.
Testing in HaCaT cells
Cell culture. The immortalized aneuploid human keratinocyte
monolayer cell line HaCaT was grown in DMEM, with
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4500 g L�1 glucose, supplemented with 10% of fetal bovine serum
(FBS), 1% w/v sodium pyruvate, 2 mM glutamine, 100 U mL�1
penicillin and 100 mg mL�1 streptomycin. Cells were grown to
90% confluence at 37 �C in a 5% CO2 atmosphere.20
MTS assay. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carbox-
ymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt),
CellTiter 96� AQueous Non-Radioactive Cell Proliferation
Assay (Promega) and PMS, phenazine methosulfate (Sigma),
were dissolved according to the manufacturer’s instructions. To
measure cell viability, HaCaT cells were resuspended at a
concentration of 1.2� 105 cells per mL and added onto a 96-well
plate (1.2 � 104 cells per well) by an 8-channel pipette. The day
after, the cells were treated with Ge–CH, Ge–Pred, or prednis-
olone. The compounds were administered at concentrations of
2 mg mL�1, 5 mg mL�1, 10 mg mL�1, 20 mg mL�1, 40 mg mL�1,
70 mg mL�1, 100 mg mL�1, and 250 mg mL�1. Control cells were
maintained in culture medium diluted with water. At 24 h after
the treatment, the viability assay was performed, maintaining a
ratio of 20 mL of combined MTS–PMS solutions per 100 mL of
culture medium, leading to final concentrations in the assay of
333 mg mL�1MTS and 25 mMPMS. The plates were incubated in
the dark for 2 h at 37 �C in a humidified, 5% CO2 atmosphere.
The absorbance of the soluble formazan was recorded at 492 nm
using a Thermo Scientific Appliskan plate reader. The back-
ground due to nonspecific absorbance was recorded at 690 nm
and subtracted from the absorbance at 492 nm.
Caspase assay. Caspases 3 and 7 activities in HaCaT cells
were measured using the Caspase-Glo� 3/7 assay (Promega).
The assay provides a luminogenic substrate, which contains the
tetra-peptide sequence DEVD, specific for caspases 3 and 7. The
cleavage leads to the release of aminoluciferin, a substrate for
luciferase. The reaction generates a luminescent signal,
proportional to the amount of caspase activity that is present.
HaCaT cells were resuspended at a concentration of 105 cells
per mL and an aliquot (100 mL) of the cell suspension was
added onto a white-walled 96-well plate (104 cells per well). The
day after, the cells were treated with Ge–CH and Ge–Pred at a
concentration of 100 mg mL�1 and with prednisolone at a
concentration of 2.4 mg mL�1. Blanks (cell culture medium
without cells) and negative control cells were treated with water
as the vehicle. After 24 h incubation, the assay was performed.
The Caspase-Glo� 3/7 Reagent was reconstituted following the
manufacturer’s instructions. The plate containing the cells was
removed from the incubator and allowed to equilibrate to room
temperature. Then, the Caspase-Glo� 3/7 Reagent was added in
a 1 : 1 ratio with the culture medium (100 mL per well). After
a gentle mixing, to aid cell lysis, the plate was incubated at room
temperature for 1 h. The luminescence of each sample
was measured in a plate-reading luminometer (Thermo Scien-
tific Appliskan). The background caspases activity can be
detected both in serum and in untreated cells. Consequently,
blank (i.e., cell culture medium without cells) and negative
control (i.e., vehicle-treated cells) experiments were performed.
Background readings from the blank reaction were subtracted
from the experimental values. Negative control cells were
used to determine the basal caspase activity of the cell culture
system.
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Statistical analysis. All values in MTS assay and caspase assay
are expressed as means � SD of at least 5 replicates. A statistical
analysis was performed with a one-way ANOVA test. Results
were considered statistically significant at P < 0.005 and P < 0.01.
Results and discussion
Preparation and characterization of the gellan gum sonicated
sample
Since the hydrophobic derivatization of the native gellan gum did
not lead to NH formation, probably due to the rigidity and the
high molecular weight of the polymer chains, we undertook a
depolymerization strategy to improve the NH size and
morphology. The gellan molecular weight was reduced by soni-
cation and the obtained polymer was then hydrophobically
modified, yielding a novel polymeric amphiphile. It is note-
worthy that the solutions of native gellan exhibited much higher
viscosities than the corresponding solutions of the sonicated
polymer. Moreover, the native polymer could be solubilized in
distilled water only at high temperatures while the corresponding
‘‘sonicated’’ polymer was easily solubilized in distilled water at
room temperature. These features represent a great advantage in
the polymer treatments during NH preparation steps and influ-
ence NH properties.
We chose ultrasonic treatment to reduce the polymer molec-
ular weight because it allowed to avoid the use of chemicals or
enzymes and the ultrasonic radiation splits the most susceptible
chemical bonds in the polymer chains without causing any
changes in the chemical structure of the macromolecule.21 The
mechanism of the ultrasound depolymerization is not still
completely clear, but, according to the most accepted hypothesis,
the shear forces generated by the ‘‘explosion’’ of microbubbles in
the solution, formed by the ultrasound waves, disrupt the poly-
meric bonds. The interactions between the polymer chains and
the eddies, generated as the bubbles cavitate and collapse, stress
the polymer chains until homolytic chemical bond cleavage
occurs.22,23 The decrease of the molecular weight of native gellan
was strongly dependent on the ultrasound treatment time. For up
to 1 h of sonication time the Mw decreased (Fig. 1a) and then
remained at a stable value for up to 100 min. A further decrease
was observed after 2 h of treatment. The Mw of gellan, starting
from about 3.7 � 106, was thus reduced �20-fold, to 1.7 � 105,
by 1 h. Sonication markedly decreased the polydispersity index
(PDI) (Fig. 1b). After 15 min and 60 min of treatment, PDI
values of 13.0 and 3.7 were obtained, respectively. This behavior
is usually observed during ultrasound polymer solution treat-
ment and it is due to the greater susceptibility of the longer
polymer chains to degradation than the shorter ones.
For the chemical derivatization and NH formation, the gellan
sample sonicated for 60 min was chosen. A detailed structural
characterization on this sample was carried out by means of
intrinsic viscosity and NMR techniques.
After 1 h of sonication, the gellan sample displayed an intrinsic
viscosity ranging from �7.18 � 1.2 dl g�1 to 0.90 � 0.02 dl g�1,
thus confirming an extensive depolymerization of gellan.
Exhaustive NMR investigations confirmed that the primary
structure of the polysaccharide was not affected by the ultra-
sound treatment. The sample was fully soluble in deuterated
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Fig. 1 Molecular weight (a) and PDI (b) dependence on the sonication
time for gellan.
Fig. 2 Schematic representation of a gellan repetitive unit and 2D1H–13CHSQCmap of 60 min sonicated gellan in D2O at 27 �C along with
the full assignment. The 1H spectrum is reported as the projection in the
f2 dimension.
Scheme 1 Reaction scheme of the Ge–Pred synthesis. (a) Synthesis of a
Br–Pred derivative; (b) Br–Pred coupling to GeTBA leading to Ge–Pred.
Unreacted Ge carboxylate groups are in the TBA salt form.
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water at room temperature, and mono- and bi-dimensional
NMR spectra were collected at 27 �C instead of at 50–90 �C as
previously reported.26 Based on the 1H–13C HSQC map of 60
min sonicated gellan (Fig. 2) and a complete assignment of
protons and carbons (Table S1†), we concluded that the primary
structure of native gellan was not affected by ultrasound treat-
ment, as previously reported.24
Synthesis of the gellan–prednisolone and gellan–cholesterol
derivatives
Attempts to synthesize the Ge–Pred derivative via carbodiimide
chemistry, by direct coupling of the prednisolone hydroxyl group
with the gellan carboxyl group, were unsuccessful, probably
because of the steric hindrance of the polysaccharide. To address
this constraint, a short spacer was introduced between gellan and
prednisolone (see Scheme 1a). The Br–Pred intermediate was
obtained by esterification of the hydroxyl group on C21 with
Br-butyric acid, via carbodiimide activation of the carboxyl
group. The NMR spectrum of Br–Pred showed the presence of
two non-equivalent CH2 protons at 4.952 ppm after esterification
with Br-butyrate, as well as the signal of methylenic CH2–Br at
3.551 ppm (see Fig. S1 in the ESI†).
The Ge–Pred derivative was then obtained by reaction
between Br–Pred and the sonicated gellan polymer, according to
the reaction reported in Scheme 1b.
The obtained Ge–Pred TBA salt was characterized by 1H-
NMR (Fig. 3). By integrating the prednisolone signals H1, H2
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and H4 at 7.332, 6.151 and 5.914 ppm with respect to the
rhamnose anomeric signal at 5.125 ppm, we determined that 6%
mol/mol derivatization had occurred, defined as the percentage
of mol of prednisolone per mol of a gellan tetrasaccharide
repetitive unit.
This result was confirmed by HPLC analysis of the hydrolyzed
product: the amount of prednisolone recovered after the de-
esterification procedure indicated polysaccharide derivatization
of 6%.
The cholesterol derivative of Ge was also prepared to have a
polymer system that can act as a nanoparticulate reference in
Soft Matter
Fig. 3 600.13 MHz 1H-NMR spectrum of a Ge–Pred derivative in
DMSO/D2O at 27 �C.
Fig. 4 Intensity ratios (I372/I383) from pyrene emission spectra as a
function of Ge–Pred concentration in distilled water.
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biological tests. Plain Ge, indeed, is not able to promote NH
formation, whereas cholesterol is largely used in the literature to
obtain amphiphilic polymers and there are several examples of
self-assembled NPs containing the cholesterol moiety.25 Ge–CH
was obtained using a two step reaction scheme similar to that
described above for the Ge–Pred synthesis. The Br–CH inter-
mediate was obtained by esterification of the hydroxyl group on
C3 with Br-butyric acid, via carbodiimide activation of the
carboxyl group. The NMR spectrum of Br–CH showed the
presence of proton 3 of cholesterol at 4.651 ppm after esterifi-
cation with Br-butyrate (Fig. S2 in the ESI†). The Ge–CH
derivative was then obtained by reaction between Br–CH and
sonicated gellan polymer using the same reaction conditions
described for the Ge–Pred derivative, obtaining an 8% mol/mol
substituted polymer (1H NMR data, not shown).
Nanohydrogel characterization
The Critical Aggregation Concentration (CAC) of Ge–Pred was
obtained by using pyrene as a fluorescent probe. Pyrene shows a
typical fluorescence emission spectrum and the ratio of the
intensities of the first (l ¼ 372 nm) and the third (l ¼ 383 nm)
peaks (I1/I3) is a sensitive indicator of the polarity of the
microenvironment around the excited pyrene. In an aqueous
solvent, the intensity ratio I1/I3 of pyrene is constant (1.71),
meaning that there is a lack of hydrophobic environment but the
ratio changes if NHs are present. When the NHs are formed the
pyrene lies close to or inside the hydrophobic microdomains and
induces a sharp transition in the value of I1/I3. In Fig. 4, I1/I3 is
plotted as a function of the Ge–Pred concentration and the CAC
corresponds to the onset of the I1/I3 transition (at 74 mg mL�1).
The Ge–Pred and Ge–CH NHs were prepared by bath-soni-
cation in aqueous media at a concentration of 1 mg mL�1. The
self-assembly of amphiphilic Ge–Pred or Ge–CH polymers in
distilled water was due to the hydrophobic interactions between
the hydrophobic core-forming prednisolone or cholesterol and
the hydrophilic interactions between the polysaccharidic shell-
forming chains. The size of the NHs decreased as the sonication
time increased, reaching a plateau value after about 30 minutes.
The size of Ge–Pred nanohydrogels measured by DLS was 320�12 nm with a polydispersity index of 0.274 � 0.035; the size of
Soft Matter
Ge–CH NHs results 340 � 13 nm with a polydispersity index of
0.311 � 0.069.
The z-potential of Ge–Pred nanohydrogels measured in
distilled water by DLS was�20.7� 1.0 mV, while the z-potential
of Ge–CH NHs under the same conditions was �26.4 � 3.2 mV.
The negatively charged shell should reflect the carboxylate
groups of the polysaccharide chains which are located in the
hydrophilic zone of the NHs. Since the charge of the nano-
hydrogels is one of the most important factors that can influence
the nanohydrogel biodistribution, organ accumulation, and the
interactions with biological barriers, it is worth noting that
the uptake of negatively charged particles can occur despite the
unfavorable interactions between the particles and the negatively
charged cell membrane.26,27 The internalization of negatively
charged nanohydrogels is believed to occur through non-specific
binding and clustering of the particles at cationic sites on the
plasma membrane (there are relatively fewer positively charged
than negatively charged domains) and their subsequent endo-
cytosis. Moreover, the negative z-potential of Ge–Pred and Ge–
CH NHs prevented the NH aggregation, permitting an electro-
static stabilization and providing long term stability in the
aqueous phase (Fig. 5). The NHs exhibited good stability on
storage for at least 30 days at room temperature and no aggre-
gation was observed after this time.
Biological investigation
To assure that the NHs of Ge–Pred were suitable for applications
as drug carriers we assessed their cytotoxicity by assay with
MTS (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-
2(4-sulfofenyl)-2H-tetrazolium, inner salt). This assay deter-
mines the metabolic activity of cells by evaluating the
bioreduction of the substrate, MTS, into a brown formazan
product by the dehydrogenase enzymes in metabolically active
cells. It was not possible to use the MTT test to evaluate Ge–CH
because of a direct interference of cholesterol with this assay. In
particular, cholesterol interferes in the MTT reduction without
affecting cellular viability. The enhanced exocytosis of formazan
along with cholesterol could represent a mechanism of inhibition
of MTT reduction.28
Before performing these biological tests, NH stability was
assessed in cell medium (DMEM) and in DMEM containing
added serum. Data reported in Fig. S3 and S4 (see ESI†) clearly
This journal is ª The Royal Society of Chemistry 2012
Fig. 5 Effect of the storage time on the size (d) of NHs stored at room
temperature measured byDLS. Data are expressed as the mean� SD of 5
observations.
Fig. 7 Luminescence from untreated control cells (CRT) or from cells
treated with prednisolone, Ge–Pred or Ge–CH. Each point represents the
mean � SD of 5 replicates. Values are blank subtracted. Results
were statistically analyzed with a one-way ANOVA test (**P < 0.005;
*P < 0.01).
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indicate that NHs are stable under the conditions adopted in the
biological test.
The MTS assay was performed on HaCaT cells, as described
above, after a 24 h treatment with Ge–Pred, Ge–CH, or pred-
nisolone alone at increasing concentrations. The administration
of Ge–CH did not induce cytotoxicity up to 100 mg mL�1 and,
moreover, the cell viability was only modestly affected at a
concentration of 250 mg mL�1. A slight decrease in cell viability
was observed both for Ge–Pred and prednisolone treatments; at
low concentrations, Ge–Pred showed a lower toxicity. However,
it must be pointed out that in no case was cell viability reduced to
less than 80% (Fig. 6). At concentrations lower than 30 mg mL�1
(range 2–70 mg mL�1), the NHs are not formed because such
concentrations are below the CAC value. These solutions of the
Ge–Pred, just as the NHs, did not perturb the cell viability and
appear to be completely cytocompatible.
Caspases are a family of cysteine aspartic acid-specific prote-
ases that play a key role in the execution of apoptosis. In
particular, the activity of the downstream caspases 3 and 7 is a
reliable indicator of cell apoptosis. Since a pro-apoptotic effect of
prednisolone has been observed in different cell lines, we assessed
the possible activation of the effector caspases due to the treat-
ment with Ge–Pred NHs, using the Caspase-Glo� 3/7 assay kit
Fig. 6 Cell viability expressed as a function of the concentration of
administered Ge–Pred (-), Ge–CH (:) or Pred (A). The viability of the
control cells (without treatment) was set at 100%. Each point is the
mean � SD of sixteen replicates.
This journal is ª The Royal Society of Chemistry 2012
(Promega).29,30 HaCaT cells were treated with Ge–CH NHs at
100 mg mL�1 and prednisolone at 3 mg mL�1, because at such a
concentration the viability of the three differently treated cells
showed the most pronounced differences (Fig. 7). The induction
of apoptosis by prednisolone was comparable to that of the
gellan derivative.
Furthermore, the larger range of luminescence values in the
NH sample compared to prednisolone treated cells suggested a
heterogeneous drug availability inside the NH treated cells. The
caspase activity in the Ge–CH treated cells did not differ
significantly from the untreated cells, indicating that the nano-
hydrogels themselves did not induce an apoptotic response.
These overall results suggest that the drug carried by the NHs
has similar bioavailability as free prednisolone, most probably
because of the cleavage of the ester bond due to the cellular
enzymes.
Conclusions
An amphiphilic polymer, based on prednisolone conjugated to
sonicated gellan, was synthesized and characterized by viscom-
etry and 1H-NMR. The sonication treatment effectively reduced
the polymer molecular weight, without causing any changes in
the chemical structure of the macromolecule, as assessed by
bi-dimensional NMR analysis. Self-assembled NHs were
obtained by suspension of the polysaccharide conjugate in water
followed by sonication. The average size of the Ge–Pred NHs
was �320 nm with a unimodal size distribution. The self-aggre-
gate stability was investigated by DLS, whereas the critical
aggregation concentration of this nanosystem was assessed by
fluorescence spectroscopy using pyrene as a probe. Biological
tests showed that the Ge–Pred NHs have no apparent cytotox-
icity and are able to promote the pro-apoptotic activity as well as
free prednisolone, suggesting a similar bioavailability of the
drug. We therefore concluded that gellan-based NHs can be
considered as efficient prednisolone carriers.
Acknowledgements
This work was financially supported by Sapienza University of
Rome ‘‘Ricerche Universitarie’’ 2011, no. C26A119N2S. The
Soft Matter
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authors are grateful to Prof. Vladimir Torchilin for his sugges-
tions during the preparation of the manuscript. The authors are
also grateful to ‘‘Enrico ed Enrica Sovena’’ Foundation, Italy, for
the financial support to Elisa Gaucci.
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