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Cite this: DOI: 10.1039/c2sm26178b

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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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