vitro evaluation of novel shell crosslinked poly

European Journal of Medicinal Chemistry 102 (2015) 132e142

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Design, characterization and in vitro evaluation of novel shell crosslinked poly(butylene adipate)-co-N-succinyl chitosan nanogels containing loteprednol etabonate: A new system for therapeutic effect enhancement via controlled drug delivery Farzaneh Hashemi Nasr, Sepideh Khoee* Polymer Laboratory, Chemistry Department, School of Science, University of Tehran, P.O. Box 14155-6455, Tehran, Iran

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 November 2014 Received in revised form 24 July 2015 Accepted 25 July 2015 Available online 1 August 2015

This study reports on the development of a novel mucoadhesive and biocompatible shell-crosslinked nanogel system based on poly(butylene adipate) (PBA) and N-succinyl chitosan (S-Cs) by coupling reaction with a new formulation method. For this purpose, two different molecular weights of dendrimerized PBA with amine terminated functional groups were synthesized separately and characterized well by FT-IR, 1HNMR and GPC. The PBA nanoparticles containing loteprednol etabonate (LPE) prepared by O/W emulsion technique were reacted immediately with modified carboxylated chitosan via carbodiimide chemistry. TEM photographs of the nanoparticles and crosslinked nanoparticles displayed a spherical morphology closely corresponding to the results obtained by DLS. On The other hand, biodegradability, biocompatibility and bioadhesiveness of the prepared nanoparticles were also studied. It is concluded that the coreeshell structured nanogels can be used as novel ocular drug delivery systems with appropriate loading capacity for slightly water soluble LPE as an anti-inflammatory drug. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Poly(butylene adipate) N-succinyl chitosan Shell-crosslinked nanogel Loteprednol etabonate Ocular therapy

1. Introduction Ophthalmic drug delivery is one of the most fascinating and challenging tasks being faced by the pharmaceutical researchers for past 20 years. The main problem in ocular drug delivery is the rapid elimination and low precorneal half-life of conventional eye drops from eye due to various types of barriers that exist in the eye [1,2]. Considering these points, the development of new drug candidates and novel delivery techniques is becoming increasingly important in treatment of ocular diseases. Drug delivery system based on nanotechnology with suitable particle size and narrow size range, confirming sufficient bioavailability and biocompatibility with ocular tissues, may prove to be the best delivery tools for pharmaceuticals treating ocular diseases, and such a system should be investigated for every suspended drug. Various nanoparticulate systems like microemulsions, nanosuspensions, polymeric nanoparticles, liposomes, niosomes, dendrimers and cyclodextrins can be utilized to explore the frontiers of ocular drug delivery and

* Corresponding author. E-mail address: [email protected] (S. Khoee). http://dx.doi.org/10.1016/j.ejmech.2015.07.045 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

therapy [3]. Among them, polymeric nanoparticles, which are prepared from inert polymeric resins, can be utilized as important drug delivery vehicles [4]. The incorporation of an ocular drug into smaller-sized dispersed polymeric nanoparticles systems can significantly increase the drug bioavailability and its permeation to the eye as well as providing controlled and prolong drug release profile [5e7]. Drug-loaded polymeric micelles (DPMs) should be carefully designed in order to deliver the drug at the site of its action. The hydrophobic core structures of polymeric micelles can solubilize hydrophobic drug. At the same time, the hydrophilic shell surrounding the micellar core can prevent intermicellar aggregation or precipitation, protein adsorption, and cell adhesion [8,9]. Since DPMs reside in the target tissues, the biocompatibility and biodegradability of the polymers used in their preparations are the two important requisites. In addition, the utilization of mucoadhesive polymers is necessary to improve ocular bioavailability and extended drug effect in targeted tissues [10]. Chitosan as the only natural and positively charged polysaccharide is capable of binding to the negatively charged mucosal layers through charge interactions [11e14]. Various investigators have studied the use of chitosan for ocular drug delivery [15e19]. One derivative of

F.H. Nasr, S. Khoee / European Journal of Medicinal Chemistry 102 (2015) 132e142

chitosan, N-succinyl-chitosan (S-Cs), synthesized by the introduction of succinyl groups into chitosan at the N-position of the glucosamine units has promising properties as a drug carrier such as biocompatibility, low toxicity and long-term retention in the body [20e22]. Up till now, the potential of polymeric micelles proposed for the ophthalmic application is less explored in comparison to their use in cancer therapy [23e26]. Increasing thermodynamic and kinetic stability of DPMs through physical or chemical approaches using chemical covalent crosslinks into the shell, core, or core-corona interface of micelles is of great importance. These, have the benefits of ensuring that the size and shape of micelles are not affected by an external parameter such as an aqueous solution saturated with amino acids, vitamins, and proteins [27e30]. Over the past decade, shell-crosslinked micelles have been extensively studied [31e33]. As a product of 'soft drug' design, Loteprednol etabonate is a corticosteroid which has been developed as a topical treatment for ocular inflammation. In contrast to other corticosteroids, LPE did not significantly increase intraocular pressure (IOP) [34,35]. Thus, LPE can safely decrease ocular inflammation caused by cataract surgery, seasonal allergic conjunctivitis or contact lens wears [36,37]. The present study focused on formulation and characterization of new biodegradable and mucoadhesive polymer nanoparticles with the aim of improving loteprednol delivery to the ocular surface in order to obtain and maintain a therapeutic level at the site of action for prolonged period of time. The synthesized PBA nanoparticles by O/W method were grafted to SCs via carbodiimde chemistry. Simultaneously, the PBA-g-Cs nanoparticles were stabilized by self-crosslinking of the S-Cs layer as the shell part. The prepared nanogels were characterized for some physical properties such as size, morphology and composition. In addition, their mucoadhesive property and biodegradation behavior were also studied to envisage their use for controlled drug targeting. These nanogels also showed low cytotoxicity to rabbit corneal epithelial cells (RCEC). The in-vitro release profiles demonstrate that the nanogels could have great potential application in delivering hydrophobic drug for improved ocular therapy. 2. Experimentals 2.1. Materials 1,4-butanediol, adipoyl chloride, triethylamine (TEA), di-tertbutyl dicarbonate and N-hydroxysuccinimide were obtained from Fluka. Acryloyl chloride (AC), diethanolamine, ethylene diamine, trifluoroacetic acid (TFA), chitosan (Cs) (3000 Da), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC), Tween 60 (polyoxyethylene sorbitan monostearate, HLB ¼ 14.9), dichloromethane (DCM), acetone and N,N-dimethylformamide (DMF) were purchased from Merck Chemical Co. Porcine stomach mucin (type III) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (methyl thiazolyl tetrazolium (MTT)) were provided by Sigma (USA). All the chemicals were of analytical grade and used without purification. 2.2. PBA synthesis (OHePBAeOH) Hydroxyl terminated polyesters with two different number average molecular weights of about 5000 and 8000 g/mol were synthesized and characterized according to our previously reported procedure [38]. Procedure steps for the synthesis of dendrimerized amine terminated polyester are as follows:

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2.3. Synthesis of G0.5 (ACePBAeAC) Dry PBA (Mw 5000 or 8000) was acrylated by reaction with triethylamine and acryloyl chloride in anhydrous DMF in a roundbottom flask equipped with a dropping funnel, a nitrogen inlet, and a magnetic stirrer. The molar ratio of PBA, AC and TEA was 1:3:3. Acryloyl chloride was added slowly to the above mixture at 0 C. Afterwards, the resulting mixture was stirred at room temperature overnight. At the end of reaction, the mixture was poured into a large amount of distilled water. The resulting precipitate was collected by centrifugation to yield purified G0.5 with 88% yield. 2.4. Synthesis of G1.0 ((OH)2ePBAe(OH)2) (ACePBAeAC) was dissolved in DMF and a solution of diethanolamine in DMF was added to it in a dropwised manner over a period of 10 min to the reaction media with vigorous magnetic stirring. The molar ratio of G0.5 and diethanolamine was 1:5. The reaction was carried out for 3 days at room temperature. Then the mixture was poured into a large amount of distilled water. The resulting precipitate was collected by centrifugation and washed well with water for 3 times (82% yield). 2.5. Synthesis of G1.5 ((AC)2ePBAe(AC)2) The purified G1.5 with 75% yield was obtained from G1, TEA and AC in DMF according to the synthesis procedure for G0.5. 2.6. Synthesis of G2 ((NH2)2ePBAe(NH2)2) The synthesis procedure for G2.0 was similar to that of G1.0 preparation. The prepared N-boc ethylenediamine was used instead of diethanolamine and the reaction was completed after 6 days. The purified product was deprotected by TFA with 76% yield according to the reported preparation method [39]. 2.7. Synthesis of N-succinyl-chitosan (S-Cs) Chitosan was succinylated according to the reported method with some modifications. Briefly 1 g of chitosan was dissolved under stirring in deionized water and the resulting solution was slowly diluted with methanol. Then, 2 g of succinic anhydride, previously dissolved in a minimum amount of acetone, was added dropwise to the polymer solution. The reaction was maintained under stirring overnight at room temperature and the mixture was precipitated in an excess of acetone. The precipitates were then filtered to remove the solvent and washed carefully with acetone. Finally, the product was dried at 40 C under vacuum condition for 24 h. The yield of the reaction was about 80% and the substitution degree of S-Cs was determined by 1HNMR. 2.8. PBA nanoparticles preparation A single oil-in-water (O/W) emulsion/solvent evaporation method was used in order to prepare amine-decorated PBA nanoparticles. In order to increase drug loading in the nanoparticles, G2 ((NH2)2ePBAe(NH2)2) (30 mg) was dissolved in DCM (2.5 ml) and then the LPE (3 mg) was dissolved in acetone (0.5 ml). The LPE in acetone was added to the PBA in DCM to form the oil phase. This oil phase was added dropwise to an aqueous solution (30 ml) containing 5% Tween 60. An oil-in-water (O/W) type emulsion was formed with a sonicator (20 KHz ± 500 Hz Ultrasonic generator, SONOPULS Ultrasonic homogenizer, Model HF-GM 3200, titanium micro tip Ms-72) employing a pulse mode (15 s “on” and 5 s “off”) for 30 s. The sample was kept in an ice bath to prevent overheating.

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Then, the organic solvent was evaporated at reduced pressure at room temperature in order to form nanoparticles with aminesurfaced groups.

2.9. Nanogel preparation Shell-crosslinked nanoparticles based on PBA and S-Cs were prepared via a carbodiimide reaction. S-Cs was dissolved in deionized water. After adjusting the pH at 4.2, equal molar amounts of EDC and NHS were added and stirred for 1 h at room temperature in order to activate the carboxylic acid groups on chitosan chain. Immediately before starting the reaction with the aminecontaining PBA nanoparticles, the pH rose to 7.2e7.5 with phosphate buffer. Subsequently, the prepared amine-decorated nanoparticles were added to the S-Cs acid activated solution in dropwise manner under magnetic stirring and then the reaction mixture was kept under agitation at room temperature for 10 h. The molar ratio of PBA: S-Cs: EDC: NHS was 1:1:0.4:0.4. At the end of the reaction, the mixture was freeze dried to produce slimy nanoparticles. Successively, the nanogels were washed with deionized water several times to remove the surfactant, placed at 70 C for 18 h, and then lyophilized to extract fine nanogels from aqueous medium and stored at 4 C.

2.10. Polymer characterization To confirm the formation of all synthesized structures, 1H NMR (Bruker, 500 MHz) and FT-IR (Bruker-equinoxss) spectra were collected. The gel permeation chromatography (GPC, Agilent GPC 1100) system was used to determine the number-average molecular weights (Mn), the weight average molecular weight (Mw) and macromolecular weight distribution of copolymers. The samples were dissolved in freshly distilled tetrahydrofuran (THF) at a concentration of 1e2 mg/ml. THF was introduced as eluent at a flow rate of 1 mL/min. To prove the potential of hydrophobic microdomain formation, the CMC values of both amine terminated PBA micelles in aqueous medium were determined using fluorescence technique (Perkin Elmer- LS50) based on partitioning of pyrene as a probe in hydrophobic and aqueous phase at 25 C as described previously [40]. CMC values are expressed as mean ± SD, which is related to three measurements.

2.11. Nanoparticle characterization Mean diameter and zeta potential of micelles and nanogels were determined using a zetasizer (Nano ZS, Malvern Instrument, UK) at 25 C. The samples were diluted to ensure free diffusion and unhindered Brownian motion of the particles. The DLS results were the mean of five test runs and the zeta potential were the mean of three test runs. The morphology of the nanogels was evaluated using transmission electron microscopy (TEM) technique (LEO 906, Germany). The nanogels were diluted with deionized water and placed on a copper grid covered with carbon film. Observation was done at 100 kV. To investigate surface morphology of degradated nanogels, scanning electron microscopy (SEM) (HITACHI S 4160) was employed. The samples were sputtered with gold before observation. X-ray diffraction pattern (XRD) analysis was used to confirm the formation of crosslinked structure of shell part of the nanogels. An X-ray diffractometer (XRD, X-pert Philips, Model No: PW 3040/60) was used for structural phase identification and crystallinity with Cu Ka (l ¼ 1.54056 A ) radiation over Bragg angles ranging from 0 to 60 .

2.12. Drug loading and encapsulation efficiency The amount of LPE entrapped within the nanogels was determined by UV spectrophotometry (Perkin Elmer, lambda 800). 1 mg of freeze-dried LPE-loaded nanogels was swelled in acetone. Then the solution was centrifuged at 10,000 rpm for 10 min, and the supernatant was analyzed by measuring the UV absorbance at 238 nm using an established calibration curve in drug/acetone solutions. At this wavelength there was no interference in the absorbance reading from the PBA and chitosan. 2.13. In-vitro release experiments The in-vitro drug release profile for drug loaded nanogels were performed in sodium phosphate buffer solution (PBS, pH 7.4). At regular time intervals, an aliquot (1.5 ml) was removed from release media, replaced with fresh PBS and measured at 265 nm according to calibration curve of LPE in PBS using UV spectrophotometry. 2.14. Mucoadhesion measurement Mucoadhesive property of nanogels was evaluated by mucin particle method [41]. Briefly, the commercially available porcine mucin was hydrated in deionized water at 4 C overnight. After that, the mucin solution was adjusted to pH 7.4 with 1 M NaOH and diluted with 0.1 M phosphate buffer (pH 7.4) to the working concentration (1% w/v) for further use. For the mucoadhesion study, the precisely controlled submicron-sized mucin suspension was prepared by ultrasonication until the mucin particle size was smaller than 1 mm. It was then centrifuged at 4000 rpm for 20 min; the supernatant portion was filtered through 0.80 mm of cellulose acetate membrane filter. The filtrate was collected and the particle size and zeta potential of mucin was measured. Various concentrations of PBA(1)-g-Cs nanogel were prepared and filtered through 0.80 mm of cellulose acetate membrane filter before use. Equal volumes of each nanogel solution and the mucin particles suspension were mixed and incubated at 37 C for 2 h. Thereafter the extent of change in zeta potential and mean particle size of the mixtures was measured by Zetasizer. Each test was measured in triplicate. 2.15. Biodegradation study of nanogels In vitro hydrolytic degradation of prepared nanogels was carried out in PBS solution (pH ¼ 7.4) at 37 C. The samples were weighted and then incubated in PBS solution in a thermostat water bath at 37 C with gentle shaking for 3 weeks. At various time points, the samples were removed, washed with distilled water and allowed to dry in air to constant weight. Based on the sample weight before and after degradation, the average percentage of mass loss was calculated. The hydrolytic degradation is reported as the weight percentage. The microscopic surface morphology of the PBA(1)-gCs nanogels during degradation was also investigated with SEM. 2.16. Cell culture and in vitro cytotoxicity study Female New Zealand white rabbit weighing 3e4 kg was used for corneal epithelium cell culture. Isolation and cultivation of rabbit corneal were accomplished according to previously published protocol [42]. The cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco #15140122) supplemented with 10% (v/v) fetal bovine serum and 1% (w/v) penicillin/streptomycin (100 U/mL penicillin and 0.1 mg/mL streptomycin). The cells were grown in 5% CO2 at 37 C. The RCEC were seeded in 96-well plates (5000 cells per well) with culture medium in 5% CO2 at 37 C overnight to


adhere. Cells were treated with different concentration of nanogels (0, 0.01, 0.1, 0.5, 1 mg/ml) for 24 h at 37 C. Subsequently, the medium was removed and gently washed with PBS (pH ¼ 7.4). A total of 100 mL of MTT (5 mg/mL in serum free DMEM medium) was supplied to each well followed by incubation at 37 C for 4 h to allow the soluble yellow MTT to be reduced into dark-blue insoluble formazan crystals by the metabolically active cells. The formazan crystals were subsequently dissolved by the addition of dimethylsulfoxide at room temperature with shaking for 20 min. Finally, the optical density (OD) of individual wells was measured at 570 nm in a microplate reader (Anthos 2020; Anthos Labtec Instruments, Wals, Austria). Nontreated cells assumed to possess 100% viability were used as control. Cell viability was expressed as the mean of three test runs ±SD. 3. Results and discussion 3.1. Nanocarrier preparation and characterization 3.1.1. Polyester diols synthesis PBA diols with two different molecular weights of approximately 5000 and 8000 g/mol were synthesized by the direct polycondensation reaction between adipoyl chloride and butylene glycol at 85 C for 24 h (Scheme 1, step 1). The structures were confirmed by FT-IR spectroscopy which showed polyester typical stretching absorption bands at 2954 cm1 (CH2), 1724 cm1 (C]O), 1256 cm1 (CeOeC) and 1163 cm1 (CeO) according to the literatures [38] (data not shown). The molar mass averages of the number (Mn) and weight (Mw) and poly dispersity index (PDI) of the synthesized polyesters were

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determined by GPC. The obtained polyesters had relatively low polydispersities (Table 1). 1H NMR (CDCl3) d ppm: 1.6 (m, 8H, inner methylenic protons: eCH2eCH2e); 2.3 (t, 4H, eCH2eC]O: methylene protons adjacent the carbonyl group); 4.1 (t, 4H, eOeCH2e) (data not shown individually).

3.1.2. Synthesis of dendrimerized amine terminated polyester The final dendrimerized structure was obtained in 5 steps. First, G0.5 was prepared by introducing acrylate segments into the OH terminus groups using acryloyl chloride as shown in Scheme 1, step 2. The appearance of the adsorption bands at 1645 cm1 and 813 cm1 assigned to C]C stretching vibration corroborates the complete conversion of hydroxyl groups to acrylate groups by reacting with acryloyl chloride (Fig. 1a). In addition, 1H NMR spectrum depicted in Fig. 2a elucidates the major characteristic peaks of vinylic groups in the range of 5.8e6.4 ppm which confirms the formation of (ACePBAeAC). In the second step (Scheme 1, step 3), G0.5 was reacted with diethanolamine by Michael-addition reaction between terminal acrylate parts and eNHe group of diethanolamine to produce ((OH)2ePBAe(OH)2). As illustrated in Fig. 1b, the presence of a wide absorption signal at 2400e3500 cm1 belonging to the hydroxyl end groups and the disappearance of 1645 cm1 band indicates almost full conversion of the vinyl group. As shown in Scheme 1, step 4, G1.5 was synthesized with the same reaction procedure for G0.5 preparation and was fully characterized by FT-IR and 1H NMR. No significant differences between characterization results of G0.5 and G1.5 were observed. (The results were the same as G0.5). Subsequently, Michael addition reaction was occurred between ((AC)2ePBAe(AC)2) and N-boc

Scheme 1. Synthetic procedure for preparation of dendrimerized PBA (left), S-Cs (a) and shell crosslinked nanogels (b) (The dashed line shows the amide bond formation between S-Cs molecules.)

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Table 1 The characteristic properties of synthesize polymers, polymer NPs and nanogels. Polymer

Mna (g/mol)

Mwa (g/mol)

PDIa

CMCb (mg/ml)

Sizec (nm)

PBA (1) PBA (2) PBA(1)-g-Cs PBA(2)-g-Cs

5500 8300 e e

7600 12,000 e e

1.38 1.4 e e

1.07 ± 0.02 0.782 ± 0.01 e e

15 36 46 93

a b c d

± ± ± ±

3.4 2.7 4.1 3.6

PDIc

Zeta potential (mV)c

Sized (nm)

DL%

EE%

Drug/copolymer ratio in feed (%)

0.25 0.2 0.3 0.3

25 23 12 14

10 34 40 82

e e 6 6.7

e e 67 70

e e 10 10

± ± ± ±

0.22 0.34 0.18 0.26

Determined using GPC (polystyrene standards). Data obtained from pyrene fluorescence probe technique. Data determined using a zetasizer (Nano ZS, Malvern Instrument, UK) at 25 C. Data presented as mean ± SD, n ¼ 5 and n ¼ 3 for size and zeta potential, respectively. Data determined by TEM.

ethylenediamine followed by deprotection reaction using TFA in order to prepare the final amine functionalized product ((NH2)2ePBAe(NH2)2) (Scheme 1, step 5). As shown in Fig. 1(c), the wide band at 3000e3350 cm1 is assigned to the NH2 stretching vibrations. The 1H NMR assignments of G2.0 were characterized as follows: 1 H NMR (CDCl3), d ppm: 1.6 (m, 8H, H-b); 2.3 (t, 4H, H-a); 2.45e2.66 (t, 8H, H-e,I,k,l); 2.95e3.04 (t, 4H, H-g,j); 3.8 (t, 2H, H-f); 4.1 (t, 4H, H-c); 4.28 (t, 2H, Heh) (Fig. 2b). The lack of proton signals at 1.37 ppm attributed to CH3 groups of tert-butyl in N-boc-ethylenediamine structure and 5.8e6.4 ppm assigned to double bands indicate the complete Michael addition and deprotection reactions.

3.1.3. Synthesis of N-succinyl-chitosan (S-Cs) S-Cs was prepared by the introduction of succinyl groups into the N-terminus of glucosamine units in chitosan (Scheme 1a). The succinylation reaction involves a condensation reaction between the polysaccharide amine group and the electrophilic carbonyl group of the anhydride, with the formation of an amidic bond by opening the anhydride ring. The degree of substitution (DS) completely depends on the reaction conditions. Herein, both the structure and DS were confirmed by 1H NMR (Fig. 2c). The 1H NMR result of S-Cs displays a broad peak at 2.43 ppm attributed to a combination of two methylene units (shown as (A)) in succinic anhydride. The ratio of this peak intensity to that of the C2 (~3.00 ppm) proton of Cs (Shown as (B)) was taken to calculate DS. According to the reaction conditions here, the calculated DS was found to be approximately 55%. 3.1.4. Determination of critical micelle concentration (CMC) The amphiphilic poly(amino-ester)-PBA-poly(amino-ester) ((NH2)2ePBAe(NH2)2) with hydrophilic dendrtic amino-ester blocks and hydrophobic linear PBA block can self-associate to form micelle like self-aggregates in an aqueous environment. To investigate the self-aggregation behavior of PBA micelles in an aqueous medium, pyrene was used as a fluorescence probe. When exposed to a polymeric micelle aqueous solution, pyrene molecules preferably participated into the hydrophobic microdomains of micelles rather than the aqueous phase. Combined with strong fluorescence illumination of pyrene in a non-polar environment, the localization, showed different photophysical characteristics depending on the concentration of micelle forming materials. Therefore, at a low concentration (C < CMC), there were negligible changes in total fluorescence emission intensity (at 375 nm). As the concentration increased, a remarkable increase of the total fluorescence intensity was observed for both PBA structures. Fig. 3 displays the ratio of intensities (I386/I375) from pyrene emission spectra as a function of log C. The CMC values of PBA micelles were in the range of 0.78e1.07 mg/mL. Depending on hydrophobic/hydrophilic balance resulting from different PBA block lengths, increasing the length of the hydrophobic portion of a micelle will lead to a decrease in its CMC. Hence, the CMC value for PBA (2) is smaller than that of PBA (1).

Fig. 1. FT-IR spectra of G0.5 (a); G 1.0 (b); G 2.0 (c).

3.1.5. PBA nanoparticles preparation The main purposes of this work were the preparation of PBA-Cs conjugate nanogels containing LPE by the reaction between carboxylic acid and amine-terminated groups in S-Cs and PBA NPs, respectively. PBA nanoparticles with amine surface functional groups were prepared by a single oil-in-water (O/W) emulsion/ solvent evaporation method. Parameters such as the amount of emulsifier, the output power and time of the sonicator were tested previously in order to obtain proper particle size and release profiles [38]. The zeta potentials, particle size and morphology of the


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Fig. 2. 1H NMR spectra of G0.5 (a), G2.0 (b), S-CS (c) (DS: degree of succinylation).

synthesized PBA NPs were studied by DLS and TEM, respectively (Table 1). In a number-averaged DLS curve, the PBA nanoparticles gave nearly a single peak with an average hydrodynamic radius between 15 and 36 nm, depending on the molecular weights of PBAs. It is clear that the obtained micelle size increased with increasing hydrophobic PBA block length. In addition, both of them result in micelles possessing low PDI values (~0.2) implying a narrow micelle size distribution. Zeta potential of micelles was measured, as indicated in Table 1. It was shown that both formulations have negative surface charges around 23 mV. As TEM provides the size distribution of dehydrated particles and DLS measurements yield an ensemble average of the particle size in solution, the average sizes of the dried PBA NPs observed by TEM were from 10 to 34 nm for PBA (1) and (2), respectively.

Fig. 3. The intensity ratio I386/I375 of the excitation spectra of pyrene in PBA solutions vs PBA concentration to determine CMC of PBA (1) (a) and PBA (2) (b) (Data were expressed as mean ± SD, n ¼ 3).

3.1.6. Nanogel preparation The synthetic procedure for Cs-g-PBA graft copolymers is shown in Scheme 1b. The EDC/NHS proceed reaction to covalently link a carboxylate (eCOOH) with an amine (eNH2) group. In this study, the amine surface functional groups of PBA NPs (~8000 and 5000 Da) were reacted with carboxyl group of S-Cs (3000 Da) by carbodiimide chemistry. According to previous studies [43,44], low-molecular-weight chitosan has lower H bonds than mediumand high-molecular-weight chitosan. Furthermore, the intermolecular hydrogen bonding interactions of S-Cs were weakened as compared with chitosan [45]. Increasing molecular-weight causes low solubility and high viscosity because of strong intermolecular H bonds. As a result, the use of low molecular weight S-Cs will increase the grafting efficiency. Once being activated by EDC/NHS known as ‘zero-length’ crossing-linker since the amide linkages are formed without leaving a spacer molecule [46], S-Cs reacted with PBA NPs to create the graft copolymer. The feed composition ratios were decided to be 1:1:0.4:0.4 for PBA: S-Cs: EDC: NHS. For the

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activation step, the molar ratio of EDC and NHS are equivalent and are less than that of S-Cs. Moreover, the pH is adjusted around 4.2, because low pHs decrease the H bonds due to the protonization of amine groups, hence, chain folding was reduced. Accordingly, gelation is avoided during the activation step. After adding PBA NPs, the graft into copolymerization occurred via amide bond formation. Most likely, not reacted activated carboxylic acid groups of S-Cs can form amide bond with free amine groups of S-Cs leading to shell crosslinked micelle structures. Different studies based on emulsion technique for preparation of biocompatible nanoparticles have been reported [47e49]. The FTIR spectra of S-Cs and PBA-g-Cs are shown in Fig. 4. Characteristic peaks of S-Cs are 3360 cm1 (OeH stretch overlapped with NeH stretch), 2920 and 2874 cm1 (CeH stretch), 1728 (C]O stretch of carboxylic acid), 1642 cm1 (amide II band, CeO stretch of acetyl group), 1543 cm1 (amide II band, NeH stretch), 1420e1377 cm1 (asymmetric CeH stretch bending of CH2 group) and 1063 cm1 (skeletal vibration involving the bridge CeO stretch) of glucosamine residue (Fig. 4a). The appearance of signals at 15501690 cm1, corresponding to amide and amine bands and a broad signal at 30003700 cm1 for the OeH and NeH stretch confirms the successful conjugation of PBA NPs and S-Cs (Fig. 4b). The particle size, zeta potential and morphological characteristics of obtained nanogels were examined using zeta sizer and TEM, respectively (Table 1). The mean hydrodynamic diameter of particles and their distribution after the grafting polymerization observed by DLS were 46 nm and 93 nm for PBA(1)-g-Cs and PBA(2)-g-Cs, respectively. For both, the decrease in negative surface charge indicates the entering of chitosan chains into PBA NPs. The TEM micrographs (Fig. 5a,b) clearly reveal the formation of well-separated and spherical nanoparticles having the well-known coreeshell nano-structure with a hydrophobic core of PBA and hydrophilic shell of Cs with size of 40 nm and 82 nm for PBA(1)-gCs and PBA(2)-g-Cs, respectively. We could observe a dark core and a relatively bright contrast region on the edge, which is typical of coreeshell particle morphology.

3.1.7. Determination of shell-crosslinked structure As illustrated in Fig. 5c,d, the WAXD peaks of pure PBA are seen at 2q ¼ 21.24 [a (110)], 22.26 [a (020)] and 24.42 [a (021)] which confirms its crystalline structure [50]. According to literatures, chitosan has two different peaks at 2q ¼ ~10 and 2q ¼ ~20 . The peak at 10 was assigned to the crystal form I and the strong peak at 20 was assigned to form II [51]. This is because the structure of chitosan molecules has certain regularity which can form crystalline regions very easily [52]. But, for succinated chitosan, the peak at 10 disappears and the reflection at 20 also significantly decreases [53]. This may occur due to the destruction of intermolecular hydrogen bonds between amine groups and hydroxyl groups of the native chitosan [54]. The Cs-gPBA graft copolymer displayed a much weaker diffraction at 2q ¼ 21.44 and 24.4 which represent the (110) and (020) reflections, respectively. Furthermore, two weak and broad peaks at 2q ¼ 22 (overlapped with (110) reflection) and 2q ¼ 42 , specify the structure change of S-Cs as the shell part of nanogel from crystalline to amorphous nature. The less peak intensity could not only be attributed to the presence of small PBA nanoparticles, but also means that large amount of S-Cs had been grafted on PBA nanoparticles. 3.2. Drug loading and encapsulation efficiency We next calculated the amount of drug in the nanogels in terms of drug loading (DL%) (Eq (1)) and encapsulation efficiency (EE%) (Eq (2)). It is well known that the hydrophobicity and molecular weight of the core-forming block have a great influence on the size and the drug loading into the core of the micelle. Hence, more amounts of drug can be incorporated into micelles formed from copolymers having larger core-forming block in comparison to a shorter hydrophobic block which leads to larger DL% and EE%. As a proof of concept, PBA(1)-g-Cs had a drug loading content of 6% indicating an encapsulation efficiency of 67%. In contrast, drug loading content for PBA(2)-g-Cs was calculated to be 6.7% reflecting a 70% encapsulation efficiency. It can be concluded that by increasing the molecular weight of hydrophobic segment, the DL% and EE% improved to some extent (Table 1).

DL% ¼

weight of encapsulated drug *100 total weight of the loaded nanoparticles

(1)

EE% ¼

weight of encapsulated drug *100 weight of feeding drug

(2)

In comparison with previous study [55], it is clearly concluded that increase in the number of interactions between drug and the polyester play an important role to have appropriate drug loading. As a result, the presence of poly(amino-ester) block causes more interactions between the hydroxyl and ester groups of LPE and the functional groups of the polyester in the nanoparticle structure. In addition, the cavities inside the dendritic structure can be useful to situate the hydrophobic drug. Hence, the drug-polymer affinity and drug-loading content were improved significantly. 3.3. In-vitro release experiments

Fig. 4. FT-IR spectra of S-Cs (a) and PBA-g-Cs (b).

In vitro release studies of the developed nanogels were carried out for 50 h and were compared with marketed conventional eye drop (Fig. 6). The nanogel formulations showed a two-step release pattern: one initial burst release followed by a second slow-release phase (extended release). An initial burst release is beneficial, helps to achieve the therapeutic concentration of drug in minimal time followed by constant release to maintain sustained and controlled


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Fig. 5. TEM photographs of the self-assembled PBA(2) nanoparticles (a) and PBA(2)-g-Cs nanogels in aqueous solution (b); XRD patterns of PBA(2) (c) and PBA(2)-g-Cs nanogel (d).

3.4. Mucoadhesion measurement In this study, the mucin particle method as a simple mucoadhesion test for polymers was used to predict the in vivo absorption of synthesized nanogels. As shown in Fig. 7, the changes in particle size and zeta potential of mucin original particles demonstrate a strong interaction between nanoparticles and mucin. The mucoadhesive property of chitosan is determined by the formation of either secondary chemical bonds such as hydrogen bonds or ionic interactions between the positively charged amino groups of chitosan and the negatively charged sialic acid residues of mucins, depending on environmental pH. The mucoadhesive performance of chitosan is significantly higher at neutral or slightly alkaline pH as in the tear film [13]. Hence, due to the presence of CS chains in shell part of nanogels structure, they showed a high affinity to mucin particles to cover their surfaces. The higher the concentration of nanogels, the more extensive the changes were in the zeta potential and particle size of mucin particles. Consequently, the capacity of nanogels to interact with the mucosal surface can be useful to prolong the contact time of drug delivery system on the ocular surface. Fig. 6. In vitro drug release profile of loteprednol etabonate loaded nanogels and marketed formulation (Data were expressed as mean ± SD, n ¼ 3).

3.5. Biodegradation study of nanogels

release of the drug. In addition, the rate of drug release is almost similar in the first stage, but it is influenced by the PBA degradation rate in the second stage. This is because of the fact that the rate of diffusion of the drug in and out of the micelle is greatly influenced by the properties of the micelle core. In other words, assuming that the hydrophilic block length is held constant, as the molecular weight of PBA block and the core size increases, the overall release rate of the entrapped agent decreases. In contrast, the marketed formulation released nearly 95% of the drug within 10 h whereas the release were sustained in nanogels and 42e57% of the drug were released in 50 h for PBA(2)-g-Cs and PBA(1)-g-Cs, respectively.

Biodegradation of PBA(1)-g-Cs nanogels was carried out at 37 C in PBS solution during 3 weeks. The weight losses of the samples at specified time points were measured gravimetrically in the degradation process for determining the biodegradability of the nanogels. The biodegradation process of aliphatic polyesters such as PBA in an aqueous medium takes place through hydrolytic scission of the ester groups which leads to the creation of monomeric and oligomeric water-soluble degradation products [56]. Degradation of semi-crystalline polyesters in aqueous media occurs in two steps. First water penetrates the amorphous, less ordered domains and then, when the amorphous regions are already degraded, the crystalline domains are attacked [57]. Chemical structures that allow water to diffuse into the polymer experience a

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Fig. 7. Particle size and zeta potential of mucin particles when mixed with various concentrations of PBA(1)-g-Cs solutions at pH ¼ 7.4 (Data were expressed as mean ± SD, n ¼ 3).

initial value which is more than pure PBA [56]. In addition to residual weight percent measurement, the surface morphology of incubated nanoparticles was also studied. The SEM images of degraded nanogels after 3 weeks’ degradation process are shown in Fig. 9. According to the images, the number of caves or channels increased with increasing degradation time. 3.6. Cell culture and in vitro cytotoxicity study

Fig. 8. Weight retention of PBA(1)-g-Cs nanogels as a function of incubation time at 37 C in phosphate buffered saline (pH ¼ 7.4) (Data were expressed as mean ± SD, n ¼ 3).

faster degradation than more hydrophobic structures [58]. In our study, the presence of two hydrophilic segments, the dendritic poly(amino-ester) block and chitosan shell, which are connected to the hydrophobic polyester, allow water to diffuse into the polyester matrix even in the inner regions, increasing the degradation rate. As shown in Fig. 8, after 21 days its weight reached about 90% of the

Excised central corneas from freshly killed albino rabbit were used to prepare the primary cultures of corneal epithelial cells. It is important to confirm that newly synthesized nanogels are not producing any cytotoxicity on ocular administration. The biocompatibility of synthesized nanogel suspensions with different concentrations were evaluated by MTT assay in triplicate using rabbit corneal epithelium cells. As shown in Fig. 10, the cell viability decreases with increasing the nanogel concentration. At or below 1 mg/mL, cell viability was approximately 90% following 24 h incubation with nanogel suspensions. As these nanoparticles possess relatively low cytotoxicity, they can therefore be considered as effective carriers for topical ocular drug delivery. 4. Conclusion Two series of shell crosslinked nanogels with different

Fig. 9. SEM images of degraded PBA(1)-g-Cs nanogels, after 5 (a), 10 (b), 15 (c) and 21 (d) days incubation in PBS (pH ¼ 7.4, at 37 C).


Fig. 10. Variation of cellular viability of the RCEC after 24 h incubation with nanogels with different concentrations (The viability of cells incubated in free nanogel medium as control was taken as 100% and all data were expressed as mean ± SD, n ¼ 3).

molecular weights of hydrophobic polyester were successfully synthesized by the amide bond formation reaction. Carboxylic acid groups of succinated chitosan were reacted with amine terminated groups on the surface of PBA nanoparticles in water without using any crosslinker at room temperature. FT-IR and XRD analysis confirmed the grafting and self-crosslinking of chitosan to PBA nanoparticle, respectively. The well-separated and spherical mucoadhesive nanogels showed appropriate size, drug loading and encapsulation efficiency. The hydrophilic modification of polyester block at both ends could significantly influence the mentioned properties (DL% and EE %) due to the increase of drug-polymer interactions. Drug releases profiles also indicate that drug release from both formulations showed a two-step release pattern: (i) An initial burst release which is necessary to achieve desired therapeutic concentration of drug in minimal time and (ii) a second slow-release phase followed by sustained and controlled release of the drug. Interestingly, the rate of drug release is almost similar in the first stage, while, it is influenced by the PBA degradation rate in the second stage. They also showed strong ability to interact with mucin through electrostatic/hydrophobic interactions that highlight their potential as mucoadhesive carriers for LPE delivery. In vitro biodegradation study of nanogels revealed that the weight loss of the nanogel was higher than that of pure PBA within the time range investigated in this study, which is related to hydrophilic parts incorporated in nanogel structure. The degradation process has also been observed by SEM which confirmed the hydrolysis of long chain molecules into shorter ones. In vitro cytotoxicity study revealed the low cytotoxicity of prepared nanogels. The results of the present study indicate that this formulation approach may have potential for topical ocular drug delivery. Acknowledgement Sincere thanks are given to Prof S. Khoei and his group in Iran University of Medical Sciences (IUMS), Tehran, Iran, for generously supporting our cell viability assay experiments. References [1] F. Behar-Cohen, Drug delivery to target the posterior segment of the eye, Med. Sci. Paris. 20 (2004) 701e706. [2] S. Duvvuri, S. Majumdar, A.K. Mitra, Drug delivery to the retina: challenges and opportunities, Expert Opin. Biol. Ther. 3 (2003) 45e56. [3] S.K. Sahoo, F. Dilnawaz, S. Krishnakumar, Nanotechnology in ocular drug delivery, Drug Discov. Today 13 (2008) 144e151. [4] R. Pignatello, C. Bucolo, G. Spedalieri, A. Maltese, G. Puglisi, Flurbiprofenloaded acrylate polymer nanosuspensions for ophthalmic application,

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