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Antioxidant Gallic Acid-Functionalized Biodegradable in Situ Gelling Copolymers for Cytoprotective Antiglaucoma Drug Delivery Systems Jui-Yang Lai*,†,‡,§,⊥ and Li-Jyuan Luo† †

Institute of Biochemical and Biomedical Engineering, ‡Biomedical Engineering Research Center, and §Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China ⊥ Center for Tissue Engineering, Chang Gung Memorial Hospital, Taoyuan, Taiwan 33305, Republic of China S Supporting Information *

ABSTRACT: In clinical ophthalmology, oxidative stress has been proposed as the initiating cause of ocular hypertension, which is one of the risk factors for glaucomatous damage and disease progression. In an attempt to improve the therapeutic efficacy of intracamerally administered pilocarpine, herein, a cytoprotective antiglaucoma drug delivery system composed of antioxidant gallic acid (GA)-functionalized gelatin-g-poly(N-isopropylacrylamide) (GN) biodegradable in situ gelling copolymer was developed for the first time. Analyses by UV−vis and Fourier transform infrared spectroscopies showed the formation of biopolymer−antioxidant covalent linkages in GNGA structures through a radical reaction in the presence of water-soluble redox initiators. The synthesized GNGA polymers with strong free radical scavenging effectiveness exhibited appropriate phase transition temperature and degradation rate as injectable bioerodible depots for minimally invasive pilocarpine delivery to the ocular anterior chamber. During the 2-week in vitro study, the sustained releases of sufficient amounts of pilocarpine for a therapeutic action in alleviating ocular hypertension could be achieved under physiological conditions. Results of cell viability, intracellular reactive oxygen species level, and intracellular calcium concentration indicated that the incorporation of antioxidant GA into GN structure can enhance cytoprotective effects of carrier materials against hydrogen peroxide-induced oxidative stress in lens epithelial cultures. Effective pharmacological responses (i.e., reduction of intraocular pressure and preservation of corneal endothelial cell morphology and density) in rabbits receiving intracameral GNGA injections containing pilocarpine were evidenced by clinical observations. The findings of in vivo studies also support the hypothesis that the GNGA carriers are more advantageous over their GN counterparts for the improvement of total antioxidant status in glaucomatous eyes with chronic ocular hypertension. The synthesized multifunctional molecules may be further used as potential polymer therapeutics for intraocular delivery of bioactive agents.

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INTRODUCTION Clinically, the elevated intraocular pressure (IOP) is one of the biggest risk factors for the diagnosis of glaucomatous progression.1 Although topical ocular drug delivery represents a convenient treatment modality with a significant impact on the management of glaucoma and intraocular hypertension, the prescribed eye drops need to be instilled several times daily because of short precorneal residence time, poor corneal penetration, and low ocular bioavailability. To overcome the limitations of this conventional ophthalmic formulation, researchers have designed new types of eye drops with enhanced drug delivery performance. Yang et al. have reported © XXXX American Chemical Society

a hybrid polyamidoamine dendrimer hydrogel/poly(lactic-coglycolic acid) nanoparticle platform for codelivery of two antiglaucoma drugs and found that this novel formulation is capable of sustaining effective IOP reduction for 4 days following the administration of one eye drop in rabbits.2 More recently, Natarajan et al. have developed a latanoprostencapsulated liposomal nanocarrier that achieves long-acting (i.e., 120 days) IOP-lowering effect in a diseased nonhuman Received: June 27, 2015 Revised: August 4, 2015

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DOI: 10.1021/acs.biomac.5b00854 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. Schematic representation of development of GA-modified GN as a novel multifunctional polymer carrier.

primate model.3 In our laboratory, the gelatin and poly(Nisopropylacrylamide) (PNIPAAm) graft copolymers are synthesized to be used for the preparation of a new intracameral drug delivery system.4 As compared to either eye drop instillation or free drug injection, the intraocular administration of proposed biodegradable in situ gelling composition containing pilocarpine can more effectively improve the therapeutic outcomes for glaucomatous rabbits. To provide an extended drug release pattern, the carboxylterminated PNIPAAm with a long chain length obtained by free radical polymerization in the presence of small amounts of mercaptoacetic acid is further grafted onto gelatin backbone molecule to slow enzymatic degradation of copolymeric carriers.5 Although the application of biomaterial carriers for ocular administration of antiglaucoma medications is found to be promising, it is generally recognized that oxidative stress may play a crucial role in IOP elevation. As reported in the literature, the ocular anterior chamber tissues are very sensitive to oxidative damage, triggering the pathogenic cascade associated with glaucoma.6 Oxidative stress signals induced by the formation of multiple reactive oxygen species (ROS) leads to decrease in local antioxidant activity, thereby contributing to increased outflow resistance in glaucomatous eyes.7 In addition, the nitric oxide levels in the aqueous humor of glaucomatous eyes are significantly higher than those of the normal eyes.8 For the treatment of glaucoma, pilocarpine is the most commonly used cholinergic agent that exhibits pharmacological effects (i.e., miosis and IOP reduction) but may not have the ability to act as a free radical scavenger and provide powerful protection against oxidative stress. This motivates us to develop an antioxidant polymeric system for intraocular delivery of antiglaucoma medications.

Gallic acid (GA), 3,4,5-trihydroxybenzoic acid, is a polyphenyl natural product that can be found in plants such as gallnut and green tea.9 It shows many biological activities such as antibacterial, antiviral, antiinflammatory, antitumorigenic, antimelanogenic, and antioxidant effects10 and therefore has attracted much attention in pharmaceutical and biomedical fields over the years. Recently, Stoddard et al. have demonstrated that GA is effective at quenching ROS in human corneal limbal epithelial cells, suggesting its potential use in protecting the corneal epithelium from oxidative damage involved in dry eye disease.11 Here, we consider the incorporation of GA molecules into the biodegradable in situ gelling copolymers to fabricate antioxidant intracameral drug delivery systems. The synthesis of antioxidant polymers by grafting of GA onto gelatin has been previously reported by the group of Iemma.12 Later, their work is extended to show that the macromolecular GA−gelatin conjugate retains the antioxidant, enzymatic, and anticancer activities of free GA.13 On the basis of these encouraging findings, we propose to synthesize a novel multifunctional GNGA polymer in which thermoresponsive PNIPAAm segments and antioxidant GA molecules are attached to the biodegradable backbone gelatin networks by covalent bonding (Figure 1). The biofunctionalization of gelatin-g-PNIPAAm (GN) with GA is achieved by radical reaction in the presence of hydrogen peroxide/ascorbic acid redox pair as an initiator. The generated hydroxyl radicals attack the residues in the protein side chains of the GN copolymers able to undergo oxidative addition processes. The resultant radical species further react with GA molecules to yield the multifunctional polymers through the formation of biopolymer−antioxidant covalent bonds. Given that disease progression strongly correlates with the local oxidative damage in the anterior chamber of glaucomatous patients, this study aims to develop an antioxidant formulation B

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capped PNIPAAm measured by end-group titration method was 7407 ± 570 Da. By means of carbodiimide chemistry, the GN was synthesized by grafting of carboxylic end-capped PNIPAAm onto the aminated gelatin.5 In brief, 10 g of carboxylic end-capped PNIPAAm was dissolved in 50 mL of MES buffer containing 2.59 g of EDC and 1.55 g of NHS under agitation for 6 h, followed by addition of 50 mL of MES buffer containing 1 g of aminated gelatin. The reaction was allowed to proceed at 25 °C for 24 h. Then, the reaction product was precipitated at 50 °C, followed by centrifugation and resuspension in deionized water. To remove unreacted components, the solution was exhaustively dialyzed (MWCO 50 000, Spectra/PorDialysis Membrane, Rancho Dominguez, CA, USA) against deionized water at 4 °C for 4 days. The GN graft copolymer was lyophilized at −50 °C and kept in a closed vessel at room temperature. Synthesis of GA-Functionalized Gelatin-g-PNIPAAm (GNGA). The synthesis of GNGA polymers was established with some modifications of the protocol used for grafting of GA onto gelatin.12 An aqueous solution was obtained by dissolution of 0.5 g of GN in 50 mL of deionized water. Then, 0.25 g of ascorbic acid and 1 mL of hydrogen peroxide were added to the above solution and kept at 25 °C for 2 h. Following the addition of 60 mg of GA, the reaction mixture was allowed to agitate for 24 h. To remove unreacted components, the solution was exhaustively dialyzed against deionized water at 4 °C for 4 days. The purified product was lyophilized at −50 °C and kept in a closed vessel at room temperature. Characterization of GA-Functionalized Gelatin-g-PNIPAAm (GNGA). To investigate the formation of biopolymer−antioxidant covalent bonds, the UV−vis spectroscopic analyses were performed. The GA, GN, and GNGA solutions (10% w/v) were prepared by dissolving the solutes in deionized water. The absorption spectra were recorded using a UV−vis spectrophotometer (Thermo Scientific, Waltham, MA, USA) operating in a spectra range of 200−500 nm. The UV absorption properties of GNGA samples were measured using GN spectrum as baseline. Difference spectra were calculated by subtracting the baseline spectrum. On the other hand, the FTIR spectroscopy of various samples was performed using a FT-730 ATRFTIR spectrophotometer (Horiba, Japan) according to the previously published method.14 The spectra were recorded between 3700 and 800 cm−1 with a resolution of 8 cm−1. Determination of Scavenging Activity against DPPH Radical. To evaluate the free radical scavenging activity of the synthesized product, the DPPH method was used. Aqueous GN and GNGA solutions were, respectively, obtained by dissolution of 250 mg of polymers in 12.5 mL of deionized water, followed by addition of equal volume of an ethanol solution containing 1 mg of DPPH radical. After incubation at 25 °C for 30 min, the absorbance of the resulting solution was measured at 517 nm using a UV−vis spectrophotometer (Thermo Scientific). The DPPH scavenging activity (%) was calculated as ((A0 − A1)/A0) × 100, where A0 is the absorbance of blank DPPH solution that was used under the same reaction conditions in the absence of synthesized polymers, and A1 is the absorbance of DPPH solution in the presence of polymer samples. Results were the average of five independent measurements. Phase-Transition Characterizations. The phase-transition property of each test sample was investigated using a DSC 2010 differential scanning calorimeter (TA Instruments, New Castle, DE, USA). The GNGA polymers were dissolved in deionized water or BSS to a concentration of 10% (w/v). After equilibration at room temperature for 1 h, the solutions (8 mg) were hermetically sealed in aluminum pans for DSC experiments. Programmed heating was carried out at 3 °C/min in the temperature range from 25−45 °C under a nitrogen gas flow. The lower critical solution temperature (LCST) was determined as the onset point of the endothermic peak. Results were averaged on four independent runs. The gross observations were also made by heating the GNGA polymers dissolved in BSS (10% w/v) from 25 to 34 °C (i.e., the aqueous humor temperature). In Vitro Degradation Tests. To measure the extent of degradation, the GNGA solutions (10% w/v) were prepared by dissolution at 25 °C and were transferred to a 34 °C thermostatically

that is suitable for use in the intraocular sustained release depot of pilocarpine. We hypothesize that the multifunctional GNGA polymer carriers composed of biodegradable and thermoresponsive GN and antioxidant GA are more advantageous over GN counterparts for the improvement of total antioxidant status in aqueous humor. To the best of our knowledge, the synthesis, characterization, and application of GNGA for a therapeutic antiglaucoma drug delivery system have not yet been reported in the literature. The GNGA polymers were prepared by redox technique at room temperature. Subsequently, the grafting reaction of GA onto GN was monitored by ultraviolet−visible (UV−vis) and Fourier transform infrared (FTIR) spectroscopies. The radical-scavenging activities of synthesized polymers were measured by using 2,2′-diphenyl-1picrylhydrazyl (DPPH) method. The effects of functionalization with GA molecules on the thermoresponsive and biodegradable properties of GN materials were investigated by determinations of phase-transition temperature and weight loss. In addition, the drug encapsulation levels and release profiles were evaluated in vitro under physiological conditions. The antioxidant activity of multifunctional GNGA polymers against oxidative stress was assessed using hydrogen peroxideinduced oxidative stress of human lens epithelial (HLE) cell model. After 24 h of pretreatment with test materials and a further incubation with hydrogen peroxide for 24 h, cell viability, intracellular ROS generation, and intracellular calcium level were measured. The GNGA polymer carriers containing pilocarpine were intracamerally administered to glaucomatous rabbits and characterized by IOP measurements and biomicroscopic examinations. At postoperative week 2, the aqueous humor was collected for biochemical analysis. The total antioxidant and nitrite levels were determined to examine whether the multifunctional GNGA polymers may have better therapeutic efficacy in vivo than the GN carrier materials.

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EXPERIMENTAL SECTION

Materials. Gelatin (type A; 300 Bloom), 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), GA, ascorbic acid, hydrogen peroxide, DPPH, matrix metalloproteinase-2 (MMP-2, EC 3.4.24.24), pilocarpine nitrate, and α-chymotrypsin were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-isopropylacrylamide (NIPAAm) and N-hydroxysuccinimide (NHS) were supplied by Acros Organics (Geel, Belgium). Before use, NIPAAm was purified by recrystallization from n-hexane. Deionized water used was purified with a Milli-Q system (Millipore, Bedford, MA, USA). The 2-(Nmorpholino)ethanesulfonic acid (MES; J.T.Baker, Phillipsburg, NJ, USA) was dissolved in deionized water to form a 0.1 M buffer solution (pH 5.0). Balanced salt solution (BSS, pH 7.4) was obtained from Alcon Laboratories (Fort Worth, TX, USA). Phosphate-buffered saline (PBS, pH 7.4) was acquired from Biochrom (Berlin, Germany). Eagle’s minimum essential medium (MEM) was purchased from Gibco-BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) and the antibiotic/antimycotic (A/A) solution (10 000 U/mL penicillin, 10 mg/mL streptomycin, and 25 μg/mL amphotericin B) were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). All the other chemicals were of reagent grade and used as received without further purification. Synthesis of Gelatin-g-PNIPAAm (GN). The aminated gelatin and carboxylic end-capped PNIPAAm were synthesized according to the protocols as described elsewhere.4 The amount of amino group in the aminated gelatin samples determined by ninhydrin assay was 48.7 ± 0.4 per molecule. By means of gel permeation chromatography, the gelatin modified with adipic acid dihydrazide was found to have a weight-average molecular weight of approximately 102 kDa. On the other hand, the number-average molecular weight of carboxylic endC

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Biomacromolecules controlled water bath for 10 min to allow gelation. Subsequently, the test samples were dried to constant weight (Wi) in vacuo and immersed at 34 °C in BSS containing 50 ng/mL MMP-2 (level of predominant gelatinase present in aqueous humor of glaucomatous eyes).15 Degradation medium was replaced weekly with fresh buffer solution containing the same concentration of enzyme. At predetermined time intervals, the degraded hydrogels were collected and further dried in vacuo. The dry weight of samples after degradation (Wd) was determined, and the percentage of weight loss (%) was calculated as ((Wi − Wd)/Wi) × 100. Results were the average of five independent measurements. In Vitro Drug Release Studies. For the evaluation of drug encapsulation levels, the GNGA solutions (10% w/v) were mixed with pilocarpine nitrate (2% w/v) at 25 °C, followed by injection into a brown colored vial containing 1.5 mL of BSS at 34 °C. The drugincorporated hydrogels were formed and then transferred to an empty vial at 25 °C. The redissolved polymer solutions were analyzed by high-performance liquid chromatography (HPLC) using a L-2400 UV detector and L-2130 pump (Hitachi, Tokyo, Japan) and a Mightysil RP-18 column (4.6 × 250 mm2) (Kanto Chemical, Tokyo, Japan). The mobile phase was a mixture of 5% monobasic potassium phosphate in Milli-Q water (pH adjusted to 2.5 with 85% phosphoric acid)/methanol (85:15 v/v) with a flow rate of 0.7 mL/min. The eluant peak was detected by measuring absorbance at 216 nm. To determine the amount of drug in each sample, photometric reading was referenced to a standard curve of peak area versus pilocarpine nitrate concentration (0.1−500 μg/mL). The drug encapsulation efficiency was calculated as the percentage of pilocarpine nitrate entrapped in the polymeric hydrogels as compared with the amount of initial drug feeding. In this study, blank experiments (GNGA solutions without drugs) were conducted simultaneously to avoid possible interference of the polymeric materials in absorbance readings. Results were averaged on five independent runs. By the temperature triggered sol−gel phase transition described earlier, the drug-incorporated GNGA samples were prepared at 34 °C and then transferred to another vial containing 1.5 mL of BSS and 75 ng of MMP-2. After incubation at 34 °C with reciprocal shaking (60 rpm) in a thermostatically controlled water bath for predetermined time periods, the release buffer was collected and analyzed by HPLC. The concentrations of pilocarpine nitrate released from the polymeric hydrogels were calculated with respect to a calibration curve. Results were the average of five independent measurements. The cumulative release percentage of drug at each time point was determined by dividing the amount of the released pilocarpine nitrate by the total amount of the loaded pilocarpine nitrate and multiplied by 100. Measurement of Antioxidant Activity against Oxidative Stress. Cell Culture and Treatment. In this study, the HLE (HLE-B3; ATCC No: CRL-11421) cell line was purchased from the American Type Cell Collection (Manassas, VA, USA). The HLE-B3 cells were maintained in MEM supplemented with 20% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 mg/mL sodium bicarbonate, and 1% A/A solution. Cultures were incubated in a humidified atmosphere of 5% CO2 at 37 °C. The hydrogen peroxideinduced oxidative stress of HLE cell model was used according to Choudhary et al.16 Incubation of HLE cells for 24 h in 200 μM hydrogen peroxide was found to cause 50% cell death. Prior to exposure to hydrogen peroxide, the HLE-B3 cells with a density of 5 × 104 cells/well were seeded in 24-well plates and incubated with 150 μL of sterile polymer solutions (10% w/v) for 24 h. Then, the cultures from GN+HP and GNGA+HP groups were treated by further incubation for 24 h in medium containing 200 μM hydrogen peroxide. To examine the biocompatibility of GN and GNGA materials, the HLE-B3 cells were exposed to the polymer samples for 24 h and subsequently incubated in medium without hydrogen peroxide for 24 h (i.e., GN and GNGA groups). The cells exposed to 0 (Ctrl group) or 200 (HP group) μM hydrogen peroxide for 24 h without 24 h of pretreatment with any polymer carrier materials were used for comparison. Measurement of Cell Viability. The qualitative and quantitative assays were performed following the earlier treatments. Cell

morphology was observed by phase-contrast microscopy (Nikon, Melville, NY, USA).17 Furthermore, cell viability was estimated using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation MTS Assay (Promega, Madison, WI, USA), in which MTS tetrazolium compound is bioreduced by cells to form a water-soluble colored formazan.18 The amount of colored product is proportional to the number of metabolically active cells. A sample of 100 μL of the combined MTS/PMS (20:1) reagent was added to each well of the 24well plate and incubated for 3 h at 37 °C in a CO2 incubator. The data of absorbance readings at 490 nm were measured using the Multiskan Spectrum Microplate Spectrophotometer (ThermoLabsystems, Vantaa, Finland). All experiments were performed in quadruplicate, and the results were expressed as relative MTS activity when compared to Ctrl groups. Measurement of Intracellular ROS. Intracellular accumulation of ROS was measured by oxidative conversion of cell-permeable 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) (Molecular Probes, Eugene, OR, USA) to fluorescent 2′,7′-dichlorofluorescein (DCF).19 The HLE cells in the culture wells were incubated with 10 μM DCFHDA solutions at 37 °C for 1 h. Then, the cells were washed three times with PBS. The DCF fluorescence imaging (Ex. 488 nm; Em. 525 nm) was acquired with a fluorescence microscope (Axiovert 200M; Carl Zeiss, Oberkochen, Germany). Furthermore, the fluorescence reading was done with a multimode microplate reader (BioTek Instruments, Winooski, VT, USA) to detect the difference in the fluorescence intensity. All experiments were performed in quadruplicate. Measurement of Intracellular Calcium. Intracellular overload of calcium was measured by using Fura-2, AM (Molecular Probes) as a Ca2+-sensitive fluorescent indicator.20 The HLE cells in the culture wells were incubated with 5 μM Fura-2, AM solutions at 37 °C for 1 h. Then, the cells were washed three times with PBS. The fluorescence imaging (Ex. 340 nm; Em. 510 nm) was acquired with a fluorescence microscope (Axiovert 200M; Carl Zeiss, Oberkochen, Germany). Furthermore, for the quantification of intracellular calcium content, the cells were collected and resuspended in N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered saline solution (containing 132 mM NaCl, 2 mM CaCl2, 3 mM KCl, 10 mM glucose, and 10 mM HEPES, pH 7.4). The fluorescence reading was done with a multimode microplate reader (BioTek Instruments, Winooski, VT, USA) to detect the difference in the fluorescence intensity. All experiments were performed in quadruplicate. Animal Studies. All animal procedures were approved by the Institutional Review Board and were carried out in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Surgical operation was performed in the single eye of animals, with the normal fellow eye. Here, six New Zealand white rabbits (National Laboratory Animal Breeding and Research Center, Taipei, Taiwan, ROC), weighing 3.0−3.5 kg and 16−20 weeks of age, were used in the first experiment to examine the therapeutic efficacy of multifunctional GNGA polymer carriers containing antiglaucoma medications. Experimental glaucoma model induced by injection of 0.1 mg/mL of α-chymotrypsin into the posterior chamber of rabbit eye was established as described by us previously.4,5 The animals were considered to be glaucomatous when the IOP was higher than 20 mmHg in the eye following 4 weeks of α-chymotrypsin injection. The glaucomatous rabbits were anesthetized intramuscularly with 2.5 mg/kg body weight of tiletamine hydrochloride/zolazepam hydrochloride mixture (Zoletil; Virbac, Carros, France) and 1 mg/kg body weight of xylazine hydrochloride (Rompun; Bayer, Leverkusen, Germany). For drug delivery to the eye, the anterior chamber was entered by a 30-gauge needle near the limbus and injected with 50 μL of a mixture of pilocarpine nitrate (2% w/v) and GNGA solutions (10% w/v). Ophthalmic evaluations were performed before and immediately after drug administration. Subsequently, we examined the bilateral eyes of six rabbits at predetermined time intervals for 2 weeks. The IOP was measured using a Schiotz tonometer (AMANN Ophthalmic Instruments, Liptingen, Germany), calibrated according to the manufacturer’s instructions.21 For each IOP determination, five readings were taken on each eye, alternating the left and right eyes, and the mean was calculated. The IOP values of the contralateral D

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Biomacromolecules normal eyes were used as baseline readings. Data were expressed as the difference from baseline values at each time point. At the end of this experiment, the morphology of the anterior segment of the eye, including corneal and lens clarity, the degree of anterior chamber activity, iris, and carrier materials were observed by slit-lamp biomicroscopy (Topcon Optical, Tokyo, Japan).22 In addition, the corneal endothelial cell density in rabbit eyes was measured by specular microscopy (Topcon Optical).23 Each data point is an average of three independent observations. To assess the effects of incorporation of GA molecules into the biodegradable in situ gelling copolymers on the antioxidant status in glaucomatous eyes, 30 New Zealand white rabbits (National Laboratory Animal Breeding and Research Center), weighing 3.0− 3.5 kg and 16−20 weeks of age, were used in the second experiment. The animals were divided into five groups, consisting of six rabbits each. The glaucomatous rabbits received intracameral injections of 50 μL of a mixture containing pilocarpine nitrate (2% w/v) and GN [GN group] or GNGA [GNGA group] solutions (10% w/v). For comparison, the animals from GNGAxPILO group received equivalent volume and concentration of GNGA injections without containing pilocarpine nitrate. Without treatment with any polymers and drugs, the glaucomatous and normal rabbits served as Ctrl and Normal groups, respectively. After drug delivery to the eye for 2 weeks, the animals were euthanized with CO2 gas. To evaluate the total antioxidant and nitrite levels, the aqueous humor from each rabbit eye was immediately aspirated using a 30-gauge needle without touching the iris, lens, and corneal endothelium. Aqueous humor specimens were collected for biochemical analysis. The level of total antioxidant was determined by the method of Koracevic et al.24 The assay could be used to measure the capacity of the biological fluids to inhibit the thiobarbituric acid reactive substances produced from sodium benzoate under the influence of the free oxygen radicals derived from Fenton’s reaction. Calibration curves were made with uric acid, ranging from 0−2 mmol/L. Each data point is an average of four independent measurements. The level of nitrite was measured by using the Griess reaction according to the method of Green et al.25 Calibration curves were made with sodium nitrite, ranging from 0−100 μmol/L. Each data point is an average of four independent measurements. Both the total antioxidant and nitrite levels were averaged from six animals in each group. Statistical Analyses. Results were expressed as mean ± standard deviation (SD). Comparative studies of means were performed using one-way analysis of variance (ANOVA). Significance was accepted with p < 0.05.

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Figure 2. (a) UV−vis spectra of GA and GNGA samples. The UV absorption properties of GNGA solutions were expressed as differences from baseline GN spectra. (b) FTIR spectra of GA, GN, and GNGA samples.

GNGA groups, the aqueous solutions showed two peaks at 231 and 278 nm, indicating the shift of absorption bands for GA molecules. The spectral differences of antioxidants due to the covalent conjugation of catechin onto insulin chain have been identified for the development of thermosensitive antioxidant hydrogels.27 Spizzirri et al. have reported in the spectrum of the free GA, the presence of two peaks at 211 and 258 nm.12 However, after radical grafting of antioxidant on gelatin chain, the wavelengths of the aromatic peaks appear at 227 and 272 nm, indicating the displacement of the absorption bands to higher wavelengths respect to free GA. In accordance with their findings, our results suggest that the two absorption bands, that is, one prominent peak and another very small peak in the aromatic spectral region, are present at higher wavelengths, mainly due to the formation of biopolymer−antioxidant covalent bonds in GNGA structures. Figure 2, panel b shows the structural characterization by recording FTIR spectra of various samples. The peaks of GA at 3496, 3360, and 3273 cm−1 correspond to modes of OH groups. The spectrum of GA also exhibited absorptions at 1706 cm−1 (CO stretch of the carboxyl groups), 1670−1613 cm−1 (β in-plane deformation of ring C−C bonds), 1543, 1429, and 1385 cm−1 (CC stretching and aliphatic CH bending), 1320 cm−1 (in-plane OH bending), 1266 and 1217 cm−1 (Ph-O

RESULTS AND DISCUSSION

Characterization of GA-Functionalized Gelatin-g-PNIPAAm (GNGA). In this study, the multifunctional polymer carriers were composed of thermoresponsive PNIPAAm, biodegradable gelatin, and antioxidant GA molecules (Figure 1). After the synthesis of PNIPAAm-grafted gelatin samples, the GA was finally connected covalently to the GN graft copolymers through biopolymer−antioxidant linkages. It has been documented that for phenolic ring free radical species, the attack at the ortho and para positions is favorable compared to that at hydroxyl groups.26 Therefore, from the viewpoint of chemical reaction, the heteroatom-centered radical species derived from amino acid residues of the GN copolymers preferentially reacts with GA at C2 position. The UV−vis and FTIR spectroscopic analyses were used to examine the binding of antioxidant molecules to the biodegradable in situ gelling copolymers. Figure 2, panel a shows the UV−vis spectra of GA and GNGA samples. An aqueous GA solution exhibited two peaks at 215 and 267 nm. To avoid the interfering absorption bands of GN, the difference spectra were calculated by subtracting the baseline GN spectra from the spectra of GNGA samples. In the E

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Biomacromolecules stretching and C−O stretching), and 1095 and 1028 cm−1 (aromatic C−H deformation). These data are in agreement with a previous FTIR study on GA.28 The materials of GN groups revealed several characteristic bands at 3281 cm−1 (N− H stretching), 2983 cm−1 (C−H stretching), 1642 (amide I, CO stretching), 1545 cm−1 (amide II, N−H bending), 1463, 1388, and 1367 cm −1 (symmetric and antisymmetric deformation of −C(CH3)2), and 1237 cm−1 (amide III, N−H bending), which are typical of those observed for biodegradable backbone gelatin networks29 and thermoresponsive PNIPAAm segments.30 Although the samples GN and GNGA showed a similar pattern of spectra, the peak intensities of O−H stretches increased after radical grafting of GA on protein side chains of the biodegradable in situ gelling copolymers, suggesting the presence of antioxidant GA molecules. To demonstrate the concept of using multifunctional polymers as intracameral drug carriers, the GN was prepared according to our previously published method.4 Here, the feed molar ratio of NH2/COOH was also controlled at 0.36, which resulted in the same grafting effectiveness for the PNIPAAm-grafted gelatin as described in the earlier report.4 The total antioxidant activity of the synthesized GNGA samples determined by phosphomolybdenum assay was 0.4 ± 0.1 mg/g of polymer (see the Supporting Information). Determination of Scavenging Activity against DPPH Radical. The DPPH method, which is based on the reduction of the stable DPPH radical, can be used for evaluation of the free radical scavenging effectiveness of antioxidant agents. We have previously quantified the peroxide formed on plasmatreated culture surface by DPPH assay.31 In this study, the radical scavenging ability of multifunctional GNGA polymers was determined by means of decolorization of stable DPPH radical in the presence of antioxidants. Figure 3, panel a shows the photographs of the reaction of DPPH reagent with synthesized GN and GNGA polymers. After incubation at 25 °C for 30 min in the absence of test polymers, the blank DPPH solution was dark purple in color. However, appreciable color changes occurred under the same reaction conditions with the addition of polymer samples. The reduction of purple chromogen radicals by GN copolymers was observed. In particular, the color of DPPH solutions in the GNGA groups turned from dark purple to light purple, indicating the additional contribution of antioxidant GA. Given that the extent of discoloration is closely related to free radical scavenging potential of test polymers,32 the decrease of absorbance at 517 nm is measured to quantify the amount of purple chromogen radicals remaining in the solution. The DPPH scavenging activities of synthesized polymers are presented in Figure 3, panel b. The GN samples showed 19.7 ± 1.3% inhibition of the DPPH radical, probably due to that the cysteine present in the gelatin was responsible for the decolorization of DPPH by its hydrogen-donating ability.12 The percentage radical inhibition in the GN groups was significantly lower than that of the GNGA (42.6 ± 2.0%) groups (p < 0.05). This result indicates that after functionalization with antioxidant GA molecules, the copolymers exhibit strong interaction with DPPH radical. Spizzirri et al. have evaluated the antioxidant activity of gelatin-GA conjugate and reported the value of 66 ± 3% inhibition of the DPPH radical.12 It is higher than that reported in the present study, suggesting the role of PNIPAAm segments in radical grafting of GA on gelatin chain and determining DPPH scavenging by the synthesized polymers.

Figure 3. (a) Photographs of the reaction of DPPH reagent with synthesized polymers (DPPH+GN and DPPH+GNGA groups). The DPPH group is the blank DPPH solution without synthesized polymers. (b) DPPH scavenging activities of GN and GNGA polymers were analyzed by UV−vis spectrophotometry. Results are expressed as percentage inhibition of the DPPH radical. Values are mean ± SD (n = 5). ∗, p < 0.05 versus all groups.

Phase-Transition Characterizations. A phase transition that is related to a change in the hydrophobic−hydrophilic balance of the thermoresponsive polymer strongly depends on the variation of external temperature. The LCST represents the temperature at which a polymer chain shows a coil-to-globule transition in aqueous solution.33 We have demonstrated that both the polymer concentration4 and molecular weight of grafted PNIPAAm chains5 may cause drastic effects on LCST of GN materials. When dissolved in deionized water, these biodegradable in situ gelling copolymers have a LCST of around 32 °C. As shown in Figure 4, panel a, the LCST of GNGA obtained by DSC analysis was 33.2 ± 0.2 °C in aqueous solution. Our results indicate that the incorporation of GA into the GN samples can lead to a higher phase-transition temperature since the hydrophilic antioxidant is responsible for the increased overall hydrophilicity of the polymers and the strong polymer−water interactions.34 On the other hand, when dissolved in physiological medium (BSS) at 25 °C, the molecules of multifunctional GNGA polymers remained attracted to the water molecules. However, apparent temperature triggered gel formation occurred at surrounding temperF

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ance.39 The formulation parameters including the weight ratio of PNIPAAm graft chains to gelatin and polymer concentration have been found to play important roles in the interrelationships between molecular architecture/internal structure of copolymeric gels/smooth muscle cell viability, and the potential usefulness of copolymer as an injectable artificial extracellular matrix.39 Here, to further understand the extent of degradation due to the functionalization of biodegradable in situ gelling copolymers with antioxidant molecules, the GNGA samples were incubated at 34 °C in physiological medium containing MMP-2. The time-course of weight loss is shown in Figure 4, panel b. During the period ranging from 8 h to 14 days, the test materials significantly degraded with time (p < 0.05). Approximately 43% of weight loss was observed over 2 weeks under gelatinase digestion. It was also noted that the remaining weight at each time point was significantly lower in the GNGA groups compared with that of the GN groups reported in our earlier study.4 This result indicates that the binding of hydrophilic GA molecules to the GN copolymers may lower the resistance to collagenolytic degradation. Polymer structure is known to be one of the most important parameters dictating its degradation behavior.40 It has been documented that the addition of substituents such as hydroxyl and carboxyl groups to the polymer backbone increases the biodegradability.41 On the basis of the aforementioned results, the hydroxyl and carboxyl groups of antioxidant agents can affect the weight loss of synthesized GNGA polymers during in vitro degradation tests. Another possible explanation for the current findings is that, following GA conjugation, the flexible hydrophilic polymer chains may be susceptible to cleavage by better fitting into the active site of the enzymes. In Vitro Drug Release Studies. At 34 °C in physiological medium, apparent temperature triggered gel formation of biodegradable and thermoresponsive GN copolymers facilitates the entrapment of pilocarpine into the carriers. The drug encapsulation efficiency analyzed by HPLC is found to be around 60%.4 In this study, the drug encapsulation level of synthesized GNGA polymers was determined to be 71.8 ± 2.3%. It was significantly higher than those of the GN materials (p < 0.05). Interestingly, although the aggregation of PNIPAAm segments due to hydrophobic interaction is reduced by introducing of hydrophilic antioxidant into GN samples, the GA grafting can increase the drug payload of multifunctional polymer carriers. It has been reported that the pilocarpine molecules are hydrophilic and carry positive charge.42 In addition, the GA is negatively charged at physiological pH (i.e., 7.4).43 Therefore, the electrostatic interaction may contribute to the observed drug loading effects. Given that the inhibited drug release from the thermoresponsive hydrogels is highly correlated to the dense surface skin formation,44 we copolymerize the gelatin and PNIPAAm to overcome the limitation of water outflow from the carrier interior. To investigate whether the functionalization of biodegradable in situ gelling copolymers with antioxidant molecules affects the capability of delivery carriers, the in vitro drug release studies were performed in physiological medium containing MMP-2. The drug release pattern of the GNGA samples can be estimated from the time-course of changes in pilocarpine concentration (Figure 5a). After 30 min, an appreciable amount of drug in BSS buffer was found, indicating the occurrence of pilocarpine desorption at the early stage. The drug concentration at 1 h of incubation was significantly decreased to 27.5 ± 6.0 μg/mL (p < 0.05), which could be

Figure 4. (a) DSC thermograms of GNGA in H2O or BSS (10% w/v). Each LCST data point represents the average of four different values. Gross morphological observation of the GNGA in BSS (10% w/v) at 25 and 34 °C. (b) Time-course of weight loss of GNGA samples after incubation at 34 °C in BSS containing MMP-2. Values are mean ± SD (n = 5). ∗, p < 0.05 versus all time point groups. Incubation time point: hour (h); day (d).

ature above the LCST (i.e., 30.3 ± 0.1 °C), mainly due to the thermal dissociation of hydrating water molecules from the polymer chains. Schmaljohann et al. have shown that the addition of electrolytes to the solution of a LCST polymer may decrease the phase-transition temperature.35 In accordance with their findings, our results suggest that the ionic interactions are crucial for the modulation of LCST of GNGA samples under physiological conditions. In Vitro Degradation Tests. It is generally recognized that the foreign carrier materials should be removed following intraocular drug delivery. The use of bioerodible and biodegradable polymers for the design of new antiglaucoma formulations may address possible concerns about the biocompatibility of drug vehicles in the ocular anterior chamber. To minimize undesirable tissue reactions, the biodegradable gelatin carriers have been previously applied in our laboratory to three areas of interest to ophthalmology: the corneal endothelial reconstruction,36 retinal sheet transplantation,37 and corneal stromal bioengineering.38 By taking advantage of this naturally occurring biopolymer, the thermoresponsive PNIPAAm can be incorporated to provide additional features that focus on enhancing polymer performG

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formulation, and the cumulative release approached 97% of the original encapsulated amount after 14 days. Measurement of Antioxidant Activity against Oxidative Stress. It is known that oxidative injury plays an important role in the pathogenesis of glaucoma. Given that the importance of antioxidant defense mechanisms in the protection against oxidative damage has been identified as crucial, a recent review paper by Aslan et al. summarizes the research on the use of various free radical scavengers in the treatment of glaucoma.46 The relationship between the chemical structure and biological activity of antioxidants has been explored from the perspective of developing a safe and effective strategy against oxidative stress. It is found that the antioxidant effect of polyphenolics is strongly dependent on the free hydroxyl groups on the aromatic ring.47 Therefore, in our laboratory, the antioxidant molecules are covalently grafted onto GN copolymers through a radical reaction in the presence of water-soluble redox initiators. The formation of biopolymer−antioxidant linkages without involving the consumption of free hydroxyl groups on the aromatic ring of GA is beneficial for GNGA products to exhibit antioxidant activity against oxidative stress. Following the determinations of free radical scavenging effectiveness of synthesized GN and GNGA samples, we further evaluated the antioxidant activity of these polymers using an in vitro model of oxidative stress. Measurement of Cell Viability. Figure 6, panel a shows representative images of HLE cell cultures photographed after 2 days. In the Ctrl groups without pretreatment with synthesized polymers and exposure to hydrogen peroxide, the cells appeared healthy and exhibited typical lens epithelial morphological characteristics. In the HP groups, a sublethal dose of hydrogen peroxide markedly decreased the cell survival rate. Additionally, the shrinkage of cells and their partial detachment from the culture substrate confirmed the alterations in the morphology of HLE cells exposed to hydrogen peroxide for 24 h. The cultures from the GN and GNGA groups were morphologically similar to the Ctrl cells, indicating good cytocompatibility of both polymers. In the GN +HP and GNGA+HP groups, the cells had a lower extent of heterogeneity in cell shape compared to those of the HP groups. Here, the loss of cellular morphology as a result of hydrogen peroxide-induced oxidative stress was prevented by the pretreatment with synthesized polymers. In particular, multifunctional GNGA polymers were more effective in maintaining the normal HLE cell morphology than their GN counterparts, thereby reflecting the importance of antioxidant molecules on the functionalization of polymer carriers. Quantitative analysis for HLE cell viability was performed following the cell proliferation MTS assay, and the results are shown in Figure 6, panel b. The mitochondrial dehydrogenase activity (MTS activity) in the Ctrl groups was defined as 100%. For the HP groups, the viability level was 49.2 ± 3.7%. In accordance with a previous study on the hydrogen peroxideinduced oxidative stress of HLE cell model,16 we found that 24 h of incubation of lens epithelial cultures with 200 μM hydrogen peroxide caused 50% cell death. After 2 days in culture, the cell growth did not show a significant difference between the Ctrl, GN (99.7 ± 2.1%), and GNGA (98.5 ± 2.6%) groups (p > 0.05), suggesting that the pretreatment with polymeric carrier materials does not affect the cell viability. The MTS activity of the GN+HP and GNGA+HP groups was significantly increased by about 11.8 and 30.2%, respectively, as compared to the HP groups (p < 0.05). Our data demonstrate

Figure 5. (a) Time-course of the concentration of pilocarpine released from GNGA samples at 34 °C in BSS containing MMP-2. An asterisk indicates statistically significant differences (∗, p < 0.05; n = 5) for the mean value of the pilocarpine concentration compared to the value at the previous time point. Incubation time point: hour (h); day (d). (b) Cumulative release percentage.

ascribed to the absence of initial burst release from the pilocarpine-loaded polymeric carriers. It was also found that compared to the value at 4 h (27.2 ± 3.7 μg/mL), the drug concentration was significantly higher at 8 h (38.1 ± 6.5 μg/ mL) (p < 0.05). A remarkable increase in the amount of released pilocarpine coincides with the onset of significant degradation of GNGA. With increasing time from 8 h to 14 days, the carriers were gradually degraded by MMP-2, thereby allowing a continual controlled release of pilocarpine. The measured drug concentration at each sampling time ranged from 30.4−45.3 μg/mL, which reaches the therapeutic level of pilocarpine used for glaucoma treatment.45 Although a sustained release profile is obtained from the multifunctional GNGA polymeric carriers, it is noteworthy that the detected drug concentrations are significantly decreased after 7 days of incubation. One potential explanation is that most of the encapsulated pilocarpine has already been released at earlier time points due to the hydrophilic antioxidant-mediated acceleration of GNGA biodegradation. As shown in Figure 5, panel b, the pilocarpine was continuously released from the H

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Figure 6. Effect of polymer carrier materials on H2O2-induced cell viability. (a) Representative phase-contrast micrographs of the HLE cells after incubation with polymer samples for 24 h and further exposure to H2O2 for 24 h. GN group, GN polymer and 0 μM H2O2; GNGA group, GNGA polymer and 0 μM H2O2; GN+HP group, GN polymer and 200 μM H2O2; GNGA+HP group, GNGA polymer and 200 μM H2O2. The cells exposed to 0 (Ctrl group) or 200 (HP group) μM H2O2 for 24 h without 24 h of pretreatment with any polymers were used for comparison. Scale bars: 200 μm. (b) Cell proliferation was measured by the MTS assay. Results of MTS activity were expressed as percentages of Ctrl groups. Values are mean ± SD (n = 4). ∗, p < 0.05 versus all groups; #, p < 0.05 versus HP, GN+HP, and GNGA+HP groups.

groups fluoresced green. For both the GN and GNGA groups, the fluorescent signal was not different in comparison with the Ctrl groups, indicating that the pretreatment with these synthesized polymer samples does not induce intracellular accumulation of ROS. In the GNGA+HP groups, the HLE cells exhibited significantly less fluorescence than those of the GN +HP groups. Our findings suggest that the binding of antioxidant GA molecules to the GN copolymers can enhance the ability of delivery carriers to inhibit hydrogen peroxideinduced ROS formation. To determine whether the qualitative data were consistent with the quantitative data, the alterations in fluorescent intensities were further analyzed by spectrofluorometer (Figure 7b). In the Ctrl, GN, and GNGA groups, the profiles were almost identical. Additionally, the ROS-induced green fluorescence of DCF in cells exposed to hydrogen peroxide was markedly stronger than those without hydrogen peroxide stimulation. The order of increasing intracellular ROS level was the following: HP > GN+HP > GNGA+HP. It is generally recognized that ROS generation is the cumulative effect of various reactive radicals.19 As demonstrated by DPPH assays, the GN samples have limited free radical scavenging capacity. This may explain the poor inhibition of ROS production

that the synthesized polymers can alleviate the oxidative damage caused by hydrogen peroxide. Furthermore, it was noted that the HLE cells exposed to GNGA materials were more metabolically active than those exposed to GN samples. The incorporation of antioxidant molecules in the structure of biodegradable in situ gelling copolymers may contribute to higher cell viability against hydrogen peroxide-induced oxidative injury by increasing antioxidant action. Measurement of Intracellular ROS. As reported in a review paper by Izzotti et al.,48 the pathogenic role of ROS in glaucoma is supported by various experimental findings. The high chemical reactivity of ROS can lead to reactions with almost all constituents of the cell and cause oxidative DNA damage, protein denaturation, and lipid peroxidation.49 Since the elevated intracellular ROS level is known to be causally connected to cell death,20 it is highly desirable to examine the ROS generation in HLE cells in response to hydrogen peroxide stimulation. The effects of synthesized polymers on intracellular ROS production were first studied by means of fluorescence microscopy (Figure 7a). For the HP groups, prominent green fluorescence of DCF, indicative of ROS generation, was detected in the cell cultures after exposure to 200 μM hydrogen peroxide for 24 h. In contrast, very few cells from the Ctrl I

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Figure 7. Effect of polymer carrier materials on H2O2-induced intracellular ROS. (a) Representative fluorescent images of the HLE cells after incubation with polymer samples for 24 h and further exposure to H2O2 for 24 h. GN group, GN polymer and 0 μM H2O2; GNGA group, GNGA polymer and 0 μM H2O2; GN+HP group, GN polymer and 200 μM H2O2; GNGA+HP group, GNGA polymer and 200 μM H2O2. The cells exposed to 0 (Ctrl group) or 200 (HP group) μM H2O2 for 24 h without 24 h of pretreatment with any polymers were used for comparison. Scale bars: 50 μm. (b) Intracellular levels of ROS were measured by the fluorescence intensity of DCFH-DA with a microplate reader. Quantification results were the mean of four independent experiments.

data demonstrate that the hydrogen peroxide-induced calcium overload in HLE cells can be effectively reduced. Figure 8, panel b also shows the results of fluorescent intensity analysis using spectrofluorometer. In the Ctrl groups, the intracellular calcium level measured at each wavelength was found to match that of the GN and GNGA groups. However, in the HP groups, the intracellular overload of calcium evaluated by the emitted fluorescent intensity was relatively very high, indicating hydrogen peroxide-stimulated alteration in calcium homeostasis. For the GNGA+HP groups, the intensity level was largely decreased by the pretreatment of HLE cells with antioxidant GA-functionalized biodegradable in situ gelling copolymers. It has been documented that the Na/Ca exchanger is one of the key factors in the regulation of intracellular calcium concentration.52 Our findings support the intracellular ROS measurements and suggest that antioxidant GA molecules of multifunctional polymer carriers interacted with Na/Ca exchanger molecules may display cytoprotective activity against hydrogen peroxide-induced oxidative stress in lens epithelial cultures. Animal Studies. It is known that the change in target IOP usually correlates with disease progression in glaucomatous patients. As reported in a clinical trial by Leske et al.,53

observed. In support of the results regarding cell viability, the detrimental effects of ROS are also not properly balanced by the GN materials. However, the GNGA polymers can potentially rescue ROS accumulation-triggered cell death, indicating the cytoprotective action of antioxidant GAfunctionalized biodegradable in situ gelling copolymers in oxidative stress. Measurement of Intracellular Calcium. Regulation of intracellular level of calcium (i.e., a chemical signal) is essential for cell survival because this mineral is critically involved in many normal cellular functions.50 In addition to an elevated intracellular ROS level, the intracellular calcium overload is commonly believed to be a contributing factor to cell death.51 Therefore, in this work, the intracellular calcium modulated by pretreatment with synthesized polymers in HLE cells was investigated by measuring Fura-2, AM fluorescence (Figure 8a). In the HP groups, significantly more blue fluorescent cells were observed as compared to the Ctrl groups, indicating elevation of intracellular calcium levels after 24 h of exposure to 200 μM hydrogen peroxide. In the GNGA+HP groups, the pretreatment with antioxidant GA-functionalized polymer samples resulted in a decrease in the number of fluorescent cells. The J

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Figure 8. Effect of polymer carrier materials on H2O2-induced intracellular calcium. (a) Representative fluorescent images of the HLE cells after incubation with polymer samples for 24 h and further exposure to H2O2 for 24 h. GN group, GN polymer and 0 μM H2O2; GNGA group, GNGA polymer and 0 μM H2O2; GN+HP group, GN polymer and 200 μM H2O2; GNGA+HP group, GNGA polymer and 200 μM H2O2. The cells exposed to 0 (Ctrl group) or 200 (HP group) μM H2O2 for 24 h without 24 h of pretreatment with any polymers were used for comparison. Scale bars: 50 μm. (b) Intracellular levels of calcium were measured by the fluorescence intensity of Fura-2, AM, with a microplate reader. Quantification results were the mean of four independent experiments.

chamber of rabbit eyes induces chronic ocular hypertension by obstructing outflow of aqueous humor through zonulolysis.54 As shown in Figure 9, panel b, the animals of the GL groups presented a very deep anterior chamber depth, indicating that the production of experimental glaucoma increases the distance between the corneal endothelium and the anterior capsule of the crystalline lens. However, in the GNGA groups, the glaucomatous rabbits receiving drug-containing polymer samples demonstrated narrowing of the anterior chamber depth. Abramson et al. have shown that a significant decrease in anterior chamber depth occurs in patients with chronic openangle glaucoma who have been treated with topically instilled pilocarpine.55 Our findings suggest that after 2 weeks of intracameral drug administration using polymeric carriers, the released pilocarpine can lead to the changes in anterior chamber depth, reflecting the pharmacological action of the drug. In addition, slit-lamp biomicroscopy revealed that there was merely a very small amount of residual GNGA materials due to in vivo degradation. The injectable polymer depots for minimally invasive pilocarpine delivery did not induce any anterior segment tissue damage, indicating good ocular biocompatibility of multifunctional drug carriers.

progression risk may decrease by 10% with every 1 mmHg of IOP reduction. To investigate whether the functionalization of biodegradable in situ gelling copolymers with antioxidant molecules affects the therapeutic efficacy of pilocarpinecontaining GNGA carriers, the in vivo animal experiments were performed by using glaucomatous rabbits. As shown in Figure 9, panel a, the normal preoperative eyes had an IOP of approximately 20 mmHg (used as baseline IOP). After 4 weeks of α-chymotrypsin injection, the animals had IOPs 21 mmHg greater than baseline values, indicating successful induction of experimental glaucoma. The elevation of IOP was significantly reduced within 8 h of intracameral administration of drugcontaining polymer samples. With increasing follow-up time from 12 h to 2 weeks, the IOPs were maintained at slightly below the baseline levels. Sustained IOP lowering effect over the study period is probably due to that gradual biodegradation of carrier materials may allow a continual release of sufficient amount of pilocarpine for a therapeutic action, as demonstrated by in vitro drug release studies. In clinical ophthalmology, chronic open-angle glaucoma is the most common form of glaucoma that is characterized by gradual blockage of drainage canals and progressive rise in IOP. Therefore, the α-chymotrypsin injection into the posterior K

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± 124 cells/mm2, which was significantly lower than that before surgery (3275 ± 106 cells/mm2; p < 0.05). The evidence in our earlier study suggests that the persistent elevation of IOP in untreated rabbits causes further cell loss.4 In contrast, IOP reduction due to ocular administration of pilocarpine may preserve the endothelial cell count. Here, at postoperative 2 weeks, the glaucomatous rabbits receiving drug-containing GNGA polymer samples presented a similar corneal endothelial cellular morphology to that of GL groups. The results of quantitative specular microscopic analysis of corneal endothelium also showed no significant difference in cell density of GNGA groups (3053 ± 95 cells/mm2) compared with the values for the animals of the GL groups (p > 0.05). These findings suggest that the drugs released from the polymer carriers can prevent IOP-mediated glaucomatous damage. According to our experience, the intracameral injection of GNGA polymer solutions without containing pilocarpine certainly produces partial pharmacological responses (i.e., IOP lowering effect and preservation of corneal endothelial cell density), which indicates the contribution of antioxidant GA molecules of multifunctional delivery carriers to the improvement of trabecular outflow. In terms of physiology, the regulation of aqueous humor circulation seems to be especially important in the control of IOP. The alterations in aqueous humor homeostasis have also been discussed in association with glaucoma pathogenesis.6 The normal aqueous humor contains a high concentration of antioxidant ascorbic acid that protects ocular tissue against oxidative damage. However, glaucomatous patients with extremely low levels of ascorbic acid in the aqueous humor become more susceptible to peroxidation by free radicals.57 In addition to reduced local antioxidant activity, the increased level of nitric oxide may be involved in a variety of glaucoma pathological processes.58 Therefore, biochemical analysis was undertaken to examine the antioxidant and nitrite levels in the aqueous humor of rabbit eyes. The measured concentrations of total antioxidant in the Normal and Ctrl groups were 0.98 ± 0.17 and 0.16 ± 0.09 mM, respectively, indicating that the induction of experimental glaucoma leads to decrease in local antioxidant activity (Figure 10a). In addition, as shown in Figure 10, panel b, the nitrite levels in the aqueous humor of glaucomatous (58.16 ± 7.05 μM) eyes were significantly higher than those of the normal (9.40 ± 1.22 μM) eyes (p < 0.05). After 2 weeks of intracameral injection of polymer solutions containing pilocarpine, the total antioxidant levels were significantly elevated in the GNGA (0.59 ± 0.10 mM) and GN (0.37 ± 0.05 mM) groups, as compared to that in the Ctrl groups (p < 0.05). Furthermore, the measured concentrations of nitrite in the GNGA (30.14 ± 2.07 μM) and GN (42.01 ± 4.38 μM) groups were significantly decreased below the values detected in Ctrl animals (p < 0.05). Our data clearly demonstrate that the delivery carriers made of different polymers have varying levels of enhanced antioxidant action, as shown by the determination of free radical scavenging potential and measurement of antioxidant activity against oxidative stress. The results of in vivo studies also support the hypothesis that the GNGA polymer carriers are more advantageous over their GN counterparts for the improvement of total antioxidant status in glaucomatous eyes. On the other hand, to clarify whether the released pilocarpine has a role in protection against oxidative stress, the glaucomatous animals from GNGAxPILO groups receiving equivalent volume and concentration of GNGA injections without containing

Figure 9. (a) Measurements of IOP after intracameral injection of GNGA solutions containing pilocarpine in rabbits with glaucoma (GL). An asterisk indicates statistically significant differences (∗, p < 0.05; n = 6) for the mean value of the IOP compared to the value at the previous time point. Follow-up time point: preoperation (Pre); hour (h); day (d). (b) Representative slit-lamp biomicroscopic images of experimental rabbit GL eyes 2 weeks after intracameral injection of GNGA solutions containing pilocarpine. Scale bars: 5 mm. (c) Typical specular microscopic images of corneal endothelium in experimental rabbit GL eyes 2 weeks after intracameral injection of GNGA solutions containing pilocarpine.

Given that the ocular hypertension is found to alter the cellular hexagonality of corneal endothelium,56 the specular microscopy is used to record the image of the endothelial monolayer and determine the cell count. As shown in Figure 9, panel c, there were some irregular shapes of cells on Descemet’s membrane in the GL groups. In addition, the corneal endothelial cell density of these glaucomatous eyes was 2991 L

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of a therapeutic antioxidant polymeric delivery system for intracameral drug administration. Here, we describe the synthesis and characterization of novel multifunctional antiglaucoma drug carriers composed of biodegradable and thermoresponsive GN copolymers and antioxidant GA molecules. The results show that the GA-functionalized GN materials may display better cytoprotective activity against hydrogen peroxide-induced oxidative stress in lens epithelial cultures than their GN counterparts, likely due to the contribution from the free radical scavenging capability of antioxidants. Although the injection of GN solutions containing pilocarpine has been previously proven to be useful in lowering the IOP, the oxidative/nitrative stresses cannot be effectively ameliorated by ocular administration of such a drug to glaucomatous rabbits. In contrast, the animals receiving GNGA polymers containing antiglaucoma medications show a significant increase of total antioxidant levels and a significant decrease of nitrite levels in aqueous humor. Overall, our findings suggest that in addition to their functions as intraocular drug delivery carriers, the antioxidant molecule-functionalized biodegradable in situ gelling materials possess cytoprotective and antioxidative actions and thereby can be considered as polymer therapeutics for ophthalmic use. The effect of GA grafting degree on the therapeutic efficacy of GNGA carriers for glaucoma treatment is worthy of further investigation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00854. Experimental methods, colorimetric measurement and absorbance of phosphomolybdenum complex (PDF)

Figure 10. Levels of (a) total antioxidant and (b) nitrite in the aqueous humor of experimental rabbit glaucoma eyes 2 weeks after intracameral injection of polymer solutions containing antiglaucoma medications. GN group, GN polymer and pilocarpine; GNGA group, GNGA polymer and pilocarpine; GNGAxPILO group, GNGA polymer. Without treatment with any polymers and drugs, the glaucomatous and normal rabbits served as Ctrl and Normal groups, respectively. Values are mean ± SD (n = 6). ∗, p < 0.05 versus all groups; #, p < 0.05 versus Normal, Ctrl, and GN groups.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +886-3-211-8800, ext. 3598. Fax: +886-3-211-8668. Email: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

antiglaucoma medications were also examined. No significant difference was noted in either total antioxidant concentration or nitrite level between GNGA and GNGAxPILO groups (p > 0.05), confirming that pilocarpine is a muscarinic agonist mainly responsible for the regulation of trabecular outflow. The present findings suggest that the antioxidant GA molecules incorporated into the structure of biodegradable in situ gelling copolymers are critically important in down-regulating the oxidative stress signals associated with the generation of multiple ROS in aqueous humor. Although ocular hypertension is a risk factor for glaucomatous damage, the normal IOP can also lead to loss of vision in glaucoma, implying other factors such as oxidative and nitrative stresses are possible initiating causes and may influence the rate of disease progression.59 For intracameral controlled drug release applications, the multifunctional GNGA polymer carriers are shown to be of benefit in the attenuation of glaucoma-induced biochemical changes.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grant No. NHRI-EX10310311EC from the National Health Research Institutes. The authors are grateful to Meng-Heng Lai and Jui-Min Ho (Institute of Biochemical and Biomedical Engineering, Chang Gung University) for technical assistance.

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REFERENCES

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CONCLUSIONS The knowledge of relationship between oxidative stress and glaucoma occurrence may potentially lead to the development M

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DOI: 10.1021/acs.biomac.5b00854 Biomacromolecules XXXX, XXX, XXX−XXX