Thermosensitive chitosan-based hydrogel as a topical ocular drug delivery system of latanoprost for glaucoma treatment

Carbohydrate Polymers 144 (2016) 390–399

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Thermosensitive chitosan-based hydrogel as a topical ocular drug delivery system of latanoprost for glaucoma treatment Yung-Hsin Cheng a,b,1 , Tung-Hu Tsai a,c,2 , Yong-Yu Jhan b,3 , Allen Wen-hsiang Chiu d,4 , Kun-Ling Tsai e,5 , Chian-Shiu Chien b,6 , Shih-Hwa Chiou b,f,∗∗ , Catherine Jui-lin Liu d,f,∗ a

Department of Education and Research, Taipei City Hospital, No.145, Zhengzhou Rd., Datong Dist., Taipei, Taiwan Department and Institute of Pharmacology, National Yang-Ming University, No. 155, Sec. 2, Linong Street, Taipei, 112, Taiwan c Institute of Traditional Medicine, National Yang-Ming University, No.155, Sec. 2, Linong Street, Taipei 112, Taiwan d National Yang-Ming University School of Medicine, No. 155, Sec. 2, Linong Street, Taipei 112, Taiwan e Department of Physical Therapy, College of Medicine, National Cheng Kung University,No. 1, University Road, Tainan City 701, Taiwan f Department of Ophthalmology, Taipei Veterans General Hospital, No. 201, Sec. 2, Shipai Rd., Beitou District, Taipei 112, Taiwan b

a r t i c l e

i n f o

Article history: Received 9 June 2015 Received in revised form 25 February 2016 Accepted 29 February 2016 Available online 3 March 2016 Keywords: Chitosan Glaucoma Latanoprost Sustained release

a b s t r a c t Ocular hypertension is a major risk factor for the development and progression of glaucoma. Frequent and long-term application of latanoprost often causes undesirable local side effects, which are a major cause of therapeutic failure due to loss of persistence in using this glaucoma medical therapy. In the present study, we developed a thermosensitive chitosan-based hydrogel as a topical eye drop formulation for the sustained release of latanoprost to control ocular hypertension. The developed formulation without preservatives may improve compliance and possibly even efficacy. The results of this study support its biocompatibility and sustained-release profile both in vitro and in vivo. After topical application of latanoprost-loaded hydrogel, triamcinolone acetonide-induced elevated intraocular pressure was significantly decreased within 7 days and remained at a normal level for the following 21 days in rabbit eyes. This newly developed chitosan-based hydrogel may provide a non-invasive alternative to traditional anti-glaucoma eye drops for glaucoma treatment. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Glaucoma is an irreversible optic neuropathy with progressive visual field loss that can eventually lead to blindness if intraocular pressure (IOP) is not well controlled. IOP-lowering

∗ Corresponding author at: National Yang-Ming University School of Medicine, No. 155, Sec. 2, Linong Street, Taipei 112, Taiwan. Fax.: +886 2 28757133. ∗∗ Corresponding author at: Department and Institute of Pharmacology, National Yang-Ming University, No. 155, Sec. 2, Linong Street, Taipei 112, Taiwan. Fax: +886 2 28202190. E-mail addresses: [email protected], [email protected] (Y.-H. Cheng), [email protected] (T.-H. Tsai), [email protected] (Y.-Y. Jhan), [email protected] (A.W.-h. Chiu), [email protected] (K.-L. Tsai), [email protected] (C.-S. Chien), [email protected] (S.-H. Chiou), [email protected] (C.J.-l. Liu). 1 Fax: +886 2 27014701. 2 Fax: +886 2 28225044. 3 Fax: +886 2 28720959. 4 Fax: +886 2 28202190. 5 Fax: +886 6 2370411. 6 Fax: +886 2 28202190. http://dx.doi.org/10.1016/j.carbpol.2016.02.080 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

medications therefore play a key role in long-term treatment of glaucoma (Bucolo et al., 2013; Lavik, Kuehn, & Kwon, 2011; Rouland, Berdeaux, & Lafuma, 2005). Almost all anti-glaucoma drugs, including ␤-blockers, prostaglandin analogs, carbonic anhydrase inhibitors, and ␣-2 agonists, are administered topically in the form of eye drops (solutions or suspensions). The limitations of using eye drops include inadequate and irregular delivery of the therapeutic agent to the eye and poor bioavailability of the drug (Knight & Lawrence, 2014; Weinreb, Aung, & Medeiros, 2014). Latanoprost, a prostaglandin analog, is a well-tolerated ocular hypotensive agent and exhibits the longest effective duration (Alm, 2014; Russo, Riva, Pizzolante, Noto, & Quaranta, 2008). The IOP-lowering effect of latanoprost is mediated by increasing aqueous outflow through the uveoscleral pathway. The recommended dosage of latanoprost is one drop (0.005% solution) applied on the eye once daily. It has been reported that less than 7% of topically administered eye drops actually reach the anterior segment due to their short residence time (Ghate & Edelhauser, 2008). Therefore, daily instillation of latanoprost is required to achieve a therapeutic level of the drug in the eye. It is well known that chronic exposure to preservatives (such as benzalkonium chloride (BAK))

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is associated with adverse effects on the ocular surface, including tearing, burning, irritation, and dryness (Alm, 2014). Frequent instillation may increase the chance of a patient developing undesirable side effects and can lead to poor patient adherence to glaucoma medication, which may result in treatment failure (Noecker et al., 2003; Rolle et al., 2012). In recent years, in situ forming hydrogels for topical ocular administration have emerged as a promising approach for longterm sustained release of drugs. Topical administration of in situ forming hydrogels can effectively prolong the retention time and prevent the rapid drainage of an instilled drug from the nasolacrimal system of the orbit (Agrawal, Das, & Jain, 2012; Almeida, Amaral, Lobao, & Lobo, 2014). Chitosan is a cationic polymer that possesses mucoadhesive properties due to the molecular attraction forces effected by its electrostatic interactions with the negatively charged mucus. Chitosan-based formulations have been widely used as topical ophthalmic drug delivery systems (Gratieri, Gelfuso, de Freitas, Rocha, & Lopez, 2011; Katiyar et al., 2014). In a previous study, we developed an injectable thermosensitive chitosan/gelatin/glycerol phosphate hydrogel that can be used as a sustained-release drug delivery system. This newly developed chitosan-based hydrogel showed promising gelation properties and biocompatibility (Cheng et al., 2014; Cheng, Yang, & Lin, 2011). Although injectable formulations are able to sustain release for at least one month, eye drops are still the least invasive and most convenient route for administration. In the present study, we aim to evaluate the feasibility of using this newly developed thermosensitive chitosan-based hydrogel as a topical eye drop formulation for the sustained release of latanoprost to control ocular hypertension. (Bron, Chiambaretta, Pouliquen, Rigal, & Rouland, 2003) In the present study, the gelation temperature and time for the latanoprost-loaded hydrogel were evaluated using a rheometer. The structure of the latanoprost-loaded hydrogel was characterized by scanning electron microscope (SEM). The in vitro and in vivo drug release profiles were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS). The biocompatibility of the latanoprost-loaded hydrogel was analyzed using cell viability assays, hemolysis tests, corneal fluorescein staining and histological examination. A rabbit model of glaucoma was established by using intravitreal injection of triamcinolone acetonide (TA). After TA-induced IOP elevation, the latanoprost-loaded hydrogel was instilled into the lower lid of the right eye. The IOP-lowering efficacy of the latanoprost-loaded hydrogel was confirmed by IOP measurement. 2. Materials and methods 2.1. Materials Chitosan (molecular weight  340,000 Da, degree of deacetylation > 95%, viscosity  581 mPa s) was purchased from Xing Cheng Biochemical Factory Nantong, China. Minimum essential medium (MEM) was purchased from Gibco, USA. Fetal bovine serum was purchased from HyClone, USA. FLUORESCITE® was purchased from Alcon, Australia. Tiletamine and zolazepam (Zoletil 50) was purchased from Virbac, France. Triamcinolone acetonide (TA, 4% of triamcinolone suspended inj.) was purchased from Tai-yu, Taiwan. Hematoxylin and eosin (H&E) was purchased from Muto Pure Chemicals, Japan. Other chemicals and reagents were purchased from Sigma–Aldrich, USA. 2.2. Preparation of a thermosensitive latanoprost-loaded hydrogel Chitosan/gelatin (C/G) solution was prepared by dissolving chitosan and type A gelatin in 0.1 M acetic acid. A combination of 2%

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(w/v) chitosan with 0.2% (w/v) gelatin (C/G solution) was sterilized by autoclave. A 44.4% (w/v) glycerol 2-phosphate disodium salt hydrate solution was sterilized by filtration through 0.22 ␮m syringe filters. The glycerol phosphate (GP) solution was added drop by drop into the C/G solution while stirring on ice, and the pH value was adjusted to 7.4. Latanoprost was then added to the chitosan/gelatin/GP (C/G/GP) solution. A C/G/GP solution containing 250 ␮g/ml of latanoprost was prepared under a laminar flow hood and stored at 4 ◦ C until further use. 2.3. Rheological analysis of the latanoprost-loaded hydrogel The sol–gel transition temperature of the latanoprost-loaded hydrogel was measured using a rheometer equipped with a Peltier plate (aluminum, 20-mm plate) in oscillatory mode (Discovery HR1, TA Instruments, New Castle, DE). The elastic modulus (G ) and viscous modulus (G ) were analyzed at a gap of 1 mm and a fixed frequency of 1.0 Hz. The sol–gel transition temperature of the samples was measured in a range from 20 ◦ C to 45 ◦ C at a rate of 1 ◦ C per minute. The gelation time of the samples was analyzed at 34 ◦ C. 2.4. Scanning electron microscopy The morphology of the latanoprost-loaded hydrogel was observed using a field-emission scanning electron microscope (FESEM, Model JSM-7600F, JEOL Ltd., Tokyo, Japan). After the lyophilization process, the samples were fixed on a metal stub and then coated with gold under vacuum by an ion sputter (JFC-1200, JEOL Ltd., Tokyo, Japan). 2.5. In vitro drug release study Latanoprost-loaded hydrogel was added to a transwell (50 ␮l/well) mounted on a 24-well plate, and 1.5 ml of PBS was added into each well. The plates were then incubated at 37 ◦ C. A 1.5 ml volume of PBS was collected from the basal side on days 1, 2, 3, 5 and 7, and 1.5 ml of fresh PBS was added after each collection. The samples were analyzed using a LC–MS/MS system equipped with an electrospray ionization source. The LC–MS/MS system consisted of a Sciex API 3000 tandem mass spectrometer and an Agilent 1100 series LC system. The separation of latanoprost was performed on a Phenomenex Luna C18 column (50 mm × 4.6 mm, 5 ␮m) at a flow rate of 0.2 ml per minute. The mobile phase was 30% by volume deionized water that contained 0.1% formic acid and 70% by volume acetonitrile. LC–MS/MS analysis was conducted using positive electrospray ionization in the multiple reaction monitoring (MRM) mode, and the transition pairs selected for latanoprost were m/z 433.5 → m/z 397.5. 2.6. In vitro and in vivo biocompatibility testing The in vitro biocompatibility of the latanoprost-loaded hydrogel was evaluated using cell viability assays, hemolysis analysis and ocular irritation test. The cytocompatibility of the latanoprostloaded hydrogel on rabbit corneal epithelial (RCE) cells (CCL-60, American Type Culture Collection (ATCC), USA) was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RCE cells were cultured in MEM containing 10% fetal bovine serum (FBS). The culture medium was refreshed every 3 days. Latanoprost-loaded hydrogel (0.1 g) was immersed in 1 ml of MEM in a 48-well plate at 37 ◦ C in a 5% carbon dioxide and 95% relative humidity environment for 72 h to prepare the extraction medium for the cytotoxicity test. RCE cells were seeded in 96-well cell culture plates at a density of 5000 cells per well and cultured in MEM. After incubation for 18 h, the cells were washed with phosphate-buffered solution (PBS), and 200 ␮l of extraction

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medium was then added into the culture well. On days 1 and 2, the cells were washed with PBS, and 500 ␮g/ml (w/v) of MTT solution (100 ␮l) was added into each culture well. After 4 h of incubation, the MTT solution was then removed, and 100 ␮l of dimethyl sulfoxide (DMSO) was added to dissolve the formazan product. The absorbance was measured using an enzyme-linked immunosorbent assay (ELISA, Sunrise remote, TECAN, USA) reader at 570 nm. Cell viability was calculated as a percentage using the following formula: Cellviability (%) =

ODexperimentalgroup − ODblank × 100 ODcontrolgroup − ODblank

Hemolysis tests were performed by placing 1.4 g of latanoprostloaded hydrogel in direct contact with whole rabbit blood in saline at 37 ◦ C for 3 h. The positive and negative controls were distilled water and PBS, respectively. The samples were centrifuged at 1000 rpm for 15 min, and then the absorbance of the supernatant in each tube was analyzed using an ELISA reader at a wavelength of 545 nm. The percentage of hemolysis was calculated using the following equation: Hemolysis (%) =

ODsamples − ODnegative × 100 ODpositive − ODnegative

Ocular irritation was evaluated by corneal fluorescein staining. All experiment protocols were approved by the Ethics Committee for Animal Research of the Taipei Veterans General Hospital. New Zealand albino rabbits (body weight = approximately 3 kg) were used and maintained in accordance with the guidelines for the care and use of laboratory animals. Three male New Zealand albino rabbits were used in this study. A volume of fifty microliters of the latanoprost-loaded hydrogel was instilled onto one of the eyes, with the untreated eye serving as a control. After topical administration of the latanoprost-loaded hydrogel, corneal fluorescein staining was performed by instillation of 10% FLUORESCITE® to the corneal surface followed by observation with a portable slit-lamp biomicroscope at 1, 6 and 24 h. 2.7. Topical application of latanoprost-loaded hydrogel and IOP measurement Under general anesthesia with 10 mg/kg of Zoletil 50, ocular hypertension was induced in rabbit eyes by intravitreal injection of TA (Kersey & Broadway, 2006; Smithen, Ober, Maranan, & Spaide, 2004; Song, Gong, Liu, Ren, & Sun, 2011; Zarei-Ghanavati, Malaekeh-Nikouei, Pourmazar, & Seyedi, 2012). Each rabbit was administered 0.1 ml of TA into the right eye on days 0, 3, and 5. IOP measurement was performed using an Icare® TONOLAB tonometer (Tiolat, Helsinki, Finland) under topical anesthesia for the duration of the experimental period. Latanoprost-loaded hydrogel (50 ␮l) was administered via eye drop onto the inferior fornix of the conjunctiva on days 7, 14, 21, and 28. The commercial latanoprost formulation (Xalatan, 0.005% eye drops solution, Pfizer, USA) was topically applied daily onto the eyes with TA injection-induced elevated IOP for 4 consecutive weeks starting on day 7 of the study period. 2.8. In vivo release study After the rabbits were anesthetized with 10 mg/kg Zoletil 50 and topical anesthesia (0.5% proparacaine hydrochloride) was applied, 100 ␮l of aqueous humor was collected using a 30-gauge needle attached to a 1 cc syringe on days 1, 2, 3, 5 and 7. Under anesthesia, 100 ␮l of aqueous humor was collected using a 30-gauge needle on days 1, 2, 3, 5 and 7. These samples were added to 300 ␮l of acetonitrile and stored at 4 ◦ C until analysis. The samples were analyzed using a LC–MS/MS system equipped with an electrospray ionization

source. The LC–MS/MS system consisted of a Sciex API 3000 tandem mass spectrometer and an Agilent 1100 series LC system. The separation of latanoprost acid was performed using a Phenomenex Luna C18 column (50 mm × 4.6 mm, 5 ␮m) at a flow rate of 0.2 ml per minute. The mobile phase was 60% by volume deionized water containing 0.1% formic acid and 40% by volume acetonitrile. LC–MS/MS analysis was conducted using negative electrospray ionization in the MRM mode, and the transition pair for latanoprost acid was selected as m/z 389.3 → m/z 345.7. 2.9. Histology of the cornea The rabbits were euthanized at the end of the study period (day 35). The eyes were fixed in 10% formalin for 24 h. The samples were dehydrated through a graded ethanol series and then embedded in paraffin blocks. The samples were cut into 5-␮m thick sections and then stained with H&E. 2.10. Statistical analysis Statistical analysis was performed using one-way analysis of variance (ANOVA). Data are reported as the means ± standard deviation (SD) of at least three experiments and were considered significant when p < 0.05. 3. Results 3.1. Characterization of latanoprost-loaded hydrogels The sol–gel transition temperature is defined as the temperature at which the elastic modulus (G ) is higher than the loss modulus (G ). As shown in Fig. 1, the sol–gel transition temperature of the latanoprost-loaded C/G/GP solution was 34.18 ± 0.67 ◦ C. The microstructure of the latanoprost-loaded hydrogel was observed by SEM. Fig. 2 shows the interconnected pores and lamellar structures of the latanoprost-loaded hydrogel that may allow small molecules to diffuse freely through the hydrogel. 3.2. In vitro drug release study The cumulative drug release (%) of latanoprost from the hydrogel was calculated from the calibration curve of latanoprost. As shown in Fig. 3, the percentages of cumulative release at 1, 2, 3, 5 and 7 days were 20.99% ± 1.77%, 35.16% ± 1.86%, 47.76% ± 1.45%, 50.85% ± 1.66%, and 51.71% ± 1.45%, respectively. 3.3. In vitro biocompatibility of latanoprost-loaded hydrogel In living cells, MTT is converted into formazan crystals by mitochondrial dehydrogenases. The level of production of colored formazan is directly proportional to the number of living cells. As shown in Fig. 4(a), cell viability in the latanoprost-loaded hydrogel group on day 1 and day 2 was 101.7% ± 2.2% and 97.3% ± 4.5%, respectively. The results showed that there was no significant difference in cell viability between the control and the latanoprostloaded hydrogel group on day 1 or 2 (n = 6, p > 0.05). The results showed that no cytotoxic effects were induced by latanoprostloaded hydrogel on RCE cells. Moreover, there were no significant differences between latanoprost-loaded hydrogel and the negative control group (Fig. 4(b)). 3.4. Corneal fluorescein staining Fluorescein staining was used to evaluate the integrity of the corneal epithelium. After topical application of the latanoprostloaded hydrogel, fluorescein staining of the corneas was negative

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Fig. 1. (a) Temperature- and (b) time-dependence of the storage modulus (G ) and the loss modulus (G ) of latanoprost-loaded hydrogel at pH 7.4. The gelation temperature was 34.18 ± 0.67 ◦ C. The gelation time was 70.72 ± 7.19 s at 34 ◦ C (n = 3). (c) Photograph of latanoprost-loaded hydrogel at 25 ◦ C (left) and at 34 ◦ C (right).

Fig. 2. Scanning electron photomicrographs of latanoprost-loaded hydrogel at magnifications of (a) 1000× and (b) 2000×.

at 1, 6 and 24 h (Fig. 5). Moreover, slit-lamp biomicroscopy showed no that abnormal findings were associated with latanoprost-loaded hydrogel instillation.

3.5. IOP-lowering effects of topical application of latanoprost-loaded hydrogel IOP was measured in both eyes during the study period (day 0–day 35). Fig. 6(a) shows that there was a significant increase in IOP, which remained elevated until day 35 after intravitreal injection of TA. Latanoprost-loaded hydrogel was topically applied weekly onto the eyes with TA injection-induced elevated IOP for 4 consecutive weeks starting on day 7 of the study period. As shown in Fig. 6(b), there were no significant differences in the mean IOPs between the latanoprost-loaded hydrogel group and the normal tension control group after day 14 of the study period. The results show that after weekly application of the latanoprostloaded hydrogel, the IOP was effectively decreased within 7 days in the ocular hypertensive rabbit eyes and remained at a normal level for the rest of the study period. As shown in Fig. 6(c), there were no significant differences in the mean IOPs between the Xalatan group and the normal tension control group after day 15 of the study period. The results showed that after daily application of the Xalatan, the IOP was effectively decreased within 8 days in the ocular hypertensive rabbit eyes and remained at a normal level for the rest of the study period. Moreover, weekly administration of latanoprost-loaded hydrogel showed the similar pattern with daily administration of Xalatan in the IOP measurement (Fig. 6(d)).

3.6. Aqueous humor levels of the drugs Latanoprost is hydrolyzed by esterases in the cornea into latanoprost acid, which is its pharmacologically active form. The concentration of latanoprost acid in aqueous humor was evaluated using LC–MS/MS. After a single topical administration of the latanoprost-loaded hydrogel, the concentration of latanoprost acid in the aqueous humor on days 1, 2, 3, 5 and 7 was 30.42 ± 14.21, 23.41 ± 10.98, 21.23 ± 18.05, 12.33 ± 9.34 and 4.35 ± 1.07 ng/ml, respectively (Fig. 7).

3.7. Histological evaluation after topical administration of latanoprost-loaded hydrogel Fig. 8 shows the results of H&E staining of corneas from the control and the latanoprost-loaded hydrogel groups. At the end of the study period, histological examination did not reveal any signs of inflammation or fibrosis in the group treated with the latanoprostloaded hydrogel (Fig. 8(b)).

4. Discussion In the present study, we developed a thermosensitive chitosanbased hydrogel that contained latanoprost to be used a topical eye drop formulation for long-term IOP control. As shown in Fig. 1(a), the sol–gel transition temperature of the developed latanoprostloaded hydrogel was 34.18 ◦ C. It has been reported that the ocular surface temperature is higher than 34 ◦ C in patients with ocular hypertension (Galassi, Giambene, Corvi, & Falaschi, 2007). Fig. 2(b)

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Fig. 3. In vitro release study. (a) Mass spectrum at a positive ionization mode of latanoprost, cumulative (b) concentration and (c) percent release of latanoprost from hydrogels in PBS at 37 ◦ C (n = 6).

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Fig. 4. In vitro biocompatibility was evaluated by (a) cell viability assays and (b) hemolysis assays. The results revealed that no cytotoxic or hemolytic effects were induced by the newly developed latanoprost-loaded hydrogel (n = 3).

Fig. 5. Images of (a) bright field and (b) fluorescein-stained corneas at 1, 6, and 24 h after topical administration of the latanoprost-loaded hydrogel.

shows that the gelation time of latanoprost-loaded hydrogel is 70.72 ± 7.19 s at 34 ◦ C. As a topical eye drop formulation, the developed latanoprostloaded hydrogel can be administered in a liquid form that turns into a hydrogel in vivo. The mechanisms involved in the sol–gel

transition include hydrophobic interactions, hydrogen bonding, electrostatic interactions and molecular chain movements. When the temperature rises above the gelling point (34.18 ◦ C), hydrophobic interactions are the main driving force during gel formation (Cheng et al., 2010; Supper et al., 2014). The results suggest

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Fig. 6. IOP was (a) increased after intravitreal injection of triamcinolone acetonide (TA) and decreased following the treatment with (b) latanoprost-loaded hydrogel or (c) Xalatan. There were (d) no significant differences in the mean IOPs between the latanoprost-loaded hydrogel and Xalatan group (n = 5, * p < 0.05).

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Fig. 7. Evaluation of in vivo drug release. (a) Mass spectrum at a negative ionization mode for latanoprost acid and (b) aqueous humor levels of latanoprost acid (n = 3).

that encapsulation of hydrophobic latanoprost (250 ␮g/ml) in the developed chitosan-based hydrogel did not significantly affect the behavior of the sol–gel transition. SEM images showed that this newly developed latanoprostloaded hydrogel presents a highly porous structure, which allows the loading and release of drugs (Fig. 2). Fig. 3(b) shows that the developed hydrogel slowly released latanoprost, with an accumulated release of 51.71% of the agent at day 7. Recent studies have indicated that this hydrophobic compound is dispersed within the hydrogel matrix and released by diffusion (Berrada et al., 2005; Ruel-Gariepy et al., 2004). The results revealed that this

newly developed latanoprost-loaded hydrogel has a sustained drug release profile over 7 days. For future clinical applications, the average dose of latanoprost that is released from the developed hydrogel may be approximately 0.9 ␮g per day following the administration of 50 ␮l of the developed hydrogel (one drop per week). The amount of latanoprost released from the developed hydrogel may be lower than the amount released from daily administration of commercial latanoprost eye drops (1.5 ␮g per day). Chitosan and gelatin are natural, biocompatible and biodegradable polymers. In recent years, chitosan and gelatin have been widely used in pharmaceutical and biomedical applications (Dash,

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Fig. 8. Corneal sections stained with hematoxylin and eosin revealed no signs of inflammation at the end of the study period (day 35) in animals that received topical administration of latanoprost-loaded hydrogel.

Chiellini, Ottenbrite, & Chiellini, 2011; Hathout & Omran, 2015). It has been reported that the biocompatibility of chitosan is strongly associated with the degree of deacetylation (DD) (Dash et al., 2011; Rodrigues, Dionisio, Lopez, & Grenha, 2012). Chitosan with DD higher than 95% was used in this study. The results of cell viability assays revealed that no cytotoxic and hemolytic effects were induced by the newly developed latanoprost-loaded hydrogel (Fig. 4). Moreover, there were no signs of eye irritation or corneal injury after topical administration of this hydrogel (Fig. 5). The results showed that the newly developed latanoprost-loaded hydrogel has good biocompatibility and might be suitable for ocular drug delivery applications. Elevated IOP is a major risk factor for glaucoma (Bucolo et al., 2013). In this study, a rabbit model of ocular hypertension was established with repeated intravitreal injections of TA, which significantly raised IOP (Fig. 6(a)) and lasted for at least 35 days. Following treatment with latanoprost-loaded hydrogel, IOP was effectively decreased within 7 days and maintained at a normal level throughout the remainder of the experiment (Fig. 6(b)). It has been reported that latanoprost is absorbed through the cornea following ocular instillation and that it is rapidly hydrolyzed into latanoprost acid (its active form). Topical application of latanoprost increases the outflow of aqueous humor via the uveoscleral pathway, which leads to a reduction in IOP (Alm, 2014). The results of the in vivo release study in this report show that latanoprost acid was detected in the aqueous humor for 7 days following a single topical administration of the developed hydrogel (Fig. 7). The level of latanoprost acid in the aqueous humor was not detectable after day 7, as measured by LC–MS/MS (data not shown). These results suggest that this newly developed latanoprost-loaded hydrogel may be administered once weekly to maintain the therapeutic level in the eye and to control IOP. Chitosan possesses mucoadhesive properties and has been shown to be a promising vehicle for topical ocular drug delivery (Fulgencio Gde et al., 2012; Gratieri et al., 2010; Hermans et al., 2014). The mucoadhesive property of chitosan increases with an increasing DD, which is associated with positively charged amino groups (Almeida et al., 2014). In this study, the newly developed hydrogel contains highly deacetylated chitosan (DD > 95%), which may result in a prolonged retention time and, therefore, greater corneal permeation. Moreover, histological analysis showed no signs of inflammation at the end of the study after repeated weekly administration of the latanoprost-loaded hydrogel (Fig. 8). These results suggest that this newly developed hydrogel significantly prolonged the residence time of the drug and may be used for potential applications for long-term glaucoma therapies.

5. Conclusion In this study, we developed a thermosensitive latanoprostloaded hydrogel as a topical eye drop formulation for the sustained release of latanoprost to control ocular hypertension. The gelling temperature of the developed hydrogel was 34.18 ◦ C. The in vitro and in vivo release studies showed that latanoprost release from the developed hydrogel displayed a sustained-release profile. The biocompatibility of the developed hydrogel was demonstrated in vitro and in vivo. In a rabbit model of glaucoma, TA-induced elevated IOP was significantly decreased within 7 days and was maintained within a normal range by a weekly topical administration of 250 ␮g/ml of latanoprost-loaded hydrogel for the remainder of the study period. The results of this study suggest that the developed hydrogel possesses promising properties that contribute to its sustained release of latanoprost. This newly developed chitosan-based formulation may provide a non-invasive alternative to traditional eye drops for long-term IOP control in the near future. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements This study was funded by the Ministry of Science and TechnologyMOST104-2325-B-010-006, and Department of Health, Taipei City Government. We also thank for the Centre for Medical Research of Taipei City Hospital for providing experimental space and facilities. References Agrawal, A. K., Das, M., & Jain, S. (2012). In situ gel systems as ‘smart’ carriers for sustained ocular drug delivery. Expert Opinion on Drug Delivery, 9(4), 383–402. Alm, A. (2014). Latanoprost in the treatment of glaucoma. Journal of Clinical Ophthalmology, 8, 1967–1985. Almeida, H., Amaral, M. H., Lobao, P., & Lobo, J. M. (2014). In situ gelling systems: a strategy to improve the bioavailability of ophthalmic pharmaceutical formulations. Drug Discovery Today, 19(4), 400–412. Berrada, M., Serreqi, A., Dabbarh, F., Owusu, A., Gupta, A., & Lehnert, S. (2005). A novel non-toxic camptothecin formulation for cancer chemotherapy. Biomaterials, 26(14), 2115–2120. Bron, A., Chiambaretta, F., Pouliquen, P., Rigal, D., & Rouland, J. F. (2003). Efficacy and safety of substituting a twice-daily regimen of timolol with a single daily instillation of nonpreserved beta-blocker in patients with chronic glaucoma or ocular hypertension. Journal Franc¸ais D’Ophtalmologie, 26(7), 668–674. Bucolo, C., Salomone, S., Drago, F., Reibaldi, M., Longo, A., & Uva, M. G. (2013). Pharmacological management of ocular hypertension: current approaches and future prospective. Current Opinion in Pharmacology, 13(1), 50–55.

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