Ocular inserts based on chitosan and brimonidine tartrate: Development, characterization and biocompatibility

Journal of Drug Delivery Science and Technology 32 (2016) 21e30

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Research paper

Ocular inserts based on chitosan and brimonidine tartrate: Development, characterization and biocompatibility Júlio Fernandes De Souza a, Kamila Nunes Maia a, Patrícia Santiago De Oliveira Patrício b, Gabriella Maria Fernandes-Cunha c, Marina Goulart Da Silva d, Carlos Eduardo De Matos Jensen d, Gisele Rodrigues Da Silva a, * ~o Joa ~o del-Rei, Avenida Sebastia ~o Gonçalves Coelho, 400, Chanadour, 35501-296, Divino polis, Minas Gerais, Brazil Biotechnology, Federal University of Sa Department of Chemistry, Federal Center of Technological Education, Avenida Amazonas, 5253, Nova Suíça, 30421-169, Belo Horizonte, Minas Gerais, Brazil c ^nio Carlos, 6627, Pampulha, 31270-901, Belo Horizonte, Minas Gerais, Brazil School of Pharmacy, Federal University of Minas Gerais, Avenida Anto d ~o Joa ~o del-Rei, Avenida Sebastia ~o Gonçalves Coelho, 400, Chanadour, 35501-296, Divino polis, Minas Gerais, School of Pharmacy, Federal University of Sa Brazil a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 November 2015 Received in revised form 13 January 2016 Accepted 15 January 2016 Available online xxx

The glaucoma is an ocular pathology characterized by the increase of intraocular pressure and possibility of retinal degeneration. The treatment is based on the administration of eye drops. However, they do not induce the permanence of drug in the eye, compromising its bioavailability. In this study, inserts based on chitosan and brimonidine tartrate were developed to treat the glaucoma by improving the drug bioavailability. Inserts were characterized using different analytical techniques FTIR, SEM, DSC and WAXS. The in vitro release profile of drug from inserts was evaluated. The in vitro biocompatibility against ARPE-19 and MIO-M1 cells was investigated. The in vivo biocompatibility using the chorioallantoic membrane was also demonstrated. Analytical techniques demonstrated that the amorphous drug was physically dispersed into the polymeric chains without chemical interactions. However, a portion of drug crystals was on the surface of systems. Inserts provided the controlled release of drug for 30 days without a burst effect. Inserts did not induce a deleterious effect to ocular cells, indicating their biocompatibility. They were also well tolerated in vivo, suggesting the absence of toxicity. These inserts could be potential delivery systems to reduce the intraocular pressure and to induce neuroprotective effects in glaucomatous patients. © 2016 Elsevier B.V. All rights reserved.

Keywords: Glaucoma Chitosan Brimonidine tartrate Ocular inserts Inserts based on chitosan and brimonidine tartrate Chorioallantoic membrane

1. Introduction The glaucoma is a long-term ocular neuropathy defined by optic disc or retinal nerve fiber structural abnormalities, which induces visual field loss and/or irreversible blindness [1]. The glaucoma is the second cause of blindness in the United States and worldwide. It affects approximately 67 million people around the world and 10% of them are bilaterally blind [2]. The most important risk factor for

~o Gonçalves Coelho, 400, Chanadour, * Corresponding author. Avenida Sebastia polis, Minas Gerais, Brazil. 35500-296, Divino E-mail addresses: [email protected] (J.F. De Souza), [email protected]. br (K.N. Maia), [email protected] (P.S. De Oliveira Patrício), [email protected] (G.M. Fernandes-Cunha), [email protected] (M.G. Da Silva), [email protected] (C.E. De Matos Jensen), [email protected] (G.R. Da Silva). http://dx.doi.org/10.1016/j.jddst.2016.01.008 1773-2247/© 2016 Elsevier B.V. All rights reserved.

progression of the glaucoma is the intraocular pressure (IOP) elevation, which leads to the optic nerve damage [3]. Eye drops are the pharmaceutical dosage forms most applied in the treatment of the glaucoma and other ocular diseases affecting the anterior segment of the eye. However, the ocular bioavailability of the drugs instilled topically is extremely poor, being lower than 5% of the total dose [4]. The inefficacy of this topical route is related to the precorneal factors such as tear turnover and drainage, dilution by tear flow, reflex blinking and lacrimation and highly selective corneal epithelial barrier [5]. The precorneal factors also lower the concentration gradient, which is the driving force for the passive absorption of drug across the cornea and conjunctiva [4,5]. These anatomical and physiological elements lead to the fluctuation of the IOP and the progression of the glaucoma, and consequently, lower the expectations of the patients to the daily therapy. To overcome the drawbacks of the conventional eye drops,

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ocular drug delivery systems, such as ocular inserts, could be applied as a therapeutic alternative to treat the glaucoma. Ocular inserts are solid or semi-solid devices, usually elaborated using natural or synthetic polymers, to be inserted in the conjunctival sac to deliver the drug in the anterior segment of the eye [6]. The polymeric matrix controls the delivery of therapeutic concentrations of the drug directly in the target tissues and provides its prolonged release, increasing its ocular residence time and bioavailability. As a consequence, the ocular inserts are capable of improving the glaucomatous patient compliance due to the efficacy of the therapy and the reduced frequency of administration. In this context, it is available, in the pharmaceutical market, the Ocusert®, an ocular device based on pilocarpine alginate enclosed by ethylene-vinyl acetate membrane. This non-biodegradable membrane delivers therapeutic concentrations of the pilocarpine for 7 consecutive days, reducing significantly the IOP in patients [7]. Additionally, many studies have been performed to demonstrate the development of ocular inserts containing different types of drugs to treat the glaucoma, including the bimatoprost [8], timolol maleate [9] and betaxolol hydrochloride [10]. In this study, ocular inserts based on chitosan and brimonidine tartrate were developed as alternative delivery systems to treat the glaucoma. The chitosan is a polycationic biopolymer obtained through the alkaline deacetylation of chitin, a natural polysaccharide found abundantly in marine crustaceans [11]. The chitosan contains a large number of hydroxy and amino groups, which provide different possibilities for derivatization or grafting of desirable substances [12]. Moreover, the chitosan is biocompatible, biodegradable, and non-toxic. It is able of interacting chemically with the mucus layer or the eye tissues, enhancing the residence time in the anterior segment of the eye, and consequently, increasing the bioavailability of the incorporated drug [13]. The brimonidine tartrate is an anti-glaucomatous drug highly selective for a-2 adrenergic receptors, which provides a potent hypotensive effect through increasing uveoscleral outflow along with decreasing aqueous humor production [6]. Moreover, it was evidenced that the brimonidine tartrate is capable of penetrating the posterior segment of the eye, leading to a neuroprotective function by promoting retinal ganglion cell survival [14,15]. The developed ocular inserts were characterized by applying different analytical techniques (FTIR, SEM, DSC and WAXS). The swelling study was performed. The in vitro release profile of the brimonidine tartrate from the chitosan inserts was also investigated for 30 consecutive days. The in vitro biocompatibility of these ocular inserts was analyzed using Müller glial cells (MIO-M1) and retinal pigment epithelial cells (ARPE-19) cultures, considering their viability and morphology; and the in vivo biocompatibility was described using the irritation test of the chorioallantoic membrane (CAM). The brimonidine tartrate-loaded chitosan inserts represented a novelty in the field of the pharmaceutical technology due to their performance in the in vitro drug release study. This hydrophilic drug was leached from the inserts for a long period without inducing a burst release. The inexistence of the burst release demonstrated the capacity of the polymeric chains to control the delivery of the drug. It have been previously showed that inserts based on chitosan did not control the release of drugs presented at the surface of the systems, leading to a significant burst release [9,16]. The brimonidine tartrate-loaded chitosan inserts represented also an innovation in the field of the pharmaceutical technology, since they were designed without using preservatives in their formulation. The preservatives, such as the benzalkonium chloride, added in the multiple-dose conventional eye drops, may induce ocular inflammation and reduce tear secretion, and consequently, disrupt the homeostasis of the ocular surface [17].

Moreover, the inserts represented also originality in the field of the ophthalmology, since, nowadays, there are not in the pharmaceutical market mucoadhesive inserts capable of controlling and prolonging the ocular delivery of a hypotensive and neuroprotective drug in order to reduce the IOP and increase the patient compliance to the therapy. 2. Materials and methods 2.1. Preparation of the inserts based on chitosan and brimonidine tartrate Inserts based on chitosan and brimonidine tartrate were produced by a casting/solvent evaporation technique [9]. Briefly, standard solution of chitosan and brimonidine tartrate was prepared by dissolving 500 mg and 56.25 mg, respectively, of these substances in 25 mL of 2% (v/v) acetic acid aqueous solution. The solution was dried at room temperature for 3 days. The dried film was cut into spheres of 4 mm diameter and 0.3 mm of thick to obtain inserts containing 1.0 mg of brimonidine tartrate. Inserts containing chitosan without drug were also produced. 2.2. Determination of the weight of inserts based on chitosan and brimonidine tartrate For the determination of the weight of the inserts based on chitosan and brimonidine tartrate, the procedure stated in the United States Pharmacopeia was followed [18]. Briefly, 20 inserts were individually weighted. The average weight and the relative standard deviation were calculated. 2.3. Determination of the content of brimonidine tartrate incorporated into chitosan inserts For the determination of content uniformity of brimonidine in the chitosan inserts, the procedure stated in the United States Pharmacopeia was followed [18]. The determination of brimonidine tartrate was performed as following: ten inserts were selected and weighted. Each insert was dissolved in 50 mL of 2% (v/v) acetic acid aqueous solution. The standard solution of the brimonidine tartrate was also prepared as described above. The absorbance of the resultant solutions was measured at 258 nm using a chitosan solution as blank. The spectrophotometric method of quantitation of brimonidine tartrate was previously validated [19]. The uniformity content of brimonidine tartrate in the inserts was expressed as the percent of the pre-indicated value (approximately 1.0 mg). The relative standard deviation was also calculated. 2.4. Characterization 2.4.1. Fourier transform infrared spectroscopy Infrared spectra were collected in a Fourier transform infrared spectrophotometer (FTIR; Perkin Elmer, model Spectrum 1000). Measurements were carried out using the attenuated total reflectance (ATR) technique. Each spectrum was a result of 32 scans with a resolution of 4 cm
1 [20]. 2.4.2. Differential scanning calorimetry Differential scanning calorimetric (DSC) thermograms were obtained on a Mettler Toledo DSC (Switzerland). Samples were put into aluminum pans. The calorimeter was calibrated for temperature and heat flow accuracy using pure indium melting (m.p. 156.6  C and DH ¼ 25.45 J g
1). The temperature ranged from 0 to 700  C with a heating rate of 25  C min
1 under nitrogen atmosphere [20].

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2.4.3. Wide angle X-ray scattering Wide angle X-ray scattering (WAXS) was performed by using a synchrotron light beam with a wavelength of 1.608 A and an exposition time of 300 s. The scattering intensity was recorded by a Pilatus (100 K, 33 mm  84 mm) detector. The sample to detector distance used was 80 mm [20]. 2.4.4. Scanning electron microscopy Scanning electron microscopy (SEM) was performed using a JEOL microscope (model JSM e 6360LV) operating at 15 kV. The inserts were fractured and mounted on aluminum stubs using double-sided adhesive tape. Prior to microscopical examination, all the samples were sputter-coated with a gold layer under argon atmosphere using a sputter apparatus (Balzers Union SCD 040 unit, Balzers, Germany). The insert surfaces were viewed at 100 magnification and the images were transferred to the computer by means of a Digital Image Transference Interface (DITI). The photomicrographs were adjusted using the software Adobe Photoshop 6.0 and Adobe Illustrator 9.01 (Adobe Systems Incorporated, 2000, USA) [20]. The inserts recently produced and after 15 and 30 days of incubation in phosphate buffer solution (PBS) (pH 7.4) were submitted to the SEM analyses. 2.4.5. Swelling study Insert swelling study was carried out in PBS (pH 7.4). Insert with and without brimonidine tartrate (n ¼ 3 for each insert) were weighed and placed in PBS for predetermined period of time (30 and 60 min, 2, 6 and 10 days). After immersion, the inserts were removed from the medium, the excess surface water was removed by using filter paper, and the pieces were weighed. Swelling index was calculated by using the equation: Swelling index ¼ [(Wt
W0)/ W0]. The weight of the swollen insert after predetermined periods of time (t) was represented by Wt. The original weight of the insert at zero time is represented by W0 [16]. 2.4.6. In vitro release of brimonidine tartrate from the chitosan inserts Five inserts based on chitosan and brimonidine tartrate were immersed inside different tubes containing 1.5 mL of PBS pH 7.4. The tubes were placed inside a shaker incubator set at 37  C and 30 rpm. At predetermined intervals, 1.5 mL of the medium was sampled and 1.5 mL of fresh medium was immediately added to each tube. The release profile was evaluated as the cumulative percentage of the drug released in the medium. The amount of brimonidine tartrate leached was measured using the spectrophotometric method previously validated [19]. 2.5. In vitro biocompatibility study 2.5.1. ARPE-19 and Müller glial cell (MIO-M1 cell) cultures ARPE-19 cells, an established but non-immortalized human RPEcell line, were graciously provided by Dr. Hjelmeland (University of California, Davis, CA) and were grown in a Dulbecco's modified eagles medium and Ham's F12 medium (DMEM/F12 Gibco BRL: Grand Island, NY) with 10% fetal bovine serum (FBS Gibco BRL: Grand Island, NY) in a 37  C humidified atmosphere of 5% CO2 and 95% air [21]. Müller glial cells (MIO-M1 cells), a spontaneously immortalized RMG cell line originated from human retina, were kindly provided by Dr. Astrid Limb (University College London, London, UK) and were grown in a Dulbecco's modified eagles medium/glutamax (DMEM/glutamax Gibco BRL: Grand Island, NY) with 10% fetal bovine serum (FBS Gibco BRL: Grand Island, NY), 0.4% gentamicin, and 0.1% amphotericin B at 37  C in a humidified atmosphere of 5% CO2 and 95% air [21].

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The culture medium of both cells was refreshed every 2 days. Upon confluence, cells were rinsed with 2 mL of a 0.05%trypsinEDTA (Gibco, Grand Island, NY) solution and incubated with 5 mL of trypsin-EDTA at 37  C in a humidified atmosphere of 5% CO2 and 95% air. Next, within 5e15 min, the trypsin enzyme activity was stopped by the addition of 5 mL of complete growth medium and centrifuged for 5 min at 1500 rpm. The supernatant was discarded, while the cells were resuspended in 13 mL of fresh medium and seeded onto culture flasks for further propagation and subsequent passages [21]. 2.5.2. ARPE-19 and MIO-M1 cell cultures in contact with the inserts based on chitosan and brimonidine tartrate The inserts based on chitosan and brimonidine tartrate were disinfected by exposure to UV light for 90 min on each side prior to cell culture. Inserts based on chitosan (without drug) were also disinfected as described above. ARPE-19 and MIO-M1 cells were plated in contact of the inserts and polyester tissue culture polystyrene (TCPS) (Costar, Cambridge, MA), as a control at a density of 4  103 cells/well [21]. 2.5.3. Cytotoxicity of the inserts based on chitosan and brimonidine tartrate After 1, 2, 5 and 10 days in the culture, the medium was aspirated, and the ARPE-19 and MIO-M1 cells in contact with inserts based on chitosan and brimonidine tartrate, inserts without drug and control TCPS were rinsed with phosphate-buffered saline (PBS). The ARPE-19 and MIO-M1 cells were incubated with 150 mL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertrazolium bromide (MTT) (1 mg/mL in PBS) (Sigma Chemical, Saint Louis,CO). After 3 h of incubation, the cells were lysed with 100 mL of isopropanol and absorbance values were measured at 570 nm versus 630 nm using a microplate reader (BioRad, San Diego, CA). The mean absorbance on the control surface was set as 100%, while the mean absorbance ± standard deviation in contact with the inserts was obtained as a percentage of the control. Data were presented as a histogram [21]. 2.5.4. Morphology of the ARPE-19 and MIO-M1 cells e immunofluorescence At 10 days of culture, the medium was aspirated, and the ARPE19 and MIO-M1 cells in contact with inserts based on chitosan and brimonidine tartrate, inserts without drug and control TCPS were rinsed with PBS and fixed in paraformaldehyde 4% (v/v) (Merck Eurolab, Fontelay Sous-Bois, France) for 15 min. Next, fixed cells were rinsed again with PBS for 5 min and immersed in PBS containing 0.3% (v/v) Triton X-100 (SigmaeAldrich) for 15 min. After rising in PBS for 5 min, the nuclei were stained with 40,6diamidino-2-phenylindole (DAPI) (SigmaeAldrich) in PBS (1:1250) for 5 min at room temperature. After nuclei staining with DAPI, F-actin fibers were labeled with Phalloidin FITC (SigmaeAldrich) in PBS (1:250) for 30 min at room temperature. Finally, the cells were washed five times at 5 min intervals with PBS and one time with water, mounted in Gel Mount (Biomeda, Burlingame, CA) and viewed using an Olympus IX70 fluorescent microscope attached to a digital camera (Olympus DP70) [21]. At 10 days of culture, for the labeling of glial fibrillary acidic protein (GFAP), the MIO-M1 cells grown in contact with inserts based on chitosan and brimonidine tartrate, inserts without drug and control TCPS were fixed with p-formaldehyde 4% (v/v) for 30 min at room temperature. The fixed cells were incubated with PBS containing Triton X-100 0.1% (v/v) for 30 min. This was followed by incubation with polyclonal rabbit antibody against GFAP (1:100) (Dako, Trappes, France) at room temperature for 3 h. After washing with PBS, an Alexa Fluor 488-conjugated goat anti-rabbit

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IgG (1:100) (Molecular Probes, Leiden, The Netherlands) was applied for 60 min in the dark. Finally, the nuclei were stained with DAPI (SigmaeAldrich) in PBS (1:1250) for 5 min at room temperature. Then, the cells were rinsed five times, mounted, and viewed using Olympus IX70 fluorescent microscope attached to a digital camera (Olympus DP70) [21]. 2.6. In vivo biocompatibility study 2.6.1. Hen's egg test-chorioallantoic membrane (HET-CAM) irritation test Fertilized hen's eggs were purchased from a poultry farm (Alimentos Rivelli, Brazil). The collected hen's eggs were incubated at 37 ± 0.5  C and 40% ± 4% relative humidity for 10 days. Eggs were turned every day during incubation but were left in a horizontal position for several minutes to assure that the embryo was properly positioned. On day 10, each egg was opened by cracking the underside of the egg against the edge of a plastic Petri dish [22]. The CAM was exposed and 300 mL of the samples were placed directly onto the CAM's surface. After 20 s, the samples were discarded and the CAM was carefully washed with HEPES buffer (pH 7.4) to ensure the total removal of the tested substance. The CAM was visually observed for 5 min (0.5, 2 and 5 min) regarding the appearing of any of the following phenomena: hyperemia, hemorrhage and coagulation for which a score was given [23]. The samples were the followed: PBS pH 7.4 (negative control), 0.1 mol/L sodium hydroxide solution (positive control), inserts based on chitosan and brimonidine tartrate, inserts based on chitosan, brimonidine tartrate leached from the chitosan inserts in PBS pH 7.4 for 7 days. 3. Results 3.1. Preparation of the inserts based on chitosan and brimonidine tartrate, weight of the inserts and content of the drug The inserts based on chitosan and brimonidine tartrate presented 5.94 ± 0.05 mm and 330 ± 10 mm of diameter and thickness, respectively. The systems showed 2.58 ± 0.01 mg of weight and the relative standard deviation was 0.45%. The brimonidine tartrate content into the chitosan inserts was 1.03 ± 0.04 mg and the standard deviation was 2.61%, indicating that the drug was uniformly distributed into the polymeric chains, as required by the pharmacopeia [18]. The inserts presented as circular flexible translucent films with a homogeneous surface without any crack.

3.2. Characterization The Fig. 1 showed the FTIR spectra of inserts based on chitosan and brimonidine tartrate (Fig. 1a), pure brimonidine tartrate (Fig. 1b), inserts based on chitosan without drug (Fig. 1c) and chitosan raw material (Fig. 1d). The FTIR spectrum of the pure brimonidine tartrate demonstrated typical infrared bands such as at 1591 cm
1 corresponding to eNH bending vibration; at 1651 cm
1 equivalent to the stretching vibration of the C]N and C]C groups; and at 1700 cm
1 corresponding to the C]O group of the tartrate salt. The obtained infrared bands of the brimonidine tartrate were similar to those previously described [6]. The FTIR spectrum of chitosan raw material showed characteristic absorption bands such as between 1540 and 1650 cm
1attributed to amide I (C]O stretching) and to NeH amine vibration overlapped to amide II (NeH vibration), respectively [8]; at 3347 cm
1 due to the stretching vibration of the OeH and NeH bonds and at 2860 cm
1 due to vibrations of the CeH bonds. The FTIR spectrum of the chitosan inserts (without drug) presented the same infrared bands of the spectrum of the pure chitosan; however the band at approximately 3347 cm
1, attributed to the stretching vibration of the OeH and NeH bonds, broadened, probably due to the reduction of the formation of intermolecular hydrogen bonding in chitosan film [24] induced by the random entanglement of the polymeric chains of the chitosan during the evaporation of the organic solvent used to design the inserts. The FTIR spectrum of the ocular inserts containing chitosan and brimonidine tartrate revealed that the typical bands of the chitosan overlapped to the bands of the drug, preventing the identification of chemical interactions between organic groups of both substances. The infrared bands of the brimonidine tartrate were masked probably due to the lower proportion of the drug related to the polymer in the ocular systems. Masking of drug peaks due to small amount of the drug in different systems was also previously reported [6]. The Fig. 2 demonstrated the DSC thermograms of pure brimonidine tartrate (Fig. 2a), chitosan raw material (Fig. 2b), inserts containing chitosan and brimonidine tartrate (Fig. 2c) and inserts based on chitosan without drug (Fig. 2d). The thermogram of the pure drug demonstrated its crystalline structure due the existence of a sharp melting endothermic peak at 215.97  C. The obtained melting point for brimonidine tartrate was similar to that previously reported [25]. Furthermore, the thermogram of the pure drug indicated an exothermic transition at 269.43  C representing the thermal decomposition of the brimonidine tartrate. The DSC curve

Fig. 1. FTIR spectra of the inserts based on chitosan and brimonidine tartrate (a), pure brimonidine tartrate (b), chitosan raw material (c) and inserts composed on chitosan without drug (d).

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Fig. 2. DSC curves of pure brimonidine tartrate (a), chitosan raw material (b), inserts containing chitosan and brimonidine tartrate (c) and inserts based on chitosan without drug (d).

of the chitosan raw material showed a broad endothermic peak at 61.19  C corresponding to the dehydration. Then, a sharp exothermic transition at 311.69  C was verified due to the thermal degradation of the polymer, including dehydration of the anhydro glucosidic ring, depolymerization and decomposition of deacetylated and acetylated chitosan unit [26]. Significant changes were not observed for the DSC curve of the inserts based on chitosan when compared with the DSC curve of the chitosan raw material. Finally, the DSC scan of the inserts containing chitosan and brimonidine tartrate showed that the sharp endothermic peak of the drug was not detected, which could be attributed to the low drug to polymer ratio, in accordance with data previously described by Aburahma and Mahmoud [6], who demonstrated the marked reduction of the intensity of the brimonidine endothermic peak due to its low amount incorporated into the polymeric chains. The inexistence of the drug sharp characteristic peak could be also attributed to the amorphization of the drug due to its well distribution into the polymer [27]. The Fig. 3 illustrated the WAXS patterns for pure brimonidine tartrate (Fig. 3a), inserts based on chitosan and brimonidine tartrate (Fig. 3b) and pure chitosan (without processing into inserts) (Fig. 3c). The diffraction patterns of brimonidine tartrate depicted several intense peaks at diffraction angles 2q of 13.5 , 13.9 , 24.8 , 29.6 and 33.5 , demonstrating the crystalline nature of the pure drug. The WAXS patterns of the chitosan indicated the semicrystalline nature of this polymer [28] due to the existence of peaks at diffraction angles 2q of 10 and 20 . These crystalline domains occurred due to high intensities of intermolecular H-bonds among OeH and NeH groups on chitosan chain segments [29]. Finally, the diffractogram of the inserts containing both substances showed a intense drug diffraction peak at 2q ¼ 29.6 ; however, the other sharp peaks of the brimonidine tartrate were not evidenced,

suggesting that the crystalline drug was partially converted to an amorphous state when incorporated into the polymeric matrix of the chitosan. The amorphization of the brimonidine tartrate was related to its dispersion within the polymer [6,30]. Moreover, the irregular molecular structure of the film that arises from the random entanglement of polymeric chains reduces drug crystalline character as well as prevents its re-crystallization during the evaporation of the organic solvent used in the manufacturing process of the ocular inserts [6,31]. This crystallographic data corroborated with the thermal behavior of the brimonidine tartrate in its entrapped form. Additionally, the diffractogram of the ocular inserts demonstrated that the chitosan diffraction peaks at 2q ¼ 10 and 2q ¼ 20 were shifted to 2q ¼ 15 and 22.5 , respectively, which suggested the reduction of the intermolecular H-bonds among OeH and NeH groups on chitosan chain segments and also the modification of the semi-crystalline structure of the polymer, producing a new semicrystalline entity [29]. The SEM analyses revealed that the inserts based on chitosan presented a homogeneous surface without pores (Fig. 4a). The photomicrograph of the inserts composed on chitosan and brimonidine tartrate demonstrated the distribution of the drug at the surface of the system in the followed manner: a portion of the brimonidine tartrate was organized as a crystalline structure and another portion of the drug was probably dispersed as an amorphous state within the polymeric matrix (Fig. 4b). The SEM results corroborated with the crystallographic data of the brimonidine tartrate in its entrapped form. The SEM photomicrographs of the inserts composed on chitosan and drug after 15 and 30 days of incubation in PBS (pH 7.4) demonstrated the erosion of the polymer. At 15-day period, the surface of the insert presented delimited degraded regions (Fig. 4c). After 30 days in direct contact with the medium, the polymer

Fig. 3. WAXS curves of pure brimonidine tartrate (a), inserts containing chitosan and brimonidine tartrate (b) and inserts based on chitosan without drug (c).

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Fig. 4. SEM photomicrographs of inserts containing chitosan without drug (a) and inserts based on chitosan and brimonidine tartrate (b) recently prepared. SEM photomicrographs of brimonidine tartrate loaded chitosan inserts after 15 days (c) and 30 days (d) of incubation in PBS pH 7.4.

showed more extended areas of degradation (Fig. 4d). The water uptake induces the swelling of the polymer, which is essential to initiate the adhesion. Following that, as the hydration becomes excessive, it leads to a sudden drop in adhesive strength as a result of disentanglement of the polymeric chains [6]. Finally, the erosion at the polymer interface occurs. However, the erosion of the chitosan depends on the solvent used, and it increases with decreasing pH due to protonation of the primary amino group [32,33]. As the PBS pH 7.4 was used in this study, which is a neutral medium, the chitosan inserts did not protonate, and consequently, eroded slower compared to the same polymer with the primary amino group protonated as a result of an acidic medium. The Fig. 5 demonstrated the swelling study of the chitosan inserts without drug and the brimonidine tartrate-loaded chitosan inserts. Chitosan inserts reached 70% of total hydration in 30 min. After that, only a slight increase in the swelling index occurred, and at 6 days, the swelling index reduced. Chitosan inserts containing brimonidine tartrate hydrated quicker than blank chitosan inserts, since more than 75% of total hydration was reached in 30 min. Then, during 2 days the swelling index increased slightly, and at 6 days, it reduced. The Fig. 6 showed the in vitro release profile of the brimonidine tartrate incorporated into chitosan inserts. The systems demonstrated a controlled release of the drug for a prolonged period (30 consecutive days) without inducing an initial burst effect. The brimonidine tartrate was released from the chitosan inserts in a constant rate at approximately 34 mg per day.

3.3. In vitro biocompatibility study The viability of the ARPE-19 and MIO-M1 cells in direct contact with the inserts containing chitosan and brimonidine tartrate, chitosan inserts, pure brimonidine and control medium was evaluated at 1, 2, 5 and 10 days in culture. The viability of the ARPE19 cells in contact with all samples and control medium was not statistically different (one-way ANOVA, p < 0.05). Similar result was obtained to the MIO-M1 cells, suggesting the absence of toxicity of the brimonidine-loaded chitosan inserts against these ocular cells (Fig. 7). The morphology of the ARPE-19 and MIO-M1 cells in direct contact with the inserts containing chitosan and brimonidine tartrate was obtained and compared to the morphology of these cells in the control group at 10 days of incubation (Fig. 8). At this period, the RPE cells reached confluence and showed high density on the surface. The actin fibers, stained with phalloidin, run in the upper part of the cytoplasm and surrounded the entire cell. Additionally, the actin fibers of adjacent cells demonstrated to be interconnected, creating a dense fiber network. The nuclei, stained with DAPI, were centrally located (Fig. 8a). The MIO-M1 cells also reached confluence during the period of experimentation. These cells presented a large number of actin filaments which delimitated each cell. The ellipsoid nuclei were centrally positioned (Fig. 8b). Additionally, the MIO-M1 cells were capable of producing glial fibrillary acidic protein (GFAP), which represents a marker of glial cytoskeletal (Fig. 8c) [34].

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6

Chitosan + Brimonidine Chitosan

5 Swelling index

27

4 3 2 1 0

0.5

1.0

48.0 144.0 Time (hours)

240.0

Fig. 5. Swelling index of inserts based on chitosan and brimonidine tartrate and inserts composed on chitosan without drug in PBS pH 7.4. Results represent mean ± standard deviation (n ¼ 3 for each sample).

based on chitosan and brimonidine tartrate and inserts based on chitosan without drug were also applied in the CAM surface; and they did not provoke any sign of hyperemia, hemorrhage and intravasal coagulation.

Cumulative brimonidine release (%)

100 80 60

4. Discussion

40 20 0

0

5

10

15

20 25 Days

30

35

40

105

105

MIO-M1 cell viability (%)

ARPE-19 cell vi abi li ty ( %)

Fig. 6. In vitro brimonidine tartrate release from chitosan inserts. Controlled and sustained release of the brimonidine tartrate was visualized during 30 days. Results represent mean ± standard deviation (n ¼ 5).

Inserts based on chitosan and brimonidine tartrate were designed as ocular drug delivery systems for the treatment of the glaucoma. The inserts were produced as solid devices; however their exposition to the ocular pH (pH 7.0) and the lacrimal fluid induces the conversion of the chitosan into a gel phase. Accordingly, the chitosan remains in the liquid state at pH 5e6 and becomes a gel at physiological pH (pH 7.4) [35]. Additionally, the chitosan interact with mucus, leading to the mucoadhesion process [36]. Therefore, these mucoadhesive inserts could present a longer residence time in the anterior segment of the eye, inducing increased ocular bioavailability of the brimonidine tartrate previ-

100

95

90

1

2

3

4

(a)

100

95

90

1

2

3

4

(b)

Fig. 7. Viability of ARPE-19 (a) and MIO-M1 cells (b) in the control medium (1) and incubated directly with inserts containing chitosan and brimonidine tartrate (2), chitosan inserts (3) and pure brimonidine (4) for 10 days (n ¼ 10 for each sample per day) (p < 0.05). The viability of the ocular cells was calculated using the control fixed at 100%.

3.4. In vivo biocompatibility study The solutions of PBS pH 7.4 (negative control) and brimonidine tartrate leached from the chitosan inserts in PBS pH 7.4 for 7 days were put in direct contact with the CAM surface. These solutions did not induce irritation to the membrane. Furthermore, inserts

ously incorporated. Nowadays, the brimonidine tartrate is administrated by glaucomatous patients using conventional eye drops. Besides the brimonidine tartrate is a potent hypotensive agent, through increasing the uveoscleral outflow and decreasing aqueous humor production [6], it needs to be instilled in the affected eye three times daily,

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Fig. 8. Photomicrographs of ARPE-19 (a) (40) and MIO-M1 cells (b) (80) incubated with inserts containing chitosan and brimonidine tartrate for 10 days. The actin filaments were stained with FITC phalloidin (green) and the nuclei were stained with DAPI (blue). GFAP (red) expressed by Müller glial cells (c) (40). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

approximately 8 h apart [37]. The lower bioavailability and the frequency of administration of this drug lead to the lower patient compliance. The devices containing chitosan and brimonidine tartrate show advantages to the conventional eye drops, since these drug delivery systems could release the brimonidine for a prolonged period (as demonstrated in the in vitro release study), as a single dose. Furthermore, the conventional ophthalmic drops could be a multiple-dose formulation, which contain necessarily preservatives to protect this liquid formulation against the contamination by microorganisms. However, these preservatives, such as the benzalkonium chloride, chlorobutanol and chlorhexidine acetate, could induce corneal and conjunctival cell death, loss of goblet cells and local inflammation [38]. By contrast, the brimonidine tartrate loaded-chitosan inserts were designed as solid devices, which do not require the incorporation of preservatives. The absence of these agents represents an advantage of the systems, whereas the toxic effects to the anterior tissues of the eye could be completely eliminated. The crystallographic and thermal analyses indicated that the brimonidine tartrate was dispersed within the chitosan network, and it partially changed its crystalline nature into an amorphous structure due to the manufacturing process of the ocular inserts. The irregular molecular structure of the inserts that emerged from the random entanglement of polymeric chains may reduce the drug crystalline character as well as prevented its re-crystallization

during the evaporation of the organic solvent used in the design of the systems [6,31]. SEM photomicrographs corroborated with WAXS and DSC results, since it provided visualization of some crystals of the brimonidine tartrate on the surface of the inserts; however, the major proportion of the drug was entrapped in the polymer network. Moreover, the FTIR results also demonstrated the dispersion of the drug into the polymeric chains of the chitosan, without presenting detectable chemical interactions. In fact, the inexistence of drugepolymer interactions could be confirmed by the swelling test: as the presence of the brimonidine tartrate induced an increase in the water uptake into the polymer when compared to the free chitosan, it was suggested that there were not intermolecular bonds between them. Therefore, the lower the number and strength of hydrogen bonding sites, the greater the diffusion of the water molecules in the hydrated matrix [39]. Contrary to this information, Franca and co-workers [8] reported that inserts containing chitosan and bimatoprost presented lower water uptake than inserts based on chitosan without drug due to the existence of H-bonds between drug and polymer. The in vitro release of brimonidine tartrate demonstrated that the chitosan inserts modulated the lixiviation of the drug for 30 days without showing an initial burst. The absence of the initial burst of the drug represents an important advantage of these systems, since the rapid release of the brimonidine tartrate could induce an extreme ocular hypotensive effect leading to toxicity.

J.F. De Souza et al. / Journal of Drug Delivery Science and Technology 32 (2016) 21e30

Additionally, the inexistence of the initial burst of this hydrophilic salt and its constant and controlled release for a prolonged period represented also an important advantage of these systems, since controlling the delivery of hydrophilic drugs by matricial inserts is extremely difficult due to the preferential partition of the drug in the aqueous physiological medium. By contrast, in some researches, which report the development of delivery systems containing brimonidine [40], the hydrophobic brimonidine free-base was selected to compose these systems rather than the hydrophilic brimonidine tartrate salt, once the drug free-base presents a lower partition in the aqueous physiological medium, becoming easier to control the delivery of these drug. The hydration of the polymer due to the penetration of the medium was related to the formation of H-bonds between water and OeH and NeH groups of the chitosan. These groups were available to form waterepolymer interactions, since it was supposed that the intermolecular H-bonds among them were reduced due to the disorganization of the polymeric structure of the chitosan, produced by the casting/solvent evaporation technique (as indicated in the crystallographic results). As a consequence of the penetration of the PBS in the inserts, the polymeric chains separated and swelled to form a gel [6]. Simultaneously to the uptake of the medium into the polymeric chains, the displacement of the entrapped drug occurred. The medium induced the dissolution of the brimonidine tartrate; however the chitosan in gel state was capable of controlling its diffusion through a long period. Concurrently, the slow erosion of the gel in the experimental period also contributed to the continuous lixiviation of the drug as a controlled manner. The slow rate of biodegradation of the polymer was associated to the neutrality of the medium pH, which did not induce the protonation of glucosamine amino groups of the chitosan [41]. The inexistence of ionization led to lower hydration rate, and consequently, decreased erosion. The eye presents the blood aqueous barrier, which is composed by tight junctions in the ciliary process non-pigmented epithelium, the endothelial cells in the iris vasculature and the inner wall endothelium of Schlemm's canal. These junctional complexes limit the diffusion of the substances to the anatomical structures of the anterior segment of the eye [42]. Moreover, the eye presents also the blood retinal barrier (BRB), which is composed by the endothelium of retinal blood vessels (inner BRB) and retinal pigment epithelium cells (RPE cells) (outer BRB). The RPE cells regulate the transport of endogenous and exogenous compounds from the choroid blood supply to the photoreceptor layer of the retina, which is avascular [14]. Therefore, the administration of drugs topically or systemically should not reach the ocular tissues due to the existence of these important barriers. However, it was previously evidenced that the brimonidine instilled in the anterior segment of the eye, using eye drops, to treat the glaucoma, was capable of penetrating the posterior segment of the eye leading to a neuroprotective function by promoting retinal ganglion cell survival [14,15]. This absorption process of the brimonidine was attributed to the existence of a carrier-mediated system facilitating the inward transport across the outer BRB represented by the RPE cells. Considering the possibility of the absorption of the brimonidine by these ocular cells after its administration in the anterior segment of the eye, in this study, the viability of the ARPE-19 cells in direct contact with the brimonidine tartrate leached from the chitosan inserts was investigated. It was observed a high survival rate of the ARPE-19 cells over a period of 10 days in in vitro conditions. It confirmed the biocompatibility of these systems and the absence of toxicity of the drug controlled released to these ocular cells of the back of the eye. Furthermore, the brimonidine tartrate leached from the polymeric implants in direct contact with the ARPE19 cells did not induce any modification in the cellular morphology.

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These RPE cells proliferated and produced a compact layer with cellecell interactions, as demonstrated by the intense network of the actin fibers from adjacent cells. Therefore, the in vitro biocompatibility of the brimonidine tartrate loaded-chitosan inserts was over again demonstrated. The MIO-M1 cells also tolerated the ocursets, since their endochylema contained a large number of actin filaments and they demonstrated functionality due to the expression of GFAP. The production of GFAP indicated that cells were not under stress, once the up-regulation of GFAP is an early event under retinal stress conditions [43]. The HET-CAM assay is based on the direct application of the samples onto the CAM and the observation of reactions such as hyperemia, hemorrhage and intravasal coagulation [44]. These signs could be clearly observed, since the CAM is highly vascularized, as the conjunctiva. However, the CAM has no sensory innervation, which implies in the absence of pain in the embryo when irritant substances are evaluated [45]. The inserts containing chitosan and brimonidine tartrate and the solution of the brimonidine tartrate leached from the chitosan inserts for 7 days demonstrated non-irritant to the CAM, since any of the signs in the blood vessels were visualized within 5 min of the test, suggesting the in vivo biocompatibility of the mucoadhesive systems and the released drug. The obtained results corroborated with those previously described by Ravindran and co-workers [46], who mentioned that ocular inserts containing timolol maleate-brimonidine tartrate presented biocompatibility in in ovo studies. Finally, our in vivo study of toxicity corroborated with the in vitro obtained results using the ocular cells, indicating the safety of the brimonidine tartrate loaded-chitosan inserts. 5. Conclusion Mucoadhesive inserts containing chitosan and brimonidine tartrate were designed to treat the glaucoma by reducing the IOP levels. The inserts showed that a portion of brimonidine tartrate was physically dispersed into the polymeric chains of the chitosan as an amorphous structure; and another portion was distributed at the surface of the inserts as a crystalline nature. Drug and polymer apparently did not interact chemically, inducing the uptake of water into the polymeric chains. The hydration led to the gel formation, which controlled the drug release. Moreover, the erosion of the polymer was not favored by the neutral pH of the medium, which also provided the control of the drug lixiviation for a prolonged period. The release pattern also revealed the absence of an initial burst of this hydrophilic drug. Finally, these mucoadhesive inserts demonstrated their in vitro biocompatibility against ARPE19 and MIO-M1 cells; and in vivo biocompatibility due to the tolerance of the CAM. The promising obtained results indicated that the brimonidine tartrate loaded-chitosan inserts could be a therapeutic alternative to the treatment of the glaucoma, whereas they could provide local pharmacological therapy, improve ocular bioavailability of the drug, reduce the fluctuation of the IOP and certainly suppress ocular toxicity due to the absence of preservatives. Acknowledgments The authors would like to thank the UFSJ (Brazil), CNPq/MCT (Brazil), Fapemig (Brazil) and CAPES (Bolsistas da CAPES-Brasília/ Brazil) for the financial support. References [1] S.J. McKinnon, L.D. Goldberg, P. Peeples, J.G. Walt, T.J. Bramley, Current management of glaucoma and the need for complete therapy, Am. J. Manag.

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Care 1 (Suppl. l) (2008) S20eS27, 14. [2] A.V. Mantravadi, N. Vadhar, Glaucoma, Prim. Care 42 (3) (2015) 437e449. [3] V.P. Costa, A. Harris, E. Stef ansson, J. Flammer, G.K. Krieglstein, N. Orzalesi, A. Heijl, J.P. Renard, L.M. Serra, The effects of antiglaucoma and systemic medications on ocular blood flow, Prog. Retin. Eye Res. 22 (6) (2003) 769e805. [4] H.K.V.R. Ananthula, M. Barot, A.K. Mitra, Bioavailability, Lippincott Williams & Wilkins, 2009. [5] R.D. Vaishya, V. Khurana, S. Patel, A.K. Mitra, Controlled ocular drug delivery with nanomicelles, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 6 (5) (2014) 422e437. [6] M.H. Aburahma, A.A. Mahmoud, Biodegradable ocular inserts for sustained delivery of brimonidine tartrate: preparation and in vitro/in vivo evaluation, AAPS PharmSciTech 12 (4) (2011), http://dx.doi.org/10.1208/s12249-0119701-3. [7] D. Karthikeyan, M. Bhowmick, V.P. Pandey, J. Nandhakumar, S. Sengottuvelu, S. Sonkar, T. Sivakumar, The concept of ocular inserts as drug delivery systems: an overview, Asian J. Pharm. 2 (4) (2008) 192e200. [8] J.R. Franca, G. Foureaux, L.L. Fuscaldi, T.G. Ribeiro, L.B. Rodrigues, R. Bravo, R.O. Castilho, M.I. Yoshida, V.N. Cardoso, S.O. Fernandes, S. Conemberger, A.J. Ferreira, A.A.G. Faraco, Bimatoprost-loaded ocular inserts as sustained release drug delivery systems for glaucoma treatment: in vitro and in vivo evaluation, Plos One 9 (4) (2014) e95461. ^ncio, F.A. Viana, R.R. Ribeiro, M.I. Yoshida, A.G. Faraco, A.S. Cunha[9] G.O. Fulge Júnior, New mucoadhesive chitosan film for ophthalmic drug delivery of timolol maleate: in vivo evaluation, J. Ocul. Pharmacol. Ther. 28 (4) (2012) 350e358. [10] H.B. Gevariya, J.K. Patel, Long acting betaxolol ocular inserts based on polymer composite, Curr. Drug Deliv. 10 (4) (2013) 384e393. [11] S.A. Agnihotri, T.M. Aminabhavi, Chitosan nanoparticles for prolonged delivery of timolol maleate, Drug Dev. Ind. Pharm. 33 (2007) 1254e1262. [12] V. Tangpasuthadol, N. Pongchaisirikul, V.P. Hoven, Surface modification of chitosan films. Effects of hydrophobicity on protein adsorption, Carbohydr. Res. 338 (9) (2003) 937e942, 22. [13] A. Ludwig, The use of mucoadhesive polymers in ocular drug delivery, Adv. Drug Deliv. Rev. 57 (2005) 1595e1639. [14] N. Zhang, R. Kannan, C.T. Okamoto, S.J. Ryan, V.H. Lee, D.R. Hinton, Characterization of brimonidine transport in retinal pigment epithelium, Investig. Ophthalmol. Vis. Sci. 47 (1) (2006) 287e294. [15] L. Wheeler, E.M. Wolde, R. Lair, Role of alpha-2 agonists in neuroprotection, Surv. Ophthalmol. 48 (Suppl. 1) (2003) S47eS51. ^ncio, T.G. Ribeiro, [16] G. Foureaux, J.R. Franca, J.C. Nogueira, G.O. Fulge R.O. Castilho, M.I. Yoshida, L.L. Fuscaldi, S.O. Fernandes, V.N. Cardoso, S. Cronemberger, A.A. Faraco, A.J. Ferreira, Ocular inserts for sustained release of the angiotensin-converting enzyme 2 activator, Diminazene Aceturate, to treat glaucoma in rats, PLoS One 10 (7) (2015) e0133149, 23. dulo, A.M. Braz, J.S. Paula, [17] L.P. Baffa, J.R. Ricardo, A.C. Dias, C.M. Mo M.L. Rodrigues, E.M. Rocha, Tear film and ocular surface alterations in chronic users of antiglaucoma medications, Arq. Bras. Oftalmol. 71 (1) (2008) 18e21. [18] United States Pharmacopeia, 32nd ed., The United States Pharmacopeial Convention, Rockville, 2009. [19] J.F. Souza, K.N. Maia, S.L. Fialho, F.P. Andrade, G.R. Silva, Development and validation of spectrophotometric method for the determination of brimonidine into ocular implants, World J. Pharm. Sci. 3 (2) (2014) 927e941. [20] A.F. Pereira, L.G. Pereira, L.A. Barbosa, S.L. Fialho, B.G. Pereira, P.S. Patricio, F.C. Pinto, G.R. Silva, Efficacy of methotrexate-loaded poly(ε-caprolactone) implants in Ehrlich solid tumor-bearing mice, Drug Deliv. 20 (3e4) (2013) 168e179. fice, G.M. Fernandes-Cunha, A. Silva-Cunha, [21] G.R. Silva, T.H. Lima, R.L. Ore M. Zhao, F. Behar-Cohen, In vitro and in vivo ocular biocompatibility of electrospun poly(Ɛ-caprolactone) nanofibers, Eur. J. Pharm. Sci. 20 (73) (2015) 9e19. [22] R.G. Alany, T. Rades, J. Nicoll, I.G. Tucker, N.M. Davies, W/O microemulsions for ocular delivery:evaluation of ocular irritation and precorneal retention, J. Control Release 111 (1e2) (2006) 145e152, 10. [23] F.A. S a, S.F. Taveira, G.M. Gelfuso, E.M. Lima, T. Gratieri, Liposomal voriconazole (VOR) formulation for improved ocular delivery, Colloids Surf. B Biointerfaces 133 (2015) 331e338, 1. [24] L.B. Rodrigues, H.F. Leite, M.I. Yoshida, J.B. Saliba, A.S. Cunha, A.A. Faraco,

[25]

[26]

[27]

[28]

[29]

[30]

[31] [32]

[33]

[34]

[35]

[36]

[37] [38]

[39]

[40]

[41] [42] [43]

[44]

[45]

[46]

In vitro release and characterization of chitosan films as dexamethasone carrier, Int. J. Pharm. 368 (1e2) (2009) 1e6, 23. S. Maiti, S. Paul, R. Mondol, S. Ray, B. Sa, Nanovesicular formulation of brimonidine tartrate for the management of glaucoma: in vitro and in vivo evaluation, AAPS PharmSciTech 12 (2) (2011) 755e763. A. Esmaeili, A. Asgari, In vitro release and biological activities of Carumcopticum essential oil (CEO) loaded chitosan nanoparticles, Int. J. Biol. Macromol. 81 (2015) 283e290, 7. T. Phromsopha, Y. Baimark, Methoxypoly(ethylene glycol)-b poly(D, L-lactide) films for controlled release of ibuprofen, Trends Appl. Sci. Res. 4 (2009) 107e115. S. Saravanan, D.K. Sameera, A. Moorthi, N. Selvamurugan, Chitosan scaffolds containing chicken feather keratin nanoparticles for bone tissue engineering, Int. J. Biol. Macromol. 62 (2013) 481e486. P.V. Bueno, P.R. Souza, H.D. Follmann, A.G. Pereira, A.F. Martins, A.F. Rubira, E.C. Muniz, N,N-Dimethyl chitosan/heparin polyelectrolyte complex vehicle for efficient heparin delivery, Int. J. Biol. Macromol. 75 (2015) 186e191. A.R. Chandak, P.R.P. Verma, Development and evaluation of HPMC based matrices for transdermal patches of Tramadol, Clin. Res. Regul. Aff. 6 (2010) 1370e1379. H. Elmotasem, Chitosan-alginate blend films for the transdermal delivery of meloxicam, Asian J. Pharm. Sci. 3 (1) (2008) 12e29. R.M. Farid, M.A. Etman, A.H. Nada, Ael A. Ebian, Formulation and in vitro evaluation of salbutamol sulphate in situ gelling nasal inserts, AAPS PharmSciTech 14 (2) (2013) 712e718. P. Aksungur, A. Sungur, S. Unal, A.B. Iskit, C.A. Squier, S. Senel, Chitosan delivery systems for the treatment of oral mucositis: in vitro and in vivo studies, J. Control Release 98 (2004) 269e279. L. Shao-Fen, M. Yu-Xiang, L. Bin, S. Wei, T. Shi-Bo, Morphological and immunocytochemical analysis of human retinal glia subtypes in vitro, Int. J. Ophthalmol. 6 (5) (2013) 559e563. H. Gupta, M. Aqil, R.K. Khar, A. Ali, A. Bhatnagar, G. Mittal, An alternative in situ gel formulation of levofloxacin eye drops for prolong ocular retention, J. Pharm. Bioallied Sci. 7 (1) (2015) 9e14. C.M. Lehr, J.A. Bouwstra, E.H. Schacht, W. Kok, A.G. De Boer, J.J. Tukker, J.C. Verhoef, D.D. Breimer, H.E. Junginger, In vitro evaluation of mucoadhesive properties of chitosan and some other natural polymers, Int. J. Pharm. 78 (1992) 43e48. http://www.allergan.com/assets/pdf/alphaganp_pi.pdf. (accessed 09.01.15). D.A. Ammar, M.Y. Kahook, Effects of glaucoma medications and preservatives on cultured human trabecular meshwork and non-pigmented ciliary epithelial cell lines, Br. J. Ophthalmol. 95 (10) (2011) 1466e1469. S.P. Panomsuk, T. Hatanaka, T. Aiba, K. Katayama, T. Koizumi, A study of the hydrophilic cellulose matrix: effect of drugs on swelling properties, Chem. Pharm. Bull. 44 (1996) 1039e1042. M.M. Ibrahim, A.H. Abd-Elgawad, M.M. Jablonski, Natural bioadhesive biodegradable nanoparticle-based topical ophthalmic formulations for management of glaucoma, 30, Transl. Vis. Sci. Technol. 4 (3) (2015) 12. eCollection. L. Noble, A.I. Gray, L. Sadiq, I.F. Uchegbu, A non-covalently cross-linked chitosan based hydrogel, Int. J. Pharm. 192 (2) (1999) 173e182, 10. M. Coca-Prados, The blood-aqueous barrier in health and disease, J. Glaucoma 8 (Suppl. 1) (2014) S36eS38, 23. A. Bringmann, I. Iandiev, T. Pannicke, A. Wurm, M. Hollborn, P. Wiedemann, N.N. Osborne, A. Reichenbach, Cellular signaling and factors involved in Muller cell gliosis: neuroprotective and detrimental effects, Prog. Retin. Eye Res. 28 (2009) 423e451. G. Abrego, H.L. Alvarado, M.A. Egea, E. Gonzalez-Mira, A.C. Calpena, M.L. Garcia, Design of nanosuspensions and freeze-dried PLGA nanoparticles as a novel approach for ophthalmic delivery of pranoprofen, J. Pharm. Sci. 103 (10) (2014) 3153e3164. A.L. Savian, D. Rodrigues, J. Weber, R.F. Ribeiro, M.H. Motta, S.R. Schaffazick, A.I. Adams, D.F. de Andrade, R.C. Beck, C.B. da Silva, Dithranol-loaded lipidcore nanocapsules improve the photostability and reduce the in vitro irritation potential of this drug, Mater. Sci. Eng. C Mater. Biol. Appl. 46 (2015) 69e76. V.K. Ravindran, S. Repala, S. Subadhra, A.K. Appapurapu, Chick chorioallantoic membrane model for in ovo evaluation of timolol maleate-brimonidine tartrate ocular inserts, Drug Deliv. 21 (4) (2014) 307e314.