Multifunctional Nanocomposites Based on Liposomes and Layered


Jun 3, 2019 - ... nasolacrimal drainage), inherent anatomical barriers of anterior eye tissues,(6) ... a classical glucocorticoid, is widely used in t...
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Article Cite This: Mol. Pharmaceutics 2019, 16, 2845−2857

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Multifunctional Nanocomposites Based on Liposomes and Layered Double Hydroxides Conjugated with Glycylsarcosine for Efficient Topical Drug Delivery to the Posterior Segment of the Eye Yan Gu,†,‡,∥ Chen Xu,†,∥ Yanyan Wang,† Xiangying Zhou,† Lei Fang,*,§ and Feng Cao*,† †

Department of Pharmaceutical, School of Pharmacy, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, China Parexel China Co., Ltd., No.488, Middle Yincheng Road, Pudong, Shanghai 200120, China § Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China

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ABSTRACT: To achieve efficient drug delivery to the posterior segment of the eye via topical instillation, novel multifunctional nanocomposites were prepared by hybridizing dexamethasone disodium phosphate (DEXP)-loaded liposome (LP) glycylsarcosine (GS)-anchored layered double hydroxides (named [email protected]) and then fully characterized. The nanocomposites exhibited sustainedrelease performance as well as prolonged precorneal retention ability. MTT assays showed that the nanocomposites were not cytotoxic to both human corneal epithelial cells (HCEpiC) and human conjunctival epithelial cells (HConEpiC) at an LDH concentration of 100 μg/mL. The [email protected] nanocomposites showed superior in vitro permeability on the HConEpiC-cell-based model. In the case of HConEpiC cells, both clathrin-mediated endocytosis and active transport by the peptide transporter-1 (PepT-1) were involved in the internalization of the nanocomposites. Fluorescent images of frozen sections of ocular tissues suggested that the possible route for the delivery of doxorubicin hydrochloride (DOX)-labeled nanocomposites from the ocular surface to the back of the eye was a non-corneal pathway. Furthermore, in rabbit eyes, the hybrid nanocomposites displayed markedly higher drug concentration in choroid-retina tissue than other single nanocarriers, such as LPs and LDH. Besides, the results of the eye irritancy test showed that nanocomposite eye drops can be classified as nonirritant, which are suitable to be used as eye drops. In a word, multifunctional nanocomposites based on LPs and LDH could be used as promising vehicles for efficient noninvasive drug delivery to the posterior segment of the eye. KEYWORDS: topical instillation, posterior segment of the eye, layered double hydroxides (LDH), liposomes, efficient ocular drug delivery

1. INTRODUCTION It is estimated that 216.6 million people may suffer from severe visual impairment globally.1 Diseases associated with the posterior segment of the eye such as age-related macular degeneration, cytomegalovirus retinitis, diabetic retinopathy (DR), and proliferative vitreoretinopathy are the leading causes of blindness.2 Despite several adverse effects including increased intraocular pressure, retinal detachment, and cataract formation,3 intravitreous injection is a clinically preferred route to treat these diseases.4 In contrast, topical administration with improved patient compliance could avoid these unwanted adverse effects. It is generally considered that there are mainly three routes for drug delivery to the back of the eye after topical instillation: (i) corneal pathway (drug penetrates cornea, vitreous, and subsequently arrives at retina), (ii) non-corneal pathway (drug penetrates conjunctiva and further diffuses into sclera, choroid, and retina), and (iii) lateral © 2019 American Chemical Society

diffusion pathway (drug penetrates cornea, anterior chamber, and lateral diffuses into uvea-sclera tissues).5 However, efficient drug diffusion into posterior eye tissues via topical administration is restricted by multiple obstacles, which involve dynamic barriers (tear turnover, nasolacrimal drainage), inherent anatomical barriers of anterior eye tissues,6 and various efflux transporters expressed in the ocular surface.7 To overcome these limitations, drug delivery strategies require prolonged residence time on the ocular surface as well as improved permeability of anterior eye tissues (cornea, conjunctiva). Received: Revised: Accepted: Published: 2845

November 4, 2018 May 22, 2019 June 3, 2019 June 3, 2019 DOI: 10.1021/acs.molpharmaceut.8b01136 Mol. Pharmaceutics 2019, 16, 2845−2857

Article

Molecular Pharmaceutics

elemental analysis. [email protected] nanocomposites were then characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), in vitro drug release, and in vivo precorneal retention. The cytotoxicity and in vitro permeability of nanocomposites were evaluated on both human corneal epithelial cells (HCEpiC) and human conjunctival epithelial cells (HConEpiC). The cellular uptake mechanism of [email protected] nanocomposites was studied on HConEpiC cells. Fluorescent frozen sections of ocular tissues were made to investigate the transport behavior of nanocomposites labeled with doxorubicin hydrochloride (DOX). Furthermore, an in vivo study including tissue distribution and eye irritancy in rabbit eyes was performed after topical administration of [email protected] nanocomposite eye drops.

Recently, several drug carriers have been designed in an attempt to achieve drug delivery to the posterior segment of the eye via topical instillation. For instance, Lajunen et al. have developed transferrin-modified small liposomes produced by a microfluidizer. The results of confocal microscopy demonstrated that liposomes could be detected in retinal pigment epithelium after topical administration.8 Mandal and his colleagues have shown the potential of B-C12-cCDF-loaded polymeric nanomicelles in delivering drug to the back of the eye in a permeability study of the cornea/retina cell model.9 In the study of Araújo et al., nanostructured lipid carriers were successfully employed to deliver the fluorescent tracker (Nile red) to the retina via eye drops.10 Nonetheless, these single nanocarriers have drawbacks such as early leakage and quick elimination in physiological environments,11 which may make it difficult for them to maintain an effective therapeutic concentration at desired sites. Considering drawbacks of those single nanocarriers, multicarriers have attracted much attention. Examples include multifunctional nanocomposites based on a cyclodextrin-inclusion complex and liposome that were investigated for improving loading,12 better targeting,13 and overcoming the drug resistance.14 Furthermore, nanobiohybrids based on micelles and layered double hydroxides (LDH) can effectively change the release of a drug.15 In order to achieve more efficient drug delivery to the back of the eye, novel nanocomposites composed of glycylsarcosine (GS)-anchored LDH and liposomes were first proposed for topical administration in this report. As a member of clay materials, LDH are promising nanomaterials with high chemical stability, advanced biocompatibility, anion exchange, and sustained release behavior.16 Recently, hybrid LDH have attracted more and more attention.17 A new application field in ocular drug delivery systems is being explored in our group.18 In our previous studies, pirenoxine sodium intercalated LDH nanoparticles hybridized with chitosan derivatives (chitosanglutathione-valine-valine (CG-VV), chitosan-glutathione-glycosyl-sarcosine (CG-GS)) showed active targeting to peptide transporter 1 (PepT-1) of the ocular surface and enhanced ocular bioavailability.19,20 Moreover, owing to abundant hydroxyl groups,16 modification on the surface of LDH could further accelerate their application, such as in fields of dye absorption21 and catalyst.22 To the best of our knowledge, the application of surface-modified LDH in the drug delivery system is still scarce. Dexamethasone disodium phosphate (DEXP), a classical glucocorticoid, is widely used in the treatment of ocular inflammation.23 However, less than 5% of DEXP eye drops could penetrate into intraocular tissues,24 which limit its application for the treatment of diseases associated with the posterior segment of the eye. DEXP was chosen as a model drug. Herein, novel nanocomposites ([email protected]) have been constructed by hybridizing DEXP-loaded liposomes with GS-anchored LDH. GS, a classical substrate for PepT-1, was modified on LDH, which is used for targeting to PepT-1 of the ocular surface. LDH, a kind of positive carrier, can promote precorneal retention via electrostatic adsorption. Liposome, with excellent ocular biocompatibility,25 may achieve relay drug delivery to the posterior segment of the eye. Combining all these advantages above, [email protected] LDH-GS nanocomposite eye drops could serve as a choice for enhanced drug bioavailability of the ocular posterior segment via topical instillation. The modification of LDH was further confirmed by the ninhydrin test, fluorescamine analysis, and

2. MATERIALS AND METHODS 2.1. Materials. Dexamethasone disodium phosphate (DEXP) was obtained from Baomanbio. Co., Ltd. (Shanghai, China). DEXP eye drops (specification: 5 mL, 1.25 mg; batch number: H42021093) were purchased from Hubei Wuhan Wujing Medicine Co., Ltd. (Hubei, China). Hydrogenated soybean phospholipids (HSPC) were purchased from Lipoid (Germany). Cholesterol, fluorenylmethyloxycarbonyl N-hydroxysuccinimide ester (Fmoc-OSu), and 3-(aminopropyl)triethoxysilane (APTES) were purchased from Aladdin (Shanghai, China). Doxorubicin hydrochloride (DOX) was purchased from Synbias Pharma Ltd. (Germany). Glycylsarcosine (GS) was obtained from Tixi Industrial Development Co., Ltd. (Shanghai, China). 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was purchased from Servicebio (China). Other reagents were of HPLC or analytical reagent grades. 2.2. Cell Culture and Animals. Both human corneal epithelial cells (HCEpiC, Cat. #6510) and human conjunctival epithelial cells (HConEpiC, Cat. #6630) were purchased from Sciencell Research Laboratories (San Diego, CA, USA). Both HCEpiC and HConEpiC were grown in a corneal epithelial cell medium (CEpiCM) supplemented with 1% penicillin/ streptomycin solution and 1% corneal epithelial cell growth supplement in a humidified atmosphere containing 5% CO2 at 37 °C. New Zealand albino rabbits (male, 2.0−3.0 kg) were purchased from Nanjing Qinglongshan farms. The animals were maintained in laboratory conditions and fed with a standard pellet diet. All animal study protocols complied with the guidelines set by the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethical Experimentation Committee of China Pharmaceutical University. 2.3. Synthesis of APTES-GS. The synthetic scheme is summarized in Figure S1. First of all, the amino group of GS was protected with Fmoc-OSu. Next, Fmoc-GS was acylated with APTES to obtain the intermediate Fmoc-GS-APTES, which was deprotected to get APTES-GS. 2.3.1. Synthesis of Fmoc-GS. NaHCO3 (4.1 g, 49.2 mmol) was dissolved in a mixture of H2O (20 mL) and dioxane (15 mL). GS (3.0 g, 20.5 mmol) was added partially into the reaction mixture and stirred for 5 min at room temperature. Fmoc-Osu (8.3 g, 24.6 mmol) in dioxane (20 mL) was then added slowly into the solution at 0 °C and stirred for 30 min at the same temperature. After that, the reaction mixture was stirred overnight at room temperature. Progress of the reaction 2846

DOI: 10.1021/acs.molpharmaceut.8b01136 Mol. Pharmaceutics 2019, 16, 2845−2857

Article

Molecular Pharmaceutics was monitored with thin layer chromatography (TLC). The solvent was removed after the completion of the reaction under reduced pressure. The pH of residue was adjusted to 3 with hydrochloric (HCL, 1 mol/L). The residue was extracted three times with ethyl acetate (50 mL), and then the organic layer was dried with anhydrous Na2SO4. The solvent of the reaction was removed under reduced pressure to get a crude product. The crude product was further purified by a silica gel column chromatograph with the eluent of dichloromethane (DCM) and methanol (MeOH) (100:1−40:1, v/v) to obtain the targeted product (Fmoc-GS). The product was characterized by ESI-MS and 1H NMR. 2.3.1.1. Fmoc-GS. Yield: 90%. ESI-MS: m/z [M−H] = 367.15. 1H NMR (300 MHz, DMSO): δ 12.72 (s, 1H), 7.89 (d, J = 7.4 Hz, 2H), 7.69 (m, 2H), 7.37 (m, 5H), 4.33−4.18 (m, 3H), 4.06 (m, 2H), 3.84 (m, 2H), 3.06−2.79 (m, 3H). 2.3.2. Synthesis of Fmoc-GS-APTES. Fmoc-GS (3 g, 8.1 mmol) was dissolved in anhydrous DCM (40 mL). Dicyclohexylcarbodiimide (DCC, 2.2 g, 10.6 mmol), Nhydroxysuccinimide (NHS, 1.1 g, 9.8 mmol), and 4dimethylaminopyridine (DMAP, Cat.) were added into the reaction mixture and then stirred for 20 min at room temperature. After that, APTES (2.7 g, 12.2 mmol) in anhydrous DCM (10 mL) was added into the reaction mixture and stirred at 35 °C overnight. Progress of the reaction was monitored with TLC. The reaction mixture was filtered to remove the insoluble solid and obtain a crude product. The crude product was further purified by a silica gel column chromatograph with the eluent of DCM and MeOH (100:0− 50:1, v/v) to obtain the targeted product (Fmoc-GS-APTES). The product was characterized by 1H NMR. 2.3.2.1. Fmoc-GS-APTES. Yield: 75%. 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 7.4 Hz, 2H), 7.60 (d, J = 7.4 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.30 (t, J = 7.4 Hz, 2H), 4.38 (d, J = 7.2 Hz, 2H), 4.36 (d, J = 11.4 Hz, 1H), 4.22 (t, J = 7.2 Hz, 1H), 4.08 (d, J = 4.5 Hz, 2H), 4.03−3.95 (m, 2H), 3.93−3.73 (m, 7H), 3.30−3.22 (m, 2H), 3.08−2.96 (m, 3H), 1.68−1.57 (m, 2H), 1.20 (t, J = 7.0 Hz, 9H), 0.65−0.57 (m, 2H). 2.3.3. Synthesis of APTES-GS. Fmoc-GS-APTES (3.2 g, 5.6 mmol) was dissolved in anhydrous dimethylformamide (DMF, 30 mL). Piperidine (1.8 g, 14 mmol) was added into the reaction mixture and stirred at 0 °C for 20 min. Progress of the reaction was monitored with TLC. The solvent of the reaction was removed under reduced pressure to get a crude product. The crude product was further purified by a silica gel column chromatograph with the eluent of DCM and MeOH (50:0− 20:1, v/v) to obtain the final product (APTES-GS). The product was characterized by ESI-MS and 1H NMR. 2.3.3.1. APTES-GS. Yield: 61.1%. ESI-MS: m/z [M + H] = 350.21 1H NMR (400 MHz, DMSO) δ 7.80 (s, 1H), 3.94− 3.84 (m, 3H), 3.73 (q, J = 7.0 Hz, 6H), 3.43 (q, J = 7.0 Hz, 2H), 3.20 (s, 1H), 2.88 (s, 2H), 2.80−2.79 (m, 3H), 1.47− 1.41 (m, 2H), 1.13 (t, J = 7.0 Hz, 9H), 0.55−0.49 (m, 2H). 2.4. Synthesis of LDH Nanocomposites. The synthesis of [email protected] nanocomposites is summarized in Figure 1. Pristine LDH was prepared by coprecipitation as our previous report.26 Briefly, a solution of metallic salts (2 mL), a mixed aqueous solution of Mg(NO3)2·6H2O (1.03 g, 4 mmol) and Al(NO3)3·9H2O (0.75 g, 2 mmol), and an aqueous solution of NaOH (0.52 g, 3 mL) were added dropwise simultaneously to 40 mL of deionized water under a N2 atmosphere followed by vigorous stirring for 1 h. The pH of the suspension was maintained at 9.0. The resultant precipitate

Figure 1. Synthesis of [email protected] nanocomposites.

was centrifuged and washed with deionized water and ethanol several times. To prepare the surface-modified LDH with GS, first of all, 300 mg of the pristine LDH was suspended in 2 mL of ethanol. 300 or 500 mg of APTES-GS (mass ratio of APTES-GS/LDH: 1:1 or 5:3) was dissolved in 30 mL of toluene. Then the prepared LDH was added into the above solution. After stirred for 12 h or 24 h at 90 °C, the resultant was collected by centrifuging at 9240 × g for 10 min and further successively washed with dichloromethane, ethanol and water for several times. DEXP-loaded liposomes (DEXP-LPs) were prepared using a thin-film hydration method.27 In brief, HSPC (100 mg) and cholesterol (30 mg) were dissolved in a mixture of chloroform/methanol (2:1, v/v). The organic phase was evaporated under reduced pressure using a rotary evaporator to form a thin film. Hydration of the lipid film was done at 55 °C in 10 mL of DEXP solution (2 mg/mL). The liposomes were processed at a pressure of 2000 bar with a microfluidizer for 6 passage times and sequentially extruded through two stacked polycarbonate filters with pore sizes of 200 and 100 nm under nitrogen pressure (Avestin, Canada). Unencapsulated DEXP was removed by a Sephadex G-50. [email protected] nanocomposites were prepared using an ion-exchange method.28 Briefly, LDH with GS were suspended in 10 mL of deionized water. 20 mL of the DEXPLPs was added into the suspension. Then the reaction was stirred at 30 °C under a N2 atmosphere for 24 h. The resultant precipitate was centrifuged and washed with 20 mL of deionized water three times. In order to testify the intercalation process, 20 μL of reaction solution and each centrifugal supernatant were analyzed with a HPLC method. 50 mg of chitosan-glutathione (CG, synthesized previously by our group29) was dissolved in 50 mL of deionized water. [email protected] nanocomposites were suspended in CG solution. After that, the suspension was processed at a pressure of 2000 bar with a microfluidizer for 5 passage times. 300 or 500 mg of APTES-GS was used in the preparation of [email protected] (1:1) nanocomposites or [email protected] LDH-GS (5:3) nanocomposites, respectively. For comparison, [email protected] (5:3) nanocomposites were prepared in the same procedure instead of using 10 mL of deionized water without DEXP in the hydration process. [email protected] nanocomposites were synthesized using the same process except that HSPC dissolved in the organic phase was added into the LDH-GS suspension. A portion of the free liposomes, pristine LDH, LDH-GS, [email protected], [email protected] (5:3), and [email protected] sample were separately freezedried, yielding powder samples for further study. Because the water solubility of doxorubicin hydrochloride (DOX) is similar to that of DEXP, [email protected] nanocomposites or 2847

DOI: 10.1021/acs.molpharmaceut.8b01136 Mol. Pharmaceutics 2019, 16, 2845−2857

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Molecular Pharmaceutics

2.8. In Vivo Precorneal Retention Study. The animals were divided into three groups randomly, each comprising three rabbits. For each rabbit, 50 μL of eye drops (commercial eye drops, [email protected] (1:1) nanocomposite eye drops, or [email protected] (5:3) nanocomposite eye drops with 250 μg/mL of DEXP) was instilled in the lower conjunctival sac of one eye separately, while the other eye was used as control and received no treatment. At predetermined time intervals (10, 30, 60, 90, 120, 180, 240, 300, and 360 min) after administration, 10 μL of tear fluid samples was withdrawn from the conjunctival sac by using a capillary pipet. Afterward, the samples were diluted with methanol to 50 μL followed by vortexing for 2 min and then centrifuging at 9240 × g for 10 min. Finally, 20 μL of supernatants was analyzed with the HPLC method. 2.9. Cytotoxicity Assay. Cell viability was analyzed by the MTT assay. Cells were seeded in 96 well culture plates at a density of 20000 cells/well. The incubation times of HCEpiC cells and HConEpiC cells were 48 and 24 h, respectively. Then the medium was replaced with 200 μL of fresh culture medium containing various concentrations of [email protected] nanocomposites, [email protected] (5:3) nanocomposites, [email protected] nanocomposites, and [email protected] (5:3) nanocomposites (LDH concentration: 25−200 μg/mL). DEXP-free LPs and DEXP-LPs were also evaluated for in vitro cytotoxicity (DEXP concentration: 1−100 μg/mL). After the exposure period (12 h), the medium was removed, and 180 μL of fresh medium with 20 μL of MTT solution in Dulbecco’s phosphate-buffered saline (DPBS, 5 mg/mL) was added to each well and incubated for 4 h at 37 °C. Then the medium was aspirated, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was recorded at 570 nm using a microplate reader (Multiskan FC; Thermo Scientific). Cells cultured with the medium alone were used as positive control, and wells without cells were used as negative control. 2.10. In Vitro Permeability Study. 2.10.1. Cultivation of HCEpiC-Cell-Based Model and HConEpiC-Cell-Based Model. Permeability of different formulations through cornea and conjunctiva was measured using the HCEpiC-cell-based model and HConEpiC-cell-based model, respectively. For the permeability studies, HCEpiC cells or HConEpiC cells were seeded onto collagen-coated polycarbonate Transwell inserts (0.4 μm pore size, 12 mm membrane diameter) at a density of 105 cells/insert. Volumes of apical and basolateral chambers were 0.5 and 1.5 mL, respectively. Transepithelial electrical resistance (TEER) was assessed at different phases of cell growth with Evom (World Precision Instruments, Sarasota, FL) to identify the integrity of HCEpiC cells and HconEpiC cells before permeability studies. The epithelial cell layer was cultured with Hank’s Balanced Salt Solution (HBSS) at 37 °C before experiments. Transport buffer (0.5 mL of HBSS with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 25 mM glucose at pH 7.4) containing cholate or monoketocholate (MKC) (0−20 mM) was added to the apical side and the basolateral side, respectively. Before the detection, the electrodes were immersed in PBS overnight and soaked in 70% ethanol for 15 min. Cells were cultivated for 23−25 days until HCEpiC cell models exhibited TEER values of >400 Ω·cm2 and HConEpiC cell models displayed TEER values of >1000 Ω·cm2. The culture medium was changed every 2 days during the cultivation.

[email protected] nanocomposites were prepared similarly except that the final concentration of DOX was 180 μg/mL. 2.5. Preparation of LDH Nanocomposite Eye Drops. To prepare [email protected] (5:3) nanocomposite eye drops, [email protected] (1:1) nanocomposite eye drops and [email protected] nanocomposite eye drops, glucose (6% w/v) and trichlorobutanol (0.25% w/v) were added into each nanocomposite dispersion as an osmotic pressure regulator and bacteriostatic agent, respectively. The final concentration of DEXP was 250 μg/mL. HPLC analysis of DEXP was performed on a Shimadzu LC-2010CHT apparatus using a reversed-phase C18 column (Dikma, 250 × 4.6 mm, CA, USA). The mobile phase consisting of triethylamine solution−methanol−acetonitrile (55:40:5, v/v/ v) was delivered at a flow rate of 1 mL/min. The triethylamine solution was composed of triethylamine (0.75%, v/v) with an appropriate amount of phosphoric acid up to pH 3.0. The detection wavelength was 240 nm. [email protected] nanocomposite eye drops were prepared similarly. 2.6. Characterization. 2.6.1. Confirmation of Surface Modification. Modification of GS on the surface of LDH was verified using the ninhydrin test.30 5 mL of ninhydrin solution (0.2%, ethanol) was added into 50 mg of modified LDH. Two drops of acetic acid were then added into the mixture and stirred for 10 min. Finally, 1 mL of the suspension was pipetted on the filter paper and further evaporated to see the change of color. The contents of grafted surface amine groups were determined by fluorescamine analysis31 and elemental analysis.32 For fluorescamine analysis, first, 0.5 mL of phosphate-buffered saline (PBS, pH 8.0) was added into 0.5 mL of GS-functionalized LDH suspension. Then the mixture was incubated with 150 μL of fluorescamine solution (0.3 g/L, acetone) for 20 min. The fluorescence intensity was measured at EX/EM = 380/470 nm using a spectrofluorophotometer (RF-5301 PC, SHIMADZU). A standard curve was prepared using a series of GS concentrations. The content of amino groups was calculated based on the standard curve. Elemental composition of samples was determined using the PE 2400 Series II analyzer (PerkinElmer, United Kingdom). 2.6.2. Particle Size, ζ Potential, TEM, and XRD. The particle size and ζ potential of samples were determined by using a ViewSizer 3000 (MANTA, USA) and ZetaSizer 3000HS (Malvern, U.K.), respectively. Transmission electron microscope (TEM) images were recorded on a HT-7700 TEM (Hitachi, Japan). Lyophilized powders of samples were analyzed by an X-ray diffractometer (Rigaku, Japan) with Cu Kα radiation (λ = 0.154 nm, range: 0−7, 2−70°). 2.7. In Vitro Drug Release. The in vitro drug release profiles of various nanocomposites were studied using the dynamic dialysis method. Briefly, 1 mL of DEXP solution, DEXP-LPs, [email protected] nanocomposites, [email protected] (1:1) nanocomposites, and [email protected] LDH-GS (5:3) nanocomposites containing 250 μg of DEXP was poured separately in a dialysis bag (MWCO of 8−14 kDa) and immersed in 50 mL of phosphate-buffered saline (PBS, pH 7.4). The entire experiment was conducted at 35 ± 0.5 °C with a shaking speed of 148 × g. At predetermined time intervals (10, 30, 45, 60, 90, 120, 180, 240, 300, and 360 min), 0.5 mL of medium was withdrawn and replaced with an equal volume of fresh PBS. The DEXP concentrations were determined by the HPLC method, expressed as a percentage of the total drug and plotted as a function of time. 2848

DOI: 10.1021/acs.molpharmaceut.8b01136 Mol. Pharmaceutics 2019, 16, 2845−2857

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Molecular Pharmaceutics

Figure 2. Influence of various inhibitors on the cellular uptake of [email protected] nanocomposites (A) and [email protected] nanocomposites (B) by HConEpiC cells; influence of GS (1, 5, 10 mM) on the cellular uptake of [email protected] nanocomposites (C) and [email protected] nanocomposites (D) by HConEpiC cells. HConEpiC cells without treatment with specific endocytic inhibitors were taken as control. (*p < 0.05, **p < 0.01, ***p < 0.001 vs control group, n = 3).

by flow cytometry (Miltenyi Biotec, Cologne, Germany; excitation wavelength: 488 nm; emission wavelength: 590 nm). The cellular uptake efficiency of the control group (without any inhibitors) was assumed as 100%. The fluorescence intensities of other nanocomposite eye drops were calculated by dividing the fluorescence intensity of the control group. 2.12. Transport Way of Nanocomposites in Ocular Tissues. To directly visualize the nanocomposite distribution in ocular tissues, DOX was used as the fluorescent tracker to label drug. 50 μL of fluorescently labeled [email protected] nanocomposite eye drops was instilled in the lower conjunctival sac of three rabbits. After a given time (5, 15, and 30 min) of instillation, the rabbits were scarified, and their eyeballs were extracted. Then the cornea, conjunctiva, and sclera-choroid-retina were isolated and frozen in dry ice. The frozen tissues were cut into 10 μm frozen sections with a cryostat. After staining with DAPI at room temperature, sections were observed with an inverted fluorescence microscope (Nikon Eclipse Ti-SR, Japan). 2.13. In Vivo Ocular Distribution. Thirty-six rabbits were divided into twelve groups randomly, each comprising three rabbits. Briefly, 50 μL of different formulations (commercial eye drops, [email protected] (5:3) nanocomposite eye drops, [email protected] (5:3) nanocomposite eye drops, DEXP-LPs, DEXP concentration: 250 μg/mL) was instilled in the lower conjunctival sac of rabbits four times at predetermined time points (0, 5, 10, and 15 min). The rabbits were euthanized at 30, 60, and 120 min after the first instillation. Then the eyeballs were enucleated and washed with normal saline. The aqueous humor was aspirated with a syringe. Conjunctiva, cornea, sclera, and choroid-retina were carefully isolated, weighed, and cut into pieces. Afterward,

2.10.2. Permeability Studies. At the initial stage of the study, both basolateral chambers and apical chambers were washed with HBSS. 1.5 mL of HBSS was added into the basolateral chamber. After that, 0.5 mL of commercial DEXP eye drops, DEXP-LPs, [email protected] nanocomposites, [email protected] (1:1) nanocomposites, and [email protected] (5:3) nanocomposites (DEXP concentration: 60 μg/mL) was added to the apical chamber separately. At predetermined time points (15, 30, 60, 120, and 180 min), 100 μL of samples was withdrawn from the basolateral chamber and replaced with the same volumes of HBSS. Samples were analyzed with the HPLC method. Permeation profiles were determined by plotting the permeated amount of DEXP versus the time. Permeability coefficients (Papp, cm/s) were calculated based on the following equation:

Papp =

dQ /dt C0A

(1)

where dQ/dt is equal to the slope of the permeation profile, A is the surface area of the Transwell insert (1.12 cm2), and C0 is the initial DEXP concentration in the apical chamber. 2.11. Cellular Uptake Mechanism. HConEpiC cells were seeded at a density of 50000 cells/well in 12 well plates and incubated for 4 days. Then cells were washed twice with DPBS and preincubated with 0.5 mL of inhibitors (sodium azide, chlorpromazine, hypertonic sucrose, genistein, nystatin, amiloride, and GS (1, 5, and 10 mM)) for 30 min. Next, 0.5 mL of DOX-labeled nanocomposites ([email protected] nanocomposites or [email protected] nanocomposites) was added, and cells were incubated for an additional 4 h. Then cells were washed twice with cold DPBS, typsinized, harvested, and resuspended in DPBS. Cells without pretreatment were used as control. The cellular uptake efficiency was determined 2849

DOI: 10.1021/acs.molpharmaceut.8b01136 Mol. Pharmaceutics 2019, 16, 2845−2857

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nanoparticles measured by the fluorescamine assay were nearly consistent with those measured by the elemental assay. It can be seen that the amount of grafted amino groups was slightly increased with the prolonged reaction time, so 24 h was selected as the reaction time in subsequent experiments. In the elemental assay, the concentration of N atoms on the LDH surface reached nearly 4% when the amount of APTES-GS was increased to 500 mg. Both fluorescamine and elemental analyses manifested that GS was successfully modified on the surface of LDH. 3.3. Particle Size, ζ Potential, TEM, and XRD. The intercalation of anionic drug into LDH usually induces aggregation, which limits the application of nanocomposites.35 In this study, [email protected] nanocomposites also revealed severe aggregation in water. To avoid this drawback, a strategy dispersing [email protected] nanocomposites in CG solution was used. The results of the stability study at room temperature for 10 days demonstrated that there was no significant change of the nanocomposites in terms of particle size and ζ potential with the help of CG (data not shown). As shown in Table 1, the ζ potential of LDH nanocomposites

these solid samples were mixed with 500 μL of normal saline and homogenized in ice bath for 3−5 min. 50 μL of aqueous humor samples or ocular tissues homogenates was directly diluted with 100 μL of methanol followed by vortexing for 2 min and then centrifuging at 9240 × g for 10 min. Aliquots of the supernatants were analyzed by the HPLC method. 2.14. Evaluation of Eye Irritancy. The ocular tolerance of [email protected] (5:3) nanocomposite eye drops (DEXP concentration: 250 μg/mL) was evaluated in New Zealand white rabbits (n = 3) following the method described by the Draize test.33 A single instillation of 50 μL of each nanocomposite formulation was instilled in the lower conjunctival sac of the left eye, and the right eye, as a control, was handled with normal saline. Moreover, nanocomposite eye drops and the control were dosed to the eyes repeatedly three times a day for 7 days to analyze the irritancy for repeated administration. The ocular tissues were evaluated according to the Draize eye scoring criteria (Table S1) at 1, 2, 4, 24, 48, and 72 h after administration. This method offers an overall scoring system for grouping the severity of ocular lesions involving the cornea (opacity) and conjunctiva (congestion, swelling, and discharge). The ocular irritation index (OII) was conducted according to the observed injuries on the basis of Table S2.34 In addition, histopathological analysis by microscopy was used to examine the changes in epithelial and basal cells. All the rabbits were euthanized, and then the cornea and conjunctiva of the ocular tissues were fixed with 10% formaldehyde. Sections were cut from the paraffin blocks at a thickness of 5 μm, stained with hematoxylin and eosin, and then observed with a light microscope. 2.15. Statistical Analysis. All experiments were conducted at least in triplicate. Statistical analysis was performed with GraphPad Prism 7 (La Jolla, CA, USA). Student’s t test was used to determine p values when the means of only two groups were compared (Figure 2, Tables 2 and 3). For a comparison among the means of more than two group means, one-way ANOVA was used. A value of p < 0.05 was considered statistically significant.

Table 1. Characterization of Different Nanocompositesa sample pristine LDH LDH-GS (1:1) LDH-GS (5:3) DEXP-LPs [email protected] (5:3) a

particle size (nm) 113.3 135.4 149.6 68.5 187.7

± ± ± ± ±

8.9 6.1 5.0 3.7 7.9

ζ potential (mV) 42.46 32.24 30.82 −10.97 24.33

± ± ± ± ±

9.51 2.56 4.23 1.23 2.56

PI 0.146 0.189 0.177 0.205 0.241

Mean ± SD, n = 3.

changed from 42.46 to 30.82 mV after modification, indicating the success of GS modification. The pristine LDH nanoparticles and [email protected] (5:3) nanocomposites were then characterized by TEM, and images are shown in Figure S5A. Pristine LDH nanoparticles exhibited a hexagonal platelet-like shape, while [email protected] (5:3) nanocomposites revealed an irregular circle morphology, which was probably due to the modification on the surface of LDH. XRD and small-angle X-ray scattering were used to determine the insertion of liposome into LDH36 (Figures S5B and S6). The reflections from the (003) basal planes stand for the thickness of the interlayer spacing plus the brucite layer.37 As shown in Figure S5B, LDH-GS nanocomposites exhibited a d-spacing of 0.50 nm, similar to that of pristine LDH nanoparticles, which means that GS modification does not alter the crystal framework. The positions of the (003) reflections of [email protected], [email protected], and [email protected] lead to interlayer distances of 2.32, 2.36, and 2.42 nm, respectively. These three nanocomposites displayed similar (003) reflection peaks accounting for the intercalation of HSPC into the LDH. Furthermore, the particle sizes of DEXPLPs and free liposome were 68.5 ± 3.7 and 40.6 ± 2.1 nm, which were much higher than the gallery heights of [email protected] and [email protected], respectively. Therefore, we propose that the inserted liposomes were destroyed during the intercalation process, which is in agreement with the previous study of Bégu et al.27 Their group confirmed that the insertion of the lipid bilayers, not the intact liposomes, occurred in the interlayer space of the LDH host when they studied the

3. RESULTS AND DISCUSSION 3.1. Characterization of APTES-GS. The ESI-MS spectra of Fmoc-GS and APTES-GS are shown in Figure S2. Figure S3 depicts 1H NMR spectra of GS, Fmoc-GS, Fmoc-GS-APTES, and APTES-GS. In comparison of spectra for GS (Figure S3d) and Fmoc-GS (Figure S3c), introduction of Fmoc-OSu to GS was confirmed from the existence of new peaks (7.37−7.89 ppm) corresponding to the aromatic protons of Fmoc-OSu in Figure S3c. It was apparent that the peaks at 1.68−1.57 ppm (m, 2H, NH−CH2−CH2) can be attributed to the segment of APTES in Figure S3b, which means that APTES was conjugated to Fmoc-GS. Successive cleavage of Fmoc and GS groups of APTES-GS was confirmed by the absence of characteristic peaks of the aromatic segment in the respective 1 H NMR spectra (Figure S3a). These results demonstrated that APTES-GS was successfully synthesized. 3.2. Surface Modification and Characterization. In this study, GS was introduced onto the LDH surface to enhance functionality of LDH.16 Figure S4 shows that the filter was stained purple after the ninhydrin test, which proved the success of the grafting process. As shown in Table S3, fluorescamine and elemental analyses were further used to quantify the amino groups on LDH-GS. According to the results, the contents of amino groups on grafted LDH 2850

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Table 2. Pharmacokinetic Parameters of Commercial Product, [email protected] (5:3), and [email protected] (1:1) Nanocomposite Eye Drops in Rabbit Tearsa sample

AUC0−6h (μg/mL min)

Cmax (μg/mL)

MRT (min)

commercial product [email protected] (5:3) [email protected] (1:1)

76.37 ± 36.15 665.64 ± 190.12a 641.37 ± 78.63a

4.11 ± 1.58 12.96 ± 1.71a 12.93 ± 1.06a

16.63 ± 3.32 42.46 ± 6.62a 45.93 ± 5.44a

Mean ± SD, n = 3. bp < 0.001, significant difference versus commercial product group.

a

clearance rate since DEXP could be detectable in tear fluid for only 90 min. Compared with commercial DEXP eye drops, [email protected] nanocomposite eye drops showed longer retention for up to 3 h. The relevant pharmacokinetic parameters calculated from the profiles are summarized in Table 2. The areas under the curve (AUC0−6h) of [email protected] (5:3) nanocomposite eye drops and [email protected] (1:1) nanocomposite eye drops were 8.71fold and 8.40-fold greater than that of commercial DEXP eye drops, respectively. These results were indicative of improved ocular bioavailability of model drug produced by [email protected] nanocomposite eye drops compared with commercial DEXP eye drops. Moreover, [email protected] nanocomposite (5:3) eye drops and [email protected] nanocomposite (1:1) eye drops exhibited similar behaviors in precorneal retention studies despite the difference in amounts of GS modified. The prolonged precorneal residence time of drug could be explained mainly by two aspects. On the one hand, positively charged nanocomposite eye drops could reinforce electrostatic interaction with a negatively charged corneal epithelium layer as well as a conjunctival epithelium layer. On the other hand, chitosan derivative CG with a high mucoadhesive property was able to further overcome the rapid removal of the drug due to disulfide bonds formed by thiol groups, which existed both on the ocular surface and CG.40 It should also be noted that the enhanced adhesion would facilitate the delivery of drug to the posterior segment of the eye for increasing period of drug exposure to the ocular surface. 3.6. Cytotoxicity Assay. The cytotoxicity of various formulations was investigated in both HCEpiC cells and HConEpiC cells via the MTT assay. As shown in Figure S9, [email protected] nanocomposites were well tolerated at an LDH concentration of 100 μg/mL after 12 h of incubation (cell viability >80%) on both HCEpiC cells and HConEpiC cells. However, in our previous study, the safe level of unmodified LDH nanoparticles was 75 μg/mL on HCEpiC cells.41 The reduced cytotoxicity of [email protected] nanocomposites compared to unmodified LDH nanoparticles might be attributed to the decreased charge density of LDH after modification and the negligible toxicity of the targeting ligand (GS). This finding was in agreement with the study. That surface modification of cationic nanocarriers with functional ligands may mitigate cytotoxicity.42 Moreover, DEXP-free LPs and DEXP-LPs had no toxicity effect toward both primary cell lines in this study. Several studies also demonstrated that liposomes were suitable for ophthalmic use due to improved safety and tolerability.11,43 Based on the results of MTT assays, [email protected] nanocomposites with good biocompatibility have promising potential for ocular drug delivery. 3.7. In Vitro Permeability Study. It has been reported that prolonged residence retention time and enhanced permeability through tissues of the ocular surface (cornea,

liposomes intercalated into LDH. Furthermore, this conclusion can be also proven by the group of Hou et al.38,39 Their group testified that betamethasone dipropionate loaded sodium cholate micelles were destroyed and cholate anions were inserted into the interlayer of LDH when they studied the intercalation process between micelles and LDH. In Figure S6, a lamellar repeat distance of free liposome and [email protected] was calculated about 5.0 nm, which was nearly 2-fold of the gallery height. Then we can suggest that the HSPC bilayer arranges in a tilt angle of 28.65° in the interlayer of the LDH.39 In addition, considering a similar d-spacing, DEXP molecules intercalated into the constrained brucite-like layers did not affect the structure of [email protected] and may disperse in the intermediate of the HSPC bilayers. In this study, the anionic exchange capacity of LDH is about 10%, which is a suitable ratio for the intercalation process according to the study of Bégu et al.27 Besides, the original DEXP concentration for ion exchange is about 2 mg/mL, and the resident DEXP concentration in the centrifugal supernatant for the first time is 50.23 ± 11.54 ng/mL. Coupled with the undetectable DEXP concentration in centrifugal supernatants for the second and third time, the intercalation of the DEXP molecules into LDH galleries is successful. In a word, as shown in Figure 1, the HSPC bilayer and DEXP molecules cointercalated into the LDH interlayer space. 3.4. In Vitro Drug Release. The release profiles of DEXP from various formulations are presented in Figure S7. The results showed that 51.41 ± 3.46% of drug was released from [email protected] (5:3) nanocomposites after 360 min, which was significantly lower than drug released from DEXP solution. The release of drug from the nanocomposites is based on the ion-exchange process in which negatively charged HSPC and DEXP were replaced by phosphate anions in the releasing media. After that, the layered structure of LDH gradually collapsed. According to the cumulative release profiles of [email protected], [email protected] (1:1), and [email protected] (5:3) nanocomposites, the amount of GS modified was inversely proportional to the cumulative release of DEXP. This may be due to the increased steric hindrance produced by the targeting ligand (GS) that hindered the diffusion of DEXP. Moreover, the chitosan derivative (CG), which served as a stabilizer to prevent aggregation, also sustained the release of drug from the nanocomposites. Additionally, the sustained release was an essential characteristic for drug delivering to the posterior segment of the eye for maintaining an effective drug concentration on the targeting site. 3.5. In Vivo Precorneal Retention Study. During the precorneal retention study, none of the signs of irritation, inflammation, or red swelling was observed, which proved that [email protected] nanocomposite eye drops were well tolerated. Figure S8 shows the precorneal DEXP concentration−time profiles after administration of different eye drops. The commercial DEXP eye drops showed a rapid ocular 2851

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noteworthy that the Papp value of [email protected] (5:3) nanocomposites for HConEpiC cells was significantly higher than the Papp value obtained for HCEpiC cells (p < 0.001). This comparison indicated that [email protected] (5:3) nanocomposites permeate through conjunctiva probably more easily than cornea. Specifically, there may be a difference in the permeation behavior of nanocomposites in ocular tissues compared with in vitro models. It could be explained by the lack of corneal stroma and conjunctiva stroma in pure corneal and conjunctival epithelial models, respectively.49 Based on the results above, [email protected] (5:3) nanocomposites were used in the further study. 3.8. Cellular Uptake Mechanism. Several inhibitors were applied in the investigation of the possible pathway involved in the cellular uptake of [email protected] nanocomposites and [email protected] nanocomposites on HConEpiC cells. The two nanocomposites at a DOX concentration of 10 μg/ mL showed little cytotoxicity (data not shown). The cellular uptake of nanocomposites is an energy-dependent process. Incubation with sodium azide or at 4 °C may lead to a reduction of energy produced by mitochondria.50 Consequently, a decreasing cellular uptake of DOX-labeled nanocomposites was observed in HConEpiC cells incubated with sodium azide as well as incubated at 4 °C as shown in Figure 2. Particularly, the cellular uptake of DOX-labeled nanocomposites on HConEpiC cells, which were pretreated with hypertonic sucrose or chlorpromazine (inhibitors of clathrinmediated endocytosis), was significantly suppressed (p < 0.05). In addition, there was no significant influence on the cellular uptake of nanocomposites after pretreatment with inhibitors of caveolae-mediated endocytosis (nystatin and genistein) and inhibitor of macropinocytosis (amiloride). These results suggested that clathrin-mediated endocytosis was a predominant cellular uptake pathway for both nanocomposites, which was consistent with our previous study.20 In addition, Oh et al. also demonstrated that the internalization mechanism of LDH nanoparticles on human osteosarcoma (MNNG/HOS) cells was clathrin-mediated endocytosis.51 To further confirm the ability of [email protected] nanocomposites to target PepT-1 expressed in HConEpiC cells, a series of GS concentrations (1, 5, and 10 mM) were also pretreated with cells. MTT assays showed that GS (1, 5, and 10 mM) has no toxicity toward HConEpiC cells (Figure S11). An obvious inhibition was observed for [email protected] LDH-GS nanocomposites when the concentration of GS increased to 10 mM (p < 0.01). However, the addition of GS did not affect the cellular uptake of [email protected] nanocomposites, which were not modified with GS. The results demonstrated that the active transport of PepT-1 might participate in the cellular uptake of [email protected] nanocomposites. Altogether, both clathrin-mediated endocytosis and active transport by PepT-1 were involved in the internalization of [email protected] nanocomposites in HConEpiC cells. 3.9. Transport Way of Nanocomposites in Ocular Tissues. To visualize the intraocular distribution of [email protected] nanocomposites, frozen sections of cornea, conjunctiva, and sclera-choroid-retina were prepared at different time points (5, 15, and 30 min) and further observed by an inverted fluorescence microscope. The model drug was tracked by DOX and emitted red fluorescence. Nuclei stained with DAPI emitted blue fluorescence.

conjunctiva) are two essential properties for noninvasive drug delivery to the back of the eye. Moreover, enhanced permeability plays an important role in decreasing drug side effects as a consequence of lessening the dose of drug.44 Drug topically administered suffers insufficient bioavailability due to inherent tissue diffusion limitations (cornea, conjunctiva) and dynamic barriers (reflex blinking, tear turnover).45 As a result, the subsequent amount of drug arriving at the posterior segment of the eye could be very low. There are mainly two routes for a drug crossing the corneal epithelium or conjunctival epithelium: transcellular route and paracellular route. For the transcellular route, drug directly crosses the cell membrane; while for the paracellular route, drug crosses the tight junctions between cells.46 The permeability of drug is influenced by several properties such as molecular weight, particle size, lipophilicity, aqueous solubility, and so on.47 In this work, permeation studies were performed on the HCEpiC-cell-based model and HConEpiCcell-based model to predict transcorneal and transconjunctival permeation of different formulations, respectively. The raw time-course data is shown in Figure S10, and the apparent permeation coefficients for all formulations based on two models are summarized in Table 3. As shown in Table 3, free Table 3. Apparent Permeation Coefficients (Papp) of DEXP in Different Formulations across the HCEpiC-Cell-Based Model and HConEpiC-Cell-Based Modela Papp (cm/s, ×10−6) formulation free DEXP DEXP LPs [email protected] nanocomposites [email protected] (1:1) nanocomposites [email protected] (5:3) nanocomposites

HCEpiC 13.40 3.47 1.49 5.95

± ± ± ±

1.73 0.79 0.32 0.86

7.44 ± 1.44

HConEpiC 14.88 4.59 1.83 7.64

± ± ± ±

1.52 0.82 0.29 0.31

13.39 ± 1.72a

Mean ± SD, n = 3 bp < 0.001, significant difference versus [email protected] (5:3) nanocomposites based on HCEpiC cells. a

drug DEXP exhibited considerable high permeability across both HCEpiC and HConEpiC cells, which can be classified into high permeable drug (Papp > 10 × 10−6 cm/s).48 We hypothesized that the probable route for free DEXP across the corneal epithelium or conjunctival epithelium was the paracellular route due to its low molecular weight (Mw: 516.41) and hydrophilic character. However, the diffusion route of free drug would be restricted by several physiological barriers and dynamic barriers as mentioned above. In comparison to [email protected] with Papp values of 1.49 ± 0.32 cm/s in HCEpiC cells and 1.83 ± 0.29 cm/s in HConEpiC cells, DEXP-LPs exhibited 2.32-fold and 2.51-fold higher Papp values than those in HCEpiC cells and HConEpiC cells, respectively. The improved permeability could be attributed to similar components of liposomes with the cell membrane. As GS is a classical substrate for PepT-1, the modification of GS on the surface of LDH could increase the recognition between nanocomposites and PepT-1, which may finally facilitate the permeation of nanocomposites across the cell membrane. This assumption was further confirmed by the calculated Papp values of [email protected], [email protected] LDH-GS (1:1), and [email protected] (5:3) nanocomposites based on HCEpiC cells and HConEpiC cells. It is 2852

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Figure 3. Fluorescent images of frozen sections of cornea (A), conjunctiva (B), and sclera-choroid-retina (C) of rabbit eyes after topical administration of [email protected] nanocomposites. Scale bar = 50 μm.

Figure 3A shows the trans-corneal fluorescence after instillation of DOX-labeled nanocomposites. The fluorescence mainly accumulated in the corneal epithelium, while negligible fluorescence could be detected in the corneal stroma. It indicated that [email protected] nanocomposites could not penetrate the corneal stroma. The cornea is mainly composed of three layers, including epithelium, stroma, and endothelium. The corneal epithelium consists of several layers of epithelium cells, which are connected by tight intercellular junctions.52 Thanks to PepT-1 expressed on the corneal epithelium cells,53 the nanocomposites could penetrate the epithelium facilitated by the targeting ligand (GS) anchored on the surface of LDH. The corneal stroma, which accounts for nearly 90% of the corneal volume, is mainly made up of hydrated collagen fibrils.52 The nanocomposites could not permeate through the stroma probably due to the barrier formed by collagen fibrils. By contrast, trans-conjunctival fluorescence showed (Figure 3B) that [email protected] nanocomposites were delivered from conjunctival epithelium to conjunctival stroma in a time-dependent manner. This can be explained by the physiological structure of the conjunctiva. For conjunctiva of human, the surface area is 17 times larger than that of cornea. In addition, conjunctiva showed superior permeability to drugs than cornea.54 Moreover, the interaction between the targeting ligand (GS) modified on the surface of LDH and PepT-1 expressed on the conjunctiva epithelium cells55 could further accelerate the permeation of the nanocomposites. The difference in permeation behaviors of nanocomposites in corneal and conjunctival tissues was

consistent with the in vitro permeability study in two cellbased models. Frozen sections of sclera-choroid-retina were prepared to observe whether the drug can be delivered to the posterior segment of the eye. As shown in Figure 3C, the fluorescent images illustrated that DOX could gradually diffuse from sclera into retina in 15 min. Due to high permeability of sclera,56 the drug could penetrate through choroid and further arrive at retina. Taking the results above into consideration, we speculated the possible route for [email protected] nanocomposite delivery from the ocular surface to the posterior segment of the eye after topical instillation. The restricted permeation behavior observed from frozen sections of cornea and several inherent barriers make it impossible for the corneal pathway and lateral diffusion pathway. Thus, the non-corneal way was considered as the principal route for the delivery of [email protected] nanocomposites. 3.10. In Vivo Ocular Distribution. DEXP is a kind of glucocorticoid for the treatment of ocular inflammation whose action site is the retina. However, it is hard to separate choroidal and retinal tissues physically and anatomically,57 so we detected the total concentrations in both choroidal and retinal tissues. After topical instillation of different formulations (commercial eye drops, [email protected] (5:3) nanocomposite eye drops, [email protected] (5:3) nanocomposite eye drops, DEXP-LPs), the biodistribution of drug in different rabbit ocular tissues was determined by the HPLC method and is shown in Figure 4. 2853

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Figure 4. Concentration of DEXP in ocular tissues at different time points (0.5, 1, 2 h) after topical administration of (A) aqueous humor, *p < 0.05, vs that of commercial products. #p < 0.05 vs that of DEXP-LPs. ##p < 0.01 vs that of [email protected] (B) cornea, *p < 0.05, **p < 0.01 vs that of commercial products. #p < 0.05, ##p < 0.01 vs that of DEXP-LPs. (C) Sclera, *p < 0.05, **p < 0.01 vs that of commercial products. ##p < 0.01 vs that of DEXP-LPs. ###p < 0.001 vs that of [email protected] (D) Conjunctiva *p < 0.05, vs that of commercial products. #p < 0.05 vs that of DEXP-LPs. ##p < 0.01 vs that of [email protected] (E) Choroid-retina. #p < 0.05, ##p < 0.01 vs that of DEXP-LPs (mean ± SD, n = 3, below level of quantification).

nanocomposite (5:3) eye drop group reached the peak in scleral and choroid-retina tissues, indicating that [email protected] nanocomposites could effectively deliver the drug to the posterior segment of the eye. We proposed that the increased amount of DEXP of the [email protected] nanocomposite (5:3) eye drop group in scleral and choroidretina tissues mainly comes from conjunctiva due to the significantly declining drug concentration in the conjunctiva (p < 0.05). Furthermore, there was a negligible change of drug concentration in the cornea with the extension of time. It indicated that [email protected] nanocomposite (5:3) eye drops only gathered on the corneal epithelium but could not achieve the deep permeation even though the drug concentration in the cornea was higher than that in the conjunctiva. This hypothesis was consistent with the results of the transport way study in ocular tissues that [email protected] LDH-GS nanocomposites could not permeate into the corneal stroma. The quantity of DEXP from [email protected] nanocomposite eye drops in the corneal tissues was significantly higher than that of commercial products (p <

After topical administration, it was obviously observed that both [email protected] (5:3) nanocomposite eye drops and [email protected] (5:3) nanocomposite eye drops exhibited strong residence on the tissues of the ocular surface (cornea and conjunctiva) compared with commercial eye drops. Electrostatic interaction between nanocomposite eye drops and the tissues of the ocular surface ensured sufficient adhesion. Moreover, the targeting ligand (GS) on the surface of LDH enhanced cellular uptake of nanocomposites via active transport by PepT-1, which played an important role in drug delivery to the posterior segment of the eye. According to the study of Johannsdottir et al.,58 the therapeutic concentration for the retinal tissues is about 50 ng/g. After an administration of 2 h, the DEXP concentration of [email protected] nanocomposite (5:3) eye drops remained at 200 ng/g in the choroidal and retinal tissues, which was higher than the therapeutic concentration of DEXP, so we speculated that the concentration of DEXP in retinal tissue was within the range of the therapeutic scope. At a 1 h time point after administration, the DEXP concentration of the [email protected] 2854

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100 μg/mL in HCEpiC cells and HConEpiC cells. The [email protected] nanocomposites showed superior in vitro permeability on the HConEpiC-cell-based model. In the case of the HConEpiC cell, both clathrin-mediated endocytosis and active transport by PepT-1 were involved in the internalization of the nanocomposites. Images of frozen sections of ocular tissues suggested that the possible route for the delivery of DOX-labeled nanocomposites from the ocular surface to the back of eye was the non-corneal pathway. Furthermore, the hybrid nanocomposites displayed higher drug concentration in choroid-retina tissue compared with other preparations composed of a single nanocarrier in the in vivo ocular distribution study. The ocular tolerance evaluation demonstrated nonirritancy of nanocomposite eye drops to organic tissues. In summary, multifunctional nanocomposites based on liposomes and layered double hydroxides could be applied as promising platforms for efficient drug delivery to the posterior segment of the eye for biologics therapy via topical instillation.

0.05). Besides, in Figure 4D, the amount of the [email protected] nanocomposite eye drop group in the conjunctiva was significantly higher than that of the [email protected] LDH-GS group at 1 h (p < 0.01). These results were consistent with the results of the in vivo permeability study that [email protected] nanocomposite eye drops were easier to permeate into ocular tissues than commercial products. As for the biodistribution of the DEXP-LP group in scleral and choroid-retina tissues, small-sized liposomes (about 68.5 nm) also had the potential to deliver drug to the back of the eye, which is consistent with the previous work by Lajunen et al.8 Furthermore, in sclera, there was a significantly higher DEXP level of [email protected] nanocomposite eye drops in comparison with that of DEXP-LP group (p < 0.01), showing that [email protected] nanocomposite eye drops can facilitate DEXP delivery to the back part of the eye. Nonetheless, a significantly higher DEXP level (p < 0.05) was found in choroid-retina tissue of the [email protected] nanocomposite eye drop group compared with the DEXPLP group, indicating that the mucoadhesive property and active transport of PepT-1 are essential for effective drug delivery to the back of the eye. For the [email protected] (5:3) eye drop group, only a small amount of DEXP could be detected in choroid-retina tissue at 30 min. It could be explained by rapid clearance of free drug that is gradually released from [email protected] nanocomposites via ion exchange. These results suggested that the nanocomposites of [email protected] showed the superior ability of transiting drug to the back of the eye than a single nanocarrier, namely, LDH or liposomes. Because the HSPC bilayer and DEXP molecules cointercalated in the LDH interlayer space, how HSPC helps DEXP molecules reach the posterior segment of the eye is a question. Bégu’s studies showed that, as the lipid bilayer is released from LDH, the lipid bilayer in an aqueous medium could spontaneously form liposomes.29 We propose that, as the concentration of HSPC is above critical aggregation concentration, liposomes may be formed in sclera and assist DEXP transport in deep ocular tissues. We are presently starting to obtain new experimental data to support this hypothesis. 3.11. Evaluation of Eye Irritancy. Considering the novel strategies of [email protected] (5:3) nanocomposite eye drops, eye irritancy was tested through the Draize test and in vitro histopathological examination, performing cell structure and tissue integrity to detect irritation. According to the results of OII shown in Table S4, the nanocomposite eye drops can be grouped into nonirritancy, which are suitable to apply in the organic tissues. The micrographs of stained tissues are shown in Figure S12. No other ocular damage or clinically abnormal signs were observed in the cornea or conjunctiva. Those results are consistent with Draize’s scale, which suggests that [email protected] (5:3) nanocomposite eye drops are biocompatible with excellent ocular tolerance.59



ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.8b01136. Synthesis process, MS spectrum, 1H NMR spectrum, ninhydrin test, characterizations, release profiles, the concentration−time curves, MTT assay results, the raw time-course data of permeability studies, cell viability, histopathology microscopy, Draize’s scale, classification of products, amine quantification, pharmacokinetic parameters, and ocular tolerance (PDF)



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] Tel: +86 25 5209 1139 (L.F.). *E-mail: [email protected] Tel: +86 25 8327 1092 (F.C.). Author Contributions ∥

Y.G. and C.X. contributed equally to this paper.

Funding

This paper was supported by the National Natural Science Foundation of China Grant No. 81373362. Notes

The authors declare no competing financial interest.



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4. CONCLUSIONS In this study, multifunctional [email protected] nanocomposites were developed via GS modification of LDH and ion-exchange method with the aim of efficient topical drug delivery to the back of the eye. Compared with commercial eye drops, the precorneal retention time of [email protected] nanocomposites was prolonged. MTT assays revealed that the nanocomposites well tolerated LDH concentration below 2855

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