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Sustained release of brimonidine from a new composite drug delivery system for treatment of glaucoma Jianguo Sun, Yuan Lei, Zhaoxing Dai, Xi Liu, Taomin Huang, Jihong Wu, Zhi Ping Xu, and Xinghuai Sun ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16509 • Publication Date (Web): 15 Feb 2017 Downloaded from http://pubs.acs.org on February 16, 2017

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ACS Applied Materials & Interfaces

Sustained release of brimonidine from a new composite drug delivery system for treatment of glaucoma Jianguo Suna,b,e,f,‡, Yuan Lei

a,e,‡

, Zhaoxing Daic, Xi Liuc, Taomin Huangg, Jihong Wua,c,d,e , Zhi

Ping Xub,*, Xinghuai Sunc,d,e,* a

Research Center, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai

200031, China b

Australian Institute for Bioengineering and Nanotechnology, ARC Centre of Excellence for

Functional Nanomaterials, The University of Queensland, Brisbane, QLD 4072, Australia c

Department of Ophthalmology & Visual Science, Eye & ENT Hospital, Shanghai Medical

College, Fudan University, Shanghai 200031, China d

State Key Laboratory of Medical Neurobiology, Institutes of Brain Science and Collaborative

Innovation Center for Brain Science, Fudan University, Shanghai 200032, China e

Key Laboratory of Myopia, NHFPC, and Shanghai Key Laboratory of Visual Impairment and

Restoration, Fudan University, Shanghai 200031, China f

State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai

200433, China g

Department of Pharmacy, Eye & ENT Hospital, Shanghai Medical College, Fudan University,

Shanghai 200031, China

KEYWORDS Layered double hydroxide (LDH) nanoparticles, Brimonidine, Thermogel, Drug delivery system (DDS), Glaucoma, Intraocular pressure (IOP)

ACS Paragon Plus Environment

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ABSTRACT A novel layered double hydroxide (LDH) nanoparticle/thermogel composite drug delivery system (DDS) for sustained release of brimonidine (Bri) has been designed, prepared and characterized in this study for treatment of severe glaucoma. Brimonidine is first loaded onto LDH (Bri@LDH) nanoparticles, which are then dispersed in the thermogel consisting of plenty of micelles based on poly-(DL-lactic acid co-glycolic acid)–polyethylene glycol–poly-(DL-lactic acid co-glycolic acid) (PLGA-PEG-PLGA) copolymer. The Bri@LDH/Thermogel DDS containing 125.0 µg/g of brimonidine has been found to sustainably release the drug for up to 144 h, significantly extending the drug release period compared to that from Bri@LDH nanoparticles. The Bri@LDH/Thermogel DDS is not cytotoxic to human corneal epithelial cells and shows good biocompatibility. In vivo drug release from the special contact lens made of Bri@LDH/Thermogel DDS has been sustained for at least 7 days, which more effectively modulates the relief of intraocular pressure (IOP). Thus the Bri@LDH/Thermogel DDS is a promising drug delivery alternative that can be used for treatment of severe glaucoma.


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1. Introduction Glaucoma is a collection of pathological conditions typically characterized by increased intraocular pressure (IOP) and the second leading cause of blindness worldwide. Long-term elevated IOP can permanently damage the optic nerve, resulting in vision impairment and even blindness. So decreasing and maintaining IOP is the most direct method to treat glaucoma. Medical therapy, such as using topical eye drops, is the first line and preferred method to treat primary open-angle glaucoma. Brimonidine is an antiglaucoma agent as an alpha adrenergic receptor agonist to benefit IOP relief by reducing aqueous humor production and increasing uveoscleral outflow1-3. In addition, brimonidine is also well known for its neuroprotective effect against retinal ganglion cell death4. However, topical application of brimonidine eye drops has low bioavailability through the cornea (1~7%) and the remaining drug that enters systemic circulation can cause side effects5. Its ocular hypotensive effect can only maintain for a few hours post-dosing and patients are often required to administer multiple eye drops daily6. As glaucoma is a chronic disease, long term therapy and rigorous administration schedule using brimonidine are necessary. However, brimonidine has a poor patient compliance, and only 31% to 67% patients can adhere the use of brimonidine eye drop for 12 months7. Thus it is difficult for some patients to administer their eye drops on a regular basis, which requires new types of ophthalmic formulations to decrease the medication time, improve patient compliance and sustain therapeutic effect on glaucoma. Brimonidine-loaded drug delivery systems (DDSs) for the effective management of glaucoma have been proposed and investigated, such as microspheres8-9, hydrogels10 and implant11-13, and many

nanoparticles

nanoparticles16,

including

albumin

charged

nanoparticles17,

nanoparticles14, eudragit

lipid

nanoparticle15,

nanoparticles18,


chitosan

polyacrylic

acid

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nanoparticles19 and poly-(DL-lactic acid co-glycolic acid) (PLGA) nanoparticle20. Recently layered double hydroxide (LDH) clay nanoparticles have attracted particular attention for their variable chemical composition, biocompatibility, anion exchange capacity, and control release characters for preparation of novel composite DDSs21-24. In general, simple DDSs based on LDH nanoparticles have a poor colloidal stability due to nanoparticle aggregation25-27 and a rapid release profile involving anionic exchange with the anion in the medium28. Rapid release profile may be suitable for anti-inflammatory applications, but this release model may not be optimal to treat chronic diseases that require drug exposure for a longer time. Therefore, development of composite LDH-drug DDSs is desirable in order to increase drug exposure time and sustain drug therapeutic effect on glaucoma. To this end, we chose a kind of new thermosensitive hydrogel based on an amphiphilic copolymer, e.g. poly-(DL-lactic acid co-glycolic acid)–polyethylene glycol–poly-(DL-lactic acid co-glycolic acid) (PLGA-PEG-PLGA), which is often used as a carrier material for drug delivery29-33 or tissue regeneration34-35. Therapeutic drugs or nanoparticles are first incorporated into the thermosensitive hydrogel (thermogel) solution at a low temperature32, 36-37, and then the corresponding formulation is injected into the in vivo target site to form a gel at the physiological temperature (37 °C) so as to control the release of loaded drugs. For example, thermogel-based topical eye drop of dexamethasone prolonged the drug contact time within the ocular tissue due to its high viscosity, which increased bioavailability and simultaneously reduced drug wastage and side effects38. Thermogel is also used to sustainably deliver drugs by intravitreal injection to treat posterior segment disease of the eyes33, 39.


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Scheme 1. (A) Schematic representation of delivering brimonidine by Bri@LDH/Thermogel DDS (brimonidine denoted as red dots is released via dual-control processes, which then enters anterior chamber through cornea (marked as red arrows)); (B) Schematic description of the external contact lenses.

The external contact lens was used to improve the spread of

Bri@LDH/Thermogel solution on rabbit eye surface which can form a smooth soft lens, and the pores were specifically designed for the convenience of aqueous humor collection (Type I) and IOP measurement (Type II).

The aim of this research is thus to develop a brimonidine-containing composite DDS that is able to sustainably release the drug for a week. For this purpose, we designed a dual-control release system, e.g. a thermogel incorporated with brimonidine-loaded LDH nanoparticles


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(Bri@LDH/Thermogel), to continuously deliver brimonidine to the ocular surface. The designed composite DDS is schematically represented in Scheme 1A as a Bri@LDH/Thermogel soft lens. The thermogel soft lens is formed once Bri@LDH/Thermogel DDS solution is topically applied on the ocular surface. Upon hydration of tear film solution, brimonidine is released from LDH nanoparticles to the thermogel matrix (Step-I) and then diffuses from the matrix to tear film solution (Step-II). In such a way, the drug release is well controlled, as supported by our in vitro and in vivo assays conducted in this research.

2. Materials and methods 2.1 Materials MgCl2·6H2O (98%), NaOH (98%), Dulbecco's Modified Eagle Media (DMEM/F12) media, fetal bovine serum (FBS), polyethylene glycol dimethacrylate (MW 700) and 2-hydroxy-1-[4(hydroxyethoxy) phenyl]-2-methl-1-propanone were all purchased from Sigma-Aldrich (Shanghai, China). AlCl3 (98%) was from Fluka (Shanghai, China) and methylthiazol tetrazolium (MTT) from Dojindo Laboratories (Kumamoto, Japan). Brimonidine tartrate (98.5%) and 2-hydroxyethyl methacrylate were bought from J&K Scientific Ltd. Triblock copolymer (PLGA1700–PEG1500–PLGA1700) was synthesized through ring-opening polymerization of lactide and glycolide in the presence of PEG (MW1500) and the catalyst stannous octoate as described previously40 (1H NMR spectrum shown in Supporting Information Figure S1). Alphagan eye drops (0.2% w/w brimonidine tartrate) was from Allergan Pharmaccuticals Ireland. Xylazine hydrochloride (Jilin Province TAT Animal Pharmaceutical Co., Ltd., Jilin, China), diazepam injection (Shanghai Xudong Haipu Pharmaceutical Co.,Ltd., Shanghai, China), ofloxacin eye ointment (Shenyang Sinqi Pharmaceutical Co., Ltd, Shenyang, China), oxybuprocaine


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hydrochloride eye drops (Santen Pharmaceutical Co., Ltd, Shanghai, China), and phosphatebuffered saline (Thermo Scientific, Waltham, MA, USA) were purchased and used as received.

2.2 Preparation of Bri@LDH/Thermogel DDS Bri@LDH nanoparticles were prepared according to a modified hydrothermal process reported previously41-42. In brief, 10 mL of a mixed solution containing MgCl2 (300 mM), AlCl3 (100 mM) and brimonidine (10 mM) was quickly added to 40 mL of an alkaline solution containing NaOH (150 mM) or plus L-tartrate (7.5 mM) under vigorous stirring. After another 10 min stirring, the precipitates (Bri@LDH nanoparticles) were collected via centrifuge separation (4,000 g, 5 min) and washed 3 times via centrifugation to remove impurities. The collected Bri@LDH nanoparticle slurry was dispersed in 20.0 mL ultrapure water and heated in an autoclave (stainless steel with a Teflon lining) at 100 °C for 6 h. The resultant Bri@LDH nanoparticle suspension was lyophilized and collected for various characterizations and subsequent usage. For simplicity, Bri@LDH and Bri@LDH(0) denoted Bri@LDH nanoparticles prepared from alkaline solution with and without L-tartrate, respectively. Subsequently, Bri@LDH/Thermogel DDS was prepared as follows. The PLGA–PEG–PLGA copolymer (1 g) was dissolved in PBS (3 g) to form 25 % (w/w) solution (thermogel solution). Then lyophilized Bri@LDH nanoparticles (20.0 mg) were added to the above thermogel solution (4.0 g) and magnetically stirred at 600 rpm and 4 °C for 2 h to get a homogeneous Bri@LDH/Thermogel suspension. All thermogel solutions used for in vitro and in vivo experiments were filtered through 0.22 µm membrane at 4 °C for sterilization and the lyophilized nanoparticles of LDH and Bri@LDH were sterilized under UV light (254 nm) for 30 min.


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As an auxiliary supply, the external normal contact lenses were prepared by UV polymerization (365 nm, 50 min, SB-100P/F, Spectronics Corporation, USA) of 2-hydroxyethyl methacrylate monomer (5 g) with assisting by crosslinker of polyethylene glycol dimethacrylate (MW 700, 0.15 g) and initiator of 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methl-1-propanone (0.075 g) under N2, as reported in our previous paper43. A polydimethylsiloxane mold was used to control the shape of these contact lenses to be suitable for rabbit eyes. The rough contact lens samples were immersed in water for 2 days and then tailored into the standard contact lens with specific morphology, as marked as Type-I and Type-II (Scheme 1B). Their photos, light transmittance and SEM image of the surface were provided in Supporting Information Figure S2-S4.

2.3 Characterization of Bri@LDH nanoparticles Dynamic light scattering (DLS) (Autosizer 4700, Malvern, Britain) was used to analyze the particle size and particle size distribution of Bri@LDH nanoparticle suspensions. Fourier transform infrared spectra (FTIR) were collected on a Nicolet 6700 FTIR (Thermofisher, USA) within 4000–400 cm-1 at a resolution of 4 cm-1 by measuring the IR absorbance of KBr disc that contained 1–2 wt% of LDH, sodium tartrate or Bri@LDH sample. Powder X-ray diffraction (XRD) patterns were recorded on a Rigaku Miniflex X-ray diffractometer with a variable slit width at a scanning rate of 2º/min with 2θ ranging from 2.5º to 80º using Cu Kα radiation (λ = 0.154 nm). Field emission scanning electron microscope (FESEM, Ultra 55, Zeiss, Germany) was used to characterize the morphology and particle size of Bri@LDH sample. The total amount of brimonidine in the Bri@LDH nanoparticle samples was determined by measuring the UV–Vis absorbance of brimonidine at 246 nm in a spectrophotometer (Thermo


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Scientific, NanoDrop 2000 Spectrophotometer) after dissolution of Bri@LDH powder (3.0 mg) in 3.0 mL solution with pH adjusted to 3.0 by HCl solution (1%). Each solution was filtered through a 0.45 µm membrane and measured three times, and the average reading was reported.

2.4 Sol-gel transition of Bri@LDH/Thermogel suspension The sol-gel transition of Bri@LDH/Thermogel suspension was investigated using a Kinexus Pro rheometer (Malvern Instrument Inc., UK) equipped with a steel parallel-plate (diameter: 40 mm, CP60, bob gap: 0.3 mm). The thermogel solution or Bri@LDH/Thermogel suspension (25%, w/w) was injected into the steel parallel-plate and then a thin layer of silicone oil was added carefully to prevent evaporation of water. The plates were equilibrated to the starting temperature of 10 °C, and temperature sweep tests were carried out at a fixed oscillatory frequency of 10 rad/s within the temperature range of 10 °C − 45 °C at the heating rate of 0.5 °C/min. The temperature was controlled using a controller with an accuracy of ± 0.05 °C (Neslab, RTε-130). Rheological test parameters, i.e. storage modulus (G') and shear loss or viscous modulus (G''), were monitored as a function of temperature. All measurements were performed in triplicate.

2.5 In vitro drug release The drug release from Bri@LDH nanoparticles or Bri@LDH/Thermogel was investigated. First of all, Bri@LDH powder (5 mg) was added into 5 mL of PBS and shaken at a speed of 50 rpm in the DKZ-3B shaker (Shanghai Yiheng Scientific instruments Co., Ltd) at 37 °C. At the time point of 0, 0.1, 0.25, 0.5, 1, 2, or 4 h, an aliquot of 0.5 mL solution was withdrawn and then refilled with 0.5 mL of fresh PBS into the solution. The withdrawn aliquot (0.5 mL) was filtered


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through a 0.45 µm membrane. The pH value of the filtered solution was adjusted to 3.0 by HCl solution (1%) for the drug concentration measurement by UV–Vis absorbance. Four parallel samples (n = 4) were used for calculation of the average result. For in vitro drug release from the Bri@LDH/Thermogel sample, Bri@LDH/Thermogel suspension (25%, 0.1 g) was first injected into a vial (15-mL volume) and was incubated in a water bath at 37°C for 30 min. After formation of hydrogel, 5 mL PBS (pH 7.4, 37 °C) was added to the vial as the release medium. The shaking rate of the water bath was set at 50 rpm. In a 144 h (6 d) period, aliquots (1 mL) of the release medium were sampled at the specific time intervals (3, 6, 24, 48, 96 and 144 h) and the same volume (1 mL) of the fresh PBS was refilled to the vial to kept PBS volume constant (5 mL). The collected release media were filtrated with a 0.45 µm filter and its pH value was adjusted to 3.0 by HCl solution (1%). The brimonidine in the release media was quantified with Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA) with a G1311A quaternary pump, a G1312A vacuum degasser, and a G1315B DAD detector. A Diamonsil C18 column (150 mm × 4.6 mm, 5 µm) was maintained at 30 °C. The mobile phase was composed of a mixture containing 10 mM phosphate buffer (pH 3.5) containing 0.5% triethlamine and methanol in the ratio of 15/85 (v/v). The flow rate of the mobile phase was set at 1 mL/min. Measurements were made with 20 µL of injection volume at 246 nm. Four parallel samples (n = 4) were investigated for in vitro drug release from Bri@LDH/Thermogel sample.

2.6 Cytotoxicity assay Cytotoxicity of Bri@LDH/Thermogel sample was investigated in the transwell cell culture method. Human corneal epithelial (HCET) cells (30000 cells in 1.5 mL culture medium per well)


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were cultured in a 12-well plate with DMEM containing 10% FBS for 24 h. Bri@LDH/Thermogel suspension (25%, 0.1 g) was placed in the transwell insert and kept at 37 °C for 30 min, and then the transwell insert with Bri@LDH/Thermogel sample was placed in 12well plate with HCET cells and fully submerged in the culture medium. The HCET cells were allowed to grow for another 48 h. The cell viability was then assessed using the Cell Counting Kit-8 (CCK-8) assay according to the manufacturer’s instructions. After removing the culture medium, diluted CCK-8 solution (10% in DMEM, 100 µL) was added to each well and the 12well plate was incubated for 1 h at 37 °C under 5% CO2. Then, the absorbance of each well was measured at 450 nm using a microplate reader (Synergy™ H1 Hybrid Reader, BioTek, Vermont, USA). The relative cell viability in thermogel, LDH/Thermogel or Bri@LDH/Thermogel group (n = 6) was assessed using the cell viability in the blank group as 100%.

2.7 In vivo drug release All rabbit experimental protocols, including transportation, care and operations, were complied with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Animal Care and Use Committee of Fudan University (Shanghai, China). In this study, mature male New Zealand rabbits were used. The external contact lens (Type-I shown in Scheme 1B) was applied to study in vivo drug release of Bri@LDH/Thermogel sample. The New Zealand white rabbits free of any ocular damages with weighing 2.0–2.5 kg were obtained from the Yin’gen Rabbits’ House (Shanghai, China). The rabbits were housed under standard conditions (25 °C and relative humidity 50%) in the animal facilities of the EENT Hospital (Fudan University) with free access to food and water.


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Twelve New Zealand rabbits were divided into two groups (six rabbits per group). All right eyes were treated groups and all left eyes as the blank control group. After general anesthesia with xylazine hydrochloride (40 mg/kg body weight) and diazepam (1 mg/kg body weight) and topical anesthesia (0.5% of oxybuprocaine hydrochloride), Bri@LDH/Thermogel suspension (25%, 0.1 mL) was dropped onto the rabbit cornea surface and a Type I external contact lens was covered on the rabbit eye surface (Bri@LDH/Thermogel group). Alphagan eye drops (250 µL, 0.2 % (w/w) brimonidine tartrate) were added drop by drop on the rabbit eye surface (Alphagan group). Assuming 2.5% (generally

Sustained Release of Brimonidine from a New ... - ACS Publications

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