Glaucoma Drainage Device Coated with Mitomycin C Loaded Opal

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Applications of Polymer, Composite, and Coating Materials

Glaucoma Drainage Device Coated with Mitomycin C Loaded Opal Shale Microparticles to Inhibit Bleb Fibrosis Aimeng Dong, Liang Han, Zhengbo Shao, Pan Fan, Xinrong Zhou, and Huiping Yuan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18551 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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ACS Applied Materials & Interfaces

Glaucoma Drainage Device Coated with Mitomycin C Loaded Opal Shale Microparticles to Inhibit Bleb Fibrosis Aimeng Dong,1,2,‡ Liang Han,3,4,‡ Zhengbo Shao,1 Pan Fan,1 Xinrong Zhou,1 Huiping Yuan1* 1 Department of Ophthalmology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150081, P. R. China 2 The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Heilongjiang Province 150081, P. R. China 3 Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, P. R. China 4 Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, P. R. China Corresponding Author

Dr. Huiping Yuan, Department of Ophthalmology, The Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin, Heilongjiang 150081, P.R. China.

* E-mail: [email protected]

‡ Aimeng Dong and Liang Han contributed equally to this publication.

Key words：Opal Shale, microparticles, drug release, Ahmed glaucoma valve, fibrosis, Mitomycin C

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Abstract Excessive fibrosis is the topmost factor for the defeat of surgical glaucoma drainage device (GDD) implantation. Adjuvant drug approaches are promising to help reduce the scar formation and excessive fibrosis. Opal shale (OS), as a natural state and non-crystalline silica substance with poriferous nature and strong adsorbability, is highly likely to undertake drug loading and delivery. Here, we employed OS microparticles (MPs) by ultrasound and centrifugation and presented an innovative and improved GDD coated with OS MPs, which were loaded with mitomycin C (MMC). MMC-loaded OS MPs were physically absorbed on the Ahmed glaucoma valve surface through OS’ adsorbability. About 5.51 μg of MMC was loaded on the modified Ahmed glaucoma valve and can be released for 18 days in vitro. MMC-loaded OS MPs inhibited fibroblast proliferation and showed low toxicity to primary Tenon’s fibroblasts. The ameliorated drainage device was well tolerated and effective in reducing the fibrous reaction in vivo. Hence, our study constructed an improved Ahmed glaucoma valve using OS MPs without disturbing aqueous humor drainage pattern over the valve surface. The modified Ahmed glaucoma valve successfully alleviated scar tissue formation after GDD implantation surgery.


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1. Introduction Glaucoma, generally acknowledged to a topmost factor of blindness, is a form of optic neuropathy.1 Currently, glaucoma may be relieved by the pharmacological agent for initial treatment or by filtration surgery for uncontrolled intraocular pressure (IOP). Glaucoma drainage device (GDD) is becoming the modality of option in glaucoma surgical treatment, especially in complicated cases that are irresponsive to trabeculectomy.2,3 Unfortunately, the GDD success rate was up to 60% to 90% at one year and decreased by 10% per year in the subsequent follow-ups.4-7 Excessive fibrosis formation, the major cause of failure, may obstruct aqueous humor escaping from the eye.8,9 Mitomycin C (MMC), as well as 5-fluorouracil (5-FU), is the most prominent agents conducted in glaucoma surgery.10,11 Due to the high biological effectuality and long-lived inhibitory effects on fibroblasts, more preference is given to MMC than 5-FU in clinic.12 Nevertheless, the intraoperative application of MMC with high concentration can lead to serious side effects.13 New approaches may be necessary to solve the problem by providing sustained delivery while reducing drug toxicity.14-18 Previous efforts mainly focused on sustained release formulations constructed of natural or synthetic biodegradable or non-degradable polymeric materials. Another possible and available option is the application of porous silicon (PSi) which is widely used in diverse disciplines, ranging from tissue engineering19 to biosensors20, and is



excellent material for drug delivery.21,22 The allowance of high level of drug adsorption and drug emancipation is benefit from the PSi pore structure, which is suitable for ocular application.23 Although the results in vivo have confirmed the biocompatibility of PSi microparticles (MPs),24 the translation from bench to bedside is still crucial issue under debate. In view of biosafety, novel silicious materials with low toxicity are highly needed. Natural opal shale (OS) is a sort of nanopore structure model mainly composed of amorphous SiO2.25 OS offers the prospects such as highly porous structure and high absorption capacity.26-28 It has been utilized for numerous applications in industry as effective carrier. Recently, Han et al. firstly proposed and validated the use of OS nanoparticles as drug carrier to deliver chemotherapeutic agents to the tumor.25 However, studies about the application of OS in medical science are limited. We happened to notice the scarcity of research about biocompatibility and drug loading capacity of OS in glaucoma therapy. Accordingly, we had the intention to fabricate a novel GDD by coating normal Ahmed glaucoma valve (AGV) with OS MPs for MMC delivery and investigate the anti-scarring effects on the outer ocular structures. 2. Material and methods 2.1. Materials Mitomycin C (MMC) was picked up out of Sigma-Aldrich (St. Louis, MO, USA). OS ultrafine powder was generous gift of Professor Han (Pharmaceutical Sciences College,


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Soochow University, China). Ahmed glaucoma valves (AGV, model FP7, Rancho Cucamonga, CA) together with medical-grade silicone sheets which were raw materials used in valve fabrication were provided by New World Medical Inc. for free. Poly (vinyl alcohol) (PVA) with specific molecular weight (30,000 to 70,000) was supplied by Sigma Chemicals Co. (St. Louis, USA). Ethyl acetate (>99%), dimethyl sulfoxide (DMSO) were acquired out of Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water (UPW, resistivity with 18.2 MΩ cm) was adopted. Phosphate-buffered saline (PBS) with pH level of 7.4 stemmed from HyClone (Logan, UT, USA). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and the antibiotic/antimycotic (A/A) solution were obtained from Biological Industries (BI) (Kibbutz Beit Haemek, Israel). 2.2 Device fabrication The preparation was achieved by emulsification solvent evaporation.25 Briefly, OS ultrafine powder (12.5 mg) were dissolved in 2 ml ethyl acetate, mixed with a solution of MMC (1.25 mg) in DMSO (50 μl) and stirred for 4-6 h. The organic mixture was added dropwise into 2 ml PVA solution (2.5% w/v) on a Vortex, subsequently, ultrasonicated six times for 10 sec at 130W in ice/water surroundings. Afterwards, the emulsion was admixed with 50 mL of PVA (0.3% w/v) and stirred overnight (200 rpm) to evaporate the ethyl acetate. The suspensions were then centrifuged (4000 rpm, 30 min, 4°C) to clear off PVA. Collected OS MPs were washed twice with ultra-purified water and resuspended in 2ml UPW. Blank AGV was submerged under the resulting suspension while avoiding



contact with the drainage tube. After 24 h immersing, the sample was left to exsiccate for another 24 h at normal temperature. Medical-grade silicone sheet, substitution of AGV, was cut into discs for in vitro cell culture experiments. Modified silicone discs were obtained by the same procedure. All the above operations were implemented at room temperature refraining from direct visible spectrum and heat. Before application in the subsequent experiments, all test samples underwent ethylene oxide fumigation for 8 h in the dark. The resulting specimens were then desiccated in an air less oven for 2 days to remove residual ethylene oxide. 2.3 Characterizations 2.3.1 Morphology and structure Surface morphology of the modified AGV and chemical composition of OS MPs were investigated via SEM-EDX (Merlin Compact, Zeiss, Germany). Samples were firstly scattered with gold under an argon atmosphere at 30 mA for a period of 10 minutes (Balzers SCD 030 gold sputter coater, Balzers, Liechenstein). The particle sizes and amorphous characteristic of OS MPs were analyzed with laser diffraction particle size analyzer (Malvern Mastersizer 2000, UK) and X-ray diffraction (XRD, Rigaku D/MAX-2200 H/PC, CuKα radiation) individually. Porosity analysis was conducted on Quadrasorb SI-KR/MP type surface area analyzer. Malvern zetasizer Nano ZS (Malvern Instruments, UK) was adopted to measure the zeta potential of OS MPs. 2.3.2 Indentation scratch test


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The adhesive strength of OS MPs coating adhered onto AGV was investigated by indentation scratch test. The hardness, as well as Young's modulus, of films was calculated after nanoindentation with Agilent Nano Indenter G200. The strain rate of Berkovich indenter applied on the OS MPs coating was 0.05 s−1. For the nanoscratch tests, drawing through the sample surface, normal load of cube corner indenter tip progressive increased to 5, 10 and 20 mN separately was applicated, with profiling and scratch velocity of 30 and 10 μm s−1.The scratched length was set as 500 μm. 2.4 Drug loading and release profiles The encapsulation efficiency and loading efficiency of MMC by OS MPs were acquired by measuring the difference between the total mass of MMC added and the mass of unloaded MMC. After preparing MMC-loaded OS MPs, the unloaded MMC concentration of the supernatant and a series of resuspended UPW solution after centrifugation were measured using spectrophotometer against blank samples. The encapsulation efficiency and loading efficiency was calculated as follows: 29 Encapsulation efficiency (%) = (Wt-Ws) / Wt × 100%; Loading efficiency (%) = (Wt-Ws) / Wo × 100%, Where Wt is the initial mass of MMC and Ws is the total mass of MMC in the supernatants. Wo is the mass of MMC-loaded OS MPs after lyophilization. In addition, the amount of MMC in each sample was extracted with DMSO for several times until MMC was assuredly absent in the extracting solution. All results were assayed in



triplicate. As to evaluate the postoperative drug release profile as approximately as possible, a syringe pump method was used to simulate the drainage of aqueous humor through AGV.15 Assuming that 1ml of daily aqueous humor flow out of the drainage device which occupies a quarter quadrant of the eyeball.30 The minimum injection rate of syringe pump is 0.1 ml/h and the surface area of syringe filter (0.22 μm) is 450 mm2, which is approximately 2.4 times the parameter of AGV. According to the drug entrapment efficiency, the filter attached to the syringe was filled with the same multiples of MMC-loaded OS MPs. The suspension was kept for 24 hours at room temperature simulating MPs adsorption process before injected into the filter. The release medium collected in a sealed glass vial was detected by fluorescence spectrophotometer at 364nm every 24 hours. After each measurement, the release medium was collected in a new vial. The averaged results were based on three independent runs in the dark. 2.5 Biocompatibility studies 2.5.1 Cytotoxicity assay of OS MPs For direct contact tests, OS MPs coated silicone discs were placed at the bottom of each well. The test specimens were secured from floating by fixation with their appropriate size. About 3000 primary fibroblasts were seeded directly on to each test sample and cultured for 24 h in incubator (5% CO2, 37 °C). Live/dead viability/cytotoxicity Kit (Molecular Probes, Eugene, OR, USA) was adopted to quantify


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fibroblast viability. After discarding the culture media and washing solution, calcein acetoxymethyl (green) and ethidium homodimer-1 (red) working solution was gingerly instilled into each culture dish. After 20 min incubation (37 °C), the samples were observed under inverted fluorescence microscopy (TE-2000U, Nikon, Japan). Development of green color indicates viability, while red color symbolizes dead cells. For dose-response studies, the seeded primary fibroblasts with same amount per sample (5000 cells in each well) were incubated for 24 h (2% FBS). The medium was then changed for steriled OS MPs solution with different concentrations. After 24 h, the cell growth was then quantified with the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). After removing the OS MPs medium, CCK-8 (10% in DMEM, 100 μL) working solution was added and incubated for 1 h and measured at 450 nm. The cytotoxicity was leaned upon the percentage of cell viability by comparison with the blank control. All operations were accomplished at least four runs. 2.5.2 Inhibition effects of MMC-loaded OS MPs The cellular responses to MMC-OS MPs coated silicone discs were dissected with transwells. In this study, primary fibroblasts (104 cells/well) were cultured with DMEM containing 10% FBS. After fibroblasts sedimentation, transwell inserts loaded with silicone discs coating with MMC-OS MPs and OS MPs were located in each well and wholly swamped in the medium. Blank silicone disc was set as the control group. After 5 days of culture, fibroblast situation was demanded with live/dead stain and CCK-8 assay



as described above. All the averaged results were based on four independent runs. 2.6 Animal studies 2.6.1 In vivo studies with modified and empty device implantation A total of thirty-one adult New Zealand white rabbits (Laboratory Animal Research Center, the 2nd Affiliated Hospital of Harbin Medical University) housed under standard conditions, carrying weight at 2.5-3 kg with either gender, were used for this study. Either a single eye was chosen for surgery while the other was untreated. Baseline intraocular pressure (IOP) was registered prior to implantation after the Tono-Pen AVIA Applanation Tonometer (Reichert Technologies, NY) measurement. In the preliminary experiment, nine rabbits divided into three groups (n=3/group) were implanted with modified AGV with different drug loadings. The mass ratio of MMC/OS MPs was controlled in 1/10, 1/20, 1/40. Formation with lowest postoperative intraocular pressure was chosen for the formal experiment. Another eighteen rabbits were randomized for subsequent study (n=6/group). The experimental group was implanted with MMC loading AGV (AGV-OS MPs-MMC), while the control groups received either AGV coated with blank OS MPs (AGV-OS MPs) or normal AGV. Finally, the durability of OS MPs-MMC coatings on AGVs was checked on four rabbits for one month. SEM of retrieved device was assessed every week. The modified drainage devices for SEM analysis were fabricated under the same conditions. In brief, all animals underwent intravenous anesthesia with 3% pentobarbital sodium (1


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ml/kg). The eyeball was treated with 2% lidocaine eye drops after insertion of a lid speculum. Conjunctival dissection was made at the limbus to improve surgical exposure. The ethylene oxide-sterilized implant was initiated by infiltration with balanced salt liquid through the tube. The ocular implant was fastened to the superior-temporal episclera sector with a continuous non-absorbable suture approximately 5 mm to the corneal edge. Next, a scleral tunnel approaching to the anterior chamber (AC) was punctured (23 G) for tube insertion (3 mm). Finally, sew together the conjunctiva tightly. The animals were handled with prednisolone acetate and ofloxacin eyedrops 3 times a day for 10 days. 2.6.2 Clinical observation Slit-lamp examination and IOP measurement were conducted in preliminary experimental groups for 2 month. Afterwards, ophthalmic evaluations were assessed over 3 months by macroscopic inspection and anterior segment optical coherence tomography (AS-OCT) in formal experiment. The morphology of the anterior sector was inspected for signs of corneal edema, infection, tube placement and implant erosion by slit-lamp biomicroscopy. IOP was put down with the Tono-Pen AVIA Applanation Tonometer (Reichert Technologies) under superficial anesthesia. Three recordings per eye were averaged. To determine the inhibitory effects, inward bleb arrangement were detected with AS-OCT (CASIA; Tomey Corp., Nagoya, Japan) by a highly experienced examiner masked to the study. According to the coronal scanning methods previously reported,31,32



maximum and minimum value of bleb wall thickness was analyzed at 3.5-mm to the edge of outlet cannula with sufficiently exposure. Interval from the first conjunctival reflective signal to the upper side of the inferior drainage interspace was regarded as bleb wall thickness.32 2.6.3 Histological examination. Animals were sacrificed by intravenous injection of air after 3 months. Bleb surrounding tissue was carefully removed and subsequently immersed in 4% paraformaldehyde for 3 days at 4 °C. After paraffin embedding, sequential pathological sections (5μm) were successively observed with hematoxylin and eosin (H&E) and Masson’s trichrome to identify thickness of covered tissue layer in different regions. Immunohistochemically, to reveal the fibroblast differentiation and proliferation activity, the average optical density (AOD) of α-SMA and PCNA positive expression was performed by means of Image-Pro Plus analysis (Media Cybernetics, Silver Spring, MD, USA).33,34 All the pathological examinations were conducted by a pathologist who was blinded to the study. 2.7 Statistical analysis Data were reported as mean ± SD and analyzed with Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s test where appropriate. Two-way ANOVA with Bonferroni post-test was applied for two-factor variables. GraphPad Prism software (version 7.0) was used for the analysis. P0.05).



Figure 5. Cytotoxicity analysis of blank OS MPs. (a) Live/dead stain of HTFs and RTFs representing the cell viability status on surfaces of blank silicone disc and OS MPs coated silicone disc. Green and red fluorescence indicated live and dead fibroblasts, respectively. White arrows: the edge of silicone disc. Scale bars are all 50 μm. (b, c) Cell viability of HTFs and RTFs after cultured in different concentrations of OS MPs, respectively. No significant difference was found among each group. Error bars represent the mean ± standard deviation (n = 4). Inhibition effects of MMC-loaded OS MPs: After 5 days in culture, representative images of HTFs and RTFs were photographed as shown in Figure 6a. Fibroblasts in AGV-OS MPs-MMC group appeared shrinkage in shape and depletion in number while were healthy in other groups. Representative photographs of HTFs and RTFs labeled with live/dead stain also showed reduction of green fluorescence in AGV-OS MPs-MMC group compared to others (Figure 6b). The relative viability of primary fibroblasts in MMC released group significantly decreased along with the incubation time (P0.05).

Figure 6. Inhibition effects of MMC-loaded OS MPs in vitro. Representative phase-contrast micrographs and cell viability (live/dead stain) of HTFs (a) and RTFs (b) after a 5-day exposure to OS MPs and MMC-loaded OS MPs modified discs. Control: absence of silicone discs. Scale bars are all 100 μm. (c, d) Survival rate of HTFs and RTFs cultures after 5 days exposure to OS MPs and MMC-loaded OS MPs modified discs, respectively. Results are presented as percentage of controls without insertion of silicone discs. **P < 0.01 as compared to controls (n=4). 3.5 Animal studies in vivo Ophthalmic Clinical Evaluation: As illustrated in Figure 7a, the group with MMC/OS MPs in 1/10 had significantly reduced the IOP at 7 days (P=0.0097 to 1/20 group, P=0.0397 to 1/40 group) and 60 days (P=0.0397 to 1/20 group ). No serious side



effects were found in each group. Therefore, MMC/OS MPs mass ratio in 1/10 was chosen for subsequent studies. The safety and efficacy of MMC-loaded AGV were studied in vivo for a period of 3 months. All the implanted devices were well tolerated with no sign of filtering bleb leakage, implant or tube extrusion or other serious complications. The most frequent side effects occurred include the chemosis and minimal flare in the anterior chambers in the early stage of post-operation. Besides, conjunctival edema was macroscopically observable in the AGV group after implantation. These surgical-related irritation symptoms gradually declined in all the eyes within 1 week. All 18 eyes of rabbits had a baseline IOP of 13.3 ± 1.5 mmHg (mean ± SD). Figure 7b shows the IOP changes versus follow-up time among different groups. A significant and rapid IOP reduction 1 d after implantation was observed. There was no significant difference in IOP at each time gap between AGV-OS MPs group and AGV group during the entire study period (P>0.05). In contrast, IOP was found to be significantly decreased at 1 day, 1 and 3 months postoperative in the AGV-OS MPs-MMC group relative to the AGV group (P

Glaucoma Drainage Device Coated with Mitomycin C Loaded Opal

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