Materials

Poly(D, L-lactic-co-glycolic acid) (PLGA; 50:50; i.v. = 0.32–0.44 dl/g) was purchased from Evonik (Germany). Hexafluoroisopropanol (HFIP) was purchased from Sigma Aldrich (St. Louis, MO). Sunitinib malate (> 99%) was purchased from LC Laboratories (Woburn, MA). Disposable syringes, 1x Dulbecco’s Phosphate-Buffered Saline (PBS), a High Capacity cDNA Reverse Transcription Kit, and TRIzol® were purchased from Thermo Fisher Scientific (Waltham, MA). 20 and 25 G blunt tip needles were purchased from Nordson EFD (East Providence, RI). Dimethyl sulfoxide (DMSO), acetonitrile, water and ethanol at analytical grade or above were purchased from Sigma Aldrich (St. Louis, MO). 10 − 0 nylon sutures were purchased from Alcon Laboratories (Fort Worth, TX).

Fabrication of nano- and micro-fiber sunitinib thin films

To prepare nanofiber thin films, PLGA was dissolved in HFIP at 20% w/w and then electrospun using a custom-built electrospinning system for 120 min at a flow rate of 250 µL/hr through a 25 G blunt tip needle, with an applied voltage of 10.5 kV at a distance of 24 cm from a grounded collector [17,18,19,20,21,22]. PLGA/sunitinib thin films were manufactured under the same conditions at a 2% sunitinib malate and 20% PLGA concentration (w/w).

The weight% of PLGA was increased to 30% in HFIP to increase fiber diameter. To prepare microfiber thin films, PLGA was dissolved in HFIP and then electrospun for 20 min at a flow rate of 1 mL/hr through a 20 G blunt tip needle, with an applied voltage of 6.5 kV, and at a distance of 13 cm from a grounded collector. Microfiber PLGA/sunitinib thin films were manufactured similarly at a 3% sunitinib and 30% PLGA concentration (w/w).

Morphological analysis

Nanofiber thin film morphology was examined using scanning electron microscopy (SEM). Thin films were sputter coated with 10 nm of Au/Pd prior to imaging at 1.5 kV on a LEO Field Emission SEM (Zeiss, Germany). Thin film thickness (n = 3, each) was measured via reflectance imaging on an LSM 510 Meta Confocal Microscope (Zeiss).

Tensile strength measurement

Thin film breaking strength was evaluated using a DMA 6800 (TA Instruments, Timonium, MD). PLGA and PLGA/sunitinib thin films (n = 5, each) were cut to 12 mm in width, clamped vertically at a length of 4.3 mm, and stretched at a force of 0.6 N/min until breaking.

Analysis of thin film permeability

Water vapor flux was used as a surrogate for corneal oxygen permeability and surface hydration. Thin film permeability was analyzed using Elcometer 5100/1 Payne permeability cups with a surface area of 10 cm2 (Elcometer, Inc., Rochester Hills, Michigan). The cups were filled with 5 mL of ultrapure water after which the PLGA and PLGA/sunitinib films (n = 3, each) were fitted to the top of the cup. The cup was then weighed and placed in a desiccator at 37 ⁰C for the duration of the experiment. The weight of each cup was measured every hour for 5 h and then at 24 h to determine permeability.

Drug loading and in vitro drug release

Sunitinib thin films were weighed and dissolved in DMSO. The solution was measured by UV-Vis at 441 nm on a BioTek Microplate Reader (Winooski, VT). The sunitinib drug concentration was calculated using a standard curve of sunitinib malate in DMSO. The drug loading (DL) and encapsulation efficiency (EE) were calculated as follows:

$$\:DL\:\left(\%\right)=\:\frac\text\text\text\text\text\text\text\text\:\text\text\text\text\text\text\:in\:thin\:film}$$

$$\:EE\:\left(\%\right)=\:\frac$$

To study the in vitro drug release profile of sunitinib thin films, 1–2 mg thin films were weighed and placed in 1.5 mL siliconized tubes. Tubes were placed on an orbital shaker in a 37 °C incubator and shaken at 120 rpm. At predetermined time points, the release media was collected and replaced with 1 mL fresh PBS. The concentration of sunitinib malate in the collected release media was measured by UV-Vis and calculated using a standard curve for sunitinib malate in PBS.

Animals

All animals were cared for in accordance with protocols approved by the Johns Hopkins University Animal Care and Use Committee, the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the National Institutes of Health guide for the care and use of laboratory animals. Male Sprague Dawley rats (6–8 weeks old) were purchased from Harlan (Indianapolis, IN). The animals were anesthetized with intramuscular injection of a mixture of ketamine (50 mg/kg) and xylazine (5 mg/kg) during experimental procedures. Topical instillation of 0.5% proparacaine and 0.5% tropicamide were used for topical anesthesia and pupil dilation, respectively. Procedures were conducted only on one eye in each rat.

Assessment of thin film biocompatibility

A 1 cm x 1 cm thin film (weight ~ 1.7–1.8 mg) was placed on the corneal surface and retained via four 10 − 0 silk suture stitches to the conjunctiva. An Elizabeth cone was attached after thin film placement to prevent removal by the rats. The animals had free access to food and water during the studies. For the biocompatibility study, films were placed for 7 days after which the thin films were removed under anesthesia and eyes imaged under slit lamp after fluorescein staining. Rats were then euthanized and eyes enucleated, fixed with 10% formalin, and embedded in paraffin. The paraffin sections were cut through the papillary optic nerve plane and stained with hematoxylin and eosin (H&E) for histological examination. Sections were examined by a masked pathologist.

Evaluation of corneal neovascularization Inhibition

Corneal neovascularization was induced by intrastromal suturing, as described previously [23, 24]. In brief, rats were anesthetized, and their pupils were dilated prior to placing two intrastromal 10 − 0 nylon suture stitches in the superior cornea under an operating microscope. The distance between the stitches and the limbus was approximately 2 mm and there was a distance of 1 mm between the two stitches. After suturing, animals were randomly assigned to four treatment groups: (1) topical instillation of PBS (10 µL, 3 times per day), (2) topical instillation of sunitinib malate free drug solution (5 mg/mL, 10 µL, 3 times per day), (3) topical application of placebo thin film (1 cm x 1 cm, weight ~ 1.7–1.8 mg, 1 time) and (4) topical application of sunitinib-eluting thin film (1 cm x 1 cm, weight ~ 1.7–1.8 mg, containing ~ 0.15 mg Sunitinib, 1 time), as shown in Fig. S1A. An Elizabeth cone was attached after thin film placement for rats receiving topical application of placebo and sunitinib-eluting thin films (Fig. S1B). Each group was comprised of n = 6 rats for a total of 6 experimental eyes. The rats were evaluated for 7 days. Afterwards, the corneas of all rats were examined by slit-lamp biomicroscope (SL120; Carl Zeiss AG, Oberkochen, Germany) and corneal photographs were taken with a digital camera by an ophthalmic photographer in a masked manner. The area and length of vascularized cornea were quantified with ImageJ software using previously described methods [23, 24]. An arc was drawn along the limbus, and the corneal neovascularization (NV) area was calculated using the following equation:

$$\:Corneal\:NV\:area=\:\frac\text\text\text\text\:\text\text\:\text\text\text\text\text\text\text\text\text\text\text\text\:\text\text\text\text\:\:}^\:area}$$

The vascularized area was evenly divided into six sections. The distance between vessel tips and the limbus at the five intersection points of the arc was measured. The five measured lengths were averaged to calculate the corneal NV length. These measurements were carried out by masked graders. At the endpoint of 7 days after treatment, three rats were sacrificed and the eyeballs were enucleated and processed for histological examination, as described before. Sections were examined by a masked pathologist. The other three rats in each group were euthanized and the corneas were collected for qPCR. Because of the limited amount of corneal tissue from individual eyes, three corneas of the same condition were pooled together to collect sufficient tissue for mRNA isolation and measurements. The mRNA expression levels of angiogenic and anti-angiogenic factors including VEGF, VEGFR1, VEGFR2, PDGFRα, PDGFRβ, VE-cadherin, Ang1, MMP2, MMP9 were quantified by RT-PCR with Fast SYBR® Green Master Mix using a 7100 Real Time PCR System (Applied Biosystems, CA). The primers are listed in Table S1. The mRNA expression levels were normalized to GAPDH. Each sample was repeated 3 times for the mRNA expression level quantification.

Statistical analysis

Thin film thickness, strength, permeability, and in vitro drug release are presented as mean ± standard error. Animal study data are presented as the average ± standard error of the mean (SEM). Two groups were compared using two-tailed Student’s t-test and three or more groups were compared using one-way ANOVA followed by Tukey’s post-hoc test. Differences were considered to be statistically significant at a level of p < 0.05. Significance for multiple comparisons: *p < 0.05; **p < 0.01; ***p < 0.001.