Functionalizing Solvent-Cast Chitosan/Polycaprolactone Membranes for Artificial Corneal Replacement

Introduction: Corneal transplantation from cadaver donors is the primary treatment for corneal blindness caused by injury, infection, or disease.[1] However, less than 2% of the global demand for cornea donor tissue is being met. Corneal allografts can also result in immunological rejection, motivating the need to develop synthetic biomaterial-based corneal replacements.[1,2] To address this, we have developed a cornea-like membrane scaffold composed of poly(caprolactone) (PCL) and chitosan (CHI) crosslinked with genipin. CHI has been used in engineering cornea but is mechanically weak.[3] Blending CHI with PCL addresses this limitation though additional functionality is required to improve cell-material and tissue-material interactions. Here, we fabricated membranes using poly(ethylene glycol) (PEG)-PCL and RGDS-PCL conjugate to prevent biofouling and enhance cell adhesion, respectively. We also optimized methods to crosslink membranes to improve mechanical stability and demonstrate that we can functionalize the membranes with PEG and RGDS without compromising mechanical properties or transparency.

Materials and

Methods: CHI was dissolved at 1% w/v in formic acid, stirred for 24 hours, and filtered using a 0.2 µm filter. PCL was dissolved at 10% w/v in formic acid and stirred for 1 hour. PEG-PCL and RGDS-PCL conjugates, synthesized with RGDS(biotin) using established methods, were separately added to the PCL solution at 1 or 5 mg/mL.[4] The CHI and PCL-based solutions were mixed at a 90:10 CHI:PCL volume ratio for 15 minutes. Genipin was added at 10 or 100 µg/mL to the CHI:PCL solution and stirred for 3 hours before casting into glass petri dishes. Membranes were formed via formic acid evaporation in a vacuum oven at 55ºC and 760 mmHg for 24 hours. Samples were punched into 8-mm circles for transparency measurements and cut into 40 mm x 7.5 mm strips for tensile testing. All samples were hydrated in phosphate-buffered saline (PBS) for 1 and 7 days before characterizing. Absorbance was measured using a SpectraMax iD3 microplate reader from 300 to 800 nm and converted to percent transmittance using the equation: % Transmittance=((1/10^Absorbance)(100)). Tensile properties were measured using a Zwick Roell tensile tester with a 100 N load cell at a 10 mm/min crosshead speed. Sample dimensions were measured using a micrometer and multiplied by a swell factor based on the width of dry and hydrated membranes to determine the cross-sectional area for converting max force (N) to max stress (MPa). A one-way ANOVA was performed to compare the membrane mechanical properties of each genipin and PEG-PCL concentration.

Results, Conclusions, and Discussions: CHI-PCL membranes were successfully fabricated for optical and mechanical testing. Membranes crosslinked with 100 µg/mL showed significantly higher max stress compared to 0 and 10 µg/mL genipin on Day 1 and compared to 0 µg/mL on Day 7 (Figure 1). Crosslinking with 100 µg/mL genipin caused a slight decrease in transparency on Days 1 and 7 (Figure 2). However, the transmittance was higher than that of eye bank cornea, ranging from approximately 50-70% from 400 to 700 nm.[5] Overall, membranes crosslinked with 100 µg/mL genipin exhibited a higher average max stress and < 60% transmittance from 300 to 800 nm on Day 1 and 7. PEG-PCL surface functionalized membranes did not compromise mechanical integrity and transparency on Day 1 (Figure 3). This study demonstrated that crosslinking CHI-PCL membranes with genipin improved mechanical stability without sacrificing transparency. Integrating PEG-PCL did not adversely affect the mechanical integrity and transparency. Ongoing work includes characterizing the mechanical properties and transparency of membranes containing RGDS-PCL conjugates and demonstrating that PEGylation prevents protein absorption and cell adhesion while RGDS enhances cell adhesion with corneal epithelial cells.

References:
1. Williams K. A., et al., Discovery Medicine, 2013
2. Moshirfar M., et al., Clinical Ophthalmology, 2021;15:2963-2973
3. Azevedo J. V., et al., Polymer International, 2013;62(10):1425-1432
4. Camacho P., et al., Biomaterials Science, 2019;7(10):4237-4247
5. Liu Y., et al., Invest. Ophthalmol. Vis. Sci., 2006;47(5):1869-1875

Acknowledgements (Optional): The authors acknowledge support from the National Science Foundation (NSF) SBIR Award (2342532 to AZ) and the Pennsylvania Infrastructure Technology Alliance (PITA) Award (C000086514 (PIT-24-08) to LWC) from the Pennsylvania Department of Community and Economic Development.