Bruch's-Mimetic Nanofibrous Membranes Functionalized with the Integrin-Binding Peptides as a Promising Approach for Human Retinal Pigment Epithelium Cell Transplantation

Abstract

Background: This study aimed to develop an ultrathin nanofibrous membrane able to, firstly, mimic the natural fibrous architecture of human Bruch's membrane (BM) and, secondly, promote survival of retinal pigment epithelial (RPE) cells after surface functionalization of fibrous membranes.

Methods: Integrin-binding peptides (IBPs) that specifically interact with appropriate adhesion receptors on RPEs were immobilized on Bruch's-mimetic membranes to promote coverage of RPEs. Surface morphologies, Fourier-transform infrared spectroscopy spectra, contact angle analysis, Alamar Blue assay, live/dead assay, immunofluorescence staining, and scanning electron microscopy were used to evaluate the outcome.

Results: Results showed that coated membranes maintained the original morphology of nanofibers. After coating with IBPs, the water contact angle of the membrane surfaces varied from 92.38 ± 0.67 degrees to 20.16 ± 0.81 degrees. RPE cells seeded on IBP-coated membranes showed the highest viability at all time points (Day 1, p < 0.05; Day 3, p < 0.01; Days 7 and 14, p < 0.001). The proliferation rate of RPE cells on uncoated poly(ε-caprolactone) (PCL) membranes was significantly lower than that of IBP-coated membranes (p < 0.001). SEM images showed a well-organized hexa/polygonal monolayer of RPE cells on IBP-coated membranes. RPE cells proliferated rapidly, contacted, and became confluent. RPE cells formed a tight adhesion with nanofibers under high-magnification SEM. Our findings confirmed that the IBP-coated PCL membrane improved the attachment, proliferation, and viability of RPE cells. In addition, in this study, we used serum-free culture for RPE cells and short IBPs without immunogenicity to prevent graft rejection and immunogenicity during transplantation.

Conclusions: These results indicated that the biomimic BM-IBP-RPE nanofibrous graft might be a new, practicable approach to increase the success rate of RPE cell transplantation.

Keywords: Bruch’s membrane; cell adhesion; electrospinning; immunogenicity; integrin; nanofibers; peptides; retinal pigment epithelial transplantation.

Figure 1

Surface characterization of PCL and IBP-coated nanofibrous membranes. (A) PCL nanofibrous membranes, ×300; (B) PCL nanofibrous membranes, ×1.00k; (C) PCL nanofibrous membranes, ×10k; (D) IBP-coated nanofibrous membranes, ×300; (E) IBP-coated nanofibrous membranes, ×1.00k; (F) IBP-coated nanofibrous membranes, ×10k. SEM images show morphologies of samples with random fibrous orientation and variably sized pores. Average diameter of IBP-coated membranes is not significantly different from uncoated PCL membranes, and pore sizes of IBP-coated membranes were also similar to uncoated PCL membranes. Distribution of fiber diameters for PCL (G) and IBP-coated (H) nanofibrous membranes. Pore sizes for PCL (I) and IBP-coated (J) nanofibrous membranes.

Figure 2

Contact angle analysis was performed to determine wettability of different membranes, including (A) PCL and (B) IBP-coated membranes. (C) Result indicated that hydrophilicity of PCL membranes was improved with IBP coating.

Figure 3

Fourier-transform infrared spectra (FTIR) of PCL membranes, IBP powder, and IBP-coated PCL membranes.

Figure 4

Biocompatibility of PCL, IBP-coated membranes, and TCP to ARPE-19 cells. (A) ARPE-19 cell morphology after growing on different groups for 1, 3, 7, and 14 days observed by laser scanning confocal microscopy. ARPE-19 cells were treated with live/dead viability test, and on Day 14, cells actin filament and nuclei were stained with AlexaFluor 488/546 Phalloidin and DAPI, respectively. Scale bars represent 200 μm. (B) ARPE-19 cells attached to different groups at comparable densities on Days 1, 3, 7, and 14 by Alamar Blue assay. ARPE-19 showed highest cell activity on IBP-coated membranes. Significant difference is denoted by: *** = p < 0.001, ** = 0.001 ≤ p ≤ 0.01, * = 0.01 ≤ p ≤ 0.05.

Figure 5

SEM images of RPE cells on the IBP-coated PCL nanofibrous membranes. (A–D) RPE cells growth on membranes on Days 1, 3, 7, and 14. The RPE cells formed a striking in vivo-like regional monolayer on Day 14 (D). With the help of integrin-binding peptides, the RPE cells formed a very tight connection with nanofibers (E–H), and the red arrows indicated the tight junctions.

Scheme 1

Fabrication of biomimic, IBP-coated, BM-mimicking nanofibrous membranes.