Development of Cell Delivery System for the Subretinal Space Using Biodegradable Nanosheets

Abstract

Age-related macular degeneration (AMD) is a major ophthalmic disease that causes visual impairment and blindness, particularly in elderly people. The developing choroidal vessels invade Bruch’s membrane and disrupt the monolayer of retinal pigment epithelial (RPE) cells. Subsequently, degeneration of photoreceptors occurs. Hence, subretinal transplantation of RPE cells to the site of degeneration would offer an ideal treatment providing the transplanted cells could generate a monolayer, reconstruct the RPE–photoreceptor interface, and inhibit further development of choroidal vessels. Although transplantation of autologous peripheral RPE cells has been tested by injection of cell suspensions using a syringe needle, limited visual improvement result due to the low viability of the injected cells, and their restricted distribution and integration into the subretinal tissue.Recently, we investigated the use of biodegradable polymeric nanosheets as cell scaffolds [1,2]. Because this system enabled the easy manipulation of the cell/nanosheet constructs with a syringe needle, it raises the possibility of transplanting cell sheets into a narrow space, such as the subretinal space, in a minimally-invasive manner. So far, poly(lactic-co-glycolic acid) (PLGA) was used as the material for the nanosheets. However, this biodegradable polymer is relatively inflammatory because it produces lactic and glycolic acids as degradation intermediates and these acid dissociation constants are about 3.8. In this study, we fabricated biodegradable nanosheets using polycaprolactone (PCL) and collagen as low inflammatory materials and investigated a minimally invasive and effective method for cell transplantation into the subretinal space (Fig.1(a)).Micropatterned nanosheets consisting of PCL, collagen and Nile red were prepared by a combination of spin-coating and micro-contact printing technique (Fig.1(b)). The diameter and thickness of the nanosheets used for the following experiments were 200 μm and 200 nm, respectively. The cell/nanosheet constructs were prepared by culturing rat retinal pigment epithelial (RPE-J) cells on the micropatterned nanosheets in a monolayer. A hand-made glass capillary needle was as thick as a 27G medical needle, and the tip angle was 30 degrees. Also, it was coated with 2-methayloxyethyl phosphorylcholine (MPC) to prevent cells from attaching the inner surface of the needle. The cell/nanosheet constructs and a sodium hyaluronate solution with the medium were loaded into the capillary needle. Subsequently, the cell/nanosheet was injected into the subretinal space through the sclera of rat. Several days after implantation, the rats were euthanized, and the eyeballs were enucleated.The micropatterned nanosheet was shown in Fig.1(c). The nanosheets had sufficient strength and flexibility to be handled in water. The shape or diameter of the nanosheets could be varied by adjusting the microstructure of the PDMS stamp. Also, the thickness of the nanosheets could be controlled by changing the concentration of the solution. As shown in Fig.1b, the cells formed a monolayer on the nanosheet of PCL/collagen. Finally, we attempted to deliver the RPE-J cell/nanosheet constructs to the subretinal space of a rat eye in vivo. The hand-made glass capillary needle shape allowed the needle insertion along the retina through the sclera. The fluorescence of PCL/collagen nanosheet and cells could be observed in the eyeballs under excitation light. Also, as shown in Fig.1(e), it was confirmed that the cell/nanosheet construct was successively delivered into the subretinal space.We fabricated biodegradable nanosheets using PCL and collagen as low inflammatory materials. Also, we developed a minimally invasive and effective method for cell transplantation into the subretinal space using capillary needles and confirmed that cell/nanosheet was successfully delivered. This method holds great promise for transplantation of monolayer cells and for the development of local cell delivery systems. In order to further improve this method, we plan to evaluate the inflammatory properties of the nanosheets and the efficacy of the delivered cells.