Biomimetic Collagen Membranes Functionalized with mRNA‐loaded Lipid Nanoparticles for Fibroblast Transdifferentiation to Endothelial Cells

Non-healing wounds are a common complication from chronic disorders such as diabetes, cardiovascular diseases and cancer, among others. Impaired vascularization during the early stages of the wound healing process is a major limitation to tissue repair, and contributes to the chronification of the injury, characterized by the deposition of fibrous tissue by fibroblasts. Targeted therapeutics aimed at enhancing vascularization at the site of injury by transdifferentiating fibroblasts to endothelial cells (EC) have the potential to promote healing and to prevent the wound from becoming chronic. Here we present a collagen membrane functionalized with phosphatidylserine (PS)-containing lipid nanoparticles (LNP) that encapsulate mRNA encoding for proteins known to drive fibroblast transdifferentiation into EC (GATA2, ETV2, FLI1, KLF4). The nanoscopic particles we developed (~70 nm in size) show highly efficient RNA encapsulation rates (over 70%) and can be taken up by fibroblasts at rates similar to other conventional transfection methods such as lipofectamine (up to 40%). Gene expression of the delivered mRNA is also significantly increased in fibroblasts treated with our LNP. Collagen membranes were functionalized with Annexin V, a protein that binds the lipid PS, as an approach to attach our LNP onto the membranes and enhance their retention. Our results also show a successful functionalization of the collagen membranes with Annexin V and our LNP. These membranes could potentially be used as implants to promote in situ wound healing by acting as a niche whereby local fibroblasts are transdifferentiated to EC via uptake of the mRNA-loaded LNP to enhance vascularization and expedite tissue repair. The biomimetic approach we propose here represents a novel alternative to the development of RNA therapeutics to concomitantly encapsulate and deliver ad hoc multiple RNAs, while protecting them from degradation and making it possible for them to exert specific biological functions in target cells. Furthermore, the versatility of these mRNA-LNP formulations provides the foundation for targeting a variety of chronic diseases, with the potential of improving patient quality of life.