Functionalized Engineered Extracellular Vesicles for targeted delivery to Intervertebral Disc Cells

Introduction: Chronic low back pain is a leading cause of disability worldwide, with intervertebral disc (IVD) degeneration being a major contributing factor [1]. The healthy IVD facilitates motion of the spinal column, and it is composed of a gelatinous core rich in proteoglycans and glycosaminoglycans known as the Nucleus Pulposus (NP) surrounded by an exterior comprised of concentric type I collagen rings known as the Annulus Fibrosus (AF). Despite its prevalence, safe and non-addictive treatment options for chronic low back pain are limited. To address this issue, we propose a novel approach that involves loading engineered extracellular vesicles (eEVs) with key developmental transcription factors (TFs) to promote a healthy, pro-anabolic phenotype in degenerate human NP and AF cells, and functionalizing them with AF- or NP-specific transmembrane ligands. This will facilitate targeted delivery to either NP or AF cells, potentially offering a promising treatment option for chronic low back pain.

Materials and

Methods: In vitro-derived eEVs loaded with the developmental TFs FOXF1 and Brachyury (T) or Mohawk (MKX) and Scleraxis (SCX) and functionalized with Netrin-1 to target NP cells or with CD11a to target AF cells, were isolated from primary human dermal fibroblasts from adult donors following electroporation [2-4]. The functionalized eEVs were characterized via Nanoparticle Tracking Analysis (NTA) to determine EVs size distribution and concentration, Super resolution microscopy to confirm the presence of the ligands on their membrane surface, and qRT-PCR to verify packing of the therapeutic TFs. Fluorescently labeled eEVs were used to characterize preferential uptake by diseased AF and NP cells isolated from human patient tissue (IRB 2015H038). The therapeutic effect of TFs was assessed through quantification of collagen and glycosaminoglycan content of degenerate human NP cells (3D hydrogel) after exposure to non-functionalized FOXF1+T eEVs in vitro and RNA sequencing of human AF cells (3D Nanofiber) after exposure to non-functionalized MKX+SCX eEVs in vitro.

Results, Conclusions, and Discussions: Our results demonstrate the potential of using eEVs loaded with key developmental TFs and functionalized with NP- or AF-specific ligands for targeted delivery to the NP and AF regions in the IVD. Characterization of the functionalized eEVs using NTA, Western blot, qRT-PCR, and Super Resolution microscopy showed the successful loading of developmental transcription factors and ligands on the eEV membrane after isolation (Fig1A-B). Confocal microscopy of co-cultures treated with eEVs indicate HDF derived eEVs loaded with FOXF1+T or MKX+SCX and functionalized with CD11a or Netrin-1 may be preferentially internalized by NP or AF cells, respectively (Fig.1D-E) while non-functionalized EVs were non-specifically captured by both NP and AF cells. Additionally, our data showed successful reprogramming of 3D cultured NP and AF cells to a healthier, pro-anabolic phenotype following exposure to non-functionalized eEVs loaded with developmental factors, as evidenced by increased collagen and glycosaminoglycan content in NP cells and decrease in immunologic/inflammatory responses and increase in matrix remodeling of AF cells (Fig1.C,F).

Our research has demonstrated the potential of functionalized eEVs as an efficient and selective delivery system to deliver developmental TFs to degenerate NP or AF cells, resulting in the reprogramming of both cell types from the IVD towards a healthier, pro-anabolic phenotype. These findings underscore the significance of advancing research and development of eEV-based nanocarriers to address IVD degeneration and chronic low back pain.

Acknowledgements (Optional):