(L-460) Functionalized Engineered Extracellular Vesicles for targeted delivery to Interverbal 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 concentrical 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 or Mohawk (MKX) 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 Nanosight to determine EVs size distribution and concentration, Western Blot and ELISA to confirm the presence of the functionalizing 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 qRT-PCR analysis of catabolic, inflammatory, and pain markers in degenerate NP (3D hydrogel) after exposure to eEVs in vitro and AF cells (2D monolayer) after direct transfection. Upregulation of desired genes was assessed after exposing NP and AF cells to CD11a or Netrin-1 functionalized eEVs loaded with FOXF1 or MKX respectively, compared to non-functionalized eEVs.


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 Nanosight, Western blot, ELISA, and qRT-PCR showed the successful loading of developmental transcription factors and ligands on the eEV membrane after isolation. eEVs loaded with FOXF1 or MKX and functionalized with CD11a or Netrin-1 were preferentially internalized by NP or AF cells, respectively (Fig.1) with increased cargo gene expression for FOXF1 (in NP cells) or MKX (in AF cells), while non-functionalized EVs were non-specifically captured by both NP and AF cells. Additionally, our data showed successful reprogramming of NP and AF cells to a healthier, pro-anabolic phenotype following exposure to non-functionalized eEVs or bulk transfection of developmental factors, as evidenced by upregulation of SOX9 and downregulation of MMP2 and Biglycan in AF cells, and increased FOXF1 and Brachyury expression in NP cells.

Our research has demonstrated the potential of functionalized eEVs as an efficient and selective delivery of 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): :

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