(WHS-I.06) SHELL OR THE CARGO? SIGNIFICANCE OF KERATINOCYTE-DERIVED EXOSOMES SURFACE MOLECULES IN TISSUE REPAIR
Wednesday, May 15, 2024
1:45 PM – 4:00 PM East Coast USA Time
Background – Exosomes, endocytically originated extracellular vesicles, play a pivotal role in cellular communication, encapsulating a myriad of biomolecules like DNA, proteins, and metabolites. This study posits that an in-depth analysis of the unique surface composition of exosomal is critical to unravel the intricacies of wound healing. Methods – Keratinocyte-derived exosomes were genetically labeled with GFP-reporter (Exoκ-GFP) using tissue nanotransfection (TNT). Exoκ-GFP were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Wound-edge keratinocyte-derived exosome uptake was blocked in mice by generating “eat me not” Exoκ-GFP-RFP using a KRT-14 promoter-driven tetraspanins plasmid connected via IRES element with “eat me not”-CD47 sequence with in-frame GFP and RFP reporter (Exoκ-GFP-RFP). Keratinocyte-targeted nanocarriers (TLNκ) were designed using pH-responsive lipid components with keratinocyte-targeting peptide sequence ASKAIQVFLLAG and loaded with siRNA of hnRNP to selectively inhibit cargo packaging within Exoκ-GFP. The isolated exosomes was characterized as per MISEV 2018 guidelines and by flow cytometry. Additionally, the surface of these exosomes was studied by various spectroscopic techniques such as Raman and FTIR measurements. Functional wound closure was evaluated using analytical histology and Transepidermal Water Loss (TEWL). Results – Our results demonstrate that both compromising cargo packaging within Exoκ-GFP without compromising uptake by blood borne wound macrophages and inhibiting Exoκ-GFP uptake results in the persistence of proinflammatory macrophages at the wound site at day 12 (n=6, n=6). The macrophages exhibited high expression of iNOS (n=4, p<0.001; n=5, p<0.001). However, although no significant difference in re-epithelialization was observed compromising cargo packaging, “eat me not” Exoκ-GFP-RFP significantly compromised wound reepithelialization (n=6, p<0.001). Surface N-glycan analysis demonstrated a high abundance of mannose on Exoκ-GFP-RFP. Raman and FTIR spectroscopic analyses demonstrated spectral differences across the protein, lipids, and nucleic acid domains. Conclusion – The findings from this research highlight the critical role of exosomal surface molecules in various physiological and pathological conditions. Expanding research on exosomal surfaces is essential for a deeper understanding of the complex healing processes at sites of injury.