Abstract 1742 Enhanced Cargo Loading into Extracellular Vesicles via Gold Nanoparticles for Precision Gene Therapy

Gene therapy is an emerging and powerful therapeutic tool for treating diverse diseases via functional genetic material delivery to targeted defective genes in cells. Extracellular vesicles (EVs) are natural carriers released by all living cells to transfer signaling molecules, which are gaining attention for usage in advancing efficiency of therapeutic delivery due to their various advantageous properties, including (i) cargo protection from enzymatic degradation, (ii) tissue-specific targeting, and (iii) high biocompatibility. EVs can be isolated from a targeted set of cells and used for transferring genes as the therapeutic delivery back to specific tissue sites. One of the leading methods for transfecting genes and cargos into EVs for therapeutic delivery is electroporation, which utilizes short-duration, voltage pulses to create temporary pores on the EV surface membrane, during which the cargo can enter. This process is limited by the maximum voltage EVs can withstand for pore formation and cargo encapsulation. We specifically use microfluidic electroporation, which utilizes water-in-oil droplet encapsulation of EVs, which we have previously observed to be the most efficient in loading. We aim to increase efficiency of gene therapy loading into EVs using gold nanoparticle (AuNP) enhanced electroporation to provide concentrated, targeted, and protected gene delivery. It has been shown that AuNPs can increase the loading into cells during electroporation by allocating the electric voltage onto the cell surface, allowing more pore formation without increasing the total voltage applied. Due to similar surface properties, we hypothesized that AuNPs would provide a similar increase in transfection of EVs. To test this hypothesis, we electroporated green fluorescence protein (GFP) into EVs with the introduction of AuNPs at various concentrations. Fluorescence analysis through Cytation 5 and EV concentration measurements with Nanoparticle Tracking Analysis (NTA) revealed a close to double increase in the GFP molecules per EV with the AuNP incorporation. Assessment with NTA and Transmission Electron Microscopy showed that the AuNPs did not significantly change the size, concentration, zeta potential, or morphology of the EVs, compared to the native EVs without electroporation, while retaining the EV's originality and integrity. Overall, AuNPs have the capacity to increase cargo transfection into EVs, allowing high-efficiency packaging for specific therapy delivery to enhance effectiveness of treatment. This research was funded by the Center for Undergraduate Research's Emerging Scholars Program at the University of Florida as well as the National Institute of Health's Undergraduate Research Fellowship Program.