Gold Nanoparticle (GNP) Enhanced Electroporation of Extracellular Vesicles to Promote Gene Loading

Abstract

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, 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 therapeutic delivery as they can travel back to the same cell type due to their unique surface proteins and markers to release the genes at the specified site. One of the leading methods for cargo transfection into EVs is electroporation, which utilizes short-duration voltage pulses to create pores on the EV surface membrane, during which the cargo can enter. The voltage creates a potential difference between the less electrically conductive EV membrane and the more electrically conductive internal and external EV environments leading to the formation of these pores. This process is limited by the maximum pulse amplitude and duration the EVs can withstand for maximum pore formation and cargo encapsulation while still permitting membrane recovery afterward. We specifically use microfluidic electroporation, which we have previously found to be the most efficient in loading. This technique utilizes water-in-oil droplet encapsulation of the EVs which are then electroporated by voltage application as they pass through parallel metal electrodes. As the droplets pass through the microfluidic device, the non-conductive oil phase creates a barrier allowing for specific consistent and short-pulsed voltage delivery for each EV-encapsulated conductive water droplet [1]. We aim to increase efficiency of gene therapy loading into EVs using gold nanoparticle (AuNP) enhanced electroporation to provide higher concentration, targeted, and protected gene delivery. It has been shown that AuNP can increase the loading into cells during electroporation by allocating the electric voltage onto the cell surface creating a pulse strength focusing effect, allowing more pore formation without increasing the total voltage applied [2]. 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 measurement 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, for retaining the EV 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 the treatment.[1] Geng, T., & Lu, C. (2013). Microfluidic electroporation for cellular analysis and delivery. Lab Chip, 13(19), 3803–3821. https://doi.org/10.1039/c3lc50566a[2] Zu, Y., Huang, S., Liao, W.-C., Lu, Y., & Wang, S. (2014). Gold Nanoparticles Enhanced Electroporation for Mammalian Cell Transfection. Journal of Biomedical Nanotechnology, 10(6), 982–992. https://doi.org/10.1166/jbn.2014.1797 Figure 1