Optimizing miRNA Loading into Platelet Exosomes via Electroporation: A Comparative Analysis of Buffer Systems and...

Exosomes, which transport miRNAs in vivo, hold significant therapeutic potential for treating diseases. Current methods for loading miRNAs into exosomes include sonication, co-incubation, kit-based transfection, and electroporation, with electroporation being the most efficient and widely used approach. However, standardized protocols for electroporation conditions remain lacking, necessitating the optimization of electroporation parameters to enhance the utility of extracellular vesicles as drug delivery vehicles in vivo. Platelets were isolated from healthy volunteer blood donors, and platelet-derived exosomes were extracted. The exosomes were labeled with specific dyes and loaded with miRNA using Bio-Rad Gene Pulser Electroporation buffer or 50 mM trehalose. Electroporation was performed at 150 V, 350 V, and 500 V. The miRNA-loaded exosomes were then co-incubated with cells. The efficiency of miRNA delivery was evaluated through fluorescence co-localization, nanoparticle tracking analysis, and qPCR. Our findings demonstrate that the Bio-Rad Gene Pulser Electroporation buffer is highly effective as an electroporation medium for exosomes. Optimal miRNA transfection efficiency and cellular uptake were achieved at 350 V, with significantly higher exosome internalization observed under these conditions. Utilizing the Bio-Rad Gene Pulser Electroporation buffer at 350 V enhances both miRNA loading efficiency into extracellular vesicles and subsequent cellular uptake. This study establishes an optimized electroporation protocol, addressing limitations in existing methodologies and advancing the potential of extracellular vesicles as a robust platform for miRNA-based therapeutic delivery.