Exploring electroporation for miRNA mimic delivery into cells with milk-derived extracellular vesicles

Milk-derived extracellular vesicles (MEVs) are widely recognized as promising natural nanocarriers for drug delivery. However, current drug-loading strategies predominantly rely on electroporation, which is often associated with low and inconsistent loading efficiency. This study aims to address these limitations. We further incorporated the widely used miRNA-carrying liposome Lipofectamine RNAiMAX to assess whether combining MEVs with liposomal delivery could enhance performance while reducing Lipofectamine-associated cytotoxicity. The outcomes of integrating these two delivery strategies were examined, and the underlying mechanisms were explored. Combining Lipofectamine with electroporated MEVs markedly improved target cell viability compared with Lipofectamine alone, although this was accompanied by a substantial reduction in loading efficiency. Despite increased cell viability, apoptosis-related gene expression remained almost unchanged. Transmission electron microscopy and the absence of notable changes in protein content after electroporation suggest that reduced Lipofectamine transfection efficiency may result from excessive membrane stacking and encapsulation. We believe this phenomenon is caused by excessive electrostatic attraction between the two membrane components, and considering that miRNAs also carry a negative charge, this may hinder the loading process. Therefore, we neutralized the electroporated MEVs with calcium chloride and then allowed the miRNAs to be loaded via passive diffusion and membrane self-repair. We demonstrate a strategy that significantly enhances and stabilizes the loading efficiency of natural MEVs without introducing exogenous components that are difficult to eliminate and could potentially elicit immune responses. This study paves the way for the future use of natural MEVs as nanomedicine carriers with low cytotoxicity, low immunogenicity, and potential homing capabilities.