qPCR-based method to measure plasmid internalization efficiency of non-viral nanoparticle carriers in adherent cell...

Non-viral nanoparticle-mediated transfection systems have gained wide acceptance in modern pharmaceutical and biotechnological applications, including RNA delivery for COVID-19 vaccines. However, these systems may show low transfection and transcription efficiencies, potentially limiting the use of reporter molecules like GFP to estimate efficacy in early development stages. Moreover, the expression of reporter molecules may not reflect internalization efficiency, which can be pertinent information for the development of these systems. Therefore, as their popularity continues to rise, more sensitive and accurate tools will be necessary. We validated a DNA extraction and qPCR assay to quantify the internalized plasmid copy number in NIH/3T3 cells transfected using LGA-PEI (lactic-co-glycolic acid-modified polyethylenimine) nanoparticles. Optimized qPCR and DNA extraction assays exhibited high linearity, sensitivity, and robustness. Additionally, we developed an appropriate extraction and extracellular plasmid digestion protocol that eliminated potential polymer interference with the qPCR reaction and increased specificity for internalized plasmid quantification. Using these optimized protocols, we estimated that transfected NIH/3T3 cells incorporated approximately 9 500 to 150 000 plasmids/cell for transfection reactions that yielded as few as 4.1 %-18.2 % GFP-positive cells. Overall, these results indicate that our optimized method is reliable for quantifying transfected plasmids in context of low-efficiency nanoparticle carrier system.