Enhancing Transfection Efficacy in Glioma Cells: A Comparison of Microfluidic

Background: Gene therapy is a promising therapeutic approach for treating various disorders by introducing modified nucleic acids to correct cellular dysfunctions or introduce new functions. Despite significant advancements in the field, the effective delivery of nucleic acids remains a challenge, due to biological barriers and the immune system's ability to target and destroy these molecules. Due to their branched structure and ability to condense negatively charged nucleic acids, cationic dendrimers have shown potential in overcoming these challenges. Despite this, standardized scalable production methods are still lacking. This study investigates the use of microfluidics to formulate generation 3-diaminobutyric polypropylenimine (DAB) dendriplexes and compares their characteristics and in vitro gene delivery efficacy to those prepared using conventional manual mixing. Methods: DAB dendriplexes were produced by both microfluidic and manual approaches and characterized. Their cellular uptake and gene expression were evaluated on C6 glioma cancer cells in vitro. Results: Dendriplexes formed using microfluidics at the optimal flow rate and ratio demonstrated enhanced DNA condensation over time, achieving up to 97% condensation at 24 hours. Both preparation methods produced positively charged dendriplexes, indicating stable formulations. However, dendriplexes prepared through hand mixing resulted in smaller particle sizes, significantly higher cellular uptake and gene expression efficacy compared to those prepared by microfluidics. Nonetheless, microfluidic preparation offers the advantage of standardized and scalable production, which is essential for future applications. Conclusion: This study highlights the potential of microfluidic technology to improve precision and scalability in gene delivery, paving the way for future advancements in gene therapy. Our findings suggest that, with further optimization, microfluidic systems could provide superior control over dendriplex formation, expanding their potential use in gene therapy applications.