Precise control of microfluidic flow conditions is critical for harnessing the in vitro transfection capability of pDNA-loaded lipid-Eudragit nanoparticles

Microfluidics is widely regarded as a leading technology for industrial-scale manufacture of multicomponent, gene-based nanomedicines in a reproducible manner. Yet, very few investigations detail the impact of flow conditions on the biological performance of the product, particularly biocompatibility and therapeutic efficiency. Herein, this study investigated the engineering of a novel lipid-Eudragit hybrid nanoparticle in a bifurcating microfluidics micromixer for plasmid DNA (pDNA) delivery. Nanoparticles of ~150 nm in size, with uniform polydispersity index (PDI = 0.2) and ξ-potential of 5-11 mV were formed across flow rate ratios (FRR, aqueous to organic phase) of 3:1 and 5:1, respectively. The hybrid nanoparticles maintained colloidal stability and structural integrity of loaded pDNA following recovery by ultracentrifugation. Importantly, in vitro testing in human embryonic kidney cell line (HEK293T) revealed significant differences in biocompatibility and transfection efficiency (TE). Lipid-Eudragit nanoparticles produced at FRR 3:1 displayed high cellular toxicity (0-30% viability), compared with nanoparticles prepared at FRR 5:1 (50-100% viability). Red fluorescent protein (RFP) expression was sustained for 24-72 h following exposure of cells to nanoparticles, indicating controlled release of pDNA and trafficking to the nucleus. Nanoparticles produced at FRR 5:1 resulted in markedly higher TE (12%) compared with those prepared at FRR 3:1 (2%). Notably, nanoparticles produced using the bench-scale nanoprecipitation method resulted in lower biocompatibility (30-90%) but higher RFP expression (25-38%). These findings emphasize the need for in-depth analysis of the effect of formulation and flow conditions on the physicochemical and biological performance of gene nanomedicines when transitioning from bench to clinic.