Microfluidic assembly and biomimetic lipid coating modulate the structure, stability, and biological interactions of P(DMAEMA-co-SMA)/DNA lipopolyplexes

In this study, we present the development and characterization of biomimetic lipopolyplexes using the pH-responsive cationic copolymer, P(DMAEMA-co-SMA), DNA, and membrane-mimicking lipids. The copolymer was synthesized via RAFT polymerization and characterized by size exclusion chromatography, ¹H NMR spectroscopy, ATR-FTIR spectroscopy, and acid-base titration for proton buffering capacity. A custom-designed 3D-printed microfluidic chip with embedded microstructures was utilized to form polyplexes under controlled flow conditions, followed by a post lipid-coating step via lipid film hydration. The statistical copolymer P(DMAEMA-co-SMA) was utilized to condense DNA 50 bp at various nitrogen-to-phosphate (N/P) ratios, yielding polyplexes with distinct physicochemical characteristics. Lipid coating of preformed polyplexes enhanced colloidal stability under storage and biorelevant conditions, highlighting its critical role in maintaining nanoparticle integrity. Cryo-TEM analysis revealed the coexistence of multiple nanostructures with small-angle X-ray scattering (SAXS) supporting these findings and demonstrating pH-dependent organization that provides insights into their structural behavior under biologically relevant conditions. In vitro cytotoxicity and hemocompatibility assays indicated that the developed P(DMAEMA-co-SMA)/DNA lipopolyplexes are well tolerated compared to polyethylenimine (PEI), the gold standard in non-viral gene delivery. Confocal microscopy showed enhanced cellular uptake, endosomal escape, and cytoplasmic distribution in HeLa cells, supporting the potential of the prepared nanocomplexes for efficient intracellular gene delivery. Overall, this study presents P(DMAEMA-co-SMA)/DNA lipopolyplexes as a stable, biocompatible, and effective gene delivery platform and demonstrates how biomimetic lipid coating can modulate the stability and biological interactions of DNA nanocomplexes.