Microfluidic co-assembly of PEG/PEG-lipid-incorporated PBAE nanoparticles for enhanced non-viral gene delivery.

Gene therapy offers significant potential for precision medicine, yet its clinical translation is often hindered by the lack of delivery systems that simultaneously achieve high efficiency, stability, and safety. Poly(β-amino esters) (PBAEs), as promising non-viral vectors, face challenges such as insufficient serum stability and limited transfection efficacy, particularly in hard-to-transfect primary immune cells. To address these limitations, this study developed a microfluidic co-assembly strategy to incorporate polyethylene glycol (PEG) or polyethylene glycol-anchored lipid (PEG-lipid) into PBAE nanoparticles. Compared to the PBAE-only controls, the optimized PEG/PEG-lipid-incorporated nanoparticles exhibited three key advantages: (1) maintained superior transfection efficiency across diverse cell types, even under stringent conditions with reduced DNA doses or lower PBAE/DNA ratios; (2) significantly enhanced serum stability; and (3) successfully overcame the transfection barrier in non-activated primary T cells, with further efficiency gains achieved through conjugation with targeting peptides and antibodies. The integrated microfluidic assembly technology and material functionalization strategy presented in this study provide robust technical support for the development of efficient and low-toxicity non-viral gene vectors. This integrated strategy offers a novel approach for advancing the clinical translation of immune cell gene therapies.