Development of Polymer–Lipid Hybrid Nanoparticles for Large-Sized Plasmid DNA Transfection

Abstract

RNA and DNA delivery technologies using lipid nanoparticles (LNPs) have advanced significantly, as demonstrated by their successful application in mRNA vaccines. To date, commercially available RNA therapeutics include Onpattro, a 21 bp siRNA, and mRNA vaccines comprising 4300 nucleotides for COVID-19. However, a significant challenge remains in achieving efficient transfection, as the size of the delivered RNA and DNA increases. In contrast to RNA transfection, plasmid DNA (pDNA) transfection requires multiple steps, including cellular uptake, endosomal escape, nuclear translocation, transcription, and translation. The low transfection efficiency of large pDNA is a critical limitation in the development of artificial cells and their cellular functionalization. Here, we introduce polymer−lipid hybrid nanoparticles designed for efficient, large-sized pDNA transfection. We demonstrated that LNPs loaded with positively charged pDNA-polycation core nanoparticles exhibited a 4-fold increase in transfection efficiency for 15 kbp pDNA compared with conventional LNPs, which encapsulate a negatively charged pDNA-polycation core. Based on assessments of the size and internal structure of the polymer−lipid nanoparticles as well as hemolysis and cellular uptake analysis, we propose a strategy to enhance large-sized pDNA transfection using LNPs. This approach holds promise for accelerating the in vivo delivery of large-sized pDNA and advancing the development of artificial cells.

Understanding the effects of ethanol on the liposome bilayer structure using microfluidic-based time-resolved small-angle X-ray scattering and molecular dynamics simulations

“…Recently, microfluidic-based ethanol injection has been widely used in producing size-controlled nanoparticles without complicated size-tuning processes. 8–15 In this method, aqueous and lipid-containing ethanol solutions are introduced into a microfluidic device, and the ethanol is then continuously diluted with the aqueous solution within the device. Lipid molecules aggregate upon ethanol dilution, followed by nanoparticle formation, and thus, ethanol is critical in nanoparticle formation in terms of nanoparticle size, structural change, and drug loading.…”