Mechanistic Insights into the Superior DNA Delivery Efficiency of Multicomponent Lipid Nanoparticles: An In Vitro and In Vivo Study

Abstract

Lipid nanoparticles (LNPs) are currently having an increasing impact on nanomedicines as delivery agents, among others, of RNA molecules (e.g., short interfering RNA for the treatment of hereditary diseases or messenger RNA for the development of COVID-19 vaccines). Despite this, the delivery of plasmid DNA (pDNA) by LNPs in preclinical studies is still unsatisfactory, mainly due to the lack of systematic structural and functional studies on DNA-loaded LNPs. To tackle this issue, we developed, characterized, and tested a library of 16 multicomponent DNA-loaded LNPs which were prepared by microfluidics and differed in lipid composition, surface functionalization, and manufacturing factors. 8 out of 16 formulations exhibited proper size and zeta potential and passed to the validation step, that is, the simultaneous quantification of transfection efficiency and cell viability in human embryonic kidney cells (HEK-293). The most efficient formulation (LNP15) was then successfully validated both in vitro, in an immortalized adult keratinocyte cell line (HaCaT) and in an epidermoid cervical cancer cell line (CaSki), and in vivo as a nanocarrier to deliver a cancer vaccine against the benchmark target tyrosine-kinase receptor HER2 in C57BL/6 mice. Finally, by a combination of confocal microscopy, transmission electron microscopy and synchrotron small-angle X-ray scattering, we were able to show that the superior efficiency of LNP15 can be linked to its disordered nanostructure consisting of small-size unoriented layers of pDNA sandwiched between closely apposed lipid membranes that undergo massive destabilization upon interaction with cellular lipids. Our results provide new insights into the structure−activity relationship of pDNA-loaded LNPs and pave the way to the clinical translation of this gene delivery technology.

Plant-derived extracellular nanovesicles: a promising biomedical approach for effective targeting of triple negative breast cancer cells

“…Endocytosis is a well-studied ED pathway for the transportation of nanoparticles across the cellular membrane, facilitating their internalization into specific cells and subsequent accumulation, which includes phagocytosis, macropinocytosis, clathrin- or caveolin-mediated endocytosis. Conversely, EI internalization mechanisms involve fusion, embedment, and direct translocation ( Quagliarini et al, 2022 ). Traditional small molecular drugs primarily enter cells through passive or active process, whereas nanomedicines enter cells via endocytosis ( Longfa et al, 2013 ).…”

Looking Back, Moving Forward: Lipid Nanoparticles as a Promising Frontier in Gene Delivery

“…Some recent studies demonstrated that DNA-LNPs are constituted by oligolamellar domains whose spatial extension is dependent on the lipid composition. 65 Despite extensive research, a clear correlation between the structure of DNA-LNPs and their lipid composition has yet to be established.…”

Formulation of Nucleic Acids by Encapsulation in Lipid Nanoparticles for Continuous Production of mRNA

“…LNP formulation for (p)DNA delivery has been reported in the literature, e.g., for LNP systems as transfection reagents for plasmid DNA (pDNA) [29], for enahnced oral delivery of DNA [30], for increased efficiency of DNA-vectored vaccines/immunoprophylaxis in animals [31], for spherical nucleic acids for intracellular DNA and RNA delivery [32], for transfection of cell model lines [33], as nanocarrier to deliver a cancer vaccine against the benchmark target tyrosine-kinase receptor HER2 in C57BL/6 mice [34], as well as in evaluating studies regarding the role of the influential Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery factors [26].…”