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Physico-Chemical Characterization of Polylipid Nanoparticles for Gene Delivery to the Liver

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Polylipid nanoparticles (PLNP) have been shown to be very effective in delivering antioxidative genes in the treatment of liver injury in mice. To build on our previous studies and to further characterize PLNP formulated from polycationic lipid (PCL) and cholesterol, we report here the synthesis of multigram quantities of PCL and employ analytical tools, such as Raman spectroscopy of single PLNP and live-cell imaging of lipofection, for the physicochemical characterization of PCL, PLNP, and the transfection process. Mass spectrometry demonstrates the characteristics of polymeric lipids. Raman spectrum of PCL reveals the polymeric structure of the polymers. The presence of cholesterol in PLNP formulation did not markedly change the Raman spectrum. PLNP-derived polyplexes exhibit Raman spectra very similar to PLNP except that the C-H out-of-plane deformation mode of the polymeric lipid is significantly suppressed, indicating the interaction with plasmid DNA. Zeta potential measurement indicates a large DNA-carrying capacity of PLNP and their stability for in vivo gene delivery. The live-cell fluorescent imaging dynamically shows that PLNP exerts transfection efficiency similar to lipofectamine in leading to early reporter gene expression in live hepatic cells. In conclusion, polylipid nanoparticles possess a high DNA carrying capacity and lipofection efficiency, rendering them suitable for testing in large animals. The employment of novel state-of-the-art technologies in the study of lipofection represents the level of physicochemical and biological characterization that is needed to best understand the key elements involved in the lipofection process.

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