(742b) Examining Intracellular Trafficking of Nucleic Acid Containing Lipid Nanoparticles for Non-Viral Gene Delivery

Lipid nanoparticles (LNPs) are promising candidates for delivering nucleic acids to target cell populations, such as hepatocytes.^(1),(2) Once LNPs are endocytosed, they must release their nucleic acid cargo into the cell cytoplasm.⁽³⁾ A further challenge, DNA must undergo nuclear uptake for transcription and therapeutic effect.⁽⁴⁾ Here, DNA encapsulated fluorescently labeled LNPs are developed to examine cellular uptake, endosomal release, and intracellular trafficking.

LNPs were developed via self-assembly using a NanoAssemblr for microfluidic mixing. A lipid mix was first prepared containing an ionizable lipid to promote nucleic acid complexation, a Cy5 labeled lipid, and helper lipids (e.g. cholesterol and lipid-conjugated PEG). Plasmid DNA (pDNA) was prepared with a 1:1 mixture of GFP producing pDNA and Cy3-labeled pDNA in buffer at pH 3. After complexation, LNPs were washed in phosphate buffered saline (PBS) and characterized for encapsulation efficiency and hydrodynamic diameter. For intracellular trafficking, HUH-7 hepatocyte cell line cells were cultured for 24 h prior to addition of LNPs. Samples were fixed with 4% paraformaldehyde and stained for cell nucleus, endosomes, and lysosomes and imaged using a confocal microscope at times ranging from 0-48 h.

LNPs were characterized with a hydrodynamic diameter of 110 nm with neutral charge. 93% of pDNA was encapsulated within the LNP (Figure 1A). Following incubation with HUH-7 cells, intracellular trafficking was monitored at 15 min, 30 min, 1 h, 2 h, 4 h, 24 h, and 48 h. A representative image for 48 h is shown in Figure 1B. GFP production was observed (dispersed green), which demonstrates nuclear uptake of pDNA. GFP production was not observed in control experiments with non-encapsulated pDNA. The Cy5-labeled LNPs showed endosomal release and dispersion within the cytoplasm as early as 15 min. Finally, tracking of Cy3 labeled pDNA and LNPs in endosomes and lysosomes was identified.

Here we demonstrate the ability to track uptake and sub-cellular fate of LNPs containing pDNA. This technique and platform allow for comparison of intracellular trafficking and nuclear uptake with different LNP formulations and ionizable lipids employed, enabling improved screening prior to in vivo studies.

References: 1. Cullis PR, Hope MJ. Lipid Nanoparticle Systems for Enabling Gene Therapies. Mol Ther. 2017;25:1467-75. 2. Munsell EV, Ross NL, Sullivan MO. Journey to the Center of the Cell: Current Nanocarrier Design Strategies Targeting Biopharmaceuticals to the Cytoplasm and Nucleus: Curr Pharm Des. 2016;22:1227-44. 3. Kowalski PS, Rudra A, Miao L, Anderson DG. Delivery the Messenger: Advances in Technologies for Therapeutic mRNA Delivery. Mol Ther. 2019;27:710-28. 4. Durymanov M, Reineke J. Non-viral delivery of nucleic acids: Insight into mechanisms of overcoming intracellular barriers. Front Pharmacol. 2018;9:1-15.