Intracellular trafficking kinetics of nucleic acid escape from lipid nanoparticles via fluorescence imaging

by Selleckchem | May 11 2023

Introduction: Lipid nanoparticles (LNPs) are one of the most clinically advanced candidates for delivering nucleic acids to target cell populations, such as hepatocytes. Once LNPs are endocytosed, they must release their nucleic acid cargo into the cell cytoplasm. For delivering messenger RNA (mRNA), delivery into the cytosol is sufficient; however, for delivering DNA, there is an added diffusional barrier needed to facilitate nuclear uptake for transcription and therapeutic effect.

Method: Here, we use fluorescence microscopy to investigate the intracellular fate of different LNP formulations to determine the kinetics of localization to endosomes and lysosomes. LNPs used in the studies were prepared via self-assembly using a NanoAssemblr for microfluidic mixing. As the content of polyethylene glycol (PEG) within the LNP formulation influences cellular uptake by hepatocyte cells, the content and hydrocarbon chain length within the formulation were assessed for their impact on intracellular trafficking. Standard LNPs were then formed using three commercially available ionizable lipids, Dlin-MC3-DMA (MC3), Dlin-KC2-DMA (KC2), and SS-OP. Plasmid DNA (pDNA) and mRNA were used, more specifically with a mixture of Cyanine 3 (Cy3)-labeled and green fluorescence protein (GFP) producing plasmid DNA (pDNA) as well as Cy5-labeled GFP producing mRNA. After formulation, LNPs were characterized for the encapsulation efficiency of the nucleic acid, hydrodynamic diameter, polydispersity, and zeta potential. All standard LNPs were ~100 nm in diameter and had neutral surface charge. All LNPs resulted in encapsulation efficiency greater than 70%. Confocal fluorescence microscopy was used for the intracellular trafficking studies, where LNPs were incubated with HuH-7 hepatocyte cells at times ranging from 0-48 h. The cells were antibody-stained for subcellular components, including nuclei, endosomes, and lysosomes.

Result: Analysis was performed to quantify localization of pDNA to the endosomes and lysosomes. LNPs with 1.5 mol% PEG and a hydrocarbon chain C14 resulted in optimal endosomal escape and GFP production. Results from this study demonstrate that a higher percentage of C14 PEG leads to smaller LNPs with limited available phospholipid binding area for ApoE, resulting in decreased cellular uptake. We observed differences in the localization kinetics depending on the LNP formulation type for SS-OP, KC2, and MC3 ionizable lipids. The results also demonstrate the technique across different nucleic acid types, where mRNA resulted in more rapid and uniform GFP production compared to pDNA delivery.

Conclusion: Here, we demonstrated the ability to track uptake and the sub-cellular fate of LNPs containing pDNA and mRNA, enabling improved screening prior to in vivo studies which would aid in formulation optimization.

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Summary:

The study aimed to investigate the intracellular fate of different lipid nanoparticles (LNPs) for delivering nucleic acids to target cell populations. The researchers used fluorescence microscopy to determine the kinetics of localization to endosomes and lysosomes. LNPs were prepared via self-assembly and characterized for encapsulation efficiency of nucleic acid, hydrodynamic diameter, polydispersity, and zeta potential. Confocal fluorescence microscopy was used to track intracellular trafficking studies, where LNPs were incubated with HuH-7 hepatocyte cells. The results showed that LNPs with 1.5 mol% PEG and a hydrocarbon chain C14 resulted in optimal endosomal escape and GFP production. The study also demonstrated the technique across different nucleic acid types, where mRNA resulted in more rapid and uniform GFP production compared to pDNA delivery. This study can aid in the formulation optimization of LNPs for in vivo studies.

Keywords: Lipid nanoparticles, nucleic acids, fluorescence microscopy, intracellular trafficking, endosomes, lysosomes, self-assembly, encapsulation efficiency, GFP, PEG, hydrocarbon chain, in vivo studies.