RESUMO
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.
RESUMO
Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.