Assistant Professor of Biomedical Engineering NYU, New York, United States
Introduction: In recent years, in vivo delivery of messenger RNA (mRNA) has emerged as a promising therapeutic approach thanks to the more favorable safety profile of mRNA compared to other gene therapies, among other reasons. To function in vivo, mRNA needs to be encapsulated in delivery vectors that protect it from degradation and facilitate cellular uptake, of which lipid nanoparticles (LNPs) are the most successful example to date. However, targeting LNPs to specific organs and cells remains a challenge. Many LNP formulations tend to accumulate in the liver, as most systemically administered nanomedicines do, which could be leveraged for the treatment of liver-specific diseases. In such cases, targeting specific cell types within the liver may be important depending on the disease to be treated. However, information on the specific cell populations within the liver that uptake specific LNP formulations is mostly lacking in the literature. In this work, we aimed to evaluate the capabilities of four new LNP formulations to transfect hepatocytes in vitro and different liver cell types (parenchymal and non-parenchymal) in vivo. We compared the performance of these formulations to FDA-approved formulation (the nanoparticle in Patisiran, Onpattro®) which is used to treat hereditary transthyretin amyloidosis.
Materials and
Methods: Four distinct LNP formulations (A1-4) were compared to an FDA-approved formulation (MC3 formulation from Onpattro). LNP lipid excipients were purchased from Avanti Polar Lipids and MedChemExpress. mCherry mRNA was transcribed in-vitro with a fully 5-methoxyuridine substitution and co-transcriptionally capped using CleanCap technology. All solvents were purchased from Fisher Scientific. All other chemical reagents were purchased from MilliporeSigma. For LNP formulation, an organic phase was prepared by solubilizing lipids in ethanol at specific molar ratios. An aqueous phase consisted of mCherry mRNA in citrate buffer (pH 4). The total lipid to mRNA weight ratio was 20:1. LNPs were formed by rapid pipette mixing at a ratio of 3:1 (aqueous: ethanol, vol: vol). The mixture was then dialyzed (Pur-A-Lyzer Midi Dialysis Kits, WMCO 3.5 kDa) against 1 x phosphate buffered saline (PBS) for 2 hours prior to usage. The particle size and polydispersity index of LNPs were measured using dynamic light scattering (DLS) (Zetasizer Pro, Malvern), and the encapsulation efficiency of mRNA in each LNP was quantified by measuring the mRNA binding following Quant-iT RiboGreen assay protocols. In vitro uptake and transfection efficiency were evaluated in Hep G2 cells (human liver cancer cell line) and in vivo biodistribution and protein expression were investigated in C57BL/6 mice. In vitro and in vivo transfection efficiency were evaluated by fluorescence imaging, bioluminescence analysis and Western Blot.
Results, Conclusions, and Discussions: All formulations tested (A01-4 and MC3 LNP) showed appropriate physicochemical characterization. Their average size measured by DLS were below 140 nm and their PDIs were between 0.03 and 0.19 on average. Encapsulation efficiency was between 79.6% and 96.2%. Transfection capabilities of these nanoparticles were first evaluated in an in vitro Hep G2 cell model using mRNA encoding for the fluorescent protein mCherry. 24 h after transfection A02 and A03 LNPs, demonstrated a significant increase in transfection efficiency compared to both the MC3 LNP control and the commercially available Lipofectamine MessengerMAX Reagent, without significant cytotoxicity. In vivo investigation to determine hepatic accumulation (by IVIS Bioluminiscence imaging) and cell type-specific uptake (via parenchymal and non-parenchymal cell isolation prior to bioluminescence analysis) is underway. Overall, our study will provide cellular resolution on the uptake and transfection efficiency of new LNP formulations that will enable rational selection of the formulation in accordance with the specific disease to treat.
Acknowledgements (Optional): Yeung Wu and this work are funded by an AHA Early Faculty Independence Award (24SCEFIA1156995).
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