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Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform
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PBAE-PEG based lipid nanoparticles for lung cell-specific gene delivery.

Bingxin Liu, Yamato Sajiki, Anusha Sridharan

    Biorxiv : the Preprint Server for Biology
    |September 4, 2024
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    This study developed novel lipid nanoparticles (LNPs) for delivering messenger RNA (mRNA) to lung cells. Different LNP formulations showed cell-specific delivery via intravenous or intratracheal routes, highlighting potential for pulmonary gene therapy.

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    Area of Science:

    • Biotechnology
    • Nanomedicine
    • Pulmonary Medicine

    Background:

    • Modified messenger RNA (mRNA) delivered via lipid nanoparticles (LNPs) shows promise for treating genetic and acquired disorders.
    • The COVID-19 pandemic highlighted the potential of mRNA-LNP technology.

    Purpose of the Study:

    • To develop and evaluate PEGylated (PBAE-PEG) and non-PEGylated (PBAE) lipid nanoparticles for mRNA delivery to pulmonary cells in vivo.
    • To assess the cell-specific transfection efficiency of different LNP formulations and administration routes in the lungs.

    Main Methods:

    • Development of PEGylated and non-PEGylated PBAE lipids formulated into LNPs with 4A3-SC8/DOPE/cholesterol/DOTAP.
    • In vitro transfection assays using HEK293T and H441 cells.
    • In vivo studies in mice involving intravenous and intratracheal administration of LNPs carrying Cre-recombinase mRNA.
    • Evaluation of Cre-recombinase expression and recombination in various lung cell types and endothelial cells.

    Main Results:

    • PBAE-PEG/LNPs demonstrated high transfection efficiency in HEK293T and H441 cells in vitro.
    • Intravenous administration of PBAE-PEG/LNPs resulted in high transfection of pulmonary vascular endothelial cells.
    • Intratracheal administration led to efficient and selective transfection of lung epithelial cells, including club cells and alveolar type 2 cells.
    • PBAE-PEG/LNPs were more effective for alveolar type 2 cells, while PBAE/LNPs favored secretory airway cells after intratracheal delivery.
    • LNPs failed to cross the pulmonary endothelial-to-epithelial barrier.
    • 5-methoxyuridine modified mRNA showed improved in vivo efficiency compared to unmodified mRNA.

    Conclusions:

    • Developed LNP formulations enable targeted mRNA delivery to specific pulmonary cell types.
    • Administration route dictates cell tropism, with intravenous targeting endothelial cells and intratracheal targeting epithelial cells.
    • These findings support the potential of LNP-mediated mRNA delivery for pulmonary gene therapies.