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    Drug Delivery

    (L-455) Reduced-Cholesterol Superhydrophilic Lipid Nanoparticles for Inhaled mRNA Delivery

    Saturday, October 14, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row L - Poster # 455

      Presenting Author(s)

    • Sabrina Meng (she/her/hers)

      Undergraduate Student
      Massachusetts Institute of Technology, United States

      • Co-Author(s)

    • AJ

      Allen Jiang (he/him/his)

      Graduate Student
      Massachusetts Institute of Technology
      Cambridge, Massachusetts, United States

    • SL

      Sushil Lathwal

      Postdoctoral Researcher
      Massachusetts Institute of Technology, United States

    • RL

      Robert Langer

      Institute Professor
      Massachusetts Institute of Technology, United States

      • Primary Investigator(s)

    • DA

      Daniel Anderson

      Professor
      Massachusetts Institute of Technology, United States

    Introduction:: Sub-Track: Nucleic Acid Delivery

    Lipid nanoparticles (LNPs) are effective vehicles for the delivery of mRNA to treat a variety of diseases. One area of interest is nebulized mRNA therapies, which involve the inhalation of aerosolized mRNA-LNP formulations for localized, non-invasive treatment of lung diseases like cystic fibrosis.     The typical composition of LNPs includes ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG)-functionalized lipid (PEG-lipid). In particular, PEG-lipids are included on the surface of LNPs to improve stability and prolong circulation in the body. While the lipid components have been optimized for systemic LNP delivery, nebulized delivery poses unique challenges: 1) nebulizers exert shear forces resulting in LNP aggregation and 2) airway mucus poses an additional barrier between LNPs and cells. While PEG-lipids have seen some success in reducing aggregation, additional barriers to nebulized delivery indicate that further optimization of LNP size and efficacy could enhance treatment success. A promising solution is the replacement of PEG with super-hydrophilic polymers (SHPs); these SHP-LNPs result in reduced particle aggregation and size compared to PEG-LNPs and offer superior nebulized delivery of mRNA to lung cells in mice.

    Materials and Methods:: A design of experiment approach, previously used to optimize PEG-containing LNP formulations for systemic delivery, was taken to optimize LNP formulations containing SHP-conjugated lipids for nebulized delivery. LNPs were prepared by microfluidic mixing with SHP-conjugated lipids or PEG-conjugated lipids as control formulations. The mRNA encapsulation efficiency of the resulting LNPs was assessed using a Ribogreen assay pre- and post-nebulization with an Aeroneb vibrating mesh nebulizer. LNP size was characterized by dynamic light scattering (DLS) measurements. Next, top-performing SHP-LNPs were tested in vivo via nebulization to C57BL/6J mice. Transfection levels in the lung were assessed 6h after nebulized delivery of FFL mRNA via IVIS imaging. Based on the results from this initial DOE screen, a secondary screen investigating the effects of cholesterol composition on PEG- and SHP-containing LNP formulations was taken following the same methodology as above.

    Results, Conclusions, and Discussions::

    In general, SHP-LNP formulations in the initial DOE screen showed comparable encapsulation efficiency (EE) to PEG-LNPs prior to nebulization. However, PEG-LNPs showed a significant drop in EE upon nebulization while no drop in EE was observed for SHP-LNPs. Pre-nebulized SHP-LNPs were generally larger compared to PEG-LNPs, but limited increase in the average SHP-LNP diameter was observed post-nebulization. In contrast, the average PEG-LNP diameter increased from approximately 100 nm to almost 200 nm after nebulization. The data indicate that SHP-LNPs retain their structural integrity during nebulization without significant mRNA loss or increase in particle diameter. Moreover, when compared to PEG-LNPs, the SHP-LNP formulation led to an order of magnitude higher transfection efficiency in the lung 6h after in vivo nebulized delivery of FFL mRNA to mice.

    However, our DOE software predicted that a reduction in cholesterol mol % would further optimize the SHP-LNP, defined as minimizing change in size, maximizing RNA encapsulation post-nebulization, and maximizing transfection efficiency. Thus, we tested three SHP-LNP formulations and their PEG counterparts containing high (H, baseline), medium (M), and low (L) cholesterol content. We found that PEG-LNPs performed poorly based on the above performance parameters compared to low-cholesterol SHP-LNPs and that M-SHP-LNP was the top performer, with the smallest post-nebulization size and the highest in vivo transfection efficiency. Generally, reducing SHP-LNP cholesterol content results in improved performance, as measured through smaller particle size, reduced mRNA loss, and increased transfection efficiency.

    In this study, we find that SHP-lipids play a key role in the stabilization and transfection efficiency of LNPs, a property that is not readily replaced by PEGs, and that low-cholesterol LNP formulations result in increased treatment performance. This finding has implications for clinically effective nebulized mRNA delivery and targeted treatment of lung diseases. Future studies may investigate a larger selection of low-cholesterol LNPs and additional characterization studies (e.g., electron microscopy and zeta potential) to better understand the mechanisms responsible for optimal SHP-LNP performance.



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