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Drug Delivery

Drug Delivery - Poster Session A

Poster J7 - Corticosteroid-Incorporated Lipid Nanoparticles for Anti-Inflammatory Delivery of mRNA

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  With the FDA approval of the siRNA drug Onpattro and the success of Moderna’s COVID-19 mRNA vaccines, lipid nanoparticles (LNPs) have emerged as the most clinically viable platform for delivery of therapeutic RNAs such as messenger RNA (mRNA) (Jung, 2022). Delivery of mRNA has extensive therapeutic potential in engineering immunotherapies, protein replacement therapies, and cell-based therapies (Qin, 2022). LNPs have been shown to be particularly effective for intracellular delivery of mRNA, promoting endosomal escape and subsequent mRNA translation for therapeutic effect (Hou, 2021). However, LNPs have been shown to be highly inflammatory in several clinical studies (Ndeupen, 2021). Additionally, LNPs have been found to exacerbate pre-existing inflammation, limiting their therapeutic effect in inflammation-causing diseases (Parhiz, 2022). This is suboptimal as increased inflammation can lead to tissue damage, immune dysregulation, as well as inhibition or degradation of therapeutic cargo (Chen, 2017). Thus, addressing this inflammatory response is crucial for maximizing the therapeutic potential of LNPs and expanding clinical applications to more inflammation-sensitive disease-types. One approach that has shown promise is the incorporation of corticosteroids either within or alongside LNPs to mitigate LNP-induced inflammation. Previous studies have shown that codelivery of anti-inflammatory steroids alongside mRNA suppresses inflammation via inhibition of proinflammatory genes (Abrams, 2010). Further research has explored LNPs encapsulating steroids to reduce LNP-induced inflammation (Zhang, 2022). However this study only tested a limited number of formulations of one steroid-incorporated LNPs. Here, we investigate a library of FDA-approved steroids to be incorporated into LNPs to mitigate inflammation and potentiate effective mRNA delivery.

Materials and

Methods:  A preliminary screening of steroid-incorporated LNPs with 0%, 10%, 50%, and 100% substitution of Dexamethasone for cholesterol was conducted and compared against free steroid delivery. The gold standard ionizable lipid C12-200 was combined with cholesterol, a helper lipid (DSPC), DMG-PEG, and steroid and microfluidically mixed to formulate LNPs encapsulating Firefly luciferase mRNA (Fig. 1B). The formulated LNPs were evaluated in RAW BLUE cells for toxicity, as well as anti-inflammatory activity via a QUANTI-Blue SEAP reporter assay linked to the inflammatory transcription factors NF-κB and AP-1. The 50% substitution ratio was identified as the most effective, leading to a second screen with LNPs formulated with six different steroids (Dexamethasone, Prednisone, Prednisolone, Methylprednisolone, Triamcinolone, and Hydrocortisone) (Fig. 1A) at 50% substitution for cholesterol. These LNPs were formulated to encapsulate luciferase mRNA and evaluated for mRNA delivery and toxicity in RAW 264.7 cells, and anti-inflammatory activity in RAW BLUE cells as outlined previously. All LNPs were characterized for size and polydispersity index (PDI) via DLS, zeta potential, encapsulation efficiency, and pKa.

Results, Conclusions, and Discussions: The preliminary screen showed that the DEX-incorporated LNPs were effective in reducing SEAP activity when compared to free DEX. However, the LNP formulated with a 100% substitution ratio of DEX for cholesterol had significantly lower mRNA encapsulation and was deemed subpar (Fig. 1C). Excitingly, the 10% and 50% DEX-substituted particles reduced SEAP activity more drastically than the free DEX control, suggesting that incorporating steroids inside LNPs facilitated their intracellular delivery and subsequent suppressive activity. The 50% ratio resulted in a balance of sufficient mRNA delivery alongside reduction in inflammation, while preserving high encapsulation efficiency. We moved forward with a second screening of six 50% substituted steroid-incorporated LNPs formulated using five new steroids. All of the LNPs formulated well, with diameters < 100 nm, polydispersity indexes (PDIs) < 0.20, and encapsulation efficiencies >80% (Fig. 1D). (Fig. 1E). Notably, all six 50% steroid-incorporated particles resulted in higher relative luminescence than the standard particle in RAW-BLUE macrophages, with the TRI-LNP demonstrating the highest relative luminescencE (Fig. 1E). All of the steroid-LNPs except the P-LNP showed significant reduction in SEAP expression when compared to the LPS control (Fig. 1F), indicating downregulation of inflammation. Additionally, the PL, MPL, and TRI LNPs showed sufficient cell viability when compared to the LPS control. Our steroid-incorporated LNPs further need to be tested for reduction of inflammation in other cell types, including dendritic cells, T cells, and monocytes. Additionally, we plan to perform multiplex ELISA assays to assess downregulation in key inflammatory cytokines from steroid-incorporated LNP delivery. We plan to optimize the TRI 50% particle by applying a design of experiments (DOE) methodology to vary excipient ratios and steroid substitution ratios to assess impact on LNP performance. Lastly, a biodistribution study will be completed in mice to assess the effect of steroid incorporation on LNP circulation, mRNA expression, and toxicity in vivo. Following this, our anti-inflammatory steroid-incorporated LNPs will be formulated to encapsulate a therapeutic mRNA cargo and applied to a mouse model of autoimmune encephalomyelitis (EAE). Together, our preliminary results demonstrate that steroid-incorporated LNPs have the potential to mediate LNP-induced inflammation while efficiently delivering mRNA for therapeutic effect.

Acknowledgements (Optional):  References
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Qin, S., Tang, X., Chen, Y. et al. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Sig Transduct Target Ther 7, 166 (2022). https://doi.org/10.1038/s41392-022-01007-w
Hou, X., Zaks, T., Langer, R., & Dong, Y. (2021). Lipid nanoparticles for mRNA delivery. Nature reviews. Materials, 6(12), 1078–1094. https://doi.org/10.1038/s41578-021-00358-0
Ndeupen, S., Qin, Z., Jacobsen, S., Bouteau, A., Estanbouli, H., & Igyártó, B. Z. (2021). The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory. iScience, 24(12), 103479. https://doi.org/10.1016/j.isci.2021.103479
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