Natural and Synthetic Nanoparticle-Based Approaches for Anti-Inflammatory Immunomodulation

Introduction: Inflammation is a complex physiological response that develops in response to harmful stimuli such as pathogens or tissue damage. Traditional pharmacological strategies for dampening severe inflammatory responses are often non-specific and carry potential for the development of opportunistic infections due to their immunosuppressive nature. Using sepsis as a model severe inflammatory condition, we have developed and mechanistically evaluated various nanoparticle-based approaches to address the multifaceted inflammatory and immunological deficits that develop. In this presentation, I will describe the approaches developed by our team, including the use of cargo-free plant-derived lipid nanoparticles (PDNP) for proinflammatory cytokine sequestration and metabolite-based immunomodulatory nanoparticles (iNP) for modulation of inflammatory immune cell trafficking and phenotypes to improve sepsis survival. Collectively, these strategies highlight the promising use of nanoparticles as multimodal anti-inflammatory therapeutics, which can be applied for the treatment of numerous inflammatory diseases and conditions.

Materials and

Methods: PDNPs were isolated from Lepidium meyenii Walp, commonly known as maca, a biennial herbal root plant indigenous to the Peruvian Andes, using density gradient ultracentrifugation (Fig. 1A). Synthetic iNPs were formulated using the single emulsion-solvent evaporation method from poly(lactic acid). Lipidomics analysis of PDNPs was conducted using LC/MS-MS to determine their composition. Bone marrow-derived macrophages were treated with NPs s at various concentrations in the presence or absence of lipopolysaccharide (LPS) stimulation. In vitro assays were conducted to evaluate toxicity, cytokine and chemokine production, and macrophage polarization. For in vivo studies, IVIS imaging and flow cytometry were performed to assess nanoparticle biodistribution and immune cell distributions, respectively. Histological examination was carried out using H&E staining. Efficacy was assessed in two mouse models of sepsis: the lethal LPS-induced endotoxemia model and the clinically relevant cecal ligation and puncture (CLP) model of polymicrobial sepsis.

Results, Conclusions, and Discussions: Dynamic light scattering analysis showed that PDNPs and iNPs had sizes of approximately 150-200 nm and 500 nm, respectively. In vitro studies indicated that neither PDNPs nor iNPs affected cell viability at the highest concentrations tested. Lipidomic analysis revealed triacylglycerols and phytoceramides as major constituents comprising PDNPs. When PDNPs were incubated with LPS-treated macrophages, there were significant reductions in proinflammatory cytokines, including IL-6 and TNFα. Incubation of PDNPs with recombinant IL-6 demonstrated that PDNPs could sequester IL-6 through direct surface binding. In a therapeutic LPS-induced endotoxemia mouse model, PDNP-treated mice demonstrated increased recovery, evidenced by a significant reduction in systemic proinflammatory cytokines, improved histological index, and increased survival (Fig 1B). Incubation of iNPs with macrophages followed by LPS stimulation resulted in significant reductions in proinflammatory cytokine secretions, in-part due to the inhibition of NF-kb and p38 MAPK signaling pathways. iNP treatment prior to a lethal LPS challenge led to a significant reduction in organ damage and improvement in survival. Co-administration of iNPs with antibiotics immediately following the CLP procedure resulted in 47% survival (Fig. 1C-D). iNP treatment also significantly reduced inflammatory monocytes in the blood, while increasing patrolling monocytes. In summary, this research highlights PDNPs and iNPs as two distinct and highly effective anti-inflammatory strategies to mitigate the severe inflammatory responses associated with sepsis.

Acknowledgements (Optional): This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM142752, and the Shock Society Faculty Research Award awarded to R.M.P.