Synthetic Nanoparticle Cytokine (SyNK) for Systemic Dosing and Immunotherapy

Abstract

Cytokines are potent proteins responsible for cell signaling and regulation of the immune system. The use of cytokines to modulate macrophage phenotype is a promising therapeutic strategy for immune-mediated diseases; however, translation of recombinant cytokines as drug products is limited by cytokines’ severe systemic side effects and nonspecific biodistribution. While immense pre-clinical effort has been invested in the development of cytokine therapies, the limited number of FDA-approved recombinant cytokine products include either the free protein or its PEGylated variant. No approved products leverage biomaterial-based drug carriers to overcome the biodistribution and toxicity limitations of the recombinant cytokine molecule. While PEGylation improves the pharmacokinetics of intravenously administered cytokine products through extended circulation half-life, it does not provide spatial or temporal control over the cytokine’s activity. Hydrogel nanoparticles (i.e., nanogels, NGs) are a promising platform to address this unmet need to precisely modulate the immune response in target tissues. Through the rational design of nanogel physicochemical and functional properties, NGs can enhance the biodistribution and stability of cytokines while minimizing off-target effects.The overarching goal of this dissertation was to develop a safe and modular platform for the delivery of immunomodulatory cytokines using NGs. We developed a synthetic poly(acrylamide-co-methacrylic acid) nanogel - based cytokine delivery system (called “Synthetic Nanoparticle cytoKine, or SyNK) designed to restrict cytokines’ systemic release under circulatory conditions while preserving bioactivity upon co-localization with, or uptake by, target immune cells. The project had three objectives: (1) characterize the biocompatibility and cellular uptake of the NG in vitro; (2) assess the pharmacokinetics and biodistribution of the NG in vivo; and (3) evaluate molecular mechanisms through which SyNK modulates macrophage phenotype in diverse immunological environments. SyNK showed significant biocompatibility with cultured macrophages and circulating immune cells, with minimal cytotoxicity and immune activation. Macrophages internalized SyNK primarily though clathrin-mediated endocytosis, while interestingly, fibroblasts and circulating whole blood-derived immune cells showed negligible uptake. These observations suggested a degree of selectivity that could facilitate macrophage-based cytokine delivery in vivo. Pharmacokinetic studies revealed that systemically administered SyNK was cleared in less than 1 hour from the circulation and accumulated primarily in the kidneys, liver, and small intestine. Within the liver, cellular distribution studies confirmed uptake primarily by endothelial cells and resident macrophages. Despite accumulation, safety studies did not reveal any toxicity to these organs. Once safety was established, SyNK was synthesized by conjugating either IFNγ, IL4, or IL10 to the nanogel, which effectively eliminated the diffusion-mediated release of free cytokine to aqueous surroundings. Macrophages treated with SyNK exhibited phenotypic changes consistent with free protein, but at a lower potency, indicating that NG-cytokine conjugation influenced bioactivity. Furthermore, the immunomodulatory effects of IL4 and IL10 were dependent on the surrounding immune microenvironment and the intrinsic properties of the NG, suggesting that the platform can provide context-dependent immune modulation. This dissertation establishes the foundation for a tunable and biocompatible delivery platform that can improve the application of cytokines for macrophage immunotherapy. Future research efforts should (1) optimize the conjugation strategy of SyNK to maximize bioactivity, (2) assess the biodistribution and activity of SyNK in relevant disease models, and (3) evaluate its therapeutic potential of SyNK, relative to freely soluble recombinant cytokines, to treat local or systemic inflammatory conditions.