Injectable Microbiome-Derived Metabolite Nano-Prodrugs For Sustained Immune Modulation in Autoimmune Arthritis

Abstract

Rheumatoid arthritis (RA) affects approximately 1% of the global population and remains a major unmet need for durable therapies that restore immune balance without global immune suppression. Indole-3-propionic acid (IPA), a microbial metabolite, has emerged as a promising immunomodulator that regulates multiple anti-inflammatory signaling pathways. However, its therapeutic potential is limited by the need for repeated oral dosing to maintain efficacy and poor lymphoid exposure.To address these limitations, we developed a long-acting nano-prodrug platform to sustain IPA exposure in lymphoid tissues regulating RA syndrome. The immunomodulator IPA was covalently added into a polymer backbone. With cleavable ester bonds, the conjugated IPA-containing polymers (pIPA) were assembled into ~90 nm nanoparticles (pIPA NP) that presented as a suspension injectable product. This design enables controlled release of IPA from the polymer that provides tunable drug loading and, when given by injection, demonstrates enhanced lymphatic IPA accumulation. Biodistribution studies in mice showed prolonged fluorescently labeled pIPA NP retention in tissues and multiple lymph nodes for up to 35 days after a single subcutaneous dose of pIPA NP. In contrast, the free fluorescence tracer without pIPA NP encapsulation was cleared by day 14 and exhibited limited lymphatic distribution. LC-MS/MS quantification of IPA in lymph nodes confirmed detectable IPA concentrations exclusively in pIPA NP-treated animals across all four time points (day 1, 3, 7, 14), reaching 3.98 ng/mg and 5.71 ng/mg in hock-draining and inguinal lymph nodes, respectively, at day 14, while undetectable levels were noted in mice treated with free IPA and control groups. The efficacy of pIPA NP for RA was evaluated with collagen antibody-induced arthritis (CAIA) mouse models in both prophylactic and therapeutic settings. Compared to PBS and free IPA controls, prophylactic pIPA NP treatment significantly reduced arthritis scores by 68.2% and 62.4%, and paw thickness by 35.6% and 32.9%, respectively, alongside reduced immune cell infiltration in joints, lower paw IL-6 levels, and dampened immune cell activation. Unformulated IPA at an equivalent dose offered no significant benefit, suggesting that sustained delivery is required for therapeutic efficacy. In an interventional study, arthritis scores were reduced by 61.0% following completion of the two-dose treatment regimen, with disease suppression sustained through the study endpoint (Day 14). Flow cytometry of both studies revealed that pIPA NP selectively suppressed activation of CD11c+ and CD11b+CD11c+ dendritic cells and upregulated checkpoint markers on T cells, with effects concentrated in the hock-draining lymph nodes rather than broadly across systemic compartments. In vivo cellular uptake studies further showed that nanoparticle internalization was highest in the CD11c+ cell populations, which exhibited the greatest functional suppression, directly linking nanoparticle-cell engagement to immunomodulatory outcome. Collectively, this work demonstrates that engineering IPA into a long-acting, lymphoid-targeted nanoparticle system enables sustained and site-specific immune modulation with improved therapeutic outcomes in CAIA. More broadly, these findings highlight how such drug delivery strategies can transform rapidly degrading, immunomodulatory molecules into effective treatments for inflammatory diseases like RA.