(146f) Immunomodulatory Nanomaterials for Imaging and Treatment of Chronic-Inflammatory Diseases.

Introduction: Inflammasomes are cytosolic protein complexes that detect pathogenic and damage-associated signals, triggering the activation of caspase-1 and the release of the inflammatory cytokines IL-1β and IL-18. Among these, the NLRP3 inflammasome is the most widely implicated in disease, responding to a diverse range of stimuli, including microbial products, environmental stress, and endogenous danger signals. Aberrant inflammasome activation is associated with chronic inflammatory conditions, including colitis, gout, psoriasis, and sepsis, making it a compelling therapeutic and diagnostic target. Despite advances in inflammasome biology, a critical need remains for non-invasive, real-time tools to monitor inflammasome activation and simultaneously assess therapeutic efficacy. Existing detection platforms, including genetically encoded fluorescent sensors, luciferase reporters, and FRET-based approaches, provide valuable mechanistic insights but lack the necessary bioavailability, retention, and translational feasibility required for dynamic in vivo imaging. These limitations hinder both basic research and clinical progress in the development of inflammasome-targeted therapies.

Results and Discussion: To address these gaps, we developed a pair of immunomodulatory nanoparticle systems with diagnostic and theranostic capabilities. The first, a reporter nanoparticle (FLTD NP), encapsulates a caspase-1-responsive peptide probe that mimics the cleavage motif in gasdermin-D or IL-1β, flanked by an infrared fluorescent dye and a quencher. This system enables spatiotemporally resolved visualization of inflammasome activation in vitro and in vivo. The second system, a theranostic nanoparticle (FLTD-MCC NP or FLTD-DSR NP), co-encapsulates the probe with a potent inflammasome inhibitor, either MCC950, which targets the NLRP3 complex upstream, or disulfiram, which blocks gasdermin-D pore formation downstream. These nanoparticles are composed of DOPC and DSPE-PEG co-lipids, which provide biocompatibility, extended circulation, and sustained release.

In vitro studies using bone marrow-derived macrophages confirmed that FLTD NPs specifically respond to LPS and Nigericin-induced caspase-1 activation. The fluorescent signal was tightly correlated with caspase-1 cleavage and IL-1β secretion and absent in caspase-1-deficient cells or in the presence of inflammasome inhibitors. When co-delivered with MCC950 or disulfiram, the theranostic nanoparticles not only attenuated inflammasome activation but also led to measurable reductions in fluorescence over time, reflecting successful suppression of caspase-1 activity. The dual-loaded nanoparticles thus provide a unique means to quantify treatment efficacy in real time. To demonstrate in vivo utility, we evaluated the reporter and theranostic nanoparticles in multiple models of inflammatory disease. In a dextran sulfate sodium (DSS)-induced colitis model, FLTD NPs accumulated in inflamed colon tissue and generated a sustained fluorescence signal corresponding to caspase-1 activity. Mice treated with FLTD-MCC NPs showed both reduced signals and improved histological markers of disease, including decreased colon shortening and crypt damage. This dual readout of inflammation and therapeutic response was similarly observed in a monosodium urate (MSU)-induced gouty arthritis model. In this case, intra-articular injection of FLTD-DSR NPs resulted in strong fluorescence localized to the inflamed joint, which declined with the progression of therapy. We further applied our system to sepsis, a complex and deadly syndrome characterized by systemic inflammation. Using a lipopolysaccharide (LPS)-induced peritonitis model, we tested nanoparticles co-loaded with MCC950 and disulfiram (MCC-DSR NPs). The rationale for this combination stems from their complementary mechanisms: MCC950 prevents NLRP3 oligomerization, blocking inflammasome assembly, while disulfiram inhibits gasdermin-D, halting downstream pyroptosis. The dual-drug nanoparticles exhibited synergistic inhibition of IL-1β secretion in vitro compared to individual drugs or free drug combinations. Notably, MCC-DSR NPs improved survival outcomes in septic mice more effectively than any single therapy, demonstrating the translational potential of this co-delivery strategy.

Beyond systemic inflammation, we sought to investigate the local modulation of inflammasome activity in chronic cutaneous inflammation. Psoriasis is a prevalent immune-mediated skin disorder characterized by abnormal keratinocyte proliferation and immune cell infiltration, including macrophages that contribute to inflammation through the activation of NLRP3 and AIM2 inflammasomes. Topical therapies targeting these inflammasomes are limited by poor skin penetration, systemic toxicity, and lack of macrophage specificity. We engineered non-spherical lipid nanorods using pyridoxine dipalmitate, a lipid with innate inflammasome-inhibiting properties, to serve as a "Trojan horse" carrier. These nanorods demonstrated enhanced uptake by macrophages, likely due to their pathogen-mimetic geometry, and were significantly more effective at reducing ASC speck formation, mitochondrial ROS, and IL-1β secretion than spherical or elliptical analogs. To enhance anti-inflammatory efficacy, we co-loaded these nanorods with a dual NLRP3/AIM2 inhibitor (NA3) and embedded them in a sprayable polymeric scaffold composed of mucin and poloxamer 407 for topical application. Mucin provides a mucosal adhesive matrix and has been reported to downregulate TLR4, further complementing inflammasome inhibition. In an imiquimod-induced psoriasis mouse model, NA3-loaded nanorods significantly reduced disease severity, as evidenced by decreased erythema, scaling, and epidermal thickness. qRT-PCR analysis of psoriatic lesions revealed substantial downregulation of NLRP3, AIM2, IL-1β, IL-17A, caspase-1, gasdermin-D, and chemokines such as CXCL2 and CCL2. This data suggests that the platform acts both locally and systemically to mitigate inflammatory signaling cascades. In parallel, we studied the uptake and trafficking of differently shaped nanoparticles in macrophages to understand how particle geometry influences inflammasome inhibition. Rod-shaped LNPs were preferentially internalized via macropinocytosis and clathrin-mediated endocytosis, bypassing phagocytic routes that could inadvertently trigger inflammasome activation. This route of entry enabled higher payload retention, sustained intracellular drug release, and prolonged suppression of the inflammasome.

Together, these studies demonstrate the utility of nanoparticles in tracking and treating inflammasome activity across multiple disease contexts. The systems enable spatiotemporal monitoring of inflammasome dynamics, correlate signal strength with disease severity, and provide real-time feedback on the success of therapeutic interventions. Importantly, these platforms operate without requiring genetically engineered cells or animals, improving their translational relevance.

Conclusions: We report on the development and validation of dual-function nanoparticle systems that integrate imaging and therapeutic control over inflammasome activation. These platforms utilize the unique capabilities of lipid nanocarriers to enhance probe stability, facilitate sustained drug release, and enable non-invasive tracking of inflammatory responses. In diseases ranging from ulcerative colitis and gouty arthritis to psoriasis and sepsis, our systems provided accurate readouts of inflammasome activation. They demonstrated potent immunomodulatory effects through delivery of NLRP3 and gasdermin-D inhibitors. By designing nanoparticles that are shape-tuned, macrophage-targeted, and responsive to disease-specific triggers, we achieved enhanced retention, cellular uptake, and therapeutic efficacy. Furthermore, we highlight the synergistic benefits of combination drug delivery in inflammasome signaling pathways, which offer superior outcomes compared to single-agent or free-drug treatments. The theranostic capability of these platforms, which provides both disease detection and tailored therapeutic responses, represents a significant step forward for personalized medicine in inflammatory diseases.