Nanoscale Strategies for Real-Time Monitoring and Therapeutic Modulation of Inflammasome

Introduction: : : Inflammasomes are cytosolic multiprotein complexes that mediate caspase-1 activation in response to pathogenic or sterile insults, triggering maturation of IL-1β/IL-18 and pyroptotic cell death. The NLRP3 inflammasome, in particular, plays a central role in numerous inflammatory diseases, including ulcerative colitis, gout, psoriasis, and sepsis. Current strategies for studying inflammasomes largely rely on endpoint biochemical assays or genetically engineered models, which fail to capture the spatiotemporal dynamics of inflammasome activation in vivo. Furthermore, small-molecule inhibitors of inflammasomes often suffer from poor bioavailability, rapid systemic clearance, and a lack of tools to monitor therapeutic response in real time. We hypothesized that a nanotechnology-based system co-delivering an activatable imaging probe and inflammasome-targeted therapeutics could simultaneously monitor and suppress inflammasome activity with high temporal and spatial resolution. To this end, we developed two classes of biocompatible lipid nanoparticles: (1) reporter nanoparticles encapsulating a caspase-1 cleavable infrared probe to visualize inflammasome activity, and (2) theranostic nanoparticles co-delivering this probe with inflammasome inhibitors such as MCC950 or disulfiram. These systems aim to overcome current limitations in inflammasome detection and provide a feedback-enabled therapeutic approach. We tested these platforms in multiple murine models of inflammatory diseases to evaluate real-time tracking of inflammasome activation, correlation with disease severity, and modulation of disease outcome. Our approach offers a non-genetic, translationally relevant platform for personalized inflammation monitoring and therapy.

Materials and

Methods: : Caspase-1-cleavable peptide sequences (FLTD and YVAD) were synthesized and conjugated with Cy5 dye and QSY21 quencher to create fluorogenic probes that are silent until enzymatically cleaved. These were encapsulated in liposomes composed of DOPC and DSPE-PEG2000 via thin-film hydration and extrusion methods to form reporter nanoparticles (FLTD NPs). For theranostic formulations, MCC950 or disulfiram was co-loaded with the probe to yield dual-function nanoparticles (FLTD-MCC and FLTD-DSR NPs). Nanoparticles were characterized for size, zeta potential, encapsulation efficiency, and release kinetics using DLS, HPLC, and fluorescence spectroscopy. In vitro studies were conducted in immortalized bone marrow-derived macrophages (iBMDMs) primed with LPS and activated with ATP. Caspase-1 activation was assessed via western blotting, ASC speck microscopy, IL-1β ELISA, and probe fluorescence. For in vivo studies, mouse models of DSS-induced colitis, MSU-induced gouty arthritis, and LPS-induced septic peritonitis were used to examine disease-specific inflammasome activation. Fluorescence imaging was performed longitudinally using IVIS Spectrum, and tissue analysis included immunofluorescence, histopathology, and qRT-PCR. Particle uptake mechanisms were evaluated using pathway inhibitors and confocal microscopy.

Results, Conclusions, and Discussions:: Results &

Discussion: To address these gaps, we developed a pair of lipid-based 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 (Fig. 1). 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. 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. 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 observed in a monosodium urate (MSU)-induced gouty arthritis model (Fig. 2). 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.

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 sepsis, our systems provided accurate readouts of inflammasome activation. They demonstrated potent immunomodulatory effects through delivery of NLRP3 and gasdermin-D inhibitors. 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.

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