Nanoparticle-mediated co-delivery of inflammasome inhibitors provides protection against sepsis.

The NLRP3 inflammasome, a multiprotein complex responsible for triggering the release of pro-inflammatory cytokines, plays a crucial role in inducing the inflammatory response associated with sepsis. While small molecule inhibitors of the NLRP3 inflammasome have been investigated for sepsis management, delivering NLRP3 inhibitors has been accompanied by several challenges, primarily related to the drug formulation, delivery route, stability, and toxicity. Many existing inflammasome inhibitors either show higher liver toxicity or require a high dosage to efficiently impede the inflammasome complex assembly. Moreover, the potential synergistic effects of combining multiple inflammasome inhibitors in sepsis therapy remain largely unexplored. Therefore, a rational approach is essential for presenting the potential administration of NLRP3 small molecule inhibitors to inhibit NLRP3 inflammasome activation effectively. In this context, we present a lipid nanoparticle-based dual-drug delivery system loaded with MCC 950 and disulfiram, demonstrating markedly higher efficiency compared to an equivalent amount of free-drug combinations and individual drug nanoparticles in vitro. This combination therapy substantially improved the in vivo survival rate of mice for LPS-induced septic peritonitis. Additionally, the synergistic approach illustrated a significant reduction in the expression of active caspase-1 as well as IL-1ß inhibition integral components in the NLRP3 pathway. This study underscores the importance of integrating combination therapies facilitated by nanoparticle delivery to address the limitations of small molecule inflammasome inhibitors.

Caspase-1 Responsive Nanoreporter for In Vivo Monitoring of Inflammasome Immunotherapy.

Inflammasomes are multimeric protein signaling complexes that are assembled in innate immune cells in response to a multitude of pathogen and damage-associated signals. They are essential for generating robust inflammatory responses to prevent pathogenic insults. However, inflammasome dysregulation can induce cascading immune responses, resulting in systemic toxicities and inflammatory disease. In this sense, there is a strong need to develop potent inflammasome inhibiting therapies as well as technologies to monitor their efficacy, yet current systems lack the ability to effectively image inflammasome activation and track therapy response early. To overcome these limitations, we report a novel nanoparticle system delivering both a caspase-1 cleavable inflammasome detecting probe and the NLRP3 inhibitor drug MCC-950, providing dual capabilities of monitoring and regulation of inflammasome activation in a biocompatible, tissue penetrating, and sustained release liposomal formulation. We observed this liposomal nanoreporter's ability to reduce and detect inflammasome activation both in vitro in immortalized bone marrow-derived macrophages and in vivo in a DSS-induced ulcerative colitis mouse model. Our results exhibited the nanoreporter's ability to penetrate inflammatory tissues and detect inflammasome activation early and in real-time for multiple days while alleviating inflammation in the groups coencapsulating imaging reporter and inflammasome inhibitor. Overall, the developed liposomal nanoreporter platform enables spatiotemporal delivery of imaging probe and inhibitor, captures early and sustained inflammasome detection, and induces inflammasome amelioration, thus establishing a novel tool for the real-time monitoring and treatment of inflammasome-mediated disease with high potential for clinical application.

Nanoreporter for Real-Time Monitoring of Inflammasome Activity and Targeted Therapy.

Inflammasome activation is associated with a myriad of inflammatory diseases. However, existing methods provides a limited understanding of spatiotemporal kinetics of inflammasome activation, with restricted scope for early detection of associated treatment efficacy. This limitation offers an opportunity for the development of biocompatible in-vivo inflammasome monitoring tools with translational prospects. To achieve this, they report developing a pair of lipid-based nanoparticle systems, a reporter nanoparticle consisting of a caspase-1 activatable probe alone, and a theranostic nanoparticle combining the probe with an inflammasome-inhibiting drug. This biocompatible platform enhances the probe's residence time in circulation by preventing its opsonization and allowing its sustained release over time. Their results demonstrate the specificity of reporter nanoparticles towards caspase-1 activity and provides early-on monitoring of inflammasome activation both in-vitro as well as in-vivo. Additionally, the delivery of disulfiram, an inflammasome-inhibiting drug, along with reporter probe using theranostic nanoparticles enables real-time tracking of treatment efficacy in the gouty-arthritis inflammatory model. In summary, they report an unparalleled pair of the inflammasome-associated reporter and theranostic platforms suited not only for diagnostic applications but can also detect inflammasome-targeted treatment efficiency in real-time. These findings establish two novel, sensitive nanotools for non-invasive evaluation of inflammasome-targeted immunotherapy.

mRNA-carrying lipid nanoparticles that induce lysosomal rupture activate NLRP3 inflammasome and reduce mRNA transfection efficiency.

In the last several years, countless developments have been made to engineer more efficient and potent mRNA lipid nanoparticle vaccines, culminating in the rapid development of effective mRNA vaccines against COVID-19. However, despite these advancements and materials approaches, there is still a lack of understanding of the resultant immunogenicity of mRNA lipid nanoparticles. Therefore, a more mechanistic, design-driven approach needs to be taken to determine which biophysical characteristics, especially related to changes in lipid compositions, drive nanoparticle immunogenicity. Here, we synthesized a panel of six mRNA lipid nanoparticle formulations, varying the concentrations of different lipid components and systematically studied their effect on NLRP3 inflammasome activation; a key intracellular protein complex that controls various inflammatory responses. Initial experiments aimed to determine differences in nanoparticle activation of NLRP3 inflammasomes by IL-1ß ELISA, which unveiled that nanoparticles with high concentrations of ionizable lipid DLin-MC3-DMA in tandem with high cationic lipid DPTAP and low cholesterol concentration induced the greatest activation of the NLRP3 inflammasome. These results were further corroborated by the measurement of ASC specks indicative of NLRP3 complex assembly, as well as cleaved gasdermin-D and caspase-1 expression indicating complex activation. We also uncovered these activation profiles to be mechanistically correlated primarily with lysosomal rupturing caused by the delayed membrane disruption capabilities of ionizable lipids until the lysosomal stage, as well as by mitochondrial reactive oxygen species (ROS) production and calcium influx for some of the particles. Therefore, we report that the specific, combined effects of each lipid type, most notably ionizable, cationic lipids, and cholesterol, is a crucial mRNA lipid nanoparticle characteristic that varies the endo/lysosomal rupture capabilities of the formulation and activate NLRP3 inflammasomes in a lysosomal rupture dependent manner. These results provide a more concrete understanding of mRNA lipid Nanoparticle-Associated Molecular Patterns for the activation of molecular-level immune responses and provide new lipid composition design considerations for future mRNA-delivery approaches.

Imaging Approaches to Monitor Inflammasome Activation.

Inflammasomes are a critical component of innate immune response which plays an important role in the pathogenesis of various chronic and acute inflammatory disease conditions. An inflammasome complex consists of a multimeric protein assembly triggered by any form of pathogenic or sterile insult, resulting in caspase-1 activation. This active enzyme is further known to activate downstream pro-inflammatory cytokines along with a pore-forming protein, eventually leading to a lytic cell death called pyroptosis. Understanding the spatiotemporal kinetics of essential inflammasome components provides a better interpretation of the complex signaling underlying inflammation during several disease pathologies. This can be attained via in-vitro and in-vivo imaging platforms, which not only provide a basic understanding of molecular signaling but are also crucial to develop and screen targeted therapeutics. To date, numerous studies have reported platforms to image different signaling components participating in inflammasome activation. Here, we review several elements of inflammasome signaling, a common molecular mechanism combining these elements and their respective imaging tools. We anticipate that future needs will include developing new inflammasome imaging systems that can be utilized as clinical tools for diagnostics and monitoring treatment responses.

Core Hydrophobicity of Supramolecular Nanoparticles Induces NLRP3 Inflammasome Activation.

Designer nanomaterials capable of delivering immunomodulators to specific immune cells have been extensively studied. However, emerging evidence suggests that several of these nanomaterials can nonspecifically activate NLRP3 inflammasomes, an intracellular multiprotein complex controlling various immune cell functions, leading to undesirable effects. To understand what nanoparticle attributes activate inflammasomes, we designed a multiparametric polymer supramolecular nanoparticle system to modulate various surface and core nanoparticle-associated molecular patterns (NAMPs), one at a time. We also investigated several underlying signaling pathways, including lysosomal rupture-cathepsin B maturation and calcium flux-mitochondrial ROS production, to gain mechanistic insights into NAMPs-mediated inflammasome activation. Here, we report that out of the four NAMPs tested, core hydrophobicity strongly activates and positively correlates with the NLRP3 assembly compared to surface charge, core rigidity, and surface hydrophobicity. Moreover, we demonstrate different signaling inclinations and kinetics followed by differential core hydrophobicity patterns with the most hydrophobic ones exhibiting both lysosomal rupture and calcium influx early on. Altogether, this study will help design the next generation of polymeric nanomaterials for specific regulation of inflammasome activation, aiding efficient immunotherapy and vaccine delivery.

Granzyme B nanoreporter for early monitoring of tumor response to immunotherapy.

Despite recent advancements in cancer immunotherapy, accurate monitoring of its efficacy is challenging due to heterogeneous immune responses. Conventional imaging techniques lack the sensitivity and specificity for early response assessment. In this study, we designed a granzyme B (GrB) nanoreporter (GNR) that can deliver an immune checkpoint inhibitor to the tumor and track time-sensitive GrB activity as a direct way to monitor initiation of effective immune responses. Anti-programmed death-ligand 1 (PD-L1) antibody-conjugated GNRs inhibited PD-1/PD-L1 interactions efficiently and induced T cell-mediated GrB release that can be imaged using activatable imaging probe. GNRs enabled real-time immunotherapy response monitoring in a tumor-bearing mice model and distinguished between highly responsive and poorly responsive tumors. Furthermore, increasing doses resulted in a better response and enhanced sensitivity in poorly responsive tumors. These findings indicate that GNR has the potential to serve as a tool for sensitive and noninvasive evaluation of immunotherapy efficacy.

Supramolecular Polysaccharide Nanotheranostics that Inhibit Cancer Cells Growth and Monitor Targeted Therapy Response.

Targeted anticancer therapies directed against specific molecular drivers of tumors are emerging as effective treatment strategies, however, monitoring their response is still challenging. Current clinical imaging techniques that measure either morphological or metabolic changes in the tumor are not always indicative of clinical outcome due to delayed or variable responses. Here, dual-stage polysaccharide-based supramolecular nanotheranostics (SPN) were designed that enable co-delivery of kinase inhibitor and an activatable imaging probe. Methods: The SPNs were prepared by supramolecular assembly of two components, polysaccharide construct conjugated with kinase inhibitor-function activatable probe and kinase inhibitor- ß-cyclodextrin conjugate. Physiochemical characterization of SPNs including size, stability, zeta potential and pH-responsiveness of the assembly was performed. The efficacy of SPNs in inducing cancer cell death by inhibition of kinase signaling and imaging the response was evaluated in murine BRAFV600E melanoma (D4M) and triple-negative breast cancer (4T1) cell lines. Finally, the in vivo efficacy was investigated in D4M melanoma tumor model. Results: The polysaccharide-constructs along with kinase inhibitor- ß-cyclodextrin conjugates self-assemble to produce SPNs of around 200 nm in diameter and were stable for over a week under physiologically relevant conditions. The SPNs exhibited enhanced cytotoxic effect and significant inhibition of kinase signaling as compared to the free inhibitor. In vitro imaging studies confirmed their enzyme-activatable therapy response tracking abilities both in cancer cells and tumor spheroids. Furthermore, SPN treated mice exhibited better tumor growth inhibition as compared to the control groups and therapy response could be imaged at both early (24-48h) and later time points. Conclusion: These findings demonstrate that the supramolecular polysaccharide nanotheranostics can not only inhibit kinase signaling pathway in aggressive tumor, but also monitor targeted therapy response early.

A Nitric Oxide (NO) Nanoreporter for Noninvasive Real-Time Imaging of Macrophage Immunotherapy.

Macrophage-centered therapeutic approaches that rely on immune modulation of tumor associated macrophages (TAMs) from a pro-tumorigenic phenotype (M2) to an anti-tumorigenic phenotype (M1) have facilitated a paradigm shift in macrophage immunotherapy. However, limited clinical success has been achieved due to the low response rates observed in different types of cancers. The ability to measure immune response in real time is critical in order to differentiate responders from non-responders; however, there are currently no platforms to monitor real-time macrophage immunotherapy response. Hence, there is an immediate need to develop imaging techniques that can longitudinally monitor macrophage immunotherapy response. Nitric oxide (NO) produced as a result of activation of macrophages to an anti-tumorigenic state is considered as a hallmark of M1 and can be a direct indication of response. In this study, a NO nanoreporter (NO-NR) is reported that enables real-time monitoring of macrophage immunotherapy drugs in vitro and in vivo. Furthermore, it is observed that sustained inhibition of colony stimulating factor 1 receptor (CSF1R) using a CSF1R inhibitor-NO-NR system leads to enhanced efficacy and better imaging signal. In conclusion, a first-of-its-kind NO nanoreporter tool is reported that can be used as an activatable imaging agent to monitor macrophage immunotherapy response in real time.

Dual inhibition of CSF1R and MAPK pathways using supramolecular nanoparticles enhances macrophage immunotherapy.

Among the numerous immune interactions, or lack-thereof, that occur during cancer progression, tumor-associated macrophages (TAMs) - cancer cell interactions have been shown to play an important role in modulating the tumor-microenvironment to an immune suppressive mode, promoting accelerated tumor growth, survival and metastatic spread. TAMs are predominantly polarized to a pro-tumorigenic M2-phenotype through macrophage colony stimulating factor 1 (MCSF) cytokines that bind to the colony-stimulating factor 1 receptor (CSF1R), a class III receptor tyrosine kinase. This MCSF-CSF1R interaction results in autophosphorylation of CSF1R and subsequent phosphorylation and activation of downstream signaling pathways including mitogen-activated protein kinase (MAPK) pathway leading to proliferation, survival and functional activity of M2 TAMs. Therapeutic inhibition of CSF1R and MAPK signaling could effectively re-polarize M2 macrophages to an anti-tumorigenic M1 phenotype; however, this is challenging. In this study, we demonstrate that concurrent and sustained inhibition of the CSF1R and MAPK signaling pathways using dual-kinase inhibitor-loaded supramolecular nanoparticles (DSNs) enhance repolarization of pro-tumorigenic M2 macrophages to the anti-tumorigenic M1 phenotype. The supramolecular nanoparticles exhibited physical stability of over 7 days during storage conditions at 4 °C and over 24 h in human serum, released the inhibitors in a sustained manner and showed significantly higher internalization and accumulation of inhibitors in the M2 macrophages even at longer time points. When tested in a highly aggressive 4T1 breast cancer model, the supramolecular nanoparticles accumulated in TAMs at a significantly higher concentration, increased M1-like phenotype at significantly higher proportion and improved anti-tumor efficacy as compared to combination of single-inhibitor nanoparticles or the small molecule inhibitors. Our data suggests that concurrent, vertical inhibition of multiple intracellular kinase signaling pathways is important for repolarization of M2 macrophages to M1 phenotype, and by utilizing dual-inhibitor loaded supramolecular nanoparticles, further increase the ability to produce more M1 macrophages as compared to M2 macrophages in the tumor microenvironment. This results in enhanced tumor growth inhibition and reduced toxicity. Therefore, vertical, co-inhibition of CSF1R and downstream signaling pathways like MAPK could be a promising macrophage immunotherapy strategy for aggressive cancers.

Dual Inhibitors-Loaded Nanotherapeutics that Target Kinase Signaling Pathways Synergize with Immune Checkpoint Inhibitor.

INTRODUCTION: Immune checkpoint inhibitors that boost cytotoxic T cell-based immune responses have emerged as one of the most promising approaches in cancer treatment. However, it is increasingly being realized that T cell activation needs to be rationally combined with molecularly targeted therapeutics for a maximal anti-tumor outcome. Currently, two oncogenic drivers, MAPK and PI3K-mTOR have emerged as the two main molecular targets for combining with immunotherapy. However, there are major challenges in enabling such combinations: first, such combinations can result in high rates of toxicity. Second, while, these molecular targets could be driving tumor progression, they are essential for activation of the immune cells. So, the kinase inhibitors and immunotherapy can antagonize each other. OBJECTIVES: We rationalized that the synergistic combination of kinase inhibitors and immunotherapy could be enabled by dual inhibitors-loaded supramolecular nanotherapeutics (DiLN) that can co-deliver PI3K- and MAPK-inhibitors to the cancer cells and activate immune response by T cell-modulating immunotherapy, resulting in greater anti-tumor efficacy while minimizing toxicity. METHODS: We engineered DiLNs by designing the amphiphilic building blocks (both drugs and co-lipids) that enables supramolecular nanoassembly. DiLNs were tested for their physiochemical properties including size, morphology, stability and drug release kinetics profiles. The efficacy of DiLNs was tested in drug-resistant cells such as BRAFV600E melanoma (D4M), Clear cell ovarian carcinoma (TOV21G) cells. The tumor inhibition efficiency of DiLNs in combination with immune checkpoint inhibitor antibody was studied in syngeneic D4M animal model. RESULTS: DiLNs were stable for over a month and released the drugs in a sustained manner. In vitro cytotoxicity studies in D4M and TOV21G cells showed that DiLNs were significantly more effective than free drugs. In vivo studies showed that the combination of DiLNs with anti PD-L1 antibody resulted in superior antitumor effect and survival. CONCLUSION: This study shows that the rational combination of DiLNs that target multiple oncogenic signaling pathways with immune checkpoint inhibitors could emerge as an effective strategy to improve immunotherapeutic response against drug resistant tumors.

CSF1R- and SHP2-Inhibitor-Loaded Nanoparticles Enhance Cytotoxic Activity and Phagocytosis in Tumor-Associated Macrophages.

Immune modulation of macrophages has emerged as an attractive approach for anti-cancer therapy. However, there are two main challenges in successfully utilizing macrophages for immunotherapy. First, macrophage colony stimulating factor (MCSF) secreted by cancer cells binds to colony stimulating factor 1 receptor (CSF1-R) on macrophages and in turn activates the downstream signaling pathway responsible for polarization of tumor-associated macrophages (TAMs) to immunosuppressive M2 phenotype. Second, ligation of signal regulatory protein α (SIRPα) expressed on myeloid cells to CD47, a transmembrane protein overexpressed on cancer cells, activates the Src homology region 2 (SH2) domain -phosphatases SHP-1 and SHP-2 in macrophages. This results in activation of "eat-me-not" signaling pathway and inhibition of phagocytosis. Here, it is reported that self-assembled dual-inhibitor-loaded nanoparticles (DNTs) target M2 macrophages and simultaneously inhibit CSF1R and SHP2 pathways. This results in efficient repolarization of M2 macrophages to an active M1 phenotype, and superior phagocytic capabilities as compared to individual drug treatments. Furthermore, suboptimal dose administration of DNTs in highly aggressive breast cancer and melanoma mouse models show enhanced anti-tumor efficacy without any toxicity. These studies demonstrate that the concurrent inhibition of CSF1-R and SHP2 signaling pathways for macrophage activation and phagocytosis enhancement could be an effective strategy for macrophage-based immunotherapy.

Platelet activation determines the severity of thrombocytopenia in dengue infection.

Thrombocytopenia is common in patients with dengue virus (DENV) infections. With a focus on understanding the possible mechanism of thrombocytopenia in DENV infections we described a direct correlation between activation and depletion of platelets in patients. Our data showed a sharp decrease in platelet counts at day 4 of fever in patients. The high DENV genome copies in platelets correlated directly with the elevated platelet activation along with increased binding of complement factor C3 and IgG on their surface at day 4. Recovery in platelet count was observed on day 10 through day 6 and 8 with simultaneous decrease in platelet activation markers. Further, our in vitro data supported the above observations describing a concentration-dependent increase in platelet activation by DENV serotype-2. The high copy number of DENV2 genome in the platelet pellet correlated directly with platelet activation, microparticle generation and clot formation. Furthermore the DENV2-activated platelets were phagocytosed in large numbers by the monocytes. The DENV2-mediated lysis and clearance of platelets were abrogated in presence of platelet activation inhibitor, prostacyclin. These observations collectively suggest that platelet activation status is an important determinant of thrombocytopenia in dengue infections. A careful strategy of inactivation of platelets may rescue them from rapid destruction during DENV infections.