NLRP3 inflammasome activation determines the fibrogenic potential of PM2.5 air pollution particles in the lung.

Airborne fine particulate matter (PM2.5) is known to cause respiratory inflammation such as chronic obstructive pulmonary disease and lung fibrosis. NLRP3 inflammasome activation has been implicated in these diseases; however, due to the complexity in PM2.5 compositions, it is difficult to differentiate the roles of the components in triggering this pathway. We collected eight real-life PM2.5 samples for a comparative analysis of their effects on NLRP3 inflammasome activation and lung fibrosis. In vitro assays showed that although the PM2.5 particles did not induce significant cytotoxicity at the dose range of 12.5 to 100 µg/mL, they induced potent TNF-α and IL-1ß production in PMA differentiated THP-1 human macrophages and TGF-ß1 production in BEAS-2B human bronchial epithelial cells. At the dose of 100 µg/mL, PM2.5 induced NLRP3 inflammasome activation by inducing lysosomal damage and cathepsin B release, leading to IL-1ß production. This was confirmed by using NLRP3- and ASC-deficient cells as well as a cathepsin B inhibitor, ca-074 ME. Administration of PM2.5 via oropharyngeal aspiration at 2 mg/kg induced significant TGF-ß1 production in the bronchoalveolar lavage fluid and collagen deposition in the lung at 21 days post-exposure, suggesting PM2.5 has the potential to induce pulmonary fibrosis. The ranking of in vitro IL-1ß production correlates well with the in vivo total cell count, TGF-ß1 production, and collagen deposition. In summary, we demonstrate that the PM2.5 is capable of inducing NLRP3 inflammasome activation, which triggers a series of cellular responses in the lung to induce fibrosis.

Nanocellulose Length Determines the Differential Cytotoxic Effects and Inflammatory Responses in Macrophages and Hepatocytes.

Nanocellulose including cellulose nanocrystal (CNC) and cellulose nanofiber (CNF) has attracted much attention due to its exceptional mechanical, chemical, and rheological properties. Although considered biocompatible, recent reports have demonstrated nanocellulose can be hazardous, including serving as drug carriers that accumulate in the liver. However, the nanocellulose effects on liver cells, including Kupffer cells (KCs) and hepatocytes are unclear. Here, the toxicity of nanocellulose with different lengths is compared, including the shorter CNCs (CNC-1, CNC-2, and CNC-3) and longer CNF (CNF-1 and CNF-2), to liver cells. While all CNCs triggered significant cytotoxicity in KCs and only CNC-2 induced toxicity to hepatocytes, CNFs failed to induce significant cytotoxicity due to their minimal cellular uptake. The phagocytosis of CNCs by KCs induced mitochondria ROS generation, caspase-3/7 activation, and apoptotic cell death as well as lysosomal damage, cathepsin B release, NLRP3 inflammasome and caspase-1 activation, and IL-1ß production. The cellular uptake of CNC-2 by hepatocytes is through clathrin-mediated endocytosis, and it induced the caspase-3/7-mediated apoptosis. CNC-2 shows the highest levels of uptake and cytotoxicity among CNCs. These results demonstrate the length-dependent mechanisms of toxicity on liver cells in a cell type-dependent fashion, providing information to safely use nanocellulose for biomedical applications.

Development of Facile and Versatile Platinum Drug Delivering Silicasome Nanocarriers for Efficient Pancreatic Cancer Chemo-Immunotherapy.

In this study a mesoporous silica nanoparticle (MSNP) based platform is developed for high-dose loading of a range of activated platinum (Pt) chemo agents that can be attached to the porous interior through the use of electrostatic and coordination chemistry under weak-basic pH conditions. In addition to the design feature for improving drug delivery, the MSNP can also be encapsulated in a coated lipid bilayer (silicasome), to improve the colloidal stability after intravenous (IV) injection. Improved pharmacokinetics and intratumor delivery of encapsulated activated oxaliplatin (1,2-diamminocyclohexane platinum(II) (DACHPt)) over free drug in an orthotopic Kras-derived pancreatic cancer (PDAC) model is demonstrated. Not only does IV injection of the DACHPt silicasome provide more efficacious cytotoxic tumor cell killing, but can also demonstrate that chemotherapy-induced cell death is accompanied by the features of immunogenic cell death (ICD) as well as a dramatic reduction in bone marrow toxicity. The added ICD features are reflected by calreticulin and high-mobility group box 1 expression, along with increased CD8+ /FoxP3+ T-cell ratios and evidence of perforin and granzyme B release at the tumor site. Subsequent performance of a survival experiment, demonstrates that the DACHPt silicasome generates a significant improvement in survival outcome, which can be extended by delayed administration of the anti-PD-1 antibody.

Dissolution of 2D Molybdenum Disulfide Generates Differential Toxicity among Liver Cell Types Compared to Non-Toxic 2D Boron Nitride Effects.

2D boron nitride (BN) and molybdenum disulfide (MoS2 ) materials are increasingly being used for applications due to novel chemical, electronic, and optical properties. Although generally considered biocompatible, recent data have shown that BN and MoS2 could potentially be hazardous under some biological conditions, for example, during, biodistribution of drug carriers or imaging agents to the liver. However, the effects of these 2D materials on liver cells such as Kupffer cells (KCs), liver sinusoidal endothelial cells, and hepatocytes, are unknown. Here, the toxicity of BN and MoS2 , dispersed in Pluronic F87 (designated BN-PF and MoS2 -PF) is compared with aggregated forms of these materials (BN-Agg and MoS2 -Agg) in liver cells. MoS2 induces dose-dependent cytotoxicity in KCs, but not other cell types, while the BN derivatives are non-toxic. The effect of MoS2 could be ascribed to nanosheet dissolution and the release of hexavalent Mo, capable of inducing mitochondrial reactive oxygen species generation and caspases 3/7-mediated apoptosis in KUP5 cells. In addition, the phagocytosis of MoS2 -Agg triggers an independent response pathway involving lysosomal damage, NLRP3 inflammasome activation, caspase-1 activation, IL-1ß, and IL-18 production. These findings demonstrate the importance of Mo release and the state of dispersion of MoS2 in impacting KC viability.

Mechanistic Differences in Cell Death Responses to Metal-Based Engineered Nanomaterials in Kupffer Cells and Hepatocytes.

The mononuclear phagocyte system in the liver is a frequent target for nanoparticles (NPs). A toxicological profiling of metal-based NPs is performed in Kupffer cell (KC) and hepatocyte cell lines. Sixteen NPs are provided by the Nanomaterial Health Implications Research Consortium of the National Institute of Environmental Health Sciences to study the toxicological effects in KUP5 (KC) and Hepa 1-6 cells. Five NPs (Ag, CuO, ZnO, SiO2 , and V2 O5 ) exhibit cytotoxicity in both cell types, while SiO2 and V2 O5 induce IL-1ß production in KC. Ag, CuO, and ZnO induced caspase 3 generated apoptosis in both cell types is accompanied by ion shedding and generation of mitochondrial reactive oxygen species (ROS) in both cell types. However, the cell death response to SiO2 in KC differs by inducing pyroptosis as a result of potassium efflux, caspase 1 activation, NLRP3 inflammasome assembly, IL-1ß release, and cleavage of gasdermin-D. This releases pore-performing peptide fragments responsible for pyroptotic cell swelling. Interestingly, although V2 O5 induces IL-1ß release and delays caspase 1 activation by vanadium ion interference in membrane Na+ /K+ adenosine triphosphate (ATP)ase activity, the major cell death mechanism in KC (and Hepa 1-6) is caspase 3 mediated apoptosis. These findings improve the understanding of the mechanisms of metal-based engineered nanomaterial (ENM) toxicity in liver cells toward comprehensive safety evaluation.

The Crystallinity and Aspect Ratio of Cellulose Nanomaterials Determine Their Pro-Inflammatory and Immune Adjuvant Effects In Vitro and In Vivo.

Nanocellulose is increasingly considered for applications; however, the fibrillar nature, crystalline phase, and surface reactivity of these high aspect ratio nanomaterials need to be considered for safe biomedical use. Here a comprehensive analysis of the impact of cellulose nanofibrils (CNF) and nanocrystals (CNC) is performed using materials provided by the Nanomaterial Health Implications Research Consortium of the National Institute of Environmental Health Sciences. An intermediary length of nanocrystals is also derived by acid hydrolysis. While all CNFs and CNCs are devoid of cytotoxicity, 210 and 280 nm fluorescein isothiocyanate (FITC)-labeled CNCs show higher cellular uptake than longer and shorter CNCs or CNFs. Moreover, CNCs in the 200-300 nm length scale are more likely to induce lysosomal damage, NLRP3 inflammasome activation, and IL-1ß production than CNFs. The pro-inflammatory effects of CNCs are correlated with higher crystallinity index, surface hydroxyl density, and reactive oxygen species generation. In addition, CNFs and CNCs can induce maturation of bone marrow-derived dendritic cells and CNCs (and to a lesser extent CNFs) are found to exert adjuvant effects in ovalbumin (OVA)-injected mice, particularly for 210 and 280 nm CNCs. All considered, the data demonstrate the importance of length scale, crystallinity, and surface reactivity in shaping the innate immune response to nanocellulose.

Toxicological Profiling of Highly Purified Single-Walled Carbon Nanotubes with Different Lengths in the Rodent Lung and Escherichia Coli.

Carbon nanotubes (CNTs) exhibit a number of physicochemical properties that contribute to adverse biological outcomes. However, it is difficult to define the independent contribution of individual properties without purified materials. A library of highly purified single-walled carbon nanotubes (SWCNTs) of different lengths is prepared from the same base material by density gradient ultracentrifugation, designated as short (318 nm), medium (789 nm), and long (1215 nm) SWCNTs. In vitro screening shows length-dependent interleukin-1ß (IL-1ß) production, in order of long > medium > short. However, there are no differences in transforming growth factor-ß1 production in BEAS-2B cells. Oropharyngeal aspiration shows that all the SWCNTs induce profibrogenic effects in mouse lung at 21 d postexposure, but there are no differences between tube lengths. In contrast, these SWCNTs demonstrate length-dependent antibacterial effects on Escherichia coli, with the long SWCNT exerting stronger effects than the medium or short tubes. These effects are reduced by Pluronic F108 coating or supplementing with glucose. The data show length-dependent effects on proinflammatory response in macrophage cell line and antibacterial effects, but not on collagen deposition in the lung. These data demonstrate that over the length scale tested, the biological response to highly purified SWCNTs is dependent on the complexity of the nano/bio interface.

The Genetic Heterogeneity among Different Mouse Strains Impacts the Lung Injury Potential of Multiwalled Carbon Nanotubes.

Genetic variation constitutes an important variable impacting the susceptibility to inhalable toxic substances and air pollutants, as reflected by epidemiological studies in humans and differences among animal strains. While multiwalled carbon nanotubes (MWCNTs) are capable of causing lung fibrosis in rodents, it is unclear to what extent the genetic variation in different mouse strains influence the outcome. Four inbred mouse strains, including C57Bl/6, Balb/c, NOD/ShiLtJ, and A/J, to test the pro-fibrogenic effects of a library of MWCNTs in vitro and in vivo are chosen. Ex vivo analysis of IL-1ß production in bone marrow-derived macrophages (BMDMs) as molecular initiating event (MIE) is performed. The order of cytokine production (Balb/c > A/J > C57Bl/6 > NOD/ShiLtJ) in BMDMs is also duplicated during assessment of IL-1ß production in the bronchoalveolar lavage fluid of the same mouse strains 40 h after oropharyngeal instillation of a representative MWCNT. Animal test after 21 d also confirms a similar hierarchy in TGF-ß1 production and collagen deposition in the lung. Statistical analysis confirms a correlation between IL-1ß production in BMDM and the lung fibrosis. All considered, these data demonstrate that genetic background indeed plays a major role in determining the pro-fibrogenic response to MWCNTs in the lung.

Reduction of pulmonary toxicity of metal oxide nanoparticles by phosphonate-based surface passivation.

BACKGROUND: The wide application of engineered nanoparticles has induced increasing exposure to humans and environment, which led to substantial concerns on their biosafety. Some metal oxides (MOx) have shown severe toxicity in cells and animals, thus safe designs of MOx with reduced hazard potential are desired. Currently, there is a lack of a simple yet effective safe design approach for the toxic MOx. In this study, we determined the key physicochemical properties of MOx that lead to cytotoxicity and explored a safe design approach for toxic MOx by modifying their hazard properties. RESULTS: THP-1 and BEAS-2B cells were exposed to 0-200 µg/mL MOx for 24 h, we found some toxic MOx including CoO, CuO, Ni2O3 and Co3O4, could induce reactive oxygen species (ROS) generation and cell death due to the toxic ion shedding and/or oxidative stress generation from the active surface of MOx internalized into lysosomes. We thus hypothesized that surface passivation could reduce or eliminate the toxicity of MOx. We experimented with a series of surface coating molecules and discovered that ethylenediamine tetra (methylene phosphonic acid) (EDTMP) could form stable hexadentate coordination with MOx. The coating layer can effectively reduce the surface activity of MOx with 85-99% decrease of oxidative potential, and 65-98% decrease of ion shedding. The EDTMP coated MOx show negligible ROS generation and cell death in THP-1 and BEAS-2B cells. The protective effect of EDTMP coating was further validated in mouse lungs exposed to 2 mg/kg MOx by oropharyngeal aspiration. After 40 h exposure, EDTMP coated MOx show significant decreases of neutrophil counts, lactate dehydrogenase (LDH) release, MCP-1, LIX and IL-6 in bronchoalveolar lavage fluid (BALF), compared to uncoated particles. The haematoxylin and eosin (H&E) staining results of lung tissue also show EDTMP coating could significantly reduce the pulmonary inflammation of MOx. CONCLUSIONS: The surface reactivity of MOx including ion shedding and oxidative potential is the dominated physicochemical property that is responsible for the cytotoxicity induced by MOx. EDTMP coating could passivate the surface of MOx, reduce their cytotoxicity and pulmonary hazard effects. This coating would be an effective safe design approach for a broad spectrum of toxic MOx, which will facilitate the safe use of MOx in commercial nanoproducts.

Long-Term Effects of Multiwalled Carbon Nanotubes and Graphene on Microbial Communities in Dry Soil.

Little is known about the long-term effects of engineered carbonaceous nanomaterials (ECNMs) on soil microbial communities, especially when compared to possible effects of natural or industrial carbonaceous materials. To address these issues, we exposed dry grassland soil for 1 year to 1 mg g(-1) of either natural nanostructured material (biochar), industrial carbon black, three types of multiwalled carbon nanotubes (MWCNTs), or graphene. Soil microbial biomass was assessed by substrate induced respiration and by extractable DNA. Bacterial and fungal communities were examined by terminal restriction fragment length polymorphism (T-RFLP). Microbial activity was assessed by soil basal respiration. At day 0, there was no treatment effect on soil DNA or T-RFLP profiles, indicating negligible interference between the amended materials and the methods for DNA extraction, quantification, and community analysis. After a 1-year exposure, compared to the no amendment control, some treatments reduced soil DNA (e.g., biochar, all three MWCNT types, and graphene; P < 0.05) and altered bacterial communities (e.g., biochar, carbon black, narrow MWCNTs, and graphene); however, there were no significant differences across the amended treatments. These findings suggest that ECNMs may moderately affect dry soil microbial communities but that the effects are similar to those from natural and industrial carbonaceous materials, even after 1-year exposure.

Mammalian Cells Exhibit a Range of Sensitivities to Silver Nanoparticles that are Partially Explicable by Variations in Antioxidant Defense and Metallothionein Expression.

While it is well known that there are interspecies differences in Ag sensitivity, differences in the cytotoxic responses of mammalian cells to silver nanoparticles (Ag NPs) are also observed. In order to explore these response outcomes, six cell lines, including epithelial cells (Caco-2, NHBE, RLE-6TN, and BEAS-2B) and macrophages (RAW 264.7 and THP-1) of human and rodent origin, are exposed to 20 nm citrate- and PVP-coated Ag NPs with Au cores, as well as 20 nm citrate-coated particles without cores. An MTS assay shows that while Caco-2 and NHBE cells are resistant to particles over a 0.1-50 µg mL(-1) dose range, RAW 264.7, THP-1, RLE-6TN, and BEAS-2B cells are more susceptible. While there are small differences in dissolution rates, there are no major differences in the cytotoxic potential of the different particles. However, differences in anti-oxidant defense and metallothionein expression among different cell types are observed, which can partially explain differential Ag NP sensitivity. So, it is important to consider these differences in understanding the potential heterogeneous effects of nano Ag on mammalian biological systems.

NADPH Oxidase-Dependent NLRP3 Inflammasome Activation and its Important Role in Lung Fibrosis by Multiwalled Carbon Nanotubes.

The purpose of this paper is to elucidate the key role of NADPH oxidase in NLRP3 inflammasome activation and generation of pulmonary fibrosis by multi-walled carbon nanotubes (MWCNTs). Although it is known that oxidative stress plays a role in pulmonary fibrosis by single-walled CNTs, the role of specific sources of reactive oxygen species, including NADPH oxidase, in inflammasome activation remains to be clarified. In this study, three long aspect ratio (LAR) materials (MWCNTs, single-walled carbon nanotubes, and silver nanowires) are used to compare with spherical carbon black and silver nanoparticles for their ability to trigger oxygen burst activity and NLRP3 assembly. All LAR materials but not spherical nanoparticles induce robust NADPH oxidase activation and respiratory burst activity in THP-1 cells, which are blunted in p22(phox) -deficient cells. The NADPH oxidase is directly involved in lysosomal damage by LAR materials, as demonstrated by decreased cathepsin B release and IL-1ß production in p22(phox) -deficient cells. Reduced respiratory burst activity and inflammasome activation are also observed in bone marrow-derived macrophages from p47(phox) -deficient mice. Moreover, p47(phox) -deficient mice have reduced IL-1ß production and lung collagen deposition in response to MWCNTs. Lung fibrosis is also suppressed by N-acetyl-cysteine in wild-type animals exposed to MWCNTs.

Differences in the Toxicological Potential of 2D versus Aggregated Molybdenum Disulfide in the Lung.

2D molybdenum disulfide (MoS2 ) has distinct optical and electronic properties compared to aggregated MoS2 , enabling wide use of these materials for electronic and biomedical applications. However, the hazard potential of MoS2 has not been studied extensively. Here, a comprehensive analysis of the pulmonary hazard potential of three aqueous suspended forms of MoS2 -aggregated MoS2 (Agg-MoS2 ), MoS2 exfoliated by lithiation (Lit-MoS2 ), and MoS2 dispersed by Pluronic F87 (PF87-MoS2 )-is presented. No cytotoxicity is detected in THP-1 and BEAS-2B cell lines. However, Agg-MoS2 induces strong proinflammatory and profibrogenic responses in vitro. In contrast, Lit- and PF87-MoS2 have little or no effect. In an acute toxicity study in mice, Agg-MoS2 induces acute lung inflammation, while Lit-MoS2 and PF87-MoS2 have little or no effect. In a subchronic study, there is no evidence of pulmonary fibrosis in response to all forms of MoS2 . These data suggest that exfoliation attenuates the toxicity of Agg-MoS2 , which is an important consideration toward the safety evaluation and use of nanoscale MoS2 materials for industrial and biological applications.

Toxicity of metal oxide nanoparticles in Escherichia coli correlates with conduction band and hydration energies.

Metal oxide nanoparticles (MOx NPs) are used for a host of applications, such as electronics, cosmetics, construction, and medicine, and as a result, the safety of these materials to humans and the environment is of considerable interest. A prior study of 24 MOx NPs in mammalian cells revealed that some of these materials show hazard potential. Here, we report the growth inhibitory effects of the same series of MOx NPs in the bacterium Escherichia coli and show that toxicity trends observed in E. coli parallel those seen previously in mammalian cells. Of the 24 materials studied, only ZnO, CuO, CoO, Mn2O3, Co3O4, Ni2O3, and Cr2O3 were found to exert significant growth inhibitory effects; these effects were found to relate to membrane damage and oxidative stress responses in minimal trophic media. A correlation of the toxicological data with physicochemical parameters of MOx NPs revealed that the probability of a MOx NP being toxic increases as the hydration enthalpy becomes less negative and as the conduction band energy approaches those of biological molecules. These observations are consistent with prior results observed in mammalian cells, revealing that mechanisms of toxicity of MOx NPs are consistent across two very different taxa. These results suggest that studying nanotoxicity in E. coli may help to predict toxicity patterns in higher organisms.

PdO doping tunes band-gap energy levels as well as oxidative stress responses to a Co3O4 p-type semiconductor in cells and the lung.

We demonstrate through PdO doping that creation of heterojunctions on Co3O4 nanoparticles can quantitatively adjust band-gap and Fermi energy levels to study the impact of metal oxide nanoparticle semiconductor properties on cellular redox homeostasis and hazard potential. Flame spray pyrolysis (FSP) was used to synthesize a nanoparticle library in which the gradual increase in the PdO content (0-8.9%) allowed electron transfer from Co3O4 to PdO to align Fermi energy levels across the heterojunctions. This alignment was accompanied by free hole accumulation at the Co3O4 interface and production of hydroxyl radicals. Interestingly, there was no concomitant superoxide generation, which could reflect the hole dominance of a p-type semiconductor. Although the electron flux across the heterojunctions induced upward band bending, the E(c) levels of the doped particles showed energy overlap with the biological redox potential (BRP). This allows electron capture from the redox couples that maintain the BRP from -4.12 to -4.84 eV, causing disruption of cellular redox homeostasis and induction of oxidative stress. PdO/Co3O4 nanoparticles showed significant increases in cytotoxicity at 25, 50, 100, and 200 µg/mL, which was enhanced incrementally by PdO doping in BEAS-2B and RAW 264.7 cells. Oxidative stress presented as a tiered cellular response involving superoxide generation, glutathione depletion, cytokine production, and cytotoxicity in epithelial and macrophage cell lines. A progressive series of acute pro-inflammatory effects could also be seen in the lungs of animals exposed to incremental PdO-doped particles. All considered, generation of a combinatorial PdO/Co3O4 nanoparticle library with incremental heterojunction density allowed us to demonstrate the integrated role of E(v), E(c), and E(f) levels in the generation of oxidant injury and inflammation by the p-type semiconductor, Co3O4.

Use of coated silver nanoparticles to understand the relationship of particle dissolution and bioavailability to cell and lung toxicological potential.

Since more than 30% of consumer products that include engineered nanomaterials contain nano-Ag, the safety of this material is of considerable public concern. In this study, Ag nanoparticles (NPs) are used to demonstrate that 20 nm polyvinylpyrrolidone (PVP or P) and citrate (C)-coated Ag NPs induce more cellular toxicity and oxidative stress than larger (110 nm) particles due to a higher rate of dissolution and Ag bioavailability. Moreover, there is also a higher propensity for citrate 20 nm (C20) nanoparticles to generate acute neutrophilic inflammation in the lung and to produce chemokines compared to C110. P110 has less cytotoxic effects than C110, likely due to the ability of PVP to complex released Ag(+) . In contrast to the more intense acute pulmonary effects of C20, C110 induces mild pulmonary fibrosis at day 21, likely as a result of slow but persistent Ag(+) release leading to a sub-chronic injury response. Interestingly, the released metallic Ag is incorporated into the collagen fibers depositing around airways and the lung interstitium. Taken together, these results demonstrate that size and surface coating affect the cellular toxicity of Ag NPs as well as their acute versus sub-chronic lung injury potential.

Reprogramming the pancreatic cancer stroma and immune landscape by a silicasome nanocarrier delivering nintedanib, a protein tyrosine kinase inhibitor.

The prevailing desmoplastic stroma and immunosuppressive microenvironment within pancreatic ductal adenocarcinoma (PDAC) pose substantial challenges to therapeutic intervention. Despite the potential of protein tyrosine kinase (PTK) inhibitors in mitigating the desmoplastic stromal response and enhancing the immune milieu, their efficacy is curtailed by suboptimal pharmacokinetics (PK) and insufficient tumor penetration. To surmount these hurdles, we have pioneered a novel strategy, employing lipid bilayer-coated mesoporous silica nanoparticles (termed "silicasomes") as a carrier for the delivery of Nintedanib. Nintedanib, a triple PTK inhibitor that targets vascular endothelial growth factor, platelet-derived growth factor and fibroblast growth factor receptors, was encapsulated in the pores of silicasomes via a remote loading mechanism for weak bases. This innovative approach not only enhanced pharmacokinetics and intratumor drug concentrations but also orchestrated a transformative shift in the desmoplastic and immune landscape in a robust orthotopic KRAS-mediated pancreatic carcinoma (KPC) model. Our results demonstrate attenuation of vascular density and collagen content through encapsulated Nintedanib treatment, concomitant with significant augmentation of the CD8+/FoxP3+ T-cell ratio. This remodeling was notably correlated with tumor regression in the KPC model. Strikingly, the synergy between encapsulated Nintedanib and anti-PD-1 immunotherapy further potentiated the antitumor effect. Both free and encapsulated Nintedanib induced a transcriptional upregulation of PD-L1 via the extracellular signal-regulated kinase (ERK) pathway. In summary, our pioneering approach involving the silicasome carrier not only improved antitumor angiogenesis but also profoundly reshaped the desmoplastic stromal and immune landscape within PDAC. These insights hold excellent promise for the development of innovative combinatorial strategies in PDAC therapy.

Zebrafish high-throughput screening to study the impact of dissolvable metal oxide nanoparticles on the hatching enzyme, ZHE1.

The zebrafish is emerging as a model organism for the safety assessment and hazard ranking of engineered nanomaterials. In this Communication, the implementation of a roboticized high-throughput screening (HTS) platform with automated image analysis is demonstrated to assess the impact of dissolvable oxide nanoparticles on embryo hatching. It is further demonstrated that this hatching interference is mechanistically linked to an effect on the metalloprotease, ZHE 1, which is responsible for degradation of the chorionic membrane. The data indicate that 4 of 24 metal oxide nanoparticles (CuO, ZnO, Cr2 O3 , and NiO) could interfere with embryo hatching by a chelator-sensitive mechanism that involves ligation of critical histidines in the ZHE1 center by the shed metal ions. A recombinant ZHE1 enzymatic assay is established to demonstrate that the dialysates from the same materials responsible for hatching interference also inhibit ZHE1 activity in a dose-dependent fashion. A peptide-based BLAST search identifies several additional aquatic species that express enzymes with homologous histidine-based catalytic centers, suggesting that the ZHE1 mechanistic paradigm could be used to predict the toxicity of a large number of oxide nanoparticles that pose a hazard to aquatic species.

Nanocarrier Co-formulation for Delivery of a TLR7 Agonist plus an Immunogenic Cell Death Stimulus Triggers Effective Pancreatic Cancer Chemo-immunotherapy.

Although toll-like receptor (TLR) agonists hold great promise as immune modulators for reprogramming the suppressive immune landscape in pancreatic ductal adenocarcinoma (PDAC), their use is limited by poor pharmacokinetics (PK) and off-target systemic inflammatory effects. To overcome these challenges as well as to attain drug synergy, we developed a lipid bilayer (LB)-coated mesoporous silica nanoparticle (silicasome) platform for co-delivery of the TLR7/8 agonist 3M-052 with the immunogenic chemotherapeutic agent irinotecan. This was accomplished by incorporating the C18 lipid tail of 3M-052 in the coated LB, also useful for irinotecan remote loading in the porous interior. Not only did the co-formulated carrier improve PK, but it strengthened the irinotecan-induced immunogenic cell death response by 3M-052-mediated dendritic cell activation at the tumor site as well as participating lymph nodes. The accompanying increase in CD8+ T-cell infiltration along with a reduced number of regulatory T-cells was associated with tumor shrinkage and metastasis disappearance in subcutaneous and orthotopic KRAS-mediated pancreatic carcinoma tumor models. Moreover, this therapeutic outcome was accomplished without drug or nanocarrier toxicity. All considered, dual-delivery strategies that combine chemo-immunotherapy with co-formulated TLR agonists or other lipid-soluble immune modulators predict successful intervention in heterogeneous PDAC immune landscapes.