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.

Nerve Growth Factor-Conjugated Mesoporous Silica Nanoparticles Promote Neuron-Like PC12 Cell Proliferation and Neurite Growth.

Therapeutic strategies to promote nerve cell growth and improve their functions or stimulate nerve fiber reconnection and ameliorate the loss of neuronal functions are in high demand. A disadvantage of current conventional methods, which includes injection of nerve growth factors (NGF) either systemically or in the affected area, is rapid clearance or degradation of NGF, thereby reducing the effective concentration of NGFs that can reach the damaged nerves to stimulate the healing process. To overcome this obstacle, a nanoparticle platform based on mesoporous silica nanoparticles (MSNs) was developed to not only prevent clearance and degradation of NGFs, but also deliver the NGF directly to nerve cells to promote nerve cell proliferation and neurite growth. We synthesized (NGF)-loaded MSN (MSN-NGF) with a diameter of 65 nm. MSN-NGF significantly promoted the differentiation of neuron-like PC12 cells and growth of neurites compared to NGF alone, as confirmed by MTS cell proliferation assay and optical microscopy analysis. This study shows that MSN-NGF could be an effective therapy to speed up nerve cell growth or recovery of function.

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.

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.

Pluronic F108 coating decreases the lung fibrosis potential of multiwall carbon nanotubes by reducing lysosomal injury.

We compared the use of bovine serum albumin (BSA) and pluronic F108 (PF108) as dispersants for multiwalled carbon nanotubes (MWCNTs) in terms of tube stability as well as profibrogenic effects in vitro and in vivo. While BSA-dispersed tubes were a potent inducer of pulmonary fibrosis, PF108 coating protected the tubes from damaging the lysosomal membrane and initiating a sequence of cooperative cellular events that play a role in the pathogenesis of pulmonary fibrosis. Our results suggest that PF108 coating could serve as a safer design approach for MWCNTs.

Use of Stromal Intervention and Exogenous Neoantigen Vaccination to Boost Pancreatic Cancer Chemo-Immunotherapy by Nanocarriers.

Despite the formidable treatment challenges of pancreatic ductal adenocarcinoma (PDAC), considerable progress has been made in improving drug delivery via pioneering nanocarriers. These innovations are geared towards overcoming the obstacles presented by dysplastic stroma and fostering anti-PDAC immune reactions. We are currently conducting research aimed at enhancing chemotherapy to stimulate anti-tumor immunity by inducing immunogenic cell death (ICD). This is accomplished using lipid bilayer-coated nanocarriers, which enable the attainment of synergistic results. Noteworthy examples include liposomes and lipid-coated mesoporous silica nanoparticles known as "silicasomes". These nanocarriers facilitate remote chemotherapy loading, as well as the seamless integration of immunomodulators into the lipid bilayer. In this communication, we elucidate innovative ways for further improving chemo-immunotherapy. The first is the development of a liposome platform engineered by the remote loading of irinotecan while incorporating a pro-resolving lipoxin in the lipid bilayer. This carrier interfered in stromal collagen deposition, as well as boosting the irinotecan-induced ICD response. The second approach was to synthesize polymer nanoparticles for the delivery of mutated KRAS peptides in conjunction with a TLR7/8 agonist. The dual delivery vaccine particle boosted the generation of antigen-specific cytotoxic T-cells that are recruited to lymphoid structures at the cancer site, with a view to strengthening the endogenous vaccination response achieved by chemo-immunotherapy.

Use of a Liver-Targeting Immune-Tolerogenic mRNA Lipid Nanoparticle Platform to Treat Peanut-Induced Anaphylaxis by Single- and Multiple-Epitope Nucleotide Sequence Delivery.

While oral desensitization is capable of alleviating peanut allergen anaphylaxis, long-term immune tolerance is the sought-after goal. We developed a liver-targeting lipid nanoparticle (LNP) platform to deliver mRNA-encoded peanut allergen epitopes to liver sinusoidal endothelial cells (LSECs), which function as robust tolerogenic antigen-presenting cells that induce FoxP3+ regulatory T-cells (Tregs). The mRNA strand was constructed by including nucleotide sequences encoding for nonallergenic MHC-II binding T-cell epitopes, identified in the dominant peanut allergen, Ara h2. These epitopes were inserted in the mRNA strand downstream of an MHC-II targeting sequence, further endowed in vitro with 5' and 3' capping sequences, a PolyA tail, and uridine substitution. Codon-optimized mRNA was used for microfluidics synthesis of LNPs with an ionizable cationic lipid, also decorated with a lipid-anchored mannose ligand for LSEC targeting. Biodistribution to the liver was confirmed by in vivo imaging, while ELISpot assays demonstrated an increase in IL-10-producing Tregs in the spleen. Prophylactic administration of tandem-repeat or a combination of encapsulated Ara h2 epitopes induced robust tolerogenic effects in C3H/HeJ mice, sensitized to and subsequently challenged with crude peanut allergen extract. In addition to alleviating physical manifestations of anaphylaxis, there was suppression of Th2-mediated cytokine production, IgE synthesis, and mast cell release, accompanied by increased IL-10 and TGF-ß production in the peritoneum. Similar efficacy was demonstrated during LNP administration postsensitization. While nondecorated particles had lesser but significant effects, PolyA/LNP-Man lacked protective effects. These results demonstrate an exciting application of mRNA/LNP for treatment of food allergen anaphylaxis, with the promise to be widely applicable to the allergy field.

Differential Toxicity of Electronic Cigarette Aerosols Generated from Different Generations of Devices In Vitro and In Vivo.

Electronic cigarettes (e-cigs) have become increasingly popular, especially among youth, raising concerns about their potential health risks. JUUL and Tank devices are two common types of e-cigs that deliver aerosols with varying nicotine levels and flavors. However, the differences in the aerosols generated from different devices and their corresponding cytotoxicity and pulmonary injury effects remain poorly understood. This study addresses these knowledge gaps by characterizing the aerosols of JUUL and Tank e-cig devices and testing their toxic effects on THP-1 and BEAS-2B human cell lines as well as the C57BL/6J mouse model. In our study, the lower-voltage device, the 3.7 V JUUL generates 2.72 mg/puff aerosols by using e-liquid containing 3% nicotine salt (i.e., nicotine benzoate), which is less than the 11.06 mg/puff aerosols generated by the 7.5 V Tank using e-liquid containing 2.4% freebase nicotine. Yet, the cytotoxicity results reveal that JUUL aerosols induced higher toxicity and increased production of pro-inflammation cytokines compared to Tank aerosols per puff. Additionally, we observed that JUUL induced more severe pulmonary inflammation and DNA damage compared to Tank after normalizing for cotinine, a nicotine metabolite, in vivo. Our findings suggest that the device design plays a more important role in e-cig aerosol-induced toxicity than the composition of the e-liquid or voltage. These results provide valuable insights into the health risks associated with various electronic-cig devices and offer an approach for evaluating them.