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.

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.

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.

Lateral size of graphene oxide determines differential cellular uptake and cell death pathways in Kupffer cells, LSECs, and hepatocytes.

As a representative two-dimensional (2D) nanomaterial, graphene oxide (GO) has shown high potential in many applications due to its large surface area, high flexibility, and excellent dispersibility in aqueous solutions. These properties make GO an ideal candidate for bio-imaging, drug delivery, and cancer therapy. When delivered to the body, GO has been shown to accumulate in the liver, the primary accumulation site of systemic delivery or secondary spread from other uptake sites, and induce liver toxicity. However, the contribution of the GO physicochemical properties and individual liver cell types to this toxicity is unclear due to property variations and diverse cell types in the liver. Herein, we compare the effects of GOs with small (GO-S) and large (GO-L) lateral sizes in three major cell types in liver, Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatocytes. While GOs induced cytotoxicity in KCs, they induced significantly less toxicity in LSECs and hepatocytes. For KCs, we found that GOs were phagocytosed that triggered NADPH oxidase mediated plasma membrane lipid peroxidation, which leads to PLC activation, calcium flux, mitochondrial ROS generation, and NLRP3 inflammasome activation. The subsequent caspase-1 activation induced IL-1ß production and GSDMD-mediated pyroptosis. These effects were lateral size-dependent with GO-L showing stronger effects than GO-S. Amongst the liver cell types, decreased cell association and the absence of lipid peroxidation resulted in low cytotoxicity in LSECs and hepatocytes. Using additional GO samples with different lateral sizes, surface functionalities, or thickness, we further confirmed the differential cytotoxic effects in liver cells and the major role of GO lateral size in KUP5 pyroptosis by correlation studies. These findings delineated the GO effects on cellular uptake and cell death pathways in liver cells, and provide valuable information to further evaluate GO effects on the liver for biomedical applications.

Combination Chemo-Immunotherapy for Pancreatic Cancer Using the Immunogenic Effects of an Irinotecan Silicasome Nanocarrier Plus Anti-PD-1.

There is an urgent need to develop new life-prolonging therapy for pancreatic ductal adenocarcinoma (PDAC). It is demonstrated that improved irinotecan delivery by a lipid bilayer coated mesoporous silica nanoparticle, also known as a silicasome, can improve PDAC survival through a chemo-immunotherapy response in an orthotopic Kras-dependent pancreatic cancer model. This discovery is premised on the weak-basic properties of irinotecan, which neutralizes the acidic lysosomal pH in PDAC cells. This effect triggers a linked downstream cascade of events that include autophagy inhibition, endoplasmic reticulum stress, immunogenic cell death (ICD), and programmed death-ligand 1 (PD-L1) expression. ICD is characterized by calreticulin expression and high-mobility group box 1 (HMGB1) release in dying Kras-induced pancreatic cancer (KPC) cells, which is demonstrated in a vaccination experiment to prevent KPC tumor growth on the contralateral site. The improved delivery of irinotecan by the silicasome is accompanied by robust antitumor immunity, which can be synergistically enhanced by anti-PD-1 in the orthotopic model. Immunophenotyping confirms the expression of calreticulin, HMGB1, PD-L1, and an autophagy marker, in addition to perforin and granzyme B deposition. The chemo-immunotherapy response elicited by the silicasome is more robust than free or a liposomal drug, Onivyde. The silicasome plus anti-PD-1 leads to significantly enhanced survival improvement, and is far superior to anti-PD-1 plus either free irinotecan or Onivyde.

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.

Arsenic, Cadmium, Lead, and Mercury in Lactation Foods and Prenatal Vitamins: Potentially Avoidable Exposure for Breastfeeding Mothers and Infants.

Background: Human milk is a biofluid that can contain heavy metals such as arsenic, cadmium, lead, and mercury. These toxins can adversely affect endocrine, respiratory, immune, and nervous systems. Infants may have higher dietary arsenic exposure than adults due to their more restricted diets and greater intake per unit body mass. We identified commonly purchased vitamins, lactation bars, and supplements, to measure the concentration of heavy metals. The goal of the study was to measure and determine if vitamins and lactation foods could be a source of exposure. Methods: We tested 9 popular vitamins and 16 lactation bars and supplements all marketed toward breastfeeding mothers to determine the presence of heavy metals. All vitamins, bars, and supplements were analyzed using inductively coupled plasma mass spectrometry, for the total concentration of arsenic, mercury, cadmium, and lead, with the lowest detection limit of 0.001 µg/L. Results: The majority of the samples had total arsenic levels below detectable quantities (detection limit of 1 part per trillion [0.001 µg/L]), one sample of syrup had a concentration of 0.112 ± 0.005, 0.132 ± 0.009, and 0.108 ± 0.010 µg of arsenic/g. We also tested nine popular prenatal vitamins for the amount of arsenic in one serving of vitamins per day and showed exposure to arsenic in one vitamin of 7.108 µg of arsenic/day. All lactation foods and vitamins tested had undetectable amounts of cadmium, mercury, and lead. Conclusion: Most of the vitamins and bars that were tested had below the detectable limit of arsenic, cadmium, lead, and mercury; we did find one vitamin and one rice syrup that had significantly higher levels. Our data suggest that it is highly feasible to manufacture vitamins and lactation foods and supplements with significantly lower concentrations of heavy metals.

Immune checkpoint inhibition in syngeneic mouse cancer models by a silicasome nanocarrier delivering a GSK3 inhibitor.

Checkpoint blocking antibodies that interfere in the PD-1/PD-L1 axis provide effective cancer immunotherapy for tumors that are immune inflamed or induced to become "hot". It has also been demonstrated that a small molecule inhibitor of the signaling hub kinase GSK3 can interfere in the PD-1/PD-L1 axis in T-cells by suppressing PD-1 expression. This provides an alternative approach to intervening in the PD-1/PD-L1 axis to provide cancer immunotherapy. In this communication, we demonstrate the remote loading of GSK3 inhibitor AZD1080 into the porous interior of mesoporous silica nanoparticles coated with a lipid bilayer (a.k.a. silicasomes). In a MC38 colon cancer model, intravenous injection (IV) of silicasome-encapsulated AZD1080 significantly improved biodistribution and drug delivery to the tumor site. The improved drug delivery was accompanied by cytotoxic MC38 tumor cell killing by perforin-releasing CD8+ T-cells, exhibiting reduced PD-1 expression. IV injection of encapsulated AZD1080 also resulted in significant tumor shrinkage in other syngeneic mouse tumor models, including another colorectal tumor (CT26), as well as pancreas (KPC) and lung (LLC) cancer models. Not only was the therapeutic efficacy of encapsulated AZD1080 similar or better than anti-PD-1 antibody, but the treatment was devoid of treatment toxicity. These results provide proof-of-principal demonstration of the feasibility of using encapsulated delivery of a GSK3 inhibitor to provide cancer immunotherapy, with the possibility to be used as a monotherapy or in combination with chemotherapy or other immunomodulatory agents.

Liposomal Delivery of Mitoxantrone and a Cholesteryl Indoximod Prodrug Provides Effective Chemo-immunotherapy in Multiple Solid Tumors.

We developed a custom-designed liposome carrier for codelivery of a potent immunogenic cell death (ICD) stimulus plus an inhibitor of the indoleamine 2,3-dioxygenase (IDO-1) pathway to establish a chemo-immunotherapy approach for solid tumors in syngeneic mice. The carrier was constructed by remote import of the anthraquinone chemotherapeutic agent, mitoxantrone (MTO), into the liposomes, which were further endowed with a cholesterol-conjugated indoximod (IND) prodrug in the lipid bilayer. For proof-of-principle testing, we used IV injection of the MTO/IND liposome in a CT26 colon cancer model to demonstrate the generation of a robust immune response, characterized by the appearance of ICD markers (CRT and HMGB-1) as well as evidence of cytotoxic cancer cell death, mediated by perforin and granzyme B. Noteworthy, the cytotoxic effects involved natural killer (NK) cell, which suggests a different type of ICD response. The immunotherapy response was significantly augmented by codelivery of the IND prodrug, which induced additional CRT expression, reduced number of Foxp3+ Treg, and increased perforin release, in addition to extending animal survival beyond the effect of an MTO-only liposome. The outcome reflects the improved pharmacokinetics of MTO delivery to the cancer site by the carrier. In light of the success in the CT26 model, we also assessed the platform efficacy in further breast cancer (EMT6 and 4T1) and renal cancer (RENCA) models, which overexpress IDO-1. Encapsulated MTO delivery was highly effective for inducing chemo-immunotherapy responses, with NK participation, in all tumor models. Moreover, the growth inhibitory effect of MTO was enhanced by IND codelivery in EMT6 and 4T1 tumors. All considered, our data support the use of encapsulated MTO delivery for chemo-immunotherapy, with the possibility to boost the immune response by codelivery of an IDO-1 pathway inhibitor.

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.

Improved Efficacy and Reduced Toxicity Using a Custom-Designed Irinotecan-Delivering Silicasome for Orthotopic Colon Cancer.

Irinotecan is a key chemotherapeutic agent for the treatment of colorectal (CRC) and pancreatic (PDAC) cancer. Because of a high incidence of bone marrow and gastrointestinal (GI) toxicity, Onivyde (a liposome) was introduced to provide encapsulated irinotecan (Ir) delivery in PDAC patients. While there is an ongoing clinical trial (NCT02551991) to investigate the use of Onivyde as a first-line option to replace irinotecan in FOLFIRINOX, the liposomal formulation is currently prescribed as a second-line treatment option (in combination with 5-fluorouracil and leucovorin) for patients with metastatic PDAC who failed gemcitabine therapy. However, the toxicity of Onivyde remains a concern that needs to be addressed for use in CRC as well. Our goal was to custom design a mesoporous silica nanoparticle (MSNP) carrier for encapsulated irinotecan delivery in a robust CRC model. This was achieved by developing an orthotopic tumor chunk model in immunocompetent mice. With a view to increase the production volume and to expand the disease applications, the carrier design was improved by using an ethanol exchange method for coating of a supported lipid bilayer (LB) that entraps a protonating agent. The encapsulated protonating agent was subsequently used for remote loading of irinotecan. The excellent irinotecan loading capacity and stability of the LB-coated MSNP carrier, also known as a "silicasome", previously showed improved efficacy and reduced toxicity when compared to an in-house liposomal carrier in a PDAC model. Intravenous injection of the silicasomes in a well-developed orthotopic colon cancer model in mice demonstrated improved pharmacokinetics and tumor drug content over free drug and Onivyde. Moreover, improved drug delivery was accompanied by substantially improved efficacy, increased survival, and reduced bone marrow and GI toxicity compared to the free drug and Onivyde. We also confirmed that the custom-designed irinotecan silicasomes outperform Onivyde in an orthotopic PDAC model. In summary, the Ir-silicasome appears to be promising as a treatment option for CRC in humans based on improved efficacy and the carrier's favorable safety profile.

Development of self-assembled multi-arm polyrotaxanes nanocarriers for systemic plasmid delivery in vivo.

Polyrotaxane (PRX) is a promising supramolecular carrier for gene delivery. Classic PRX exhibits a linear structure in which the amine-functionalized α-cyclodextrin (CD) is threaded along the entire polyethylene glycol (PEG) backbone. While promising in vitro, the absence of free PEG moieties after CD threading compromised the in vivo implementation, due to the unfavorable pharmacokinetics (PK) and biodistribution profile. Herein, we developed a multi-arm PRX nanocarrier platform, which has been designed for protective nucleic acid encapsulation, augmented biodistribution and PK, and suitable for intravenous (IV) administration. A key design was to introduce cationic CD rings onto a multi-arm PEG backbone in a spatially selective fashion. The optimal structural design was obtained through iterative rounds of experimentation to determine the appropriate type and density of cationic charge on CD ring, the degree of PEGylation, the size and structure of polymer backbone, etc. This allowed us to effectively deliver large size reporter and therapeutic plasmids in cancer mouse models. Post IV injection, we demonstrated that our multi-arm polymer design significantly enhanced circulatory half-life and PK profile compared to the linear PRX. We continued to use the multi-arm PRX to formulate a therapeutic plasmid encoding an immunomodulatory cytokine, IL-12. When tested in a colon cancer syngeneic mouse model with same background, the IL-12 plasmid was protected by the multi-arm PRX and delivered through the tail vein to the tumor site, leading to a significant tumor inhibition effect. Moreover, our delivery system was devoid of major systemic toxicity.

Toxicological Profiling of Metal Oxide Nanoparticles in Liver Context Reveals Pyroptosis in Kupffer Cells and Macrophages versus Apoptosis in Hepatocytes.

The liver and the mononuclear phagocyte system are a frequent target for engineered nanomaterials, either as a result of particle uptake and spread from primary exposure sites or systemic administration of therapeutic and imaging nanoparticles. In this study, we performed a comparative analysis of the toxicological impact of 29 metal oxide nanoparticles (NPs), some commonly used in consumer products, in transformed or primary Kupffer cells (KCs) and hepatocytes. We not only observed differences between KCs and hepatocytes, but also differences in the toxicological profiles of transition-metal oxides (TMOs, e. g., Co3O4) versus rare-earth oxide (REO) NPs ( e. g., Gd2O3). While pro-oxidative TMOs induced the activation of caspases 3 and 7, resulting in apoptotic cell death in both cell types, REOs induced lysosomal damage, NLRP3 inflammasome activation, caspase 1 activation, and pyroptosis in KCs. Pyroptosis was accompanied by cell swelling, membrane blebbing, IL-1ß release, and increased membrane permeability, which could be reversed by knockdown of the pore forming protein, gasdermin D. Though similar features were not seen in hepatocytes, the investigation of the cytotoxic effects of REO NPs could also be seen to affect macrophage cell lines such as J774A.1 and RAW 264.7 cells as well as bone marrow-derived macrophages. These phagocytic cell types also demonstrated features of pyroptosis and increased IL-1ß production. Collectively, these findings demonstrate important mechanistic considerations that can be used for safety evaluation of metal oxides, including commercial products that are developed from these materials.

Surface Oxidation of Graphene Oxide Determines Membrane Damage, Lipid Peroxidation, and Cytotoxicity in Macrophages in a Pulmonary Toxicity Model.

While two-dimensional graphene oxide (GO) is used increasingly in biomedical applications, there is uncertainty on how specific physicochemical properties relate to biocompatibility in mammalian systems. Although properties such as lateral size and the colloidal properties of the nanosheets are important, the specific material properties that we address here is the oxidation state and reactive surface groups on the planar surface. In this study, we used a GO library, comprising pristine, reduced (rGO), and hydrated GO (hGO), in which quantitative assessment of the hydroxyl, carboxyl, epoxy, and carbon radical contents was used to study the impact on epithelial cells and macrophages, as well as in the murine lung. Strikingly, we observed that hGO, which exhibits the highest carbon radical density, was responsible for the generation of cell death in THP-1 and BEAS-2B cells as a consequence of lipid peroxidation of the surface membrane, membrane lysis, and cell death. In contrast, pristine GO had lesser effects, while rGO showed extensive cellular uptake with minimal effects on viability. In order to see how these in vitro effects relate to adverse outcomes in the lung, mice were exposed to GOs by oropharyngeal aspiration. Animal sacrifice after 40 h demonstrated that hGO was more prone than other materials to generate acute lung inflammation, accompanied by the highest lipid peroxidation in alveolar macrophages, cytokine production (LIX, MCP-1), and LDH release in bronchoalveolar lavage fluid. Pristine GO showed less toxicity, whereas rGO had minimal effects. We demonstrate that the surface oxidation state and carbon radical content play major roles in the induction of toxicity by GO in mammalian cells and the lung.

Nano-enabled pancreas cancer immunotherapy using immunogenic cell death and reversing immunosuppression.

While chemotherapy delivery by nanocarriers has modestly improved the survival prospects of pancreatic ductal adenocarcinoma (PDAC), additional engagement of the immune response could be game changing. We demonstrate a nano-enabled approach for accomplishing robust anti-PDAC immunity in syngeneic mice through the induction of immunogenic cell death (ICD) as well as interfering in the immunosuppressive indoleamine 2,3-dioxygenase (IDO) pathway. This is accomplished by conjugating the IDO inhibitor, indoximod (IND), to a phospholipid that allows prodrug self-assembly into nanovesicles or incorporation into a lipid bilayer that encapsulates mesoporous silica nanoparticles (MSNP). The porous MSNP interior allows contemporaneous delivery of the ICD-inducing chemotherapeutic agent, oxaliplatin (OX). The nanovesicles plus free OX or OX/IND-MSNP induce effective innate and adaptive anti-PDAC immunity when used in a vaccination approach, direct tumor injection or intravenous biodistribution to an orthotopic PDAC site. Significant tumor reduction or eradication is accomplishable by recruiting cytotoxic T lymphocytes, concomitant with downregulation of Foxp3+ T cells.

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.

Enhanced Immune Adjuvant Activity of Aluminum Oxyhydroxide Nanorods through Cationic Surface Functionalization.

Aluminum-salt-based vaccine adjuvants are prevailingly used in FDA-approved vaccines for the prevention of infectious diseases for over eighty years. Despite their safe applications, the mechanisms regarding how the material characteristics affect the interactions at nano-bio interface and immunogenicity remain unclear. Recently, studies have indicated that the activation of NLRP3 inflammasome plays a critical role in inducing adjuvant effects that are controlled by the inherent shape and hydroxyl contents of aluminum oxyhydroxide (AlOOH) nanoparticles; however, the detailed relationship between surface properties and adjuvant effects for these materials remains unknown. Thus, we engineered AlOOH nanorods (ALNRs) with controlled surface functionalization and charge to assess their effects on the activation of NLRP3 inflammasome in vitro and the potentiation of immunogenicity in vivo. It is demonstrated that NH2-functionalized ALNRs exhibited higher levels of cellular uptake, lysosomal damage, oxidative stress, and NLRP3 inflammasome activation than pristine and SO3H-functionalized ALNRs in cells. This structure-activity relationship also correlates with the adjuvant activity of the material using ovalbumin (OVA) in a mouse vaccination model. This study demonstrates that surface functionalization of ALNRs is critical for rational design of aluminum-based adjuvants to boost antigen-specific immune responses for more effective and long-lasting vaccination.

Agglomeration Determines Effects of Carbonaceous Nanomaterials on Soybean Nodulation, Dinitrogen Fixation Potential, and Growth in Soil.

The potential effects of carbonaceous nanomaterials (CNMs) on agricultural plants are of concern. However, little research has been performed using plants cultivated to maturity in soils contaminated with various CNMs at different concentrations. Here, we grew soybean for 39 days to seed production in soil amended with 0.1, 100, or 1000 mg kg-1 of either multiwalled carbon nanotubes (MWCNTs), graphene nanoplatelets (GNPs), or carbon black (CB) and studied plant growth, nodulation, and dinitrogen (N2) fixation potential. Plants in all CNM treatments flowered earlier (producing 60% to 372% more flowers when reproduction started) than the unamended controls. The low MWCNT-treated plants were shorter (by 15%) with slower leaf cover expansion (by 26%) and less final leaf area (by 24%) than the controls. Nodulation and N2 fixation potential appeared negatively impacted by CNMs, with stronger effects at lower CNM concentrations. All CNM treatments reduced the whole-plant N2 fixation potential, with the highest reductions (by over 91%) in the low and medium CB and the low MWCNT treatments. CB and GNPs appeared to accumulate inside nodules as observed by transmission electron microscopy. CNM dispersal in aqueous soil extracts was studied to explain the inverse dose-response relationships, showing that CNMs at higher concentrations were more agglomerated (over 90% CNMs settled as agglomerates >3 µm after 12 h) and therefore proportionally less bioavailable. Overall, our findings suggest that lower concentrations of CNMs in soils could be more impactful to leguminous N2 fixation, owing to greater CNM dispersal and therefore increased bioavailability at lower concentrations.