A novel humanized mouse model for HIV and tuberculosis co-infection studies.

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to be a major public health problem worldwide. The human immunodeficiency virus (HIV) is another equally important life-threatening pathogen. HIV infection decreases CD4+ T cell levels markedly increasing Mtb co-infections. An appropriate animal model for HIV/Mtb co-infection that can recapitulate the diversity of the immune response in humans during co-infection would facilitate basic and translational research in HIV/Mtb infections. Herein, we describe a novel humanized mouse model. Methods: The irradiated NSG-SGM3 mice were transplanted with human CD34+ hematopoietic stem cells, and the humanization was monitored by staining various immune cell markers for flow cytometry. They were challenged with HIV and/or Mtb, and the CD4+ T cell depletion and HIV viral load were monitored over time. Before necropsy, the live mice were subjected to pulmonary function test and CT scan, and after sacrifice, the lung and spleen homogenates were used to determine Mtb load (CFU) and cytokine/chemokine levels by multiplex assay, and lung sections were analyzed for histopathology. The mouse sera were subjected to metabolomics analysis. Results: Our humanized NSG-SGM3 mice were able to engraft human CD34+ stem cells, which then differentiated into a full-lineage of human immune cell subsets. After co-infection with HIV and Mtb, these mice showed decrease in CD4+ T cell counts overtime and elevated HIV load in the sera, similar to the infection pattern of humans. Additionally, Mtb caused infections in both lungs and spleen, and induced granulomatous lesions in the lungs. Distinct metabolomic profiles were also observed in the tissues from different mouse groups after co-infections. Conclusion: The humanized NSG-SGM3 mice are able to recapitulate the pathogenic effects of HIV and Mtb infections and co-infection at the pathological, immunological and metabolism levels and are therefore a reproducible small animal model for studying HIV/Mtb co-infection.

Caveolin-1-derived peptide attenuates cigarette smoke-induced airway and alveolar epithelial injury.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease with no effective treatment that can reduce mortality or slow the disease progression. COPD is the third leading cause of global death and is characterized by airflow limitations due to chronic bronchitis and alveolar damage/emphysema. Chronic cigarette smoke (CS) exposure damages airway and alveolar epithelium and remains a major risk factor for the pathogenesis of COPD. We found that the expression of caveolin-1, a tumor suppressor protein; p53; and plasminogen activator inhibitor-1 (PAI-1), one of the downstream targets of p53, was markedly increased in airway epithelial cells (AECs) as well as in type II alveolar epithelial (AT2) cells from the lungs of patients with COPD or wild-type mice with CS-induced lung injury (CS-LI). Moreover, p53- and PAI-1-deficient mice resisted CS-LI. Furthermore, treatment of AECs, AT2 cells, or lung tissue slices from patients with COPD or mice with CS-LI with a seven amino acid caveolin-1 scaffolding domain peptide (CSP7) reduced mucus hypersecretion in AECs and improved AT2 cell viability. Notably, induction of PAI-1 expression via increased caveolin-1 and p53 contributed to mucous cell metaplasia and mucus hypersecretion in AECs, and reduced AT2 viability, due to increased senescence and apoptosis, which was abrogated by CSP7. In addition, treatment of wild-type mice having CS-LI with CSP7 by intraperitoneal injection or nebulization via airways attenuated mucus hypersecretion, alveolar injury, and significantly improved lung function. This study validates the potential therapeutic role of CSP7 for treating CS-LI and COPD. NEW & NOTEWORTHY Chronic cigarette smoke (CS) exposure remains a major risk factor for the pathogenesis of COPD, a debilitating disease with no effective treatment. Increased caveolin-1 mediated induction of p53 and downstream plasminogen activator inhibitor-1 (PAI-1) expression contributes to CS-induced airway mucus hypersecretion and alveolar wall damage. This is reversed by caveolin-1 scaffolding domain peptide (CSP7) in preclinical models, suggesting the therapeutic potential of CSP7 for treating CS-induced lung injury (CS-LI) and COPD.

The urinary proteome infers dysregulation of mitochondrial, lysosomal, and protein reabsorption processes in chronic kidney disease of unknown etiology (CKDu).

Chronic kidney disease (CKD) of uncertain etiology (CKDu) is a global health concern affecting tropical farming communities. CKDu is not associated with typical risk factors (e.g., diabetes) and strongly correlates with environmental drivers. To gain potential insights into disease etiology and diagnosis, here we report the first urinary proteome comparing patients with CKDu and non-CKDu controls from Sri Lanka. We found 944 differentially abundant proteins. In silico analyses identified 636 proteins of likely kidney and urogenital origin. As expected, renal tubular injury in patients with CKDu was evinced by increases in albumin, cystatin C, and ß2-microglobulin. However, several proteins typically elevated under CKD, including osteopontin and α-N-acetylglucosaminidase, were decreased in patients with CKDu. Furthermore, urinary excretion of aquaporins found higher in CKD was lower in CKDu. Comparisons with previous CKD urinary proteome datasets revealed a unique proteome for CKDu. Notably, the CKDu urinary proteome was relatively similar to that of patients with mitochondrial diseases. Furthermore, we report a decrease in endocytic receptor proteins responsible for protein reabsorption (megalin and cubilin) that correlated with an increase in abundance of 15 of their cognate ligands. Functional pathway analyses identified kidney-specific differentially abundant proteins in patients with CKDu denoted significant changes in the complement cascade and coagulation systems, cell death, lysosomal function, and metabolic pathways. Overall, our findings provide potential early detection markers to diagnose and distinguish CKDu and warrant further analyses on the role of lysosomal, mitochondrial, and protein reabsorption processes and their link to the complement system and lipid metabolism in CKDu onset and progression.NEW & NOTEWORTHY CKDu is a global health concern debilitating a number of tropical rural farming communities. In the absence of typical risk factors like diabetes and hypertension and the lack of molecular markers, it is crucial to identify potential early disease markers. Here, we detail the first urinary proteome profile to distinguish CKDu from CKD. Our data and in silico pathway analyses infer the roles of mitochondrial, lysosomal, and protein reabsorption processes in disease onset and progression.

Costimulatory CD226 Signaling Regulates Proliferation of Memory-like NK Cells in Healthy Individuals with Latent Mycobacterium tuberculosis Infection.

It is now widely accepted that NK cells can acquire memory, and this makes them more effective to protect against some pathogens. Prior reports indicate memory-like NK cells (mlNKs) in murine model of Mycobacterium tuberculosis (Mtb) as well as in healthy individuals with latent TB infection (LTBI). The increased expression of CD226 was evident in mlNKs from LTBI+ people after stimulation with γ-irradiated Mtb (γ-Mtb). We thus evaluated the contribution of costimulatory CD226 signaling in the functionality of mlNKs in LTBI+ people. We found that blockade of CD226 signaling using the antibody- or CRISPR/Cas9-mediated deletion of the CD226 gene in NK cells diminished the proliferation of mlNKs from LTBI+ people. Blocking CD226 signaling also reduced the phosphorylation of FOXO1 and cMyc expression. Additionally, cMyc inhibition using a chemical inhibitor reduced proliferation by mlNKs from LTBI+ people. Moreover, blocking CD226 signaling reduced glycolysis in NK cells, and the inhibition of glycolysis led to reduced effector function of mlNKs from LTBI+ people. Overall, our results provide a role for CD226 signaling in mlNK responses to Mtb.

Caveolin-1 scaffolding domain peptide abrogates autophagy dysregulation in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and fatal form of interstitial lung disease. IPF is characterized by irreversible scarring of the lungs leading to lung function decline. Although the etiology remains poorly understood, dysregulated autophagy in alveolar-epithelial cells (AECs) together with interplay between apoptotic-AECs and proliferative-myofibroblasts have been strongly implicated in IPF pathogenesis. Recent studies have revealed that a caveolin-1-derived 7-mer peptide, CSP7, mitigates established PF at least in part by improving AEC viability. In the present study, we aimed to determine whether and how CSP7 regulates autophagy in fibrotic-lung AECs. We found that p53 and autophagic proteins were markedly upregulated in AECs from mice with single/multi-doses of bleomycin-or silica-induced PF. This was abolished following treatment of PF-mice with CSP7. Further, CSP7 abrogated silica- or bleomycin-induced p53 and autophagy proteins in AECs. Immunoprecipitation further revealed that CSP7 abolishes the interaction of caveolin-1 with LC3BII and p62 in AECs. AEC-specific p53-knockout mice resisted silica- or bleomycin-induced changes in autophagy proteins, or CSP7 treatment. Our findings provide a novel mechanism by which CSP7 inhibits dysregulated autophagy in injured AECs and mitigates existing PF. These results affirm the potential of CSP7 for treating established PF, including IPF and silicosis.

Realistic biomarkers from plasma extracellular vesicles for detection of beryllium exposure.

PURPOSE: Exposures related to beryllium (Be) are an enduring concern among workers in the nuclear weapons and other high-tech industries, calling for regular and rigorous biological monitoring. Conventional biomonitoring of Be in urine is not informative of cumulative exposure nor health outcomes. Biomarkers of exposure to Be based on non-invasive biomonitoring could help refine disease risk assessment. In a cohort of workers with Be exposure, we employed blood plasma extracellular vesicles (EVs) to discover novel biomarkers of exposure to Be. METHODS: EVs were isolated from plasma using size-exclusion chromatography and subjected to mass spectrometry-based proteomics. A protein-based classifier was developed using LASSO regression and validated by ELISA. RESULTS: We discovered a dual biomarker signature comprising zymogen granule protein 16B and putative protein FAM10A4 that differentiated between Be-exposed and -unexposed subjects. ELISA-based quantification of the biomarkers in an independent cohort of samples confirmed higher expression of the signature in the Be-exposed group, displaying high predictive accuracy (AUROC = 0.919). Furthermore, the biomarkers efficiently discriminated high- and low-exposure groups (AUROC = 0.749). CONCLUSIONS: This is the first report of EV biomarkers associated with Be exposure and exposure levels. The biomarkers could be implemented in resource-limited settings for Be exposure assessment.

Memory like NK cells display stem cell like properties after Zika virus infection.

NK cells have been shown to display adaptive traits such as memory formation akin to T and B lymphocytes. Here we show that Zika virus infection induces memory like NK cells that express CD27. Strikingly, these cells exhibit stem-like features that include expansion capacity, self-renewal pathway, differentiation into effector cells, longer telomeres and gene signature associated with hematopoietic stem cell (HSC) progenitors. This subset shared transcriptional and epigenetic changes with memory CD8 T cells, stem cells and stem like T cells. These NK cells with memory and stem cell features, which we term "NK memory stem cells", demonstrated greater antiviral potential than CD27- or naïve CD27+ NK when adoptively transferred to Zika infected mice. Our results also suggest a role for the transcription factor TCF-1 in memory and stemness features of this NK subset. This study defines a unique TCF1hi CD27+ NK subset with memory capacity and stem cell features that play a role in antiviral immunity.

Age-Dependent Translocation of Gold Nanoparticles across the Air-Blood Barrier.

Do immature lungs have air-blood barriers that are more permeable to inhaled nanoparticles than those of fully developed mature lungs? Data supporting this notion and explaining the underlying mechanisms do not exist as far as we know. Using a rat model of postnatal lung development, here the data exactly supporting this notion, that is, significantly more gold nanoparticles (NPs) cross from the air space of the lungs to the rest of the body in neonates than in adults, are presented. Moreover, in neonates the translocation of gold NPs is not size dependent, whereas in adult animals smaller NPs cross the air-blood lung barrier much more efficiently than larger NPs. This difference in air-blood permeability in neonate versus adult animals suggests that NP translocation in the immature lungs may follow different rules than in mature lungs. Supporting this notion, we propose that the paracellular transport route may play a more significant role in NP translocation in immature animals, as suggested by protein expression studies. Findings from this study are critical to design optimal ways of inhalation drug delivery using NP nanocarriers for this age group, as well as for better understanding of the potential adverse health effects of nanoparticle exposures in infants and young children.

Fate of Barium Sulfate Nanoparticles Deposited in the Lungs of Rats.

We have shown that barium [from BaSO4 nanoparticles (NPs)] was cleared from the lungs faster than other poorly soluble NPs and translocated mostly to bone. We now studied barium biokinetics in rats during Study 1: two-year inhalation exposure to 50 mg/m3 BaSO4 NP aerosols, and Study 2: single intratracheal (IT) instillation of increasing doses of BaSO4 NPs or BaCl2. Study 1 showed that lung barium content measured by inductively coupled plasma mass spectrometry increased during 360 days of BaSO4 NP aerosol exposures. An equilibrium was established from that time until 2 years. Barium concentrations in BaSO4-exposed animals were in the order (lungs > lymph nodes > hard bone > bone marrow > liver). In Study 2, there was an increase in lung barium post-IT instillation of BaSO4 NPs while barium from BaCl2 was mostly cleared by day 28. Transmission electron microscopy showed intact BaSO4 NPs in alveolar macrophages and type II epithelial cells, and in tracheobronchial lymph nodes. Using stimulated Raman scattering microscopy, specific BaSO4 Raman spectra were detected in BaSO4 NP-instilled lungs and not in other organs. Thus, we posit that barium from BaSO4 NPs translocates from the lungs mainly after dissolution. Barium ions are then incorporated mostly into the bone and other organs.

Nanoparticle Wettability Influences Nanoparticle-Phospholipid Interactions.

We explored the influence of nanoparticle (NP) surface charge and hydrophobicity on NP-biomolecule interactions by measuring the composition of adsorbed phospholipids on four NPs, namely, positively charged CeO2 and ZnO and negatively charged BaSO4 and silica-coated CeO2, after exposure to bronchoalveolar lavage fluid (BALf) obtained from rats, and to a mixture of neutral dipalmitoyl phosphatidylcholine (DPPC) and negatively charged dipalmitoyl phosphatidic acid (DPPA). The resulting NP-lipid interactions were examined by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). Our data show that the amount of adsorbed lipids on NPs after incubation in BALf and the DPPC/DPPA mixture was higher in CeO2 than in the other NPs, qualitatively consistent with their relative hydrophobicity. The relative concentrations of specific adsorbed phospholipids on NP surfaces were different from their relative concentrations in the BALf. Sphingomyelin was not detected in the extracted lipids from the NPs despite its >20% concentration in the BALf. AFM showed that the more hydrophobic CeO2 NPs tended to be located inside lipid vesicles, whereas less hydrophobic BaSO4 NPs appeared to be outside. In addition, cryo-TEM analysis showed that CeO2 NPs were associated with the formation of multilamellar lipid bilayers, whereas BaSO4 NPs with unilamellar lipid bilayers. These data suggest that the NP surface hydrophobicity predominantly controls the amounts and types of lipids adsorbed, as well as the nature of their interaction with phospholipids.

Protein corona: implications for nanoparticle interactions with pulmonary cells.

BACKGROUND: We previously showed that cerium oxide (CeO2), barium sulfate (BaSO4) and zinc oxide (ZnO) nanoparticles (NPs) exhibited different lung toxicity and pulmonary clearance in rats. We hypothesize that these NPs acquire coronas with different protein compositions that may influence their clearance from the lungs. METHODS: CeO2, silica-coated CeO2, BaSO4, and ZnO NPs were incubated in rat lung lining fluid in vitro. Then, gel electrophoresis followed by quantitative mass spectrometry was used to characterize the adsorbed proteins stripped from these NPs. We also measured uptake of instilled NPs by alveolar macrophages (AMs) in rat lungs using electron microscopy. Finally, we tested whether coating of gold NPs with albumin would alter their lung clearance in rats. RESULTS: We found that the amounts of nine proteins in the coronas formed on the four NPs varied significantly. The amounts of albumin, transferrin and α-1 antitrypsin were greater in the coronas of BaSO4 and ZnO than that of the two CeO2 NPs. The uptake of BaSO4 in AMs was less than CeO2 and silica-coated CeO2 NPs. No identifiable ZnO NPs were observed in AMs. Gold NPs coated with albumin or citrate instilled into the lungs of rats acquired the similar protein coronas and were cleared from the lungs to the same extent. CONCLUSIONS: We show that different NPs variably adsorb proteins from the lung lining fluid. The amount of albumin in the NP corona varies as does NP uptake by AMs. However, albumin coating does not affect the translocation of gold NPs across the air-blood barrier. A more extensive database of corona composition of a diverse NP library will develop a platform to help predict the effects and biokinetics of inhaled NPs.

An integrated methodology for assessing the impact of food matrix and gastrointestinal effects on the biokinetics and cellular toxicity of ingested engineered nanomaterials.

BACKGROUND: Engineered nanomaterials (ENMs) are increasingly added to foods to improve their quality, sensory appeal, safety and shelf-life. Human exposure to these ingested ENMs (iENMS) is inevitable, yet little is known of their hazards. To assess potential hazards, efficient in vitro methodologies are needed to evaluate particle biokinetics and toxicity. These methodologies must account for interactions and transformations of iENMs in foods (food matrix effect) and in the gastrointestinal tract (GIT) that are likely to determine nano-biointeractions. Here we report the development and application of an integrated methodology consisting of three interconnected stages: 1) assessment of iENM-food interactions (food matrix effect) using model foods; 2) assessment of gastrointestinal transformations of the nano-enabled model foods using a three-stage GIT simulator; 3) assessment of iENMs biokinetics and cellular toxicity after exposure to simulated GIT conditions using a triculture cell model. As a case study, a model food (corn oil-in-water emulsion) was infused with Fe2O3 (Iron(III) oxide or ferric oxide) ENMs and processed using this three-stage integrated platform to study the impact of food matrix and GIT effects on nanoparticle biokinetics and cytotoxicity . METHODS: A corn oil in phosphate buffer emulsion was prepared using a high speed blender and high pressure homogenizer. Iron oxide ENM was dispersed in water by sonication and combined with the food model. The resulting nano-enabled food was passed through a three stage (mouth, stomach and small intestine) GIT simulator. Size distributions of nano-enabled food model and digestae at each stage were analyzed by DLS and laser diffraction. TEM and confocal imaging were used to assess morphology of digestae at each phase. Dissolution of Fe2O3 ENM along the GIT was assessed by ICP-MS analysis of supernatants and pellets following centrifugation of digestae. An in vitro transwell triculture epithelial model was used to assess biokinetics and toxicity of ingested Fe2O3 ENM. Translocation of Fe2O3 ENM was determined by ICP-MS analysis of cell lysates and basolateral compartment fluid over time. RESULTS: It was demonstrated that the interactions of iENMs with food and GIT components influenced nanoparticle fate and transport, biokinetics and toxicological profile. Large differences in particle size, charge, and morphology were observed in the model food with and without Fe2O3 and among digestae from different stages of the simulated GIT (mouth, stomach, and small intestine). Immunoflorescence and TEM imaging of the cell culture model revealed markers and morphology of small intestinal epithelium including enterocytes, goblet cells and M cells. Fe2O3 was not toxic at concentrations tested in the digesta. In biokinetics studies, translocation of Fe2O3 after 4 h was <1% and ~2% for digesta with and without serum, respectively, suggesting that use of serum proteins alters iENMs biokinetics and raises concerns about commonly-used approaches that neglect iENM - food-GIT interactions or dilute digestae in serum-containing media. CONCLUSIONS: We present a simple integrated methodology for studying the biokinetics and toxicology of iENMs, which takes into consideration nanoparticle-food-GIT interactions. The importance of food matrix and GIT effects on biointeractions was demonstrated, as well as the incorporation of these critical factors into a cellular toxicity screening model. Standardized food models still need to be developed and used to assess the effect of the food matrix effects on the fate and bioactivity of iENMs since commercial foods vary considerably in their compositions and structures.

Pulmonary distribution of nanoceria: comparison of intratracheal, microspray instillation and dry powder insufflation.

Particles can be delivered to the respiratory tract of animals using various techniques. Inhalation mimics environmental exposure but requires large amounts of aerosolized NPs over a prolonged dosing time, varies in deposited dose among individual animals, and results in nasopharyngeal and fur particle deposition. Although less physiological, intratracheal (IT) instillation allows quick and precise dosing. Insufflation delivers particles in their dry form as an aerosol. We compared the distribution of neutron-activated 141CeO2 nanoparticles (5 mg/kg) in rats after (1) IT instillation, (2) left intrabronchial instillation, (3) microspraying of nanoceria suspension and (4) insufflation of nanoceria dry powder. Blood, tracheobronchial lymph nodes, liver, gastrointestinal tract, feces and urine were collected at 5 min and 24 h post-dosing. Excised lungs from each rat were dried at room temperature while inflated at a constant 30 cm water pressure. Dried lungs were then sliced into 50 pieces. The radioactivity of each lung piece and other organs was measured. The evenness index (EI) of each lung piece was calculated [EI = (µCi/mgpiece)/(µCi/mglung)]. The degree of EI value departure from 1.0 is a measure of deposition heterogeneity. We showed that the pulmonary distribution of nanoceria differs among modes of administration. Dosing by IT or microspraying resulted in similar spatial distribution. Insufflation resulted in significant deposition in the trachea and in more heterogeneous lung distribution. Our left intrabronchial instillation technique yielded a concentrated deposition into the left lung. We conclude that animal dosing techniques and devices result in varying patterns of particle deposition that will impact biokinetic and toxicity studies.

Surface modification of zinc oxide nanoparticles with amorphous silica alters their fate in the circulation.

Nanoparticle (NP) pharmacokinetics and biological effects are influenced by many factors, especially surface physicochemical properties. We assessed the effects of an amorphous silica coating on the fate of zinc after intravenous (IV) injection of neutron activated uncoated (65)ZnO or silica-coated (65)ZnO NPs in male Wistar Han rats. Groups of IV-injected rats were sequentially euthanized, and 18 tissues were collected and analyzed for (65)Zn radioactivity. The protein coronas on each ZnO NP after incubation in rat plasma were analyzed by SDS-PAGE gel electrophoresis and mass spectrometry of selected gel bands. Plasma clearance for both NPs was biphasic with rapid initial and slower terminal clearance rates. Half-lives of plasma clearance of silica-coated (65)ZnO were shorter (initial - <1 min; terminal - 2.5 min) than uncoated (65)ZnO (initial - 1.9 min; terminal - 38 min). Interestingly, the silica-coated (65)ZnO group had higher (65)Zn associated with red blood cells and higher initial uptake in the liver. The (65)Zn concentrations in all the other tissues were significantly lower in the silica-coated than uncoated groups. We also found that the protein corona formed on silica-coated ZnO NPs had higher amounts of plasma proteins, particularly albumin, transferrin, A1 inhibitor 3, α-2-hs-glycoprotein, apoprotein E and α-1 antitrypsin. Surface modification with amorphous silica alters the protein corona, agglomerate size, and zeta potential of ZnO NPs, which in turn influences ZnO biokinetic behavior in the circulation. This emphasizes the critical role of the protein corona in the biokinetics, toxicology and nanomedical applications of NPs.

Silica coating influences the corona and biokinetics of cerium oxide nanoparticles.

BACKGROUND: The physicochemical properties of nanoparticles (NPs) influence their biological outcomes. METHODS: We assessed the effects of an amorphous silica coating on the pharmacokinetics and pulmonary effects of CeO2 NPs following intratracheal (IT) instillation, gavage and intravenous injection in rats. Uncoated and silica-coated CeO2 NPs were generated by flame spray pyrolysis and later neutron-activated. These radioactive NPs were IT-instilled, gavaged, or intravenously (IV) injected in rats. Animals were analyzed over 28 days post-IT, 7 days post-gavage and 2 days post-injection. RESULTS: Our data indicate that silica coating caused more but transient lung inflammation compared to uncoated CeO2. The transient inflammation of silica-coated CeO2 was accompanied by its enhanced clearance. Then, from 7 to 28 days, clearance was similar although significantly more (141)Ce from silica-coated (35%) was cleared than from uncoated (19%) (141)CeO2 in 28 days. The protein coronas of the two NPs were significantly different when they were incubated with alveolar lining fluid. Despite more rapid clearance from the lungs, the extrapulmonary (141)Ce from silica-coated (141)CeO2 was still minimal (<1%) although lower than from uncoated (141)CeO2 NPs. Post-gavage, nearly 100% of both NPs were excreted in the feces consistent with very low gut absorption. Both IV-injected (141)CeO2 NP types were primarily retained in the liver and spleen. The silica coating significantly altered the plasma protein corona composition and enhanced retention of (141)Ce in other organs except the liver. CONCLUSION: We conclude that silica coating of nanoceria alters the biodistribution of cerium likely due to modifications in protein corona formation after IT and IV administration.

Bioavailability, distribution and clearance of tracheally-instilled and gavaged uncoated or silica-coated zinc oxide nanoparticles.

BACKGROUND: Nanoparticle pharmacokinetics and biological effects are influenced by several factors. We assessed the effects of amorphous SiO2 coating on the pharmacokinetics of zinc oxide nanoparticles (ZnO NPs) following intratracheal (IT) instillation and gavage in rats. METHODS: Uncoated and SiO2-coated ZnO NPs were neutron-activated and IT-instilled at 1 mg/kg or gavaged at 5 mg/kg. Rats were followed over 28 days post-IT, and over 7 days post-gavage. Tissue samples were analyzed for 65Zn radioactivity. Pulmonary responses to instilled NPs were also evaluated at 24 hours. RESULTS: SiO2-coated ZnO elicited significantly higher inflammatory responses than uncoated NPs. Pulmonary clearance of both 65ZnO NPs was biphasic with a rapid initial t1/2 (0.2 - 0.3 hours), and a slower terminal t1/2 of 1.2 days (SiO2-coated ZnO) and 1.7 days (ZnO). Both NPs were almost completely cleared by day 7 (>98%). With IT-instilled 65ZnO NPs, significantly more 65Zn was found in skeletal muscle, liver, skin, kidneys, cecum and blood on day 2 in uncoated than SiO2-coated NPs. By 28 days, extrapulmonary levels of 65Zn from both NPs significantly decreased. However, 65Zn levels in skeletal muscle, skin and blood remained higher from uncoated NPs. Interestingly, 65Zn levels in bone marrow and thoracic lymph nodes were higher from coated 65ZnO NPs. More 65Zn was excreted in the urine from rats instilled with SiO2-coated 65ZnO NPs. After 7 days post-gavage, only 7.4% (uncoated) and 6.7% (coated) of 65Zn dose were measured in all tissues combined. As with instilled NPs, after gavage significantly more 65Zn was measured in skeletal muscle from uncoated NPs and less in thoracic lymph nodes. More 65Zn was excreted in the urine and feces with coated than uncoated 65ZnO NPs. However, over 95% of the total dose of both NPs was eliminated in the feces by day 7. CONCLUSIONS: Although SiO2-coated ZnO NPs were more inflammogenic, the overall lung clearance rate was not affected. However, SiO2 coating altered the tissue distribution of 65Zn in some extrapulmonary tissues. For both IT instillation and gavage administration, SiO2 coating enhanced transport of 65Zn to thoracic lymph nodes and decreased transport to the skeletal muscle.

Impaired clearance and enhanced pulmonary inflammatory/fibrotic response to carbon nanotubes in myeloperoxidase-deficient mice.

Advancement of biomedical applications of carbonaceous nanomaterials is hampered by their biopersistence and pro-inflammatory action in vivo. Here, we used myeloperoxidase knockout B6.129X1-MPO (MPO k/o) mice and showed that oxidation and clearance of single walled carbon nanotubes (SWCNT) from the lungs of these animals after pharyngeal aspiration was markedly less effective whereas the inflammatory response was more robust than in wild-type C57Bl/6 mice. Our results provide direct evidence for the participation of MPO - one of the key-orchestrators of inflammatory response - in the in vivo pulmonary oxidative biodegradation of SWCNT and suggest new ways to control the biopersistence of nanomaterials through genetic or pharmacological manipulations.

Carbon nanotubes degraded by neutrophil myeloperoxidase induce less pulmonary inflammation.

We have shown previously that single-walled carbon nanotubes can be catalytically biodegraded over several weeks by the plant-derived enzyme, horseradish peroxidase. However, whether peroxidase intermediates generated inside human cells or biofluids are involved in the biodegradation of carbon nanotubes has not been explored. Here, we show that hypochlorite and reactive radical intermediates of the human neutrophil enzyme myeloperoxidase catalyse the biodegradation of single-walled carbon nanotubes in vitro, in neutrophils and to a lesser degree in macrophages. Molecular modelling suggests that interactions of basic amino acids of the enzyme with the carboxyls on the carbon nanotubes position the nanotubes near the catalytic site. Importantly, the biodegraded nanotubes do not generate an inflammatory response when aspirated into the lungs of mice. Our findings suggest that the extent to which carbon nanotubes are biodegraded may be a major determinant of the scale and severity of the associated inflammatory responses in exposed individuals.

Phosphatidylserine targets single-walled carbon nanotubes to professional phagocytes in vitro and in vivo.

Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid "eat-me" signal, phosphatidylserine (PS), makes them recognizable in vitro by different phagocytic cells - murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells, and rat brain microglia. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells.