Preparation and in Vitro Antitumor Study of Two-Dimensional Muscovite Nanosheets.

Inorganic nanosheets are endowed with many two-dimensional (2D) morphological features including ultra-high specific surface area, ultra-thin thickness, easy functionalization, and so on. They push forward an immense influence on effective cancer diagnosis and therapy, overcoming the inherent limitations of traditional treatment methods. However, long-term toxicity and poor biocompatibility are the critical issues for most inorganic nanosheets, which hinder their further oncological applications and clinical translations. Muscovite, also named white mica (WM), an aluminosilicate, is a major component of traditional Chinese medicine, which can be exfoliated into 2D nanosheets and expected to be a potential drug carrier. In this study, WM powder was exfoliated to prepare WM nanosheets (WMNs) through a polyamine intercalation method. In addition, doxorubicin hydrochloride (Dox) was loaded to WMNs via physical adsorption and electrostatic interaction to prepare Dox-loaded WMNs (Dox@WMNs). Then, we studied that Dox@WMNs released Dox in phosphate buffer saline. We also studied the cellular uptake and cytotoxicity of Dox@WMNs in vitro. The results illustrated that Dox@WMNs cumulatively released Dox much faster and more at acidic pH (6.0 and 4.6) compared with that at physiological pH. In addition, WMNs showed selective cytotoxicity. Within a certain concentration range, WMNs were cytotoxic to Hela cells but non-cytotoxic to RAW 264.7 cells. Compared with cytotoxicity at pH 7.4, the cytotoxicity of Dox@WMNs was significantly enhanced at pH 6.4 and 4.6. WMNs mainly promoted the immunostimulatory polarization of RAW 264.7 cells into M1 macrophages.

Dendritic Mesoporous Silica Nanoparticle Adjuvants Modified with Binuclear Aluminum Complex: Coordination Chemistry Dictates Adjuvanticity.

Aluminum-containing adjuvants used in vaccine formulations suffer from low cellular immunity, severe aggregation, and accumulation in the brain. Conventional aluminosilicates widely used in the chemical industry focus mainly on acidic sites for catalytic applications, but they are rarely used as adjuvants. Reported here is an innovative "ligand-assisted steric hindrance" strategy to create a high density of six-coordinate VI Al-OH groups with basicity on dendritic mesoporous silica nanoparticles as new nanoadjuvants. Compared to four-coordinate IV Al-modified counterparts, VI Al-OH-rich aluminosilicate nanoadjuvants enhance cellular delivery of antigens and provoke stronger cellular immunity. Moreover, the aluminum accumulation in the brain is more reduced than that with a commercial adjuvant. These results show that coordination chemistry can be used to control the adjuvanticity, providing new understanding in the development of next-generation vaccine adjuvants.

The effect of two novel cholesterol-lowering agents, disodium ascorbyl phytostanol phosphate (DAPP) and nanostructured aluminosilicate (NSAS) on the expression and activity of P-glycoprotein within Caco-2 cells.

BACKGROUND: Many drugs are substrates for P-glycoprotein (P-gp) and interactions involving P-gp may be relevant to clinical practice. Co-administration with P-gp inhibitors or inducers changes the absorption profile as well as the risk for drug toxicity, therefore it is important to evaluate possible P-gp alterations. The purpose of this study was to investigate the effect of two novel cholesterol-lowering agents, disodium ascorbyl phytostanol phosphate (DAPP) and nanostructured aluminium silicate (NSAS), a protonated montmorillonite clay, on mdr-1 gene expression and its protein, P-glycoprotein (P-gp) within Caco-2 cells. METHODS: The effects of DAPP and NSAS on the regulation of mdr-1 gene, P-gp protein expression and activity within Caco-2 cells, were determined using cell viability and cytotoxicity tests, RT-PCR, Western Blot analysis and bi-directional transport studies. RESULTS: We observed a significant down-regulation of mdr-1 mRNA (e.g. 38.5 ± 17% decrease vs. control at 5 µM DAPP and 61.2 ± 25% versus control at 10 µM DAPP; n = 6, P* < 0.05) within Caco-2 cells. Western Blot analysis of P-gp expression showed that changes in mdr-1 gene expression lead to correlating changes in P-gp protein expression. This down-regulation of P-glycoprotein also resulted in decreased activity of P-glycoprotein compared to untreated control. In contrast, when Caco-2 cells were treated with NSAS, no changes in mdr-1 gene expression, P-gp protein expression nor P-gp activity were observed. CONCLUSIONS: DAPP but not NSAS decreases P-gp mediated drug efflux through decreased mdr-1 gene expression and consequently decreased P-gp protein expression. These findings have to be taken into consideration when DAPP is concurrently given with other drugs that are substrates for P-gp since drug-drug interactions harbour a safety issue and alter bioavailability profiles.NSAS does not have any P-gp altering properties and therefore might not affect drug-drug interactions. We conclude from this study that NSAS might make a safer drug candidate compared to DAPP for lowering LDL-cholesterol.

Total allowable concentrations of monomeric inorganic aluminum and hydrated aluminum silicates in drinking water.

Maximum contaminant levels are used to control potential health hazards posed by chemicals in drinking water, but no primary national or international limits for aluminum (Al) have been adopted. Given the differences in toxicological profiles, the present evaluation derives total allowable concentrations for certain water-soluble inorganic Al compounds (including chloride, hydroxide, oxide, phosphate and sulfate) and for the hydrated Al silicates (including attapulgite, bentonite/montmorillonite, illite, kaolinite) in drinking water. The chemistry, toxicology and clinical experience with Al materials are extensive and depend upon the particular physical and chemical form. In general, the water solubility of the monomeric Al materials depends on pH and their water solubility and gastrointestinal bioavailability are much greater than that of the hydrated Al silicates. Other than Al-containing antacids and buffered aspirin, food is the primary source of Al exposure for most healthy people. Systemic uptake of Al after ingestion of the monomeric salts is somewhat greater from drinking water (0.28%) than from food (0.1%). Once absorbed, Al accumulates in bone, brain, liver and kidney, with bone as the major site for Al deposition in humans. Oral Al hydroxide is used routinely to bind phosphate salts in the gut to control hyperphosphatemia in people with compromised renal function. Signs of chronic Al toxicity in the musculoskeletal system include a vitamin D-resistant osteomalacia (deranged membranous bone formation characterized by accumulation of the osteoid matrix and reduced mineralization, reduced numbers of osteoblasts and osteoclasts, decreased lamellar and osteoid bands with elevated Al concentrations) presenting as bone pain and proximal myopathy. Aluminum-induced bone disease can progress to stress fractures of the ribs, femur, vertebrae, humerus and metatarsals. Serum Al ≥100 µg/L has a 75-88% positive predictive value for Al bone disease. Chronic Al toxicity is also manifest in the hematopoietic system as an erythropoietin-resistant microcytic hypochromic anemia. Signs of Al toxicity in the central nervous system (speech difficulty to total mutism to facial grimacing to multifacial seizures and dyspraxia) are related to Al accumulation in the brain and these symptoms can progress to frank encephalopathy. There are four groups of people at elevated risk of systemic Al intoxication after repeated ingestion of monomeric Al salts: the preterm infant, the infant with congenital uremia and children and adults with kidney disease. There is a dose-dependent increase in serum and urinary Al in people with compromised renal function, and restoration of renal function permits normal handling of systemically absorbed Al and resolution of Al bone disease. Clinical experience with 960 mg/day of Al(OH)(3) (~5 mg Al/kg-day) given by mouth over 3 months to men and women with compromised renal function found subclinical reductions in hemoglobin, hematocrit and serum ferritin. Following adult males and females with reduced kidney function found that ingestion of Al(OH)(3) at 2.85 g/day (~40 mg/kg-day Al) over 7 years increased bone Al, but failed to elicit significant bone toxicity. There was one report of DNA damage in cultured lymphocytes after high AlCl(3) exposure, but there is no evidence that ingestion of common inorganic Al compounds presents an increased carcinogenic risk or increases the risk for adverse reproductive or developmental outcomes. A number of studies of Al exposure in relation to memory in rodents have been published, but the results are inconsistent. At present, there is no evidence to substantiate the hypothesis that the pathogenesis of Alzheimer's Disease is caused by Al found in food and drinking water at the levels consumed by people living in North America and Western Europe. Attapulgite (palygorskite) has been used for decades at oral doses (recommended not to exceed two consecutive days) of 2,100 mg/day in children of 3-6 years, 4,200 mg/day in children of 6-12 years, and 9,000 mg/day in adults. Chronic ingestion of insoluble hydrated Al silicates (in kg) can result in disturbances in iron and potassium status, primarily as a result of clay binding to intestinal contents and enhanced fecal iron and zinc elimination. Sufficiently high doses of ingested Al silicates (≥50 g/day) over prolonged periods of time can elicit a deficiency anemia that can be corrected with oral Fe supplements. There is essentially no systemic Al uptake after ingestion of the hydrated Al silicates. Rats fed up to 20,000 ppm Ca montmorillonite (equivalent to 1,860 ppm total Al as the hydrated Al silicate) for 28 weeks failed to develop any adverse signs. The results of dietary Phase I and II clinical trials conducted in healthy adult volunteers over 14 days and 90 days with montmorillonite found no adverse effects after feeding up to 40 mg/kg-day as Al. Since the Al associated with ingestion of hydrated Al silicates is not absorbed into the systemic circulation, the hydrated Al silicates seldom cause medical problems unless the daily doses consumed are substantially greater than those used clinically or as dietary supplements. A no-observable-adverse-effect-level (NOAEL) of 13 mg/kg-day as total Al can be identified based on histologic osteomalacia seen in adult hemodialysis patients given Al hydroxide for up to 7 years as a phosphate binder. Following U.S. EPA methods for calculation of an oral reference dose (RfD), an intraspecies uncertainty factor of 10x was applied to that value results in a chronic oral reference dose (RfD) of 1.3 mg Al/kg-day; assuming a 70-kg adult consumes 2 L of drinking water per day and adjusting for a default 20% relative source contribution that value corresponds to a drinking water maximum concentration of 9 mg/L measured as total Al. A chronic NOAEL for montmorillonite as representative of the hydrated Al silicates was identified from the highest dietary concentration (20,000 ppm) fed in a 28-week bioassay with male and female Sprague-Dawley rats. Since young rats consume standard laboratory chow at ~23 g/day, this concentration corresponds to 56 mg Al/kg-day. Application of 3x interspecies uncertainty factor and a 3x factor to account for study duration results in a chronic oral RfD of 6 mg Al/kg-day. Of note, this RfD is 5-10 fold less than oral doses of Al silicates consumed by people who practice clay geophagy and it corresponds to a maximum drinking water concentration of 40 mg Al/L. To utilize the values derived here, the risk manager must recognize the particular product (e.g., alum) or source (e.g., groundwater, river water, clay or cement pipe) of the Al found in tap water, apply the appropriate analytical methods (atomic absorption, energy dispersive X-ray diffraction, infrared spectral analysis and/or scanning transmission electron microscopy) and compare the results to the most relevant standard. The drinking water concentrations derived here are greater than the U.S. EPA secondary maximum contaminant level (MCL) for total Al of 0.05-0.2 mg/L [40 CFR 143.3]. As such, domestic use of water with these concentrations is likely self-limiting given that its cloudy appearance will be greater than the maximum permitted (0.5-5.0 nephalometric turbidity units; 40 CFR Parts 141 and 142). Therefore, the organoleptic properties of Al materials in water determine public acceptance of potable water as contrast to any potential health hazard at the concentrations ordinarily present in municipal drinking water.

Pulmonary inflammatory and fibrotic responses in Fischer 344 rats after intratracheal instillation exposure to Libby amphibole.

Increased incidences of asbestosis have been reported in workers from Libby, MT, associated with exposures to amphibole-contaminated vermiculite. In this study pulmonary and histopathological changes were investigated following Libby amphibole (LA) exposure in a rat model. Rat respirable fractions of LA and amosite (aerodynamic diameter <2.5 µm) were prepared by water elutriation. Male F344 rats were exposed to single doses of either saline (SAL), amosite (0.65 mg/rat), or LA (0.65 or 6.5 mg/rat) by intratracheal instillation. At times from 1 d to 3 mo after exposure, bronchoalveolar lavage (BAL) was performed and right and left lungs were removed for reverse-transcription polymerase chain reaction (RT-PCR) and histopathological analysis, respectively. Data indicated that 0.65 mg amosite resulted in a higher degree of pulmonary injury, inflammation, and fibrotic events than LA at the same mass dose. Exposure to either amosite or high dose LA resulted in higher levels of cellular permeability and injury, inflammatory enzymes, and iron binding proteins in both BAL fluid and lung tissue at most time points when compared to SAL controls. However, mRNA expression for some growth factors (e.g., platelet-derived growth factor [PDGF]-A and transforming growth factor [TGF]-1ß), which contribute to fibrosis, were downregulated at several time points. Furthermore, histopathological examination showed notable thickening of interstitial areas surrounding the alveolar ducts and terminal bronchioles. On a mass dose basis, amosite produced a greater acute and persistent lung injury for at least 3 mo after exposure. However, further testing and analysis of LA are needed with regard to the dose metric to fully evaluate its potential fibrogenicity and carcinogenicity.

What makes a natural clay antibacterial?

Natural clays have been used in ancient and modern medicine, but the mechanism(s) that make certain clays lethal against bacterial pathogens has not been identified. We have compared the depositional environments, mineralogies, and chemistries of clays that exhibit antibacterial effects on a broad spectrum of human pathogens including antibiotic resistant strains. Natural antibacterial clays contain nanoscale (<200 nm), illite-smectite and reduced iron phases. The role of clay minerals in the bactericidal process is to buffer the aqueous pH and oxidation state to conditions that promote Fe(2+) solubility. Chemical analyses of E. coli killed by aqueous leachates of an antibacterial clay show that intracellular concentrations of Fe and P are elevated relative to controls. Phosphorus uptake by the cells supports a regulatory role of polyphosphate or phospholipids in controlling Fe(2+). Fenton reaction products can degrade critical cell components, but we deduce that extracellular processes do not cause cell death. Rather, Fe(2+) overwhelms outer membrane regulatory proteins and is oxidized when it enters the cell, precipitating Fe(3+) and producing lethal hydroxyl radicals.

Toxicological studies of "Chondrokola Rosh", an Ayurvedic preparation on liver function tests of rats.

Chondrokola Rosh (CKR) is a traditional metallic Ayurvedic preparation widely used by the rural and ethnic people of Bangladesh in dysuria. It is a preparation of various roasted metals (Hg and Cu), non-metal (sulphur and Mica) and medicinal herbs. Considering the controversy over the risk of toxic heavy metals in Ayurvedic herbo-mineral preparations, toxicological parameters on liver functions were investigated. A single dose of 100mg/kg body weight of the preparation was administered orally to the rats of both sexes for ninety days. In this evaluation a statistically significant (p<0.001) increase of serum albumin levels in male (17%) and female (15%) rat groups were observed. On the other hand, the plasma bilirubin level was decreased 50% and 28% respectively in both rats groups. But no remarkable differences were observed in plasma protein, sGPT, sGOT and ALP activities from their corresponding control values. This study showed that CKR had no remarkable toxic effect on liver of the animals despite the presence of traces of transformed heavy metals.

Pulmonary tumor types induced in Wistar rats of the so-called "19-dust study".

The incidences of primary lung tumor types histologically diagnosed in 28 groups of Wistar rats of the so-called "19-dust study" are described, the total study having been already presented by Pott and Roller (Carcinogenicity study with nineteen granular dusts in rats. Eur J Oncol, 2005; 10: 249-81). Each exposed group was repeatedly instilled intratracheally with a suspension of one type and dose of 13 non-mining dusts differing in at least one of the following properties: chemical composition, density, specific surface area, and mean particle size. Eleven of the 13 dusts were classified as respirable granular bio-durable particles without known significant specific toxicity (abbreviation of the nine-word definition: GBP). In 579 (58%) lungs of 1002 rats which survived more than 26 weeks after the first instillation of GBP, at least one primary lung tumor type was observed, and in 306 (31%) at least two types. Three benign tumor types were diagnosed in the 579 tumor-bearing rats: bronchiolo-alveolar adenoma in 46%, cystic keratinizing epithelioma in 53%, and non-keratinizing epithelioma in 2.6% of the rats. Two of three malignant tumor types (bronchiolo-alveolar carcinoma and squamous cell carcinoma) occurred in 46% and 31% of the tumor-bearing rats, respectively, and adenosquamous carcinoma was diagnosed in 0.9%. Numerous lungs with a malignant tumor also showed one or more benign tumor types. In addition, single or multiple metastases from primary tumors of other sites (mainly carcinoma of the uterus) were diagnosed in 14% of the 1002 lungs. The proportionate incidences of the four predominantly diagnosed tumor types were compared with three summarized experimental groups which were exposed either to carbon black (two size classes), to titanium dioxide (two size classes), or to the total of the other nine GBP. A significant difference was not detected. The combination of dust volume with particle size correlated best with the carcinogenic effect, in contrast to dust mass and surface area.

[Pharmacological study on the compatibility of cortex Cinnamomi with Halloysitum Rubrum].

The decoction of Cortex Cinnamomi (CC, 1 g/kg p.o.) and Halloysitum Rubrum (HR, 3 g/kg p.o.) or the combination of the two drugs (4 g/kg p.o., CC 1 g/kg, HR 3 g/kg) could antagonize the diarrhea caused by p.o. water ex tract of Radix et Rhizoma Rhei in mice; and inhibit the platelet aggregation induced by ADP in vitro. Meanwhile, the effect of the combination of the two drugs was not different from that of each single one. In addition, CC was able to inhibit the spontaneous movement of intestine in situ and showed an analgesic effect (hot-plate method) in mice; HR was ineffective in these aspects and did not reduce the effect of CC. CC(20 g/kg p.o., i.p. or i.v.) exhibited very strong toxicity in mice, while HR(60 g/kg p.o., i.p. or i.v.) was nontoxic. When the two drugs were used together, the toxicity was markedly reduced.

Interfacial reactions of osteoblasts to dental and implant materials.

Scanning electron microscopy was used to study the response of osteoblasts to various surfaces including ceramics and glasses as well as steel and titanium. Hydroxylapatite, tricalcium phosphate, bioglass, steel, and titanium supported cell adhesion. However, the toxic effects of the In-Ceram (Vita, Bad Säckingen, Germany), and feldspar ceramic and glaze were severe enough to cause verrucous necrosis that was identifiable after 2 days of culture. After 10 days in culture only the peripheral portions of these specimens were still occupied by cells; the cells in the central portion of the circular specimens had succumbed to necrosis. It was concluded that scanning electron microscopy is useful in identifying the response of cells to materials. Pathologic changes are not recognizable if they are limited to the internal structure of the cell, but readily discernible when they impinge on the morphologic integrity of the cell surface.

The effects of intrapleural injections of alumina and aluminosilicate (ceramic) fibres.

Groups of rats, 24 male and 24 female, approximately 8 weeks old, were dosed by a single intrapleural injection with a saline suspension of refractory alumina fibres (Saffil fibres ICI plc) either as manufactured or after extensive thermal ageing; or one of two aluminosilicate ('ceramic') fibres with different diameter distributions. Similar groups were dosed with a suspension of UICC chrysotile A asbestos or saline solution to serve as positive and negative controls respectively. Rats were maintained to 85% mortality and all decedents and terminal sacrifices were closely examined for the presence of mesothelioma. Malignant mesothelioma was diagnosed in ten rats, seven dosed with asbestos and three dosed with aluminosilicate fibre B. No mesothelioma was detected in any rat dosed with Saffil fibres or aluminosilicate fibre A or in negative controls. The results support the predicted inert nature of Saffil alumina fibres and provide further evidence for the importance of fibre dimension in the induction of mesothelioma. The implication of the results for inhalation exposures is discussed.

Ranking toxicity of industrial dusts by bronchoalveolar lavage fluid analysis.

Female wistar rats were inoculated intratracheally with 10 mg/ml suspensions of various dusts, viz: quartz, fly ash, mica and corundum in physiological saline. Biochemical markers of bronchoalveolar lavage fluid (BALF) were analysed 8 days after the instillation of the dusts. Elevated levels of proteins, sialic acid and phospholipid contents and the activity of lactate dehydrogenase correlated well with the degree of the known fibrogenic potential of different dusts in the lungs in the following order, quartz greater than fly ash greater than mica greater than corundum. beta-glucuronidase activity, was however, only elevated in the quartz inoculated group of rats. It is suggested that biochemical constituents of BALF analysed shortly after the exposure to different dusts can be useful to mirror alterations in the tissue response to mineral dusts.

Alterations in in vitro functional activities of alveolar macrophages exposed in vivo to mineral dusts.

To determine whether macrophages exposed to mineral dusts are altered, rats were exposed intratracheally to one of several mineral dusts, held 8 days, their lungs washed and the cellular composition of the fluid characterized morphologically and functionally. The number of cells recovered from lung washings of exposed rats increased 2 to 5 times relative to control rats; however, the percentage of such cells that were macrophages, or were capable of phagocytosis, adherence to glass or metabolism of carbohydrates via the hexose monophosphate shunt as indicated by reduction of nitroblue tetrazolium, were reduced. Silica dust produced the greatest effect, corresponding qualitatively to earlier in vivo studies.