Rawiwan Maniratanachote

Determination of silver nanoparticle release from antibacterial fabrics into artificial sweat

Silver nanoparticles have been used in numerous commercial products, including textiles, to prevent bacterial growth. Meanwhile, there is increasing concern that exposure to these nanoparticles may cause potential adverse effects on humans as well as the environment. This study determined the quantity of silver released from commercially claimed nanosilver and laboratory-prepared silver coated fabrics into various formulations of artificial sweat, each made according to AATCC, ISO and EN standards. For each fabric sample, the initial amount of silver and the antibacterial properties against the model Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria on each fabric was investigated. The results showed that silver was not detected in some commercial fabrics. Furthermore, antibacterial properties of the fabrics varied, ranging from 0% to greater than 99%. After incubation of the fabrics in artificial sweat, silver was released from the different fabrics to varying extents, ranging from 0 mg/kg to about 322 mg/kg of fabric weight. The quantity of silver released from the different fabrics was likely to be dependent on the amount of silver coating, the fabric quality and the artificial sweat formulations including its pH. This study is the unprecedented report on the release of silver nanoparticles from antibacterial fabrics into artificial sweat. This information might be useful to evaluate the potential human risk associated with the use of textiles containing silver nanoparticles.

Silver nanoparticles induce toxicity in A549 cells via ROS-dependent and ROS-independent pathways

Silver nanoparticles (AgNPs) are incorporated into a large number of consumer and medical products. Several experiments have demonstrated that AgNPs can be toxic to the vital organs of humans and especially to the lung. The present study evaluated the in vitro mechanisms of AgNP (<100 nm) toxicity in relationship to the generation of reactive oxygen species (ROS) in A549 cells. AgNPs caused ROS formation in the cells, a reduction in their cell viability and mitochondrial membrane potential (MMP), an increase in the proportion of cells in the sub-G1 (apoptosis) population, S phase arrest and down-regulation of the cell cycle associated proliferating cell nuclear antigen (PCNA) protein, in a concentration- and time-dependent manner. Pretreatment of the A549 cells with N-acetyl-cysteine (NAC), an antioxidant, decreased the effects of AgNPs on the reduced cell viability, change in the MMP and proportion of cells in the sub-G1population, but had no effect on the AgNP-mediated S phase arrest or down-regulation of PCNA. These observations allow us to propose that the in vitro toxic effects of AgNPs on A549 cells are mediated via both ROS-dependent (cytotoxicity) and ROS-independent (cell cycle arrest) pathways.

Mechanistic study on the biological effects of silver and gold nanoparticles in Caco-2 cells--induction of the Nrf2/HO-1 pathway by high concentrations of silver nanoparticles.

The most commonly used metal nanoparticles (NPs) across diverse applications, including in agro-food applications, include silver (AgNPs) and gold (AuNPs). In the present study, we aimed to investigate the biological responses and possible toxicological effects of AgNPs and AuNPs in the Caco-2 cells as an in vitro human GI tract model. Both AgNPs and AuNPs were internalized into the cytoplasm of Caco-2 cells, but not within the nucleus and only exposure to high concentrations of AgNPs, but not AuNPs, caused acute cytotoxicity and depolarization of the mitochondrial membrane potential. In addition, only AgNPs significantly depleted the total intracellular glutathione level, induced the activation of the stress-responsive gene, Nrf2, and dramatically increased the expression of heme oxygenase-1 (HO-1). Furthermore, siRNA silencing of Nrf2 transcripts significantly reduced the AgNP-induced HO-1 mRNA induction, suggesting a key role for Nrf2 in the control of HO-1 expression. Taken together, AgNPs but not AuNPs induced acute cytotoxicity and cellular responses via the oxidative stress-related activation of Nrf2/HO-1 signaling pathway in Caco-2 cells. The expression of HO-1 transcripts may be useful as a sensitive marker for safety evaluation of AgNPs in the GI tract of humans.

Effects of silver nanoparticles on rat hepatic cytochrome P450 enzyme activity.

Silver nanoparticles (AgNPs) are increasingly used in various products and consequentially the potential adverse effects associated with exposure to them are of concern. This study investigated the effects of AgNPs on the hepatic drug-metabolizing enzymes of the cytochrome P450 (CYP) families 1, 2 and 3, using both in vitro and in vivo biological assays. AgNPs were orally administered to Sprague-Dawley rats at various concentrations (0–1000 mg/kg body weight/day) for 2 weeks. No effect was found on the plasma levels of ALT, AST and ALP in all treated rat groups, and no significant change in the activities of CYP1A, CYP2C, CYP2D, CYP2E1 and CYP3A was observed for all tested AgNP doses. The results correlated with the observation that no AgNPs were detected in the liver sections of the tested rats. However, the in vitro system using rat liver microsomes demonstrated a strong inhibition of CYP2C (IC50 = 28 µg/mL) and CYP2D (IC50 = 23 µg/mL) activities, but not of CYP1A, CYP2E1 and CYP3A activities (IC50 > 100 µg/mL) at concentrations up to 100 µg/mL of AgNPs. The inhibitory effect of AgNPs on these CYPs indicates the possibility of the AgNP-drug interaction when co-administered with some medicines and this may cause adverse effects to patients.

Mechanisms of antibiotic resistance in bacteria mediated by silver nanoparticles

ABSTRACT Silver nanoparticles (AgNPs) are widely used in industry, consumer products, and medical appliances due to their efficient antimicrobial properties. However, information on environmental toxicity and bacterial impact of these particles is not completely elucidated. Results showed that AgNPs produced growth inhibition and oxidative stress in bacteria Escherichia coli (gram negative) and Staphylococcus aureus (gram positive), with half-maximal inhibitory concentrations (IC50) of 12 and 7 mg/L, respectively. Surprisingly, bacteria pre-exposed to sublethal dose of AgNPs exhibited increased resistance toward antibiotics (ampicillin and Pen-Strep) with IC50 elevated by 3–13-fold. Further, AgNP pre-exposure raised the minimal inhibitory concentration and minimal biocidal concentration by two- to eightfold when cells were challenged with antibiotics with diverse mechanisms of action (penicillin, chloramphenicol, and kanamycin). Interestingly, we found that upon exposure to ampicillin, strains pretreated with AgNPs exhibited lower levels of membrane damage and oxidative stress, together with elevated levels of intracellular ATP relative to untreated cells. Bacterial reverse mutation assay (Ames test) showed that AgNPs are highly mutagenic, consistent with further assays demonstrating abiotic reactive oxygen species (ROS) generation and intrinsic DNA cleavage activity in vitro of AgNPs. Overall, our results suggest that AgNPs enhance bacterial resistance to antibiotics by promoting stress tolerance through induction of intracellular ROS. Our data suggest potential consequences of incidental environmental exposure of bacteria to AgNPs and indicate the need to regulate use and disposal of AgNPs in industry and consumer products.

Oil-in-water emulsions stabilized by sodium phosphorylated chitosan

Oil-in-water (O/W) emulsions with sodium phosphorylated chitosan (PCTS) were obtained via simple emulsification. PCTS in aqueous solution was amphiphilic with a hydrophilic-lipophilic balance (HLB) of 19 and a critical aggregation concentration (CAC) of 0.13% w/v. The emulsifying efficiency and emulsion stability of PCTS over oil droplets were evaluated in terms of the droplet size, droplet size distribution and microscopic observation using confocal laser scanning microscopy. PCTS preferred to cover oil droplets to produce an O/W emulsion and formed long term stable particles (90 days storage at room temperature) when using PCTS concentrations from above the CAC to 3% w/v. However, emulsions formed from PCTS concentrations below the CAC or over 3% w/v were unstable with particle agglomeration by flocculation after only 7 days storage, although they reverted to individual droplets that retained their integrity in acidic conditions. Overall, PCTS forms effective stable O/W encapsulated particles with potential applications in lipophilic drug encapsulation via a simple emulsion system.

Human primary erythroid cells as a more sensitive alternative in vitro hematological model for nanotoxicity studies: Toxicological effects of silver nanoparticles.

Although immortalized cells established from cancerous cells have been widely used for studies in nanotoxicology studies, the reliability of the results derived from immortalized cells has been questioned because of their different characteristics from normal cells. In the present study, human primary erythroid cells in liquid culture were used as an in vitro hematological cell model for investigation of the nanotoxicity of silver nanoparticles (AgNPs) and comparing the results to the immortalized hematological cell lines HL60 and K562. The AgNPs caused significant cytotoxic effects in the primary erythroid cells, as shown by the decreased cell viability and induction of intracellular ROS generation and apoptosis, whereas they showed much lower cytotoxic and apoptotic effects in HL60 and K562 cells and did not induced ROS generation in these cell lines. Scanning electron microcopy revealed an interaction of AgNPs to the cell membrane in both primary erythroid and immortalized cells. In addition, AgNPs induced hemolysis in the primary erythroid cells in a dose-dependent manner, and transmission electron microcopy analysis revealed that AgNPs damaged the erythroid cell membrane. Taken together, these results suggest that human primary erythroid cells in liquid culture are a more sensitive alternative in vitro hematological model for nanotoxicology studies.

Interaction evaluation of silver and dithizone complexes using DFT calculations and NMR analysis

Silver has distinct antibacterial properties and has been used as a component of commercial products with many applications. An increasing number of commercial products cause risks of silver effects for human and environment such as the symptoms of Argyria and the release of silver to the environment. Therefore, the detection of silver in the aquatic environment is important. The colorimetric chemosensor is designed by the basic of ligand interactions with metal ion, leading to the change of signals for the naked-eyes which is very useful method to this application. Dithizone ligand is considered as one of the effective chelating reagents for metal ions due to its high selectivity and sensitivity of a photochromic reaction for silver as well as the linear backbone of dithizone affords the rotation of various isomeric forms. The present study is focused on the conformation and interaction of dithizone with silver using density functional theory (DFT). The interaction parameters were determined in term of binding energy of complexes and the geometry optimization, frequency of the structures and calculation of binding energies using density functional approaches B3LYP and the 6-31G(d,p) basis set. Moreover, the interaction of silver-dithizone complexes was supported by UV-Vis spectroscopy, FT-IR spectrum that were simulated by using B3LYP/6-31G(d,p) and (1)H NMR spectra calculation using B3LYP/6-311+G(2d,p) method compared with the experimental data. The results showed the ion exchange interaction between hydrogen of dithizone and silver atom with minimized binding energies of silver-dithizone interaction. Therefore, the results can be the useful information for determination of complex interaction using the analysis of computer simulations.

Shape and surface properties of titanate nanomaterials influence differential cellular uptake behavior and biological responses in THP-1 cells

We investigated cellular uptake behavior and biological responses of spherical and fibrous titanate nanomaterials in human monocyte THP-1 cells. Two titanate nanofibers (TiNFs), namely TF-1 and TF-2, were synthesized from anatase TiO2 nanoparticles (TNPs) via hydrothermal treatment. The synthesized TiNFs and TNPs were thoroughly characterized for their size, crystallinity, surface area and surface pH. TF-1 (∼2 µm in length) was amorphous with an acidic surface, while TF-2 (∼7 µm in length) was brookite with a basic surface. The results demonstrated that none of these titanate nanomaterials resulted in significant cytotoxicity, even at the highest doses tested (50 µg/ml), consistent with an absence of ROS generation and lack of change of mitochondrial membrane potential. While no cytotoxic effect was found in the titanate nanomaterials, TF-2 tended to decrease the proliferation of THP-1 cells. Furthermore, TF-2 resulted in an inflammatory cytokine response, as evidenced by dramatic induction of IL-8 and TNF-α release in TF2 but not TF-1 nor TNPs. These results suggest that shape of titanate nanomaterials plays an important role in cellular internalization, while surface pH may play a prominent role in inflammatory response in THP-1 cells.

Biocompatibility study of quaternized chitosan on the proliferation and differentiation of Caco-2 cells as an in vitro model of the intestinal barrier

The development of different chitosan derivatives for medical applications has increased recently. Among these chitosan derivatives, quaternized chitosan was designed to improve the solubility of chitosan in biological fluids for oral drug delivery while retaining the cationic character for mucoadhesion. However, the biocompatibility of quaternized chitosan on the human intestine is unknown. In this study, we aimed to examine the potential biological effects of quaternized chitosan on the intestinal barrier, in terms of cell proliferation and cell differentiation, using the Caco-2 cell line as an in vitro model. The lower the degree of substitution of quaternized chitosan, the lower the cytotoxic and anti-proliferative effect on Caco-2 cells. In addition, the anti-proliferative effect of quaternized chitosan might induce a cell cycle disturbance and differentiation delay. Long-term continuous exposure (9 days) to quaternized chitosan caused a delay in differentiation of the Caco-2 cells even at non-cytotoxic quaternized chitosan doses (0.005% (w/v)), as shown by the low level of alkaline phosphatase in the quaternized chitosan–treated group compared to the control cells. In contrast, short-term discontinuous exposure to quaternized chitosan (0.005% (w/v) for 4 h/day over 9 days) that more realistically mimics the daily intestinal exposure did not inhibit the intestinal differentiation of Caco-2 cells. Thus, the use of a low degree of substitution and a low concentration of quaternized chitosan resulted in a good biocompatibility to the intestinal barrier supporting the potential usefulness of quaternized chitosan in the application of an oral drug delivery system.