Suresh Valiyaveettil

Cytotoxicity and Genotoxicity of Silver Nanoparticles in Human Cells

Silver nanoparticles (Ag-np) are being used increasingly in wound dressings, catheters, and various household products due to their antimicrobial activity. The toxicity of starch-coated silver nanoparticles was studied using normal human lung fibroblast cells (IMR-90) and human glioblastoma cells (U251). The toxicity was evaluated using changes in cell morphology, cell viability, metabolic activity, and oxidative stress. Ag-np reduced ATP content of the cell caused damage to mitochondria and increased production of reactive oxygen species (ROS) in a dose-dependent manner. DNA damage, as measured by single cell gel electrophoresis (SCGE) and cytokinesis blocked micronucleus assay (CBMN), was also dose-dependent and more prominent in the cancer cells. The nanoparticle treatment caused cell cycle arrest in G(2)/M phase possibly due to repair of damaged DNA. Annexin-V propidium iodide (PI) staining showed no massive apoptosis or necrosis. The transmission electron microscopic (TEM) analysis indicated the presence of Ag-np inside the mitochondria and nucleus, implicating their direct involvement in the mitochondrial toxicity and DNA damage. A possible mechanism of toxicity is proposed which involves disruption of the mitochondrial respiratory chain by Ag-np leading to production of ROS and interruption of ATP synthesis, which in turn cause DNA damage. It is anticipated that DNA damage is augmented by deposition, followed by interactions of Ag-np to the DNA leading to cell cycle arrest in the G(2)/M phase. The higher sensitivity of U251 cells and their arrest in G(2)/M phase could be explored further for evaluating the potential use of Ag-np in cancer therapy.

Toxicity of silver nanoparticles in zebrafish models

This study was initiated to enhance our insight on the health and environmental impact of silver nanoparticles (Ag-np). Using starch and bovine serum albumin (BSA) as capping agents, silver nanoparticles were synthesized to study their deleterious effects and distribution pattern in zebrafish embryos (Danio rerio). Toxicological endpoints like mortality, hatching, pericardial edema and heart rate were recorded. A concentration-dependent increase in mortality and hatching delay was observed in Ag-np treated embryos. Additionally, nanoparticle treatments resulted in concentration-dependent toxicity, typified by phenotypes that had abnormal body axes, twisted notochord, slow blood flow, pericardial edema and cardiac arrhythmia. Ag(+) ions and stabilizing agents showed no significant defects in developing embryos. Transmission electron microscopy (TEM) of the embryos demonstrated that nanoparticles were distributed in the brain, heart, yolk and blood of embryos as evident from the electron-dispersive x-ray analysis (EDS). Furthermore, the acridine orange staining showed an increased apoptosis in Ag-np treated embryos. These results suggest that silver nanoparticles induce a dose-dependent toxicity in embryos, which hinders normal development.

Anti-proliferative activity of silver nanoparticles

BackgroundNanoparticles possess exceptional physical and chemical properties which led to rapid commercialisation. Silver nanoparticles (Ag-np) are among the most commercialised nanoparticles due to their antimicrobial potential. Ag-np based cosmetics, therapeutic agents and household products are in wide use, which raised a public concern regarding their safety associated with human and environmental use. No safety regulations are in practice for the use of these nanomaterials. The interactions of nanomaterials with cells, uptake mechanisms, distribution, excretion, toxicological endpoints and mechanism of action remain unanswered.ResultsNormal human lung fibroblasts (IMR-90) and human glioblastoma cells (U251) were exposed to different doses of Ag-nps in vitro. Uptake of Ag-nps occurred mainly through endocytosis (clathrin mediated process and macropinocytosis), accompanied by a time dependent increase in exocytosis rate. The electron micrographs revealed a uniform intracellular distribution of Ag-np both in cytoplasm and nucleus. Ag-np treated cells exhibited chromosome instability and mitotic arrest in human cells. There was efficient recovery from arrest in normal human fibroblasts whereas the cancer cells ceased to proliferate. Toxicity of Ag-np is mediated through intracellular calcium (Ca2+) transients along with significant alterations in cell morphology and spreading and surface ruffling. Down regulation of major actin binding protein, filamin was observed after Ag-np exposure. Ag-np induced stress resulted in the up regulation of metallothionein and heme oxygenase -1 genes.ConclusionHere, we demonstrate that uptake of Ag-np occurs mainly through clathrin mediated endocytosis and macropinocytosis. Our results suggest that cancer cells are susceptible to damage with lack of recovery from Ag-np-induced stress. Ag-np is found to be acting through intracellular calcium transients and chromosomal aberrations, either directly or through activation of catabolic enzymes. The signalling cascades are believed to play key roles in cytoskeleton deformations and ultimately to inhibit cell proliferation.

Health impact and safety of engineered nanomaterials

Many engineered nanomaterials (NMs) are being synthesized and explored for potential use in consumer and medical products. Already, nanoparticles (NPs) of titanium dioxide (TiO(2)), zinc oxide (ZnO), silver (Ag) and other metals or their oxides are present in commercial products such as sunscreens, cosmetics, wound dressings, surgical tools, detergents, automotive paints and tires. More recent and advanced FDA-approved use of NMs includes quantum dots (QDs) in live cell imaging, zirconium oxides in bone replacement and prosthetic devices and nanocarriers in drug delivery. The benefits from nanotechnology are aplenty, comprising antimicrobial activities, scratch- and water-resistance, long-lasting shine, improved processor speeds and better display resolution, to name a few. While developers of these products often focus on the exciting beneficial aspects of their products, safety and toxicity issues are often not discussed in detail. Long-term effects such as chronic exposure and environmental pollution are even less documented. Along with widespread manufacture and use of NMs, concerns for occupational hazards, proper handling, disposal, storage, shipping and clean up are expected to rise. This review focus on the possible biological impact of engineered NPs, serving as a reminder that nanomaterials can become a double-edged sword if not properly handled.

DNA damage and p53-mediated growth arrest in human cells treated with platinum nanoparticles

AIM Platinum-based therapeutic agents are widely used in medicine. Thus, a thorough understanding of their mechanism of action in cells is warranted. This study investigates the uptake and bioactivity (e.g., cytotoxicity, genotoxicity and protein expression) of platinum nanoparticles (Pt-NPs, approximately 5-8 nm in size) in human cells. MATERIALS & METHODS Pt-NPs capped with polyvinyl alcohol were synthesized, characterized and incubated with human cells. Uptake and the biological properties were evaluated through metabolic activity, genome integrity, cell cycle and protein expression. RESULTS Pt-NPs entered the cells through diffusion, and localized inside the cytoplasm. Exposure to the Pt-NP increased DNA damage, accumulation of cells at the S-phase of the cell cycle and apoptosis. A significant number of cells recovered from the stress and formed colonies. Protein-expression levels uncovered upregulation of p53, phosphorylated p53, p21 and downregulation of proliferating cell nuclear antigen following Pt-NP treatment. Pro-caspase 3 and poly-ADP ribose polymerase and cyclin B levels were not altered in both the cell types after Pt-NP exposure. CONCLUSION The results suggest p53 activation in Pt-NP-treated cells due to genotoxic stress, with subsequent activation of p21 leading to a proliferating cell nuclear antigen-mediated growth arrest and apoptosis. This study recommends development of Pt-NP-based anticancer agents by appropriate surface modifications to augment its innate anticancer activity.

Investigation of the role of ansocalcin in the biomineralization in goose eggshell matrix

The role of proteins in biomineralization and the mechanism of eggshell formation are not well understood. We have isolated and purified the major protein, ansocalcin from goose eggshell matrix. The amino acid sequence study indicates that ansocalcin is homologous to the chicken eggshell protein, ovocleidin 17, and C-type lectins. Ansocalcin nucleates polycrystalline aggregates of calcite crystals in in vitro mineralization experiments. The polycrystalline aggregates obtained at higher concentration of ansocalcin appears to be similar to the crystals observed at the mamillary layer of the eggshell.

Differential regulation of intracellular factors mediating cell cycle, DNA repair and inflammation following exposure to silver nanoparticles in human cells.

BackgroundInvestigating the cellular and molecular signatures in eukaryotic cells following exposure to nanoparticles will further our understanding on the mechanisms mediating nanoparticle induced effects. This study illustrates the molecular effects of silver nanoparticles (Ag-np) in normal human lung cells, IMR-90 and human brain cancer cells, U251 with emphasis on gene expression, induction of inflammatory mediators and the interaction of Ag-np with cytosolic proteins.ResultsWe report that silver nanoparticles are capable of adsorbing cytosolic proteins on their surface that may influence the function of intracellular factors. Gene and protein expression profiles of Ag-np exposed cells revealed up regulation of many DNA damage response genes such as Gadd 45 in both the cell types and ATR in cancer cells. Moreover, down regulation of genes necessary for cell cycle progression (cyclin B and cyclin E) and DNA damage response/repair (XRCC1 and 3, FEN1, RAD51C, RPA1) was observed in both the cell lines. Double strand DNA damage was observed in a dose dependant manner as evidenced in γH2AX foci assay. There was a down regulation of p53 and PCNA in treated cells. Cancer cells in particular showed a concentration dependant increase in phosphorylated p53 accompanied by the cleavage of caspase 3 and PARP. Our results demonstrate the involvement of NFκB and MAP kinase pathway in response to Ag-np exposure. Up regulation of pro-inflammatory cytokines such as interleukins (IL-8, IL-6), macrophage colony stimulating factor, macrophage inflammatory protein in fibroblasts following Ag-np exposure were also observed.ConclusionIn summary, Ag-np can modulate gene expression and protein functions in IMR-90 cells and U251 cells, leading to defective DNA repair, proliferation arrest and inflammatory response. The observed changes could also be due to its capability to adsorb cytosolic proteins on its surface.

Effect of substituents on the electron transport properties of bay substituted perylene diimide derivatives

Electron donating substituents such as 3,4,5-trimethoxy phenyl (PDI1), thiophene (PDI2), 6-methoxy naphthyl (PDI3), 5-hexyl dithiophene (PDI4) and thioanthrenyl (PDI5) moieties incorporated at the bay region of N,N′-dodecylperylene diimide (PDI) derivatives have been synthesized and characterised. The absorption maxima of PDI1–5 showed a bathochromic shift with considerable peak broadening and merging of vibronic fine structure as compared to that of PDI. The photoinduced intramolecular charge transfer (ICT) from electron donating substituents to the perylene-acceptor has been observed from fluorescence self-quenching. The molecules are stable up to 400 °C and amorphous in nature. The cyclic voltammetry results revealed that the oxidation potential can be tuned by the electron donating capacity of the substituents and the dithiophene-attached perylene derivative (PDI4) showed a very low band gap of 1.57 eV. The electron transporting properties have been studied using xerographic time-of-flight method and the highest electron mobility reached up to 4.4 × 10−4 cm2V−1 s−1 at 6.4 × 105 V cm−1 for 1,7-di(3,4,5-trimethoxyphenyl)perylene diimide (PDI1) under ambient conditions.

Surface modified electrospun poly(vinyl alcohol) membranes for extracting nanoparticles from water

Contamination of water from nanomaterials will be an emerging problem in the future due to incorporation of nanomaterials in many commercial products and improper disposal of waste materials. In this report, electrospun polyvinyl alcohol nanofibers (PVA NFs) with diameters ranging between 300 and 500 nm were used for the extraction of nanosized contaminants from the aqueous environment. To obtain the best extraction efficiency, surface hydroxyl groups of PVA NFs were chemically modified with functional groups, such as thiols and amines. Two model nanoparticles (silver and gold) dissolved in water were used for adsorption studies. Depending on the nature of the surface functionalities, the fibers showed unique ability to adsorb nanoparticles. The extraction studies revealed that the amine and thiol modified PVA NFs showed 90% extraction efficiency for both silver and gold nanoparticles. The thiol and amine functionalized PVA NFs showed maximum adsorption capacities (Q(t)) towards Au NPs, which were around 79-84 mg g(-1). Similarly for Ag NP extraction, amine functionalized PVA NFs showed a value for Q(t) at 56 mg g(-1). Our results highlight that functionalized nanofibers have high extraction efficiency for dissolved nanoparticles in water and can be used for removal of the nanocontaminants from the aqueous environment.

Structural Characterization of Myotoxic Ecarpholin S From Echis carinatus Venom

Phospholipase A(2) (PLA(2)), a common toxic component of snake venom, has been implicated in various pharmacological effects. Ecarpholin S, isolated from the venom of the snake Echis carinatus sochureki, is a phospholipase A(2) (PLA(2)) belonging to the Ser(49)-PLA(2) subgroup. It has been characterized as having low enzymatic but potent myotoxic activities. The crystal structures of native ecarpholin S and its complexes with lauric acid, and its inhibitor suramin, were elucidated. This is the first report of the structure of a member of the Ser(49)-PLA(2) subgroup. We also examined interactions of ecarpholin S with phosphatidylglycerol and lauric acid, using surface plasmon resonance, and of suramin with isothermal titration calorimetry. Most Ca(2+)-dependent PLA(2) enzymes have Asp in position 49, which plays a crucial role in Ca(2+) binding. The three-dimensional structure of ecarpholin S reveals a unique conformation of the Ca(2+)-binding loop that is not favorable for Ca(2+) coordination. Furthermore, the endogenously bound fatty acid (lauric acid) in the hydrophobic channel may also interrupt the catalytic cycle. These two observations may account for the low enzymatic activity of ecarpholin S, despite full retention of the catalytic machinery. These observations may also be applicable to other non-Asp(49)-PLA(2) enzymes. The interaction of suramin in its complex with ecarpholin S is quite different from that reported for the Lys(49)-PLA(2)/suramin complex(,) where the interfacial recognition face (i-face), C-terminal region, and N-terminal region of ecarpholin S play important roles. This study provides significant structural and functional insights into the myotoxic activity of ecarpholin S and, in general, of non-Asp(49)-PLA(2) enzymes.