Advances in the Toxicity Assessment of Silver Nanoparticles Derived from a Sphagnum fallax Extract for Monolayers and Spheroids.

The production of nanomaterials through environmentally friendly methods is a top priority in the sustainable development of nanotechnology. This paper presents data on the synthesis of silver nanoparticles using an aqueous extract of Sphagnum fallax moss at room temperature. The morphology, stability, and size of the nanoparticles were analyzed using various techniques, including transmission electron microscopy, Doppler laser velocimetry, and UV-vis spectroscopy. In addition, Fourier transform infrared spectroscopy was used to analyze the presence of moss metabolites on the surface of nanomaterials. The effects of different concentrations of citrate-stabilized and moss extract-stabilized silver nanoparticles on cell viability, necrosis induction, and cell impedance were compared. The internalization of silver nanoparticles into both monolayers and three-dimensional cells spheroids was evaluated using dark-field microscopy and hyperspectral imaging. An eco-friendly method for the synthesis of silver nanoparticles at room temperature is proposed, which makes it possible to obtain spherical nanoparticles of 20-30 nm in size with high bioavailability and that have potential applications in various areas of human life.

Magnetic Nanoparticles for Biomedical and Imaging Applications.

Magnetic nanoparticles (MNPs) are a class of nanomaterials composed of metals such as cobalt, nickel, and iron with paramagnetic, ferromagnetic, or superparamagnetic properties [...].

Comparative Characterization of Iron and Silver Nanoparticles: Extract-Stabilized and Classical Synthesis Methods.

Synthesis of silver nanoparticles using extracts from plants is an advantageous technological alternative to the traditional colloidal synthesis due to its simplicity, low cost, and the inclusion of environmentally friendly processes to obtain a new generation of antimicrobial compounds. The work describes the production of silver and iron nanoparticles using sphagnum extract as well as traditional synthesis. Dynamic light scattering (DLS) and laser doppler velocimetry methods, UV-visible spectroscopy, transmission electron microscopy (TEM) combined with energy dispersive X-ray spectroscopy (EDS), atomic force microscopy (AFM), dark-field hyperspectral microscopy, and Fourier-transform infrared spectroscopy (FT-IR) were used to study the structure and properties of synthesized nanoparticles. Our studies demonstrated a high antibacterial activity of the obtained nanoparticles, including the formation of biofilms. Nanoparticles synthesized using sphagnum moss extracts likely have high potential for further research.

DNA/Magnetic Nanoparticles Composite to Attenuate Glass Surface Nanotopography for Enhanced Mesenchymal Stem Cell Differentiation.

Mesenchymal stem cells (MSCs) have extensive pluripotent potential to differentiate into various cell types, and thus they are an important tool for regenerative medicine and biomedical research. In this work, the differentiation of hTERT-transduced adipose-derived MSCs (hMSCs) into chondrocytes, adipocytes and osteoblasts on substrates with nanotopography generated by magnetic iron oxide nanoparticles (MNPs) and DNA was investigated. Citrate-stabilized MNPs were synthesized by the chemical co-precipitation method and sized around 10 nm according to microscopy studies. It was shown that MNPs@DNA coatings induced chondrogenesis and osteogenesis in hTERT-transduced MSCs. The cells had normal morphology and distribution of actin filaments. An increase in the concentration of magnetic nanoparticles resulted in a higher surface roughness and reduced the adhesion of cells to the substrate. A glass substrate modified with magnetic nanoparticles and DNA induced active chondrogenesis of hTERT-transduced MSC in a twice-diluted differentiation-inducing growth medium, suggesting the possible use of nanostructured MNPs@DNA coatings to obtain differentiated cells at a reduced level of growth factors.

Biogenic Silver Nanoparticles: Synthesis and Application as Antibacterial and Antifungal Agents.

The importance and need for eco-oriented technologies has increased worldwide, which leads to an enhanced development of methods for the synthesis of nanoparticles using biological agents. This review de-scribes the current approaches to the preparation of biogenic silver nanoparticles, using plant extracts and filtrates of fungi and microorganisms. The peculiarities of the synthesis of particles depending on the source of biocomponents are considered as well as physico-morphological, antibacterial and antifungal properties of the resulting nanoparticles which are compared with such properties of silver nanoparticles obtained by chemical synthesis. Special attention is paid to the process of self-assembly of biogenic silver nanoparticles.

Pharmaceuticals Removal by Adsorption with Montmorillonite Nanoclay.

The problem of purifying domestic and hospital wastewater from pharmaceutical compounds is becoming more and more urgent every year, because of the continuous accumulation of chemical pollutants in the environment and the limited availability of freshwater resources. Clay adsorbents have been repeatedly proposed as adsorbents for treatment purposes, but natural clays are hydrophilic and can be inefficient for catching hydrophobic pharmaceuticals. In this paper, a comparison of adsorption properties of pristine montmorillonite (MMT) and montmorillonite modified with stearyl trimethyl ammonium (hydrophobic MMT-STA) towards carbamazepine, ibuprofen, and paracetamol pharmaceuticals was performed. The efficiency of adsorption was investigated under varying solution pH, temperature, contact time, initial concentration of pharmaceuticals, and adsorbate/adsorbent mass ratio. MMT-STA was better than pristine MMT at removing all the pharmaceuticals studied. The adsorption capacity of hydrophobic montmorillonite to pharmaceuticals decreased in the following order: carbamazepine (97%) > ibuprofen (95%) > paracetamol (63-67%). Adsorption isotherms were best described by Freundlich model. Within the pharmaceutical concentration range of 10-50 µg/mL, the most optimal mass ratio of adsorbates to adsorbents was 1:300, pH 6, and a temperature of 25 °C. Thus, MMT-STA could be used as an efficient adsorbent for deconta×ating water of carbamazepine, ibuprofen, and paracetamol.

Revisiting the Cytotoxicity of Cationic Polyelectrolytes as a Principal Component in Layer-by-Layer Assembly Fabrication.

Polycations are an essential part of layer-by-layer (LbL)-assembled drug delivery systems, especially for gene delivery. In addition, they are used for other related applications, such as cell surface engineering. As a result, an assessment of the cytotoxicity of polycations and elucidation of the mechanisms of polycation toxicity is of paramount importance. In this study, we examined in detail the effects of a variety of water-soluble, positively charged synthetic polyelectrolytes on in vitro cytotoxicity, cell and nucleus morphology, and monolayer expansion changes. We have ranked the most popular cationic polyelectrolytes from the safest to the most toxic in relation to cell cultures. 3D cellular cluster formation was disturbed by addition of polyelectrolytes in most cases in a dose-dependent manner. Atomic force microscopy allowed us to visualize in detail the structures of the polyelectrolyte-DNA complexes formed due to electrostatic interactions. Our results indicate a relationship between the structure of the polyelectrolytes and their toxicity, which is necessary for optimization of drug and gene delivery systems.

Comparative Toxicity of Fly Ash: An In Vitro Study.

Fly ash produced during coal combustion is one of the major sources of air and water pollution, but the data on the impact of micrometer-size fly ash particles on human cells is still incomplete. Fly ash samples were collected from several electric power stations in the United States (Rockdale, TX; Dolet Hill, Mansfield, LA; Rockport, IN; Muskogee, OK) and from a metallurgic plant located in the Russian Federation (Chelyabinsk Electro-Metallurgical Works OJSC). The particles were characterized using dynamic light scattering, atomic force, and hyperspectral microscopy. According to chemical composition, the fly ash studied was ferro-alumino-silicate mineral containing substantial quantities of Ca, Mg, and a negligible concentration of K, Na, Mn, and Sr. The toxicity of the fly ash microparticles was assessed in vitro using HeLa cells (human cervical cancer cells) and Jurkat cells (immortalized human T lymphocytes). Incubation of cells with different concentrations of fly ash resulted in a dose-dependent decrease in cell viability for all fly ash variants. The most prominent cytotoxic effect in HeLa cells was produced by the ash particles from Rockdale, while the least was produced by the fly ash from Chelyabinsk. In Jurkat cells, the lowest toxicity was observed for fly ash collected from Rockport, Dolet Hill and Muscogee plants. The fly ash from Rockdale and Chelyabinsk induced DNA damage in HeLa cells, as revealed by the single cell electrophoresis, and disrupted the normal nuclear morphology. The interaction of fly ash microparticles of different origins with cells was visualized using dark-field microscopy and hyperspectral imaging. The size of ash particles appeared to be an important determinant of their toxicity, and the smallest fly ash particles from Chelyabinsk turned out to be the most cytotoxic to Jukart cells and the most genotoxic to HeLa cells.

Biocompatibility of magnetic nanoparticles coating with polycations using A549 cells.

Fe3O4 nanoparticles were obtained by chemical coprecipitation of iron chloride and sodium hydroxide. The morphology and sizes of the obtained nanoparticles were characterized using laser Doppler velocimetry, transmission electron and atomic force microscopy. Then the nanoparticles were stabilized by three polycations (polyethylenimine (PEI), poly(allylamine hydrochloride) (PAH), poly(diallyldimethylammonium chloride) (PDADMAC)) to increase their biocompatibility. The cytotoxicity of the obtained polymer-stabilized nanoparticles was studied using a human lung carcinoma cell line (A549). The biodistribution of nanoparticles stabilized by polycations in human lung carcinoma cells was analyzed by transmission electron microscopy, and the toxicity of nanomaterials was evaluated using toxicity tests and flow cytometry. As a result, the most biocompatible nanoparticle-biopolymer complex was identified. PAH stabilized magnetic nanoparticles demonstrated the best biocompatibility, and the PEI-magnetic nanoparticle complex was the most toxic.

Bidirectional alterations in antibiotics susceptibility in Staphylococcus aureus-Pseudomonas aeruginosa dual-species biofilm.

In mixed infections, the bacterial susceptibility differs significantly compared to monocultures of bacteria, and generally the concentrations of antibiotics required for the treatment increases drastically. For S. aureus and P. aeruginosa dual species biofilms, it has been numerously reported that P. aeruginosa decreases S. aureus susceptibility to a broad range of antibiotics, including beta-lactams, glycopeptides, aminoglycosides, macrolides, while sensitizes to quinolones via secretion of various metabolites. Here we show that S. aureus also modulates the susceptibility of P. aeruginosa to antibiotics in mixed cultures. Thus, S. aureus-P. aeruginosa consortium was characterized by tenfold increase in susceptibility to ciprofloxacin and aminoglycosides compared to monocultures. The same effect could be also achieved by the addition of cell-free culture of S. aureus to P. aeruginosa biofilm. Moreover, similar increase in antibiotics efficacy could be observed following addition of S. aureus suspension to the P. aeruginosa mature biofilm, compared to P. aeruginosa monoculture, and vice versa. These findings open promising perspectives to increase the antimicrobial treatment efficacy of the wounds infected with nosocomial pathogens by the transplantation of the skin residential microflora.

Facile Fabrication of Natural Polyelectrolyte-Nanoclay Composites: Halloysite Nanotubes, Nucleotides and DNA Study.

Complexation of biopolymers with halloysite nanotubes (HNTs) can greatly affect their applicability as materials building blocks. Here we have performed a systematic investigation of fabrication of halloysite nanotubes complexes with nucleotides and genomic DNA. The binding of DNA and various nucleotide species (polyAU, UMP Na2, ADP Na3, dATP Na, AMP, uridine, ATP Mg) by halloysite nanotubes was tested using UV-spectroscopy. The study revealed that binding of different nucleotides to the nanoclay varied but was low both in the presence and absence of MgCl2, while MgCl2 facilitated significantly the binding of longer molecules such as DNA and polyAU. Modification of the nanotubes with DNA and nucleotide species was further confirmed by measurements of ζ-potentials. DNA-Mg-modified nanotubes were characterized using transmission electron (TEM), atomic force (AFM) and hyperspectral microscopies. Thermogravimetric analysis corroborated the sorption of DNA by the nanotubes, and the presence of DNA on the nanotube surface was indicated by changes in the surface adhesion force measured by AFM. DNA bound by halloysite in the presence of MgCl2 could be partially released after addition of phosphate buffered saline. DNA binding and release from halloysite nanotubes was tested in the range of MgCl2 concentrations (10-100 mM). Even low MgCl2 concentrations significantly increased DNA sorption to halloysite, and the binding was leveled off at about 60 mM. DNA-Mg-modified halloysite nanotubes were used for obtaining a regular pattern on a glass surface by evaporation induced self-assembly process. The obtained spiral-like pattern was highly stable and resisted dissolution after water addition. Our results encompassing modification of non-toxic clay nanotubes with a natural polyanion DNA will find applications for construction of gene delivery vehicles and for halloysite self-assembly on various surfaces (such as skin or hair).

Nanoarchitectonics meets cell surface engineering: shape recognition of human cells by halloysite-doped silica cell imprints.

Cell surface engineering, as a practical manifestation of nanoarchitectonics, is a powerful tool to modify and enhance properties of live cells. In turn, cells may serve as sacrificial templates to fabricate cell-mimicking materials. Herein we report a facile method to produce cell-recognising silica imprints capable of the selective detection of human cells. We used HeLa cells to template silica inorganic shells doped with halloysite clay nanotubes. The shells were destroyed by sonication resulting in the formation of polydisperse hybrid imprints that were used to recognise HeLa cells in liquid media supplemented with yeast. We believe that methodology reported here will find applications in biomedical and clinical research.

Kaolin alleviates the toxicity of graphene oxide for mammalian cells.

The development of novel nanoscale vehicles for drug delivery promotes the growth of interest in investigations of interaction between nanomaterials. In this paper, we report the in vitro studies of eukaryotic cell physiological response to incubation with graphene oxide and planar kaolin nanoclay. Graphene family materials, including graphene oxide (GO), hold promise for numerous applications due to their unique electronic properties. However, graphene oxide reveals toxicity to some cell lines through an unidentified mechanism. Thus, methods and agents reducing the toxicity of graphene oxide can widen its practical application. We used a colorimetric test, flow cytometry and cell index assay methods to evaluate the effects of separate and combined application of graphene oxide and kaolin on mammalian cells. We have shown that the joint application of graphene oxide and kaolin reduced the negative effects of graphene by almost 20%, most likely because of coagulation of the nanoparticles with each other, which was detected by atomic force microscopy.

Unraveling the Molecular Mechanism of Selective Antimicrobial Activity of 2(5H)-Furanone Derivative against Staphylococcus aureus.

Staphylococcus aureus causes various infectious diseases, from skin impetigo to life-threatening bacteremia and sepsis, thus appearing an important target for antimicrobial therapeutics. In turn, the rapid development of antibiotic resistance and biofilm formation makes it extremely robust against treatment. Here, we unravel the molecular mechanism of the antimicrobial activity of the recently unveiled F105 consisting of three pharmacophores: chlorinated 2(5H)-furanone, sulfone, and l-menthol moieties. F105 demonstrates highly selective activity against Gram-positive bacteria and biofilm-embedded S. aureus and exhibits low risk of resistance development. We show explicitly that the fluorescent analogue of F105 rapidly penetrates into Gram-positive bacteria independently of their cell integrity and viability and accumulates there. By contrast, Gram-negative bacteria remain impermeable and, therefore, insusceptible to F105. Apparently, in bacterial cells, F105 induces reactive oxygen species (ROS) formation and nonspecifically interacts with a number of proteins, including ROS-utilizing ones. Using native and 2D PAGE, we confirm that F105 changes the charge of some proteins by either oxidation or direct interaction with them. Therefore, it seems justified to conclude that being simultaneously a ROS inducer and damaging proteins responsible for ROS utilization, F105 impairs the cellular anti-ROS defense representing a prospective ROS-inducing antibacterial agent.

Organic-nanoclay composite materials as removal agents for environmental decontamination.

Here we overview the recent advances in the fabrication of sustainable composite nanomaterials with decontamination capacity towards inorganic and organic pollutants. In this regards, we present the development of hybrid systems based on clay nanoparticles with different shapes (such as kaolinite nanosheets and halloysite nanotubes) and organic molecules (biopolymers, surfactants, cucurbituril) as efficient removal agents for both aliphatic and aromatic hydrocarbons. Due to their high specific surface area, clay nanoparticles have been successfully employed as fillers for composite membranes with excellent filtration capacity. The preparation of composite gel beads based on biopolymers (alginate and pectin) and halloysite nanotubes has been discussed and their adsorption capacities towards both heavy metals and organic dyes have been highlighted. We describe the successful preparation of kaolinite/graphene composites as well as tubular inorganic micelles obtained by the select functionalization of the halloysite cavity with anionic surfactants. Finally, recent research on Pickering emulsions (for oil spill remediation) and bioremediation technologies has been discussed.

Fluorescence and Cytotoxicity of Cadmium Sulfide Quantum Dots Stabilized on Clay Nanotubes.

Quantum dots (QD) are widely used for cellular labeling due to enhanced brightness, resistance to photobleaching, and multicolor light emissions. CdS and CdxZn1-xS nanoparticles with sizes of 6⁻8 nm were synthesized via a ligand assisted technique inside and outside of 50 nm diameter halloysite clay nanotubes (QD were immobilized on the tube's surface). The halloysite⁻QD composites were tested by labeling human skin fibroblasts and prostate cancer cells. In human cell cultures, halloysite⁻QD systems were internalized by living cells, and demonstrated intense and stable fluorescence combined with pronounced nanotube light scattering. The best signal stability was observed for QD that were synthesized externally on the amino-grafted halloysite. The best cell viability was observed for CdxZn1-xS QD immobilized onto the azine-grafted halloysite. The possibility to use QD clay nanotube core-shell nanoarchitectures for the intracellular labeling was demonstrated. A pronounced scattering and fluorescence by halloysite⁻QD systems allows for their promising usage as markers for biomedical applications.

Nanohydrogel Formation within the Halloysite Lumen for Triggered and Sustained Release.

An easy strategy to obtain nanohydrogels within the halloysite nanotube (HNTs) lumen was investigated. Inorganic reverse micelles based on HNTs and hexadecyltrimethylammonium bromides were dispersed in chloroform, and the hydrophilic cavity was used as a nanoreactor to confine the gel formation based on alginate cross-linked by calcium ions. Spectroscopy and electron microscopy experiments proved the confinement of the polymer into the HNT lumen and the formation of calcium-mediated networks. Biological tests proved the biocompatibility of the hybrid hydrogel. The nanogel in HNTs was suitable for drug loading and sustained release with the opportunity of triggered burst release by chemical stimuli. Here, we propose a new strategy based on inorganic reverse micelles for nanohydrogel formation, which are suitable for industrial and biological applications as well as for selective and triggered adsorption and/or release.

Antimicrobial Effects of Sulfonyl Derivative of 2(5H)-Furanone against Planktonic and Biofilm Associated Methicillin-Resistant and -Susceptible Staphylococcus aureus.

The gram-positive opportunistic bacterium Staphylococcus aureus is one of the most common causatives of a variety of diseases including skin and skin structure infection or nosocomial catheter-associated infections. The biofilm formation that is an important virulence factor of this microorganism renders the antibiotic therapy ineffective, because biofilm-embedded bacteria exhibit strongly increased tolerance to antimicrobials. Here, we describe a novel 3-chloro-5(S)-[(1R,2S,5R)-2-isopropyl-5-methylcyclohexyloxy]-4-[4-methylphenylsulfonyl]-2(5H)-furanone (F105), possessing a sulfonyl group and l-menthol moiety. Minimal inhibitory and bactericidal concentration values (MIC and MBC) of F105 were 10 and 40 mg/L, respectively, suggesting F105 biocidal properties. F105 exhibits pronounced activity against biofilm-embedded S. aureus and increases the efficacy of aminoglycosides (amikacin, gentamicin, and kanamycin) and benzalkonium chloride with fractional inhibitory concentration index values of 0.33-0.44 and 0.29, respectively, suggesting an alternative external treatment option, e.g., for wound infections. Moreover, low concentrations (0.5-1.3 mg/L) of F105 reduced the MICs of these antimicrobials twofold. By using confocal laser scanning microscopy and CFU counting, we show explicitly that F105 also restores the antimicrobial activity of gentamicin and ampicillin against S. aureus biofilms by several orders of magnitude. Biofilm structures were not destroyed but sterilized, with embedded cells being almost completely killed at twofold MBC. While F105 is quite toxic (CC50/MBC ratio 0.2), our data suggest that the F105 chemotype might be a promising starting point for the development of complex topical agents for combined anti-staphylococcal biofilm-therapies restoring the efficacy of some antibiotics against difficult to treat S. aureus biofilm.

Binase Immobilized on Halloysite Nanotubes Exerts Enhanced Cytotoxicity toward Human Colon Adenocarcinoma Cells.

Many ribonucleases (RNases) are considered as promising tools for antitumor therapy because of their selective cytotoxicity toward cancer cells. Binase, the RNase from Bacillus pumilus, triggers apoptotic response in cancer cells expressing RAS oncogene which is mutated in a large percentage of prevalent and deadly malignancies including colorectal cancer. The specific antitumor effect of binase toward RAS-transformed cells is due to its direct binding of RAS protein and inhibition of downstream signaling. However, the delivery of proteins to the intestine is complicated by their degradation in the digestive tract and subsequent loss of therapeutic activity. Therefore, the search of new systems for effective delivery of therapeutic proteins is an actual task. This study is aimed to the investigation of antitumor effect of binase immobilized on natural halloysite nanotubes (HNTs). Here, we have developed the method of binase immobilization on HNTs and optimized the conditions for the enzyme loading and release (i); we have found the non-toxic concentration of pure HNTs which allows to distinguish HNTs- and binase-induced cytotoxic effects (ii); using dark-field and fluorescent microscopy we have proved the absorption of binase-loaded HNTs on the cell surface (iii) and demonstrated that binase-halloysite nanoformulations possessed twice enhanced cytotoxicity toward tumor colon cells as compared to the cytotoxicity of binase itself (iv). The enhanced antitumor activity of biocompatible binase-HNTs complex confirms the advisability of its future development for clinical practice.

Halloysite Nanotubes: Controlled Access and Release by Smart Gates.

Hollow halloysite nanotubes have been used as nanocontainers for loading and for the triggered release of calcium hydroxide for paper preservation. A strategy for placing end-stoppers into the tubular nanocontainer is proposed and the sustained release from the cavity is reported. The incorporation of Ca(OH)2 into the nanotube lumen, as demonstrated using transmission electron microscopy (TEM) imaging and Energy Dispersive X-ray (EDX) mapping, retards the carbonatation, delaying the reaction with CO2 gas. This effect can be further controlled by placing the end-stoppers. The obtained material is tested for paper deacidification. We prove that adding halloysite filled with Ca(OH)2 to paper can reduce the impact of acid exposure on both the mechanical performance and pH alteration. The end-stoppers have a double effect: they preserve the calcium hydroxide from carbonation, and they prevent from the formation of highly basic pH and trigger the response to acid exposure minimizing the pH drop-down. These features are promising for a composite nanoadditive in the smart protection of cellulose-based materials.