Biocompatibility and Cytotoxicity of Gold Nanoparticles: Recent Advances in Methodologies and Regulations.

Recent advances in the synthesis of metal nanoparticles (MeNPs), and more specifically gold nanoparticles (AuNPs), have led to tremendous expansion of their potential applications in different fields, ranging from healthcare research to microelectronics and food packaging. The properties of functionalised MeNPs can be fine-tuned depending on their final application, and subsequently, these properties can strongly modulate their biological effects. In this review, we will firstly focus on the impact of MeNP characteristics (particularly of gold nanoparticles, AuNPs) such as shape, size, and aggregation on their biological activities. Moreover, we will detail different in vitro and in vivo assays to be performed when cytotoxicity and biocompatibility must be assessed. Due to the complex nature of nanomaterials, conflicting studies have led to different views on their safety, and it is clear that the definition of a standard biosafety label for AuNPs is difficult. In fact, AuNPs' biocompatibility is strongly affected by the nanoparticles' intrinsic characteristics, biological target, and methodology employed to evaluate their toxicity. In the last part of this review, the current legislation and requirements established by regulatory authorities, defining the main guidelines and standards to characterise new nanomaterials, will also be discussed, as this aspect has not been reviewed recently. It is clear that the lack of well-established safety regulations based on reliable, robust, and universal methodologies has hampered the development of MeNP applications in the healthcare field. Henceforth, the international community must make an effort to adopt specific and standard protocols for characterisation of these products.

Influence of Bacillus subtilis and Trichoderma harzianum on Penthiopyrad Degradation under Laboratory and Field Studies.

In plant protection, biological preparations are used alternately with chemical pesticides. The applied microorganism can influence the concentration of chemical substances. Laboratory and field studies were conducted to assess the influence of Bacillus subtilis and Trichoderma harzianum on the penthiopyrad concentration. In laboratory studies, the effectiveness of penthiopyrad degradation by B. subtilis was approximately 5% during 14 days of the experiment. For penthiopyrad treated with T. harzianum strains, the degradation effectiveness ranged from 34.2% on Day 3 to 56.9% on Day 14. In experiments testing the effects of mixed culture of microorganisms, the effectiveness of penthiopyrad degradation ranged from 23.7% on Day 3 to 29.1% on Day 14. After treatment of apple trees of Gala and Golden Delicious varieties with a biological preparation, a maximum degradation of penthiopyrad of 20% was found in both varieties. Samples of apples were prepared by the quick, easy, cheap, effective, rugged and safe (QuEChERS) method, and penthiopyrad was analyzed by gas chromatography with a mass detector. A determined value of the chronic exposure to penthiopirad was 1.02% of the acceptable daily intake, both for children and for adults. The acute exposure amounted to 7.2% and 1.9% of the acute reference dose for children and adults, respectively. These values were considered to be acceptable and not threatening to health.

A Non-Vector Approach to Increase Lipid Levels in the Microalga Planktochlorella nurekis.

Microalgae are freshwater and marine unicellular photosynthetic organisms that utilize sunlight to produce biomass. Due to fast microalgal growth rate and their unique biochemical profiles and potential applications in food and renewable energy industries, the interest in microalgal research is rapidly increasing. Biochemical and genetic engineering have been considered to improve microalgal biomass production but these manipulations also limited microalgal growth. The aim of the study was the biochemical characterization of recently identified microalgal strain Planktochlorella nurekis with elevated cell size and DNA levels compared to wild type strain that was achieved by a safe non-vector approach, namely co-treatment with colchicine and cytochalasin B (CC). A slight increase in growth rate was observed in twelve clones of CC-treated cells. For biochemical profiling, several parameters were considered, namely the content of proteins, amino acids, lipids, fatty acids, ß-glucans, chlorophylls, carotenoids, B vitamins and ash. CC-treated cells were characterized by elevated levels of lipids compared to unmodified cells. Moreover, the ratio of carotenoids to chlorophyll a and total antioxidant capacity were slightly increased in CC-treated cells. We suggest that Planktochlorella nurekis with modified DNA levels and improved lipid content can be considered to be used as a dietary supplement and biofuel feedstock.

Green pyomelanin-mediated synthesis of gold nanoparticles: modelling and design, physico-chemical and biological characteristics.

BACKGROUND: Synthesis of nanoparticles (NPs) and their incorporation in materials are amongst the most studied topics in chemistry, physics and material science. Gold NPs have applications in medicine due to their antibacterial and anticancer activities, in biomedical imaging and diagnostic test. Despite chemical synthesis of NPs are well characterized and controlled, they rely on the utilization of harsh chemical conditions and organic solvent and generate toxic residues. Therefore, greener and more sustainable alternative methods for NPs synthesis have been developed recently. These methods use microorganisms, mainly yeast or yeast cell extract. NPs synthesis with culture supernatants are most of the time the preferred method since it facilitates the purification scheme for the recovery of the NPs. Extraction of NPs, formed within the cells or cell-wall, is laborious, time-consuming and are not cost effective. The bioactivities of NPs, namely antimicrobial and anticancer, are known to be related to NPs shape, size and size distribution. RESULTS: Herein, we reported on the green synthesis of gold nanoparticles (AuNPs) mediated by pyomelanin purified from the yeast Yarrowia lipolytica. A three levels four factorial Box-Behnken Design (BBD) was used to evaluate the influence of temperature, pH, gold salt and pyomelanin concentration on the nanoparticle size distribution. Based on the BBD, a quadratic model was established and was applied to predict the experimental parameters that yield to AuNPs with specific size. The synthesized nanoparticles with median size value of 104 nm were of nanocrystalline structure, mostly polygonal or spherical. They exhibited a high colloidal stability with zeta potential of - 28.96 mV and a moderate polydispersity index of 0.267. The absence of cytotoxicity of the AuNPs was investigated on two mammalian cell lines, namely mouse fibroblasts (NIH3T3) and human osteosarcoma cells (U2OS). Cell viability was only reduced at AuNPs concentration higher than 160 µg/mL. Moreover, they did not affect on the cell morphology. CONCLUSION: Our results indicate that different process parameters affect significantly nanoparticles size however with the mathematical model it is possible to define the size of AuNPs. Moreover, this melanin-based gold nanoparticles showed neither cytotoxicity effect nor altered cell morphology.

Green synthesis and antibacterial effects of aqueous colloidal solutions of silver nanoparticles using camomile terpenoids as a combined reducing and capping agent.

Green synthesis method using camomile extract was applied to synthesize silver nanoparticles to tune their antibacterial properties merging the synergistic effect of camomile and Ag. Scanning transmission electron microscopy revealed that camomile extract (CE) consisted of porous globular nanometer sized structures, which were a perfect support for Ag nanoparticles. The Ag nanoparticles synthesized with the camomile extract (AgNPs/CE) of 7 nm average sizes, were uniformly distributed on the CE support, contrary to the pure Ag nanoparticles synthesized with glucose (AgNPs/G), which were over 50 nm in diameter and strongly agglomerated. The energy dispersive X-ray spectroscopy chemical analysis showed that camomile terpenoids act as a capping and reducing agent being adsorbed on the surface of AgNPs/CE enabling their reduction from Ag(+) and preventing them from agglomeration. Fourier transform infrared and ultraviolet-visible spectroscopy measurements confirmed these findings, as the spectra of AgNPs/CE, compared to pure CE, did not contain the 1109 cm(-1) band, corresponding to -C-O groups of terpenoids and the peaks at 280 and 320 nm, respectively. Antibacterial tests using four bacteria strains showed that the AgNPs/CE performed five times better compared to CE AgNPs/G samples, reducing totally all the bacteria in 2 h.

ChIP - Does it work correctly? The optimization steps of chromatin immunoprecipitation.

The proteins interaction with DNA is one of the key regulatory elements of many biological processes; including gene transcription, epigenetic modification or cell differentiation. Immunoprecipitation of chromatin; ChIP; is a method used to assess the interaction of the protein with a DNA sequence, and determines the localization of specific locus in the genome. The main steps of this method are fixation, sonication, immunoprecipitation and analysis of DNA. Although the immunoprecipitation assay is a multipurpose tool applied in biochemistry and biotechnology, it requires optimization. This paper describes several critical parameters that should be taken into account when immunoprecipitation assay is applied.

Biosynthesis of the antioxidant γ-glutamyl-cysteine with engineered Yarrowia lipolytica.

The dipeptide γ-glutamylcysteine (γ-GC), the first intermediate of glutathione (GSH) synthesis, is considered as a promising drug to reduce or prevent plethora of age-related disorders such as Alzheimer and Parkinson diseases. The unusual γ-linkage between the two constitutive amino acids, namely cysteine and glutamate, renders its chemical synthesis particularly challenging. Herein, we report on the metabolic engineering of the non-conventional yeast Yarrowia lipolytica for efficient γ-GC synthesis. The yeast was first converted into a γ-GC producer by disruption of gene GSH2 encoding GSH synthase and by constitutive expression of GSH1 encoding glutamylcysteine ligase. Subsequently genes involved in cysteine and glutamate anabolism, namely MET4, CYSE, CYSF, and GDH1 were overexpressed with the aim to increase their intracellular availability. With such a strategy, a γ-GC titer of 464 nmol mg-1 protein (93 mg gDCW-1 ) was obtained within 24 h of cell growth.

Impact of heat shock transcription factor 1 on global gene expression profiles in cells which induce either cytoprotective or pro-apoptotic response following hyperthermia.

BACKGROUND: Elevated temperatures induce activation of the heat shock transcription factor 1 (HSF1) which in somatic cells leads to heat shock proteins synthesis and cytoprotection. However, in the male germ cells (spermatocytes) caspase-3 dependent apoptosis is induced upon HSF1 activation and spermatogenic cells are actively eliminated. RESULTS: To elucidate a mechanism of such diverse HSF1 activity we carried out genome-wide transcriptional analysis in control and heat-shocked cells, either spermatocytes or hepatocytes. Additionally, to identify direct molecular targets of active HSF1 we used chromatin immunoprecipitation assay (ChIP) combined with promoter microarrays (ChIP on chip). Genes that are differently regulated after HSF1 binding during hyperthermia in both types of cells have been identified. Despite HSF1 binding to promoter sequences in both types of cells, strong up-regulation of Hsps and other genes typically activated by the heat shock was observed only in hepatocytes. In spermatocytes HSF1 binding correlates with transcriptional repression on a large scale. HSF1-bound and negatively regulated genes encode mainly for proteins required for cell division, involved in RNA processing and piRNA biogenesis. CONCLUSIONS: Observed suppression of the transcription could lead to genomic instability caused by meiotic recombination disturbances, which in turn might induce apoptosis of spermatogenic cells. We propose that HSF1-dependent induction of cell death is caused by the simultaneous repression of many genes required for spermatogenesis, which guarantees the elimination of cells damaged during heat shock. Such activity of HSF1 prevents transmission of damaged genetic material to the next generation.

Effect of microorganisms on degradation of fluopyram and tebuconazole in laboratory and field studies.

Nowadays, chemical pesticides are the most widespread measure used to control crop pests and diseases. However, their negative side effects prompted the researchers to search for alternative options that were safer for the environment and people. Pesticide biodegradation by microorganisms seems to be the most reasonable alternative. The aim of the laboratory studies was to assess the influence of Bacillus subtilis and Trichoderma harzianum, used separately and combined together, on fluopyram and tebuconazole degradation. In field studies, the degradation of fluopyram and tebuconazole after the application of the biological preparation in apples was investigated. The results from the laboratory studies show that the greatest decomposition of fluopyram and tebuconazole was observed in tests with T. harzianum in a range of 74.3-81.5% and 44.5-49.2%, respectively. The effectiveness of fluopyram degradation by B. subtilis was 7.5%, while tebuconazole inhibited bacterial cell growth and no degradation was observed. The mixture of microorganisms affected the degradation of fluopyram in a range of 8.3-24.1% and tebuconazole in a range of 6.1-23.3%. The results from the field studies show that degradation increased from 3.1 to 30.8% for fluopyram and from 0.4 to 14.3% for tebuconazole when compared to control samples. The first-order kinetics models were used to simulate the residue dissipation in apples. For the determination of pesticide residues, the QuEChERS method for apple sample preparation was performed, followed by GC-MS/MS technique. Immediately after the treatments, the maximum residue level (MRL) values for tebuconazole were exceeded, and it was equal to 100.7% MRL for the Red Jonaprince variety and 132.3% MRL for the Gala variety. Thus, preharvest time is recommended to obtain apples in which the concentration of pesticides is below the MRL and which can be recognized as safe for humans.

Biomaterial composed of chitosan, riboflavin, and hydroxyapatite for bone tissue regeneration.

Biomaterial engineering approaches involve using a combination of miscellaneous bioactive molecules which may promote cell proliferation and, thus, form a scaffold with the environment that favors the regeneration process. Chitosan, a naturally occurring biodegradable polymer, possess some essential features, i.e., biodegradability, biocompatibility, and in the solid phase good porosity, which may contribute to promote cell adhesion. Moreover, doping of the materials with other biocompounds will create a unique and multifunctional scaffold that will be useful in regenerative medicine. This study is focused on the manufacturing and characterization of composite materials based on chitosan, hydroxyapatite, and riboflavin. The resulting films were fabricated by the casting/solvent evaporation method. Morphological and spectroscopy analyses of the films revealed a porous structure and an interconnection between chitosan and apatite. The composite material showed an inhibitory effect on Staphylococcus aureus and exhibited higher antioxidant activity compared to pure chitosan. In vitro studies on riboflavin showed increased cell proliferation and migration of fibroblasts and osteosarcoma cells, thus demonstrating their potential for bone tissue engineering applications.

Treasure on the Earth-Gold Nanoparticles and Their Biomedical Applications.

Recent advances in the synthesis of metal nanoparticles (NPs) have led to tremendous expansion of their potential applications in different fields, ranging from healthcare research to microelectronics and food packaging. Among the approaches for exploiting nanotechnology in medicine, gold nanomaterials in particular have been found as the most promising due to their unique advantages, such as in sensing, image enhancement, and as delivery agents. Although, the first scientific article on gold nanoparticles was presented in 1857 by Faraday, during the last few years, the progress in manufacturing these nanomaterials has taken an enormous step forward. Due to the nanoscale counterparts of gold, which exhibit distinct properties and functionality compared to bulk material, gold nanoparticles stand out, in particular, in therapy, imaging, detection, diagnostics, and precise drug delivery. This review summarizes the current state-of-the-art knowledge in terms of biomedical applications of gold nanoparticles. The application of AuNPs in the following aspects are discussed: (i) imaging and diagnosing of specific target; (ii) treatment and therapies using AuNPs; and (iii) drug delivery systems with gold nanomaterials as a carrier. Among the different approaches in medical imaging, here we either consider AuNPs as a contrast agent in computed tomography (CT), or as a particle used in optical imaging, instead of fluorophores. Moreover, their nontoxic feature, compared to the gadolinium-based contrast agents used in magnetic resonance imaging, are shown. The tunable size, shape, and functionality of gold nanoparticles make them great carriers for targeted delivery. Therefore, here, we summarize gold-based nanodrugs that are FDA approved. Finally, various approaches to treat the specific diseases using AuNPs are discussed, i.e., photothermal or photodynamic therapy, and immunotherapy.

[Finding targets of transcriptional regulators--chromatin immunoprecipitation assay (ChIP)]. / W poszukiwaniu miejsc wiazania regulatorów transkrypcji--technika immunoprecypitacji chromatyny (ChIP).

The protein-DNA interactions play a crucial role in many cellular processes such as DNA replication, recombination, repair, or gene transcription. Here we describe a chromatin immunoprecipitation (ChIP) technique, a tool for identifying DNA regions, which are in vivo associated with protein of interest. The main steps of ChIP are: fixation, chromatin fragmentation, immunoprecipitation with a specific antibody, and analysis of the immunoprecipitated DNA. Different analytical tools combined with ChIP (ChIP-chip, ChIP-seq) enable researchers to map protein binding in a truly genome-wide manner with high resolution.

Noncytotoxic silver nanoparticles as a new antimicrobial strategy.

Drug-resistance of bacteria is an ongoing problem in hospital treatment. The main mechanism of bacterial virulency in human infections is based on their adhesion ability and biofilm formation. Many approaches have been invented to overcome this problem, i.e. treatment with antibacterial biomolecules, which have some limitations e.g. enzymatic degradation and short shelf stability. Silver nanoparticles (AgNPs) may be alternative to these strategies due to their unique and high antibacterial properties. Herein, we report on yeast Saccharomyces cerevisiae extracellular-based synthesis of AgNPs. Transmission electron microscopy (TEM) revealed the morphology and structure of the metallic nanoparticles, which showed a uniform distribution and good colloid stability, measured by hydrodynamic light scattering (DLS). The energy dispersive X-ray spectroscopy (EDS) of NPs confirms the presence of silver and showed that sulfur-rich compounds act as a capping agent being adsorbed on the surface of AgNPs. Antimicrobial tests showed that AgNPs inhibit the bacteria growth, while have no impact on fungi growth. Moreover, tested NPs was characterized by high inhibitory potential of bacteria biofilm formation but also eradication of established biofilms. The cytotoxic effect of the NPs on four mammalian normal and cancer cell lines was tested through the metabolic activity, cell viability and wound-healing assays. Last, but not least, ability to deep penetration of the silver colloid to the root canal was imaged by scanning electron microscopy (SEM) to show its potential as the material for root-end filling.

Characterization of a nontoxic pyomelanin pigment produced by the yeast Yarrowia lipolytica.

In this study, we report on the ability of the yeast Yarrowia lipolytica W29 to produce an extracellular melanin-like brown pigment at high yield (0.5 mg/ml) in culture medium supplemented with L-tyrosine. This pigment has been characterized as pyomelanin and its synthesis was found to occur by the so-called HGA-melanin pathway. The purified pyomelanin was found embedded with antioxidant properties as it exhibited a radical scavenging activity toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 of 230 µg/ml. It was also characterized as noncytotoxic toward two mammalian cell lines, namely the mouse fibroblast NIH3T3 and human keratinocytes HaCaT. When blended with different commercial sunscreens, the purified pyomelanin increased significantly the sun protection factor (SPF) value, highlighting its potential utilization as UV-filter in cosmetic preparations.

Efficient NIR energy conversion of plasmonic silver nanostructures fabricated with the laser-assisted synthetic approach for endodontic applications.

Silver nanoparticles were synthesized with the laser-assisted wet chemical approach at room temperature. The effect of light exposure on the silver nanoparticles' spatial parameters shaping the localized surface plasmon resonance has been evaluated. The optical, structural and morphological characterizations of the Ag nanostructures were conducted with UV-VIS-NIR spectrophotometry, DLS and TEM techniques. The ability of the light-modified Ag nanostructures for energy conversion under the influence of 445 and 880 nm laser radiation is estimated. We have found that the most efficient heat generation can be achieved using triangular Ag nanostructures under the NIR irradiation (880 nm). The temperature effect on the Ag nanostructures' antibacterial properties has been evaluated against the Staphylococcus aureus population. The prospects of triangular Ag nanostructures' application on modern endodontics for the rapid nano-laser disinfection of the root canal system of the human tooth have been demonstrated.

Correction: Efficient NIR energy conversion of plasmonic silver nanostructures fabricated with the laser-assisted synthetic approach for endodontic applications.

[This corrects the article DOI: 10.1039/D0RA06614A.].

Alternative Approach for Fighting Bacteria and Fungi: Use of Modified Fluorapatite.

The recent studies on fluorapatite and hydroxyapatite, in the context of their medical applications, have shown that the former has relatively higher thermal stability and better mechanical properties than the latter. Moreover, the presence of fluoride ions is relevant for protection of teeth from dental caries since they stimulate processes of mineralization and crystallization. In this report, a silver modified fluorapatite (FAP/Ag), considered as a novel biomedical compound, was tested for its bactericidal, fungicidal and cytotoxicity activity. All these features were compared to the impact of pure FAP and were evaluated against the bacteria and fungi strains, which constitute main pathogenic species among orthopedic clinical isolates of implant-associated infections. Generally, considerable increase in the antimicrobial activity was observed when silver modified fluorapatite was compared to the pure material. This was manifested among others by disturbance of cell growth pattern and various deformations in the final cell shape as revealed using atomic force microscopy (AFM). Regarding toxicity nowadays as a major issue in implantation, we additionally examined whether the Ag+ ions have an impact on human keratinocytes and mouse fibroblasts using a cytotoxicity assay. To conclude, owing to a great bactericidal potential without collateral cytotoxicity effect the fluorapatite doped with silver ions may be considered a promising biocomponent useful in medical and healthcare applications.

Development of noncytotoxic silver-chitosan nanocomposites for efficient control of biofilm forming microbes.

Severe bacterial and fungal infections have become a major clinical and public health concern. Nowadays, additional efforts are needed to develop effective antimicrobial materials that are not harmful to human cells. This work describes the synthesis and characterization of chitosan-ascorbic acid-silver nanocomposites as films exhibiting high antimicrobial activity and non-cytotoxicity towards human cells. The reductive and stabilizing activity of both the biocompatible polymer chitosan and ascorbic acid were used in the synthesis of silver nanoparticles (AgNPs). Herein, we propose an improved composite synthesis based on medium average molecular weight chitosan with a high deacetylation degree, that together with ascorbic acid gave films with a uniform distribution of small AgNPs (<10 nm) exhibiting high antimicrobial activity against biofilm forming bacterial and fungal strains of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. At the same time, the resulting solid nanocomposites showed, at the same doses, reduced or totally excluded cytotoxicity on mammalian somatic and tumoral cells. Data obtained in the present study suggest that adequately designed chitosan-silver nanocomposites are powerful and promising materials for reducing pathogenic microorganism-associated infections without harmful effects towards mammalian cells.

Development of noncytotoxic chitosan-gold nanocomposites as efficient antibacterial materials.

This work describes the synthesis and characterization of noncytotoxic nanocomposites either colloidal or as films exhibiting high antibacterial activity. The biocompatible and biodegradable polymer chitosan was used as reducing and stabilizing agent for the synthesis of gold nanoparticles embedded in it. Herein, for the first time, three different chitosan grades varying in the average molecular weight and deacetylation degree (DD) were used with an optimized gold precursor concentration. Several factors were analyzed in order to obtain antimicrobial but not cytotoxic nanocomposite materials. Films based on chitosan with medium molecular weight and the highest DD exhibited the highest antibacterial activity against biofilm forming strains of Staphylococcus aureus and Pseudomonas aeruginosa. The resulting nanocomposites did not show any cytotoxicity against mammalian somatic and tumoral cells. They produced a disruptive effect on the bacteria wall while their internalization was hindered on the eukaryotic cells. This selectivity and safety make them potentially applicable as antimicrobial coatings in the biomedical field.

Simple assay of trehalose in industrial yeast.

Trehalose is an essential chemical marker to control a quality of the industrial yeast strains and to assess a tolerance of the yeasts products to different physical stresses. A high-performance liquid chromatography analysis with charged aerosol detection (HPLC-CAD) was developed for trehalose determination in industrial yeasts. The method offers a linearity in the range of 5.0-15 mM with linear regression coefficient R(2)=0.9995, a good reproducibility and relatively short analysis time (7 min). Trehalose can be detected at concentrations as low as 0.07 mM, and limit of precise quantification is 0.2 mM. The coefficient of variation (CV%) is 0.3%. The developed method is more sensitive compared with conventional chromatography procedure with UV absorbance detection. It was shown that the proposed method can be used in baker's industry to control a quality of the yeast products and to assess biotechnological significance of the yeast strains.