Farnesol and Selected Nanoparticles (Silver, Gold, Copper, and Zinc Oxide) as Effective Agents Against Biofilms Formed by Pathogenic Microorganisms.

Purpose: Biofilms, which are created by most microorganisms, are known for their widely developed drug resistance, even more than planktonic forms of microorganisms. The aim of the study was to assess the effectiveness of agents composed of farnesol and nanoparticles (silver, gold, copper, and zinc oxide) in the degradation of biofilms produced by pathogenic microorganisms. Methods: Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans were used to create the biofilm structure. Colloidal suspensions of silver, gold, copper, and zinc oxide (Ag, Au, Cu, ZnO) with the addition of farnesol (F) were used as the treatment factor. The size distribution of those composites was analyzed, their zeta potential was measured, and their structure was visualized by transmission electron microscopy. The viability of the microorganism strains was assessed by an XTT assay, the ability to form biofilms was analyzed by confocal microscopy, and the changes in biofilm structure were evaluated by scanning electron microscopy. The general toxicity toward the HFFF2 cell line was determined by a neutral red assay and a human inflammation antibody array. Results: The link between the two components (farnesol and nanoparticles) caused mutual stability of both components. Planktonic forms of the microorganisms were the most sensitive when exposed to AgF and CuF; however, the biofilm structure of all microorganism strains was the most disrupted (both inhibition of formation and changes within the structure) after AgF treatment. Composites were not toxic toward the HFFF2 cell line, although the expression of several cytokines was higher than in the not-treated group. Conclusion: The in vitro studies demonstrated antibiofilm properties of composites based on farnesol and nanoparticles. The greatest changes in biofilm structure were triggered by AgF, causing an alteration in the biofilm formation process as well as in the biofilm structure.

Ciprofloxacin-, Cefazolin-, and Methicilin-Soaked Graphene Paper as an Antibacterial Medium Suppressing Cell Growth.

This paper presents the results of research on the impact of graphene paper on selected bacterial strains. Graphene oxide, from which graphene paper is made, has mainly bacteriostatic properties. Therefore, the main goal of this research was to determine the possibility of using graphene paper as a carrier of a medicinal substance. Studies of the degree of bacterial inhibition were performed on Staphylococcus aureus and Pseudomonas aeruginosa strains. Graphene paper was analyzed not only in the state of delivery but also after the incorporation of the antibiotics ciprofloxacin, cefazolin, and methicillin into its structures. In addition, Fourier-Transform Infrared Spectroscopy, contact angle, and microscopic analysis of bacteria on the surface of the examined graphene paper samples were also performed. Studies have shown that graphene paper with built-in ciprofloxacin had a bactericidal effect on the strains of Staphylococcus aureus and Pseudomonas aeruginosa. In contrast, methicillin, as well as cefazolin, deposited on graphene paper acted mainly locally. Studies have shown that graphene paper can be used as a carrier of selected medicinal substances.

Influence of C60 Nanofilm on the Expression of Selected Markers of Mesenchymal-Epithelial Transition in Hepatocellular Carcinoma.

The epithelial-mesenchymal transition (EMT) is a process in which epithelial cells acquire the ability to actively migrate via a change to the mesenchymal phenotype. This mechanism occurs in an environment rich in cytokines and reactive oxygen species but poor in nutrients. The aim of this study was to demonstrate that the use of a fullerene C60 nanofilm can inhibit liver cancer cell invasion by restoring their non-aggressive, epithelial phenotype. We employed epithelial and mesenchymal HepG2 and SNU-449 liver cancer cells and non-cancerous mesenchymal HFF2 cells in this work. We used enzyme-linked immunosorbent assays (ELISAs) to determine the content of glutathione and transforming growth factor (TGF) in cells. We measured the total antioxidant capacity with a commercially available kit. We assessed cell invasion based on changes in morphology, the scratch test and the Boyden chamber invasion. In addition, we measured the effect of C60 nanofilm on restoring the epithelial phenotype at the protein level with protein membranes, Western blotting and mass spectrometry. C60 nanofilm downregulated TGF and increased glutathione expression in SNU-449 cells. When grown on C60 nanofilm, invasive cells showed enhanced intercellular connectivity; reduced three-dimensional invasion; and reduced the expression of key invasion markers, namely MMP-1, MMP-9, TIMP-1, TIMP-2 and TIMP-4. Mass spectrometry showed that among the 96 altered proteins in HepG2 cells grown on C60 nanofilm, 41 proteins are involved in EMT and EMT-modulating processes such as autophagy, inflammation and oxidative stress. The C60 nanofilm inhibited autophagy, showed antioxidant and anti-inflammatory properties, increased glucose transport and regulated the ß-catenin/keratin/Smad4/snail+slug and MMP signalling pathways. In conclusion, the C60 nanofilm induces a hybrid mesenchymal-epithelial phenotype and could be used in the prevention of postoperative recurrences.

Nanostructured graphene oxide enriched with metallic nanoparticles as a biointerface to enhance cell adhesion through mechanosensory modifications.

Nanostructuring is a process involving surface manipulation at the nanometric level, which improves the mechanical and biological properties of biomaterials. Specifically, it affects the mechanotransductive perception of the microenvironment of cells. Mechanical force conversion into an electrical or chemical signal contributes to the induction of a specific cellular response. The relationship between the cells and growth surface induces a biointerface-modifying cytophysiology and consequently a therapeutic effect. In this study, we present the fabrication of graphene oxide (GO)-based nanofilms decorated with metallic nanoparticles (NPs) as potential coatings for biomaterials. Our investigation showed the effect of decorating GO with metallic NPs for the modification of the physicochemical properties of nanostructures in the form of nanoflakes and nanofilms. A comprehensive biocompatibility screening panel revealed no disturbance in the metabolic activity of human fibroblasts (HFFF2) and bone marrow stroma cells (HS-5) cultivated on the GO nanofilms decorated with gold and copper NPs, whereas a significant cytotoxic effect of the GO nanocomplex decorated with silver NPs was demonstrated. The GO nanofilm decorated with gold NPs beneficially managed early cell adhesion as a result of the transient upregulation of α1ß5 integrin expression, acceleration of cellspreading, and formation of elongated filopodia. Additionally, the cells, sensing the substrate derived from the nanocomplex enriched with gold NPs, showed reduced elasticity and altered levels of vimentin expression. In the future, GO nanocomplexes decorated with gold NPs can be incorporated in the structure of architecturally designed biomimetic biomaterials as biocompatible nanostructuring agents with proadhesive properties.

Polyhydroxylated Fullerene C60(OH)40 Nanofilms Promote the Mesenchymal-Epithelial Transition of Human Liver Cancer Cells via the TGF-ß1/Smad Pathway.

Background: The various growth factors change the phenotype of neoplastic cells from sedentary (epithelial) to invasive (mesenchymal), which weaken intercellular connections and promote chemotaxis. It can be assumed that the use of anti-inflammatory polyhydroxyfull nanofilms will restore the sedentary phenotype of neoplastic cells in the primary site of the tumor and, consequently, increase the effectiveness of the therapy. Methods: The studies were carried out on liver cancer cells HepG2, C3A and SNU-449, and non-cancer hepatic cell line THLE-3. Transforming growth factor (TGF), epidermal growth factor and tumor necrosis factor were used to induce the epithelial-mesenchymal transition. C60(OH)40 nanofilm was used to induce the mesenchymal-epithelial transition. Obtaining an invasive phenotype was confirmed on the basis of changes in the morphology using inverted light microscopy. RT-PCR was used to confirm mesenchymal or epithelial phenotype based on e-cadherin, snail, vimentin expression or others. Water colloids at a concentration of 100 mg/L were used to create nanofilms of fullerene, fullerenol, diamond and graphene oxide. The ELISA test for the determination of TGF expression and growth factor antibody array were used to select the most anti-inflammatory carbon nanofilm. Mitochondrial activity and proliferation of cells were measured by XTT and BrdU tests. Results: Cells lost their natural morphology of cells growing in clusters and resembled fibroblast cells after adding a cocktail of factors. Among the four allotropic forms of carbon tested, only the C60(OH)40 nanofilm inhibited the secretion of TGF in all the cell lines used and inhibited the secretion of other factors, including insulin-like growth factor system. Nanofilm C60(OH)40 was non-toxic to liver cells and inhibited the TGF-ß1/Smad pathway of invasive cells treated with the growth factor cocktail. Conclusion: The introduction of an anti-inflammatory, nontoxic component that can induce the mesenchymal-epithelial transition of cancer cells may represent a future adjuvant therapy after tumor resection.

Influence of GO-Antisense miRNA-21 on the Expression of Selected Cytokines at Glioblastoma Cell Lines.

Introduction: Graphene oxide (GO) is a single layer of carbon atoms with unique properties, which are beneficial due to its surface functionalisation by miRNA. miRNAs are a non-coding small form of RNA that suppress the expression of protein-coding genes by translational repression or degradation of messenger RNA. Antisense miRNA-21 is very promising for future investigation in cancer therapy. This study aimed to detect cytokine expression levels after the administration of GO-antisense miRNA-21 into U87, U118, U251 and T98 glioma cell lines. Methods: U87, U118, U251 and T98 glioma cell line were investigated in term of viability, human cytokine expression level at protein and genes after treatment with GO, GO-antisense miRNA-21 and antisense miRNA-21. The delivery of antisense miRNA-21 into the glioma cell at in vitro investigation were conducted by GO based transfection and electroporation. Results: The results of the protein microarray and gene expression profile showed that complexes of GO-antisense miRNA-21 modified the metallopeptidase inhibitor 2 (TIMP-2), interleukin-6 (IL-6), interleukin 8 (IL-8), intercellular adhesion molecule 1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) expression level compared to transfection by electroporation of antisense miRNA-21 at investigated glioblastoma cell lines. The TIMP-2 protein and gene expression level was upregulated after antisense miRNA-21 delivery by GO complex into U87, U251 and T98 glioblastoma cell lines comparing to the non-treated control group. The downregulation at protein expression level of ICAM - 1 was observed at U87, U118, U251 and T98 glioma cell lines. Moreover, the IL-8 expression level at mRNA for genes and protein was decreased significantly after delivery the antisense-miRNA-21 by GO compared to electroporation as a transfection method. Discussion: This work demonstrated that the graphene oxide complexes with antisense miRNA-21 can effectively modulate the cytokine mRNA and protein expression level at U87, U118, U251 and T98 glioma cell lines.

Antimicrobial Properties and Assessment of the Content of Bioactive Compounds Lavandula angustifolia Mill. Cultivated in Southern Poland.

Lavender is a valued plant due to its cosmetic, perfumery, culinary, and health benefits. A wide range of applications is related to the composition of bioactive compounds, the quantity and quality of which is determined by various internal and external factors, i.e., variety, morphological part of the plant, and climatic and soil conditions during vegetation. In the presented work, the characterization of antimicrobial properties as well as the qualitative and quantitative assessment of bioactive compounds in the form of polyphenols in ethanol extracts from leaves and flowers of Lavandula angustifolia Mill. intended for border hedges, cultivated in the region of southern Poland, were determined. The composition of the fraction of volatile substances and antioxidant properties were also assessed. The conducted research shows that extracts from leaves and flowers significantly affected the viability of bacterial cells and the development of mold fungi. A clear decrease in the viability of bacteria and C. albicans cells was shown in the concentration of 0.32% of extracts. Leaf extracts were characterized by a much higher content of polyphenols and antioxidant properties than flower extracts. The composition of volatiles measured by GC-MS was significantly different between the extracts. Linalyl acetate and ocimene isomers mix dominated in flower extracts, whereas coumarin, γ-cadinene, and 7-methoxycoumarin were identified as dominant in leaf extracts.

Dependence of diamond nanoparticle cytotoxicity on physicochemical parameters: comparative studies of glioblastoma, breast cancer, and hepatocellular carcinoma cell lines.

Reports on the cytotoxicity of diamond nanoparticles (ND) are ambiguous and depend on the physicochemical properties of the material and the tested cell lines. Thus, the aim of this research was to evaluate the influence of thirteen types of diamond nanoparticles, differing in production method, size, and surface functional groups, on their cytotoxicity against four tumor cell lines (T98G, U-118 MG, MCF-7, and Hep G2) and one non-tumor cell line (HFF-1). In order to understand the dependence of diamond nanoparticles on physicochemical properties, the following parameters were analyzed: viability, cell membrane damage, morphology, and the level of intracellular general ROS and mitochondrial superoxide. The performed analyses revealed that all diamond nanoparticles showed no toxicity to MCF-7, Hep G2, and HFF-1 cells. In contrast, the same nanomaterials were moderately toxic for the glioblastoma T98G and U-118 MG cell lines. In general, the effect of the production method did not influence ND toxicity. Some changes in cell response after treatment with modified nanomaterials were observed, with the presence of carboxyl groups having a more detrimental effect than the presence of other functional groups. Although nanoparticles of different sizes caused similar toxicity, nanomaterials with bigger particles caused a more pronounced effect.

Effect of Melittin Complexes with Graphene and Graphene Oxide on Triple-Negative Breast Cancer Tumors Grown on Chicken Embryo Chorioallantoic Membrane.

One of the components of bee venom is melittin (M), which has strong lysing properties on membranes. M has high toxicity to cancer cells, but it also affects healthy cells, making it necessary to use methods for targeted delivery to ensure treatment. This research is a continuation of previous studies using graphene nanomaterials as M carriers to breast cancer cells. The studies described below are conducted on a more organized biological structure than what is found in vitro cells, namely, cancerous tumors grown on a chicken embryo chorioallantoic membrane. Caspase 3 and 8 levels are analyzed, and the level of oxidative stress markers and changes in protein expression for cytokines are examined. The results show that M complexes with nanomaterials reduce the level of oxidative stress more than M alone does, but the use of graphene (GN) as a carrier increases the level of DNA damage to a greater extent than the increase caused by M alone. An analysis of cytokine levels shows that the use of the M and GN complex increases the level of proteins responsible for inhibiting tumor progression to a greater extent than the increase occasioned by a complex with graphene oxide (GO). The results suggest that the use of GN as an M carrier may increase the toxic effect of M on structures located inside a cell.

Life in an optical fiber: Monitoring of cell cultures with microcavity in-line Mach-Zehnder interferometer.

Monitoring cell adhesion and growth are crucial for various applications involving drug screening, cytotoxicity, and cytocompatibility studies. However, acquiring accurate information about the growing state and responsiveness to a treatment of a cell system in a real-time and label-free manner is still a challenge. This work presents the first research on direct, real-time, and label-free adherent cell culture monitoring using a microcavity in-line Mach-Zehnder interferometer (µIMZI) fabricated in an optical fiber. The sensing solution based on µIMZI offers a great advantage over many other monitoring concepts tracking the changes taking place on the microcavity's bottom surface and within its volume, thus offering a greater penetration depth. In this study, we verified performance of the approach using a non-cancer bone marrow stromal cell line HS-5. The results demonstrate that the changes of the acquired signal are closely related to the different states of cells' adhesion, proliferation, morphology, and variation of mass. Thus, this label-free, real-time µIMZI-based monitoring technique gives a great promise to the analysis or monitoring of relevant new treatments in future scientific, as well as clinical applications.

Nanocomposites of Graphene Oxide-Silver Nanoparticles for Enhanced Antibacterial Activity: Mechanism of Action and Medical Textiles Coating.

The resistance of microorganisms to antibiotics is a crucial problem for which the application of nanomaterials is among a growing number of solutions. The aim of the study was to create a nanocomposite (composed of graphene oxide and silver nanoparticles) with a precise mode of antibacterial action: what enables textiles to be coated in order to exhibit antibacterial properties. A characterization of nanomaterials (silver nanoparticles and graphene oxide) by size distribution, zeta potential measurements, TEM visualization and FT-IR was performed. The biological studies of the nanocomposite and its components included the toxicity effect toward two pathogenic bacteria species, namely Pseudomonas aeruginosa and Staphylococcus aureus, interaction of nanomaterials with the outer layer of microorganisms, and the generation of reactive oxygen species and lipid peroxidation. Afterwards, antibacterial studies of the nanocomposite's coated textiles (cotton, interlining fabric, polypropylene and silk) as well as studies of the general toxicity towards a chicken embryo chorioallantoic membrane model were conducted. The toxicity of the nanocomposite used was higher than its components applied separately (zones of growth inhibition for P. aeruginosa for the final selected concentrations were as follows: silver nanoparticles 21 ± 0.7 mm, graphene oxide 14 ± 1.9 mm and nanocomposite 23 ± 1.6 mm; and for S. aureus were: silver nanoparticles 27 ± 3.8 mm, graphene oxide 14 ± 2.1 mm, and nanocomposite 28 ± 0.4 mm. The viability of P. aeruginosa and S. aureus after treatment with selected GO-Ag decreased to 27% and 31%, respectively, compared to AgNPs, when the viability of both species was 31% and 34%, accordingly). The coated textiles showed encouraging antibacterial features without general toxicity towards the chicken embryo chorioallantoic membrane model. We demonstrated that graphene oxide might constitute a functional platform for silver nanoparticles, improving the antibacterial properties of bare silver. Due to the application of the nanocomposite, the textiles showed promising antibacterial features with a low general toxicity, thereby creating a wide possibility for them to be used in practice.

MicroRNA Delivery by Graphene-Based Complexes into Glioblastoma Cells.

Glioblastoma (GBM) is the most common primary and aggressive tumour in brain cancer. Novel therapies, despite achievements in chemotherapy, radiation and surgical techniques, are needed to improve the treatment of GBM tumours and extend patients' survival. Gene delivery therapy mostly uses the viral vector, which causes serious adverse events in gene therapy. Graphene-based complexes can reduce the potential side effect of viral carries, with high efficiency of microRNA (miRNA) or antisense miRNA delivery to GBM cells. The objective of this study was to use graphene-based complexes to induce deregulation of miRNA level in GBM cancer cells and to regulate the selected gene expression involved in apoptosis. The complexes were characterised by Fourier transform infrared spectroscopy (FTIR), scanning transmission electron microscopy and zeta potential. The efficiency of miRNA delivery to the cancer cells was analysed by flow cytometry. The effect of the anticancer activity of graphene-based complexes functionalised by the miRNA sequence was analysed using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide salt (XTT) assays at the gene expression level. The results partly explain the mechanisms of miRNA deregulation stress, which is affected by graphene-based complexes together with the forced transport of mimic miR-124, miR-137 and antisense miR-21, -221 and -222 as an anticancer supportive therapy.

Diamond Nanofilm Normalizes Proliferation and Metabolism in Liver Cancer Cells.

PURPOSE: Surgical resection of hepatocellular carcinoma can be associated with recurrence resulting from the degeneration of residual volume of the liver. The objective was to assess the possibility of using a biocompatible nanofilm, made of a colloid of diamond nanoparticles (nfND), to fill the side after tumour resection and optimize its contact with proliferating liver cells, minimizing their cancerous transformation. METHODS: HepG2 and C3A liver cancer cells and HS-5 non-cancer cells were used. An aqueous colloid of diamond nanoparticles, which covered the cell culture plate, was used to create the nanofilm. The roughness of the resulting nanofilm was measured by atomic force microscopy. Mitochondrial activity and cell proliferation were measured by XTT and BrdU assays. Cell morphology and a scratch test were used to evaluate the invasiveness of cells. Flow cytometry determined the number of cells within the cell cycle. Protein expression in was measured by mass spectrometry. RESULTS: The nfND created a surface with increased roughness and exposed oxygen groups compared with a standard plate. All cell lines were prone to settling on the nanofilm, but cancer cells formed more relaxed clusters. The surface compatibility was dependent on the cell type and decreased in the order C3A >HepG2 >HS-5. The invasion was reduced in cancer lines with the greatest effect on the C3A line, reducing proliferation and increasing the G2/M cell population. Among the proteins with altered expression, membrane and nuclear proteins dominated. CONCLUSION: In vitro studies demonstrated the antiproliferative properties of nfND against C3A liver cancer cells. At the same time, the need to personalize potential therapy was indicated due to the differential protein synthetic responses in C3A vs HepG2 cells. We documented that nfND is a source of signals capable of normalizing the expression of many intracellular proteins involved in the transformation to non-cancerous cells.

Comparison of the Toxicity of Pristine Graphene and Graphene Oxide, Using Four Biological Models.

There are numerous applications of graphene in biomedicine and they can be classified into several main areas: delivery systems, sensors, tissue engineering and biological agents. The growing biomedical field of applications of graphene and its derivates raises questions regarding their toxicity. We will demonstrate an analysis of the toxicity of two forms of graphene using four various biological models: zebrafish (Danio rerio) embryo, duckweed (Lemna minor), human HS-5 cells and bacteria (Staphylococcus aureus). The toxicity of pristine graphene (PG) and graphene oxide (GO) was tested at concentrations of 5, 10, 20, 50 and 100 µg/mL. Higher toxicity was noted after administration of high doses of PG and GO in all tested biological models. Hydrophilic GO shows greater toxicity to biological models living in the entire volume of the culture medium (zebrafish, duckweed, S. aureus). PG showed the highest toxicity to adherent cells growing on the bottom of the culture plates-human HS-5 cells. The differences in toxicity between the tested graphene materials result from their physicochemical properties and the model used. Dose-dependent toxicity has been demonstrated with both forms of graphene.

Effect of Elaeagnus umbellata (Thunb.) fruit extract on H2O2-induced oxidative and inflammatory responses in normal fibroblast cells.

BACKGROUND: Elaeagnus umbellata is a plant commonly used in traditional Asian medicine for its many health benefits and strong antioxidative activity. Its therapeutic potential is believed to be connected to its effect on fibroblasts. This study aimed to investigate E. umbellata methanol-acetone extract's (EUE) defense against hydrogen peroxide (H2O2)-induced fibroblast damage. METHODS: Because the main biologically active compounds of E. umbellata are water-insoluble, we evaluated the effects of methanol-acetone fruit extracts using liquid chromatography (for ascorbic acid and beta-carotene) and spectrophotometry (for lycopene and total phenolics). The extract's antioxidative activity was measured using DPPH radical inhibition, and EUE's effect on human fibroblasts was also evaluated. We assessed the metabolic activity and apoptosis of HFFF-2 fibroblasts exposed to EUE and/or H2O2using the XTT test and flow cytometry, respectively. Superoxide dismutase activity and reactive oxygen species (ROS) production were evaluated using colorimetric and fluorometric assays, respectively. We measured pro-inflammatory cytokine (MIF, fractalkine, MCP-4, BLC, GCP-2, NAP-2, Eotaxin-2, and Eotaxin-3) expression in HFFF-2 cells using immunocytochemistry. RESULT: The extract increased HFFF-2 cell proliferation and reduced cell death caused by H2O2-induced stress. H2O2-treated fibroblasts had greater ROS production than cells treated with both H2O2 and EUE. Additionally, the group treated with H2O2 alone showed higher pro-inflammatory cytokine (MIF, MCP-4, NAP-2, Eotaxin-2, and Eotaxin-3) expression. CONCLUSION: EUE protected human fibroblasts from H2O2-induced oxidative stress and reduced the fibroblast-mediated inflammatory response triggered by ROS.

Graphene Oxide Scaffold Stimulates Differentiation and Proangiogenic Activities of Myogenic Progenitor Cells.

The physiological process of muscle regeneration is quite limited due to low satellite cell quantity and also the inability to regenerate and reconstruct niche tissue. The purpose of the study was to examine whether a graphene oxide scaffold is able to stimulate myogenic progenitor cell proliferation and the endocrine functions of differentiating cells, and therefore, their active participation in the construction of muscle tissue. Studies were carried out using mesenchymal cells taken from 6-day-old chicken embryos and human umbilical vein endothelial cells (HUVEC) were used to assess angiogenesis. The graphene scaffold was readily colonized by myogenic progenitor cells and the cells dissected from heart, brain, eye, and blood vessels did not avoid the scaffold. The scaffold strongly induced myogenic progenitor cell signaling pathways and simultaneously activated proangiogenic signaling pathways via exocrine vascular endothelial growth factor (VEGF) secretion. The present study revealed that the graphene oxide (GO) scaffold initiates the processes of muscle cell differentiation due to mechanical interaction with myogenic progenitor cell.

Silver and Graphenic Carbon Nanostructures Differentially Influence the Morphology and Viability of Cardiac Progenitor Cells.

The characteristic features of nanomaterials provide rich opportunities for a broad range of applications due to their different physicochemical properties. Nanocolloidal silver and graphenic carbon materials differ in most physicochemical characteristics, except for their nanodimensions. Since there is a growing demand for stem cell therapies for coronary disorders, examining cardiac progenitor cells (CPC) in terms of their response to nanostructure treatment seems to be a reasonable approach. Morphological studies and viability assessments were performed with CPC in vitro, treated with small concentrations of silver nanoparticles (AgNP), hierarchical nanoporous graphenic carbon (HNC) and their mixtures. A viability test confirmed the morphological assessment of CPC treated with AgNP and HNC; moreover, the action of both nanomaterials was time-dependent and dose-dependent. For AgNP, between the two of the applied concentrations lies a border between their potential beneficial effect and toxicity. For HNC, at a lower concentration, strong stimulation of cell viability was noted, whereas a higher dosage activated their differentiation. It is necessary to perform further research examining the mechanisms of the action of AgNP and especially of unexplored HNC, and their mixtures, on CPC and other cells.

Influence of Selected Carbon Nanostructures on the CYP2C9 Enzyme of the P450 Cytochrome.

Carbon nanostructures have recently gained significant interest from scientists due to their unique physicochemical properties and low toxicity. They can accumulate in the liver, which is the main expression site of cytochrome P450 (CYP450) enzymes. These enzymes play an important role in the metabolism of exogenous compounds, such as drugs and xenobiotics. Altered activity or expression of CYP450 enzymes may lead to adverse drug effects and toxicity. The objective of this study was to evaluate the influence of three carbon nanostructures on the activity and expression at the mRNA and protein levels of CYP2C9 isoenzyme from the CYP2C subfamily: Diamond nanoparticles, graphite nanoparticles, and graphene oxide platelets. The experiments were conducted using two in vitro models. A microsome model was used to assess the influence of the three-carbon nanostructures on the activity of the CYP2C9 isoenzyme. The CYP2C9 gene expression at the mRNA and protein levels was determined using a hepatoma-derived cell line HepG2. The experiments have shown that all examined nanostructures inhibit the enzymatic activity of the studied isoenzymes. Moreover, a decrease in the expression at the mRNA and protein levels was also observed. This indicates that despite low toxicity, the nanostructures can alter the enzymatic function of CYP450 enzymes, and the molecular pathways involved in their expression.

Use of Selected Carbon Nanoparticles as Melittin Carriers for MCF-7 and MDA-MB-231 Human Breast Cancer Cells.

Despite advanced techniques in medicine, breast cancer caused the deaths of 627,000 women in 2018. Melittin, the main component of bee venom, has lytic properties for many types of cells, including cancer cells. To increase its toxic effect, carbon nanoparticles, graphene oxide, pristine graphene, and diamond were used as carriers of melittin to breast cancer cells. To date, the effects of carbon nanoparticles as carriers of melittin on cancer cells have not been studied. The present study was carried out on MCF-7 and MDA-MB-231 cell lines. The investigation consisted of structural analysis of complexes using transmission electron microscopy, zeta potential measurements, evaluation of cell morphology, assessment of cell viability and membrane integrity, investigation of reactive oxygen species production, and investigation of mitochondrial membrane potential. Cell death was examined by flow cytometry and a membrane test for 43 apoptotic proteins. The results indicate that melittin complex with nanographene oxide has a stronger toxic effect on breast cancer cells than melittin alone. Moreover, nanodiamonds can protect cells against the lytic effects of melittin. All complexes reduced, but not completely eliminated the level of necrosis, compared to melittin. Thus, results suggest that the use of carbon nanoparticles as carriers for melittin may find use in medicine in the future.

Mechano-signalling, induced by fullerene C60 nanofilms, arrests the cell cycle in the G2/M phase and decreases proliferation of liver cancer cells.

INTRODUCTION AND OBJECTIVE: Degradation of the extracellular matrix (ECM) changes the physicochemical properties and dysregulates ECM-cell interactions, leading to several pathological conditions, such as invasive cancer. Carbon nanofilm, as a biocompatible and easy to functionalize material, could be used to mimic ECM structures, changing cancer cell behavior to perform like normal cells. METHODS: Experiments were performed in vitro with HS-5 cells (as a control) and HepG2 and C3A cancer cells. An aqueous solution of fullerene C60 was used to form a nanofilm. The morphological properties of cells cultivated on C60 nanofilms were evaluated with light, confocal, electron and atomic force microscopy. The cell viability and proliferation were measured by XTT and BrdU assays. Immunoblotting and flow cytometry were used to evaluate the expression level of proliferating cell nuclear antigen and determine the number of cells in the G2/M phase. RESULTS: All cell lines were spread on C60 nanofilms, showing a high affinity to the nanofilm surface. We found that C60 nanofilm mimicked the niche/ECM of cells, was biocompatible and non-toxic, but the mechanical signal from C60 nanofilm created an environment that affected the cell cycle and reduced cell proliferation. CONCLUSION: The results indicate that C60 nanofilms might be a suitable, substitute component for the niche of cancer cells. The incorporation of fullerene C60 in the ECM/niche may be an alternative treatment for hepatocellular carcinoma.