The cytocompatibility of graphene oxide as a platform to enhance the effectiveness and safety of silver nanoparticles through in vitro studies.

The increasing emergence of antibiotic-resistant bacteria and the need to reduce the use of antibiotics call for the development of safe alternatives, such as silver nanoparticles. However, their potential cytotoxic effect needs to be addressed. Graphene oxide provides a large platform that can increase the effectiveness and safety of silver nanoparticles. Graphene oxide and silver nanoparticles complex applied as a part of an innovative material might have direct contact with human tissues, such as skin, or might be inhaled from aerosol or exfoliated pieces of the complex. Thereby, the safety of the prepared complex has to be evaluated carefully, employing a range of methods. We demonstrated the high cytocompatibility of graphene oxide and the graphene oxide-silver nanoparticles complex toward human cell lines, fetal foreskin fibroblasts (HFFF2), and lung epithelial cells (A549). The supporting platform of graphene oxide also neutralized the slight toxicity of bare silver nanoparticles. Finally, in studies on Staphylococcus aureus and Pseudomonas aeruginosa, the number of bacteria reduction was observed after incubation with silver nanoparticles and the graphene oxide-silver nanoparticles complex. Our findings confirm the possibility of employing a graphene oxide-silver nanoparticles complex as a safe agent with reduced silver nanoparticles' cytotoxicity and antibacterial properties.

A Comprehensive Assessment of the Biocompatibility and Safety of Diamond Nanoparticles on Reconstructed Human Epidermis.

Diamond nanoparticles, also known as nanodiamonds (NDs), exhibit remarkable, awe-inspiring properties that make them suitable for various applications in the field of skin care products. However, a comprehensive assessment of their compatibility with human skin, according to the irritation criteria established by the Organization for Economic Cooperation and Development (OECD), has not yet been conducted. The purpose of this study was to evaluate if diamond nanoparticles at a concentration of 25 µg/mL, incubated with reconstituted human epidermis (EpiDermTM) for 18 h, conform to the OECD TG439 standard used to classify chemical irritants. For this purpose, a cell viability test (MTT assay), histological assessment, and analysis of pro-inflammatory cytokine expression were performed. The results indicated that NDs had no toxic effect at the tested concentration. They also did not adversely affect tissue structure and did not lead to a simultaneous increase in protein and mRNA expression of the analyzed cytokines. These results confirm the safety and biocompatibility of NDs for application in skincare products, thereby creating a wide range of possibilities to exert an impact on the advancement of contemporary cosmetology in the future.

Changes in novel anti-infalmmatory cytokine concetration in the bood of endurance and race horses at different levels of training.

Several anti-inflammatory cytokines have been proposed as markers for exercise monitoring in humans such as the interleukin 1 receptor agonist (IL-ra), or interleukin 13 (IL-13). Equine athletes may be considered a model for human exercise physiology research, however there is a lack of such studies of this species. Thus, we decided to examine the changes of IL-1ra and IL-13 in serum concentration during aerobic (endurance) and anaerobic (race) exercise in horses of different fitness levels in comparison with the well-known anti-inflammatory cytokine interleukin 10 (IL-10). The group of endurance horses (n = 13) consisted of animals competing over 100 (n = 7) and 120 km (n = 6) rides. The group of racehorses (n = 18) consisted of trained (n = 9) and untrained (n = 9) animals. The blood samples were obtained before and after the exercise. The ELISA test was performed to evaluate the changes of IL-1ra, IL-13 and IL-10 during different types of exercise. In endurance horses there was an increase in IL-13 (p = 0.0012) after the 100 km ride and in IL-1ra (p = 0.0411) after the 120 km ride. In race horses there was a higher IL-13 basal serum concentration in the untrained group, as well as a decrease of IL-13 after exercise (p = 0.0188). In trained racehorses there was an increase in IL-1ra (p < 0.0001) and IL-13 after exercise (p = 0.0028). In conclusion, the reaction of IL-1ra and IL-13 to different types of exercise differ from each other. Thus, in future, they may be helpful in monitoring the fitness of horses, however more research is needed.

Reduced Graphene Oxide Modulates the FAK-Dependent Signaling Pathway in Glioblastoma Multiforme Cells In Vitro.

Aggressive invasiveness is a common feature of malignant gliomas, despite their high level of tumor heterogeneity and possible diverse cell origins. Therefore, it is important to explore new therapeutic methods. In this study, we evaluated and compared the effects of graphene (GN) and reduced graphene oxides (rGOs) on a highly invasive and neoplastic cell line, U87. The surface functional groups of the GN and rGO flakes were characterized by X-ray photoelectron spectroscopy. The antitumor activity of these flakes was obtained by using the neutral red assay and their anti-migratory activity was determined using the wound healing assay. Further, we investigated the mRNA and protein expression levels of important cell adhesion molecules involved in migration and invasiveness. The rGO flakes, particularly rGO/ATS and rGO/TUD, were found highly toxic. The migration potential of both U87 and Hs5 cells decreased, especially after rGO/TUD treatment. A post-treatment decrease in mobility and FAK expression was observed in U87 cells treated with rGO/ATS and rGO/TUD flakes. The rGO/TUD treatment also reduced ß-catenin expression in U87 cells. Our results suggest that rGO flakes reduce the migration and invasiveness of U87 tumor cells and can, thus, be used as potential antitumor agents.

Equid Alphaherpesvirus 1 Modulates Actin Cytoskeleton and Inhibits Migration of Glioblastoma Multiforme Cell Line A172.

Equid alphaherpesvirus 1 (EHV-1) causes respiratory diseases, abortion, and neurological disorders in horses. Recently, the oncolytic potential of this virus and its possible use in anticancer therapy has been reported, but its influence on cytoskeleton was not evaluated yet. In the following study, we have examined disruptions in actin cytoskeleton of glioblastoma multiforme in vitro model-A172 cell line, caused by EHV-1 infection. We used three EHV-1 strains: two non-neuropathogenic (Jan-E and Rac-H) and one neuropathogenic (EHV-1 26). Immunofluorescent labelling, confocal microscopy, real-time cell growth analysis and OrisTM cell migration assay revealed disturbed migration of A172 cells infected with the EHV-1, probably due to rearrangement of actin cytoskeleton and the absence of cell projections. All tested strains caused disruption of the actin network and general depolymerization of microfilaments. The qPCR results confirmed the effective replication of EHV-1. Thus, we have demonstrated, for the first time, that EHV-1 infection leads to inhibition of proliferation and migration in A172 cells, which might be promising for new immunotherapy treatment.

Effects of Metallic and Carbon-Based Nanomaterials on Human Pancreatic Cancer Cell Lines AsPC-1 and BxPC-3.

Pancreatic cancer, due to its asymptomatic development and drug-resistance, is difficult to cure. As many metallic and carbon-based nanomaterials have shown anticancer properties, we decided to investigate their potential use as anticancer agents against human pancreatic adenocarcinoma. The objective of the study was to evaluate the toxic properties of the following nanomaterials: silver (Ag), gold (Au), platinum (Pt), graphene oxide (GO), diamond (ND), and fullerenol (C60(OH)40) against the cell lines BxPC-3, AsPC-1, HFFF-2, and HS-5. The potential cytotoxic properties were evaluated by the assessment of the cell morphology, cell viability, and cell membrane damage. The cancer cell responses to GO and ND were analysed by determination of changes in the levels of 40 different pro-inflammatory proteins. Our studies revealed that the highest cytotoxicity was obtained after the ND treatment. Moreover, BxPC-3 cells were more sensitive to ND than AsPC-1 cells due to the ND-induced ROS production. Furthermore, in both of the cancer cell lines, ND caused an increased level of IL-8 and a decreased level of TIMP-2, whereas GO caused only decreased levels of TIMP-2 and ICAM-1 proteins. This work provides important data on the toxicity of various nanoparticles against pancreatic adenocarcinoma cell lines.

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.

The Effect of the Clenbuterol-ß2-Adrenergic Receptor Agonist on the Peripheral Blood Mononuclear Cells Proliferation, Phenotype, Functions, and Reactive Oxygen Species Production in Race Horses In Vitro.

Clenbuterol, the ß2-adrenoceptor agonist, is gaining growing popularity because of its effects on weight loss (i.e., chemical liposuction). It is also popular in bodybuilding and professional sports, due to its effects that are similar to anabolic steroids. However, it is prohibited by anti-doping control. On the other hand, it is suggested that clenbuterol can inhibit the inflammatory process. The cells from 14 untrained and 14 well-trained race horses were collected after acute exercise and cultured with clenbuterol. The expressions of CD4, CD8, FoxP3, CD14, MHCII, and CD5 in PBMC, and reactive oxygen species (ROS) production, as well as cell proliferation, were evaluated by flow cytometry. In addition, IL-1ß, IL-4, IL-6, IL-10, IL-17, INF-γ and TNF-α concentrations were evaluated by ELISA. ß2-adrenoceptor stimulation leads to enhanced anti-inflammatory properties in well-trained horses, as do low doses in untrained animals. In contrast, higher clenbuterol doses create a pro-inflammatory environment in inexperienced horses. In conclusion, ß2-adrenoceptor stimulation leads to a biphasic response. In addition, the immune cells are more sensitive to drug abuse in inexperienced individuals under physical training.

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.

Reduced Graphene Oxides Modulate the Expression of Cell Receptors and Voltage-Dependent Ion Channel Genes of Glioblastoma Multiforme.

The development of nanotechnology based on graphene and its derivatives has aroused great scientific interest because of their unusual properties. Graphene (GN) and its derivatives, such as reduced graphene oxide (rGO), exhibit antitumor effects on glioblastoma multiforme (GBM) cells in vitro. The antitumor activity of rGO with different contents of oxygen-containing functional groups and GN was compared. Using FTIR (fourier transform infrared) analysis, the content of individual functional groups (GN/exfoliation (ExF), rGO/thermal (Term), rGO/ammonium thiosulphate (ATS), and rGO/ thiourea dioxide (TUD)) was determined. Cell membrane damage, as well as changes in the cell membrane potential, was analyzed. Additionally, the gene expression of voltage-dependent ion channels (clcn3, clcn6, cacna1b, cacna1d, nalcn, kcne4, kcnj10, and kcnb1) and extracellular receptors was determined. A reduction in the potential of the U87 glioma cell membrane was observed after treatment with rGO/ATS and rGO/TUD flakes. Moreover, it was also demonstrated that major changes in the expression of voltage-dependent ion channel genes were observed in clcn3, nalcn, and kcne4 after treatment with rGO/ATS and rGO/TUD flakes. Furthermore, the GN/ExF, rGO/ATS, and rGO/TUD flakes significantly reduced the expression of extracellular receptors (uPar, CD105) in U87 glioblastoma cells. In conclusion, the cytotoxic mechanism of rGO flakes may depend on the presence and types of oxygen-containing functional groups, which are more abundant in rGO compared to GN.

Effects of Graphene Oxide Nanofilm and Chicken Embryo Muscle Extract on Muscle Progenitor Cell Differentiation and Contraction.

Finding an effective muscle regeneration technique is a priority for regenerative medicine. It is known that the key factors determining tissue formation include cells, capable of proliferating and/or differentiating, a niche (surface) allowing their colonization and growth factors. The interaction between these factors, especially between the surface of the artificial niche and growth factors, is not entirely clear. Moreover, it seems that the use of a complex of complementary growth factors instead of a few strictly defined ones could increase the effectiveness of tissue maturation, including muscle tissue. In this study, we evaluated whether graphene oxide (GO) nanofilm, chicken embryo muscle extract (CEME), and GO combined with CEME would affect the differentiation and functional maturation of muscle precursor cells, as well as the ability to spontaneously contract a pseudo-tissue muscle. CEME was extracted on day 18 of embryogenesis. Muscle cells obtained from an 8-day-old chicken embryo limb bud were treated with GO and CEME. Cell morphology and differentiation were observed using different microscopy methods. Cytotoxicity and viability of cells were measured by lactate dehydrogenase and Vybrant Cell Proliferation assays. Gene expression of myogenic regulatory genes was measured by Real-Time PCR. Our results demonstrate that CEME, independent of the culture surface, was the main factor influencing the intense differentiation of muscle progenitor cells. The present results, for the first time, clearly demonstrated that the cultured tissue-like structure was capable of inducing contractions without externally applied impulses. It has been indicated that a small amount of CEME in media (about 1%) allows the culture of pseudo-tissue muscle capable of spontaneous contraction. The study showed that the graphene oxide may be used as a niche for differentiating muscle cells, but the decisive influence on the maturation of muscle tissue, especially muscle contractions, depends on the complexity of the applied growth factors.

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.

Effect of Juice and Extracts from Saposhnikovia divaricata Root on the Colon Cancer Cells Caco-2.

Colorectal cancer ranks 3rd in terms of cancer incidence. Growth and development of colon cancer cells may be affected by juice and extracts from Saposhnikovia divaricata root. The objective of the research was to analyze the effect of S. divaricata juice and extracts on the viability, membrane integrity and types of cell death of Caco-2 cells. Juice and extracts were analyzed using Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) and in respect of the presence of antioxidants, total carbohydrates, protein, fat and polyphenols. The contents of cimifugin ß-D-glucopyranoside, cimifugin, 4'-O-glucopyranosyl-5-O-methylvisamminol, imperatorin and protein were the highest in juice. 50% Hydroethanolic extract had the greatest antioxidant potential, concentration of polyphenols and fat. Water extract was characterized by the highest content of glutathione. Juice and 75% hydroethanolic extract contained the most carbohydrates. After the application of juice, 50% extract and the juice fraction containing the molecules with molecular weights >50 kDa, a decrease of the cell viability was noted. Juice and this extract exhibited the protective properties in relation to the cell membranes and they induced apoptosis. The knowledge of further mechanisms of anticancer activity of the examined products will allow to consider their use as part of combination therapy.

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.

Anti-Inflammatory State in Arabian Horses Introduced to the Endurance Training.

Development of an anti-inflammatory state during physical training has been postulated in both human and equine athletes, but it is not completely understood. The aim of this study was to investigate whether endurance training changes pro- and anti-inflammatory cytokine profiles within a 20-week training season in young inexperienced endurance horses. Nine Arabian horses were examined in this prospective 20-week follow-up study. Blood samples were analysed 5 times monthly, at rest and after training sessions. Routine haematological examinations were performed. Cytokine patterns including IL-1ß, IL-6, TNF-α, IL-10 mRNA expression using Real Time-PCR, and serum concentrations of IL-1ß, IL-2, IL-4, IL-6, IL-17, INFγ, TNF-α, and IL-10 by ELISA test were determined. During endurance training, the most significant decrease in post-exercise cytokine type 1 levels (TNFα and IL-ß) occurred within 20 weeks, beginning from the 3rd month of training. IL-6 serum level decreased after the 4th month. The results suggest that endurance training can induce advanced overall anti-inflammatory response as an adaptation to increasing workload.

Changes in Serum Amyloid A (SAA) Concentration in Arabian Endurance Horses During First Training Season.

Sport training leads to adaptation to physical effort that is reflected by the changes in blood parameters. In equine endurance athletes, blood testing is accepted as a support in training, however, only the changes before versus after exercise in creatine phosphokinase activity (CPK) and basic blood parameters are usually measured. This study is the first longitudinal investigation of the changes in routinely measured blood parameters and, additionally, serum amyloid A (SAA), during seven months, in Arabian horses introduced to endurance training and competing in events for young horses. It has been determined that CPK, aspartate aminotransferase (AST), packed cell volume (PCV), hemoglobin concentration, red blood cell count (RBC), and concentration of total serum protein (TSP) slightly increased after training sessions and competitions in similar manner. The increase in white blood cell (WBC) count was higher after competitions and SAA increased only after competitions. Total protein concentration was the only parameter that increased with training during a 7-month program. SAA indicated only in the case of heavy effort, and, it thus may be helpful in the monitoring of training in young horses. In an optimal program, its concentration should not increase after a training session but only after heavy effort, which should not be repeated too often.

Diamond Nanoparticles Downregulate Expression of CycD and CycE in Glioma Cells.

Our previous studies have shown that diamond nanoparticles (NDs) exhibited antiangiogenic and proapoptotic properties in vitro in glioblastoma multiforme (GBM) cells and in tumors in vivo. Moreover, NDs inhibited adhesion, leading to the suppression of migration and invasion of GBM. In the present study, we hypothesized that the NDs might also inhibit proliferation and cell cycle in glioma cells. Experiments were performed in vitro with the U87 and U118 lines of GBM cells, and for comparison, the Hs5 line of stromal cells (normal cells) after 24 h and 72 h of treatment. The analyses included cell morphology, cell death, viability, and cell cycle analysis, double timing assay, and gene expression (Rb, E2F1, CycA, CycB, CycD, CycE, PTEN, Ki-67). After 72 h of ND treatment, the expression level of Rb, CycD, and CycE in the U118 cells, and E2F1, CycD, and CycE in the U87 cells were significantly lower in comparison to those in the control group. We observed that decreased expression of cyclins inhibited the G1/S phase transition, arresting the cell cycle in the G0/G1 phase in glioma cells. The NDs did not affect the cell cycle as well as PTEN and Ki-67 expression in normal cells (Hs5), although it can be assumed that the NDs reduced proliferation and altered the cell cycle in fast dividing cells.

Evaluation of in vitro effect of selected contact lens solutions conjugated with nanoparticles in terms of preventive approach to public health risk generated by Acanthamoeba strains.

INTRODUCTION: Various Acanthamoeba species are free-living organisms widely distributed in the human environment. Amphizoic amoebae as facultative parasites may cause vision-threatening eye disease - Acanthamoeba keratitis, mostly among contact lens wearers. As the number of cases is increasing, and applied therapy often unsuccessful, proper hygienic measures and effective contact lenses disinfection are crucial for the prevention of this disease. Available contact lens solutions are not fully effective against amphizoic amoebae; there is a need to enhance their disinfecting activity to prevent amoebic infections. The use of developing nanotechnology methods already applied with success in the prevention, diagnostic and therapy of other infectious diseases might be helpful regarding amoebic keratitis. This study assesses the in vitro effect of selected contact lens solutions conjugated with nanoparticles against Acanthamoeba trophozoites. MATERIAL AND METHODS: Three selected contact lens solutions conjugated with silver and gold nanoparticles in concentration of 0.25-2.5 ppm were used in vitro against the axenically cultured ATCC 30010 type Acanthamoeba castellanii strain. The anti-amoebic efficacy was examined based on the oxido-reduction of AlamarBlue. The cytotoxicity tests based on the measurement of lactate dehydrogenase (LDH) activity were performed using a fibroblast HS-5 cell line. RESULTS: Enhancement of the anti-amoebic activity of contact lens solutions conjugated with selected nanoparticles expressed in the dose dependent amoebic growth inhibition with a low cytotoxicity profile was observed. CONCLUSIONS: Results of the study showed that conjugation of selected contact lens solutions with silver nanoparticles might be a promising approach to prevent Acanthamoeba keratitis among contact lens users.