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.

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.

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.

Degradation of Mitochondria and Oxidative Stress as the Main Mechanism of Toxicity of Pristine Graphene on U87 Glioblastoma Cells and Tumors and HS-5 Cells.

Due to the development of nanotechnologies, graphene and graphene-based nanomaterials have attracted immense scientific interest owing to their extraordinary properties. Graphene can be used in many fields, including biomedicine. To date, little is known about the impact graphene may have on human health in the case of intentional exposure. The present study was carried out on U87 glioma cells and non-cancer HS-5 cell lines as in vitro model and U87 tumors cultured on chicken embryo chorioallantoic membrane as in vivo model, on which the effects of pristine graphene platelets (GPs) were evaluated. The investigation consisted of structural analysis of GPs using transmission electron microscopy, Fourier transmission infrared measurements, zeta potential measurements, evaluation of cell morphology, assessment of cell viability, investigation of reactive oxygen species production, and investigation of mitochondrial membrane potential. The toxicity of U87 glioma tumors was evaluated by calculating the weight and volume of tumors and performing analyses of the ultrastructure, histology, and protein expression. The in vitro results indicate that GPs have dose-dependent cytotoxicity via ROS overproduction and depletion of the mitochondrial membrane potential. The mass and volume of tumors were reduced in vivo after injection of GPs. Additionally, the level of apoptotic and necrotic markers increased in GPs-treated tumors.

Silver Nanoparticles and Graphene Oxide Complex as an Anti-Inflammatory Biocompatible Liquid Nano-Dressing for Skin Infected with Staphylococcus aureus.

Background: Bacterial skin infections, including Staphylococcus aureus, are a powerful and still not fully resolved problem. The aim of this research was to determine the possibility of using a complex of graphene oxide (GO) encrusted with silver nanoparticles as an effective antibacterial agent against S. aureus and to assess its pro-inflammatory properties. Methods: The tests were carried out in vitro on EpiDerm™ Skin, an artificial skin model (MatTek in vitro Life Science Laboratories, Slovak Republic), and the fibroblast cell line (HFF-2 from ATCC, USA). Both models were infected with S. aureus bacteria (ATCC 25923) and then treated with antibiotics or our experimental factors: silver nanoparticles (AgNPs, Nano-koloid, Poland), graphene oxide (GO, NanoPoz, Poland), and complex AgNP-GO (hydrocolloid created by self-assembly). Results: The antibacterial effectiveness of the AgNP-GO complex was equivalent to that of the antibiotic. In addition, an increase in the level of pro-inflammatory cytokines was observed under the influence of antibiotic administration, in contrast to the effect of AgNP-GO, which showed very limited pro-inflammatory activity. Conclusion: Hydrocolloid of the AgNP-GO complex, administered in the form of a liquid dressing, may act as an antibacterial agent and also reduce inflammation induced by S. aureus infection.

Graphene Oxide Decreases Pro-Inflammatory Proteins Production in Skeletal Muscle Cells Exposed to SARS-CoV-2 Spike Protein.

Aim: The experiments aimed to document the presence of the ACE2 receptor on human muscle cells and the effects of the interaction of these cells with the spike protein of the SARS-CoV-2 virus in terms of induction of pro-inflammatory proteins, as well as to assess the possibility of reducing the pool of these proteins with the use of graphene oxide (GO) flakes. Methods: Human Skeletal Myoblast (HSkM), purchased from Gibco were maintained in standard condition according to the manufacturer's instruction. The cells were divided into 4 groups; 1. C-control, 2. S-with addition of spike protein, 3. GO-with the addition of graphene oxide, 4. GO-S-with addition of GO followed by the addition of S protein. Protein S (PX-COV-P049) was purchased from ProteoGenix (France). GO was obtained from Advanced Graphene Products (Zielona Gora, Poland). The influence of all the factors on the morphology of cells was investigated using light and confocal microscopy. ACE2 protein expression on muscle cells was visualized and 40 pro-inflammatory cytokines were investigated using the membrane antibody array method. The protein profile of the lysate of cells from individual groups was also analyzed by mass spectrometry. Conclusion: The experiments confirmed the presence of the ACE2 receptor in human skeletal muscle cells. It has also been documented that the SARS-CoV-2 virus spike protein influences the activation of selected pro-inflammatory proteins that promote cytokine storm and oxidative stress in muscle cells. The use of low levels of graphene oxide does not adversely affect muscle cells, reducing the levels of most proteins, including pro-inflammatory proteins. It can be assumed that GO may support anti-inflammatory therapy in muscles by scavenging proteins that activate cytokine storm.

Delivery of Melittin as a Lytic Agent via Graphene Nanoparticles as Carriers to Breast Cancer Cells.

Melittin, as an agent to lyse biological membranes, may be a promising therapeutic agent in the treatment of cancer. However, because of its nonspecific actions, there is a need to use a delivery method. The conducted research determined whether carbon nanoparticles, such as graphene and graphene oxide, could be carriers for melittin to breast cancer cells. The studies included the analysis of intracellular pH, the potential of cell membranes, the type of cellular transport, and the expression of receptor proteins. By measuring the particle size, zeta potential, and FT-IT analysis, we found that the investigated nanoparticles are connected by electrostatic interactions. The level of melittin encapsulation with graphene was 86%, while with graphene oxide it was 78%. A decrease in pHi was observed for all cell lines after administration of melittin and its complex with graphene. The decrease in membrane polarization was demonstrated for all lines treated with melittin and its complex with graphene and after exposure to the complex of melittin with graphene oxide for the MDA-MB-231 and HFFF2 lines. The results showed that the investigated melittin complexes and the melittin itself act differently on different cell lines (MDA-MB-231 and MCF-7). It has been shown that in MDA-MD-231 cells, melittin in a complex with graphene is transported to cells via caveolin-dependent endocytosis. On the other hand, the melittin-graphene oxide complex can reach breast cancer cells through various types of transport. Other differences in protein expression changes were also observed for tumor lines after exposure to melittin and complexes.

Bacterial Surface Disturbances Affecting Cell Function during Exposure to Three-Compound Nanocomposites Based on Graphene Materials.

Combating pathogenic microorganisms in an era of ever-increasing drug resistance is crucial. The aim of the study was to evaluate the antibacterial mechanism of three-compound nanocomposites that were based on graphene materials. To determine the nanomaterials' physicochemical properties, an analysis of the mean hydrodynamic diameter and zeta potential, transmission electron microscope (TEM) visualization and an FT-IR analysis were performed. The nanocomposites' activity toward bacteria species was defined by viability, colony forming units, conductivity and surface charge, cell wall integrity, ATP concentration, and intracellular pH. To ensure the safe usage of nanocomposites, the presence of cytokines was also analyzed. Both the graphene and graphene oxide (GO) nanocomposites exhibited a high antibacterial effect toward all bacteria species (Enterobacter cloacae, Listeria monocytogenes, Salmonella enterica, and Staphylococcus aureus), as well as exceeded values obtained from exposure to single nanoparticles. Nanocomposites caused the biggest membrane damage, along with ATP depletion. Nanocomposites that were based on GO resulted in lower toxicity to the cell line. In view of the many aspects that must be considered when investigating such complex structures as are three-component nanocomposites, studies of their mechanism of action are crucial to their potential antibacterial use.

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.

Effect of Muscle Extract and Graphene Oxide on Muscle Structure of Chicken Embryos.

The effects of CEME and it complex with GO injected in ovo on the growth and development of chicken embryo hindlimb muscle were investigated. First, the preliminary in vitro study on primary muscle precursor cell culture obtained from a nine-day-old chicken embryo was performed to assess toxicity (MTT assay) of CEME, GO (100 ppm) and it complex with different concentrations (1, 2, 5, and 10 wt.%). The effect on cell proliferation was investigated by BrdU assay. CEME at concentrations 1-5% increased cell proliferation, but not the complex with GO. In vitro cytotoxicity was highest in 10% and GO groups. Next, the main experiment with chicken embryos was performed with CEME, GO and it complex injected in ovo on day one of embryogenesis. On day 20 of embryogenesis survival, morphological development, histological structure of the muscle, and biochemical parameters of blood serum of the embryos were measured. No negative effect on mortality, body weight, or biochemistry of blood after use of CEME or GO-CEME complexes was observed. Interestingly, the slight toxicity of GO, observed in in vitro studies, was not observed in vivo. The use of CEME at the levels of 2% and 5% improved the structure of the lower limb muscle by increasing the number of cells, and the administration of 2% CEME increased the number of nuclei visible in the stained cross-section of the muscle. The complex GO-CEME did not further improve the muscle structure. The results indicate that CEME can be applied as an in ovo enhancer of muscle development in broilers.

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.

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.

Analysis of the cytotoxicity of hierarchical nanoporous graphenic carbon against human glioblastoma grade IV cells.

A newly produced hierarchical, nanoporous carbon (HNC) material is studied for the first time in a biological model. The material consists of uniform particles and is characterized by a mean diameter <150 nm, a high specific surface area of 1,000 m2/g, well-developed porosity, and high electrical conductivity. These unique properties and ability to transfer charge create a possibility of employing HNC as a moderator of tumor cell growth. As the charge of HNC may interfere with cell membranes by adhesion and by bonding with cell receptors, it may block the supply of nutrients. The interactions of HNC with the U87 cells can also lead to the excessive generation of reactive oxygen species (ROS) and activate apoptotic mechanisms in cancer cells. The investigation was performed using U87 human glioblastoma and PCS-201-010 normal fibroblast cell lines, where cell morphology and ultrastructure, viability, ROS production, type of cell death, mitochondrial transmembrane potential, and the expression of genes engaged in apoptosis pathways are studied. The results demonstrate that cytotoxicity of HNC particles increases with concentration from 5 to 100 µg/mL by activation of apoptosis through the mitochondrial pathway, without inducing necrosis. Our research indicates the potential applicability of HNC in cancer therapy.