The Influence of Graphene Oxide-Fe3O4 Differently Conjugated with 10-Hydroxycampthotecin and a Rotating Magnetic Field on Adenocarcinoma Cells.

Nanoparticles (e.g., graphene oxide, graphene oxide-Fe3O4 nanocomposite or hexagonal boron nitride) loaded with anti-cancer drugs and targeted at cancerous cells allowed researchers to determine the most effective in vitro conditions for anticancer treatment. For this reason, the main propose of the present study was to determine the effect of graphene oxide (GO) with iron oxide (Fe3O4) nanoparticles (GO-Fe3O4) covalently (c-GO-Fe3O4-HCPT) and non-covalently (nc-GO-Fe3O4-HCPT) conjugated with hydroxycamptothecin (HCPT) in the presence of a rotating magnetic field (RMF) on relative cell viability using the MCF-7 breast cancer cell line. The obtained GO-Fe3O4 nanocomposites demonstrated the uniform coverage of the graphene flakes with the nanospheres, with the thickness of the flakes estimated as ca. 1.2 nm. The XRD pattern of GO-Fe3O4 indicates that the crystal structure of the magnetite remained stable during the functionalization with HCPT that was confirmed with FTIR spectra. After 24 h, approx. 49% and 34% of the anti-cancer drug was released from nc-GO-Fe3O4-HCPT and c-GO-Fe3O4-HCPT, respectively. The stronger bonds in the c-GO-Fe3O4-HCPT resulted in a slower release of a smaller drug amount from the nanocomposite. The combined impact of the novel nanocomposites and a rotating magnetic field on MCF-7 cells was revealed and the efficiency of this novel approach has been confirmed. However, MCF-7 cells were more significantly affected by nc-GO-Fe3O4-HCPT. In the present study, it was found that the concentration of nc-GO-Fe3O4-HCPT and a RMF has the highest statistically significant influence on MCF-7 cell viability. The obtained novel nanocomposites and rotating magnetic field were found to affect the MCF-7 cells in a dose-dependent manner. The presented results may have potential clinical applications, but still, more in-depth analyses need to be performed.

Application of PCR-HRM method for microsatellite polymorphism genotyping in the LDHA gene of pigeons (Columba livia).

High-resolution melting (HRM) is a post-PCR method that allows to discriminate genotypes based on fluorescence changes during the melting phase. HRM is used to detect mutations or polymorphisms (e.g. microsatellites, SNPs, indels). Here, the (TTTAT)3-5 microsatellite polymorphism within intron 6 of the LDHA gene in pigeons was analysed using the HRM method. Individuals (123 homing pigeons) were genotyped using conventional PCR. Birds were classified into groups based on genotype type and the results were tested by qPCR-HRM and verified using sequencing. Based on the evaluated protocol, five genotypes were identified that vary in the number of TTTAT repeat units (3/3, 4/4, 3/4, 4/5, and 5/5). Sequencing have confirmed the results obtained with qPCR-HRM and verified that HRM is a suitable method for identification of three-allele microsatellite polymorphisms. It can be concluded that the high-resolution melting (HRM) method can be effectively used for rapid (one-step) discrimination of the (TTTAT)3-5 microsatellite polymorphism in the pigeon's LDHA gene.

Modulation of Cellular Response to Different Parameters of the Rotating Magnetic Field (RMF)-An In Vitro Wound Healing Study.

Since the effect of MFs (magnetic fields) on various biological systems has been studied, different results have been obtained from an insignificant effect of weak MFs on the disruption of the circadian clock system. On the other hand, magnetic fields, electromagnetic fields, or electric fields are used in medicine. The presented study was conducted to determine whether a low-frequency RMF (rotating magnetic field) with different field parameters could evoke the cellular response in vitro and is possible to modulate the cellular response. The cellular metabolic activity, ROS and Ca2+ concentration levels, wound healing assay, and gene expression analyses were conducted to evaluate the effect of RMF. It was shown that different values of magnetic induction (B) and frequency (f) of RMF evoke a different response of cells, e.g., increase in the general metabolic activity may be associated with the increasing of ROS levels. The lower intracellular Ca2+ concentration (for 50 Hz) evoked the inability of cells to wound closure. It can be stated that the subtle balance in the ROS level is crucial in the wound for the effective healing process, and it is possible to modulate the cellular response to the RMF in the context of an in vitro wound healing.

Initial assessment of suitability of MCF-7 and HepG2 cancer cell lines for AQP3 research in cancer biology.

Cancer cell lines are widely used as in vitro models to elucidate biological processes in cancer, and as a tool to evaluate anticancer agents. In fact, the use of an appropriate cancer cell line in cancer research is crucial for investigating new, potential factors involved in carcinogenesis. One of them is aquaporin-3 (AQP3), which is a small, hydrophobic, integral membrane protein with a predominant role in water and glycerol transport. Recently, altered expression of AQP3 has been reported in many types of cancer. Increasing evidence strongly suggests that AQP3 plays a key role in cancer cell proliferation, migration and invasion. In this study, we performed an insightful characteristic of AQP3 location and its protein expression in MCF-7 human breast adenocarcinoma and HepG2 hepatocellular carcinoma cell lines in the context of cancer biology using immunocytochemistry, immunofluorescence and Western blot analyses. AQP3 was found to be located in the cell membrane and cytoplasm of MCF-7 cells, and in the cytoplasm and nuclear membrane of HepG2 cells. Immunoblotting of proteins derived from both cell lines revealed a clear band with a molecular weight of approx. 30 kDa representing an unglycosylated form of AQP3. However, the expression of this protein was higher in MCF-7 than in HepG2. Concluding, our results clearly indicated variability in both the expression levels and subcellular location of the AQP3 protein in MCF-7 and HepG2 cell lines. This leads to the possibility that the expression patterns and subcellular location of AQP3 in the tested cancer cell lines are tissue-of-origin specific, and may be related to the aggressiveness of cancer cells and their mobility.

Size-Dependent in Vitro Biocompatibility and Uptake Process of Polymeric Carbon Nitride.

Polymeric carbon nitride (PCN), which demonstrates unique properties, has been widely explored, mostly in photocatalysis; however, the evaluation of its biocompatibility is still needed. Herein, the cytocompatibility of PCN with different lateral size distributions (A-PCN with 160 nm, B-PCN with 20 nm, and C-PCN with 10 nm dominating lateral sizes) was investigated. The viability of three cell lines (L929, MCF-7, and HepG2) has been determined using cell counting kit-8 (CCK-8), neutral red uptake (NRU), and lactate dehydrogenase (LDH) leakage assays. It was found that the highest cytotoxicity of PCN was observed for flakes with a lateral size of ∼20 nm (B-PCN) in three cell lines after 48 h of exposition. The uptake process of B-PCN sheets labeled with fluorescein isothiocyanate (FITC) by cells was also the most effective. Confocal laser scanning microscopy and atomic force microscopy revealed the nanomaterial distribution throughout the cytoplasm and perinuclear region. The results demonstrated the correlation among size, internalization process, and cytocompatibility of the tested polymeric carbon nitride structures.

Non-cytotoxic hydroxyl-functionalized exfoliated boron nitride nanoflakes impair the immunological function of insect haemocytes in vivo.

To induce the water solubility of hexagonal boron nitride (h-BN), we exfoliated and functionalized bulk h-BN with hydroxyl groups (h-BN-OH-n). Short-term studies showed that h-BN-OH-n induced low cytotoxicity in different models: insect haemocytes (in vivo), human erythrocytes and mouse fibroblasts (in vitro). We also demonstrated that Alexa Fluor 647-h-BN-OH-n administered topically to the insects passed through the cuticle barrier and was phagocytosed by haemocytes. Nanoflakes did not affect the haemocyte cell membrane and did not interfere with the phagocytosis of latex beads. Long-term immunoassays showed that h-BN-OH-n, despite not inducing haemocytotoxicity, impaired nodulation, the most important cellular immune response in insects. The haemocytes exposed to h-BN-OH-n and then to bacteria differed in morphology and adhesiveness from the haemocytes exposed only to bacteria and exhibited the same morphology and adhesiveness as the control haemocytes. The h-BN-OH-n-induced decrease in nodulation can therefore result from the reduced ability of haemocytes to recognize bacteria, migrate to them or form microaggregates around them, which can lead to dysfunction of the immune system during pathogen infection. Long-term in vivo studies with animal models are still necessary to unambiguously confirm that h-BN is biocompatible and useful for application as a platform for drug delivery or for bioimaging.

Hexagonal Boron Nitride Functionalized with Au Nanoparticles-Properties and Potential Biological Applications.

Hexagonal boron nitride is often referred to as white graphene. This is a 2D layered material, with a structure similar to graphene. It has gained many applications in cosmetics, dental cements, ceramics etc. Hexagonal boron nitride is also used in medicine, as a drug carrier similar as graphene or graphene oxide. Here we report that this material can be exfoliated in two steps: chemical treatment (via modified Hummers method) followed by the sonication treatment. Afterwards, the surface of the obtained material can be efficiently functionalized with gold nanoparticles. The mitochondrial activity was not affected in L929 and MCF-7 cell line cultures during 24-h incubation, whereas longer incubation (for 48, and 72 h) with this nanocomposite affected the cellular metabolism. Lysosome functionality, analyzed using the NR uptake assay, was also reduced in both cell lines. Interestingly, the rate of MCF-7 cell proliferation was reduced when exposed to h-BN loaded with gold nanoparticles. It is believed that h-BN nanocomposite with gold nanoparticles is an attractive material for cancer drug delivery and photodynamic therapy in cancer killing.

Effect of GO-Fe3O4 and rotating magnetic field on cellular metabolic activity of mammalian cells.

The effect of hybrid material-graphene flakes with Fe3O4 nanospheres (GO-Fe3O4), graphene oxide (GO) and magnetite nanospheres (Fe3O4) in rotating magnetic field on mammalian cells metabolism has been studied. Several reports shown that exposure to magnetic field may have influence on cellular membrane permeability. Thus, the aim of presented study was to determine the cellular response of L929 fibroblast cells to nanomaterials and rotating magnetic field for 8-h exposure experiment. The GO had tendency to adsorb proteins, thus cell metabolism was decreased and the effect of that mechanism was enhanced by impact of nanospheres and rotating magnetic field. The highest reduction of cellular metabolism was recorded for WST-1 and NR assays at concentration 100 µg/mL of all tested nanomaterials and magnetic induction value 10.06 mT. The lactate dehydrogenase leakage assay has shown significant changes in membrane permeability. Further studies need to be carried out to precisely determine the mechanism of that process.