Ehsan Hashemi

Cyto and genotoxicities of graphene oxide and reduced graphene oxide sheets on spermatozoa

Concentration-dependent cyto and genotoxicities of graphene oxide (GO) and reduced GO (rGO) sheets on spermatozoa were studied. rGO sheets with various surface chemical states were achieved using hydrazine (N2H4) hydrothermal (HT) reactions and green tea polyphenols (GTPs). Although 0.1 μg mL−1 graphene could not change sperm viability and kinetic parameters, <40% and 20% of spermatozoa were viable and progressively motile, after 2 h incubation with 400 μg mL−1 GO or rGO, respectively. All the graphene nanomaterials induced concentration-dependent reductions of adenosine triphosphate and NAD+/NADH produced by spermatozoa for motility and metabolic activity. While GO, N2H4–rGO, and HT-rGO sheets caused increasing reactive oxygen species and sperm nitric oxide production, GO sheets reduced by antioxidant GTPs decreased them. Hence, physical trapping of spermatozoa by graphene (particularly GTP–rGO) is one of the important mechanisms describing the cytotoxicity, in addition to the other reactions, resulting in the inactivation and/or death of spermatozoa. Graphene genotoxicity was initiated by 1.0 μg mL−1 of N2H4–rGO and HT-rGO and 10 μg mL−1 of GO and GTP–rGO sheets. The extremely sharp edge and/or high mobility of N2H4–rGO provided easy penetration of the sheets into spermatozoa to interact with cell nuclei. In contrast, the steric effect induced by GTPs attached on rGO caused a lower genotoxicity.

DNA and RNA extractions from eukaryotic and prokaryotic cells by graphene nanoplatelets

Graphene nanoplatelets with lateral dimensions of ∼50–200 nm and thicknesses <2 nm were utilized for the extraction of nucleic acids (NAs) from eukaryotic and prokaryotic cells. The graphene nanoplatelets (both chemically exfoliated graphene oxide nanoplatelets and hydrazine-reduced graphene oxide nanoplatelets) successfully extracted plasmid DNA (pDNA) from Escherichia coli bacteria, comparable to a conventional phenol–chloroform (PC) method. Furthermore, it was found that the yield of graphene nanoplatelets in genomic DNA (gDNA) and RNA extractions from embryonic stem cells (ESCs) was also comparable to the yield of the conventional methods. The effects of the graphene nanoplatelets on restriction enzyme digestion of the pDNA and gene amplification of all the extracted NAs (including pDNA, gDNA and RNA) were also investigated in order to confirm the quality of the extractions. These results not only demonstrated an easy gene extraction capability of graphene nanoplatelets with a high gene amplification, but also provide an easy, fast, inexpensive and biocompatible DNA/RNA extraction method.

Synthesis and cyto-genotoxicity evaluation of graphene on mice spermatogonial stem cells

The present study analyzed the dose-dependent cyto- and genotoxicity of graphene oxide and reduced graphene oxide on spermatogonial stem cells (SSCs) for the first time. The results showed that graphene oxide significantly increased oxidative stress at concentrations of 100 and 400μg/ml, while low concentrations did not have a significant effect. In addition, according to the MTT assay, the cell number decreased in high-concentration (100 and 400μg/ml) graphene oxide-treated samples compared to untreated cells. However, a reduced graphene-treated sample demonstrated a significant increase in cell number. Moreover, microscopic analysis found high concentrations of graphene nanosheets in cell culture medium that reduced the number of colonies and colony forming cells. We conclude that a high concentration of graphene can be toxic to SSCs. However, such toxicity can be reduced by the surface modification of graphene nanomaterials.

Influence of heavy nanocrystals on spermatozoa and fertility of mammals

In recent years, quantum dots (QDs) have been widely used in upcoming nanotechnology-based solar cells, light-emitting diodes and even bioimaging, due to their tunable optical properties and excellent quantum yields. But, such nanostructures are currently constituted by heavy elements which can threat the human health and living environment. Hence, in this work, the in vivo effects of CdTe nanocrystals (NCs) (as one of the promising QDs) on spermatozoa of male mice and subsequently on fertility of female mice were investigated, for the first time. To do this, CdTe NCs were synthesized through an environment-friendly (aqueous-based solution) method. The sperm cells presented a high potential for uptake of the heavy QDs. Meantime, the NCs exhibited concentration-dependent adverse effects on morphology, viability, kinetic characteristics and DNA of the spermatozoa. At low concentration of 0.1μg/mL, the NCs showed a moderate toxicity (~25% reduction in viability and motility of the spermatozoa), while remarkable toxicities were observed at higher concentrations of 1.0-100μg/mL (~67% reduction in viability and motility for 100μg/mL). Furthermore, significant in vitro DNA fragmentation of the spermatozoa was observed at CdTe concentrations ≥10μg/mL. In vivo toxicity of the NCs was found lower than the in vitro toxicity. Nevertheless, the in vivo destructive effects of the NCs still caused ~34% reduction in viability as well as motility and ~5% damages in DNA of male mice spermatozoa. These resulted in ~26% decrease in fertility and gestation of female mice, along with an overall hormone secretion during the pregnancy, and ~39% reduction in viability of pups/pregnant females.

Toward Chemical Perfection of Graphene-Based Gene Carrier via Ugi Multicomponent Assembly Process

The graphene-based materials with unique, versatile, and tunable properties have brought new opportunities for the leading edge of advanced nanobiotechnology. In this regard, the use of graphene in gene delivery applications is still at early stages. In this study, we successfully designed a new complex of carboxylated-graphene (G-COOH) with ethidium bromide (EtBr) and used it as a nanovector for efficient gene delivery into the AGS cells. G-COOH, with carboxyl functions on its surface, in the presence of EtBr, formaldehyde, and cyclohexylisocyanide were participated in Ugi four component reaction to fabricate a stable amphiphilic graphene-EtBr (AG-EtBr) composite. The coupling reaction was confirmed by further analyses with FT-IR, AFM, UV-vis, Raman, photoluminescence, EDS, and XPS. The AG-EtBr nanocomposite was able to interact with a plasmid DNA (pDNA). This nanocomposite has been applied for transfection of cultured mammalian cells successfully. Moreover, the AG-EtBr composites showed a remarkable decreased cytotoxicity in compared to EtBr. Interestingly, the advantages of AG-EtBr in cell transfection are more dramatic (3-fold higher) than Lipofectamine2000 as a commercial nonviral vector. To the best of our knowledge, this is the first report in which EtBr is used as an intercalating agent along with graphene to serve as a new vehicle for gene delivery application.

Graphene oxide for rapid determination of testosterone in the presence of cetyltrimethylammonium bromide in urine and blood plasma of athletes.

Electro-reduction behavior of testosterone at reduced graphene oxide/glassy carbon electrode (rGO/GCE) was studied. Cationic surfactant cetyltrimethylammonium bromide (CTAB) enhanced the reduction peak of testosterone. In borate buffer (pH5.4) CTAB-testosterone showed a reduction peak at -1.1 V (versus, Ag/AgCl). The increment of peak current obtained by deducting the reduction peak current of the CTAB-testosterone was rectilinear with testosterone concentration in the range of 2.0 to 210.0 nM, with a detection limit of 0.1 nM. The sensor was used for quantification of testosterone in biological fluids and drug.

Effect of blastocoel fluid reduction before vitrification on gene expression in mouse blastocysts

Artificial collapse of the blastocoel cavity before vitrification can improve the quality of warmed embryos, yet how reduction of blastocoel fluid impacts formation of the blastocyst cell lineages is not clear. The present study assessed the effect of pre‐vitrification blastocoel fluid reduction on the survival, hatching rate, and the expression of genes related to apoptosis (Tp53), pluripotency (Pou5f1, Nanog), and differentiation (Cdx2, Eomes, Gata6) in mouse blastocysts. In vivo‐produced blastocysts were randomly divided into three groups: The first group was vitrified and warmed; the second group underwent artificial collapse of the blastocoel cavity prior to vitrification and warming; the third group served as the control, in which neither vitrification or artificial collapse was performed. The survival rate of treatment groups was similar to the control group, whereas the hatching rate of artificial collapse/vitrified blastocysts was significantly higher than vitrified blastocysts. Quantitative reverse‐transcription PCR analysis revealed a considerable reduction in the expression of Cdx2, Eomes, Gata6, Grb2, and Tp53 transcripts following artificial collapse/vitrification in comparison to the vitrification‐alone group; the abundance of Pou5f1 and Nanog, however, did not change. These results suggest that artificial collapse of the blastocoel cavity before vitrification leads to relatively normal expression of apoptosis and development‐related genes plus higher hatching rates. Mol. Reprod. Dev. 83: 735–742, 2016

Cell attachment evaluation of the immobilized bioactive peptide on a nanographene oxide composite

The immobilization of bioactive peptides as key molecules in numerous biological and physiological functions holds promise for designing advanced biomaterials. Graphene and its derivatives, having unique physicochemical properties, have brought considerable attention in the life sciences. In this regard, the chemical manipulation of the graphene surface with bioactive peptides opens a new horizon to design bioactive materials for a variety of future nanobiotechnologies. In this study, the first straightforward strategy for the covalent immobilization of the cell-adhesion peptide onto the graphene surface based on the Ugi four-component assembly process (Ugi 4-CAP) will be presented. The modified adhesion motif peptide, as an amine component in the presence of formaldehyde, cyclohexylisocyanide and carboxylated-graphene (G-COOH), was adopted in a four component reaction to fabricate a peptide-graphene (Peptide-G) biomaterial in water as a green solvent at an ambient temperature. The amino functional groups corresponded to the modified adhesion motif peptide and were immobilized onto the graphene sheets, which were quantified by the Kaiser test. The sheets were characterized by further analyses with FT-IR, AFM, UV-vis, Raman and thermogravimetric analyses. The Peptide-G biomaterial showed excellent biocompatibility. In addition, the Peptide-G treated surface, due to the presence of RGD on the surface of the graphene, significantly accelerated the proliferation of human mesenchymal stem cells (hMSCs) at a better rate regarding the tissue plate.

Effect of graphene oxide nanosheets on visible light-assisted antibacterial activity of vertically-aligned copper oxide nanowire arrays

In the present work, the effect of graphene oxide (GO) nanosheets on the antibacterial activity of CuO nanowire arrays under visible light irradiation is shown. A combined thermal oxidation/electrophoretic deposition technique was employed to prepare three-dimensional networks of graphene oxide nanosheets hybridized with vertically aligned CuO nanowires. With the help of standard antibacterial assays and X-ray photoelectron spectroscopy, it is shown that the light-activated antibacterial response of the hybrid material against gram-negative Escherichia coli is significantly improved as the oxide functional groups of the GO nanosheets are reduced. In order to explore the physicochemical mechanism behind this behavior, ab-initio simulations based on density functional theory were performed and the effect of surface functional groups and hybridization were elucidated. Supported by the experiments, a three-step photo-antibacterial based mechanism is suggested: (i) injection of an electron from CuO into rGO, (ii) localization of the excess electron on rGO functional groups, and (iii) release of reactive oxygen species lethal to bacteria. Activation of new photoactive and physical mechanisms in the hybrid system makes rGO-modified CuO nanowire coatings as promising nanostructure devices for antimicrobial applications in particular for dry environments.

Apoptotic and anti-apoptotic genes transcripts patterns of graphene in mice

Recent studies showed that a large amount of graphene oxide accumulated in kidney and liver when it injected intravenously. Evaluation of lethal and apoptosis gene expression in these tissues, which are under stress is very important. In this paper the in vivo dose-dependent effects of graphene oxide and reduced graphene oxide nanoplatelets on kidney and liver of mice were studied. Balb/C mice were treated by 20mg/kg body weight of nanoplatelets. Molecular biology analysis showed that graphene nanoplatelets injected intravenously lead to overexpression of BAX gene in both kidney and liver tissues (P≥0.01). In addition these nanoparticles significantly increase BCL2 gene expression in both kidney and liver tissues (P≥0.05). Graphene significantly increase level of SGPT in groups 1 (220.64±13), 2 (164.44±9.3) in comparison to control group (P≤0.05). Also in comparison with control group (148.11±10.4), (P≤0.05), the level of SGOT in groups 1(182.01±12.6) and 2 (178.2±2.2) significantly increased.