Gaurav Lalwani

Cell Specific Cytotoxicity and Uptake of Graphene Nanoribbons

The synthesis of oxidized graphene nanoribbons (O-GNR) via longitudinal unzipping of carbon nanotubes opens avenues for their further development for a variety of biomedical applications. Evaluation of the cyto- and bio-compatibility is necessary to develop any new material for in vivo biomedical applications. In this study, we report the cytotoxicity screening of O-GNRs water-solubilized with PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]), using six different assays, in four representative cell lines; Henrietta Lacks cells (HeLa) derived from cervical cancer tissue, National Institute of Health 3T3 mouse fibroblast cells (NIH-3T3), Sloan Kettering breast cancer cells (SKBR3) and Michigan cancer foundation-7 breast cancer cells (MCF7). These cell lines significantly differed in their response to O-GNR-PEG-DSPE formulations; assessed and evaluated using various endpoints (lactate dehydrogenase (LDH) release, cellular metabolism, lysosomal integrity and cell proliferation) for cytotoxicity. In general, all the cells showed a dose-dependent (10-400 μg/ml) and time-dependent (12-48 h) decrease in cell viability. However, the degree of cytotoxicity was significantly lower in MCF7 or SKBR3 cells compared to HeLa cells. These cells were 100% viable upto 48 h, when incubated at 10 μg/ml O-GNR-PEG-DSPE concentration, and showed decrease in cell viability above this concentration with ~78% of cells viable at the highest concentration (400 μg/ml). In contrast, significant cell death (5-25% cell death depending on the time point, and the assay) was observed for HeLa cells even at a low concentration of 10 μg/ml. The decrease in cell viability was steep with increase in concentration with the CD(50) values ≥ 100 μg/ml depending on the assay, and time point. Transmission electron microscopy of the various cells treated with the O-GNR solutions show higher uptake of the O-GNR-PEG-DSPEs into HeLa cells compared to other cell types. Additional analysis indicates that this increased uptake is the dominant cause of the significantly higher toxicity exhibited by HeLa cells. The results suggest that water-solubilized O-GNR-PEG-DSPEs have a heterogenous cell-specific cytotoxicity, and have significantly different cytotoxicity profile compared to graphene nanoparticles prepared by the modified Hummers method (graphene nanoparticles prepared by oxidation of graphite, and its mechanical exfoliation) or its variations.

Two-Dimensional Nanostructure- Reinforced Biodegradable Polymeric Nanocomposites for Bone Tissue Engineering

This study investigates the efficacy of two-dimensional (2D) carbon and inorganic nanostructures as reinforcing agents for cross-linked composites of the biodegradable and biocompatible polymer polypropylene fumarate (PPF) as a function of nanostructure concentration. PPF composites were reinforced using various 2D nanostructures: single- and multiwalled graphene oxide nanoribbons (SWGONRs, MWGONRs), graphene oxide nanoplatelets (GONPs), and molybdenum disulfide nanoplatelets (MSNPs) at 0.01-0.2 weight% concentrations. Cross-linked PPF was used as the baseline control, and PPF composites reinforced with single- or multiwalled carbon nanotubes (SWCNTs, MWCNTs) were used as positive controls. Compression and flexural testing show a significant enhancement (i.e., compressive modulus = 35-108%, compressive yield strength = 26-93%, flexural modulus = 15-53%, and flexural yield strength = 101-262% greater than the baseline control) in the mechanical properties of the 2D-reinforced PPF nanocomposites. MSNP nanocomposites consistently showed the highest values among the experimental or control groups in all the mechanical measurements. In general, the inorganic nanoparticle MSNP showed a better or equivalent mechanical reinforcement compared to carbon nanomaterials, and 2D nanostructures (GONPs, MSNPs) are better reinforcing agents compared to one-dimensional (1D) nanostructures (e.g., SWCNTs). The results also indicated that the extent of mechanical reinforcement is closely dependent on the nanostructure morphology and follows the trend nanoplatelets > nanoribbons > nanotubes. Transmission electron microscopy of the cross-linked nanocomposites indicated good dispersion of nanomaterials in the polymer matrix without the use of a surfactant. The sol-fraction analysis showed significant changes in the polymer cross-linking in the presence of MSNP (0.01-0.2 wt %) and higher loading concentrations of GONP and MWGONR (0.1-0.2 wt %). The analysis of surface area and aspect ratio of the nanostructures taken together with the above results indicated differences in nanostructure architecture (2D vs 1D nanostructures), and the chemical compositions (inorganic vs carbon nanostructures), number of functional groups, and structural defects for the 2D nanostructures may be key properties that affect the mechanical properties of 2D nanostructure-reinforced PPF nanocomposites and the reason for the enhanced mechanical properties compared to the controls.

The Effects of Graphene Nanostructures on Mesenchymal Stem Cells

We report the effects of two-dimensional graphene nanostructures; graphene nano-onions (GNOs), graphene oxide nanoribbons (GONRs), and graphene oxide nanoplatelets (GONPs) on viability, and differentiation of human mesenchymal stem cells (MSCs). Cytotoxicity of GNOs, GONRs, and GONPs dispersed in distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), on adipose derived mesenchymal stem cells (adMSCs), and bone marrow-derived mesenchymal stem cells (bmMSCs) was assessed by AlamarBlue and Calcein AM viability assays at concentrations ranging from 5 to 300 μg/ml for 24 or 72 h. Cytotoxicity of the 2D graphene nanostructures was found to be dose dependent, not time dependent, with concentrations less than 50 μg/ml showing no significant differences compared to untreated controls. Differentiation potential of adMSCs to adipocytes and osteoblasts, - characterized by Oil Red O staining and elution, alkaline phosphatase activity, calcium matrix deposition and Alizarin Red S staining - did not change significantly when treated with the three graphene nanoparticles at a low (10 μg/ml) and high (50 μg/ml) concentration for 24 h. Transmission electron microscopy (TEM) and confocal Raman spectroscopy indicated cellular uptake of only GNOs and GONPs. The results lay the foundation for the use of these nanoparticles at potentially safe doses as ex vivo labels for MSC-based imaging and therapy.

Physicochemical characterization of a novel graphene-based magnetic resonance imaging contrast agent.

We report the synthesis and characterization of a novel carbon nanostructure-based magnetic resonance imaging contrast agent (MRI CA); graphene nanoplatelets intercalated with manganese (Mn2+) ions, functionalized with dextran (GNP-Dex); and the in vitro assessment of its essential preclinical physicochemical properties: osmolality, viscosity, partition coefficient, protein binding, thermostability, histamine release, and relaxivity. The results indicate that, at concentrations between 0.1 and 100.0 mg/mL, the GNP-Dex formulations are hydrophilic, highly soluble, and stable in deionized water, as well as iso-osmolar (upon addition of mannitol) and iso-viscous to blood. At potential steady-state equilibrium concentrations in blood (0.1–10.0 mg/mL), the thermostability, protein-binding, and histamine-release studies indicate that the GNP-Dex formulations are thermally stable (with no Mn2+ ion dissociation), do not allow non-specific protein adsorption, and elicit negligible allergic response. The r1 relaxivity of GNP-Dex was 92 mM−1s−1 (per-Mn2+ ion, 22 MHz proton Larmor frequency); ~20- to 30-fold greater than that of clinical gadolinium (Gd3+)- and Mn2+-based MRI CAs. The results open avenues for preclinical in vivo safety and efficacy studies with GNP-Dex toward its development as a clinical MRI CA.

Tungsten disulfide nanotubes reinforced biodegradable polymers for bone tissue engineering

In this study, we have investigated the efficacy of inorganic nanotubes as reinforcing agents to improve the mechanical properties of poly(propylene fumarate) (PPF) composites as a function of nanomaterial loading concentration (0.01-0.2 wt.%). Tungsten disulfide nanotubes (WSNTs) were used as reinforcing agents in the experimental group. Single- and multi-walled carbon nanotubes (SWCNTs and MWCNTs) were used as positive controls, and crosslinked PPF composites were used as the baseline control. Mechanical testing (compression and three-point bending) shows a significant enhancement (up to 28-190%) in the mechanical properties (compressive modulus, compressive yield strength, flexural modulus and flexural yield strength) of WSNT-reinforced PPF nanocomposites compared to the baseline control. In comparison to the positive controls, significant improvements in the mechanical properties of WSNT nanocomposites were also observed at various concentrations. In general, the inorganic nanotubes (WSNTs) showed mechanical reinforcement better than (up to 127%) or equivalent to that of carbon nanotubes (SWCNTs and MWCNTs). Sol fraction analysis showed significant increases in the crosslinking density of PPF in the presence of WSNTs (0.01-0.2 wt.%). Transmission electron microscopy (TEM) analysis on thin sections of crosslinked nanocomposites showed the presence of WSNTs as individual nanotubes in the PPF matrix, whereas SWCNTs and MWCNTs existed as micron-sized aggregates. The trend in the surface area of nanostructures obtained by Brunauer-Emmett-Teller (BET) surface area analysis was SWCNTs>MWCNTs>WSNTs. The BET surface area analysis, TEM analysis and sol fraction analysis results taken together suggest that chemical composition (inorganic vs. carbon nanomaterials), the presence of functional groups (such as sulfide and oxysulfide) and individual dispersion of the nanomaterials in the polymer matrix (absence of aggregation of the reinforcing agent) are the key parameters affecting the mechanical properties of nanostructure-reinforced PPF composites and the reason for the observed increases in the mechanical properties compared to the baseline and positive controls.

Synthesis, characterization, in vitro phantom imaging, and cytotoxicity of a novel graphene- based multimodal magnetic resonance imaging- X-ray computed tomography contrast agent

Graphene nanoplatelets (GNPs), synthesized using potassium permanganate-based oxidation and exfoliation followed by reduction with hydroiodic acid (rGNP-HI), have intercalated manganese ions within the graphene sheets, and upon functionalization with iodine, show excellent potential as biomodal contrast agents for magnetic resonance imaging (MRI) and computed tomography (CT). Structural characterization of rGNP-HI nanoparticles with low- and high-resolution transmission electron microscope (TEM) showed disc-shaped nanoparticles (average diameter, 200 nm, average thickness, 3 nm). Energy dispersive X-ray spectroscopy (EDX) analysis confirmed the presence of intercalated manganese. Raman spectroscopy and X-ray diffraction (XRD) analysis of rGNP-HI confirmed the reduction of oxidized GNPs (O-GNPs), absence of molecular and physically adsorbed iodine, and the functionalization of graphene with iodine as polyiodide complexes (I3- and I5-). Manganese and iodine content were quantified as 5.1 ± 0.5 and 10.54 ± 0.87 wt% by inductively-coupled plasma optical emission spectroscopy and ion-selective electrode measurements, respectively. In vitro cytotoxicity analysis, using absorbance (LDH assay) and fluorescence (calcein AM) based assays, performed on NIH3T3 mouse fibroblasts and A498 human kidney epithelial cells, showed CD50 values of rGNP-HI between 179-301 µg/ml, depending on the cell line and the cytotoxicity assay. CT and MRI phantom imaging of rGNP-HI showed high CT (approximately 3200% greater than HI at equimolar iodine concentration) and MRI (approximately 59% greater than equimolar Mn2+ solution) contrast. These results open avenues for further in vivo safety and efficacy studies towards the development of carbon nanostructure-based multimodal MRI-CT contrast agents.

Graphene-based contrast agents for photoacoustic and thermoacoustic tomography §

In this work, graphene nanoribbons and nanoplatelets were investigated as contrast agents for photoacoustic and thermoacoustic tomography (PAT and TAT). We show that oxidized single- and multi-walled graphene oxide nanoribbons (O-SWGNRs, O-MWGNRs) exhibit approximately 5–10 fold signal enhancement for PAT in comparison to blood at the wavelength of 755 nm, and approximately 10–28% signal enhancement for TAT in comparison to deionized (DI) water at 3 GHz. Oxidized graphite microparticles (O-GMPs) and exfoliated graphene oxide nanoplatelets (O-GNPs) show no significant signal enhancement for PAT, and approximately 12–29% signal enhancement for TAT. These results indicate that O-GNRs show promise as multi-modal PAT and TAT contrast agents, and that O-GNPs are suitable contrast agents for TAT.

Toxicology of graphene-based nanomaterials.

Graphene based nanomaterials possess remarkable physiochemical properties suitable for diverse applications in electronics, telecommunications, energy and healthcare. The human and environmental exposure to graphene-based nanomaterials is increasing due to advancements in the synthesis, characterization and large-scale production of graphene and the subsequent development of graphene based biomedical and consumer products. A large number of in vitro and in vivo toxicological studies have evaluated the interactions of graphene-based nanomaterials with various living systems such as microbes, mammalian cells, and animal models. A significant number of studies have examined the short- and long-term in vivo toxicity and biodistribution of graphene synthesized by variety of methods and starting materials. A key focus of these examinations is to properly associate the biological responses with chemical and morphological properties of graphene. Several studies also report the environmental and genotoxicity response of pristine and functionalized graphene. This review summarizes these in vitro and in vivo studies and critically examines the methodologies used to perform these evaluations. Our overarching goal is to provide a comprehensive overview of the complex interplay of biological responses of graphene as a function of their physiochemical properties.

MULTIFUNCTIONAL FULLERENE- AND METALLOFULLERENE-BASED NANOBIOMATERIALS

Recent advances in nanotechnology have enabled the synthesis and characterization of nanomaterials suitable for applications in the field of biology and medicine. Due to their unique physico-chemical properties, carbon-based nanomaterials such as fullerenes, metallofullerenes, carbon nanotubes and graphene have been widely investigated as multifunctional materials for applications in tissue engineering, molecular imaging, therapeutics, drug delivery and biosensing. In this review, we focus on the multifunctional capabilities of fullerenes and metallofullerenes for diagnosis and therapy. Specifically, we review recent advances toward the development of fullerene- and metallofullerene-based magnetic resonance imaging (MRI) and X-ray imaging contrast agents, drug and gene delivery vehicles, and photodynamic therapy agents. We also discuss in vitro and in vivo toxicity, and biocompatibility issues associated with the use of fullerenes and metallofullerenes for biomedical applications.

Enzymatic degradation of oxidized and reduced graphene nanoribbons by lignin peroxidase

The expanding use of graphene for various industrial and biomedical applications requires efficient remediation strategies during their disposal into waste streams. Additionally, the interactions of graphene with the biota need thorough evaluation. In this study, we investigated the interactions of oxidized and reduced graphene oxide nanoribbons (GONRs and rGONRs) with lignin peroxidase (LiP), a ligninolytic enzyme released from white rot fungus. GONRs and rGONRs were treated with LiP in the presence and absence of veratryl alcohol (VA; an electron transfer mediator and secondary metabolite of white rot fungi). Transmission electron microscopy showed the formation of large defects (holes) in the graphene sheet, which increased in diameter with increased degradation time. Raman spectroscopic analysis indicated that, within 96 hours, in the presence of hydrogen peroxide and VA, the GONRs and rGONRs were completely and partially degraded by LiP, respectively. Comparisons between groups with or without VA showed that degradation of GONRs was accelerated in the presence of VA. These results indicated that LiP could efficiently degrade GONRs and rGONRs in the presence of VA, suggesting that VA may be an essential factor needed to degrade rGONRs via LiP treatment. Thus, the wide presence of white rot fungi, and thereby LiP, in nature, could lead to efficient degradation of graphene present in the environment. Additionally, LiP, which has a higher theoretical redox potential compared to horseradish peroxidases and myeloperoxidases, could be a better candidate for the environmental remediation of graphene.