Sayan Mullick Chowdhury

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.

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.

Dose ranging, expanded acute toxicity and safety pharmacology studies for intravenously administered functionalized graphene nanoparticle formulations.

Graphene nanoparticle dispersions show immense potential as multifunctional agents for in vivo biomedical applications. Herein, we follow regulatory guidelines for pharmaceuticals that recommend safety pharmacology assessment at least 10-100 times higher than the projected therapeutic dose, and present comprehensive single dose response, expanded acute toxicology, toxicokinetics, and respiratory/cardiovascular safety pharmacology results for intravenously administered dextran-coated graphene oxide nanoplatelet (GNP-Dex) formulations to rats at doses between 1 and 500 mg/kg. Our results indicate that the maximum tolerable dose (MTD) of GNP-Dex is between 50 mg/kg ≤ MTD < 125 mg/kg, blood half-life < 30 min, and majority of nanoparticles excreted within 24 h through feces. Histopathology changes were noted at ≥250 mg/kg in the heart, liver, lung, spleen, and kidney; we found no changes in the brain and no GNP-Dex related effects in the cardiovascular parameters or hematological factors (blood, lipid, and metabolic panels) at doses < 125 mg/kg. The results open avenues for pivotal preclinical single and repeat dose safety studies following good laboratory practices (GLP) as required by regulatory agencies for investigational new drug (IND) application.

Graphene Nanoribbons as a Drug Delivery Agent for Lucanthone Mediated Therapy of Glioblastoma Multiforme

We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24h. However, their uptake was ~38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1.

Graphene-based platforms for cancer therapeutics

Graphene is a multifunctional carbon nanomaterial and could be utilized to develop platform technologies for cancer therapies. Its surface can be covalently and noncovalently functionalized with anticancer drugs and functional groups that target cancer cells and tissue to improve treatment efficacies. Furthermore, its physicochemical properties can be harnessed to facilitate stimulus responsive therapeutics and drug delivery. This review article summarizes the recent literature specifically focused on development of graphene technologies to treat cancer. We will focus on advances at the interface of graphene based drug/gene delivery, photothermal/photodynamic therapy and combinations of these techniques. We also discuss the current understanding in cytocompatibility and biocompatibility issues related to graphene formulations and their implications pertinent to clinical cancer management.

Graphene nanoribbons elicit cell specific uptake and delivery via activation of epidermal growth factor receptor enhanced by human papillomavirus E5 protein.

Ligands such as peptides, antibodies or other epitopes bind and activate specific cell receptors, and are employed for targeted cellular delivery of pharmaceuticals such as drugs, genes and imaging agents. Herein, we show that oxidized graphene nanoribbons, non-covalently functionalized with PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[amino(polyethyleneglycol)]) (O-GNR-PEG-DSPE) activate epidermal growth factor receptors (EGFRs). This activation generates a predominantly dynamin-dependent macropinocytosis-like response, and results in significant O-GNR-PEG-DSPE uptake into cells with high EGFR expression. Cells with an integrated human papillomavirus (HPV) genome also show increased uptake due to the modulation of the activated EGFR by the viral protein E5. We demonstrate that this cell specific uptake of O-GNR-PEG-DSPE can be exploited to achieve significantly enhanced drug efficacies even in drug resistant cells. These results have implications for the development of active targeting and delivery agents without ligand functionalization for use in the diagnosis and treatment of pathologies that overexpress EGFR or mediated by HPV.

Towards An Advanced Graphene-Based Magnetic Resonance Imaging Contrast Agent: Sub-acute Toxicity and Efficacy Studies in Small Animals.

Current clinical Gd3+-based T1 magnetic resonance imaging (MRI) contrast agents (CAs) are suboptimal or unsuitable, especially at higher magnetic fields (>1.5 Tesla) for advanced MRI applications such as blood pool, cellular and molecular imaging. Herein, towards the goal of developing a safe and more efficacious high field T1 MRI CA for these applications, we report the sub-acute toxicity and contrast enhancing capabilities of a novel nanoparticle MRI CA comprising of manganese (Mn2+) intercalated graphene nanoparticles functionalized with dextran (hereafter, Mangradex) in rodents. Sub-acute toxicology performed on rats intravenously injected with Mangradex at 1, 50 or 100 mg/kg dosages 3 times per week for three weeks indicated that dosages ≤50 mg/kg could serve as potential diagnostic doses. Whole body 7 Tesla MRI performed on mice injected with Mangradex at a potential diagnostic dose (25 mg/kg or 455 nanomoles Mn2+/kg; ~2 orders of magnitude lower than the paramagnetic ion concentration in a typical clinical dose) showed persistent (up to at least 2 hours) contrast enhancement in the vascular branches (Mn2+ concentration in blood at steady state = 300 ppb, per voxel = 45 femtomoles). The results lay the foundations for further development of Mangradex as a vascular and cellular/ molecular MRI probe.

Vasoactive effects of stable aqueous suspensions of single walled carbon nanotubes in hamsters and mice

Abstract Single-walled carbon nanotubes synthesized with iron (Fe-SWCNT) or gadolinium (Gd-SWCNT) show promise as high performance multimodal contrast and drug-delivery agents. Our purpose was to evaluate potential vasoactive effects of SWCNT. Stable aqueous solutions of Fe-SWCNTs or Gd-SWCNTs were made using the biocompatible amphiphilic polymer N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoylsn-glycero-3- phosphoethanolamine (PEG-DSPE). Both aggregated and non-aggregated (sonicated) formulations were tested. The initial vasoactivity of the formulations and their potential for inducing pro-inflammatory endothelial dysfunction were investigated in the hamster cheek pouch and murine cremaster muscle intravital microscopy models. These models provide an assay to test several formulations/dosages in a paired fashion. Abluminal exposure to small arterioles exposes both endothelial and vascular smooth muscle cells. Using abluminal exposures of dosages that would approximate the first pass of an i.v. bolus injection, both Fe-SWCNTs and Gd-SWCNTs were immediately vasoactive. Aggregated formulations induced dilation and non-aggregated formulations induced constriction in both hamsters and mice. Endothelial dysfunction was evident after exposure to either aggregated or non-aggregated forms. General loss of dilator capability was seen after exposure to non-aggregated but not aggregated forms. Thus concentrations mimicking bolus dosing of PEG-DSPE coated SWCNT induce both acute and chronic vascular responses.

Structural disruption increases toxicity of graphene nanoribbons

The increased utilization of graphene nanoribbons (GNRs) for biomedical and material science applications necessitates the thorough evaluation of potential toxicity of these materials under both intentional and accidental exposure scenarios. We here investigated the effects of structural disruption of GNRs (induced by low‐energy bath and high‐energy probe sonication) to in vitro (human cell lines), and in vivo (Oryzias latipes embryo) biological systems. Our results demonstrate that low concentration (20 µg ml−1) suspensions of GNRs prepared by as little as 1 min of probe sonication can cause significant decreases in the overall metabolic state of cells in vitro, and increased embryo/larval mortality in vivo, as compared to bath sonicated or unsonicated suspensions. Structural analysis indicates that probe sonication leads to disruption in GNR structure and production of smaller carbonaceous debris, which may be the cause of the toxicity observed. These results point out the importance of assessing post‐production structural modifications for any application using nanomaterials. Copyright

Interaction of graphene nanoribbons with components of the blood vascular system

Aim: The systemic administration of graphene nanoribbons for a variety of in vivo biomedical applications will result in their interaction with cellular and protein components of the circulatory system. The aim of this study was to assess the in vitro effects of graphene nanoribbons (O-GNR) noncovalently functionalized with PEG-DSPE (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N [amino (polyethylene glycol)]) on some of the key hematological and vascular components of the circulatory system. Methods: Transmission electron microscopy was used to characterize the nanoparticles. ELISA-based assays, bright-field microscopy, transmission electron microscopy and colorimetric assays were used to assess toxicological effects. Results: Our findings taken together indicate that low concentrations of O-GNR-PEG-DSPE (<80 µg/ml) are relatively nontoxic to the hematological components, and could be employed for diagnostic and therapeutic applications especially for diseases of the circulatory system.