Abhilash Sasidharan

Differential nano-bio interactions and toxicity effects of pristine versus functionalized graphene

We report the effect of carboxyl functionalization of graphene in pacifying its strong hydrophobic interaction with cells and associated toxic effects. Pristine graphene was found to accumulate on the cell membrane causing high oxidative stress leading to apoptosis, whereas carboxyl functionalized hydrophilic graphene was internalized by the cells without causing any toxicity.

Hemocompatibility and Macrophage Response of Pristine and Functionalized Graphene

Graphene and its derivatives are being proposed for several important biomedical applications including drug delivery, gene delivery, contrast imaging, and anticancer therapy. Most of these applications demand intravenous injection of graphene and hence evaluation of its hemocompatibility is an essential prerequisite. Herein, both pristine and functionalized graphene are extensively characterized for their interactions with murine macrophage RAW 264.7 cells and human primary blood components. Detailed analyses of the potential uptake by macrophages, effects on its metabolic activity, membrane integrity, induction of reactive oxygen stress, hemolysis, platelet activation, platelet aggregation, coagulation cascade, cytokine induction, immune cell activation, and immune cell suppression are performed using optimized protocols for nanotoxicity evaluation. Electron microscopy, confocal Raman spectral mapping, and confocal fluorescence imaging studies show active interaction of both the graphene systems with macrophage cells, and the reactive oxygen species mediated toxicity effects of hydrophobic pristine samples are significantly reduced by surface functionalization. In the case of hemocompatibility, both types of graphene show excellent compatibility with red blood cells, platelets, and plasma coagulation pathways, and minimal alteration in the cytokine expression by human peripheral blood mononuclear cells. Further, both samples do not cause any premature immune cell activation or suppression up to a relatively high concentration of 75 μg mL(-1) after 72 h of incubation under in vitro conditions. This study clearly suggests that the observed toxicity effects of pristine graphene towards macrophage cells can be easily averted by surface functionalization and both the systems show excellent hemocompatibility.

Rapid dissolution of ZnO nanocrystals in acidic cancer microenvironment leading to preferential apoptosis

The microenvironment of cancer plays a very critical role in the survival, proliferation and drug resistance of solid tumors. Here, we report an interesting, acidic cancer microenvironment-mediated dissolution-induced preferential toxicity of ZnO nanocrystals (NCs) against cancer cells while leaving primary cells unaffected. Irrespective of the size-scale (5 and 200 nm) and surface chemistry differences (silica, starch or polyethylene glycol coating), ZnO NCs exhibited multiple stress mechanisms against cancer cell lines (IC(50)∼150 μM) while normal human primary cells (human dermal fibroblast, lymphocytes, human umbilical vein endothelial cells) remain less affected. Flow cytometry and confocal microscopy studies revealed that ZnO NCs undergo rapid preferential dissolution in acidic (pH ∼5-6) cancer microenvironment causing elevated ROS stress, mitochondrial superoxide formation, depolarization of mitochondrial membrane, and cell cycle arrest at S/G2 phase leading to apoptosis. In effect, by elucidating the unique toxicity mechanism of ZnO NCs, we show that ZnO NCs can destabilize cancer cells by utilizing its own hostile acidic microenvironment, which is otherwise critical for its survival.

Highly biocompatible TiO2:Gd3+ nano-contrast agent with enhanced longitudinal relaxivity for targeted cancer imaging

We report the development of a novel magnetic nano-contrast agent (nano-CA) based on Gd(3+) doped amorphous TiO(2) of size ∼25 nm, exhibiting enhanced longitudinal relaxivity (r(1)) and magnetic resonance (MR) contrasting together with excellent biocompatibility. Quantitative T1 mapping of phantom samples using a 1.5 T clinical MR imaging system revealed that the amorphous phase of doped titania has the highest r(1) relaxivity which is ∼2.5 fold higher than the commercially used CA Magnevist™. The crystalline (anatase) samples formed by air annealing at 250 °C and 500 °C showed significant reduction in r(1) values and MR contrast, which is attributed to the loss of proton-exchange contribution from the adsorbed water and atomic re-arrangement of Gd(3+) ions in the crystalline host lattice. Nanotoxicity studies including cell viability, plasma membrane integrity, reactive oxygen stress and expression of pro-inflammatory cytokines, performed on human primary endothelial cells (HUVEC), human blood derived peripheral blood mononuclear cells (PBMC) and nasopharyngeal epidermoid carcinoma (KB) cell line showed excellent biocompatibility up to relatively higher doses of 200 μg ml(-1). The potential of this nano-CA to cause hemolysis, platelet aggregation and plasma coagulation were studied using human peripheral blood samples and found no adverse effects, illustrating the possibility of the safe intravenous administration of these agents for human applications. Furthermore, the ability of these agents to specifically detect cancer cells by targeting molecular receptors on the cell membrane was demonstrated on folate receptor (FR) positive oral carcinoma (KB) cells, where the folic acid conjugated nano-CA showed receptor specific accumulation on cell membrane while leaving the normal fibroblast cells (L929) unstained. This study reveals that the Gd(3+) doped amorphous TiO(2) nanoparticles having enhanced magnetic resonance contrast and high biocompatibility is a promising candidate for molecular receptor targeted MR imaging.

Radiofrequency Ablation of Drug‐Resistant Cancer Cells Using Molecularly Targeted Carboxyl‐Functionalized Biodegradable Graphene

Under ultralow radiofrequency (RF) power, transferrin-conjugated graphene nanoparticles can thermally ablate drug- or radiation-resistant cancer cells very effectively. The results suggest that graphene-based RF hyperthermia can be an efficient method to manage drug-/radiation-resistant cancers.

Cellular and molecular mechanistic insight into the DNA-damaging potential of few-layer graphene in human primary endothelial cells

Despite graphene being proposed for a multitude of biomedical applications, there is a dearth in the fundamental cellular and molecular level understanding of how few-layer graphene (FLG) interacts with human primary cells. Herein, using human primary umbilical vein endothelial cells as model of vascular transport, we investigated the basic mechanism underlying the biological behavior of graphene. Mechanistic toxicity studies using a battery of cell based assays revealed an organized oxidative stress paradigm involving cytosolic reactive oxygen stress, mitochondrial superoxide generation, lipid peroxidation, glutathione oxidation, mitochondrial membrane depolarization, enhanced calcium efflux, all leading to cell death by apoptosis/necrosis. We further investigated the effect of graphene interactions using cDNA microarray analysis and identified potential adverse effects by down regulating key genes involved in DNA damage response and repair mechanisms. Single cell gel electrophoresis assay/Comet assay confirmed the DNA damaging potential of graphene towards human primary cells.

Computational simulations and experimental validation of structure- physicochemical properties of pristine and functionalized graphene: Implications for adverse effects on p53 mediated DNA damage response

Recent reports indicated DNA damaging potential of few-layer graphene in human cell systems. Here, we used computational technique to understand the interaction of both pristine (pG) or carboxyl functionalized graphene (fG) of different sizes (1, 6, and 10nm) with an important DNA repair protein p53. The molecular docking study revealed strong interaction between pG and DNA binding domains (DBD) of p53 with binding free energies (BE) varying from -12.0 (1nm) to -34 (6nm)kcal/mol, while fG showed relatively less interaction with BE varying from -6.7 (1nm) to -11.1 (6nm)kcal/mol. Most importantly, pG or fG bound p53-DBDs could not bind to DNA. Further, microarray analysis of human primary endothelial cells revealed graphene intervention on DNA damage and its structure-properties effect using comet assay studies. Thus, computational and experimental results revealed the structure-physicochemical property dependent adverse effects of graphene in DNA repair protein p53.