Balaji Sitharaman

In vivo biocompatibility of ultra-short single-walled carbon nanotube/biodegradable polymer nanocomposites for bone tissue engineering

Scaffolds play a pivotal role in the tissue engineering paradigm by providing temporary structural support, guiding cells to grow, assisting the transport of essential nutrients and waste products, and facilitating the formation of functional tissues and organs. Single-walled carbon nanotubes (SWNTs), especially ultra-short SWNTs (US-tubes), have proven useful for reinforcing synthetic polymeric scaffold materials. In this article, we report on the in vivo biocompatibility of US-tube reinforced porous biodegradable scaffolds in a rabbit model. US-tube nanocomposite scaffolds and control polymer scaffolds were implanted in rabbit femoral condyles and in subcutaneous pockets. The hard and soft tissue response was analyzed with micro-computed tomography (micro CT), histology, and histomorphometry at 4 and 12 weeks after implantation. The porous US-tube nanocomposite scaffolds exhibited favorable hard and soft tissue responses at both time points. At 12 weeks, a three-fold greater bone tissue ingrowth was seen in defects containing US-tube nanocomposite scaffolds compared to control polymer scaffolds. Additionally, the 12 week samples showed reduced inflammatory cell density and increased connective tissue organization. No significant quantitative difference in polymer degradation was observed among the various groups; qualitative differences between the two time points were consistent with expected degradation due to the progression of time. Although no conclusions can be drawn from the present study concerning the osteoinductivity of US-tube nanocomposite scaffolds, the results suggest that the presence of US-tubes may render nanocomposite scaffolds bioactive assisting osteogenesis.

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.

Superparamagnetic gadonanotubes are high-performance MRI contrast agents

We report the nanoscale loading and confinement of aquated Gd3+n-ion clusters within ultra-short single-walled carbon nanotubes (US-tubes); these Gd3+n@US-tube species are linear superparamagnetic molecular magnets with Magnetic Resonance Imaging (MRI) efficacies 40 to 90 times larger than any Gd3+-based contrast agent (CA) in current clinical use.

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.

Single-walled carbon nanotubes as a multimodal-thermoacoustic and photoacoustic-contrast agent

We have developed a novel carbon nanotube-based contrast agent for both thermoacoustic and photoacoustic tomography. In comparison to deionized water, single-walled carbon nanotubes exhibited more than twofold signal enhancement for thermoacoustic tomography at 3 GHz. In comparison to blood, they exhibited more than sixfold signal enhancement for photoacoustic tomography at 1064 nm wavelength. The large contrast enhancement of single-walled carbon nanotubes was further corroborated by tissue phantom imaging studies.

Toward Carbon-Nanotube-Based Theranostic Agents for Microwave Detection and Treatment of Breast Cancer: Enhanced Dielectric and Heating Response of Tissue-Mimicking Materials

The experimental results reported in this paper suggest that single-walled carbon nanotubes (SWCNTs) have the potential to enhance dielectric contrast between malignant and normal tissue for microwave detection of breast cancer and facilitate selective heating of malignant tissue for microwave hyperthermia treatment of breast cancer. In this study, we constructed tissue-mimicking materials with varying concentrations of SWCNTs and characterized their dielectric properties and heating response. At SWCNT concentrations of less than 0.5% by weight, we observed significant increases in the relative permittivity and effective conductivity. In microwave heating experiments, we observed significantly greater temperature increases in mixtures containing SWCNTs. These temperature increases scaled linearly with the effective conductivity of the mixtures. This work is a first step towards the development of functionalized, tumor-targeting SWCNTs as theranostic (integrated therapeutic and diagnostic) agents for microwave breast cancer detection and treatment.

Water-Soluble Fullerene (C60) Derivatives as Nonviral Gene-Delivery Vectors

A new class of water-soluble C60 transfecting agents has been prepared using Hirsch−Bingel chemistry and assessed for their ability to act as gene-delivery vectors in vitro. In an effort to elucidate the relationship between the hydrophobicity of the fullerene core, the hydrophilicity of the water-solubilizing groups, and the overall charge state of the C60 vectors in gene delivery and expression, several different C60 derivatives were synthesized to yield either positively charged, negatively charged, or neutral chemical functionalities under physiological conditions. These fullerene derivatives were then tested for their ability to transfect cells grown in culture with DNA carrying the green fluorescent protein (GFP) reporter gene. Statistically significant expression of GFP was observed for all forms of the C60 derivatives when used as DNA vectors and compared to the ability of naked DNA alone to transfect cells. However, efficient in vitro transfection was only achieved with the two positively charged C60 derivatives, namely, an octa-amino derivatized C60 and a dodeca-amino derivatized C60 vector. All C60 vectors showed an increase in toxicity in a dose-dependent manner. Increased levels of cellular toxicity were observed for positively charged C60 vectors relative to the negatively charged and neutral vectors. Structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized C60 compounds, the C60 vector/DNA complexes, their physical attributes (aggregation, charge) and their transfection efficiencies. Recently, similar Gd@C60-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging (MRI). Thus, the successful demonstration of intracellular DNA uptake, intracellular transport, and gene expression from DNA using C60 vectors suggests the possibility of developing analogous Gd@C60-based vectors to serve simultaneously as both therapeutic and diagnostic agents.

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.