Baoyun Sun

Understanding the Toxicity of Carbon Nanotubes

Because of their unique physical, chemical, electrical, and mechanical properties, carbon nanotubes (CNTs) have attracted a great deal of research interest and have many potential applications. As large-scale production and application of CNTs increases, the general population is more likely to be exposed to CNTs either directly or indirectly, which has prompted considerable attention about human health and safety issues related to CNTs. Although considerable experimental data related to CNT toxicity at the molecular, cellular, and whole animal levels have been published, the results are often conflicting. Therefore, a systematic understanding of CNT toxicity is needed but has not yet been developed. In this Account, we highlight recent investigations into the basis of CNT toxicity carried out by our team and by other laboratories. We focus on several important factors that explain the disparities in the experimental results of nanotoxicity, such as impurities, amorphous carbon, surface charge, shape, length, agglomeration, and layer numbers. The exposure routes, including inhalation, intravenous injection, or dermal or oral exposure, can also influence the in vivo behavior and fate of CNTs. The underlying mechanisms of CNT toxicity include oxidative stress, inflammatory responses, malignant transformation, DNA damage and mutation (errors in chromosome number as well as disruption of the mitotic spindle), the formation of granulomas, and interstitial fibrosis. These findings provide useful insights for de novo design and safe application of carbon nanotubes and their risk assessment to human health. To obtain reproducible and accurate results, researchers must establish standards and reliable detection methods, use standard CNT samples as a reference control, and study the impact of various factors systematically. In addition, researchers need to examine multiple types of CNTs, different cell lines and animal species, multidimensional evaluation methods, and exposure conditions. To make results comparable among different institutions and countries, researchers need to standardize choices in toxicity testing such as that of cell line, animal species, and exposure conditions. The knowledge presented here should lead to a better understanding of the key factors that can influence CNT toxicity so that their unwanted toxicity might be avoided.

The scavenging of reactive oxygen species and the potential for cell protection by functionalized fullerene materials

We demonstrated that three different types of water-soluble fullerenes materials can intercept all of the major physiologically relevant ROS. C(60)(C(COOH)(2))(2), C(60)(OH)(22), and Gd@C(82)(OH)(22) can protect cells against H(2)O(2)-induced oxidative damage, stabilize the mitochondrial membrane potential and reduce intracellular ROS production with the following relative potencies: Gd@C(82)(OH)(22)> or =C(60)(OH)(22)>C(60)(C(COOH)(2))(2). Consistent with their cytoprotective abilities, these derivatives can scavenge the stable 2,2-diphenyl-1-picryhydrazyl radical (DPPH), and the reactive oxygen species (ROS) superoxide radical anion (O(2)(*-)), singlet oxygen, and hydroxyl radical (HO(*)), and can also efficiently inhibit lipid peroxidation in vitro. The observed differences in free radical-scavenging capabilities support the hypothesis that both chemical properties, such as surface chemistry induced differences in electron affinity, and physical properties, such as degree of aggregation, influence the biological and biomedical activities of functionalized fullerenes. This represents the first report that different types of fullerene derivatives can scavenge all physiologically relevant ROS. The role of oxidative stress and damage in the etiology and progression of many diseases suggests that these fullerene derivatives may be valuable in vivo cytoprotective and therapeutic agents.

Metallofullerene nanoparticles circumvent tumor resistance to cisplatin by reactivating endocytosis

Cisplatin is a chemotherapeutic drug commonly used in clinics. However, acquired resistance confines its application in chemotherapeutics. To overcome the acquired resistance to cisplatin, it is reasoned, based on our previous findings of mediation of cellular responses by [Gd@C82(OH)22]n nanoparticles, that [Gd@C82(OH)22]n may reverse tumor resistance to cisplatin by reactivating the impaired endocytosis of cisplatin-resistant human prostate cancer (CP-r) cells. Here we report that exposure of the CP-r PC-3-luc cells to cisplatin in the presence of nontoxic [Gd@C82(OH)22]n not only decreased the number of surviving CP-r cells but also inhibited growth of the CP-r tumors in athymic nude mice as measured by both optical and MRI. Labeling the CP-r PC-3 cells with transferrin, an endocytotic marker, demonstrated that pretreatment of the CP-r PC-3-luc cells with [Gd@C82(OH)22]n enhanced intracellular accumulation of cisplatin and formation of cisplatin-DNA adducts by restoring the defective endocytosis of the CP-r cancer cells. The results suggest that [Gd@C82(OH)22]n nanoparticles overcome tumor resistance to cisplatin by increasing its intracellular accumulation through the mechanism of restoring defective endocytosis. The technology can be extended to other challenges related to multidrug resistance often found in cancer treatments.

Molecular mechanism of pancreatic tumor metastasis inhibition by Gd@C82(OH)22 and its implication for de novo design of nanomedicine

Pancreatic adenocarcinoma is the most lethal of the solid tumors and the fourth-leading cause of cancer-related death in North America. Matrix metalloproteinases (MMPs) have long been targeted as a potential anticancer therapy because of their seminal role in angiogenesis and extracellular matrix (ECM) degradation of tumor survival and invasion. However, the inhibition specificity to MMPs and the molecular-level understanding of the inhibition mechanism remain largely unresolved. Here, we found that endohedral metallofullerenol Gd@C82(OH)22 can successfully inhibit the neoplastic activity with experiments at animal, tissue, and cellular levels. Gd@C82(OH)22 effectively blocks tumor growth in human pancreatic cancer xenografts in a nude mouse model. Enzyme activity assays also show Gd@C82(OH)22 not only suppresses the expression of MMPs but also significantly reduces their activities. We then applied large-scale molecular-dynamics simulations to illustrate the molecular mechanism by studying the Gd@C82(OH)22–MMP-9 interactions in atomic detail. Our data demonstrated that Gd@C82(OH)22 inhibits MMP-9 mainly via an exocite interaction, whereas the well-known zinc catalytic site only plays a minimal role. Steered by nonspecific electrostatic, hydrophobic, and specific hydrogen-bonding interactions, Gd@C82(OH)22 exhibits specific binding modes near the ligand-specificity loop S1′, thereby inhibiting MMP-9 activity. Both the suppression of MMP expression and specific binding mode make Gd@C82(OH)22 a potentially more effective nanomedicine for pancreatic cancer than traditional medicines, which usually target the proteolytic sites directly but fail in selective inhibition. Our findings provide insights for de novo design of nanomedicines for fatal diseases such as pancreatic cancer.

Bio-distribution and metabolic paths of silica coated CdSeS quantum dots

With the rapid development of quantum dot (QD) technology, water-soluble QDs have the prospect of being used as a biological probe for specific diagnoses, but their biological behaviors in vivo are little known. Our recent in vivo studies concentrated on the bio-kinetics of QDs coated by hydroxyl group modified silica networks (the QDs are 21.3+/-2.0 nm in diameter and have maximal emission at 570 nm). Male ICR mice were intravenously given the water-soluble QDs with a single dose of 5 nmol/mouse. Inductively coupled plasma-mass spectrometry was used to measure the (111)Cd content to indicate the concentration of QDs in plasma, organs, and excretion samples collected at predetermined time intervals. Meanwhile, the distribution and aggregation state of QDs in tissues were also investigated by pathological examination and differential centrifugation. The plasma half-life and clearance of QDs were 19.8+/-3.2 h and 57.3+/-9.2 ml/h/kg, respectively. The liver and kidney were the main target organs for QDs. The QDs metabolized in three paths depending on their distinct aggregated states in vivo. A fraction of free QDs, maintaining their original form, could be filtered by glomerular capillaries and excreted via urine as small molecules within five days. Most QDs bound to protein and aggregated into larger particles that were metabolized in the liver and excreted via feces in vivo. After five days, 8.6% of the injected dose of aggregated QDs still remained in hepatic tissue and it was difficult for this fraction to clear.

The effect of Gd@C82(OH)22 nanoparticles on the release of Th1/Th2 cytokines and induction of TNF-α mediated cellular immunity

It is known that down-regulation of the immune response may be associated with the progenesis, development and prognosis of cancer or infectious diseases. Up-regulating the immune response in vivo is therefore a desirable strategy for clinical treatment. Here we report that poly-hydroxylated metallofullerenol (Gd@C(82)(OH)(22)) has biomedical functions useful in anticancer therapy arising from immunomodulatory effects observed both in vivo and in vitro. We found that metallofullerenol can inhibit the growth of tumors, and shows specific immunomodulatory effects on T cells and macrophages. These effects include polarizing the cytokine balance towards Th1 (T-helper cell type 1) cytokines, decreasing the production of Th2 cytokines (IL-4, IL-5 and IL-6), and increasing the production of Th1 cytokines (IL-2, IFN-gamma and TNF-alpha) in the serum samples. Immune-system regulation by this nanomaterial showed dose-dependent behavior: at a low concentration, Gd@C(82)(OH)(22) nanoparticles slightly affected the activity of immune cells in vitro, while at a high concentration, they markedly enhanced immune responses and stimulated immune cells to release more cytokines, helping eliminate abnormal cells. Gd@C(82)(OH)(22) nanoparticles stimulated T cells and macrophages to release significantly greater quantities of TNF-alpha, which plays a key role in cellular immune processes. Gd@C(82)(OH)(22) nanoparticles are more effective in inhibiting tumor growth in mice than some clinical anticancer drugs but have negligible side effects. The underlying mechanism for high anticancer activity may be attributed to the fact that this water-soluble nanomaterial effectively triggers the host immune system to scavenge tumor cells.

Inhibition of tumor growth by endohedral metallofullerenol nanoparticles optimized as reactive oxygen species scavenger.

Intraperitoneal injection of [Gd@C82(OH)22]n nanoparticles decreased activities of enzymes associated with the metabolism of reactive oxygen species (ROS) in the tumor-bearing mice. Several physiologically relevant ROS were directly scavenged by nanoparticles, and lipid peroxidation was inhibited in this study. [Gd@C82(OH)22]n nanoparticles significantly reduced the electron spin resonance (ESR) signal of the stable 2,2-diphenyl-1-picryhydrazyl radical measured by ESR spectroscopy. Like-wise, studies using ESR with spin-trapping demonstrated efficient scavenging of superoxide radical anion, hydroxyl radical, and singlet oxygen (1O2) by [Gd@C82(OH)22]n nanoparticles. In vitro studies using liposomes prepared from bovine liver phosphatidylcholine revealed that nanoparticles also had a strong inhibitory effect on lipid peroxidation. Consistent with their ability to scavenge ROS and inhibit lipid peroxidation, we determined that [Gd@C82(OH)22]n nanoparticles also protected cells subjected in vitro to oxidative stress. Studies using human lung adenocarcinoma cells or rat brain capillary endothelial cells demonstrated that [Gd@C82(OH)22]n nanoparticles reduced H2O2-induced ROS formation and mitochondrial damage. [Gd@C82(OH)22]n nanoparticles efficiently inhibited the growth of malignant tumors in vivo. In summary, the results obtained in this study reveal antitumor activities of [Gd@C82(OH)22]n nanoparticles in vitro and in vivo. Because ROS are known to be implicated in the etiology of a wide range of human diseases, including cancer, the present findings demonstrate that the potent inhibition of [Gd@C82(OH)22]n nanoparticles on tumor growth likely relates with typical capacity of scavenging reactive oxygen species.

Gd-metallofullerenol nanomaterial as non-toxic breast cancer stem cell-specific inhibitor

The contemporary use of nanomedicines for cancer treatment has been largely limited to serving as carriers for existing therapeutic agents. Here, we provide definitive evidence that, the metallofullerenol nanomaterial Gd@C82(OH)22, while essentially not toxic to normal mammary epithelial cells, possesses intrinsic inhibitory activity against triple-negative breast cancer cells. Gd@C82(OH)22 blocks epithelial-to-mesenchymal transition with resultant efficient elimination of breast cancer stem cells (CSCs) resulting in abrogation of tumour initiation and metastasis. In normoxic conditions, Gd@C82(OH)22 mediates these effects by blocking TGF-β signalling. Moreover, under hypoxic conditions found in the tumour microenvironment, cellular uptake of Gd@C82(OH)22 is facilitated where it functions as a bi-potent inhibitor of HIF-1α and TGF-β activities, enhancing CSC elimination. These studies indicate that nanomaterials can be engineered to directly target CSCs. Thus, Gd-metallofullerenol is identified as a kind of non-toxic CSC specific inhibitors with significant therapeutic potential.

Gadolinium metallofullerenol nanoparticles inhibit cancer metastasis through matrix metalloproteinase inhibition: imprisoning instead of poisoning cancer cells

UNLABELLED The purpose of this work is to study the antimetastasis activity of gadolinium metallofullerenol nanoparticles (f-NPs) in malignant and invasive human breast cancer models. We demonstrated that f-NPs inhibited the production of matrix metalloproteinase (MMP) enzymes and further interfered with the invasiveness of cancer cells in tissue culture condition. In the tissue invasion animal model, the invasive primary tumor treated with f-NPs showed significantly less metastasis to the ectopic site along with the decreased MMP expression. In the same animal model, we observed the formation of a fibrous cage that may serve as a physical barrier capable of cancer tissue encapsulation that cuts the communication between cancer- and tumor-associated macrophages, which produce MMP enzymes. In another animal model, the blood transfer model, f-NPs potently suppressed the establishment of tumor foci in lung. Based on these data, we conclude that f-NPs have antimetastasis effects and speculate that utilization of f-NPs may provide a new strategy for the treatment of tumor metastasis. FROM THE CLINICAL EDITOR In this study utilizing metallofullerenol nanoparticles, the authors demonstrate antimetastasis effects and speculate that utilization of these nanoparticles may provide a new strategy in metastatic tumor therapy.

Metallofullerol nanoparticles with low toxicity inhibit tumor growth by induction of G0/G1 arrest

AIMS [Gd@C(82)(OH)(22)](n) is a new type of nanoparticle with potent antineoplastic activity and low toxicity compared with traditional drugs. In this study, we explored, for the first time, the effect of [Gd@C(82)(OH)(22)](n) on the cell cycle using human breast cancer MCF-7 and human umbilical vein endothelial ECV304 cell lines by flow cytometry. METHODS Cell viability was assessed through CCK-8 assay, and MCF-7 tumor-bearing mice were examined after 2 weeks of treatment with [Gd@C(82)(OH)(22)](n). Cell cycle-related gene expression was detected by microarray and confirmed by real-time PCR and RNAi. RESULTS Cell viability studies confirmed that [Gd@C(82)(OH)(22)](n) inhibits breast cancer effectively with very low toxicity. Flow cytometric data and microarray results reveal that [Gd@C(82)(OH)(22)](n) mediates G0/G1 arrest in both cell lines by regulating the expression of several genes, such as cyclin D2, cyclin E and CDK4, among others, in the related cell cycle. CONCLUSION Results further demonstrated that [Gd@C(82)(OH)(22)](n) could inhibit tumor growth by inducing tumor cell and vein endothelial cell G0/G1 arrest, which may explain the low toxicity of [Gd@C(82)(OH)(22)](n).