Man Yang

Immune responses of BALB/c mice to subcutaneously injected multi-walled carbon nanotubes

Abstract Carbon nanotubes have been shown to have the ability to transport therapeutic and detective reagents into cells. However, the rapid advances in new carbon nanotube-based materials and technologies have raised concerns about their safety. Such concerns require a fundamental understanding of the toxicological properties of carbon nanotubes. In particular, the use of carbon nanotubes as drug or probe delivery platforms may depend on the prevention of stimulatory side-effects to the immune system. In this study, we investigated the immunological properties of oxidized water dispersible multi-walled carbon nanotubes (MWCNTs) in healthy BALB/c mice. We injected the MWCNTs subcutaneously, and the immune responses of the mice were monitored over time. We show that the MWCNTs induce complement activation and the production of pro-inflammatory cytokines early after injection of the mice, and that the levels of complement and cytokines return to normal levels over time. With the exception of the lymph nodes, there was no obvious accumulation of MWCNTs observed in the liver, spleen, kidney, or heart. In addition, we did not observe injury in the organs or lymph nodes. Our results indicate that local, subcutaneous administration of MWCNTs induces obvious short-term immunological reactions, which can be eliminated over time.

Characterization of multiwalled carbon nanotubes dispersing in water and association with biological effects

Biomedical application potentials of carbon nanotubes-based materials have been investigated intensively in recent years; however, characterization and metrology are still facing great technical challenges when the materials are intended to be used as carriers for therapeutics in aqueous solutions. Systematic characterization on the dispersing carbon nanotubes is urgently required and therefore of significance. In this paper multiwalled carbon nanotubes (MWCNTs) with different average lengths or with different oxidation degrees were dispersed in water and characterized systematically by applying UV spectroscopy, SEM, DLS, TGA, XPS, and FTIR. In particular, the characteristic absorption of the carbon nanotubes was analyzed using resolution-fitting technique to establish relations of wavelength and absorption intensity to the size distribution and surface chemistry. Results indicated that the absorption spectra of MWCNTs could reflect the variation of surface chemistry and length distribution of carbon nanotubes dispersed in water by combining with the other measurements. A vascular endothelium cell line was taken as a model to figure out association between physicochemical features and cytotoxicity of the carbon nanotubes. It was showed that the multiwalled carbon nanotubes with different oxidation degrees and similar length distribution exhibited different interaction files to the cells proliferation in a manner of time dependence and concentration dependence.

Preparation, characterization and fluorescent imaging of multi-walled carbon nanotube–porphyrin conjugate

In order to track carbon nanotubes (CNTs) in vitro or in vivo, many technologies have already been applied. However, the potential toxic effects of technologies such as radio and quantum dot labeling are the major problems. Therefore, the need to search for a new kind of biocompatible material to label CNTs has become urgent. In this work, one kind of porphyrin derivative with a single amino group was synthesized and covalently conjugated to oxidized water-soluble multi-walled carbon nanotubes (MWCNTs). The physiochemical properties of the oxidized MWCNTs and the fluorescent characteristics of their conjugate were carefully investigated. The experimental results indicated that the carboxyl group was introduced to the oxidized MWCNTs, allowing reaction with the amino group of the porphyrin, while the tube-like structure was well retained. The conjugate of the amino porphyrin and the MWCNTs exhibited strong red fluorescence emissions at 670 nm and 730 nm when excited with the optimal excitation wavelength of 420 nm. An aqueous solution containing the conjugate was injected subcutaneously into mice and was then imaged in vivo and ex vivo. Results showed that the conjugate was mainly engulfed by macrophages under the skin, and most of the conjugates accumulated in subcutaneous connective mucosa and a small amount in the liver, over a long period.

Multiwalled Carbon Nanotubes Interact with Macrophages and Influence Tumor Progression and Metastasis

Macrophages are one of the most important types of immune effector cells and are closely associated with tumor progression and metastasis. In this work, we investigated the influences of oxidized multiwalled carbon nanotubes (o-MWCNT) on macrophages that are resting in the normal subcutis tissue or in the tumor microenvironment in vivo as well as on the macrophage cell line of RAW 264.7 treated with combination of IL4, IL10 and IL13 in vitro. The o-MWCNT were characterized with SEM, DLS, FTIR, TGA, and UV-vis-NIR spectroscopy, and their effects on the RWA 264.7 cell line and breast cancer tumor-bearing mice were analyzed using the MTS assay, flow cytometry analysis, and histological and immunohistochemical observations. Our experimental results showed that subcutaneously injected o-MWCNT not only induced phagocytosis of the local resident macrophages, but also competitively recruited macrophages from other tissues. These interactions resulted in macrophage reduction and decreased vessel density around the tumor mass, which together inhibited tumor progression and metastasis in the lung. In the cell line model, the o-MWCNT inhibited the ability of the interleukin treated RAW macrophages to promote tumor cell migration as well as decreased their proliferation rate.

Concentration control of carbon nanotubes in aqueous solution and its influence on the growth behavior of fibroblasts.

This work investigated the biological influence of water-soluble multiwalled carbon nanotubes (wsMWCNTs) on fibroblast cell growth as a function of concentration control in an aqueous solution. The wsMWCNTs were prepared by an optimal procedure of ultrasonication/concentrated acids oxidation. The concentration of wsMWCNT in the solution was quantified by an established calibration line. A stable concentration of 0.3mg/ml was obtained in the surfactant-free water. The physicochemical properties of wsMWCNTs were characterized using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), UV/VIS/NIR spectroscopy, and dynamic light scattering (DLS). Cell proliferation and the cell cycle were examined by MTS assay, flow cytometry and TEM respectively. Experimental results showed that the oxidation degree was a key factor that determined the concentration and stability of wsMWCNTs in the aqueous solution. The wsMWCNTs were able to enter into the cells and mainly accumulated in the cytoplasm. The wsMWCNTs-induced variations in cell proliferation and the cell cycle were concentration dependent. Cells cultivated with wsMWCNTs of 0.3mg/ml underwent a dramatic apoptosis. The proliferation was clearly suppressed when the cells were cultivated with wsMWCNTs of 0.03 mg/ml. There were no obvious influences on cell proliferation and the cell cycle when the concentration of wsMWNTs decreased to 0.01 mg/ml.

Carbon Nanotubes Activate Limulus Amebocyte Lysate Coagulation by Interface Adsorption

Limulus amebocyte lysate (LAL) assay is worldwide requested in the assessment of endotoxin contamination for biomaterials. As carbon nanotubes are one major nanomaterial with multiple potentials in biomedical application, here we investigate whether oxidized multiwalled carbon nanotubes (O-MWCNT) interferes the assessment by LAL assays. We showed that the endotoxin free O-MWCNT dispersing in aqueous solutions could activate both the gel-clotting and the end-point chromogenic LAL assay by converting coagulogen into coagulin through interfacial interactions between O-MWCNT and enzymes in the assays. In conclusion, the O-MWCNT could induce false positive results by activating the enzyme cascade of LAL.

Abstract 403: Interaction of carbon nanotubes with macrophages in tumor microenvironment

Macrophages take on different phenotypes and play different roles in tumor progress as well as anti-tumor therapy depending on the environmental signals that they receive. We have previously reported that subcutaneously injected oxidized multiwalled carbon nanotubes (o-MWCNTs) in normal BABL/c mice were largely engulfed by resident macrophages around the injection site, inducing complement activation and short-term upregulation of inflammatory cytokines [Jie Meng, Man Yang, Zhen Xu, Fumin Jia, Hua Kong, and Haiyan Xu. Immune responses of BALB/c mice to subcutaneously injected multi-walled carbon nanotubes. Nanotoxicology, 2010, DOI:10.3109/17435390.2010.523483]; subcutaneous injection of o-MWCNTs in tumor bearing BALB/c mice upregulated the expression of GM-CSF and IL12p70 in the serum over 2-fold increase and inhibited the tumor progression to a certain extent [Jie Meng, Man Yang, Fumin Jia, Hua Kong, Weiqi Zhang, ChaoyingWang, Jianmin Xing, Sishen Xie and Haiyan Xu. Subcutaneous injection of water-soluble multi-walled carbon nanotubes in tumor-bearing mice boosts the host immune activity. Nanotechnology 2010; 21: 145104]. When o-MWCNTs were used as a carrier of tumor lysate, subcutaneous injection of this conjugate in tumor-bearing mice effectively increased cytotoxicity of the lymphocytes to the tumor cells [Jie Meng, Jie Meng, Jinhong Duan, Hua Kong, Chen Wang, Sishen Xie, Shuchang Chen, Haiyan Xu and Xian-Da Yang. Carbon Nanotubes Conjugated to Tumor Lysate Protein Enhance the Efficacy of an Anti-tumor Immunotherapy. Small 2008; 4(9): 1364-1370]. These findings imply that the inhibitory effect of o-MWCNTs on tumor progression may be associated with their interactions with macrophages. The present work focused on the effect of o-MWCNTs on macrophages phenotypes and functions in the tumor environment. The o-MWCNTs were physicochemically characterized with SEM, DLS, FTIR, TGA, and UV-vis-NIR spectroscopy; and the effects on RWA 264.7 cell line or macrophages in breast cancer tumor-bearing mice were investigated using MTS assay, flow cytometry analysis, and histological and immunohistochemical observations. Experimental results showed that subcutaneously injected o-MWCNTs not only attracted resident macrophages in tissue and induced phagocytosis, but also competitively recruited macrophages from circulating monocytes, which led to reduction of macrophages and vessel density around the tumor mass and inhibition of tumor progression and metastasis in the lung. Additionally, o-MWCNTs inhibited the ability of alternatively activated RAW macrophages to promote tumor cells migration as well as decreased their proliferation rate. Acknowledgement: This work was supported by National Key Scientific Projects of China (2011CB933504, 2010CB934002) and Beijing Municipal Natural Science Foundation (2011022). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 403. doi:1538-7445.AM2012-403

Preparation, Characterization and Cellular Biological Impact of Water Soluble Multiwalled Carbon Nanotubes

Biological effect and multiple application potentials of carbon nanotubes have been becoming important issues attracting intense research interests and concerns. In this work, water dispersible oxidized multiwalled carbon nanotubes (o-MWNTs) were prepared by an optimal combination of ultrasonication and concentrated acids oxidation; stable concentrations of 0.3mg/ml were obtained in deionized water of surfactant free. The o-MWNTs dispersing in water displayed a strong characteristic adsorption at 253nm in the spectra of UV/VIS/NIR. XPS analysis showed the presence of carboxylated groups on the o-MWNTs. SEM observation indicated that after the oxidation, the as received MWNTs were cut short; the average length was around 500nm-800nm, the maximum effective size obtained by DLS measurement was about 340nm. It was inferred that the oxidation degree was one major key factor to determine the stable dispersion concentration of o-MWNTs in water. The o-MWNTs dispersing in water entered into the cells and located in the cytoplasm. Cells proliferation and living cycle were affected by the o-MWNTs dispersing in the culture medium in a concentration dependant manner; the cells cultivated in the medium with high concentration of o-MWNTs underwent apoptosis to a point determined by flowcytometry and TEM respectively, while low concentration of o-MWNTs induced light suppression of cells proliferation examined with cell viability assay.