Hua Kong

Carbon Nanotubes Conjugated to Tumor Lysate Protein Enhance the Efficacy of an Antitumor Immunotherapy

The biomedical applications of carbon nanotubes (CNTs) have attracted deep interest in recent years. Antitumor immunotherapy has the potential to improve the prognosis of cancer treatment but the efficacy of current immunotherapy generally needs further improvement. Multi-walled CNTs conjugated to tumor lysate protein are investigated as to whether they would enhance the efficacy of an immunotherapy employing a tumor-cell vaccine in a mouse model bearing the H22 liver cancer. The tumor cure rate is found to be markedly improved by CNTs conjugated to tumor lysate protein. The cellular antitumor immune reaction is also enhanced. Moreover, the observed antitumor immune response is relatively specific against the tumor intended for treatment. These findings suggest that CNTs may have a prospective role in the development of new antitumor immunotherapies.

Super-paramagnetic responsive nanofibrous scaffolds under static magnetic field enhance osteogenesis for bone repair in vivo

A novel nanofibrous composite scaffold composed of super-paramagnetic γ-Fe2O3 nanoparticles (MNP), hydroxyapatite nanoparticles (nHA) and poly lactide acid (PLA) was prepared using electrospinning technique. The scaffold well responds extern static magnetic field with typical saturation magnetization value of 0.049 emu/g as well as possesses nanofibrous architecture. The scaffolds were implanted in white rabbit model of lumbar transverse defects. Permanent magnets are fixed in the rabbit cages to provide static magnetic field for the rabbits post surgery. Results show that MNP incorporated in the nanofibers endows the scaffolds super-paramagnetic responsive under the applied static magnetic field, which accelerates new bone tissue formation and remodeling in the rabbit defect. The scaffold also exhibits good compatibility of CK, Cr, ALT and ALP within normal limits in the serum within 110 days post implantation. In conclusion, the super-paramagnetic responding scaffold with applying of external magnetic field provides a novel strategy for scaffold-guided bone repair.

Enhancement of nanofibrous scaffold of multiwalled carbon nanotubes/polyurethane composite to the fibroblasts growth and biosynthesis.

In this work, the effect of nanofibrous structure and multiwalled carbon nanotubes (MWNTs) incorporation in the polyurethane (PU) on the fibroblasts growth behavior was studied. The nanofibrous scaffold of multiwalled carbon nanotubes and polyurethane composite (MWNT/PU) with an average fiber diameter of 300-500 nm was fabricated by electrospinning technique. The nanofibrous scaffold of PU, smooth film of PU, and MWNT/PU were also prepared as controls. Cell viability assay, laser confocal microscopy, and scanning electron microscopy were applied to evaluate cell adhesion, proliferation, and cytoskeletal development on the scaffolds, respectively. Cell-released protein was analyzed by Bradford protein assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometry, and transwell assay, respectively. Experimental results demonstrated that the scaffold with nanofibrous structure and MWNTs incorporation exhibited highest enhancement not only to the cell adhesion and proliferation but also to the cell migration and aggregation. Besides, cells cultured on the nanofibrous scaffold of MWNT/PU released the largest amount of proteins including collagen in comparison with those on the other substrates. Hence, the nanofibrous architecture and MWNTs incorporation provided favorite interactions to the cells, which implied the application potentials of the nanofibrous composite for tissue repair and regeneration.

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.

Effects of long and short carboxylated or aminated multiwalled carbon nanotubes on blood coagulation.

In this work the effects of four different multiwalled carbon nanotubes (MWCNTs), including long carboxylated (L-COOH), short carboxylated (S-COOH), long aminated (L-NH2) and short aminated (S-NH2) ones, on the integrity of red blood cells, coagulation kinetics and activation of platelets were investigated with human whole blood. We found that the four MWCNTs induced different degrees of red blood cell damage as well as a mild level of platelet activation (10–25%). L-COOH and L-NH2 induced a higher level of platelet activation than S-COOH and S-NH2 respectively; meanwhile L-NH2 caused marked reductions in platelet viability. The presence of the four MWCNTs led to earlier fibrin formation, L-NH2 increased the clots hardness significantly, while L-COOH and S-NH2 made the clots become softer. It was concluded that the four MWCNTs affected blood coagulation process and the clots mechanical properties; they also altered the integrity of the red blood cells and the viability of the platelets, as well as induced platelets activation. The effects of MWCNTs depended on the size and chemistry of the nanotubes and the type of cells they contacted.

Electrospun aligned nanofibrous composite of MWCNT/polyurethane to enhance vascular endothelium cells proliferation and function

Aligned or random nanofibrous meshes of multiwalled carbon nanotubes/polyurethane composite (MWCNT/PU) were fabricated by electrospinning and characterized by scanning electron microscopy (SEM). The regulatory effects of nanofibrous structure and MWCNT on the growth and anticoagulant function of human umbilical vein endothelial cells (HUVECs) were investigated by examining proliferation, type IV collagen secretion, tissue factor and plasminogen activator inhibitor-1 (PAI-1) release, and cytoskeleton arrangement, as well as via pull-down analysis. We show that aligned nanofibrous structure and MWCNT can function as extracellular signals to stimulate cell growth, proliferation, and extracellular collagen secretion, in addition to preserving anticoagulant function. The nanofibrous structures played important roles in the activation of Rac and Cdc42, while CNT regulated the activation of Rho. These two features synergistically activated Rho GTPases that transmitted cell-substrate signals to the cytoplasm. These signals were then relayed to the nucleus by the MAP kinase pathway to direct cytoskeletal arrangement and cell orientation.

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.