| Single-walled carbon nanotube (SWNTs) is one-dimensional tube-shaped carbon nanomaterials, which is crimped by a single layer of graphite. SWNTs have a series of excellent optical, electrical, physical and chemical properties, their potential applications in biomedical nano drug carrier, near-infrared fluorescence imaging, diagnosis and photothermal cancer therapy. Related studies are of currently one of the important research hotspots in the world. In recent years, people in the field of biomedical applications of SWNTs have carried out a lot of research works and have achieved remarkable results.With SWNTs extensive research and applications in various fields and its potential biological toxicity were more and more get attention from the people, the researchers carried out a large number of experiments on animals, cell and molecular level upon biotoxicity. As SWNTs are ultrafine particles, can spread easily into the respiratory tract by atmosphere, therefore the lungs toxicity caused by the trachea drip SWNTs and its mechanism of the research is one of the most popular and much reported in literatures. However, whether as a drug carrier or applied to near-infrared fluorescence imaging for diagnosis in the body, SWNTs dosing method is priority with intravenous injection. A large number of studies have shown that after entering the blood circulation, SWNTs mainly accumulate in the liver, kidney and lung. Therefore, the study of intravenous SWNTs to the liver, kidney and lung fibrosis and other toxic effects, not only provides a basis for toxicity in biomedical applications of SWNTs, but also helps to modify SWNTs to improve the biological compatibility.Objective:1. To evaluate the induced effect on fibrosis of liver, lungs and kidney by intravenous injection of carboxyl single-walled carbon nanotubes (SWNT-COOH) in SD rat tails.2. Fluorescent quantitative PCR method was used to study the liver fibrosis in rats induced by SWNT-COOH and related mRNA gene expression.3. immunohistochemical assay was used to explore the liver fibrosis in rats1 induced by SWNT-COOH and related protein expression.4.To evaluate the effect of SWNT-COOH induced fibrosis on lung and kidney by intravenous injection of carboxyl single-walled carbon nanotubes in SD rat tails and to explore the related proteins expression.Methods:1. The preparation of SWNT-COOH and characterization before the animal experiment:followed by heat oxidation of the mixed acid (nitric acid and sulfuric acid), ultrasonic oscillatory shear of SWNTs, preparation of SWNT-COOH good dispersion in water, removing impurity by dialysis, ultrafiltration concentration and Co60 gamma-ray irradiation disinfection. With transmission electron microscope (TEM) and laser nanometer particle size analyzer (LPSA), the SWNT-COOH morphology and particle size were observed and determined respectively, the inductive coupled plasma atomic emission spectrometer (icp-oes) was used to detect the metal content in SWNT-COOH dispersion, the ultraviolet-visible spectrophotometer (UV-Vis) was used to determine the concentration of SWNT-COOH.2. Animal grouping and cycle setting:96 SD rats were randomly divided into two groups as normal control group and SWNT-COOH treated group, with half male and female in each group. They will set up a total of 4 cycles as 1 month,2 month,3 month and 4 month, among them rats in 4 month cycles received drugs for 3 months and the last month was withdrawal. All the rats were given by intravenous injection of tails.Daily observed and recorded the rat survival condition and weighted them once a week. At the end of each cycle, total of 24 rats from each group were selected randomly with half male and female and given anesthesia for anatomy and collected their specimens.3. SD rats with fibrosis induced by SWNT-COOH:we used hematoxylin-eosin staining (HE staining) method to evaluate the pathological changes in rats liver, lung and kidney induced by SWNT-COOH; by TEM method, we observed the effect of SWNT-COOH in rat liver cells ultrastructure.4. Real-time quantitative PCR (qPCR) was used to analyze the relative mRNA protein expression of rat liver tissues in normal control group and SWNT-COOH group including smooth muscle excited protein alpha 2 (ACTA-2), transforming growth factor beta (TGF-beta), I type collagen (Col Ⅰ) and type Ⅲ collagen (Col Ⅲ), etc.5. Immunohistochemical method was used to detect and analyze the relative protein expression of rats liver, lungs and kidney in control group and SWNT-COOH treated group including smooth muscle excited protein alpha 2 (ACTA-2), transforming growth factor beta (TGF-β), I type collagen (Col Ⅰ) and type Ⅲ collagen (Col Ⅲ), matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase inhibitory factor 2 (TIMP-2).Results:1. SWNT-COOH showed good dispersion in water after oxidation, shear and purification, TEM observation and LPSA test showed that the SWNT-COOH particle size distribution was uniform, reunion and narrow; Z particle size of SWNT-COOH was significantly shorter,94.7 nm. ICP-OES test results showed that the sample of SWNT-COOH in animal experiments could lower the metal contents of Fe, Co, AL, Cr, Cu, etc.2. Compare with the normal control group, SD rats in the SWNT-COOH treated group had 2 deaths in 2 month cycle and only 1 death in 4 month cycle and regarded as totally 3 deaths in treatment group. Obvious abdominal swelling and abdominal effusion were found in death rats. With the extension of drug delivery cycle, the black dye in rat liver and lungs were deeper and a large number of SWNT-COOH in liver and lung was deposited, the black dye became faint after 1 month withdrawal of drug.3. Rat liver specimens stained with HE were observed under optical microscope. SWNT-COOH treated group showed perivascular inflammatory cell infiltration accompanied by fibrosis, and exacerbated with the extension of drug delivery cycle, cycle dependent. From TEM result, the nucleolus in normal control group was obvious with abundant organelles and sharp edge; in treatment group, mitochondria were damaged, a large number of cytoplasmic vacuoles were appeared and SWNT-COOH were dissolved in lysosome with bundle of collagen fiber in intercellular space.4. Real-time qPCR results showed that compared with the normal control group, SWNT-COOH treated groups of ACTA-2, TGF-p, Col I and Col III mRNA expression levels increased significantly.5. Immunohistochemical staining results show that compared with the normal control group, the relative protein expression of ACTA-2, TGF-β, Col I, Col Ⅲ, MMP-2 and TIMP-2 in fibrosis of rat liver, lungs and kidney tissue were raised in SWNT-COOH treated groups. Conclusion:1.The SWNT-COOH can damage the organs such as liver, lungs and kidney of rats, and biological toxicity effect of inducing fibrosis.2.1ts possible mechanism is that large amount of SWNT-COOH is deposit in the rat liver, lungs and kidneys and lead to swallow of tissue cells, damage mitochondria, lysosome and cause chronic inflammation, and the increased expression of fibrosis related gene and protein also result in fibrosis. |
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