Construction And Combination With Photothermal Therapy Of Water-soluble Single-walled Carbon Nanotubes As Tumor Targeting Drug Delivery System

Single-walled carbon nanotubes (SWNTs) are special nano-materials which exhibit unique physical, chemical and biology properties. Since SWNTs were discovered, it caused widespread concern. In recent years, applications of SWNTs has spanned many fields and efforts have also been devoted to exploring the potential biological applications of SWNTs. However, the surface of SWNTs is highly hydrophobic and insoluble in aqueous solutions and common organic reagents, poor biocompatibility severely hindered the development of SWNTs in biomedical applications. Therefore, improving the biocompatibility of SWNTs is the most important thing of which to promote the biomedical applications of them.Due to the ability to cross cell membranes, SWNTs with choicely designed which are able to enter cells without toxicity as carriers of a variety of organic or inorganic particles, such as proteins, nucleic acids and chemical drugs. The traditional non-covalent method can make SWNTs dispersed in aqueous solutions, but this method is often accompanied by high toxicity of surfactants, easy to depolymerization and so on which limited the applications of SWNTs in biological system. In summary, to establish a tumor-targeting drug delivery system of SWNTs with covalent functionalized has a wide range of scientific significance and prospects.Biological systems are highly transparent to 700～1,100nm near-infrared (NIR) light. High optical absorbance of SWNTs in this biological window can be utilized for photothermal therapy. In addition, the combination of drug therapy and photothermal therapy can be more effective for tumor treatment.This research mainly studies convalent functionalition of SWNTs and synergistic enhancement of anti-tumor activity in vivo and in vitro using a combination of siRNA and photothermal therapy, main content as following:1. Construction and characterization of SWNT-PEI. The SWNT-PEI was produced by PEI polymerization after SWNTs were purificated, carboxylated and amino-modified. The resulting product was systematically characterized by fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), transmission electron microscopy (TEM), atomic force microscopy (AFM), thermal gravimetric analysis (TGA) and particle size analysis(PSD) technique.It is demonstrated that PEI was grafted on the surface of functionalized SWNTs and there was no significant changes in the basic structure. The aqueous solution of SWNT-PEI was with good particle size distribution and remained stable in a few weeks even a few months.2. Preparation of SWNT-PEI/siRNA/NGR tumor-targeting drug delivery system. SWNT-PEI/siRNA/NGR tumor-targeting delivery system was obtained by firstly adding a certain amout of DSPE-PEG2000-Maleimide to attach SWNTs byπ-πattacking which used to connect peptides containing the NGR tumor targeted motif on SWNT-PEI, then binding negatively charged hTERT siRNA by electrostatic interactions. AGE and ultraviolet wavelength scanning were used to determine the encapsulation of siRNA; the connection of NGR motif and SWNTs was analysed by fluorescence spectrophotometer; the stability and biocompatibility of SWNT-PEI/siRNA/NGR were investigated. The results showed that SWNT-PEI/siRNA/NGR encapsulated siRNA completely, and the SWNTs delivery system successfully connected with NGR motif. SWNT-PEI/siRNA/NGR tumor-targeting drug delivery system exhibited good stability and biocompatibility. 3. Investigation of anti-tumor activity of SWNT-PEI/siRNA/NGR tumor-targeting drug delivery system in PC-3 human prostate cancer cells. The toxicity of SWNT-PEI and the cell viability of SWNT-PEI/siRNA/NGR was determined by SRB method; the cell uptake of the system was detected by fluorescence microscope; the transfection and apoptosis efficiency was analysed by FCM; and used RT-PCR and Western Blotting to determine hTERT mRNA and protein expression in cells. The results indicated that there was no obviously toxictiy to cells in the dose range of SWNT-PEI; the transfection of the system was about 2 times higher than PEI alone; delivery system can uptake by cells in 4 hours and siRNA transported by SWNTs can significantly inhibit hTERT mRNA and protein in cells; the system has been shown to promote apoptosis in PC-3 cells.4. Examination of the toxicity, biodistribution and antitumor effects of SWNT-PEI/siRNA/NGR in vivo. Using tumor-bearing nude mice as model, HE staining result showed that SWNT-PEI/siRNA/NGR delivery system in the major organs of nude mice without apparent toxicity in heart, liver, spleen, lung, kidney within the dose range. SWNT-PEI labeled by QDs was investigated on biodistribution in nude mice; the tumor growth curve was showed that SWNT-PEI/siRNA/NGR can significantly inhibit the tumor volume; the results of RT-PCR, Western Blotting and apoptosis experiments indicated the tumor-targeting delivery system can efficiently inhibit the expression of hTERT mRNA and protein and promote the apoptosis in tumor.5. Evaluation of the anti-tumor activity under 808nm NIR photothermal therapy in vitro and in vivo. The result of heating effect under 808nm NIR Laser irradiation ex vitro showed that the heating effect of SWNTs in aqueous solution is concentration dependent and time dependent; compared with the other groups SWNT-PEI/siRNA/NGR+Laser group is proved to have more effectives on suppresion tumor cells, significant photothermal therapeutical effect in votro and in vivo; the tumor growth curve was showed that SWNT-PEI/siRNA/NGR+Laser group can more significantly inhibit the tumor volume than SWNTs delivery system without laser.In summary, preparation of SWNT-PEI/siRNA/NGR tumor-targeting delivery system with the method above makes highly efficient anti-tumor activity, and the combination of RNAi and photothermal therapy is more effective for tumor treatment.