Application Of MWCNT/DOX/TC Drug Delivery System Combined With Hyperthermia In The Treatment Of Liver Cancer

Carbon nanotubes(CNTs)is a common drug carrier in recent years,due to its unique size and structure,which has a good application prospect in biology.CNTs not only can pass through a variety of cell membrane passively,be taken by tumor cell,but also can absorb near-infrared(Near Infrared,808nm NIR)to induce tumor cell death selectively,without harming normal cells,so as to achieve the synergistic effect of chemotherapy and hyperthermia.However,since the carbon nanotube itself has disadvantages such as poor dispersibility and forming clusters easily,which has limited its application in the biomedical field,therefore,selecting suitable materials to improve its dispersion has important significance.Chitosan(Chitosan,CS)is a natural cationic polysaccharide with good water solubility and biocompatibility and biodegradability.It can be combined with carbon nanotubes by non-covalent adsorption to improve the dispersion of carbon nanotubes.TAT peptide(TAT peptides,TATp)is a kind of cell penetrating peptide,which is mainly composed of basic amino acid arginine and lysine,with lots of positive charge,has good water solubility,can penetrate the cell membrane into the cytoplasm or nucleus.This paper adopts the TAT to covalently modify low molecular weight chitosan(MW=5000?8000,deacetylation degree>90%)by the method covalent cross-linking to form TAT-CS(hereinafter referred to as TC),and using TC to modify carboxylated multi-walled carbon nanotubes(MWCNT-COOH),in order to enhance the dispersion and cell membrane permeability,combine carbon nanotubes and chemotherapeutic drugs DOX by non-covalent adsorption,and modified by TC,prepare MWCNT/DOX/TC drug delivery system,using carbon nanotubes itself has the characteristics of selective absorption of near infrared laser,combine chemotherapy with hyperthermia,to explore its therapeutic effect on hepatocellular carcinoma.The main contents are as follows:1.Preparation and characterization of MWCNT/DOX/TC complexwe mix MWCNT with DOX using different ratio,and combine TC to the surface of MWCNT/DOX complex through non-covalent adsorption,calculate entrapment efficiency and drug loading capacity of different feed ratio by dialysis method;and investigate release behavior in different conditions in vitro,the results show that when the feed ratio is DOX/MWCNT=3/1,the loading capacity reaches maximum;and the in vitro release behavior has pH response.The measurement results of Zeta potential value show that,DOX or TC's modification can increase the Zeta potential value of MWCNT-COOH;transmission electron microscope was used to observe the MWCNTs,MWCNTs/DOX and MWCNTs/DOX/TC,the results showed that the surface morphology of the three are basically the same;Raman spectroscopy analysis showed that the three I(D)/I(G)(1315/1599 cm-1)there has no obvious difference,the aboved results indicate that the load of DOX or TC had no obvious effect on the structure of MWCNTs.The results of UV-scanning and FTIR analysis show that DOX can be effectively adsorbed on carbon nanotubes.2.In vivo image tracking of MWCNT/DOX/TC drug delivery systemBy using laser confocal microscopy,we observed the distribution of drug carrier system in liver cancer cells BEL-7402,and the results showed that MWCNTs/DOX/TC can be effectively absorbed by liver cancer cells,and can be released in the cytoplasm,DOX enters into the nucleus to play its biological functions.In order to observe the cytotoxicity of drug delivery system,the hepatoma cells BEL-7402 and DOX,MWCNTs/DOX,MWCNTs/TC or MWCNTs/DOX/TC were co-cultured and the cell viability was observed,results show that the MWCNTs/TC itself only slightly inhibited the viability of hepatoma cells;on the MWCNTs/DOX/TC,MWCNTs/DOX or DOX group,the inhibition on cell viability has certain enhancement effect.The nude mouse model of liver cancer was established by subcutaneous injection of luciferase BEL-7402,and the inhibitory effect of different doses of MWCNT/DOX/TC on tumor was observed by In vivo image tracking.The results show that the drug delivery system can effectively enhance the residence time of DOX in the tumor and play a slow release effect.3.Effect of MWCNT/DOX/TC drug delivery system in combination of chemotherapy and hyperthermiaIn order to observe whether NIR irradiation will stimulate the release of DOX from the drug carrier,we studied the release of DOX in different pH and with or without NIR,the results show that the presence of NIR has no obvious influence on the release of DOX;in vitro experiments,CCK-8 cytotoxicity testing results show that the presence of MWCNT/TC under the condition of NIR irradiation group the cell viability was significantly lower than that of no NIR irradiation group,indicating the presence of drug carrier MWCNT/TC under NIR irradiation can inhibit cell growth;In order to observe the effect of chemotherapy combined with hyperthermia on tumor growth inhibition,we observed the presence of DOX in tumors by In vivo animal imaging based on the characteristics of the red fluorescence of DOX.The results showed that MWCNT/DOX/TC could significantly prolong the residence time of DOX in tumor tissues,and NIR had no significant effect on the release of DOX in drug delivery system.We observed the anti-tumor effect of the nano drug delivery system by using bioluminescence imaging in vivo.The observation time was 25 days.The results showed that the antitumor effect of MWCNT/DOX/TC combined with NIR irradiation was the most significant in all groups.Based on the above results,we can conclude that MWCNT-COOH can effectively adsorb the chemotherapeutic drug DOX,and the TAT-CS can significantly improve the dispersion and cell membrane permeability,and reduce the toxicity of carbon nanotubes.In vitro experiments we confirm this drug carrier has no obvious side effects on cells,and this vector can effectively make doxorubicin release controlled and sustained in cells;in vivo experiments also showed that this drug carrier can effectively prolong the residence time of drug in the body;at the same time also proved that NIR irradiation can significantly improve the inhibition effect,these findings suggest that the study of MWCNT/DOX/TC drug delivery system has important significance in the combination treatment of tumor.