| Objective:1.Study the feasibility and effectivity of magnetic fluid methotrexate in carcinoma magnetic induction thermochemotherapy, and discuss the coordinated sensitization of thermotherapy and chemotherapy, providing possibility for magnetic induction of thermochemotherapy in tumour treatment.2.Prepare the magnetic nano-drug with methotrexate for cancer therapy, and discuss the application of the magnetic nano ranged medicine for drug delivery, slow release, chemotherapy and thermotherapy.Methods:1.Preparation of magnetic fluid and surface modification:optimal chemical coprecipitation was used for magnetic fluid preparation, which was then modified by surfactant 3-aminopropyltriethoxysilane (APTES). The product was analysed by TEM, FTIR, TGA, XRD, magnetization curve and thermo function. Cells were sterilised by magnetic fluid Cobalt 60. The structure and component of the cells were then tested through the physical characterisations.2.In vitro cell cytotoxicity test:human breast cancer cell MCF-7 was used for CCK8 cell vitality test. Cell proliferation was recorded for magnetic field induction, and the optimal concentration of methotrexate for thermochemotherapy was determined by cell cytotoxicity. Lethal effects of methotrexate associating with water bath thermotherapy and magnetic induction were discussed for synergistic application.3.In vivo anti-tumour activity:Model of breast cancer tumour was established by subcutaneous injection of MCF-7 in nude mouse with inoculum size of 107 cells each. The bearing cancer nude mice were randomly divided into four groups when the tumour sizes reached 500 mm3. Four groups were treated with thermotherapy, chemotherapy, thermochemotherapy and without treatment specifically, and monitored for the growth of both nude mice and the bearing tumour. Mouse was proved to death when the tumour size was more than 3000 mm3. Dead mouse were executed by over anesthetize, and the main organs were dissected and slicing. HE staining was used for pathological analyse and prussian blue for analysing the distribution of magnetic fluid.4.Magnetic nano-drug construction:amino silane was added in the surface of magnetic fluid, which reacts with carboxyl group of methotrexate under EDC and NHS activation and forms amide linkage. Nano particles were analysed by TEM, XRD, FTIR, thermo characterisation and in vitro release.Results:1.Physical characterisations of amino silane modified magnetic nano particles presents in subsphaeroidal shape with lOnm in diameter. The crystal structure is consistence with Fe3O4 form, with superparamagnetism and great capability for rising temperature. Co60 radiation sterilisation breaks the modified magnetic fluid which indicates a method for cell applied sterilisation.2.In vitro cell experiments presents the exposure of MCF-7 cell in magnetic field will not affect the vitality of the cells.3.In vitro thermochemotherapy results in synergism and sensitisation in thermotherapy with methotrexate, and more damage for breast cancer cells with association dosage.4.In vivo thermochemotherapy shows that comparing with the control group, the growth of bearing tumour in nude mouse is inhibited obviously, with slow tumour grow speed and longer mouse life time. When comparison with the chemotherapy group, the thermochemotherapy enhances the anti-tumour activity with longer live time results but no significant difference has been seen between thermochemotherapy group and thermotherapy group.5.FTIR results show the MTX could coupled with the amino silane modified magnetic fluid surface, and the in vitro temperature test presents good capability for raising temperature. The particles also indicates slow release effect for chemotherapeutics, and is favourable in mimical lysosomal environment.Conclusion:1.The in vitro experiment indicates that the methotrexate combined thermotherapy contains certain anti-tumour synergistic reaction. Although the in vivo experiment does not support the synergism for thermochemotherapy, evidence shows that the thermochemotherapy results in obvious inhibition in tumour cell growth and the extension of nude mouse live time, which indicates the feasibility and effectivity of magnetic fluid combined methotrexate for breast cancer thermochemotherapy. 2.The magnetic nano drug with methotrexate has been made successfully, with favourable capacity of raising temperature in alternative magnetic fields, which approach the possibility for magnetic nano drug use in tumour multi-modelling treatment. |
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