| Cancer is currently one of the major diseases affecting human health, clinically, the traditional chemotherapy is the main way for cancer treatment, however, due to the low bioavailability and multi-resistant of the single chemotherapy drugs, combined drug therapy is used in the clinical treatment. Paclitaxel (PTX) is a kind of antitumor drug with a taxane structure, and it can promote the formation of microtubules and inhibit the depolymerization of microtubules. Thereby, PTX hinders the division of normal cells and inhibit the proliferation of tumor cells. In the clinical treatment, paclitaxel is used to treat breast cancer, prostate cancer, ovarian cancer and non-small cell lung cancer, etc. Doxorubicin (DOX) is an anthracycline-based antitumor drug. It can embed into the structure of the DNA molecule, and increase the distance between the base pairs in the DNA molecule, thus the rupture and damage are found in the structure of the DNA. Clinically, doxorubicin is applicable to non-hodgkin’s lymphoma. breast cancer, lung cancer, acute leukemia, etc. PTX combined DOX can produce synergistic effect and improve the effect of treatment. The different mechanisms of PTX and DOX, to a certain extent, prevent the multi-resistant, and now PTX combined DOX are as the first method used in the treatment of advanced breast cancer. However. due to the greatly different characters and structures of DOX and PTX, it is the biggest difficulty and challenge to realize the load of the two different drugs in a carrier and control the synchronous release of the drugs.In the project, we had synthesized the CAD which was sensitive to an acid environment. Then F127-CS/CAD/PTX polyion complex micelles were prepared, in which the F127-CS copolymer was as the carrier, to realize the co-delivery of DOX and PTX. Later, the in vivo and in vitro evaluation were investigated and the main works were detailed as follows:1. Synthesis and structure identification of CAD, and the amount of amino group replaced in the F127-CS were measuredCAD had been designed and synthesized according to the literature, and the structure was then identified. The figure of high performance liquid chromatography (HPLC) displayed that two new peaks appeared at the retention time of 6.15 min and 7.18 min, which respectively belonged to the synthetic product of CAD-1 and CAD-2; In the infrared spectrogram of CAD, the stretching vibration of amine hydrogen atoms at 3334.54 cm-1 3532.16 cm-1 disappeared, instead, a new unimodal peak appeared at 3416.36 cm-1 and an amide bond carbonyl absorption peak appeared at 1647.06 cm-1. In the NMR spectrum of CAD, typical peaks of the hydrogen atom in -COCH= (6.05ppm and 6.24ppm) appeared. All the results indicated the successful synthesis of CAD.2. Preparation and physicochemical characteristics of F127-CS/CAD/PTX micellesThis subject prepared F127-CS/CAD/PTX micelles by the membrane dispersion, and drug loading capacity was used as indicators to select the optimal prescription of F127-CS/CAD/PTX micelles from the ratio of the carrier material and drugs, the ratio of the two drugs, ultrasonic time and ultrasonic temperature. The cytotoxicity experiment showed that when the rate of DOX and PTX was 0.6-1, the synergic effect of the two drugs was the best. Spherical micelles were observed by TEM. The average particle diameter of the micelles was 179.3nm and the average potential was at-0.27mV. What’s more, the critical micelle concentration was 0.039mg/mL by the pyrene fluorescence probe method.3. In vitro release of F127-CS/CAD/PTX micellesIn vitro drug release behavior of micelles was investigated by the dialysis bag method, and pH5.0 and pH7.4 citric acid buffer containing 0.5%(w/v) tween-80 were used as the release medium. Results showed that the release of DOX and PTX were dependent on the pH. The lower the pH was, the faster the drug released. What’s more, the release rate of DOX was significantly faster than that of PTX.4. Anticancer activity of micellesAnticancer activity of F127-CS/CAD/PTX micelles and PTX+DOX mixture in B16 cells was examined using the MTT assay. Besides, cell uptake of the F127-CS/CAD micelle and intake of DOX solution were investigated by the fluorescence microscope and flow cytometry. Results showed the IC50 value of F127-CS/CAD/PTX micelles was much less than that of the DOX+PTX solution, which indicated that micelles performed a stronger anti-tumor activity compared to the solution. The experiment of cell uptake showed that compared to DOX solution, F127-CS/CAD micelles were easier to be took in by tumor cells.5. Pharmacokinetics studies of F127-CS/CAD/PTX micelles in ratsPharmacokinetics characteristics of F127-CS/CAD/PTX micelles and PTX+ DOX solution were studied in rats by the administration of femoral vein and carotid sinus for blood. Results showed that the AUC(0-∞) values of PTX and DOX after administration of the mixed solution were 2.628 mg/L*h and 0.512 mg/L*h, while AUC(0-∞) values of PTX and DOX in micelles were 5.450 mg/L*h and 1.081 mg/L*h, which were significantly higher than the mixed solution group. The half-life time (t1/2) of PTX in micelles was 8.29h, which increased 4.5 times than that of PTX in the mixed solution, similarly, the t1/2 of DOX in micelles was also higher than that of DOX in the mixed solution. PTX and DOX loaded in micelles could reduce their clearance and extend the time in the body.6. In vivo pharmacodynamics studies of F127-CS/CAD/PTX micellesA tumor-burdened mice model was established using B16 celles. Then changes of the tumor volume and weight in mice were used as indicators to examine the tumor inhibition of F127-CS/CAD/PTX micelles and PTX+DOX solution. Results showed that both the micelles and mixture solution had a strong inhibitory effect on tumors in mice. However, the weight of the mice after administration of the mixture solution changed obviously. The 14th day after administration, the weight of the mixture group was 78.44% to that of the saline group, which indicated the toxicity of PTX + DOX solution. While, there was no obvious changes in body weight for micelles, which showed the toxicity of F127-CS/CAD/PTX micelles was much lower than the solution. |
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