紫杉醇阿霉素共载药胶束的制备及体内外评价

发布时间：2018-01-09 07:30

本文关键词：紫杉醇阿霉素共载药胶束的制备及体内外评价　出处：《山东大学》2016年硕士论文　论文类型：学位论文

【摘要】：癌症是目前影响人类健康的主要疾病之一。传统化疗药物是临床肿瘤治疗的主体,但是,单一化疗药物由于生物利用度低、易产生多药耐药,临床上多采用联合用药的方式。紫杉醇(Paclitaxel, PTX)是一类具有紫杉烷结构的抗肿瘤药物,它能够促进微管的形成并抑制微管的解聚,阻碍细胞的正常分裂,进而抑制肿瘤细胞的增殖。临床上常用来治疗乳腺癌、前列腺癌、卵巢癌、非小细胞肺癌等。阿霉素(Doxorubicin, DOX)是一种蒽环类的抗肿瘤药,它能够嵌入到DNA分子的结构中,使碱基对之间的距离增大,进而引起DNA链的断裂和结构的破坏。临床上适用于淋巴癌、乳腺癌、肺癌、急性白血病等。紫杉醇联合阿霉素使用能够产生协同效应,提高治疗效果。而且,由于这两种化疗药物的作用机制不同,可在一定程度上预防多药耐药的产生,现已作为一线方案用于治疗晚期乳腺癌。但是,由于阿霉素和紫杉醇两种药物的性质有极大的不同,如何实现同时负载这两种不同的药物以及控制不同药物同步释放是研究者们目前面临的最大困难和挑战。本课题合成了具有pH敏感性的乌头酸酐-阿霉素(CAD)分子,然后以普郎尼克F127-壳聚糖(F127-CS)为载体,构建了F127-CS/CAD/PTX胶束,实现DOX与PTX的共递送。并对该载药胶束进行体外体内的相关评价,具体内容如下：1.CAD的合成、表征及F127-CS聚合物取代度的测定：根据文献设计合成了CAD,并对其结构进行验证。高效液相色谱图显示在保留时间6.15min和7.18min新出现了两个色谱峰,其分别为合成产物CAD-1和CAD-2的色谱峰；在CAD红外光谱图上,3532.16cm-1和3334.54cm-1波长处伯氨基上氢原子的伸缩振动消失,而在3416.36cm-1波长处出现较强的单峰吸收,同时,在1647.06cm-1处出现酰胺键的羰基吸收峰；核磁共振图谱显示在6.05ppm和6.24ppm处出现-COCH=基团上氢原子的特征位移。所有结果均显示成功合成了CAD分子。采用三硝基苯磺酸法建立了游离氨基测定的方法,测得F127-CS聚合物氨基的取代度为：每1moL壳聚糖中平均有0.832±0.119个氨基被取代。2.共载药胶束的制备及理化性质的考察：本课题采用薄膜分散法制备了F127-CS/CAD/PTX共载药胶束,并分别从载体材料与药物质量比、两种药物的质量比、超声时间以及超声温度四个方面对影响胶束载药量的因素进行考察,确定制备载药胶束的最佳工艺。同时通过细胞毒性实验筛选最佳药物配比,当阿霉素与紫杉醇的比例范围为0.6-1时,两种药物协同效果最佳。通过透射电镜观察到胶束呈圆整球形,无粘连。胶束的粒径为179.3nm,粒径分布均匀, Zeta电位为2.37mV。芘荧光探针法测定胶束的临界胶束浓度为0.039mg/mL。3.载药胶束的体外释放实验：采用透析袋法,以含0.5%的吐温-80的pH5.0和pH7.4的柠檬酸缓冲液为释放介质考察载药胶束在体外的释放行为。结果显示阿霉素和紫杉醇的释放均呈现依赖pH的性质,pH越低药物释放的就越快,而且阿霉素的释放速率要明显快于紫杉醇。4.载药胶束的体外细胞实验：以黑色素瘤B16细胞为模型,采用MTT法,考察F127-CS/CAD/PTX共载药胶束和PTX+DOX混合溶液对肿瘤细胞的毒性作用。随后采用荧光显微镜及流式细胞术考察肿瘤细胞对F127-CS/CAD胶束和DOX溶液的摄取情况。结果显示F127-CS/CAD/PTX胶束的1C50值小十DOX+PTX溶液,载药胶束对肿瘤细胞的毒性高于混合溶液。细胞摄取实验显示与DOX溶液相比,F127-CS/CAD胶束更容易被细胞摄取。5.载药胶束的体内药动力学实验：本实验以Wistar大鼠为实验动物,通过股静脉给药,于大鼠颈静脉窦取血,考察F127-CS/CAD/PTX共载药胶束和PTX+DOX混合溶液大鼠体内的药动力学特征。结果显示混合溶液组中PTX和DOX的AUC(o-∞)值分别为2.628 mg/L*h和0.512 mg/L*h,而胶束组中PTX和DOX的AUC(o-∞)值分别为5.450 mg/L*h和1.081 mg/L*h,明显高于混合溶液组。载药胶束组中PTX的体内半衰期(t1/2)为8.29h,比混合溶液中PTX的t1/2增加了4.5倍,载药胶束中的DOX的t1/2比混合溶液中的也有所增加。将PTX和DOX包载于胶束中能降低其在体内的清除率延长在体内的时间。6.载药胶束的体内药效学实验：本实验以B16细胞为模型建立荷瘤小鼠模型。通过尾静脉注射给药,以小鼠瘤体积变化及体重变化为指标,考察F127-CS/CAD/PTX共载药胶束和PTX+DOX混合溶液对肿瘤的抑制作用。实验结果显示载药胶束组和混合溶液组对小鼠肿瘤均具有较强的抑制作用,但是混合溶液组小鼠体重变化明显,第14日,混合溶液组小鼠体重是生理盐水组78.44%,毒性较大,而载药胶束组小鼠体重无明显变化,毒性较小。
[Abstract]:Cancer is one of the major diseases that affect human health. At present, traditional chemotherapy drugs is the main clinical treatment of tumors but single chemotherapy because of low bioavailability, easy to produce multi drug resistance, clinical use of combination therapy. Paclitaxel (Paclitaxel, PTX) is a kind of taxane anticancer structure the drug, which can promote microtubule formation and inhibit the depolymerization of microtubules, hinder the normal division of cells, and then inhibit the proliferation of tumor cells. Clinically to treat breast cancer, prostate cancer, ovarian cancer, non-small cell lung cancer. Adriamycin (Doxorubicin, DOX) is an anthracycline anticancer drug and it can be embedded into the molecular structure of DNA, the distance between the base pairs increases, which lead to fracture and the structure of the DNA chain destruction. Clinically applicable for lymphatic cancer, breast cancer, lung cancer, acute leukemia. Combined with paclitaxel The use of doxorubicin can produce synergistic effect, improve the therapeutic effect. Moreover, because the mechanism of these two kinds of chemotherapeutic drugs, to a certain extent, prevent the generation of multi drug resistance, has been used as a first-line chemotherapy for the treatment of advanced breast cancer. However, due to the nature of doxorubicin and paclitaxel two drugs are greatly different. How to realize the load of the two different drugs and different drug release synchronization is the biggest difficulty and challenge researchers are currently facing control. With the pH sensitivity of adriamycin - aconitic anhydride synthesis in this paper (CAD) molecules, then chitosan with Pluronic F127- shell (F127-CS) as the carrier, to construct the F127-CS/CAD/PTX micelles, CO delivery the realization of DOX and PTX. The correlation was evaluated in vitro and in vivo of the micelle, the specific contents are as follows: 1.CAD synthesis, characterization and determination of polymer from generation F127-CS: According to the synthesis of CAD document design, and validate its structure. HPLC showed that the retention time of 6.15min and 7.18min appeared two peaks, the peaks were synthesized CAD-1 and CAD-2; CAD in the infrared spectrum, 3532.16cm-1 and 3334.54cm-1 wavelength on primary amino hydrogen atom the stretching vibration disappeared, and at the 3416.36cm-1 wavelength of strong absorption peak appeared at 1647.06cm-1, at the same time, carbonyl amide absorption peak; NMR spectra showed the characteristics of hydrogen atom displacement on -COCH= group in 6.05ppm and 6.24ppm. All the results showed that CAD was successfully synthesized. The molecular method was established for determination of free amino groups using three trinitrobenzene sulfonic acid method, measured F127-CS polymer amino substitution: each chitosan in 1moL with an average of 0.832 + 0.119 amino substituted.2. micelle preparation Investigation and physicochemical properties: This study prepared by film dispersion method of F127-CS/CAD/PTX co micelles, and separately from the carrier material and drug quality ratio, quality of two kinds of drug ratio, ultrasonic time and ultrasonic temperature factors in four aspects of the effect of micellar drug loading for an investigation, to determine the optimum process for preparing drug at the same time. By micellar cytotoxicity screening the best drug ratio, when doxorubicin and paclitaxel ratio in the range of 0.6-1, two kinds of synergistic effect is the best. It was observed that the micelles showed spherical micelles, no adhesion. The diameter of 179.3nm, uniform particle size distribution, Zeta potential for critical micelle concentration determination of micellar 2.37mV. fluorescent probe method for drug loaded 0.039mg/mL.3. micelles in vitro release experiments using dialysis bag method, using citric acid buffer containing 0.5% -80 pH5.0 and Twain pH7.4 as the release medium Quality investigation of micelle dissolution behavior in vitro. The results showed that doxorubicin and paclitaxel release showed properties of pH dependent pH, lower drug release is faster, and the in vitro release rate of adriamycin is significantly faster than paclitaxel.4. micelles in melanoma B16 cells as a model and using the method of MTT toxicity on tumor cells of F127-CS/CAD/PTX micelles and PTX+DOX mixed solution. Then by fluorescence microscope and flow cytometry study of F127-CS/CAD micelles and DOX solution uptake of tumor cells. The results showed that F127-CS/CAD/PTX micelles 1C50 value ten DOX+PTX solution, the toxicity of the drug loaded micelles on tumor cells higher than that of the mixed solution. The cellular uptake test showed that compared with DOX solution, F127-CS/CAD micelles are more likely to be in vivo pharmacokinetic experiments of drug loaded cell uptake of.5. micelles: in this experiment, Wi Star rats as the experimental animal, administered through the femoral vein, venous sinus in the neck of blood in rats, to investigate the characteristics of pharmacokinetics in rats F127-CS/CAD/PTX drug loaded micelles and PTX+DOX mixed solution. The results show that mixed PTX and DOX solution in the AUC group (o- -) respectively 2.628 mg/L*h and 0.512 mg/L*h. While PTX and DOX micelles in the AUC group (o- -) values were 5.450 mg/L*h and 1.081 mg/L*h, was significantly higher than that of the mixed solution group. The drug loaded micelles in group PTX (t1/2) in vivo half-life of 8.29h, than in the mixed solution of PTX t1/2 increased by 4.5 times, the drug loaded micelles in DOX than t1/2 in the mixed solution is also increased. PTX and DOX will be loaded in the micelle can reduce the rate of extension in vivo in vivo.6. micelles the in vivo clearance: the experimental model of tumor bearing mice model with B16 cell. Administered by intravenous injection in mice The change of tumor volume and weight changes as the index, inhibition of PTX+DOX micelles and mixed solution of F127-CS/CAD/PTX drug on tumor were investigated. The experimental results show that the micelle solution group and mixed group has a strong inhibitory effect on tumor in mice, but the mixed solution of mice body weight change was fourteenth, the mixed solution of group body weight of mice is the saline group 78.44%, toxicity, and drug loaded micelles were without significant changes in body weight, less toxic.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R943

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