透明质酸多功能聚合物胶束的制备及抗肿瘤作用研究
发布时间：2019-02-15 09:45
【摘要】：本研究旨在构建CD44靶向、pH敏感释药和克服肿瘤多药耐药的多功能透明质酸聚合物胶束,用于抗肿瘤药物阿霉素(Doxorubicin,DOX)的传递,以期克服DOX水溶性差,生物利用度低,易产生多药耐药,毒副作用大等缺点。以具有CD44靶向的透明质酸(Hyaluronic acid,HA)作为胶束的亲水链,首先制备了5种不同取代度的透明质酸-十八胺聚合物,探讨了不同取代度的HA胶束与细胞摄取之间的关系。其次,设计了具有pH敏感的原酸酯基的化合物2-十八烷氧基-(1,3)二恶烷-5-基胺(OD),以其作为胶束的疏水端,制备得到pH敏感的透明质酸聚合物胶束(HOD)。对载药HOD胶束体外pH敏感性能,药物释放情况,细胞毒性,细胞摄取,摄取机制和体内靶向性进行评价。而后,合成经腙键连接的pH敏感DOX-NN-VES前药,以HOD将其包载,构建了DNVM药物传递系统,以MCF-7/ADR细胞为模型,考察其克服肿瘤多药耐药作用和逆转机制,并构建MCF-7/ADR荷瘤裸鼠模型,对其体内药效学进行考察。合成了取代度为12%~51%的5种透明质酸-十八胺聚合物,并制备DOX/HOA透明质酸胶束,其粒径随着取代度的增加而增加,且具有较好的血清稳定性和药物控制释放性能。细胞毒性和细胞摄取实验结果表明,取代度为23%的DOX/HOA胶束(DOX/HOA23)具有适宜的粒径和取代度,药物摄取效率最高,抗肿瘤活性最佳,说明取代度和粒径都会影响基于透明质酸胶束的抗肿瘤活性和细胞摄取效率。制备了pH敏感和肿瘤靶向的HOD胶束(以无pH敏感的HOA胶束作为对照),通过动态光散射法和动态透析法对胶束的体外pH敏感性进行考察,结果表明HOD胶束有明显的pH敏感性。以MCF-7细胞为模型,进行体外细胞学实验。结果显示,DOX/HOD胶束的细胞毒性明显大于DOX/HOA胶束;通过CD44受体介导的内吞,显著提高了胞内药物的摄取量;可在溶酶体中快速释放药物。摄取机制实验显示HOD胶束的内吞途径主要是以小窝蛋白介导的内吞形式。小动物活体成像实验表明HOD胶束在肿瘤部位富集,具有较好的肿瘤靶向性。制备经酸敏感的腙键连接的DOX-NN-VES前药,通过HOD载体将其包载,构建得到DNVM多功能药物传递系统,以包载DOX-VES(通过酰胺键连接)的HOD胶束(DVSM)和单纯载DOX的HOD胶束(DOXM)作为对照。DNVM细胞毒性和细胞摄取量都显著高于DVSM和DOXM,具有更好的克服肿瘤多药耐药的作用。细胞内定位实验表明DNVM有pH敏感触发释药和溶酶体逃逸功能,可有效地将药物传递到肿瘤细胞内发挥功效。逆转机制结果表明,相比于DVSM和DOXM,DNVM可更好地抑制药物外排,使细胞产生更多的活性氧,增加促凋亡蛋白的产生量,抑制P-gp的表达,从而克服肿瘤多药耐药。体内药效学实验结果表明,DNVM抑制肿瘤生长效果显著,且在实验期间小鼠无明显的体重变化,说明胶束在提高抗肿瘤药物治疗效果的同时,还显著降低其毒副作用。
[Abstract]:The aim of this study was to construct multifunctional hyaluronic acid polymer micelles targeting CD44, pH sensitive drug release and tumor multidrug resistance for the delivery of adriamycin (Doxorubicin,DOX), in order to overcome the poor water solubility and low bioavailability of DOX. Easy to produce multi-drug resistance, toxic side effects and other shortcomings. Five kinds of hyaluronic acid-octadecylamine polymers with different degree of substitution were prepared by using hyaluronic acid (Hyaluronic acid,HA) with CD44 targeting as hydrophilic chain. The relationship between HA micelles with different degree of substitution and cellular uptake was investigated. Secondly, the compound 2-octadecyloxy- (1keto3) dioxane-5-amine (OD), which has pH sensitive orthoester group, was designed as the hydrophobic end of the micelle, and the pH sensitive hyaluronic acid polymer micelle (HOD). Was prepared by using it as the hydrophobic end of the micelle. The in vitro pH sensitivity, drug release, cytotoxicity, cellular uptake, uptake mechanism and in vivo targeting of HOD micelles were evaluated. Then, pH sensitive DOX-NN-VES prodrugs linked with Hydrazone bond were synthesized and encapsulated with HOD to construct a drug delivery system for DNVM. Using MCF-7/ADR cells as a model, the mechanism of overcoming multidrug resistance and reversing the drug resistance was investigated. A nude mouse model of MCF-7/ADR bearing tumor was established and its pharmacodynamics was investigated in vivo. Five kinds of hyaluronic acid-octadecylamine polymers with a degree of substitution of 12% were synthesized, and DOX/HOA hyaluronic acid micelles were prepared. The particle size of the micelles increased with the increase of the degree of substitution. The results of cytotoxicity and cell uptake experiments showed that the DOX/HOA micelles (DOX/HOA23) with 23% degree of substitution had the appropriate particle size and degree of substitution, the drug uptake efficiency was the highest, and the antitumor activity was the best. Both the degree of substitution and the particle size affect the antitumor activity and cell uptake efficiency based on hyaluronic acid micelles. PH sensitive and tumor-targeting HOD micelles (compared with HOA micelles without pH sensitivity) were prepared. The in vitro pH sensitivity of HOD micelles was investigated by dynamic light scattering and dynamic dialysis. The results showed that HOD micelles had obvious pH sensitivity. Using MCF-7 cells as model, in vitro cytological experiments were carried out. The results showed that the cytotoxicity of DOX/HOD micelles was significantly higher than that of DOX/HOA micelles, the uptake of intracellular drugs was significantly increased by CD44 receptor mediated endocytosis, and the drug could be released rapidly in lysosomes. Uptake mechanism showed that the endocytosis pathway of HOD micelles was mainly mediated by fossa protein. In vivo imaging of small animals showed that HOD micelles were rich in tumor site and had good tumor targeting. The DOX-NN-VES prodrugs with acid-sensitive Hydrazone bond were prepared and encapsulated with HOD carrier to construct a multifunctional drug delivery system for DNVM. The cytotoxicity and cell uptake of DNVM were significantly higher than that of DVSM and DOXM, in comparison with HOD micelle (DVSM) and HOD micelle (DOXM), which were encapsulated with DOX-VES (via amide bond) and HOD micelle (DOXM), respectively. Both DNVM and DOXM, had better effects on overcoming multidrug resistance of tumor than those of DVSM and DOXM,. The cytotoxicity and cell intake of DNVM were significantly higher than those of DVSM and DOXM,. Intracellular localization experiments showed that DNVM could trigger drug release and lysosome escape with pH sensitivity, which could effectively transfer the drug to tumor cells. The reverse mechanism showed that compared with DVSM and DOXM,DNVM, it could inhibit drug efflux, produce more reactive oxygen species, increase the production of pro-apoptotic protein, inhibit the expression of P-gp, and overcome the multidrug resistance of tumor. The results of in vivo pharmacodynamics experiment showed that DNVM inhibited tumor growth significantly, and there was no obvious weight change in mice during the experiment, which indicated that micelle not only improved the therapeutic effect of antitumor drugs, but also significantly reduced its toxicity and side effects.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96
本文编号：2423209
[Abstract]:The aim of this study was to construct multifunctional hyaluronic acid polymer micelles targeting CD44, pH sensitive drug release and tumor multidrug resistance for the delivery of adriamycin (Doxorubicin,DOX), in order to overcome the poor water solubility and low bioavailability of DOX. Easy to produce multi-drug resistance, toxic side effects and other shortcomings. Five kinds of hyaluronic acid-octadecylamine polymers with different degree of substitution were prepared by using hyaluronic acid (Hyaluronic acid,HA) with CD44 targeting as hydrophilic chain. The relationship between HA micelles with different degree of substitution and cellular uptake was investigated. Secondly, the compound 2-octadecyloxy- (1keto3) dioxane-5-amine (OD), which has pH sensitive orthoester group, was designed as the hydrophobic end of the micelle, and the pH sensitive hyaluronic acid polymer micelle (HOD). Was prepared by using it as the hydrophobic end of the micelle. The in vitro pH sensitivity, drug release, cytotoxicity, cellular uptake, uptake mechanism and in vivo targeting of HOD micelles were evaluated. Then, pH sensitive DOX-NN-VES prodrugs linked with Hydrazone bond were synthesized and encapsulated with HOD to construct a drug delivery system for DNVM. Using MCF-7/ADR cells as a model, the mechanism of overcoming multidrug resistance and reversing the drug resistance was investigated. A nude mouse model of MCF-7/ADR bearing tumor was established and its pharmacodynamics was investigated in vivo. Five kinds of hyaluronic acid-octadecylamine polymers with a degree of substitution of 12% were synthesized, and DOX/HOA hyaluronic acid micelles were prepared. The particle size of the micelles increased with the increase of the degree of substitution. The results of cytotoxicity and cell uptake experiments showed that the DOX/HOA micelles (DOX/HOA23) with 23% degree of substitution had the appropriate particle size and degree of substitution, the drug uptake efficiency was the highest, and the antitumor activity was the best. Both the degree of substitution and the particle size affect the antitumor activity and cell uptake efficiency based on hyaluronic acid micelles. PH sensitive and tumor-targeting HOD micelles (compared with HOA micelles without pH sensitivity) were prepared. The in vitro pH sensitivity of HOD micelles was investigated by dynamic light scattering and dynamic dialysis. The results showed that HOD micelles had obvious pH sensitivity. Using MCF-7 cells as model, in vitro cytological experiments were carried out. The results showed that the cytotoxicity of DOX/HOD micelles was significantly higher than that of DOX/HOA micelles, the uptake of intracellular drugs was significantly increased by CD44 receptor mediated endocytosis, and the drug could be released rapidly in lysosomes. Uptake mechanism showed that the endocytosis pathway of HOD micelles was mainly mediated by fossa protein. In vivo imaging of small animals showed that HOD micelles were rich in tumor site and had good tumor targeting. The DOX-NN-VES prodrugs with acid-sensitive Hydrazone bond were prepared and encapsulated with HOD carrier to construct a multifunctional drug delivery system for DNVM. The cytotoxicity and cell uptake of DNVM were significantly higher than that of DVSM and DOXM, in comparison with HOD micelle (DVSM) and HOD micelle (DOXM), which were encapsulated with DOX-VES (via amide bond) and HOD micelle (DOXM), respectively. Both DNVM and DOXM, had better effects on overcoming multidrug resistance of tumor than those of DVSM and DOXM,. The cytotoxicity and cell intake of DNVM were significantly higher than those of DVSM and DOXM,. Intracellular localization experiments showed that DNVM could trigger drug release and lysosome escape with pH sensitivity, which could effectively transfer the drug to tumor cells. The reverse mechanism showed that compared with DVSM and DOXM,DNVM, it could inhibit drug efflux, produce more reactive oxygen species, increase the production of pro-apoptotic protein, inhibit the expression of P-gp, and overcome the multidrug resistance of tumor. The results of in vivo pharmacodynamics experiment showed that DNVM inhibited tumor growth significantly, and there was no obvious weight change in mice during the experiment, which indicated that micelle not only improved the therapeutic effect of antitumor drugs, but also significantly reduced its toxicity and side effects.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96
【参考文献】
相关期刊论文 前1条
1 浦龙健;刘浩;蒋琛琛;;肿瘤多药耐药机制及逆转策略的研究进展[J];蚌埠医学院学报;2014年03期
相关硕士学位论文 前1条
1 施瑜;硼酸调控的pH响应型多糖纳米药物载体的设计、制备及表征[D];江南大学;2016年
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