透明質(zhì)酸多功能聚合物膠束的制備及抗腫瘤作用研究
發(fā)布時間：2019-02-15 09:45
【摘要】：本研究旨在構(gòu)建CD44靶向、pH敏感釋藥和克服腫瘤多藥耐藥的多功能透明質(zhì)酸聚合物膠束,用于抗腫瘤藥物阿霉素(Doxorubicin,DOX)的傳遞,以期克服DOX水溶性差,生物利用度低,易產(chǎn)生多藥耐藥,毒副作用大等缺點。以具有CD44靶向的透明質(zhì)酸(Hyaluronic acid,HA)作為膠束的親水鏈,首先制備了5種不同取代度的透明質(zhì)酸-十八胺聚合物,探討了不同取代度的HA膠束與細胞攝取之間的關(guān)系。其次,設(shè)計了具有pH敏感的原酸酯基的化合物2-十八烷氧基-(1,3)二惡烷-5-基胺(OD),以其作為膠束的疏水端,制備得到pH敏感的透明質(zhì)酸聚合物膠束(HOD)。對載藥HOD膠束體外pH敏感性能,藥物釋放情況,細胞毒性,細胞攝取,攝取機制和體內(nèi)靶向性進行評價。而后,合成經(jīng)腙鍵連接的pH敏感DOX-NN-VES前藥,以HOD將其包載,構(gòu)建了DNVM藥物傳遞系統(tǒng),以MCF-7/ADR細胞為模型,考察其克服腫瘤多藥耐藥作用和逆轉(zhuǎn)機制,并構(gòu)建MCF-7/ADR荷瘤裸鼠模型,對其體內(nèi)藥效學進行考察。合成了取代度為12%~51%的5種透明質(zhì)酸-十八胺聚合物,并制備DOX/HOA透明質(zhì)酸膠束,其粒徑隨著取代度的增加而增加,且具有較好的血清穩(wěn)定性和藥物控制釋放性能。細胞毒性和細胞攝取實驗結(jié)果表明,取代度為23%的DOX/HOA膠束(DOX/HOA23)具有適宜的粒徑和取代度,藥物攝取效率最高,抗腫瘤活性最佳,說明取代度和粒徑都會影響基于透明質(zhì)酸膠束的抗腫瘤活性和細胞攝取效率。制備了pH敏感和腫瘤靶向的HOD膠束(以無pH敏感的HOA膠束作為對照),通過動態(tài)光散射法和動態(tài)透析法對膠束的體外pH敏感性進行考察,結(jié)果表明HOD膠束有明顯的pH敏感性。以MCF-7細胞為模型,進行體外細胞學實驗。結(jié)果顯示,DOX/HOD膠束的細胞毒性明顯大于DOX/HOA膠束;通過CD44受體介導的內(nèi)吞,顯著提高了胞內(nèi)藥物的攝取量;可在溶酶體中快速釋放藥物。攝取機制實驗顯示HOD膠束的內(nèi)吞途徑主要是以小窩蛋白介導的內(nèi)吞形式。小動物活體成像實驗表明HOD膠束在腫瘤部位富集,具有較好的腫瘤靶向性。制備經(jīng)酸敏感的腙鍵連接的DOX-NN-VES前藥,通過HOD載體將其包載,構(gòu)建得到DNVM多功能藥物傳遞系統(tǒng),以包載DOX-VES(通過酰胺鍵連接)的HOD膠束(DVSM)和單純載DOX的HOD膠束(DOXM)作為對照。DNVM細胞毒性和細胞攝取量都顯著高于DVSM和DOXM,具有更好的克服腫瘤多藥耐藥的作用。細胞內(nèi)定位實驗表明DNVM有pH敏感觸發(fā)釋藥和溶酶體逃逸功能,可有效地將藥物傳遞到腫瘤細胞內(nèi)發(fā)揮功效。逆轉(zhuǎn)機制結(jié)果表明,相比于DVSM和DOXM,DNVM可更好地抑制藥物外排,使細胞產(chǎn)生更多的活性氧,增加促凋亡蛋白的產(chǎn)生量,抑制P-gp的表達,從而克服腫瘤多藥耐藥。體內(nèi)藥效學實驗結(jié)果表明,DNVM抑制腫瘤生長效果顯著,且在實驗期間小鼠無明顯的體重變化,說明膠束在提高抗腫瘤藥物治療效果的同時,還顯著降低其毒副作用。
[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.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R943;R96
本文編號：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.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R943;R96
【參考文獻】
相關(guān)期刊論文 前1條
1 浦龍健;劉浩;蔣琛琛;;腫瘤多藥耐藥機制及逆轉(zhuǎn)策略的研究進展[J];蚌埠醫(yī)學院學報;2014年03期
相關(guān)碩士學位論文 前1條
1 施瑜;硼酸調(diào)控的pH響應(yīng)型多糖納米藥物載體的設(shè)計、制備及表征[D];江南大學;2016年
，本文編號：2423209
本文鏈接：http://sikaile.net/yixuelunwen/yiyaoxuelunwen/2423209.html
最近更新
教材專著
