叶酸受体靶向熊果酸长循环脂质体的制备及其抑瘤作用的研究

发布时间：2018-01-20 19:17

本文关键词： 熊果酸隐形纳米脂质体叶酸受体靶向组织分布促凋亡人口腔表皮样癌抑瘤作用　出处：《华中科技大学》2014年博士论文　论文类型：学位论文

【摘要】：研究目的：熊果酸(Urosolic acid, UA)具有广泛的生物学效应和良好的抗癌效果,但因其水溶性差且生物利用度低,使其广泛应用受到限制。本研究首先制备熊果酸长循环脂质体,再利用叶酸与叶酸受体之间的特异性结合能力和某些肿瘤细胞表面叶酸受体过度表达的特点,用叶酸修饰的脂质材料制备叶酸靶向熊果酸脂质体,极大地提高了熊果酸脂质体在肿瘤部位的富集,在解决熊果酸溶解性和生物利用度问题的同时,增加肿瘤靶区组织里的药物浓度,延长其作用时间,提高抗癌效果,降低药物的毒副作用,为临床肿瘤的有效治疗提供一种新的思路和方法。研究方法：在本课题的研究中,首先采用经典的脂质体制备方法薄膜分散法分别制备非靶向熊果酸长循环脂质体(non-targeted PEGylated liposomal UA, PL-UA)和叶酸受体靶向熊果酸长循环脂质体(folate-receptor targeted liposomal UA, FTL-UA),同时考察熊果酸脂质体的理化性质如粒径、载药量、包封率、电位和稳定性等各项因素。选择合适的肿瘤靶向细胞,探讨熊果酸脂质体的细胞靶向性及体外抑瘤作用,分析PL-UA和FTL-UA对人口腔表皮样癌(KB)细胞抑制作用上的差异性：利用细胞增殖抑制实验(MTT)分析熊果酸脂质体对KB肿瘤细胞生长抑制作用；Hoechst33258荧光染色分析细胞凋亡的形态学变化；流式细胞术分析熊果酸脂质体对细胞凋亡率的影响和细胞周期分布的变化；通过Annexin V-FITC/PI双染的细胞凋亡试验,定量分析药物诱导细胞凋亡的具体情况。考察UA脂质体在小鼠体内药物代谢动力学及组织分布的特征：通过对小鼠血浆、心、肝、脾、胃、肾等组织的药物含量进行分析,考察熊果酸隐形纳米脂质体、游离药物在体内长循环作用、组织分布上的差异性,为进一步研究叶酸受体靶向脂质体在肿瘤治疗中的应用提供依据。考察熊果酸脂质体叶酸靶向治疗荷瘤鼠人口腔表皮样癌的作用：在前述实验研究的基础上进一步证实FTL-UA是否具有良好的抗肿瘤效果,利用KB细胞表面大量表达叶酸受体的特征,以balb/c裸鼠为研究对象,建立荷瘤鼠人口腔表皮样癌KB肿瘤模型,通过对该模型尾静脉注射FTL-UA,观察其靶向治疗荷瘤鼠人口腔表皮样癌的效果及对荷瘤鼠生存周期的影响,为FTL-UA进一步应用于临床肿瘤靶向治疗提供依据。实验结果：采用薄膜分散法制备的FTL-UA及PL-UA的平均粒径分别为(160.1±12.5) nm,(151.4士16.1)m;分散系数为0.196±0.052,0.168±0.066,粒度分布窄,大小均匀；其电位分别为(-21.24±4.2)mV,(-23.15±6.7)mV;包封率为(88.9±7.9)%,(86.7土12.1)%。在4℃放置3个月后,能维持较好的稳定性。在体外,MTT结果显示FTL-UA对KB细胞有明显的杀伤作用,其抑制率呈明显时间-剂量依赖性,FTL-UA组的IC50值低于PL-UA组(作用72h后的IC50值分别为22.05μM和146.3μM)；细胞周期分布的结果显示FTL-UA主要使KB细胞阻滞于S期,即肿瘤细胞的DNA合成期；另外,荧光染色和流式细胞术分析也证实了FTL-UA能诱导KB细胞凋亡。给6周龄左右小鼠尾静脉注射20mg/Kg药物,通过药物代谢动力学各参数和组织分布的分析,熊果酸脂质体的半衰期、药-时曲线下面积、表观分布容积、平均滞留时间和清除率较单纯给予游离熊果酸有显著性差异,表现出明显的体内缓释和长循环效应。同时,熊果酸脂质体在肝、肾中含量高于其他组织,在其他组织中分布很少,仅有少量组织残留。建立balb/c裸鼠KB肿瘤模型,尾静脉分别注射4.5mg/Kg的游离UA.PL-UA及FTL-UA,每隔一天注射一次,连续给药5次。其结果显示FTL-UA组中每只荷瘤鼠的肿瘤表面有明显的坏死空洞形成,瘤体逐渐萎缩,明显抑制肿瘤生长,生存周期也高于其他组。实验结论：本实验研究结果提示,FTL-UA有望作为新型的化疗药物在人口腔癌化疗方面发挥积极的作用。除了改善熊果酸的溶解性外,还可增加药物疗效,延长体内循环时间,显著抑制肿瘤生长,发挥其抗肿瘤效应。本研究中所获得的实验结果也为FTL-UA作为化疗药物进一步应用于临床肿瘤的治疗奠定了基础。通过对该新剂型的研究,化疗药物的叶酸受体靶向治疗有可能成为肿瘤综合治疗的新方法之一。
[Abstract]:The purpose of the study is:
Ursolic acid (Urosolic acid UA) has a wide range of biological effects and good anticancer effect, but because of its poor water solubility and low bioavailability, its application was limited. Ursolic acid was firstly prepared long circulating liposomes in this study, using specific characteristics between folate and folate receptor binding ability and some tumor cell surface receptor overexpression with folic acid, folic acid modified lipid material preparation of folate targeted liposomes of ursolic acid, greatly improve the ursolic acid liposome in the enrichment of the site of the tumor, in the solution of ursolic acid solubility and bioavailability of the problem at the same time, increase the drug concentration in the tumor tissues in the target area and the role of time, improve the anti-cancer effect, reduce the side effects of drugs, to provide a new idea and method for effective clinical treatment of tumors.
Research methods:
In this study, firstly, the classic preparation methods of liposome were prepared by film dispersion method and non targeting of ursolic acid long circulating liposomes (non-targeted PEGylated liposomal UA, PL-UA) and folate receptor targeting of ursolic acid long circulating liposomes (folate-receptor targeted liposomal UA, FTL-UA), and the effects of ursolic acid liposome the physicochemical properties such as particle size, drug loading, encapsulation efficiency, various factors of potential and stability.
Choose appropriate to the tumor target cells, target cells of ursolic acid liposomes to the antitumor effect and in vitro analysis of PL-UA and FTL-UA on human oral epidermoid carcinoma (KB) cell difference inhibition: inhibition of cell proliferation (MTT) analysis of the inhibitory effects of ursolic acid liposome on KB tumor cells growth; morphological changes of cell apoptosis analysis by Hoechst33258 staining; analysis of ursolic acid liposome on apoptosis and changes of cell cycle distribution by flow cytometry; cell apoptosis test Annexin V-FITC/PI double staining, quantitative analysis of the specific situation of drug induced apoptosis.
The effects of UA liposomes in vivo drug metabolism and pharmacokinetics characteristics of mice and tissue distribution in mice by plasma, heart, liver, spleen, stomach, kidney and other tissues of the drug content of ursolic acid, stealth nano liposomes and free drug in the body long cycle, differences in tissue distribution, provide the basis for to the application of liposome in the treatment of tumor and further study on folate receptor targeting.
To the treatment of nude mice bearing human oral epidermoid carcinoma of the effects of ursolic acid liposome target: folic acid based on the experimental study on further confirm whether FTL-UA has a good anti-tumor effect by KB cell surface expression characteristics of folate receptor, balb/c in nude mice as the research object, the establishment of human oral epidermoid tumor bearing mice kind of KB cancer model, based on the model of tail vein injection of FTL-UA to observe the targeted treatment of nude mice bearing human oral epidermoid carcinoma and its effect on the survival period of tumor bearing mice, FTL-UA for the further clinical application of tumor targeted therapy provides the basis.
Experimental results:
The average particle by film dispersion of FTL-UA and PL-UA prepared by the diameters of (160.1 + 12.5) nm, (151.4 + 16.1) m; dispersion coefficient is 0.196 + 0.052,0.168 + 0.066, narrow size distribution and uniform size; the potential respectively (-21.24 + 4.2) mV, (-23.15 + 6.7) mV package; was (88.9 + 7.9)% and (86.7 + 12.1)%. For 3 months at 4 DEG C, can maintain good stability.
In vitro, MTT results showed that FTL-UA has obvious killing effect on KB cells, the inhibition rate was time dose dependent, FTL-UA group's IC50 value is lower than that of group PL-UA (72h after the IC50 values were 22.05 M and 146.3 M); cell cycle distribution showed that FTL-UA KB cells. The arrest in S phase, DNA synthesis of tumor cells; in addition, fluorescence staining and flow cytometry analysis also confirmed that FTL-UA could induce apoptosis of KB cells.
For about 6 week old mice tail vein injection of 20mg/Kg drugs, through the analysis of pharmacokinetic parameters and tissue distribution, liposome half-life, area under concentration time curve, the apparent volume of distribution, the average residence time and clearance were treated free of ursolic acid has significant differences, showing sustained release in vivo the obvious and long circulating effect. At the same time, liposome in the liver, kidney was higher than that in other tissues, rarely found in other tissues, only a small amount of tissue residues.
The establishment of balb/c KB nude mice tumor model, free UA.PL-UA and FTL-UA 4.5mg/Kg were injected into the tail vein injection, every day once, continuous administration of 5 times. The results showed that the surface of the tumor in the FTL-UA group each mice had obvious necrosis of tumor cavity, gradually atrophic, inhibit tumor growth, survival period is also higher than other groups.
Experimental conclusions:
The results of this study suggest that FTL-UA may be a novel chemotherapeutic drugs play a positive role in human oral cancer chemotherapy. In addition to improve the solubility of ursolic acid, but also can increase the curative effect, prolong the circulation time in vivo, inhibit tumor growth, exert its anti-tumor effect. The experimental results obtained in this study also laid the foundation for the treatment of FTL-UA as a drug for clinical application of tumor. Through the study of the new formulation, target chemotherapy to folate receptor therapy may become a new method of tumor therapy.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96;R94

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