【作者】 许东航；

【导师】 梁文权；

【作者基本信息】 浙江大学 ， 药理学， 2008， 博士

【摘要】 肿瘤细胞对抗肿瘤药物产生多药耐药性(multidrug resistance,MDR)是导致肿瘤化疗失败的重要原因,也是化疗介入肿瘤治疗以来一直悬而未决的重要问题。克服MDR的策略主要包括化疗增敏剂如维拉帕米等的开发,肿瘤药物进行结构改造,生物技术相关的逆转方法如基因治疗等,这些策略均存在一定不足。药物载体作为一种新型的MDR逆转策略,既可提高化疗药物作用的敏感性,又可不增加化疗药物对正常组织的毒副作用,还可突破抗肿瘤药物的剂量限制,可有效克服上述克服MDR策略的不足而受到较大关注。本论文分别制备了阿霉素(doxorubicin,DOX)脂质体、阿霉素固体脂质纳米粒、阿霉素非离子型表面活性剂囊泡等三种纳米制剂,并分别用非离子型表面活性剂、阳离子材料等修饰脂质体、固体脂质纳米粒,同时将脂质体和维拉帕米联合应用,对阿霉素的细胞积聚及其胞内动力学特征,细胞摄取、外排机制和克服肿瘤细胞多药耐药作用的可能机制进行了探讨,同时研究了Pluronic P85修饰阿霉素脂质体的小鼠药代药动学和药效学以及阿霉素非离子型表面活性剂囊泡的小鼠药效学。论文首先建立了耐药细胞模型和分析测定方法。结果显示K562/DOX细胞P-gp高表达,对阿霉素耐药指数达107.50,而维拉帕米(Ver)可部分逆转耐药性。K562/DOX细胞可以作为以阿霉素为模型药的纳米制剂克服肿瘤细胞耐药作用及机理研究的细胞模型。以荧光分光光度计法测定DOX的浓度,DOX浓度在10～1000ng/mL范围内呈良好线性关系,相关系数为0.9998;该方法日内、日间精密度、回收率符合测定要求。并通过在测定过程添加0.1%十二烷基硫酸钠孵育30min,有效地解决了阿霉素测定过程的荧光淬灭。脂质体可部分克服肿瘤多药耐药作用。首先采用硫酸铵梯度法制备了阿霉素脂质体,药物稳定,渗漏少。K562/DOX细胞摄取结果显示,阿霉素脂质体(DOX-L)较同浓度的溶液(DOX-I)有更多被耐药细胞摄取。而耐药细胞外排阿霉素脂质体较溶液缓慢,说明阿霉素脂质体具有延缓药物外排的作用。药物敏感试验(MTT)检测脂质体的耐药指数降低为3.30,说明阿霉素脂质体可部分克服多药耐药。低温和内吞抑制剂(叠氮钠、甘露醇和氯喹)均显著降低K562/DOX细胞内DOX的积聚,说明耐药细胞摄取脂质体是通过能量依赖性的内吞方式。为了进一步了解DOX的细胞动力学及其DOX在亚细胞器的分布,我们采用HPLC建立了细胞内亚细胞器(细胞核)的含量测定方法。DOX-L和DOX-I在亚细胞器的分布结果显示,与对照DOX-I相比,相同药物量的DOX-L与细胞孵育后,能显著提高细胞核内药物量。而且脂质体减慢消除速率,延长细胞内药物滞留时间,改变药物在细胞内的动力学特征和分布,提高了细胞核内药物量;说明阿霉素脂质体有趋核分布的特点。非离子型表面活性剂修饰脂质体后,将对细胞摄取、外排行为和克服多药耐药产生影响。采用非离子型表面活性剂pluronic、Span、Tween和Cremophor EL修饰脂质体,包封率均达到90%以上,粒径与阿霉素脂质体相似。脂质体渗漏结果显示,随着非离子型表面活性剂HLB值的降低,渗漏加快。对于同系列的非离子型表面活性剂,2h时K562/DOX摄取药物量随非离子型表面活性剂的HLB值降低,摄取增加。但对不同系列的表面活性剂修饰的脂质体,摄取量除与HLB值相关外,还可能与表面活性剂的性质相关。MTT检测结果显示修饰后的细胞毒作用增加,随HLB值降低,细胞毒作用增加,证实了非离子型表面活性剂修饰的阿霉素脂质体可更有效克服多药耐药。阳离子脂质体可促进阿霉素趋核,但可能降低耐药细胞摄取药物量。采用DC-Chol和CTAB制备了阳离子脂质体,发现进入细胞的药物量较普通脂质体有所下降,但细胞核的药物量却和脂质体相当,细胞核百分比高于普通脂质体。维拉帕米核阿霉素脂质体具有协同抗多药耐药作用。为了评价维拉帕米对阿霉素脂质体摄取和外排行为的影响,试制了5种阿霉素制剂,结果发现DOX脂质体与维拉帕米溶液混合液(DOX-L-FV)摄取速率快,2h摄取药物量最大。而阿霉素和维拉帕米共同包被脂质体(DOX-L-LV)摄取速率相对慢,但达稳态的时间延长,在4h摄取药物量接近DOX-L-FV。由于DOX-L-LV可降低阿霉素和维拉帕米的心脏毒性,有望被开发为新剂型。细胞内药物外排结果显示含维拉帕米组明显减缓了外排速率。MTT检测结果显示:DOX-L-LV＜DOX-L-FV＜DOX-I-FV＜DOX-L＜DOX-I,显示脂质体和维拉帕米有良好的协同作用。采用S180肿瘤模型评价了Pluronic P85修饰的脂质体(DOX-L-Pluronic P85)在小鼠的药代动力学和药效学。首先建立了小鼠血浆和组织样品中DOX含量测定的HPLC方法。体内药动学研究结果显示,DOX-L-Pluronic P85可显著延长DOX的体内循环时间,提高血浆中药物浓度,其消除半衰期为DOX-I的6.72倍,AUC为DOX-I的6.21倍。DOX-L-Pluronic P85可增加DOX在肿瘤内的分布,其AUC和峰浓度分别为DOX-I组的4.65和1.44倍。其各器官(包括心、肾、肝、脾、肺、肠)的相对靶向效率均大于1,心、肾的相对靶向效率更是10倍以上,说明DOX-L-Pluronic P85的小鼠心、肾毒性显著低于DOX-I组(p＜0.01)。DOX-L-Pluronic P85 2mg/kg时其抑瘤率为30.83%,与同剂量的DOX-I抑瘤率相当(32.09%);1mg/Kg和4mg/kg的DOX-L-Pluronic P85的抑瘤率分别为28.53%和51.91%,显示较好的剂量依赖性。DOX-I用药后体重较对照组有所降低,而采用DOX-L-PLURONIC P85治疗,体重较对照组增加,显示了DOX-L-Pluronic P85毒性低.肿瘤病理切片显示,DOX-L-PLURONIC P85对肿瘤细胞抑制作用显著,对心肌毒性小。固体脂质纳米粒(SLN)也可有效克服肿瘤多药耐药。通过阴离子聚合物络合法有效地解决了水溶性细胞毒药物阿霉素的固体脂质纳米粒包封率。与阿霉素溶液相比,固体脂质纳米粒可以显著提高细胞对药物的摄取,说明固体脂质纳米粒具有克服肿瘤细胞耐药性的作用,而外排试验说明固体脂质纳米粒可以延缓药物细胞内消除。我们分别采用Pluronic F87、F88和P85三种不同Pluronic制备了DOX-SLN,其粒径和包封率相似,但细胞摄取试验显示DOX-SLN-P85在各时间点进入细胞的量比DOX-SLN-F87和DOX-SLN-F88多。这可能由于Pluronic组成不同,导致耐药细胞K562/DOX的细胞膜的流动性不同,从而影响SLN进入细胞的量。另外制备了CTAB修饰的DOX-SLN,发现阳离子CTAB修饰的SLN具有细胞核靶向性。MTT实验证实了DOX-SLN可部分克服多药耐药,低温和内吞抑制试验说明细胞摄取DOX-SLN为通过能量依赖的内吞途径。非离子型表面活性剂囊泡具有克服部分肿瘤多药耐药的作用。采用薄膜分散法制备了DOX非离子型表面活性剂囊泡(DOX-N)。DOX-N和耐药细胞K562/DOX孵育后摄取进入细胞的量显著高于阿霉素溶液,4h进入细胞的药物为阿霉素溶液的1.4倍。内吞抑制剂和低温摄取实验显示K562/DOX细胞摄取非离子型表面活性剂囊泡也主要通过内吞途径。MTT实验提示DOX-N可部分克服多药耐药的机制可能抑制P-gp表达采用K562/DOX细胞嫁接法建立了动物耐药模型,随时间的延长,DOX-N组与DOX-I组的肿瘤体积相比明显减小,差异有显著性。DOX-N组的抑瘤率高达为67.97%,远高于DOX-I组(16.12%)。体外细胞和在体实验说明,DOX-N有效克服了多药耐药。本论文的研究,将为这些制剂克服肿瘤多药耐药作用的进一步研究提供理论参考和实验依据。细胞药动学和亚细胞器的分布研究将为纳米载体的设计提供新的手段。非离子型表面活性剂在克服多药耐药上的研究,将促进对耐药机制的理解。更多还原

【Abstract】 Drug resistance is a major problem that limits the effectiveness of chemotherapies used to treat cancer. There are a variety of strategies and approaches to inhibit or circurnvent MDR, including chemical agents which reverse increased efflux such as verapamil, new anticancer drugs that are not substrates of drug transporters, strategies on biotechnology such as gene treatment. But it is one of the significant missions in the area of overcoming MDR. Drug carrier is a novel approach to overcoming MDR in cancer nowadays. The main aims of this research were to investigate nanocarriers to overcome MDR in cancer, based on doxorubicin, which will provide the basis for future studies of overcoming drug resistance and ultimately impoving chemotherapy and the outcome of cancer patients.The contents of this thesis were including （1） Preparation and characterization of liposome, SLN and niosomes of doxorubicin; （2）evaluation of overcoming MDR effect of the nanocarriers with the resistanct cnacer cells K562/DOX; （3） evaluation of overcoming MDR effect of the vehicles such as non-ionic surfactant, cationic with K562/DOX cells; （3）investigation of the mechanism of overcoming MDR with the resistanct cnacer cells K562/DOX; （4）study of Phamacokineties, biodistribution and pharmacodynamics of the liposome and niosomes of doxorubicin.The model of resistant cell and determination of doxorubicin in cells were established. The K562/DOX cells expressed high activity of P-glycoprotein（P-gp） and showed an obviously resistance to K562/DOX. The resistance index（RI） of K562/DOX cells was up to 107.90, and Verapamil（VER） as P-glycoprotein substrate could partially reverse this resistance. The results of uptake of DOX further proved that K562/DOX cells had significant resistance to DOX, and VER can partially reverse the resistance and increase the uptake of drug in resistant cells. The method of spectrofluorometer was set up to determine the concentration of DOX. The assay exhibited a linear range of 10-1000 ng/ml and gave a correlation coefficient （r） of 0.9998 or better. The intra-assay precision , inter-assay precision , recovery were validated according to the FDA guidelines on bioanalytical method validation. It is effective to avoid the fluorescence quence when incubated with 0.1% SDS during determination.Liposome can partially overcome multidrug resistance. Doxorubicin liposome （DOX-L） was encapsulated by the ammonium sulfate gradient-driven method. It is stable and the leakage is slow. DOX-L enhanced the drug accumulation in K562/DOX cells, and improved DOX retention in cells after withdrawal. It showed that liposome can improve the intracellular DOX concentration, which showed DOX-L can partially overcome the drug resistance. The uptake of DOX-L was energy-dependent and was influenced by temperature. Endocytosis inhibitors （sodium azide, mannitol and chloroquine） decreased significantly accumulation of DOX （p<0.01）. The result revealed that DOX-L was uptaken through the endocytosis of energy-dependent.To study the intracellular kinetics behavior and distribution of DOX, reversed-phase HPLC mothod was established to determine nuclear DOX concentration. The cellular uptake kinetics study showed that DOX-L could significantly increase intracellular accumulation and nuclear delivery of DOX-L compared with DOX-I at same dose. The nuclei levels of liposome rose slowly and reached the plateau after 2h incubation, whereas the free drug reached the plateau in 15min, suggesting that it takes times for the liposome to transport from cytoplasm to nuclei. Our results also demonstrated that liposome extended the retention in nuclei when exposed to resistance cells.Pharmaceutical technique of modifying on the surface of liposomes will have effect on cellular uptaken and effluxed and overcoming MDR. A series of non-ionic surfactnat including Pluronic （F68, F87, F88, F108, P85）, Span （20, 40, 60, 80, 85）, Tween （20, 80） and Cremophor EL were incorporating into DOX-L. The effect of expicients on the properties of liposomes and cellular uptake were investigated. Results showed that non-ionic surfactant modifying DOX-L caused significant increasing of cellular uptake. And after 2 h exposure to non-ionic surfactant modified DOX-L, the intracellular DOX concentration was correspond to HLB of non-ionic surfactant. The intracellular DOX level of different series non-ionic surfactant modified DOX-L was not only correlate to the HLB, but to property of surfactant.The cationic liposomes increased DOX allocate into nuclei but decreased the drug amount in cells. The cationic liposomes were prepared by DC-Choi or CTAB. We found that, when compared to DOX-L, the drug amount in intact cells decreased, but the DOX concentration in nuclei was similar to that of DOX-L. Percentage of nuclei in k562/DOX cells exposed to the cationic liposomes was high than that of DOX-L. The IC₅₀ of DOX-L-DC-Chol and DOX-L-CTAB was 8.629±0.808μg and 9.653±0.889μg, repectively, lower than that of DOX-L（15.475±2.392μg）.There was synergistic effect of DOX-L and verapamil on overcoming MDR. In order to estimate the influence of verapamil（VER） on the uptaken and effluxed of DOX-L, a new liposome（DOX-L-LV） co-encapsualting DOX and VER into liposome was developed. The data of DOX-I-FV demonstrated a rapid accumulation and reached a plateau within 0.75h, whereas in cells treated with DOX-L-FV, the cellular DOX levels continued to increase up to 2 h. After 2 h of treatment, nearly 1.25-fold of DOX delivered by DOX-I-FV was transported into cells compared that produced by DOX-L-LV. The amount of drug uptaken by K562/DOX cell exposed to DOX-L-LV was significant lower than that of DOX-L-FV at 2 h, but it continued to increase and get close that of DOX-L-FV at 4 h. The IC50 of DOX-L-LV as measured by MTT assay in K562/DOX cells was 0.510±0.012μg/mL, 5.4 fold less than that for liposomal DOX with free VER （DOX-I-FV 2.739±0.457μg/mL）, but only about 1.6 times less than DOX-L-FV（0.821±0.182μg/mL）. The IC₅₀ cytotoxicityon K562/DOX cells of the various formulations was as follows: DOX-L-LV<DOX-L-FV< DOX-I-FV<DOX-L< DOX-L The expression of P-gp showed the same results: DOX-L-LV< DOX-L-FV< DOX-I-FV<DOX-L<DOX-I.The pharmacokintics and pharmacodynamics of DOX-L-Pluronic P85 was eatablished using S-180 tumor bearing mice. It turned out that DOX-L-Pluronic P85 could extend the half-life of DOX-I by 6.72 times, when AUC was enhanced by 6.21 folds, respectively. DOX-L-Pluronic P85 enhanced DOX content of tumor, and had significantly tumor targeting effects. The AUC and C_max of DOX-L-Pluronic P85 in tumor were 4.65 and 1.44 folds higher than that of DOX-I, respectively. The relative tumor tissue exposure （Re） （contain heart, kidney, liver, spleen, lung, interine） were above 1 and the heart, kidney tumor targeting efficient were above 10. It showed that DOX-L-Pluronic P85 could reduce the mice cardiac and renal toxicity compare with DOX-I. Pharmacological test showed that DOX-L-Pluronic P85 at 2 mg/kg induced a 30.83% tumor inhibition rate, Similar with DOX-I at same dose showed tumor inhibiton rate of 32.09%. DOX-L-Pluronic P85 at 1 mg/kg and 4 mg/kg had tumor inhibiton rate of 28.53 % and 51.91 %, respectively. The latter was about 2 folds higher than that of DOX-I at 2 mg/kg. Pathologic slice of tumor tissue also showed the effect of antitumor was higher than that of DOX-I, and the effect of antitumor was lower than that of DOX-I.Solid lipid nanoparticle （SLN） could effectively overcome MDR. We developed a SLN preparation which attempted the use of organic anions to form ion pairs with Dox for the improvement of drug lipophilicity and drug loading. SLN could significantly improve DOX accumulated in K562/DOX cells. And we were interested to find that SLN uptaken by K562/DOX relatively slowly and effluxed also slowly. It showed that SLN could improve DOX accumulated in resistant cells and SLN could partially overcome MDR. We prepared 3 different SLN by different non-ionic surfactant: Pluronic F87, F88 and P85, repectively. And found that the DOX concentration when K562/DOX cells exposed to DOX-SLN-P85 was more than that of DOX-SLN-F87 and DOX-SLN-F88 at different time point. It might because the EPO of Pluronic P85 was smaller that that of Pluronic F87 or F88, which might influence the fluidity of cellular membrane when SLN incubated with K562/DOX cells. The uptake of DOX-SLN was also through the endocytosis of energy-dependent and was influenced by temperature.DOX niosomes could also partially overcome MDR. DOX niosomes were prepared by film dispersion method. The amount of drug uptaken by K562/DOX cell exposed to DOX niosomes was significantly higher than that of Dox solution （p < 0.05） at the same drug concentration. After 4 h exposure to DOX-N, the intracellular DOX concentration was 1.4 times higher than that incubated with free drug. The uptake of DOX-N was also energy-dependent and was influenced by temperature. Endocytosis inhibitors （sodium azide, mannitol and chloroquine） decreased significantly accumulation of DOX （p<0.01）. The result revealed that DOX-N was uptaken through the endocytosis of energy-dependent.更多还原