阿霉素纳米制剂克服肿瘤多药耐药作用的研究
|
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。
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 IC_(50) 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 IC_(50) cytotoxicityon K562/DOX cells of the various formulations was as follows: DOX-L-LV 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.
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。
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 IC_(50) 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 IC_(50) cytotoxicityon K562/DOX cells of the various formulations was as follows: DOX-L-LV
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.
引文
[1] Longley DB, Johnston PGMolecular mechanisms of drug resistance[J]. J Pathol. 2005 Jan;205(2):275-92.
[2] Nobili S, Landini I, Giglioni B, Mini E. Pharmacological strategies for overcoming multidrug resistance[J]. Curr Drug Targets. 2006 Jul;7(7):861-79.
[3] Foji T, Bates S. Strategies for reversing drug resistance[J]. Oncogene, 2003, 22(47):7512-7523.
[4] Castaing M, Loiseau A, Cornish-Bowden A. Synergy between verapamil and other multidrug -resistance modulators in model membranes[J]. J Biosci. 2007 Jun;32(4):737-46.
[5] Krishna R, Mayer LD. Multidrug resistance (MDR) in cancer. Mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs[J]. Eur J Pharm Sci. 2000; 11:265-283.
[6] Advani R, Fisher GA, Lum BL, et al. A phase I trial of doxorubicin,paclitaxel, and valspodar (PSC 833), a modulator of multidrug resistance[J]. Clin Cancer Res. 2001;7:1221-1229.
[7] Baekelandt M, Lehne G,Trope CG, et al. Phase I/II trial of the multidrug-resistance modulator valspodar combined with cisplatin and doxorubicin in refractory ovarian cancer[J]. J Clin Oncol.2001;19:2983-2993.
[8] Pauli-Magnus C, Kroetz DL. Functional implications of genetic polymorphisms in the multidrug resistance gene MDR1 (ABCB1) [J]. Pharm Res. 2004; 21(6):904-13.
[9] Pakunlu RI, Cook TJ, Minko T. Simultaneous Modulation of Multidrug Resistance and Antiapoptotic Cellular Defense by MDR1 and BCL-2 Targeted Antisense Oligonucleotides Enhances the Anticancer Efficacy of Doxorubicin[J]. Pharm Res. 2003,20(3):351-359.
[10] Bjelogrlic SK, Radulovic S, Babovic N. Molecular targeting agents in renal cell carcinoma: present strategies and future perspectives[J]. Curr Pharm Des. 2008; 14(11): 1058-77.
[11] Xiong XB, Huang Y, Lu WL, et al. Enhanced intracellular delivery and improved antitumor efficacy of doxorubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic[J]. J Control Release. 2005;107(2):262-75.
[12] Pan XQ , Wang H , Shukla S , et al . Boron-containing folate receptor-targeted liposomes as potential delivery agents for neutron capture therapy[J]. Bioconjug Chem, 2002 ,13 :435-442.
[13] Mastrobattista E , Crommelin DJ , Wilschut J , et al . Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumor cells[J]. J Liposome Res, 2002 , 12 : 57-65.
[14] Kabanov AV, Batrakova EV, Alakhov VY. An essential relationship between ATP depletion and chemosensitizing activity of Pluronic block copolymers[J]. J Control Release. 2003;91(1-2):75-83.
[15] Sommer K, Kaiser S, Krylova OO, et al. Influence of amphiphilic block copolymer induced changes in membrane ion conductance on the reversal of multidrug resistance[J]. J Med Chem. 2008;51(14):4253-9.
[16] Wang Y, Yu L, Han L, et al. Difunctional Pluronic copolymer micelles for paclitaxel delivery: synergistic effect of folate-mediated targeting and Pluronic-mediated overcoming multidrug resistance in tumor cell lines[J]. Int J Pharm. 2007;337(l-2):63-73.
[17] Batrakova EV, Kelly DL, Li S, et al. Alteration of genomic responses to doxorubicin and prevention of MDR in breast cancer cells by a polymer excipient: pluronic P85[J]. Mol Pharm. 2006;3(2):113-23.
[18] Demina T, Grozdova I, Krylova O, et al. Relationship between the structure of amphiphilic copolymers and their ability to disturb lipid bilayers. Biochemistry. 2005 ;44(10):4042-54.
[19] Batrakova EV, Li S, Li Y, et al. Effect of pluronic P85 on ATPase activity of drug efflux transporters. Pharm Res. 2004;21(12):2226-33.
[20] Wong HL, Bendayan R, Rauth AM, et al. A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system[J]. J Pharmacol Exp Then 2006 ;317(3): 1372-81.
[21] Wong HL, Rauth AM, Bendayan R, et al. Ramaswamy M, Liu Z, Erhan SZ, Wu XY. A new polymer-lipid hybrid nanoparticle system increases cytotoxicity of doxorubicin against multidrug-resistant human breast cancer cells[J].Pharm Res.2006;23(7):1574-85.
[22]Wong HL,Bendayan R,Rauth AM,et al.Simultaneous delivery of doxorubicin and GG918(Elacridar) by new polymer-lipid hybrid nanoparticles(PLN) for enhanced treatment of multidrug-resistant breast cancer[J].J Control Release.2006;116(3):275-84.
[23]Wong HL,Bendayan R,Rauth AM,et al.Development of solid lipid nanoparticles containing ionically complexed chemotherapeutic drugs and chemosensitizers[J].J Pharm Sci.2004;93(8):1993-2008.
[24]Wong HL,Rauth AM,Bendayan R,et al.In vivo evaluation of a new polymer-lipid hybrid nanoparticle(PLN) formulation of doxorubicin in a murine solid tumor model[J].Eur J Pharm Biopharm.2007;65(3):300-8.
[25]Vyas SP,Katare YK,Mishra V,et al.Ligand directed macrophage targeting of amphotericin B loaded liposomes[J].Int J Pharm,2000,210:1-14.
[26]Takeuchi H,Kojima H,Yamamoto H,et al.Polymer coating of liposomes with a modified polyvinyl alcohol and their systemic circulation and RES uptake in rats[J].J Control Release,2000,68:195-205.
[27]Cocera M,Lopez O,Coderch L,et al.Partitioning of SDS in liposomes coated by the exopolymer excreted by Pseudoalteromonas antarctica NF3 as a measure of vesicle protection against this surfactant[J].J Biomater Sci Polym Ed,2001,12:255-266.
[28]Wells J,Sen A,Hui SW.Localized delivery to CT-26 tumors in mice using thermosensitive liposomes[J].Int J.Pharm,2003,261:105-114.
[29]Rosa GD,Iommelli R,La Rotonda MI,et al.Influence of the co-encapsulation of different non-ionic surfactants on the properties of PLGA insulin-loaded microspheres[J].J Control Release,2000,69:283-295.
[30]Singh J,Robinson DH.Controlled release captopril microcapsules:effect of non-ionic surfactants on release from ethyl cellulose microcapsules[J].J Microencapsul.,1988,5:129-137.
[31]Barreiro-Iglesias R,Alvarez-Lorenzo C,Concheiro A.Incorporation of small quantities of surfactants as a way to improve the rheological and diffusional behavior of carbopol gels[J].J Control Release,2001,77:59-75.
[32]Regev R,Katzir H,Yeheskely-Hayon D,et al.Modulation of P-glyeoprotein-mediated multidrug resistance by acceleration of passive drug permeation across the plasma membrane[J].FEBS J.2007;274(23):6204-14.
[33]Lo YL.Relationships between the hydrophilic-lipophilie balance values of pharmaceutical excipients and their multidrug resistance modulating effect in Caco-2 cells and rat intestines[J].J Control Release.2003;90(1):37-48.
[34]Bogman K,Eme-Brand F,Alsenz J,et al.The role of surfactants in the reversal of active transport mediated by multidrug resistance proteins[J].J Pharm Sci.2003;92(6):1250-61.
[35]朱蕾,卢兴国,张晓红,等。巨核细胞脱核和胞核胞质连体分离的形态学研究[J]。中华检验医学杂志200;28(2):163-166.
[36]韩丽妹。基于透转肿瘤多药耐药性的共聚物胶束给药体系的研究。博士学位论文。复旦大学。2006。
[37]Dabholkar RD,Sawant RM,Mongayt DA,et al.Polyethylene glycol-phosphatidylethanolamine conjugate(PEG-PE)-based mixed micelles:some properties,loading with paclitaxel,and modulation of P-glycoprotein-mediated efflux[J].Int J Pharm.2006,315(1-2):148-157.
[38]Chan HS,Haddad G,Zheng L,et al.Sensitive immunofluoreseence detection of the expression of P-glycoprotein in malignant cells[J].Cytometry.1997,29(1):65-75.
[39]Beck WT,Grogan TM,Willman CL,et al.Methods to detect P-glycoprotein-associated multidrug resistance in patients' tumors:consensus recommendations[J].Cancer Res.1996,56(13):3010-3020.
[40]Ren Y,Wei D.Quantification intracellular levels of oligodeoxynucleotide-doxorubicin conjugate in human carcinoma cells in situ[J].J Pharm Biomed Anal.2004 Oct 29;36(2):387-91.
[41]张洪妍,沈朋,栾连军,等.一种测定MDR肿瘤细胞内外阿霉素浓度的方法J].化学学报.2004,62(12):1162-1165.
[42]Alakhov VYu,Moskaleva EYu,Batrakova EV,et al.Hypersensitization of multidrug resistant human ovarian carcinoma cells by pluronic P85 block copolymer[J].Bioconjug Chem.1996;7(2):209-16.
[43]Xiong G,Chen Y,Arriaga EA.Measuring the doxorubicin content of single nuclei by micellar electrokinetie capillary chromatography with laser-induced fluorescence detection[J].Anal Chem.2005;77(11):3488-93.
[44]van Dalen EC,Michiels EM,Caron HN,et al.Different anthracycline derivates for reducing cardiotoxicity in cancer patients[J].Cochrane Database Syst Rev.2006;(4):CD005006.
[45]陈涛,王昭,韩欢牛,等.离子梯度载药法制备脂质体药物的研究进展J].世界最新医学信息文摘,2004,3(4):1204-1209。
[46]赵嘉惠,张华屏,王春芳.MTT法在检测细胞增殖方面的探讨J].山西医科大学学报,2007,38(3):262-263.
[47]Kumi-Diaka J,Nguyen V,Butler A.Cytotoxic potential of the phytochemical genistein isoflavone(4',5',7-trihydroxyisoflavone) and certain environmental chemical compounds on testicular cells[J],Biol Cell,1999,91:515-523.
[48]Sauer I,Dunay IR,Weisgraber K,et al.An apolipoprotein E-derived peptide mediates uptake of sterically stabilized liposomes into brain capillary endothelial cells[J].Biochemistry.2005,44(6):2021-2029.
[49]Bajoria R,Sooranna SR,Contractor SF.Endocytotic uptake of small unilamellar liposomes by human trophoblast cells in culture[J].Hum Reprod.1997,12(6):1343-1348.
[50]Muller WJ,Zen K,Fisher AB,et al.Pathways for uptake of fluorescently labeled liposomes by alveolar type Ⅱ cells in culture[J].Am J Physiol.1995,269(1 Pt 1):L11-L19.
[51]Nielsen HM,Aemisegger C,Burmeister G,et al.Effect of oil-in-water emulsions on 5-aminolevulinic acid uptake and metabolism to PpⅨ in cultured MCF-7 cells[J].Pharm Res.2004,21(12):2253-2260.
[52]Managit C,Kawakami S,Yamashita F,et al.Uptake characteristics of galactosylated emulsion by HepG2 hepatoma cells[J].Int J Pharm.2005,301(1-2):255-261.
[53]Duffy CF,McEathron AA,Arriaga EA.Determination of individual microsphere properties by capillary electrophoresis with laser-induced fluorescence detection[J].Electrophoresis.2002;23(13):2040-7.
[54]Duffy CF,Gafoor S,Richards DP,et al.Determination of properties of individual liposomes by capillary electrophoresis with postocolumn laser-induced fluorescence detection[J].Anal Chem.2001;73(8):1855-61.
[55]Alakhov VYu,Moskaleva EYu,Batrakova EV,et al.Hypersensitization of multidrug resistant human ovarian carcinoma cells by pluronic P85 block copolymer[J].Bioconjug Chem.1996;7(2):209-16.
[56]Alakhov V,Klinski E,Li S,et al.Block copolymer-based formulation of doxorubicin.From cell screen to clinical trials[J].Colloids and Surfaces B:Biointerfaces,1999,16(1-4):113-134.
[57]许东航,高建青,梁文权,等。高效液相色谱法测定细胞核内多柔比星浓度J]。中国药学杂志.2008;43(16):1264-1267.
[58]Ren YH,Wei DZ.Quantification intracellular levels of oligodeoxynucleotide doxorubicin conjugate in human carcinoma cells in situ[J].J Pharm Biomed Anal.2004,36(2):387-391.
[59]Rodriguez AM,Sastre S,Ribot J,et al.Beta-carotene uptake and metabolism in human lung bronchial epithelial cultured cells depending on delivery vehicle[J].Biochim Biophys Acta.2005,1740(2):132-138.
[60]Harashima H,Hirai N,Kiwada H.Kinetic modelling of liposome degradation in peritoneal maerophages[J].Biopharm Drug Dispos.1995,16(2):113-123.
[61]Kabanov AV,Batrakova EV,Alakhov VY.Pluronic block copolymers as novel polymer therapeutics for drug and gene delivery[J].J Control Release,2002,82:189-212.
[62]Rowe RC,Sheskey PL,Weller PJ,et al.药物辅料手册[M].郑俊民主译.北京:化学工业出版社,2004.
[63]Bergstrom M.Thermodynamics of Unilamellar Vesicles:Influence of Mixing on the Curvature Free Energy of a Vesicle Bilayer,J Colloid Interface Sci,240(2001) 294-306.
[64]黄永焯。非离子表面活性剂在胶体基因给药系统中的应用博士论文]。杭州 浙江大学。2005年.
[65]Liu,Y.,Yang,S.F.,Li,Y.,Xu,H.,Qin,L.,Tay,J.H.,The influence of cell and substratum surface hydrophobicities on microbial attachment,J Biotechnol.,110(2004) 251-256.
[66]Groth D,Keil O,Lehmarm C,Schneider M,Rudolph M,Reszka R.Preparation and characterization of a new lipospermine for gene delivery into various cell-lines.Int J Pharm.1998,162(1-2):143-157.
[67]Cortesi R,Esposito E,Menegatti E,et al.Effect of cationic liposome composition on in vitro cytotoxicity and protective effect on carried DNA[J].Int J Pharm.1996,139(1-2):69-78.
[68]Heurtault B,Saulnier P,Pech B,et al.Physico-chemical stability of colloidal lipid particles[J],Biomaterials,24(2003) 4283-4300.
[69]Tsuruo T,Iida H,Tsukagoshi S,et al.Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil[J].Cancer Res.1981,41(5):1967-1972.
[70]王坚成,刘晓岩,吕万良,等.新型阿霉素抗耐药性隐形脂质体的体外细胞毒和体内毒性研究[J].药学学报 2005,40(5):475 480.
[71]王增寿,计佩影,张华,等.高效液相色谱法测定人血中表阿霉素的浓度[J].中国医院药学杂志 2006;26(5):527-528
[72]徐叔云,卞如濂,陈修.药理实验方法学(第二版)[M].北京:人民卫生出版社,1994:180-192.
[73]王桂玲,魏树礼,孙华东,等.阿霉素隐形脂质体的研制及其在小鼠体内的组织分布J].中国药学杂志 1999;34(5):310-312.
[74]齐宪荣,肖瑜,魏树礼,等.阿霉素脂质体对小鼠的抗肿瘤活性比较J].中国药学杂志1997;32(4):207-210.
[75]吕万良,齐宪荣,孙华东,等.盐酸多柔比星隐形脂质体给药后在小鼠组织中质量分数的测定J].北京医科大学学报 1999;31(5):467-468.
[76]耿芹,王子淑,邹方东,等.电脉冲结合盐酸阿霉素对小鼠S180肉瘤的抑制作用J].四川大学学报(自然科学版).2003;40(3):586-589.
[77]Han HD,Lee A,Song CK,et al.In vivo distribution and antitumor activity of heparin-stabilized doxorubicin-loaded liposomes[J].Int J Pharm.2006;313(1-2):181-8.
[78]Gabizon A.Liposomes as in vivo carriers of adriamycin reduced cardiac uptake and preserved antitumor activity in mice[J].Cancer Res,1982,42:4734-4738.
[79]Van Hoesel Q GCM,St terenbery,PA Gommelin BJA,et al.Reduced cardio toxicity and nephrotoxicity with p reservation of antitumor activity of doxorubicin entrapped in stable liposomes[J].Cancer Res,1984,44:3698-3703.
[80]孙淑英,顾学裘.脂质体作为抗癌抗生素载体的研究概况J].沈阳药学院学报,1989,6:73-75.
[81]Rahman A,Carmichael D,Harris M,et al.Comparative pharmaeokinetics of free doxorubicin and doxorubicin entrapped in cardiolipin liposomes[J].Cancer Res.1986 May;46(5):2295-9.
[82]Raham A,More N,Schein PS.Doxorubicin induced chronic cardiotoxicity and its protection by liposomal administration[J].Cancer Res,1982,42:1817-1825.
[83]Rahman A,Fumagalli A,Schein PS.Potential of liposomes to ameliorate anthraycline induced cardiotoxicity[J].Semin Oncol,1984,11:44-49.
[84]Gallo JM,Gupta PK,Hung CT,et al.Evaluation of drug delivery following the administration of magnetic albumin microspheres containing adriamycin to the rat[J].J Pharm Sci.1989,78(3):190-194.
[85]张弘炜,范健.脂质体阿霉素与阿霉素对小鼠毒性作用的比较研究J].铁道医学 2000;28(6):362-364.
[86]Gabizon A,Meshorer A,Bareaholz Y,et al.Comparative long-term study of the toxicity of free and liposome-associated doxorubicin in mice after intravenous administration[J].JNCI,1986,77(2):459-467.
[87]Rahamn A,White G,More N,et al.Pharmacological,toxicological and therapeutic evaluation in mice of doxorubicin entrapped in cardiolipin liposomes[J].Cancer Res,1985,45:796-801.
[88]Herman E H,Rahman A,Ferrans V J,et al.Prevention of chronic dox2orubicin cardiotoxicity in beagles by liposomal encapsulation[J].Cancer Res,1983,43:5427-31.
[89]Gregorio D,Ronald K,Joseph A T,et al.A phase Ⅰ/Ⅱ study of intraperitoneally administered doxorubicin entrapped in cardiolipin liposomes in patients with ovarian cancer[J].AmJ Obstet Gynecol,1989,160(4):812-9.
[90]Villani F,Piccinini F,Nerelli P,et al.Influence of adriamycin on caleinm exchangeability in cardiac muscle and its modification by ouabain[J].Biochem Pharmacol,1978,27:985-7.
[91]Rahman A,Kessur A,More N,et al.Liposomal protection of adriamycin- induced cardiotoxicity in mice[J].Cancer Res,1980,40:1532-35.
[92]Gabizon A,Dagan A,Goren D,et al.Liposome as in vivo carders of adriamycin:reduced cardiac uptake and preserved antitumor activity in mice[J].Cancer Res,1982,42:4734-9.
[93]马秉亮,吴玉林,刘国卿.P-gp及对抗P-gp介导多药耐药的研究现状J].中国临床药理学与治疗学,2006,11(1):14-19.
[94]段明华,王东凯,刘红,等.固体脂质纳米粒制备方法简介J].中国医院药学杂志,2004,24(7):432-433.
[95]李欣玮,孙立新,林晓宏,等.固体脂质纳米粒作为药物载体[J].化学进展,2007,19(1):87-92.
[96]Uchegbu IF,Double JA,Turton JA,et al.Distribution,metabolism and tumoricidal activity of doxorubicin administered in sorbitan monostearate(Span 60) niosomes in the mouse[J],Pharm Res,1995,12:1019-1024.
[97]Hao Y,Zhao F,Li N,et al.Studies on a high encapsulation of colchicine by a niosome system,Int J Pharm,2002,244:73-80.
[98]Jain CP,Vyas SP.Preparation and characterization of niosomes containing rifampicin for lung targeting[J],J Microencapsul.,1995,12:401-407.
[99]Fang JY,Yu SY,Wu PC,et al,In vitro skin permeation of estradiol from various proniosome formulations[J],Int J Pharm,2001,215:91-99.
[100]Pardakhty A,Varshosaz J,Rouholamini A,et al.In vitro studyof polyoxyethylene alkyl ether niosomes for delivery of insulin[J].Int J Pharm,2007,328(2):130-141.
[101]Fang JY,Hong CT,Chiu WT,et al.Effect of liposomes andniosomes on skin permeation of enoxacin[J].Int J Pharm,2001,219(1-2):61-72.
[102]Pardakhty A,Varshosaz J,Rouholamini A,et al.In vitro studyof polyoxyethylene alkyl ether niosomes for delivery of insulin[J].Int J Pharm,2007,328(2):130-141.
[103]Uchegbu IF,Vyas SP,Non-ionic surfactant based vesicles(niosomes) in drug delivery[J].Int.J.Pharm.,172(1998) 33-70.
[104]张磊,潘弘,刘敏,陆伟跃。阿霉素的不同盐型对其脂质体体外药物泄漏和体内长循环的影响J].药学学报.2004,39(12):1018-1022
[105]王黎,侯宝光,侯新朴,于美丽,杨江山。氢化与非氢化卵磷脂对阿霉素脂质体体内外稳定性的影响[J].药学学报。2001.36(6):444-447.
[106]柯爱武,李羲。脂质体逆转肿瘤多药耐药研究进展J]。国外医学预防诊断治疗用生物制品分册 2005;28(2):75-80.
[107]王永中.紫杉醉嵌段共聚物胶束给药系统克服种瘤多药耐药性的研究[博士学位论文].上海复旦大学.2007。1-2.
[1]Krishna R,Mayer LD.Multidrug resistance(MDR) in cancer.Mechanisms,reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs[J].Eur J Pharm Sci.2000;11:265-283.
[2]Advani R,Fisher GA,Lum BL,et al.A phase Ⅰ trial of doxorubicin,paclitaxel,and valspodar (PSC 833),a modulator of multidrug resistance[J].Clin Cancer Res.2001;7:1221-1229.
[3]Baekelandt M,Lehne G,Trope CG,et al.Phase Ⅰ/Ⅱ trial of the multidrug-resistance modulator valspodar combined with cisplatin and doxorubicin in refractory ovarian cancer[J].J Clin Oncol.2001;19:2983-2993.
[4]Chico I,Kang MH,Bergan R,et al.Phase Ⅰ study of infusional paclitaxel in combination with the P-glycoprotein antagonist PSC833[J].J Clin Oncol.2001;19:832-842.
[5]Xiong XB,Huang Y,Lu WL,et al.Enhanced intracellular delivery and improved antitumor efficacy of doxorubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic[J].J Control Release.2005;107(2):262-75.
[6]Pan XQ,Wang H,Shukla S,et al.Boron-containing folate receptor-targeted liposomes as potential delivery agents for neutron capture therapy[J].Bioconjug Chem,2002,13:435-442.
[7]Mastrobattista E,Crommelin DJ,Wilschut J,et al.Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumor cells[J].J Liposome Res,2002,12:57-65.
[8]马秉亮,吴玉林,刘国卿.P-gp及对抗P-gp介导多药耐药的研究现状[J].中国临床药理学与治疗学,2006,11(1):14-19.
[9]Wong HL,Bendayan R,Rauth AM,et al.A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system[J].J Pharmacol Exp Ther.2006 Jun;317(3):1372-81.
[10]Wong HL,Rauth AM,Bendayan R,et al.A new polymer-lipid hybrid nanoparticle system increases cytotoxicity of doxorubicin against multidrug-resistant human breast cancer cells[J].Pharm Res.2006 Jul;23(7):1574-85.
[11]应小英,胡富强,袁弘.卡马西平硬脂酸固体脂质纳米粒的制备与理化性质研究[J].中国医药工杂志,2002,33(11):543-346.
[12]瞿文,陈庆华,朱宝全.固体脂质纳米粒的研究进展J].中国医药工业杂志,2001,32(9):424-429.
[13]Cavalli R,Caputo,Gasco M.R,Solid lipospheres of doxorubicin and idarubicin[J],Int.J.Pharm.1993,89:R9-R12.
[14]Wong HL,Bendayan R,Rauth AM,et al.Simultaneous delivery of doxorubicin and GG918(Elacridar) by new polymer-lipid hybrid nanoparticles(PLN) for enhanced treatment of multidrug-resistant breast cancer[J].J Control Release.2006 Dec 1;116(3):275-84.
[15]Serpe L,Catalano MG,Cavalli R,et al.Cytotoxicity of anticancer drugs incorporated in solid lipid nanoparticles on HT-29 colorectal cancer cell line[J].Eur J Pharm Biopharm.2004;58(3):673-80.
[16]Miglietta A,Cavalli R,Bocca C,et al.Cellular uptake and cytotoxicity of solid lipid nanospheres(SLN) incorporating doxorubicin or paclitaxel[J]._Int J Pharm.2000;210(1-2):61-7.
[17]Serpe L,Catalano MG,Cavalli R,et al.Cytotoxicity of anticancer drugs incorporated in solid lipid nanoparticles on HT-29 colorectal cancer cell line[J].Eur J Pharm Biopharm.2004;58(3):673-80.
[18]Fundarb A,Cavalli R,Bargoni A,et al.Non-stealth and stealth solid lipid nanoparticles(SLN)carrying doxorubicin:pharmaeokinetics and tissue distribution after i.v.administration to rats[J].Pharmaeol Res.2000;42(4):337-43.
[19]Wong H.L,Bendayan R,Rauth.A.M,et al.A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle(PLN) system[J].Pharmcol.Exp.Ther 2006,317:1372-1381.
[20]刘扬,吕万良,张强.脂质体及纳米粒药物递送系统的研究进展[J].中国医学科学院学报,2006,28(4):583-589.
[2] Nobili S, Landini I, Giglioni B, Mini E. Pharmacological strategies for overcoming multidrug resistance[J]. Curr Drug Targets. 2006 Jul;7(7):861-79.
[3] Foji T, Bates S. Strategies for reversing drug resistance[J]. Oncogene, 2003, 22(47):7512-7523.
[4] Castaing M, Loiseau A, Cornish-Bowden A. Synergy between verapamil and other multidrug -resistance modulators in model membranes[J]. J Biosci. 2007 Jun;32(4):737-46.
[5] Krishna R, Mayer LD. Multidrug resistance (MDR) in cancer. Mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs[J]. Eur J Pharm Sci. 2000; 11:265-283.
[6] Advani R, Fisher GA, Lum BL, et al. A phase I trial of doxorubicin,paclitaxel, and valspodar (PSC 833), a modulator of multidrug resistance[J]. Clin Cancer Res. 2001;7:1221-1229.
[7] Baekelandt M, Lehne G,Trope CG, et al. Phase I/II trial of the multidrug-resistance modulator valspodar combined with cisplatin and doxorubicin in refractory ovarian cancer[J]. J Clin Oncol.2001;19:2983-2993.
[8] Pauli-Magnus C, Kroetz DL. Functional implications of genetic polymorphisms in the multidrug resistance gene MDR1 (ABCB1) [J]. Pharm Res. 2004; 21(6):904-13.
[9] Pakunlu RI, Cook TJ, Minko T. Simultaneous Modulation of Multidrug Resistance and Antiapoptotic Cellular Defense by MDR1 and BCL-2 Targeted Antisense Oligonucleotides Enhances the Anticancer Efficacy of Doxorubicin[J]. Pharm Res. 2003,20(3):351-359.
[10] Bjelogrlic SK, Radulovic S, Babovic N. Molecular targeting agents in renal cell carcinoma: present strategies and future perspectives[J]. Curr Pharm Des. 2008; 14(11): 1058-77.
[11] Xiong XB, Huang Y, Lu WL, et al. Enhanced intracellular delivery and improved antitumor efficacy of doxorubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic[J]. J Control Release. 2005;107(2):262-75.
[12] Pan XQ , Wang H , Shukla S , et al . Boron-containing folate receptor-targeted liposomes as potential delivery agents for neutron capture therapy[J]. Bioconjug Chem, 2002 ,13 :435-442.
[13] Mastrobattista E , Crommelin DJ , Wilschut J , et al . Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumor cells[J]. J Liposome Res, 2002 , 12 : 57-65.
[14] Kabanov AV, Batrakova EV, Alakhov VY. An essential relationship between ATP depletion and chemosensitizing activity of Pluronic block copolymers[J]. J Control Release. 2003;91(1-2):75-83.
[15] Sommer K, Kaiser S, Krylova OO, et al. Influence of amphiphilic block copolymer induced changes in membrane ion conductance on the reversal of multidrug resistance[J]. J Med Chem. 2008;51(14):4253-9.
[16] Wang Y, Yu L, Han L, et al. Difunctional Pluronic copolymer micelles for paclitaxel delivery: synergistic effect of folate-mediated targeting and Pluronic-mediated overcoming multidrug resistance in tumor cell lines[J]. Int J Pharm. 2007;337(l-2):63-73.
[17] Batrakova EV, Kelly DL, Li S, et al. Alteration of genomic responses to doxorubicin and prevention of MDR in breast cancer cells by a polymer excipient: pluronic P85[J]. Mol Pharm. 2006;3(2):113-23.
[18] Demina T, Grozdova I, Krylova O, et al. Relationship between the structure of amphiphilic copolymers and their ability to disturb lipid bilayers. Biochemistry. 2005 ;44(10):4042-54.
[19] Batrakova EV, Li S, Li Y, et al. Effect of pluronic P85 on ATPase activity of drug efflux transporters. Pharm Res. 2004;21(12):2226-33.
[20] Wong HL, Bendayan R, Rauth AM, et al. A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system[J]. J Pharmacol Exp Then 2006 ;317(3): 1372-81.
[21] Wong HL, Rauth AM, Bendayan R, et al. Ramaswamy M, Liu Z, Erhan SZ, Wu XY. A new polymer-lipid hybrid nanoparticle system increases cytotoxicity of doxorubicin against multidrug-resistant human breast cancer cells[J].Pharm Res.2006;23(7):1574-85.
[22]Wong HL,Bendayan R,Rauth AM,et al.Simultaneous delivery of doxorubicin and GG918(Elacridar) by new polymer-lipid hybrid nanoparticles(PLN) for enhanced treatment of multidrug-resistant breast cancer[J].J Control Release.2006;116(3):275-84.
[23]Wong HL,Bendayan R,Rauth AM,et al.Development of solid lipid nanoparticles containing ionically complexed chemotherapeutic drugs and chemosensitizers[J].J Pharm Sci.2004;93(8):1993-2008.
[24]Wong HL,Rauth AM,Bendayan R,et al.In vivo evaluation of a new polymer-lipid hybrid nanoparticle(PLN) formulation of doxorubicin in a murine solid tumor model[J].Eur J Pharm Biopharm.2007;65(3):300-8.
[25]Vyas SP,Katare YK,Mishra V,et al.Ligand directed macrophage targeting of amphotericin B loaded liposomes[J].Int J Pharm,2000,210:1-14.
[26]Takeuchi H,Kojima H,Yamamoto H,et al.Polymer coating of liposomes with a modified polyvinyl alcohol and their systemic circulation and RES uptake in rats[J].J Control Release,2000,68:195-205.
[27]Cocera M,Lopez O,Coderch L,et al.Partitioning of SDS in liposomes coated by the exopolymer excreted by Pseudoalteromonas antarctica NF3 as a measure of vesicle protection against this surfactant[J].J Biomater Sci Polym Ed,2001,12:255-266.
[28]Wells J,Sen A,Hui SW.Localized delivery to CT-26 tumors in mice using thermosensitive liposomes[J].Int J.Pharm,2003,261:105-114.
[29]Rosa GD,Iommelli R,La Rotonda MI,et al.Influence of the co-encapsulation of different non-ionic surfactants on the properties of PLGA insulin-loaded microspheres[J].J Control Release,2000,69:283-295.
[30]Singh J,Robinson DH.Controlled release captopril microcapsules:effect of non-ionic surfactants on release from ethyl cellulose microcapsules[J].J Microencapsul.,1988,5:129-137.
[31]Barreiro-Iglesias R,Alvarez-Lorenzo C,Concheiro A.Incorporation of small quantities of surfactants as a way to improve the rheological and diffusional behavior of carbopol gels[J].J Control Release,2001,77:59-75.
[32]Regev R,Katzir H,Yeheskely-Hayon D,et al.Modulation of P-glyeoprotein-mediated multidrug resistance by acceleration of passive drug permeation across the plasma membrane[J].FEBS J.2007;274(23):6204-14.
[33]Lo YL.Relationships between the hydrophilic-lipophilie balance values of pharmaceutical excipients and their multidrug resistance modulating effect in Caco-2 cells and rat intestines[J].J Control Release.2003;90(1):37-48.
[34]Bogman K,Eme-Brand F,Alsenz J,et al.The role of surfactants in the reversal of active transport mediated by multidrug resistance proteins[J].J Pharm Sci.2003;92(6):1250-61.
[35]朱蕾,卢兴国,张晓红,等。巨核细胞脱核和胞核胞质连体分离的形态学研究[J]。中华检验医学杂志200;28(2):163-166.
[36]韩丽妹。基于透转肿瘤多药耐药性的共聚物胶束给药体系的研究。博士学位论文。复旦大学。2006。
[37]Dabholkar RD,Sawant RM,Mongayt DA,et al.Polyethylene glycol-phosphatidylethanolamine conjugate(PEG-PE)-based mixed micelles:some properties,loading with paclitaxel,and modulation of P-glycoprotein-mediated efflux[J].Int J Pharm.2006,315(1-2):148-157.
[38]Chan HS,Haddad G,Zheng L,et al.Sensitive immunofluoreseence detection of the expression of P-glycoprotein in malignant cells[J].Cytometry.1997,29(1):65-75.
[39]Beck WT,Grogan TM,Willman CL,et al.Methods to detect P-glycoprotein-associated multidrug resistance in patients' tumors:consensus recommendations[J].Cancer Res.1996,56(13):3010-3020.
[40]Ren Y,Wei D.Quantification intracellular levels of oligodeoxynucleotide-doxorubicin conjugate in human carcinoma cells in situ[J].J Pharm Biomed Anal.2004 Oct 29;36(2):387-91.
[41]张洪妍,沈朋,栾连军,等.一种测定MDR肿瘤细胞内外阿霉素浓度的方法J].化学学报.2004,62(12):1162-1165.
[42]Alakhov VYu,Moskaleva EYu,Batrakova EV,et al.Hypersensitization of multidrug resistant human ovarian carcinoma cells by pluronic P85 block copolymer[J].Bioconjug Chem.1996;7(2):209-16.
[43]Xiong G,Chen Y,Arriaga EA.Measuring the doxorubicin content of single nuclei by micellar electrokinetie capillary chromatography with laser-induced fluorescence detection[J].Anal Chem.2005;77(11):3488-93.
[44]van Dalen EC,Michiels EM,Caron HN,et al.Different anthracycline derivates for reducing cardiotoxicity in cancer patients[J].Cochrane Database Syst Rev.2006;(4):CD005006.
[45]陈涛,王昭,韩欢牛,等.离子梯度载药法制备脂质体药物的研究进展J].世界最新医学信息文摘,2004,3(4):1204-1209。
[46]赵嘉惠,张华屏,王春芳.MTT法在检测细胞增殖方面的探讨J].山西医科大学学报,2007,38(3):262-263.
[47]Kumi-Diaka J,Nguyen V,Butler A.Cytotoxic potential of the phytochemical genistein isoflavone(4',5',7-trihydroxyisoflavone) and certain environmental chemical compounds on testicular cells[J],Biol Cell,1999,91:515-523.
[48]Sauer I,Dunay IR,Weisgraber K,et al.An apolipoprotein E-derived peptide mediates uptake of sterically stabilized liposomes into brain capillary endothelial cells[J].Biochemistry.2005,44(6):2021-2029.
[49]Bajoria R,Sooranna SR,Contractor SF.Endocytotic uptake of small unilamellar liposomes by human trophoblast cells in culture[J].Hum Reprod.1997,12(6):1343-1348.
[50]Muller WJ,Zen K,Fisher AB,et al.Pathways for uptake of fluorescently labeled liposomes by alveolar type Ⅱ cells in culture[J].Am J Physiol.1995,269(1 Pt 1):L11-L19.
[51]Nielsen HM,Aemisegger C,Burmeister G,et al.Effect of oil-in-water emulsions on 5-aminolevulinic acid uptake and metabolism to PpⅨ in cultured MCF-7 cells[J].Pharm Res.2004,21(12):2253-2260.
[52]Managit C,Kawakami S,Yamashita F,et al.Uptake characteristics of galactosylated emulsion by HepG2 hepatoma cells[J].Int J Pharm.2005,301(1-2):255-261.
[53]Duffy CF,McEathron AA,Arriaga EA.Determination of individual microsphere properties by capillary electrophoresis with laser-induced fluorescence detection[J].Electrophoresis.2002;23(13):2040-7.
[54]Duffy CF,Gafoor S,Richards DP,et al.Determination of properties of individual liposomes by capillary electrophoresis with postocolumn laser-induced fluorescence detection[J].Anal Chem.2001;73(8):1855-61.
[55]Alakhov VYu,Moskaleva EYu,Batrakova EV,et al.Hypersensitization of multidrug resistant human ovarian carcinoma cells by pluronic P85 block copolymer[J].Bioconjug Chem.1996;7(2):209-16.
[56]Alakhov V,Klinski E,Li S,et al.Block copolymer-based formulation of doxorubicin.From cell screen to clinical trials[J].Colloids and Surfaces B:Biointerfaces,1999,16(1-4):113-134.
[57]许东航,高建青,梁文权,等。高效液相色谱法测定细胞核内多柔比星浓度J]。中国药学杂志.2008;43(16):1264-1267.
[58]Ren YH,Wei DZ.Quantification intracellular levels of oligodeoxynucleotide doxorubicin conjugate in human carcinoma cells in situ[J].J Pharm Biomed Anal.2004,36(2):387-391.
[59]Rodriguez AM,Sastre S,Ribot J,et al.Beta-carotene uptake and metabolism in human lung bronchial epithelial cultured cells depending on delivery vehicle[J].Biochim Biophys Acta.2005,1740(2):132-138.
[60]Harashima H,Hirai N,Kiwada H.Kinetic modelling of liposome degradation in peritoneal maerophages[J].Biopharm Drug Dispos.1995,16(2):113-123.
[61]Kabanov AV,Batrakova EV,Alakhov VY.Pluronic block copolymers as novel polymer therapeutics for drug and gene delivery[J].J Control Release,2002,82:189-212.
[62]Rowe RC,Sheskey PL,Weller PJ,et al.药物辅料手册[M].郑俊民主译.北京:化学工业出版社,2004.
[63]Bergstrom M.Thermodynamics of Unilamellar Vesicles:Influence of Mixing on the Curvature Free Energy of a Vesicle Bilayer,J Colloid Interface Sci,240(2001) 294-306.
[64]黄永焯。非离子表面活性剂在胶体基因给药系统中的应用博士论文]。杭州 浙江大学。2005年.
[65]Liu,Y.,Yang,S.F.,Li,Y.,Xu,H.,Qin,L.,Tay,J.H.,The influence of cell and substratum surface hydrophobicities on microbial attachment,J Biotechnol.,110(2004) 251-256.
[66]Groth D,Keil O,Lehmarm C,Schneider M,Rudolph M,Reszka R.Preparation and characterization of a new lipospermine for gene delivery into various cell-lines.Int J Pharm.1998,162(1-2):143-157.
[67]Cortesi R,Esposito E,Menegatti E,et al.Effect of cationic liposome composition on in vitro cytotoxicity and protective effect on carried DNA[J].Int J Pharm.1996,139(1-2):69-78.
[68]Heurtault B,Saulnier P,Pech B,et al.Physico-chemical stability of colloidal lipid particles[J],Biomaterials,24(2003) 4283-4300.
[69]Tsuruo T,Iida H,Tsukagoshi S,et al.Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil[J].Cancer Res.1981,41(5):1967-1972.
[70]王坚成,刘晓岩,吕万良,等.新型阿霉素抗耐药性隐形脂质体的体外细胞毒和体内毒性研究[J].药学学报 2005,40(5):475 480.
[71]王增寿,计佩影,张华,等.高效液相色谱法测定人血中表阿霉素的浓度[J].中国医院药学杂志 2006;26(5):527-528
[72]徐叔云,卞如濂,陈修.药理实验方法学(第二版)[M].北京:人民卫生出版社,1994:180-192.
[73]王桂玲,魏树礼,孙华东,等.阿霉素隐形脂质体的研制及其在小鼠体内的组织分布J].中国药学杂志 1999;34(5):310-312.
[74]齐宪荣,肖瑜,魏树礼,等.阿霉素脂质体对小鼠的抗肿瘤活性比较J].中国药学杂志1997;32(4):207-210.
[75]吕万良,齐宪荣,孙华东,等.盐酸多柔比星隐形脂质体给药后在小鼠组织中质量分数的测定J].北京医科大学学报 1999;31(5):467-468.
[76]耿芹,王子淑,邹方东,等.电脉冲结合盐酸阿霉素对小鼠S180肉瘤的抑制作用J].四川大学学报(自然科学版).2003;40(3):586-589.
[77]Han HD,Lee A,Song CK,et al.In vivo distribution and antitumor activity of heparin-stabilized doxorubicin-loaded liposomes[J].Int J Pharm.2006;313(1-2):181-8.
[78]Gabizon A.Liposomes as in vivo carriers of adriamycin reduced cardiac uptake and preserved antitumor activity in mice[J].Cancer Res,1982,42:4734-4738.
[79]Van Hoesel Q GCM,St terenbery,PA Gommelin BJA,et al.Reduced cardio toxicity and nephrotoxicity with p reservation of antitumor activity of doxorubicin entrapped in stable liposomes[J].Cancer Res,1984,44:3698-3703.
[80]孙淑英,顾学裘.脂质体作为抗癌抗生素载体的研究概况J].沈阳药学院学报,1989,6:73-75.
[81]Rahman A,Carmichael D,Harris M,et al.Comparative pharmaeokinetics of free doxorubicin and doxorubicin entrapped in cardiolipin liposomes[J].Cancer Res.1986 May;46(5):2295-9.
[82]Raham A,More N,Schein PS.Doxorubicin induced chronic cardiotoxicity and its protection by liposomal administration[J].Cancer Res,1982,42:1817-1825.
[83]Rahman A,Fumagalli A,Schein PS.Potential of liposomes to ameliorate anthraycline induced cardiotoxicity[J].Semin Oncol,1984,11:44-49.
[84]Gallo JM,Gupta PK,Hung CT,et al.Evaluation of drug delivery following the administration of magnetic albumin microspheres containing adriamycin to the rat[J].J Pharm Sci.1989,78(3):190-194.
[85]张弘炜,范健.脂质体阿霉素与阿霉素对小鼠毒性作用的比较研究J].铁道医学 2000;28(6):362-364.
[86]Gabizon A,Meshorer A,Bareaholz Y,et al.Comparative long-term study of the toxicity of free and liposome-associated doxorubicin in mice after intravenous administration[J].JNCI,1986,77(2):459-467.
[87]Rahamn A,White G,More N,et al.Pharmacological,toxicological and therapeutic evaluation in mice of doxorubicin entrapped in cardiolipin liposomes[J].Cancer Res,1985,45:796-801.
[88]Herman E H,Rahman A,Ferrans V J,et al.Prevention of chronic dox2orubicin cardiotoxicity in beagles by liposomal encapsulation[J].Cancer Res,1983,43:5427-31.
[89]Gregorio D,Ronald K,Joseph A T,et al.A phase Ⅰ/Ⅱ study of intraperitoneally administered doxorubicin entrapped in cardiolipin liposomes in patients with ovarian cancer[J].AmJ Obstet Gynecol,1989,160(4):812-9.
[90]Villani F,Piccinini F,Nerelli P,et al.Influence of adriamycin on caleinm exchangeability in cardiac muscle and its modification by ouabain[J].Biochem Pharmacol,1978,27:985-7.
[91]Rahman A,Kessur A,More N,et al.Liposomal protection of adriamycin- induced cardiotoxicity in mice[J].Cancer Res,1980,40:1532-35.
[92]Gabizon A,Dagan A,Goren D,et al.Liposome as in vivo carders of adriamycin:reduced cardiac uptake and preserved antitumor activity in mice[J].Cancer Res,1982,42:4734-9.
[93]马秉亮,吴玉林,刘国卿.P-gp及对抗P-gp介导多药耐药的研究现状J].中国临床药理学与治疗学,2006,11(1):14-19.
[94]段明华,王东凯,刘红,等.固体脂质纳米粒制备方法简介J].中国医院药学杂志,2004,24(7):432-433.
[95]李欣玮,孙立新,林晓宏,等.固体脂质纳米粒作为药物载体[J].化学进展,2007,19(1):87-92.
[96]Uchegbu IF,Double JA,Turton JA,et al.Distribution,metabolism and tumoricidal activity of doxorubicin administered in sorbitan monostearate(Span 60) niosomes in the mouse[J],Pharm Res,1995,12:1019-1024.
[97]Hao Y,Zhao F,Li N,et al.Studies on a high encapsulation of colchicine by a niosome system,Int J Pharm,2002,244:73-80.
[98]Jain CP,Vyas SP.Preparation and characterization of niosomes containing rifampicin for lung targeting[J],J Microencapsul.,1995,12:401-407.
[99]Fang JY,Yu SY,Wu PC,et al,In vitro skin permeation of estradiol from various proniosome formulations[J],Int J Pharm,2001,215:91-99.
[100]Pardakhty A,Varshosaz J,Rouholamini A,et al.In vitro studyof polyoxyethylene alkyl ether niosomes for delivery of insulin[J].Int J Pharm,2007,328(2):130-141.
[101]Fang JY,Hong CT,Chiu WT,et al.Effect of liposomes andniosomes on skin permeation of enoxacin[J].Int J Pharm,2001,219(1-2):61-72.
[102]Pardakhty A,Varshosaz J,Rouholamini A,et al.In vitro studyof polyoxyethylene alkyl ether niosomes for delivery of insulin[J].Int J Pharm,2007,328(2):130-141.
[103]Uchegbu IF,Vyas SP,Non-ionic surfactant based vesicles(niosomes) in drug delivery[J].Int.J.Pharm.,172(1998) 33-70.
[104]张磊,潘弘,刘敏,陆伟跃。阿霉素的不同盐型对其脂质体体外药物泄漏和体内长循环的影响J].药学学报.2004,39(12):1018-1022
[105]王黎,侯宝光,侯新朴,于美丽,杨江山。氢化与非氢化卵磷脂对阿霉素脂质体体内外稳定性的影响[J].药学学报。2001.36(6):444-447.
[106]柯爱武,李羲。脂质体逆转肿瘤多药耐药研究进展J]。国外医学预防诊断治疗用生物制品分册 2005;28(2):75-80.
[107]王永中.紫杉醉嵌段共聚物胶束给药系统克服种瘤多药耐药性的研究[博士学位论文].上海复旦大学.2007。1-2.
[1]Krishna R,Mayer LD.Multidrug resistance(MDR) in cancer.Mechanisms,reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs[J].Eur J Pharm Sci.2000;11:265-283.
[2]Advani R,Fisher GA,Lum BL,et al.A phase Ⅰ trial of doxorubicin,paclitaxel,and valspodar (PSC 833),a modulator of multidrug resistance[J].Clin Cancer Res.2001;7:1221-1229.
[3]Baekelandt M,Lehne G,Trope CG,et al.Phase Ⅰ/Ⅱ trial of the multidrug-resistance modulator valspodar combined with cisplatin and doxorubicin in refractory ovarian cancer[J].J Clin Oncol.2001;19:2983-2993.
[4]Chico I,Kang MH,Bergan R,et al.Phase Ⅰ study of infusional paclitaxel in combination with the P-glycoprotein antagonist PSC833[J].J Clin Oncol.2001;19:832-842.
[5]Xiong XB,Huang Y,Lu WL,et al.Enhanced intracellular delivery and improved antitumor efficacy of doxorubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic[J].J Control Release.2005;107(2):262-75.
[6]Pan XQ,Wang H,Shukla S,et al.Boron-containing folate receptor-targeted liposomes as potential delivery agents for neutron capture therapy[J].Bioconjug Chem,2002,13:435-442.
[7]Mastrobattista E,Crommelin DJ,Wilschut J,et al.Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumor cells[J].J Liposome Res,2002,12:57-65.
[8]马秉亮,吴玉林,刘国卿.P-gp及对抗P-gp介导多药耐药的研究现状[J].中国临床药理学与治疗学,2006,11(1):14-19.
[9]Wong HL,Bendayan R,Rauth AM,et al.A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system[J].J Pharmacol Exp Ther.2006 Jun;317(3):1372-81.
[10]Wong HL,Rauth AM,Bendayan R,et al.A new polymer-lipid hybrid nanoparticle system increases cytotoxicity of doxorubicin against multidrug-resistant human breast cancer cells[J].Pharm Res.2006 Jul;23(7):1574-85.
[11]应小英,胡富强,袁弘.卡马西平硬脂酸固体脂质纳米粒的制备与理化性质研究[J].中国医药工杂志,2002,33(11):543-346.
[12]瞿文,陈庆华,朱宝全.固体脂质纳米粒的研究进展J].中国医药工业杂志,2001,32(9):424-429.
[13]Cavalli R,Caputo,Gasco M.R,Solid lipospheres of doxorubicin and idarubicin[J],Int.J.Pharm.1993,89:R9-R12.
[14]Wong HL,Bendayan R,Rauth AM,et al.Simultaneous delivery of doxorubicin and GG918(Elacridar) by new polymer-lipid hybrid nanoparticles(PLN) for enhanced treatment of multidrug-resistant breast cancer[J].J Control Release.2006 Dec 1;116(3):275-84.
[15]Serpe L,Catalano MG,Cavalli R,et al.Cytotoxicity of anticancer drugs incorporated in solid lipid nanoparticles on HT-29 colorectal cancer cell line[J].Eur J Pharm Biopharm.2004;58(3):673-80.
[16]Miglietta A,Cavalli R,Bocca C,et al.Cellular uptake and cytotoxicity of solid lipid nanospheres(SLN) incorporating doxorubicin or paclitaxel[J]._Int J Pharm.2000;210(1-2):61-7.
[17]Serpe L,Catalano MG,Cavalli R,et al.Cytotoxicity of anticancer drugs incorporated in solid lipid nanoparticles on HT-29 colorectal cancer cell line[J].Eur J Pharm Biopharm.2004;58(3):673-80.
[18]Fundarb A,Cavalli R,Bargoni A,et al.Non-stealth and stealth solid lipid nanoparticles(SLN)carrying doxorubicin:pharmaeokinetics and tissue distribution after i.v.administration to rats[J].Pharmaeol Res.2000;42(4):337-43.
[19]Wong H.L,Bendayan R,Rauth.A.M,et al.A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle(PLN) system[J].Pharmcol.Exp.Ther 2006,317:1372-1381.
[20]刘扬,吕万良,张强.脂质体及纳米粒药物递送系统的研究进展[J].中国医学科学院学报,2006,28(4):583-589.