紫杉醇嵌段共聚物胶束给药系统克服肿瘤多药耐药性的研究
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摘要
肿瘤多药耐药性(Multidrug resistance，MDR)是临床上肿瘤化学治疗的最大障碍，也是化疗失败的主要原因之一，在肿瘤内科治疗中是一个亟待解决的问题。迄今，许多抗肿瘤药物在临床上都出现了多药耐药性。从给药系统的角度研究逆转肿瘤MDR是克服肿瘤多药耐药性的一种新思路。本文以聚合物胶束和紫杉醇(PTX)作为研究对象，旨在制备一种新型紫杉醇给药系统，既能增溶难溶性的紫杉醇，同时又具有克服肿瘤细胞耐药性的能力，以解决紫杉醇临床应用的两大问题：水难溶性与耐药性，为探索逆转肿瘤多药耐药性的新途径提供实验依据。
本文的主要内容包括：制备与表征了4种不同的紫杉醇聚合物胶束给药系统；体外评价了这些紫杉醇聚合物胶束制剂对人乳腺癌细胞或人卵巢癌细胞耐药性的逆转作用：同时以人卵巢癌耐药细胞为细胞模型，探讨了它们逆转肿瘤MDR的机理；最后考察了紫杉醇共聚物胶束系统的体内药动学、组织分布以及药效学。
首先，采用固体分散-水化法制备了紫杉醇Pluronic P105胶束(P105／PTX)，并在单因素考察的基础上，以包封率、载药量和胶束溶液中药物浓度为指标，采用三因素、五水平的星点设计-效应面优化法进行了PTX胶束处方优化，获得较优处方：P105为300mg，PTX为6mg，水相量为5mL，温度为70℃。优化后胶束的载药量大约为1％，药物浓度为650μg／ml，DLS测定的平均粒径为24nm左右。为了提高Pluronic P105对PTX的增溶能力，从两个角度出发对其进行了修饰：(1)在研究Pluronic P105与L101在水溶液中相互作用的基础上，确定以P105／L101=8：1的比例制备混合胶束；(2)采用聚己内酯(PCL)嵌段对PluronicP105进行了修饰，合成了三种PCL修饰的P105共聚物：P105／PCL5，P105／PCL20，P105／PCL50，以制备不同PCL嵌段修饰的P105胶束。进一步采用固体分散-水化法与透析法分别制备了P105／L101混合胶束、PCL修饰的Pluronic P105胶束(P105／PCL50／PTX)，两者对PTX的增溶能力均有较大的提高，制得的两种胶束载药量分别为2％和5％左右，药物浓度都可以达1000μg／mL，同时两者的粒径也有所增大，分别为185nm与150nm左右。体外释放实验表明，市售的Taxol注射液的PTX释放行为类似于PTX储备液，其在6h内累积释放了95.2％，而3种PTX胶束制剂(P105／PTX胶束，P105／L101／PTX混合胶束，P105／PCL50／PTX胶束)均具有一定的缓释作用。P105／PTX胶束在6 h内累积释放仅为45.4％(P＜0.05)。与P105胶束比较，P105／PCL50胶束与P105／L101混合胶束的释放加快，但与Taxol注射液比较，两者的释放则显著减慢(P＜0.05)。它们的缓释效果的大小顺序为：P105／PTX＞P105／PCL50／PTX≈P105／L101／PTX＞Taxol。
其次，采用MTT法，对紫杉醇聚合物胶束逆转人乳腺癌细胞和人卵巢癌细胞耐药性的作用进行了体外评价。结果表明，Pluronic P105／PTX和P105／L101／PTX胶束可显著提高PTX对人乳腺癌耐药细胞与人卵巢癌耐药细胞的细胞毒性，能不同程度地逆转二者的耐药性。对于人乳腺癌敏感细胞MCF-7，PTX溶液、Taxol、P105／PTX以及P105／L101／PTX的IC_(50)值分别在10.8-12.4ng/ml之间；对于人乳腺癌耐药细胞MCF-7／ADR，四者分别为425.7±69.8，88.4±6.5．30.1±4.4，19.3±5.4 ng／ml，后三者的耐药逆转指数(RRI)分别为4.82，14.10，22.10。对于人卵巢癌敏感细胞SKOV-3，四者IC_(50)值分别在0.11-0.17μg／ml之间：对于人卵巢癌耐药细胞SKOV-3/PTX，四者的IC_(50)值分别为11.1±2.9，5.11±1.78，1.144±0.07，0.47±0.11μg／ml，后三者的RR1分别为2.15，9.65，23.40。3种紫杉醇P105／PCL胶束亦可显著提高PTX对耐药细胞的细胞毒性，能不同程度地逆转人卵巢癌耐药细胞SKOV-3／PTX对PTX的耐药性。三种P105／PCL胶束制剂对SKOV-3的IC_(50)值在0.06-0.17μg／ml之间。对于SKOV-3／PTX而言，P105／PCL50／PTX、P105／PCL20／PTX以及P105／PCL5／PTX的IC_(50)值分别为0.20±0.05，0.41±0.08，0.67±0.24μg／ml。三者的RR1分别为55.00，26.83，16.42。
在以上研究的基础上，为了考察靶向胶束系统增强Pluronic胶束逆转肿瘤MDR的能力，制备了两种叶酸介导的Pluronic给药系统：FOL-P105/PTX与FOL-P105／L101／PTX。研究结果显示：与普通胶束比较，两种叶酸介导靶向胶束制剂(FOL-Pluronic／PTX)可以显著促进人乳腺癌耐药细胞MCF-7／ADR摄取PTX(P＜0.05)。1mM游离叶酸在90min时可以显著降低叶酸介导胶束的PTX摄取(P＜0.05)，但对普通胶束或PTX溶液的PTX摄取没有显著性的影响。两种叶酸介导胶束(FOL-Pluronic／PTX)对敏感细胞(MCF-7)和耐药细胞(MCF-7／ADR)的抑制作用都要比普通胶束强，可显著提高PTX对耐药细胞的细胞毒性(P＜0.05)。对敏感细胞和耐药细胞的IC_(50)值分别为6.4±0.6，9.4±2.6ng／ml(FOL-P105／PTX)以及5.8±2.6，7.5±1.5 ng／ml(FOL-P105／L101／PTX)。
以人卵巢癌耐药细胞(SKOV-3／PTX)为细胞模型，探讨了紫杉醇聚合物胶束制剂逆转肿瘤MDR的机理。研究结果显示，五种Pluronic载体材料(P105，P105／L101，P105／PCL5，P105／PCL20，P105／PCL50)都可以在一定浓度范围内不同程度地促进罗丹明-123(R-123)在耐药细胞内的蓄积。R123蓄积的增加是由于耐药细胞的P-糖蛋白(Pgp)外排活性被载体材料抑制所致。一方面，在较高浓度范围内(0.01％-1.0％)，5种载体材料都可以显著减低线粒体跨膜电位，耗竭细胞内ATP，从而进一步影响耐药细胞膜上Pgp的外排功能；另一方面，5种载体材料在低浓度范围(0.001-0.01％)以及高浓度范围(0.01％-1.0％)，都可以通过增强细胞膜的流动性来影响Pgp的外排活性。两种途径双管齐下，从而提高了R123的胞内蓄积量。进一步研究显示，P105／L101混合胶束制剂(PTX=10μg／mL Pluronic=0.25％)可以促进PTX诱导的耐药细胞凋亡，这也是胶束制剂增加耐药肿瘤细胞药敏性的另一机制。由载体材料导致的线粒体跨膜电位的降低，可能是其促进PTX诱导耐药细胞凋亡的内在原因。
为了进一步了解紫杉醇Pluronic胶束制剂逆转肿瘤MDR的分子机制，采用药物基因组学的研究方法考察了Pluronic P105／L101／PTX混合胶束制剂(PTX=10μg／mL Pluronic=0.25％)对耐药细胞SKOV-3／PTX全基因表达谱的影响。初步结果显示，Pluronic P105／L101胶束制剂对耐药细胞SKOV-3／PTX的基因表达谱的影响较大，四组样品共有2293个基因的表达发生了改变。对其中3类基因(细胞凋亡相关、耐药／代谢相关、细胞周期调控相关)进一步分析发现，Pluronic P105／L101胶束制剂虽然在一定程度上调节了一些有利于逆转耐药性的基因表达，同时又调节了另一些不利于逆转耐药性的基因表达。但是，总体而言，前者占的比重较大。
最后，进行了紫杉醇共聚物胶束制剂的体内研究。大鼠药动学研究结果表明，与Taxol制剂比较，紫杉醇的三种胶束制剂都具有不同的长循环作用。药时曲线下面积(AUC)的大小顺序为：P105／PTX＞P105／L101／PTX＞P105／PCL50／PTX＞Taxol；清除率(CL)的大小顺序为：Taxol＞P105／PCL50／PTX＞P105／L101／PTX＞P105／PTX；t_(1/2α)的大小顺序为：P105／PCL50／PTX＞P105／L101／PTX＞P105／PTX＞Taxol，t_(1/2β)的大小顺序为：P105／L101／PTX＞P105／PTX＞P105／PCL50／PTX＞Taxol。三种胶束制剂在体外的释药行为基本上与它们在体内的长循环作用具有一定的相关性。小鼠组织分布研究结果显示，与Taxol制剂比较，紫杉醇的三种胶束制剂在小鼠体内的分布不尽相同。相同之处在于：在小鼠血浆中的蓄积量都有不同程度的提高，在肝脏中有不同程度的降低，这进一步说明它们具有一定的缓释作用和长循环的特点。P105／PTX与其物理混合胶束P105／L101／PTX的分布比较接近，在肺、脾以及肾脏中分布较多。PCL修饰的P105胶束(P105／PCL50／PTX)的分布与本课题组前期研究的Pluronic P123／PTX胶束比较接近，在卵巢／子宫的蓄积都有较大的增加，在肺、脾与肾脏的分布也有不同程度的提高。
进一步采用PCL修饰的P105胶束(P105／PCL50／PTX)进行了体内逆转肿瘤MDR的药效学评价。初步研究结果显示，以12mg／kg剂量给药后，胶束制剂对耐药肿瘤SKOV-3／PTX的抑制作用比Taxol强，两者具有显著性差异(P＜0.05)。相对生理盐水组的平均瘤重，Taxol组对肿瘤生长抑制率为43.9％，PTX胶束组为63.4％．初步判断，P105／PCL50／PTX胶束具有一定程度的逆转肿瘤耐药性的作用。
Multidrug resistance of cancer cells is a major cause for the failure of anti-cancerchemotherapy in the treatment of cancer patients. So far, various anticancer drugswhich are most frequently associated with multidrug resistance (MDR) includetaxanes, vinca alkaloids, anthracyclines, epipodophyllotoxins, antimetabolites,topotecan, dactinomycin and mitomycin C. Establishment of various strategies andapproaches to inhibit or circumvent MDR and hence to enhance the efficacy ofanticancer drugs is one of the significant missions in the area of tumor therapy. Drugdelivery system (DDS) is a novel approach to overcoming MDR in cancer nowadays.The main aims of this research were to investigate a novel mieellar delivery system toovercome MDR in cancer, based on Pluronic and paxlitaxel, which will provide thebasis for future studies of overcoming drug resistance and ultimately improvingchemotherapy and the outcome of cancer patients.
The contents of this thesis were including (1) preparation and characterization ofpolymeric mieellar delivery systems of paelitaxel; (2) evaluation of reversing MDReffect of the polymeric micelles with the resistant cancer cell lines MCF-7/ADR orSKOV-3/PTX; (3) investigation of the mechanism of overcoming MDR with theresistant cancer cell lines SKOV-3/PTX; (4) study of pharmaeokineties,biodistribution and in vivo anticaneer efficacy of the micellar delivery system ofpaclitaxel.
PTX-loaded polymeric micelles were prepared with Pluronic P105 by thin-filmhydration methods. Based on the results of single factor experiments, with theevaluation index of entrapment efficiency, drug loading coefficient and drugconcentration of the micelle solution, micelle formulation was optimized employingthe central composite design-response surface methodology. The final optimizedformulation was 6mg of PTX, 300mg cartier, 5 mL of water phase and 70℃temperature of hydration. Studies of the physieo-chemical property of micellesshowed that the mieelle size was about ca. 24 nm with drug loading coefficient of ca.1％and PTX concentration of ca. 650μg/ml. In order to improve the ability ofsolubilizing PTX, Pluronic P105 was modified with two strategies. One approach wasto prepare a mixed P105/L101 micelle with P105:L101=8:1(v/v) based on the resultof interaction of binary Pluronic P105 and L101 in aqueous solution. Another approach was to synthesis PCL-P105-PCL and then to prepare the micelles with threedifferent PCL-P 105-PCL block copolymers(P 105/PCL5, P 105/PCL20, P 105/PCL50).The mixed P105/L101 micelle showed high solubilization capacity for PTX with drugloading coefficient of ca. 2％, PTX concentration of ca. 1000μg/ml and micelle size ofca. 185nm. The P105/PCL50 micelle showed higher solubilization capacity for PTXwith drug loading coefficient of ca. 5％, PTX concentration of ca. 1000μg/ml andmicelle size of ca. 150nm. The in vitro release experiment showed that three micellarPTXs (P105/PTX, P105/L101/PTX P105/PCL50/PTX) sustained the release of PTXfrom micelles. The release profile of PTX from Taxol was similar with the PTX stocksolution. The cumulative release amount of PTX from Taxol in 6 h was around 95.2％.Pluronic P105 micelles released only 45.4％PTX in 6 h (P＜0.05) and 79.6％PTX in24 h, which was much slower than Taxol. The release of PTX from P105/PCL50/PTXand P105/L101/PTX were faster than P105/PTX, but they were much slower thanTaxol(P＜0.05). The release rates of four PTX formulations in an ascending order wereP105/PTX, P105/PCL50/PTX, P105/L101/PTX, Taxol.
The cytotoxicity of polymeric micellar PTX was assessed against human breastcancer cell line (MCF-7, MCF-7/ADR) or human ovarian cancer cell line (SKOV-3,SKOV-3/PTX) by a standard MTT assay. The results demonstrated that Pluronicmicellar PTX (P105/PTX or P105/L101/PTX) were able to reverse resistance to PTXin MCF-7/ADR and SKOV-3/PTX tumor cells compared with free PTX solution. Inthe case of human ovarian cancer cell, the IC_(50) of PTX solution(1％DMSO), Taxol,P105/PTX and P105/L101/PTX against MCF-7/ADR were ca 425.7±69.8, 88.4±6.5,30.1±4.4, 19.3±5.4ng/ml, respectively, comparing with the IC_(50) value ranging from10.8 to 12.4ng/ml against MCF-7. The resistance reversion indexes (RRI) of Taxol,P105/PTX and P105/L101/PTX against MCF-7/ADR were ca. 4.82, 14.10, 22.10,respectively. In the ease of human ovarian cancer cell line, the IC_(50) of PTXsolution(1％DMSO), Taxol, P105/PTX and P105/L101/PTX against SKOV-3/PTXwere ca 11.1±2.9, 5.11±1.78, 1.14±0.07, 0.47±0.11μg/ml, respectively, comparingwith the IC_(50) value ranging from 0.11-0.17μg/ml against SKOV-3. The resistancereversion index (RRI) of Taxol, P105/PTX and P105/L101/PTX againstSKOV-3/PTX was ca. 2.15, 9.65, 23.40, respectively. Three P 105-PCL micellar PTXs(i.e. P105/PCL5/PTX, P105/PCL20/PTX or P105/PCL50 /PTX) were also able toreverse resistance to PTX in SKOV-3/PTX tumor cells compared with free PTXsolution. IC_(50) of P105-PCL50/PTX, P105-PCL20/PTX and P105-PCL5/PTX against SKOV-3/PTX were ca 0.20±0.05, 0.41±0.08, 0.67±0.24μg/ml, respectively,comparing with the IC_(50) ranging from 0.06-0.17μg/ml against SKOV-3. Theresistance reversion indexes (RRI) of three micelles against SKOV-3/PTX were ca.55.00, 26.83, 16.42, respectively.
Based on the experimental results mentioned above, two folate-mediatedPluronic/PTX micelles (FOL-P105/PTX, FOL-PI05/L101/PTX) were prepared inorder to investigate whether folate-mediated micellar PTXs increase theircytotoxicities in tumor cells. The results demonstrated that FOL-micellar PTX had asignificant increase in cellular uptake compared with a plain micellar PTX during 2.5h incubation with MCF-7/ADR cells (P＜0.05). 1 mM free folic acid significantlyreduced the PTX uptake in MCF-7/ADR cells incubated with FOL-micellar PTX for90 min (P＜0.05), but had no significant effect on PTX uptake in the case of plainmicellar PTX or free PTX. Two FOL-micellar PTXs significantly enhanced thecytotoxicity of PTX against MCF-7 and MCF-7/ADR cells compared with plainmicellar PTX or free PTX solution. IC_(50) of FOL-micellar PTXs against MCF-7 andMCF-7/ADR cells were 6.4±0.6, 9.4±2.6 ng/ml (FOL-P105/PTX) and 5.8±2.6,7.5±1.5 ng/ml(FOL-P105/L101/PTX), respectively.
With PTX-resistant SKOV-3 tumor cells as an in vitro model of MDR tumor cells,a combination of experiments examining effects of five carriers (P 105, P 105/L 101,P105/PCL5, P105/PCL20, P105/PCL50 ) on R123 accumulation, membranemicroviscosity, Pgp ATPase activity, intracellular ATP and mitochondrialtransmembrane potential in SKOV-3/PTX tumor cells was used to uncover themechanism of action of the micellar formulation based on Pluronic P105. The resultsshowed the five carriers over the different experimental concentration range enhancedthe R123 accumulation in SKOV-3/PTX tumor cells, which was believed to correlatewith inhibition of Pgp in SKOV3/PTX cells, because R123, a substrate of Pgp, wascommonly used for evaluation of the Pgp mediated drug efflux in MDR cancer cells.Five carriers not only caused a dramatic decrease in the intracellular ATP levels and aloss in mitochondrial transmembrane potential, but also induced drastic decrease inthe membrane microviscosity and inhibitory effect on Pgp ATPase activity over thedifferent experimental concentration range. These results indicated that themechanism of reversal of the MDR cancer cells by these polymers involved ATPdepletion, the decrease in membrane microviscosity, and the interaction of thepolymer molecules with cell membranes accompanied by significant inhibition of Pgp ATPase activity, which all had a combined result of potent inhibition of Pgp inSKOV-3/PTX tumor cells. In addition to inhibition of Pgp efflux system, PluronicP105/L101/PTX formulation (PTX=10μg/mL Pluronic=0.25％) induced apoptosis inthe resistant SKOV-3/PTX cancer cells, which was believed to be another mechanismof action of the micellar formulation sensitizing MDR tumor cells. Decrease ofmitochondrial transmembrane potential was believed to be one of the earlyintracellular events of apoptosis induced by Pluronic P 105/L101 micelles.
In order to understand the mechanisms of Pluronic effects in various cells better,further studies were carried out to correlate the global gene expression profiles ofSKOV-3/PTX and sensitizing responses to Pluronic formulations applying thepharmacogenomic approach. The results showed that there were significantlydifferent changes in the global gene expression profiles of SKOV-3/PTX exposure tomixed P105/L101/PTX micelle (PTX=10μg/mL Pluronic=0.25％), PTX solution(1％DMSO) or blank control (DMEM medium). There were 2293 differentlyexpressed genes with four samples. Further analysis was carried out to investigate thedifferently expressed genes involved in apoptosis, drug resistance/metabolism and cellcycle. The results demonstrated that formulation of the drug with Pluronic P 105/L101could affect the expression of those genes, of which most of genes as a growth-suppressing signal could inhibit tumor growth and enhanced apoptosis, butsimultaneously some genes as a growth-enhancing signal could enhance tumorgrowth.
Pharmacokinetics and biodistribution of the three polymeric micelles (P105/PTX,P105/L101/PTX, P105/PCL50/PTX) were assessed by i.v. administration of Taxolinjection and the three polymeric micelles to rats and mice, respectively. The resultsof pharmacokinetics study in rats indicated that the three polymeric micelles couldalter the parameters of pharmacokinetics of paclitaxel. The blood circulation time ofpaclitaxel loaded in the three polymeric micelles was significantly prolongedcompared to Taxol injection. The AUCs of four PTX formulations in a descendingorder were P105/PTX＞P105/L101/PTX＞P105/PCL50/PTX＞Taxol; The CLs in adescending order were Taxol＞P105/PCL50/PTX＞P105/L101/PTX＞P105/PTX; t_(/2a)in a descending order were P105/PCL50/PTX＞P105/L101/PTX＞P105/PTX＞Taxol, t_(1/2β) in a descending order were P105/L101/PTX＞P105/PTX＞P105/PCL50/PTX＞Taxolo The in vitro release behaviors of the three polymericmicelles were basically correlated to the prolonged blood circulation time of three micellar paclitaxel in vivo. The study of biodistrabution in mice showed the drugaccumulation of the three polymeric micelles in plasma was increased and the drugaccumulation of the three in liver was decreased, which also indicated the sustainedrelease of the three micelles in vivo. The drug accumulation of P105/PTX andP105/L101/PTX in lung, spleen and kindey was significantly increased. Like theP123/PTX, the drug accumulation of P105/PCL50/PTX in lung, spleen, kindey andovary/uterus was increased.
Further studies were carried out to investigate whether P105/PCL50/PTX couldinhibit the growth of s.c. human ovarian SKOV-3/PTX carcinoma xenografts inBALB/c nude mice compared with reference Taxol injection. The results indicatedthat the polymeric micellar PTX showed more potent inhibition of tumor growth thanTaxol injection with 12 mg/kg PTX equivalent dose (P＜0.05). Inhibition ratio(％) oftumor growth was 43.9％and 63.4％for Taxol injection and the polymeric micellarPTX, respectively, which demonstrated that P105/PCL50/PTX could significantlyinhibit the growth of resistant SKOV-3/PTX tumor in BALB/c nude mice.
本文的主要内容包括：制备与表征了4种不同的紫杉醇聚合物胶束给药系统；体外评价了这些紫杉醇聚合物胶束制剂对人乳腺癌细胞或人卵巢癌细胞耐药性的逆转作用：同时以人卵巢癌耐药细胞为细胞模型，探讨了它们逆转肿瘤MDR的机理；最后考察了紫杉醇共聚物胶束系统的体内药动学、组织分布以及药效学。
首先，采用固体分散-水化法制备了紫杉醇Pluronic P105胶束(P105／PTX)，并在单因素考察的基础上，以包封率、载药量和胶束溶液中药物浓度为指标，采用三因素、五水平的星点设计-效应面优化法进行了PTX胶束处方优化，获得较优处方：P105为300mg，PTX为6mg，水相量为5mL，温度为70℃。优化后胶束的载药量大约为1％，药物浓度为650μg／ml，DLS测定的平均粒径为24nm左右。为了提高Pluronic P105对PTX的增溶能力，从两个角度出发对其进行了修饰：(1)在研究Pluronic P105与L101在水溶液中相互作用的基础上，确定以P105／L101=8：1的比例制备混合胶束；(2)采用聚己内酯(PCL)嵌段对PluronicP105进行了修饰，合成了三种PCL修饰的P105共聚物：P105／PCL5，P105／PCL20，P105／PCL50，以制备不同PCL嵌段修饰的P105胶束。进一步采用固体分散-水化法与透析法分别制备了P105／L101混合胶束、PCL修饰的Pluronic P105胶束(P105／PCL50／PTX)，两者对PTX的增溶能力均有较大的提高，制得的两种胶束载药量分别为2％和5％左右，药物浓度都可以达1000μg／mL，同时两者的粒径也有所增大，分别为185nm与150nm左右。体外释放实验表明，市售的Taxol注射液的PTX释放行为类似于PTX储备液，其在6h内累积释放了95.2％，而3种PTX胶束制剂(P105／PTX胶束，P105／L101／PTX混合胶束，P105／PCL50／PTX胶束)均具有一定的缓释作用。P105／PTX胶束在6 h内累积释放仅为45.4％(P＜0.05)。与P105胶束比较，P105／PCL50胶束与P105／L101混合胶束的释放加快，但与Taxol注射液比较，两者的释放则显著减慢(P＜0.05)。它们的缓释效果的大小顺序为：P105／PTX＞P105／PCL50／PTX≈P105／L101／PTX＞Taxol。
其次，采用MTT法，对紫杉醇聚合物胶束逆转人乳腺癌细胞和人卵巢癌细胞耐药性的作用进行了体外评价。结果表明，Pluronic P105／PTX和P105／L101／PTX胶束可显著提高PTX对人乳腺癌耐药细胞与人卵巢癌耐药细胞的细胞毒性，能不同程度地逆转二者的耐药性。对于人乳腺癌敏感细胞MCF-7，PTX溶液、Taxol、P105／PTX以及P105／L101／PTX的IC_(50)值分别在10.8-12.4ng/ml之间；对于人乳腺癌耐药细胞MCF-7／ADR，四者分别为425.7±69.8，88.4±6.5．30.1±4.4，19.3±5.4 ng／ml，后三者的耐药逆转指数(RRI)分别为4.82，14.10，22.10。对于人卵巢癌敏感细胞SKOV-3，四者IC_(50)值分别在0.11-0.17μg／ml之间：对于人卵巢癌耐药细胞SKOV-3/PTX，四者的IC_(50)值分别为11.1±2.9，5.11±1.78，1.144±0.07，0.47±0.11μg／ml，后三者的RR1分别为2.15，9.65，23.40。3种紫杉醇P105／PCL胶束亦可显著提高PTX对耐药细胞的细胞毒性，能不同程度地逆转人卵巢癌耐药细胞SKOV-3／PTX对PTX的耐药性。三种P105／PCL胶束制剂对SKOV-3的IC_(50)值在0.06-0.17μg／ml之间。对于SKOV-3／PTX而言，P105／PCL50／PTX、P105／PCL20／PTX以及P105／PCL5／PTX的IC_(50)值分别为0.20±0.05，0.41±0.08，0.67±0.24μg／ml。三者的RR1分别为55.00，26.83，16.42。
在以上研究的基础上，为了考察靶向胶束系统增强Pluronic胶束逆转肿瘤MDR的能力，制备了两种叶酸介导的Pluronic给药系统：FOL-P105/PTX与FOL-P105／L101／PTX。研究结果显示：与普通胶束比较，两种叶酸介导靶向胶束制剂(FOL-Pluronic／PTX)可以显著促进人乳腺癌耐药细胞MCF-7／ADR摄取PTX(P＜0.05)。1mM游离叶酸在90min时可以显著降低叶酸介导胶束的PTX摄取(P＜0.05)，但对普通胶束或PTX溶液的PTX摄取没有显著性的影响。两种叶酸介导胶束(FOL-Pluronic／PTX)对敏感细胞(MCF-7)和耐药细胞(MCF-7／ADR)的抑制作用都要比普通胶束强，可显著提高PTX对耐药细胞的细胞毒性(P＜0.05)。对敏感细胞和耐药细胞的IC_(50)值分别为6.4±0.6，9.4±2.6ng／ml(FOL-P105／PTX)以及5.8±2.6，7.5±1.5 ng／ml(FOL-P105／L101／PTX)。
以人卵巢癌耐药细胞(SKOV-3／PTX)为细胞模型，探讨了紫杉醇聚合物胶束制剂逆转肿瘤MDR的机理。研究结果显示，五种Pluronic载体材料(P105，P105／L101，P105／PCL5，P105／PCL20，P105／PCL50)都可以在一定浓度范围内不同程度地促进罗丹明-123(R-123)在耐药细胞内的蓄积。R123蓄积的增加是由于耐药细胞的P-糖蛋白(Pgp)外排活性被载体材料抑制所致。一方面，在较高浓度范围内(0.01％-1.0％)，5种载体材料都可以显著减低线粒体跨膜电位，耗竭细胞内ATP，从而进一步影响耐药细胞膜上Pgp的外排功能；另一方面，5种载体材料在低浓度范围(0.001-0.01％)以及高浓度范围(0.01％-1.0％)，都可以通过增强细胞膜的流动性来影响Pgp的外排活性。两种途径双管齐下，从而提高了R123的胞内蓄积量。进一步研究显示，P105／L101混合胶束制剂(PTX=10μg／mL Pluronic=0.25％)可以促进PTX诱导的耐药细胞凋亡，这也是胶束制剂增加耐药肿瘤细胞药敏性的另一机制。由载体材料导致的线粒体跨膜电位的降低，可能是其促进PTX诱导耐药细胞凋亡的内在原因。
为了进一步了解紫杉醇Pluronic胶束制剂逆转肿瘤MDR的分子机制，采用药物基因组学的研究方法考察了Pluronic P105／L101／PTX混合胶束制剂(PTX=10μg／mL Pluronic=0.25％)对耐药细胞SKOV-3／PTX全基因表达谱的影响。初步结果显示，Pluronic P105／L101胶束制剂对耐药细胞SKOV-3／PTX的基因表达谱的影响较大，四组样品共有2293个基因的表达发生了改变。对其中3类基因(细胞凋亡相关、耐药／代谢相关、细胞周期调控相关)进一步分析发现，Pluronic P105／L101胶束制剂虽然在一定程度上调节了一些有利于逆转耐药性的基因表达，同时又调节了另一些不利于逆转耐药性的基因表达。但是，总体而言，前者占的比重较大。
最后，进行了紫杉醇共聚物胶束制剂的体内研究。大鼠药动学研究结果表明，与Taxol制剂比较，紫杉醇的三种胶束制剂都具有不同的长循环作用。药时曲线下面积(AUC)的大小顺序为：P105／PTX＞P105／L101／PTX＞P105／PCL50／PTX＞Taxol；清除率(CL)的大小顺序为：Taxol＞P105／PCL50／PTX＞P105／L101／PTX＞P105／PTX；t_(1/2α)的大小顺序为：P105／PCL50／PTX＞P105／L101／PTX＞P105／PTX＞Taxol，t_(1/2β)的大小顺序为：P105／L101／PTX＞P105／PTX＞P105／PCL50／PTX＞Taxol。三种胶束制剂在体外的释药行为基本上与它们在体内的长循环作用具有一定的相关性。小鼠组织分布研究结果显示，与Taxol制剂比较，紫杉醇的三种胶束制剂在小鼠体内的分布不尽相同。相同之处在于：在小鼠血浆中的蓄积量都有不同程度的提高，在肝脏中有不同程度的降低，这进一步说明它们具有一定的缓释作用和长循环的特点。P105／PTX与其物理混合胶束P105／L101／PTX的分布比较接近，在肺、脾以及肾脏中分布较多。PCL修饰的P105胶束(P105／PCL50／PTX)的分布与本课题组前期研究的Pluronic P123／PTX胶束比较接近，在卵巢／子宫的蓄积都有较大的增加，在肺、脾与肾脏的分布也有不同程度的提高。
进一步采用PCL修饰的P105胶束(P105／PCL50／PTX)进行了体内逆转肿瘤MDR的药效学评价。初步研究结果显示，以12mg／kg剂量给药后，胶束制剂对耐药肿瘤SKOV-3／PTX的抑制作用比Taxol强，两者具有显著性差异(P＜0.05)。相对生理盐水组的平均瘤重，Taxol组对肿瘤生长抑制率为43.9％，PTX胶束组为63.4％．初步判断，P105／PCL50／PTX胶束具有一定程度的逆转肿瘤耐药性的作用。
Multidrug resistance of cancer cells is a major cause for the failure of anti-cancerchemotherapy in the treatment of cancer patients. So far, various anticancer drugswhich are most frequently associated with multidrug resistance (MDR) includetaxanes, vinca alkaloids, anthracyclines, epipodophyllotoxins, antimetabolites,topotecan, dactinomycin and mitomycin C. Establishment of various strategies andapproaches to inhibit or circumvent MDR and hence to enhance the efficacy ofanticancer drugs is one of the significant missions in the area of tumor therapy. Drugdelivery system (DDS) is a novel approach to overcoming MDR in cancer nowadays.The main aims of this research were to investigate a novel mieellar delivery system toovercome MDR in cancer, based on Pluronic and paxlitaxel, which will provide thebasis for future studies of overcoming drug resistance and ultimately improvingchemotherapy and the outcome of cancer patients.
The contents of this thesis were including (1) preparation and characterization ofpolymeric mieellar delivery systems of paelitaxel; (2) evaluation of reversing MDReffect of the polymeric micelles with the resistant cancer cell lines MCF-7/ADR orSKOV-3/PTX; (3) investigation of the mechanism of overcoming MDR with theresistant cancer cell lines SKOV-3/PTX; (4) study of pharmaeokineties,biodistribution and in vivo anticaneer efficacy of the micellar delivery system ofpaclitaxel.
PTX-loaded polymeric micelles were prepared with Pluronic P105 by thin-filmhydration methods. Based on the results of single factor experiments, with theevaluation index of entrapment efficiency, drug loading coefficient and drugconcentration of the micelle solution, micelle formulation was optimized employingthe central composite design-response surface methodology. The final optimizedformulation was 6mg of PTX, 300mg cartier, 5 mL of water phase and 70℃temperature of hydration. Studies of the physieo-chemical property of micellesshowed that the mieelle size was about ca. 24 nm with drug loading coefficient of ca.1％and PTX concentration of ca. 650μg/ml. In order to improve the ability ofsolubilizing PTX, Pluronic P105 was modified with two strategies. One approach wasto prepare a mixed P105/L101 micelle with P105:L101=8:1(v/v) based on the resultof interaction of binary Pluronic P105 and L101 in aqueous solution. Another approach was to synthesis PCL-P105-PCL and then to prepare the micelles with threedifferent PCL-P 105-PCL block copolymers(P 105/PCL5, P 105/PCL20, P 105/PCL50).The mixed P105/L101 micelle showed high solubilization capacity for PTX with drugloading coefficient of ca. 2％, PTX concentration of ca. 1000μg/ml and micelle size ofca. 185nm. The P105/PCL50 micelle showed higher solubilization capacity for PTXwith drug loading coefficient of ca. 5％, PTX concentration of ca. 1000μg/ml andmicelle size of ca. 150nm. The in vitro release experiment showed that three micellarPTXs (P105/PTX, P105/L101/PTX P105/PCL50/PTX) sustained the release of PTXfrom micelles. The release profile of PTX from Taxol was similar with the PTX stocksolution. The cumulative release amount of PTX from Taxol in 6 h was around 95.2％.Pluronic P105 micelles released only 45.4％PTX in 6 h (P＜0.05) and 79.6％PTX in24 h, which was much slower than Taxol. The release of PTX from P105/PCL50/PTXand P105/L101/PTX were faster than P105/PTX, but they were much slower thanTaxol(P＜0.05). The release rates of four PTX formulations in an ascending order wereP105/PTX, P105/PCL50/PTX, P105/L101/PTX, Taxol.
The cytotoxicity of polymeric micellar PTX was assessed against human breastcancer cell line (MCF-7, MCF-7/ADR) or human ovarian cancer cell line (SKOV-3,SKOV-3/PTX) by a standard MTT assay. The results demonstrated that Pluronicmicellar PTX (P105/PTX or P105/L101/PTX) were able to reverse resistance to PTXin MCF-7/ADR and SKOV-3/PTX tumor cells compared with free PTX solution. Inthe case of human ovarian cancer cell, the IC_(50) of PTX solution(1％DMSO), Taxol,P105/PTX and P105/L101/PTX against MCF-7/ADR were ca 425.7±69.8, 88.4±6.5,30.1±4.4, 19.3±5.4ng/ml, respectively, comparing with the IC_(50) value ranging from10.8 to 12.4ng/ml against MCF-7. The resistance reversion indexes (RRI) of Taxol,P105/PTX and P105/L101/PTX against MCF-7/ADR were ca. 4.82, 14.10, 22.10,respectively. In the ease of human ovarian cancer cell line, the IC_(50) of PTXsolution(1％DMSO), Taxol, P105/PTX and P105/L101/PTX against SKOV-3/PTXwere ca 11.1±2.9, 5.11±1.78, 1.14±0.07, 0.47±0.11μg/ml, respectively, comparingwith the IC_(50) value ranging from 0.11-0.17μg/ml against SKOV-3. The resistancereversion index (RRI) of Taxol, P105/PTX and P105/L101/PTX againstSKOV-3/PTX was ca. 2.15, 9.65, 23.40, respectively. Three P 105-PCL micellar PTXs(i.e. P105/PCL5/PTX, P105/PCL20/PTX or P105/PCL50 /PTX) were also able toreverse resistance to PTX in SKOV-3/PTX tumor cells compared with free PTXsolution. IC_(50) of P105-PCL50/PTX, P105-PCL20/PTX and P105-PCL5/PTX against SKOV-3/PTX were ca 0.20±0.05, 0.41±0.08, 0.67±0.24μg/ml, respectively,comparing with the IC_(50) ranging from 0.06-0.17μg/ml against SKOV-3. Theresistance reversion indexes (RRI) of three micelles against SKOV-3/PTX were ca.55.00, 26.83, 16.42, respectively.
Based on the experimental results mentioned above, two folate-mediatedPluronic/PTX micelles (FOL-P105/PTX, FOL-PI05/L101/PTX) were prepared inorder to investigate whether folate-mediated micellar PTXs increase theircytotoxicities in tumor cells. The results demonstrated that FOL-micellar PTX had asignificant increase in cellular uptake compared with a plain micellar PTX during 2.5h incubation with MCF-7/ADR cells (P＜0.05). 1 mM free folic acid significantlyreduced the PTX uptake in MCF-7/ADR cells incubated with FOL-micellar PTX for90 min (P＜0.05), but had no significant effect on PTX uptake in the case of plainmicellar PTX or free PTX. Two FOL-micellar PTXs significantly enhanced thecytotoxicity of PTX against MCF-7 and MCF-7/ADR cells compared with plainmicellar PTX or free PTX solution. IC_(50) of FOL-micellar PTXs against MCF-7 andMCF-7/ADR cells were 6.4±0.6, 9.4±2.6 ng/ml (FOL-P105/PTX) and 5.8±2.6,7.5±1.5 ng/ml(FOL-P105/L101/PTX), respectively.
With PTX-resistant SKOV-3 tumor cells as an in vitro model of MDR tumor cells,a combination of experiments examining effects of five carriers (P 105, P 105/L 101,P105/PCL5, P105/PCL20, P105/PCL50 ) on R123 accumulation, membranemicroviscosity, Pgp ATPase activity, intracellular ATP and mitochondrialtransmembrane potential in SKOV-3/PTX tumor cells was used to uncover themechanism of action of the micellar formulation based on Pluronic P105. The resultsshowed the five carriers over the different experimental concentration range enhancedthe R123 accumulation in SKOV-3/PTX tumor cells, which was believed to correlatewith inhibition of Pgp in SKOV3/PTX cells, because R123, a substrate of Pgp, wascommonly used for evaluation of the Pgp mediated drug efflux in MDR cancer cells.Five carriers not only caused a dramatic decrease in the intracellular ATP levels and aloss in mitochondrial transmembrane potential, but also induced drastic decrease inthe membrane microviscosity and inhibitory effect on Pgp ATPase activity over thedifferent experimental concentration range. These results indicated that themechanism of reversal of the MDR cancer cells by these polymers involved ATPdepletion, the decrease in membrane microviscosity, and the interaction of thepolymer molecules with cell membranes accompanied by significant inhibition of Pgp ATPase activity, which all had a combined result of potent inhibition of Pgp inSKOV-3/PTX tumor cells. In addition to inhibition of Pgp efflux system, PluronicP105/L101/PTX formulation (PTX=10μg/mL Pluronic=0.25％) induced apoptosis inthe resistant SKOV-3/PTX cancer cells, which was believed to be another mechanismof action of the micellar formulation sensitizing MDR tumor cells. Decrease ofmitochondrial transmembrane potential was believed to be one of the earlyintracellular events of apoptosis induced by Pluronic P 105/L101 micelles.
In order to understand the mechanisms of Pluronic effects in various cells better,further studies were carried out to correlate the global gene expression profiles ofSKOV-3/PTX and sensitizing responses to Pluronic formulations applying thepharmacogenomic approach. The results showed that there were significantlydifferent changes in the global gene expression profiles of SKOV-3/PTX exposure tomixed P105/L101/PTX micelle (PTX=10μg/mL Pluronic=0.25％), PTX solution(1％DMSO) or blank control (DMEM medium). There were 2293 differentlyexpressed genes with four samples. Further analysis was carried out to investigate thedifferently expressed genes involved in apoptosis, drug resistance/metabolism and cellcycle. The results demonstrated that formulation of the drug with Pluronic P 105/L101could affect the expression of those genes, of which most of genes as a growth-suppressing signal could inhibit tumor growth and enhanced apoptosis, butsimultaneously some genes as a growth-enhancing signal could enhance tumorgrowth.
Pharmacokinetics and biodistribution of the three polymeric micelles (P105/PTX,P105/L101/PTX, P105/PCL50/PTX) were assessed by i.v. administration of Taxolinjection and the three polymeric micelles to rats and mice, respectively. The resultsof pharmacokinetics study in rats indicated that the three polymeric micelles couldalter the parameters of pharmacokinetics of paclitaxel. The blood circulation time ofpaclitaxel loaded in the three polymeric micelles was significantly prolongedcompared to Taxol injection. The AUCs of four PTX formulations in a descendingorder were P105/PTX＞P105/L101/PTX＞P105/PCL50/PTX＞Taxol; The CLs in adescending order were Taxol＞P105/PCL50/PTX＞P105/L101/PTX＞P105/PTX; t_(/2a)in a descending order were P105/PCL50/PTX＞P105/L101/PTX＞P105/PTX＞Taxol, t_(1/2β) in a descending order were P105/L101/PTX＞P105/PTX＞P105/PCL50/PTX＞Taxolo The in vitro release behaviors of the three polymericmicelles were basically correlated to the prolonged blood circulation time of three micellar paclitaxel in vivo. The study of biodistrabution in mice showed the drugaccumulation of the three polymeric micelles in plasma was increased and the drugaccumulation of the three in liver was decreased, which also indicated the sustainedrelease of the three micelles in vivo. The drug accumulation of P105/PTX andP105/L101/PTX in lung, spleen and kindey was significantly increased. Like theP123/PTX, the drug accumulation of P105/PCL50/PTX in lung, spleen, kindey andovary/uterus was increased.
Further studies were carried out to investigate whether P105/PCL50/PTX couldinhibit the growth of s.c. human ovarian SKOV-3/PTX carcinoma xenografts inBALB/c nude mice compared with reference Taxol injection. The results indicatedthat the polymeric micellar PTX showed more potent inhibition of tumor growth thanTaxol injection with 12 mg/kg PTX equivalent dose (P＜0.05). Inhibition ratio(％) oftumor growth was 43.9％and 63.4％for Taxol injection and the polymeric micellarPTX, respectively, which demonstrated that P105/PCL50/PTX could significantlyinhibit the growth of resistant SKOV-3/PTX tumor in BALB/c nude mice.
引文
1. Filipits, M., Mechanisms of cancer: multidrug resistance. Drug Discovery Today: Disease Mechanisms, 2004. 1(2): p. 229-234.
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