姜黄素聚氰基丙烯酸正丁酯纳米粒抗癌活性及逆转多药耐药研究
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摘要
目的：
目前,抗肿瘤药物普遍存在药物靶向性差和全身毒性问题,限制了药物疗效的发挥和长期使用。而且普遍存在的肿瘤多药耐药(mu1tidrug resistance, MDR)也是治疗失败的主要原因。目前,体外逆转MDR的药物有很多,但是存在着体内不稳定,肿瘤靶点性差,毒副作用大等缺点。
近年来国内外学者开始从中药中筛选提取低毒的可逆转MDR的有效成分。姜黄素(curcumin, CUR)是常用中药姜黄(curcuma)中一个生物活性成分,具有多方面的药理作用,被认为是理想的抗癌化疗药物之一,具有逆转MDR的作用。作为中药逆转剂又较其他化药逆转剂有很大的优越性。但是姜黄素存在水溶性差,体内吸收少、不稳定,易降解等缺陷,因此限制了其应用。
生物纳米载体的应用不仅改善药物的溶解度,提高药物的生物利用度,而且还具有缓释性,延长药物体内滞留时间和靶向性,从而降低药物的毒性。
本研究的目的就是基于生物纳米载体的优势以及姜黄素的药学特点,筛选最佳的生物纳米材料,制备载姜黄素的纳米粒,并考察该纳米粒的抗癌活性及对人乳腺癌阿霉素耐药细胞MCF-7/ADR的逆转作用机制,为制备高效、长效、靶向和低毒的姜黄素奠定基础,为探索逆转MDR提供一条思路。
方法：
(1) CUR-PBCA-NPs的制备及表征
采用阴离子乳化聚合法制备CUR-PBCA-NPs,通过单因素实验结合正交试验对CUR-PBCA-NPs的制备工艺进行了优化,考察稳定剂的种类、反应过程的pH值、单体的用量、稳定剂的用量及药物浓度对纳米粒的粒径、表面电位和包封率的影响,确定最佳制备工艺。
透射电子显微镜和激光粒度分析仪分别对纳米粒的外形、粒径和表面电位进行表征,紫外分光光度计和高效液相色谱的方法在420nm处测定药物的载药量和包封率,运用体外动态透析的方法研究了纳米粒中姜黄素的体外释药。
(2)体内外抗癌活性及药代动力学研究
MTT法比较CUR-PBCA-NPs、空白纳米粒(PBCA-NPs)和游离姜黄素(CUR)对人肝癌细胞HepG2、Be17402、Huh7和正常肝细胞L02存活率的影响,流式细胞仪和荧光显微镜检测不同浓度的载药纳米粒对HepG2细胞细胞周期和细胞凋亡的影响,western blotting方法比较了三者对HepG2细胞中的血管内皮生长因子(vascular endothelial growth factor, VEGF)和环氧合酶-2(cyclooxygenase, COX-2)表达的变化。
建立裸鼠移植性肝癌动物模型,尾静脉注射PBCA-NPs、CUR-PBCA-NPs和生理盐水后测定肿瘤大小,免疫组化法检测肿瘤组织中的VEGF和COX-2蛋白的表达。通过测定SD大鼠尾静脉注射CUR和CUR-PBCA-NPs后的血液中姜黄素的药物浓度来比较其主要的药物动力学参数。
(3)逆转多药耐药的研究
选用人乳腺癌阿霉素耐药细胞株MCF-7/ADR作为耐药细胞模型。MTT法比较CUR、CUR-PBCA-NPs和PBCA-NPs对MCF-7/ADR的逆转倍数,流式细胞仪测定CUR-PBCA-NPs对MCF-7/ADR细胞周期及细胞摄取阿霉素的影响。Western blotting方法比较CUR、CUR-PBCA-NPs和PBCA-NPs对MCF-7/ADR细胞中P-糖蛋白(P-glycoprotein, P-gp)、多药耐药相关蛋白1 (multidrug resistance-associated protein, MRP1)、乳腺癌耐药蛋白(breast cancer resistance protein, ABCG2)、DNA拓扑异构酶Ⅱα亚型(TopoisomeraseⅡα, TopoⅡα)多药耐药蛋白的表达,对姜黄素纳米粒逆转多药耐药的机制进行了探讨。
(4)阿霉素-姜黄素聚氰基丙烯酸正丁酯复方纳米粒(DOX-CUR-PBCA-NPs)的研制及逆转多药耐药的作用
采取乳化聚合法制备DOX-CUR-PBCA-NPs复方纳米粒,通过单因素实验考察姜黄素加入的时间、单体和壳聚糖的量、姜黄素和阿霉素的量对DOX-CUR-PBCA-NPs的粒径、表面电位、载药量和包封率的影响,确定最佳制备工艺。应用透射电子显微镜、傅里叶变换红外光谱、凝胶渗透色谱、示差扫描量热法来表征DOX-CUR-PBCA-NPs,运用体外动态透析的方法研究了复方纳米粒的体外释放,MTT实验和western blotting方法研究姜黄素与阿霉素联用的各种纳米制剂及单一药物纳米制剂对MCF-7/ADR的逆转效应。
结果：
(1)优化的CUR-PBCA-NPs制备条件为：10mL反应体系,pH为1.22, BCA选择1.0%(V/V%),壳聚糖选择0.1%(W/V%),姜黄素的加入量为1mg。制备的姜黄素纳米粒的粒径约为185±13.12nm,表面电位为+50.1±2.08mV,载药量为1.078±0.05%,包封率为94.54±1.54%。制得的胶体溶液半年未发现有相分离和絮凝发生,其载药纳米粒粒径和分布无明显变化。CUR-PBCA-NPs的体外释药符合双相动力学释药规律,前30min有50%的突释,其余50%到第7天全部释放出来。
(2) CUR-PBCA-NPs的IC50 (250ng/mL,以游离姜黄素计算)远小于游离姜黄素的IC50 (15μg/mL),且对肿瘤细胞的生长抑制呈时间和浓度依赖性。CUR-PBCA-NPs减少了游离姜黄素本身对正常肝细胞L02的杀伤,在对正常肝细胞杀伤不大的同时能有效地抑制肝癌细胞的增殖。荧光显微镜、Annexin V-FITC/PI方法检测发现经姜黄素纳米粒处理后的HepG2细胞呈现明显的凋亡和坏死状态,细胞阻滞在G2／M期,与游离姜黄素呈现出相同周期的阻滞。体内实验表明：姜黄素纳米粒给药组治疗后的肿瘤体积的抑制率为56.46%,与生理盐水对照组和空白纳米粒对照组(抑制率约为7.69%)相比有显著性差异(P<0.05%)。
CUR-PBCA-NPs治疗后使VEGF和COX-2在肿瘤组织的表达明显减少,肿瘤组织切片中COX-2和VEGF细胞染色为弱阳性,与强阳性的生理盐水组和空白纳米粒组相比,具有统计学差异(P<0.05%)。同样,体外实验证明,20μg/mL CUR-PBCA-NPs(以纳米粒的浓度计算)处理HepG2细胞48h后,比等量的游离姜黄素更能抑制血管生成,使COX-2和VEGF的表达下调增加。
(3)通过SD大鼠尾静脉注射姜黄素纳米粒的水溶液和游离姜黄素药物的生理盐水溶液后,测定了不同时间点的血药浓度,比较得出,前者的平均滞留时间(MRT0-∞)比后者的长很多,表观容积分布Vd是后者的51倍,血药浓度-时间曲线下面积AUC0-∞约为后者的2倍,消除相消除半衰期(t1/2β)约为后者的52倍,说明姜黄素纳米粒具有比游离姜黄素更长的血药半衰期,生物利用度有了一定的提高,延长了姜黄素在循环系统内的滞留时间。
(4)姜黄素纳米粒相对于游离姜黄素的逆转效应增强了近两倍,10μg/mL DOX与阴性对照组对MCF-7/ADR的细胞周期的影响无显著性差异(P>0.05%),而同时加入10μg/mL DOX与50μg/mL CUR-PBCA-NPs后,细胞出现G2/M期阻滞,同时加入40μg/mL的DOX与50μg/mL CUR-PBCA-NPs后,细胞则呈现S期阻滞。MCF-7/ADR经50μg/mL CUR-PBCA-NPs与5、10、20μg/mL DOX同时处理后,细胞内阿霉素的荧光强度明显高于相对应的阿霉素试验组。
CUR-PBCA-NPs能有效逆转MCF-7/ADR中P-gp的表达,随着孵育时间的递增,表达明显下降,处理5d时,P-gp的表达开始下降,到第7d时,就基本上检测不到P-gp的表达了。MRP1的下调程度不大。但TopoⅡα和ABCG2蛋白的表达与阴性对照组的细胞相比没有显著性的差异。说明姜黄素纳米粒逆转MDR的机制可能是逆转能量依赖性药物转出泵P-gp。(5)采用同样的乳化聚合法制备DOX-CUR-PBCA-NPs复方纳米粒,该纳米粒平均粒径为133±5.34nm, Zeta电位为+32.23±4.56mV, DOX和CUR的包封率分别为49.98±3.32%,94.52±3.14%。复方纳米粒中姜黄素和阿霉素的体外释放与单一药物纳米粒非常相似,说明两种药物共存并不影响各自的释放。MTT实验结果和Western Blotting实验结果均表明,DOX-CUR-PBCA-NPs与CUR-PBCA-NPs+ DOX-PBCA-NPs体外对MCF-7/ADR细胞的生长抑制活性相当,下调MCF-7/ADR细胞中P-gp的表达也相当,较没有用PBCA纳米粒包载的游离药物、单一药物的纳米制剂及其他形式的制剂联用的抗肿瘤活性及逆转多药耐药的性能都显著增强。说明利用PBCA纳米粒同时包裹抗癌药物阿霉素与中药逆转剂姜黄素协同用药可以增强克服MDR的疗效。
结论：
制备的这种带正电荷的CUR-PBCA-NPs很好地保留且增强了姜黄素的抗癌活性和逆转MCF-7/ADR细胞中P-gp介导的多药耐药。改善了姜黄素本身的水不溶性,提高了它的生物利用度,延长了其在循环系统内的滞留时间。将抗癌药物阿霉素包载在中药逆转剂姜黄素的PBCA纳米粒中形成复方纳米粒协同用药,可以进一步增强克服MDR的疗效。载药纳米技术用于逆转肿瘤多药耐药是很有前途的应用之一,这一方法给解决MDR提供了新策略,较其它的策略更为可靠易行,可为临床上对化疗产生耐药的患者提供一条新的治疗方法,具有广阔的应用开发前景。
Purpose:
Nowadays, most anticancer drugs have poor selectivity and high toxicity, which limits their applications. The multi-drug resistance (MDR) is one of the major causes of unsuccessful therapy. Many kinds of reversing MDR agents have disadvantages of in vivo unstability, poor tumor targeting and high toxic side effects.
Scientists all over the worlds have begun to scan some effective ingredients of low toxicity and revering MDR from Chinese drugs in recent years. Curcumin is a major effective ingredient of commonly-used traditional Chinese drug curcuma. It has many kinds of pharmacological activities and is described as one of an ideal antitumour agent. And acted as a Chinese herbal reversal agent, it has much more advantages than other chemical reversal agents. Despite great therapeutic potential of curcumin utilization for a variety of diseases, its clinical development has been hindered due to its poor water solubility, low bioavailability, fast metabolism and degradation.
Biological nanocarriers can not only improve the solubility and bioavailability of drug, but also control the drug released and attenuate the toxic side effects.
Based on the advantages of biological nanocarriers and the merits of curcumin, the purpose of present study was to produce CUR-PBCA-NPs by examining the anticancer activity and reversal mechanism in MCF-7/ADR, provide a basis of producing high effective, long-term, targeting and low toxicity curcumin and establish a new technological method for multi-drug resistance reversion.
Methods:
(1)Preparation and characterization of CUR-PBCA-NPs
CUR-PBCA-NPs had been prepared by using anionic emulsion polymerization. The size, zeta potential and encapsulation ratio of the CUR-PBCA-NPs were evaluated to optimize the parameters of stabilizer, pH value, the concentration ofα-BCA and curcumin.
The size, morphology and Zeta potential were characterized by transmission electron microscopy (TEM) and Zetasizer Nano system. The encapsulation ratio and drug loading of curcumin were measured with UV-VIS spectrometer and HPLC at 420nm. The study of drug release behavior of CUR-PBCA-NPs with chitosan in vitro was performed by dialysis method.
(2) Study of pharmacokinetics, anticancer activity in vitro and in vivo
The influences of growth inhibition of different concentrations of CUR-PBCA-NPs, PBCA-NPs and CUR on three kinds of hepatoma carcinoma cells like HepG2, Be17402 and Huh7 cell lines were investigated by MTT assay. The effect of CUR-PBCA-NPs, PBCA-NPs and CUR on cell cycles and apoptosis of HepG2 were studied with flow cytometry and fluorescent microscopy. The VEGF and COX-2 expression of HepG2 effected by CUR-PBCA-NPs, PBCA-NPs and CUR was measured by western blotting.
The HCC nude mice models were established by injecting subcutaneously HepG2 cells into BALB/c-nu nude mice. The preparation of drugs like CUR-PBCA NPs, PBCA NPs and physiological saline were injected i.v. into the tail vein. The tumors were detected and the VEGF and COX-2 protein were measured by immunohistochemical method. The pharmacokinetics of CUR and CUR-PBCA NPs were studied in SD rats by determining the concentration of curcumin in serum.
(3) Evaluation of overcoming multidrug resistance (MDR) effect
The MCF-7/ADR cells were selected as the model of resistant cell. The resistance index (RI) of CUR, CUR-PBCA-NPs and PBCA-NPs were studied by MTT assay. The effects of uptake of DOX and cell cycles of different concentration CUR-PBCA-NPs in MCF-7/ADR cells were investigated by flow cytometry. The expression of MDR1、MRP1、ABCG2 and TopoⅡαproteins in MCF-7/ADR cells dealed with CUR, CUR-PBCA-NPs and PBCA-NPs were determined by western blotting, and the mechanism of reversing the MDR was studied.
(4) Preparation of DOX-CUR-PBCA-NPs and study of drug resistance
The PBCA nanoparticles co-encapsulated of DOX and CUR were prepared by emulsion polymerization. The size, zeta potential, drug loading and encapsulation ratio of the DOX-CUR-PBCA-NPs were evaluated to optimize the following parameters:the time intervals of added CUR from the initiation the polymerization reaction, the concentration ofα-BCA, chitosan, doxorubicin and curcumin. DOX-CUR-PBCA-NPs were characterized by TEM, FTIR, GPC and DSC. The drug release behavior of DOX-CUR-PBCA-NPs in vitro was performed by dialysis method. Reversion efficiency of the formulations and various combination approaches were assessed using MTT assay and western blotting.
Results:
(1) When reaction volume was 10mL,the optimized emulsion polymerization to prepare CUR-PBCA NPs was as follows:pH=1.22, 100αL BCA monomer, 10mg chitosan, 1mg curcumin power, temperature 25℃, stirring time 6h. In this condition, the CUR-PBCA NPs were spherical in shape and narrow in distribution. The average size of CUR-PBCA NPs was about 185±13.12nm in diameter, the drug loading and encapsulation ratio were 1.078±0.05% and 94.54±1.54%, respectively, and the zeta potential was +50.1±2.08mV. The drug release of CUR-PBCA NPs in vitro was according to the second phase release. Curcumin released from CUR-PBCA NPs was faster in the first 30min, then entered into release platform phase, and almost released completely after 7days. The obtained colloid solution were stabilized for at least half a year, there were no phase separation and deposition. The particle size and size distributions of had no significant change after half a year.
(2) Both CUR-PBCA NPs and free CUR killed the HepG2, Be17402 and Huh7 cells were in a concentration and time dependent manner in vitro. While at the same concentration, CUR-PBCA NPs had more potent killing function than that of free CUR, which presented in different cell lines. IC50 of free CUR to HepG2 cells were 60 fold of CUR-PBCA NPs. According to flow cytometry and fluorescent microscopy, CUR-PBCA NPs induced HepG2 cells apoptosis at a time and dose dependent manner. When treated with CUR-PBCA NPs for 4h or longer time, HepG2 cells turned to circle, fell down from wall and proliferated slowly. Cell apoptosis percentage was gradually increased along with CUR-PBCA NPs concentration rising. The apoptosis rate at 10μg/mL,20μg/mL,40μg/mL,60μg/mL is about 4.68%, 14.25%,89.75%,99.95% respectively. After treatment with CUR-PBCA NPs, HepG2 cells were observed to block cell cycle in G2/M phase, which is similar to free CUR. The anticancer activity of CUR-PBCA NPs in vivo was studied in nude nice transplanted subcutaneously by HepG2 cell lines. The inhibitory effect of CUR-PBCA NPs on tumor growth 56.46% was stronger than that of normal saline group and PBCA NPs group (7.69%, P< 0.05).
CUR-PBCA NPs significantly down-regulated protein level of VEGF and COX-2 in HepG2 cells and HepG2 xenografts. The levels of VEGF and COX-2 protein decreased significantly in the cells co-incubated with CUR-PBCA NPs at 20μg/mL for 48 h as compared with those treated with the same concentration of free CUR and in the control cells.
(3) The drug concentration-time curves of CUR-PBCA NPs and CUR in mice blood were determined by HPLC, and the difference between the CUR-PBCA NPs and free CUR in vivo was observed. The pharmocokinetic parameters of CUR-PBCA NPs and free CUR were analyzed with DAS 2.1.1. The results showed that the area under the curve(AUC0-∞)and the elimination half-life (t 1/2β) were 2 and 52 times bigger than that of free CUR, the mean residence time (MRT0-∞) of CUR-PBCA NPs (63.787 h) was longer than that of free CUR (0.159h). There was a substantial increase in the volume of distribution (51-fold). This illustrated that the properties of particles (such as shape, size, charge, and hydrophilicity) can prolong the retention of CUR in the blood compartment.
(4) The reversal index of CUR-PBCA NPs was almost two times of that of free CUR in MCF-7/ADR resistant cell lines. After co-administration of 10μg/mL DOX and 50μg/mL CUR-PBCA NPs, MCF-7/ADR cells were observed to arrest at G2/M phase. And blocked at S phase when co-administration of 40μg/mL DOX and 50μg/mL CUR-PBCA NPs. These differences are evidently remarkable compared with PBCA NPs and free DOX group. The amount of drug uptaken by MCF-7/ADR cells exposed to 50μg/mL CUR-PBCA NPs was significantly higher than that of free DOX solution (P< 0.05) at the same drug concentration.
The CUR-PBCA NPs can cause down-regulated the expression of P-gp in MCF-7/ADR cell lines at a time dependent. After treatment with 50μg/mL CUR-PBCA NPs, the expression of P-gp began to decline on the 5th day and when the 7th day no P-gp could be measured. The MRP1 protein was decreased slower than that of MDR1, while there were no evident alternations of Topo Ila and ABCG2 protein. The results revealed that the mechanism of reversing the multi-drug resistance caused by CUR-PBCA NPs was likely to down-regulate the MDR expression but not to influence TopoⅡαand ABCG2 expression obviously.
(5) Co-encapsulated doxorubicin and curcumin in PBCA nanoparticles were prepared with emulsion polymerization. The mean particle size and the zeta potential of DOX-CUR-PBCA-NPs were 133±5nm in diameter and +32.23±4.56 mV, respectively. The entrapment efficiencies of DOX and CUR were 49.98±3.32% and 94.52±3.14%, respectively. The presence of associated DOX/CUR did not alter the release profile of CUR/DOX from the nanoparticles. Anticancer activities and reversal efficacy of the formulations and various combination approaches were assessed using MTT assay and western blotting. The results showed that the dual-agent loaded PBCA-NPs system had the similar cytotoxicity to co-administration of two single-agent loaded PBCA-NPs (DOX-PBCA-NPs + CUR-PBCA-NPs), which was slightly higher than that of the free drug combination (DOX-CUR) and one free drug/another agent loaded PBCA-NPs combination (DOX+CUR-PBCA-NPs or CUR+DOX-PBCA-NPs). The simultaneous administration of DOX and CUR could achieve the highest reversal efficacy, and down-regulate of P-gp in MCF-7/ADR cell lines. Multidrug-resistant can be enhanced by treated combination of encapsulated cytotoxic drugs and reversal agents.
Conclusions:
The novel cationic CUR-PBCA-NPs maintain and even enhance the anticancer activity and reversion P-gp mediated MDR in MCF-7/ADR of free curcumin. The solubility and bioavailability of curcumin have been improved. These colloidal delivery systems have been shown to increase the circulation time of curcumin in the serum. Co-encapsulation of anticancer drug DOX and reversal agent CUR can be more effective in reversing multi-drug resistance than the other formulations and might cause lower normal tissue drug toxicity and fewer drug-drug interactions. Nanodrugs have not only opened a new horizon to the treatment of disease, but also provided an idea for producing safe, reasonable and effective anticancer agents. It is anticipated that drug delivery system could contribute greatly to reversing MDR. Being a reliable and safe way to reverse MDR, drug delivery system represents a promising prospect both in research and application in recent years.
目前,抗肿瘤药物普遍存在药物靶向性差和全身毒性问题,限制了药物疗效的发挥和长期使用。而且普遍存在的肿瘤多药耐药(mu1tidrug resistance, MDR)也是治疗失败的主要原因。目前,体外逆转MDR的药物有很多,但是存在着体内不稳定,肿瘤靶点性差,毒副作用大等缺点。
近年来国内外学者开始从中药中筛选提取低毒的可逆转MDR的有效成分。姜黄素(curcumin, CUR)是常用中药姜黄(curcuma)中一个生物活性成分,具有多方面的药理作用,被认为是理想的抗癌化疗药物之一,具有逆转MDR的作用。作为中药逆转剂又较其他化药逆转剂有很大的优越性。但是姜黄素存在水溶性差,体内吸收少、不稳定,易降解等缺陷,因此限制了其应用。
生物纳米载体的应用不仅改善药物的溶解度,提高药物的生物利用度,而且还具有缓释性,延长药物体内滞留时间和靶向性,从而降低药物的毒性。
本研究的目的就是基于生物纳米载体的优势以及姜黄素的药学特点,筛选最佳的生物纳米材料,制备载姜黄素的纳米粒,并考察该纳米粒的抗癌活性及对人乳腺癌阿霉素耐药细胞MCF-7/ADR的逆转作用机制,为制备高效、长效、靶向和低毒的姜黄素奠定基础,为探索逆转MDR提供一条思路。
方法：
(1) CUR-PBCA-NPs的制备及表征
采用阴离子乳化聚合法制备CUR-PBCA-NPs,通过单因素实验结合正交试验对CUR-PBCA-NPs的制备工艺进行了优化,考察稳定剂的种类、反应过程的pH值、单体的用量、稳定剂的用量及药物浓度对纳米粒的粒径、表面电位和包封率的影响,确定最佳制备工艺。
透射电子显微镜和激光粒度分析仪分别对纳米粒的外形、粒径和表面电位进行表征,紫外分光光度计和高效液相色谱的方法在420nm处测定药物的载药量和包封率,运用体外动态透析的方法研究了纳米粒中姜黄素的体外释药。
(2)体内外抗癌活性及药代动力学研究
MTT法比较CUR-PBCA-NPs、空白纳米粒(PBCA-NPs)和游离姜黄素(CUR)对人肝癌细胞HepG2、Be17402、Huh7和正常肝细胞L02存活率的影响,流式细胞仪和荧光显微镜检测不同浓度的载药纳米粒对HepG2细胞细胞周期和细胞凋亡的影响,western blotting方法比较了三者对HepG2细胞中的血管内皮生长因子(vascular endothelial growth factor, VEGF)和环氧合酶-2(cyclooxygenase, COX-2)表达的变化。
建立裸鼠移植性肝癌动物模型,尾静脉注射PBCA-NPs、CUR-PBCA-NPs和生理盐水后测定肿瘤大小,免疫组化法检测肿瘤组织中的VEGF和COX-2蛋白的表达。通过测定SD大鼠尾静脉注射CUR和CUR-PBCA-NPs后的血液中姜黄素的药物浓度来比较其主要的药物动力学参数。
(3)逆转多药耐药的研究
选用人乳腺癌阿霉素耐药细胞株MCF-7/ADR作为耐药细胞模型。MTT法比较CUR、CUR-PBCA-NPs和PBCA-NPs对MCF-7/ADR的逆转倍数,流式细胞仪测定CUR-PBCA-NPs对MCF-7/ADR细胞周期及细胞摄取阿霉素的影响。Western blotting方法比较CUR、CUR-PBCA-NPs和PBCA-NPs对MCF-7/ADR细胞中P-糖蛋白(P-glycoprotein, P-gp)、多药耐药相关蛋白1 (multidrug resistance-associated protein, MRP1)、乳腺癌耐药蛋白(breast cancer resistance protein, ABCG2)、DNA拓扑异构酶Ⅱα亚型(TopoisomeraseⅡα, TopoⅡα)多药耐药蛋白的表达,对姜黄素纳米粒逆转多药耐药的机制进行了探讨。
(4)阿霉素-姜黄素聚氰基丙烯酸正丁酯复方纳米粒(DOX-CUR-PBCA-NPs)的研制及逆转多药耐药的作用
采取乳化聚合法制备DOX-CUR-PBCA-NPs复方纳米粒,通过单因素实验考察姜黄素加入的时间、单体和壳聚糖的量、姜黄素和阿霉素的量对DOX-CUR-PBCA-NPs的粒径、表面电位、载药量和包封率的影响,确定最佳制备工艺。应用透射电子显微镜、傅里叶变换红外光谱、凝胶渗透色谱、示差扫描量热法来表征DOX-CUR-PBCA-NPs,运用体外动态透析的方法研究了复方纳米粒的体外释放,MTT实验和western blotting方法研究姜黄素与阿霉素联用的各种纳米制剂及单一药物纳米制剂对MCF-7/ADR的逆转效应。
结果：
(1)优化的CUR-PBCA-NPs制备条件为：10mL反应体系,pH为1.22, BCA选择1.0%(V/V%),壳聚糖选择0.1%(W/V%),姜黄素的加入量为1mg。制备的姜黄素纳米粒的粒径约为185±13.12nm,表面电位为+50.1±2.08mV,载药量为1.078±0.05%,包封率为94.54±1.54%。制得的胶体溶液半年未发现有相分离和絮凝发生,其载药纳米粒粒径和分布无明显变化。CUR-PBCA-NPs的体外释药符合双相动力学释药规律,前30min有50%的突释,其余50%到第7天全部释放出来。
(2) CUR-PBCA-NPs的IC50 (250ng/mL,以游离姜黄素计算)远小于游离姜黄素的IC50 (15μg/mL),且对肿瘤细胞的生长抑制呈时间和浓度依赖性。CUR-PBCA-NPs减少了游离姜黄素本身对正常肝细胞L02的杀伤,在对正常肝细胞杀伤不大的同时能有效地抑制肝癌细胞的增殖。荧光显微镜、Annexin V-FITC/PI方法检测发现经姜黄素纳米粒处理后的HepG2细胞呈现明显的凋亡和坏死状态,细胞阻滞在G2／M期,与游离姜黄素呈现出相同周期的阻滞。体内实验表明：姜黄素纳米粒给药组治疗后的肿瘤体积的抑制率为56.46%,与生理盐水对照组和空白纳米粒对照组(抑制率约为7.69%)相比有显著性差异(P<0.05%)。
CUR-PBCA-NPs治疗后使VEGF和COX-2在肿瘤组织的表达明显减少,肿瘤组织切片中COX-2和VEGF细胞染色为弱阳性,与强阳性的生理盐水组和空白纳米粒组相比,具有统计学差异(P<0.05%)。同样,体外实验证明,20μg/mL CUR-PBCA-NPs(以纳米粒的浓度计算)处理HepG2细胞48h后,比等量的游离姜黄素更能抑制血管生成,使COX-2和VEGF的表达下调增加。
(3)通过SD大鼠尾静脉注射姜黄素纳米粒的水溶液和游离姜黄素药物的生理盐水溶液后,测定了不同时间点的血药浓度,比较得出,前者的平均滞留时间(MRT0-∞)比后者的长很多,表观容积分布Vd是后者的51倍,血药浓度-时间曲线下面积AUC0-∞约为后者的2倍,消除相消除半衰期(t1/2β)约为后者的52倍,说明姜黄素纳米粒具有比游离姜黄素更长的血药半衰期,生物利用度有了一定的提高,延长了姜黄素在循环系统内的滞留时间。
(4)姜黄素纳米粒相对于游离姜黄素的逆转效应增强了近两倍,10μg/mL DOX与阴性对照组对MCF-7/ADR的细胞周期的影响无显著性差异(P>0.05%),而同时加入10μg/mL DOX与50μg/mL CUR-PBCA-NPs后,细胞出现G2/M期阻滞,同时加入40μg/mL的DOX与50μg/mL CUR-PBCA-NPs后,细胞则呈现S期阻滞。MCF-7/ADR经50μg/mL CUR-PBCA-NPs与5、10、20μg/mL DOX同时处理后,细胞内阿霉素的荧光强度明显高于相对应的阿霉素试验组。
CUR-PBCA-NPs能有效逆转MCF-7/ADR中P-gp的表达,随着孵育时间的递增,表达明显下降,处理5d时,P-gp的表达开始下降,到第7d时,就基本上检测不到P-gp的表达了。MRP1的下调程度不大。但TopoⅡα和ABCG2蛋白的表达与阴性对照组的细胞相比没有显著性的差异。说明姜黄素纳米粒逆转MDR的机制可能是逆转能量依赖性药物转出泵P-gp。(5)采用同样的乳化聚合法制备DOX-CUR-PBCA-NPs复方纳米粒,该纳米粒平均粒径为133±5.34nm, Zeta电位为+32.23±4.56mV, DOX和CUR的包封率分别为49.98±3.32%,94.52±3.14%。复方纳米粒中姜黄素和阿霉素的体外释放与单一药物纳米粒非常相似,说明两种药物共存并不影响各自的释放。MTT实验结果和Western Blotting实验结果均表明,DOX-CUR-PBCA-NPs与CUR-PBCA-NPs+ DOX-PBCA-NPs体外对MCF-7/ADR细胞的生长抑制活性相当,下调MCF-7/ADR细胞中P-gp的表达也相当,较没有用PBCA纳米粒包载的游离药物、单一药物的纳米制剂及其他形式的制剂联用的抗肿瘤活性及逆转多药耐药的性能都显著增强。说明利用PBCA纳米粒同时包裹抗癌药物阿霉素与中药逆转剂姜黄素协同用药可以增强克服MDR的疗效。
结论：
制备的这种带正电荷的CUR-PBCA-NPs很好地保留且增强了姜黄素的抗癌活性和逆转MCF-7/ADR细胞中P-gp介导的多药耐药。改善了姜黄素本身的水不溶性,提高了它的生物利用度,延长了其在循环系统内的滞留时间。将抗癌药物阿霉素包载在中药逆转剂姜黄素的PBCA纳米粒中形成复方纳米粒协同用药,可以进一步增强克服MDR的疗效。载药纳米技术用于逆转肿瘤多药耐药是很有前途的应用之一,这一方法给解决MDR提供了新策略,较其它的策略更为可靠易行,可为临床上对化疗产生耐药的患者提供一条新的治疗方法,具有广阔的应用开发前景。
Purpose:
Nowadays, most anticancer drugs have poor selectivity and high toxicity, which limits their applications. The multi-drug resistance (MDR) is one of the major causes of unsuccessful therapy. Many kinds of reversing MDR agents have disadvantages of in vivo unstability, poor tumor targeting and high toxic side effects.
Scientists all over the worlds have begun to scan some effective ingredients of low toxicity and revering MDR from Chinese drugs in recent years. Curcumin is a major effective ingredient of commonly-used traditional Chinese drug curcuma. It has many kinds of pharmacological activities and is described as one of an ideal antitumour agent. And acted as a Chinese herbal reversal agent, it has much more advantages than other chemical reversal agents. Despite great therapeutic potential of curcumin utilization for a variety of diseases, its clinical development has been hindered due to its poor water solubility, low bioavailability, fast metabolism and degradation.
Biological nanocarriers can not only improve the solubility and bioavailability of drug, but also control the drug released and attenuate the toxic side effects.
Based on the advantages of biological nanocarriers and the merits of curcumin, the purpose of present study was to produce CUR-PBCA-NPs by examining the anticancer activity and reversal mechanism in MCF-7/ADR, provide a basis of producing high effective, long-term, targeting and low toxicity curcumin and establish a new technological method for multi-drug resistance reversion.
Methods:
(1)Preparation and characterization of CUR-PBCA-NPs
CUR-PBCA-NPs had been prepared by using anionic emulsion polymerization. The size, zeta potential and encapsulation ratio of the CUR-PBCA-NPs were evaluated to optimize the parameters of stabilizer, pH value, the concentration ofα-BCA and curcumin.
The size, morphology and Zeta potential were characterized by transmission electron microscopy (TEM) and Zetasizer Nano system. The encapsulation ratio and drug loading of curcumin were measured with UV-VIS spectrometer and HPLC at 420nm. The study of drug release behavior of CUR-PBCA-NPs with chitosan in vitro was performed by dialysis method.
(2) Study of pharmacokinetics, anticancer activity in vitro and in vivo
The influences of growth inhibition of different concentrations of CUR-PBCA-NPs, PBCA-NPs and CUR on three kinds of hepatoma carcinoma cells like HepG2, Be17402 and Huh7 cell lines were investigated by MTT assay. The effect of CUR-PBCA-NPs, PBCA-NPs and CUR on cell cycles and apoptosis of HepG2 were studied with flow cytometry and fluorescent microscopy. The VEGF and COX-2 expression of HepG2 effected by CUR-PBCA-NPs, PBCA-NPs and CUR was measured by western blotting.
The HCC nude mice models were established by injecting subcutaneously HepG2 cells into BALB/c-nu nude mice. The preparation of drugs like CUR-PBCA NPs, PBCA NPs and physiological saline were injected i.v. into the tail vein. The tumors were detected and the VEGF and COX-2 protein were measured by immunohistochemical method. The pharmacokinetics of CUR and CUR-PBCA NPs were studied in SD rats by determining the concentration of curcumin in serum.
(3) Evaluation of overcoming multidrug resistance (MDR) effect
The MCF-7/ADR cells were selected as the model of resistant cell. The resistance index (RI) of CUR, CUR-PBCA-NPs and PBCA-NPs were studied by MTT assay. The effects of uptake of DOX and cell cycles of different concentration CUR-PBCA-NPs in MCF-7/ADR cells were investigated by flow cytometry. The expression of MDR1、MRP1、ABCG2 and TopoⅡαproteins in MCF-7/ADR cells dealed with CUR, CUR-PBCA-NPs and PBCA-NPs were determined by western blotting, and the mechanism of reversing the MDR was studied.
(4) Preparation of DOX-CUR-PBCA-NPs and study of drug resistance
The PBCA nanoparticles co-encapsulated of DOX and CUR were prepared by emulsion polymerization. The size, zeta potential, drug loading and encapsulation ratio of the DOX-CUR-PBCA-NPs were evaluated to optimize the following parameters:the time intervals of added CUR from the initiation the polymerization reaction, the concentration ofα-BCA, chitosan, doxorubicin and curcumin. DOX-CUR-PBCA-NPs were characterized by TEM, FTIR, GPC and DSC. The drug release behavior of DOX-CUR-PBCA-NPs in vitro was performed by dialysis method. Reversion efficiency of the formulations and various combination approaches were assessed using MTT assay and western blotting.
Results:
(1) When reaction volume was 10mL,the optimized emulsion polymerization to prepare CUR-PBCA NPs was as follows:pH=1.22, 100αL BCA monomer, 10mg chitosan, 1mg curcumin power, temperature 25℃, stirring time 6h. In this condition, the CUR-PBCA NPs were spherical in shape and narrow in distribution. The average size of CUR-PBCA NPs was about 185±13.12nm in diameter, the drug loading and encapsulation ratio were 1.078±0.05% and 94.54±1.54%, respectively, and the zeta potential was +50.1±2.08mV. The drug release of CUR-PBCA NPs in vitro was according to the second phase release. Curcumin released from CUR-PBCA NPs was faster in the first 30min, then entered into release platform phase, and almost released completely after 7days. The obtained colloid solution were stabilized for at least half a year, there were no phase separation and deposition. The particle size and size distributions of had no significant change after half a year.
(2) Both CUR-PBCA NPs and free CUR killed the HepG2, Be17402 and Huh7 cells were in a concentration and time dependent manner in vitro. While at the same concentration, CUR-PBCA NPs had more potent killing function than that of free CUR, which presented in different cell lines. IC50 of free CUR to HepG2 cells were 60 fold of CUR-PBCA NPs. According to flow cytometry and fluorescent microscopy, CUR-PBCA NPs induced HepG2 cells apoptosis at a time and dose dependent manner. When treated with CUR-PBCA NPs for 4h or longer time, HepG2 cells turned to circle, fell down from wall and proliferated slowly. Cell apoptosis percentage was gradually increased along with CUR-PBCA NPs concentration rising. The apoptosis rate at 10μg/mL,20μg/mL,40μg/mL,60μg/mL is about 4.68%, 14.25%,89.75%,99.95% respectively. After treatment with CUR-PBCA NPs, HepG2 cells were observed to block cell cycle in G2/M phase, which is similar to free CUR. The anticancer activity of CUR-PBCA NPs in vivo was studied in nude nice transplanted subcutaneously by HepG2 cell lines. The inhibitory effect of CUR-PBCA NPs on tumor growth 56.46% was stronger than that of normal saline group and PBCA NPs group (7.69%, P< 0.05).
CUR-PBCA NPs significantly down-regulated protein level of VEGF and COX-2 in HepG2 cells and HepG2 xenografts. The levels of VEGF and COX-2 protein decreased significantly in the cells co-incubated with CUR-PBCA NPs at 20μg/mL for 48 h as compared with those treated with the same concentration of free CUR and in the control cells.
(3) The drug concentration-time curves of CUR-PBCA NPs and CUR in mice blood were determined by HPLC, and the difference between the CUR-PBCA NPs and free CUR in vivo was observed. The pharmocokinetic parameters of CUR-PBCA NPs and free CUR were analyzed with DAS 2.1.1. The results showed that the area under the curve(AUC0-∞)and the elimination half-life (t 1/2β) were 2 and 52 times bigger than that of free CUR, the mean residence time (MRT0-∞) of CUR-PBCA NPs (63.787 h) was longer than that of free CUR (0.159h). There was a substantial increase in the volume of distribution (51-fold). This illustrated that the properties of particles (such as shape, size, charge, and hydrophilicity) can prolong the retention of CUR in the blood compartment.
(4) The reversal index of CUR-PBCA NPs was almost two times of that of free CUR in MCF-7/ADR resistant cell lines. After co-administration of 10μg/mL DOX and 50μg/mL CUR-PBCA NPs, MCF-7/ADR cells were observed to arrest at G2/M phase. And blocked at S phase when co-administration of 40μg/mL DOX and 50μg/mL CUR-PBCA NPs. These differences are evidently remarkable compared with PBCA NPs and free DOX group. The amount of drug uptaken by MCF-7/ADR cells exposed to 50μg/mL CUR-PBCA NPs was significantly higher than that of free DOX solution (P< 0.05) at the same drug concentration.
The CUR-PBCA NPs can cause down-regulated the expression of P-gp in MCF-7/ADR cell lines at a time dependent. After treatment with 50μg/mL CUR-PBCA NPs, the expression of P-gp began to decline on the 5th day and when the 7th day no P-gp could be measured. The MRP1 protein was decreased slower than that of MDR1, while there were no evident alternations of Topo Ila and ABCG2 protein. The results revealed that the mechanism of reversing the multi-drug resistance caused by CUR-PBCA NPs was likely to down-regulate the MDR expression but not to influence TopoⅡαand ABCG2 expression obviously.
(5) Co-encapsulated doxorubicin and curcumin in PBCA nanoparticles were prepared with emulsion polymerization. The mean particle size and the zeta potential of DOX-CUR-PBCA-NPs were 133±5nm in diameter and +32.23±4.56 mV, respectively. The entrapment efficiencies of DOX and CUR were 49.98±3.32% and 94.52±3.14%, respectively. The presence of associated DOX/CUR did not alter the release profile of CUR/DOX from the nanoparticles. Anticancer activities and reversal efficacy of the formulations and various combination approaches were assessed using MTT assay and western blotting. The results showed that the dual-agent loaded PBCA-NPs system had the similar cytotoxicity to co-administration of two single-agent loaded PBCA-NPs (DOX-PBCA-NPs + CUR-PBCA-NPs), which was slightly higher than that of the free drug combination (DOX-CUR) and one free drug/another agent loaded PBCA-NPs combination (DOX+CUR-PBCA-NPs or CUR+DOX-PBCA-NPs). The simultaneous administration of DOX and CUR could achieve the highest reversal efficacy, and down-regulate of P-gp in MCF-7/ADR cell lines. Multidrug-resistant can be enhanced by treated combination of encapsulated cytotoxic drugs and reversal agents.
Conclusions:
The novel cationic CUR-PBCA-NPs maintain and even enhance the anticancer activity and reversion P-gp mediated MDR in MCF-7/ADR of free curcumin. The solubility and bioavailability of curcumin have been improved. These colloidal delivery systems have been shown to increase the circulation time of curcumin in the serum. Co-encapsulation of anticancer drug DOX and reversal agent CUR can be more effective in reversing multi-drug resistance than the other formulations and might cause lower normal tissue drug toxicity and fewer drug-drug interactions. Nanodrugs have not only opened a new horizon to the treatment of disease, but also provided an idea for producing safe, reasonable and effective anticancer agents. It is anticipated that drug delivery system could contribute greatly to reversing MDR. Being a reliable and safe way to reverse MDR, drug delivery system represents a promising prospect both in research and application in recent years.
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