载多西紫杉醇脂质超声微泡联合超声靶向微泡破裂对肝癌作用的实验研究
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摘要
原发性肝癌是世界第六大常见的恶性肿瘤,我国的肝癌发病率和死亡率居全球之首,因此,对于肝癌治疗的研究显得尤其重要和必要。目前肝癌首选的治疗方案仍是手术切除,但手术切除率低,术后复发率高,化疗常常是可供选择的主要治疗方法和手段,然而,化疗药物大多为细胞毒性药物,对肿瘤和正常组织缺乏靶向性,具有较大的毒副作用。因此,寻求一种有效、安全、靶向性高、毒副作用小的治疗手段迫在眉睫。近年来,超声微泡作为一种药物或基因载体,逐渐受到广泛的关注;载药微泡注入体内后,可以通过血液循环到达靶器官,利用超声可监控微泡到达靶组织的情况,并利用一定强度的超声经体表定位辐照使微泡在靶组织内破裂,从而达到药物定位释放的目的,同时,微泡破裂产生的一系列效应,可增加药物吸收,提高疗效。
多西紫杉醇(Docetaxel)是常用的疗效较好的化疗药物之一。由于多西紫杉醇水溶性差,目前市售制剂采用吐温-80增溶、乙醇助溶,易产生溶血及过敏等不良反应,临床使用极为不便;研究表明,将紫杉1烷类药物载入脂质后可明显降低药物的毒性,提高机体耐受性,且能保持与市售注射液相同的抗肿瘤活性。因此,在本实验中,我们将脂质微泡作为一种药物载体,将多西紫杉醇载入了微泡,制备了载多西紫杉醇脂质超声微泡,检测其特性;运用超声靶向微泡破裂使药物定位释放在靶组织,并通过体外和体内实验,探讨载多西紫杉醇微泡联合超声靶向微泡破裂对肝癌的作用及相关机理。本研究为构建一种新的药物剂型提供新策略,载药微泡联合UTMD为肿瘤的靶向治疗提供了新的思路。本课题主要包括以下三部分内容:
第一部分载多西紫杉醇脂质微泡的制备及一般特性研究
目的制备一种载多西紫杉醇的脂质超声微泡并测定其物理特性。方法采用机械振荡法制备载多西紫杉醇脂质微泡,测定其浓度、粒径分布范围、电位、包封率及载药量等特性;观察60Coγ射线灭菌前后微泡特性的差异及不同储存条件下微泡特性的改变;检测超声辐照后载药微泡内药物的释放。结果载多西紫杉醇微泡的浓度为2.2×109～3.2×109个/ml;粒径分布范围为473.4nm～706.6nm,平均粒径为623.1nm;微泡的包封率高于70%,载药量高于17.5%;Zeta电位为-(3.1±0.9)mV; 60Coγ射线灭菌前后微泡的特性无显著性差异,微泡在4℃、-20℃储存7天后其特性有所改变;超声辐照微泡溶液后,药物可释放。结论本实验成功制备了载多西紫杉醇脂质超声微泡,其包封率较高、粒径均匀、较稳定,超声辐照可使微泡破裂,促使微泡中的药物释放。
第二部分载多西紫杉醇脂质微泡联合超声靶向微泡破裂对人SMMC-7721肝癌细胞作用的实验研究
目的探索人SMMC-7721肝癌细胞株的体外培养条件及多西紫杉醇体外治疗的适宜浓度。探讨载多西紫杉醇脂质微泡联合超声靶向微泡破裂对SMMC- 7721细胞的增殖抑制、形态学影响、细胞周期的影响、诱导凋亡作用及相关蛋白和基因表达水平的影响。方法体外培养SMMC-7721细胞,观察其生长情况,并用MTT法检测其生长特性,描绘生长曲线。MTT法检测不同浓度多西紫杉醇对SMMC-7721细胞的增殖抑制率,并以此确定适宜的多西紫杉醇体外作用浓度。实验分为6组:空白对照组(C)、单纯药物组(Doc)、载药微泡组(LMLD)、药物+超声组(Doc+US)、单纯微泡+超声组(PLM+US)、载药微泡+超声组(LMLD+US)。MTT法检测不同时间点各组SMMC-7721细胞的增殖抑制率;透射电镜观察其细胞形态和超微结构变化;流式细胞仪检测各组细胞周期分布; Annexin V-FITC荧光染色检测细胞凋亡;实时定量-PCR和Western Blotting法分别检测Caspase3、Bcl-2、Bax在基因和蛋白水平的表达情况。结果在SMMC-7721细胞培养过程中,确定以1×105个/ml细胞数接种。应用一定浓度多西紫杉醇能抑制SMMC-7721细胞生长,呈明显的浓度-时间-效应关系。与其它组比较,LMLD+US组对SMMC-7721细胞的增殖抑制作用最强,细胞凋亡率最高,并可将SMMC-7721细胞明显阻滞在G2/M期。LMLD+US组可见较多凋亡细胞及凋亡小体形成,Doc+US组可见早期凋亡细胞。LMLD+US组Caspase3和Bax在基因和蛋白水平的表达高于其它各组,而Bcl-2在基因和蛋白水平的表达明显低于其它各组。结论多西紫杉醇对SMMC-7721细胞的增殖有抑制作用,确定多西紫杉醇后续实验药物浓度为1×10-9 mol/L。载多西紫杉醇脂质微泡联合超声靶向微泡破裂对SMMC-7721细胞具有明显的增殖抑制作用,并可将细胞明显阻滞在G2/M期、诱导细胞凋亡,其作用机制可能是通过影响细胞凋亡有关的多个基因及蛋白的表达水平。
第三部分载多西紫杉醇脂质微泡体内显影观察及其联合超声靶向微泡破裂对兔VX2肝癌的作用
目的利用超声检查监测肿瘤情况,并探讨载多西紫杉醇脂质微泡体内的显影效果。研究载多西紫杉醇脂质微泡联合UTMD介导药物靶向释放对兔VX2肝癌的治疗效果。方法90只新西兰大白兔,采用瘤块种植法于肝左叶内植入VX2肿瘤组织块,建立兔肝癌模型。用2D、CDFI及微泡造影观察肿瘤的大小、回声及血流灌注,并和CT检查结果相比较,从而评估超声在兔VX2肝癌模型监测中的应用价值。病理解剖验证VX2瘤株在兔肝脏的成瘤情况。实验动物随机分成6组,每组15只,包括空白对照组(C)、载药微泡组(LMLD)、单纯药物组(Doc)、药物+超声组(Doc+US)、单纯微泡+超声组(PLM+US)、载药微泡+超声组(LMLD+ US)。治疗前后用超声和CT观察肿瘤并测量其大小,计算其体积和肿瘤抑制率;免疫组化法检测肿瘤组织CD34、MMP2及PCNA的表达;TUNEL法检测肿瘤细胞凋亡;原位杂交法检测MMP2和Caspase3 mRNA表达水平;比较各组动物的存活时间和转移情况。
结果采用瘤块种植法成功建立了兔肝VX2肝癌模型,成瘤率为100%。超声能较准确地测量肿瘤的大小,能实时、准确地监测肝内肿瘤的回声变化及肿瘤血流情况,超声与CT测量的肿瘤大小无显著性差异(P>0.05);静脉注射载药微泡后,兔肝实质见良好、持续的增强显影,肝癌可见典型的“快进快出”显影表现。LMLD+ US组的肿瘤抑制率明显高于其他各组。治疗后肿瘤内部多呈混合回声,CDFI显示肿瘤的血流明显减少。LMLD+ US组的凋亡指数(AI)显著高于其它各组(P<0.01);LMLD+ US组的肿瘤增殖受到明显抑制,其增殖指数明显低于其它各组(P<0.01);LMLD+ US组MMP2蛋白及mRNA表达水平明显低于其它各组,而Caspase3 mRNA表达明显高于其它各组;LMLD+ US组的动物存活时间明显长于其它组。各组动物均见有腹腔转移,但LMLD+US组的远处转移最少。结论瘤块种植法建立兔VX2肝癌模型操作较简单、成瘤率高。载药微泡对肝实质及肿瘤具有良好的显影效果,超声检查能无创、方便、较准确地监测肝脏VX2肿瘤的大小、形态等;超声检查为利用VX2肝癌模型评价载多西紫杉醇脂质微泡联合UTMD的疗效及实时监控下的治疗提供了有效的监测方法。载多西紫杉醇脂质微泡联合UTMD能抑制兔VX2肝癌的增殖、促进其凋亡,对VX2肝癌具有显著的抗肿瘤作用,为寻求一种有效、安全、靶向性高的肿瘤治疗手段提供了新的思路,为研究一种基于超声微泡的靶向定位控释技术提供了依据。
Hepatocellular carcinoma (HCC) is the sixth most common tumor worldwide, and the incidence and mortality in China are highest around the world, for these reasons, research on liver carcinoma treatment is important and essential. At present, surgical resection is the main treatment method for it, but because of the low rate of surgical resection and the high recurrence rate, chemotherapy is usually the main available treatment method, however, most of chemotherapy drugs are cytotoxic, lacking of targeting to cancer cells, and causing severely side effects to body. Therefore, it is imminent to explore a kind of effective, safe, high-targeting and low toxicity treatment method. Recently, ultrasound microbubbles as a kind of drug or gene delivery vehicles are paid an ever increasing attention. After intravenous injection, microbubbles can arrive at the target tissues through blood circulation, ultrasound can be used to monitor the appearance of microbubbles in target organs and certain intensity of ultrasound could make microbubbles rupture, and then, the drug could be released locally in target tissues and absorbed more by target cells, by this way, the treatment effects could be improved and the side effects on other tissues could be decreased. Docetaxel is an extensively used chemotherapy drug in clinical trials, which has been evaluated as one of the better effective chemotherapy drug, however, water solubility of docetaxel is so poor that it is solubilized in polysorbate 80 (Tween 80) and alcohol for clinical use, which usually causes adverse events such as haemolysis and anaphylaxis, therefore, clinical application of docetaxel is inconvenient and limited. Current studies showed loading taxane drugs into lipid materials could lower toxic effects, improve the tolerance of patients, and the same antitumor activity as injection could be kept. In this study, lipid ultrasoud microbubbles was used as a drug vehicle, and UTMD was applied to release the drug locally in target tissues. Lipid ultrasoud microbubbles loaded docetaxel were prepared, and the properties of which were determined. We explored the effect of LMLD combined with UTMD on liver tumor and related mechanisms in vitro and in vivo level. This research may give a new strategy for composing pharmaceutical dosage form, and drug-loaded microbubbles combined with UTMD may provide a novel thought for tumor targeted-treatment. The study consisted of the following three parts:
PART I The preparation and general properties of lipid ultrasoud microbubbles loaded docetaxel
Objective To prepare a kind of lipid ultrasoud microbubbles loaded docetaxel, study its physical properties, and evaluate as a new ultrasound contrast agent for chemotherapeutic drug delivery. Methods The LMLD were made using mechanic vibration technique. Properties were studied contained concentration, size distribution, zeta potential, drug entrapment efficiency and drug-loading amount. Primary stability experiments were performed including comparison of the properties of lipid ultrasoud microbubbles loaded docetaxel under different storage condition, before and after sterilizing with 60Coγirradiation. Drug released with ultrasound was observed.Results LMLD had a concentration of approximately 2.2×109~3.2×109/ml, a mean size of 623.1nm and a zeta potential of–(3.1±0.9)mV. Size distribution was 473.4~706.6nm. Drug entrapment efficiency was more than 70% and drug-loading amount was more than 17.5%. There was no significant difference in properties between before and after sterilizing with 60Co irradiation, properties were changed a little after being stored at 4℃and -20℃for 7 days. Certain intensity of ultrasound energy can rupture microbubbles and release docetaxel. Conclusions In this study, we successfully prepared lipid ultrasoud microbubbles loaded docetaxel, and this kind of microbubbles had high drug entrapment efficiency, the size was well-distributed and it was stable,docetaxel could be released from microbubbles by ultrasound irradiation.
PART II Effect of lipid ultrasoud microbubbles loaded docetaxel combined with ultrasound-targeted microbubble destruction on SMMC-7721 hepatoma carcinoma cells
Objective To study the cultivation parameters and suitable docetaxel treatment concentration on hepatoma carcinoma cell strain SMMC-7721 in vitro. To investigate the effects of lipid ultrasoud microbubbles loaded docetaxel combined with ultrasound-targeted microbubble destruction on cells proliferation, ultrastructure, cell cycle distribution, apoptosis and expression of apoptosis-related proteins and genes. Methods SMMC-7721cells were cultured in vitro,cells growth characteristics were determined by morphologic observation and cell counting.The suitable treatment concentration was decided according to the proliferating inhibiton rate of SMMC-7721 cells in different dose groups, the proliferating inhibiton rate was detected by MTT assay. SMMC-7721 cells were divided into 6 groups:blank control group (C),docetaxel group(Doc),lipid microbubbles loaded docetaxel group(LMLD), docetaxel+ultrasound group(Doc+US), pure lipid microbubbles+ultrasound group(PLM+US) and lipid microbubbles loaded docetaxel+ultrasound group(LMLD+US). Proliferating inhibition rates at different time point were detected with MTT assay. Ultrastructural changes were observed by transmission electron microscope(TEM). Cell cycle distribution was observed by flow cytometry(FCM). Cell apoptosis was detected with Annexin V-FITC technology. Protein and mRNA expression of Caspase3, Bcl-2, Bax were separately detected by Western Blotting and real-time PCR. Results During the course of cultivation,SMMC-7721cells were inoculated with a concentration of 1×105/ml. Certain concentration of docetaxel could inhibit the growth of SMMC-7721 cells, the inhibition effects were positively related with the concentration of docetaxel and time, which showed an obvious dose-time-response relation. Compared with other groups, the proliferating inhibition effect of LMLD+US group was strongest and the apoptotic rate was highest; percentage of cells blocked in G2/M phase was highest in LMLD+US group. Many apoptosis bodies were observed in LMLD+US group, early stage apoptosis cells could be seen in Doc+US group. The expression of Caspase3 and Bax in LMLD+US group was significantly higher than other groups at protein and mRNA level, the Bcl-2 expression in LMLD+US group was lowest among all groups. Conclusions Docetaxel could inhibit the growth of SMMC-7721 cells in vitro, 1×10-9 mol/L was chosen as the suitable concentration in following experiments. Lipid microbubbles loaded docetaxel combined with ultrasound-targeted microbubble destruction could obviously inhibit the proliferation and induce apoptosis of SMMC-7721 cells,and also blocked SMMC-7721 cells in G2/M phase;the possible mechanisms of which may be lie in influencing the expression of apoptosis-related proteins and genes.
PART III Tumor imaging with lipid microbubbles loaded docetaxel and effect of lipid microbubbles loaded docetaxel combined with ultrasound-targeted microbubble destruction on rabbit VX2 liver tumor
Objective To evaluate the ultrasound imaging quality of tumor with LMLD; to explore the antitumor effect of LMLD combined with UTMD on rabbit VX2 liver tumor. Methods VX2 liver tumor models were established in 90 rabbits. Size, echo and blood flow of tumors were detected by 2D, CDFI and CEUS, the axes values detected by ultrasound were compared with CT measurements. The successful establishment of VX2 tumor was testified by pathologic biopsy. 90 rabbits were randomly divided into 6 groups: blank control group (C),docetaxel group(Doc),lipid microbubbles loaded docetaxel group(LMLD), docetaxel+ultrasound group(Doc+US), pure lipid microbubbles+ultrasound group(PLM+US) and lipid microbubbles loaded docetaxel+ultrasound group(LMLD+US). Tumor sizes were observed by ultrasound and CT before and after treatment; tumor volume(TV) and the tumor inhibition rate(TIR) were calculated and compared. Apoptosis was detected by TUNEL. Protein expression of CD34, PCNA and MMP2 was detected by immunohistochemistry(IHC). mRNA expression of Caspase3 and MMP2 was detected by hybridization histochemistry (ISH). Tumor metastasis rate and survival time of animals were compared. Results The rabbit VX2 liver tumor models were successfully established with tumor block transplanting method, and the successful rate was 100%. Ultrasound could detect the tumors accurately and provide real-time monitoring, there was no significant difference between the axes measured by ultrasound and CT(P>0.05). Liver imaging of rabbits could be enhanced obviously and persistently,“fast in and out”phenomenon was typical of VX2 liver tumor. TIR and apoptotic index (AI) of LMLD+US group were highest among all groups(P<0.01) and proliferating labelling index (LI) was lowes(tP<0.01).MVD of LMLD+US group was lowest among all groups(P<0.01).Protein and mRNA expression of MMP2 in LMLD+US group were lowest(P<0.01), and Caspase3 mRNA expression in LMLD+US group was highest among all groups(P<0.01).Abdominal cavity metastasis was found in all groups, extensive metastasis rate was lowest in LMLD+US group, and survival time of animals in LMLD+US group was longest. Conclusions Tumor block transplanting method is a simple way to establish rabbit VX2 liver tumor models with high successful rate. Ultrasonography is a non-invasive, convenient and accurate method to monitor the tumor size,shape,and blood flow in tumors, which provides an effective monitoring method for estimating the treatment effect of real-time monitoring treatments. LMLD combined with UTMD could inhibit the growth of rabbit VX2 liver tumor by inhibiting proliferation and promoting apoptosis, which may give a novel strategy for exploring an effective, safe and high-targeting way for cancer therapy and provide the foundation for study on a kind of ultrasound microbubbles-based locally targeted-releasing technology.
多西紫杉醇(Docetaxel)是常用的疗效较好的化疗药物之一。由于多西紫杉醇水溶性差,目前市售制剂采用吐温-80增溶、乙醇助溶,易产生溶血及过敏等不良反应,临床使用极为不便;研究表明,将紫杉1烷类药物载入脂质后可明显降低药物的毒性,提高机体耐受性,且能保持与市售注射液相同的抗肿瘤活性。因此,在本实验中,我们将脂质微泡作为一种药物载体,将多西紫杉醇载入了微泡,制备了载多西紫杉醇脂质超声微泡,检测其特性;运用超声靶向微泡破裂使药物定位释放在靶组织,并通过体外和体内实验,探讨载多西紫杉醇微泡联合超声靶向微泡破裂对肝癌的作用及相关机理。本研究为构建一种新的药物剂型提供新策略,载药微泡联合UTMD为肿瘤的靶向治疗提供了新的思路。本课题主要包括以下三部分内容:
第一部分载多西紫杉醇脂质微泡的制备及一般特性研究
目的制备一种载多西紫杉醇的脂质超声微泡并测定其物理特性。方法采用机械振荡法制备载多西紫杉醇脂质微泡,测定其浓度、粒径分布范围、电位、包封率及载药量等特性;观察60Coγ射线灭菌前后微泡特性的差异及不同储存条件下微泡特性的改变;检测超声辐照后载药微泡内药物的释放。结果载多西紫杉醇微泡的浓度为2.2×109～3.2×109个/ml;粒径分布范围为473.4nm～706.6nm,平均粒径为623.1nm;微泡的包封率高于70%,载药量高于17.5%;Zeta电位为-(3.1±0.9)mV; 60Coγ射线灭菌前后微泡的特性无显著性差异,微泡在4℃、-20℃储存7天后其特性有所改变;超声辐照微泡溶液后,药物可释放。结论本实验成功制备了载多西紫杉醇脂质超声微泡,其包封率较高、粒径均匀、较稳定,超声辐照可使微泡破裂,促使微泡中的药物释放。
第二部分载多西紫杉醇脂质微泡联合超声靶向微泡破裂对人SMMC-7721肝癌细胞作用的实验研究
目的探索人SMMC-7721肝癌细胞株的体外培养条件及多西紫杉醇体外治疗的适宜浓度。探讨载多西紫杉醇脂质微泡联合超声靶向微泡破裂对SMMC- 7721细胞的增殖抑制、形态学影响、细胞周期的影响、诱导凋亡作用及相关蛋白和基因表达水平的影响。方法体外培养SMMC-7721细胞,观察其生长情况,并用MTT法检测其生长特性,描绘生长曲线。MTT法检测不同浓度多西紫杉醇对SMMC-7721细胞的增殖抑制率,并以此确定适宜的多西紫杉醇体外作用浓度。实验分为6组:空白对照组(C)、单纯药物组(Doc)、载药微泡组(LMLD)、药物+超声组(Doc+US)、单纯微泡+超声组(PLM+US)、载药微泡+超声组(LMLD+US)。MTT法检测不同时间点各组SMMC-7721细胞的增殖抑制率;透射电镜观察其细胞形态和超微结构变化;流式细胞仪检测各组细胞周期分布; Annexin V-FITC荧光染色检测细胞凋亡;实时定量-PCR和Western Blotting法分别检测Caspase3、Bcl-2、Bax在基因和蛋白水平的表达情况。结果在SMMC-7721细胞培养过程中,确定以1×105个/ml细胞数接种。应用一定浓度多西紫杉醇能抑制SMMC-7721细胞生长,呈明显的浓度-时间-效应关系。与其它组比较,LMLD+US组对SMMC-7721细胞的增殖抑制作用最强,细胞凋亡率最高,并可将SMMC-7721细胞明显阻滞在G2/M期。LMLD+US组可见较多凋亡细胞及凋亡小体形成,Doc+US组可见早期凋亡细胞。LMLD+US组Caspase3和Bax在基因和蛋白水平的表达高于其它各组,而Bcl-2在基因和蛋白水平的表达明显低于其它各组。结论多西紫杉醇对SMMC-7721细胞的增殖有抑制作用,确定多西紫杉醇后续实验药物浓度为1×10-9 mol/L。载多西紫杉醇脂质微泡联合超声靶向微泡破裂对SMMC-7721细胞具有明显的增殖抑制作用,并可将细胞明显阻滞在G2/M期、诱导细胞凋亡,其作用机制可能是通过影响细胞凋亡有关的多个基因及蛋白的表达水平。
第三部分载多西紫杉醇脂质微泡体内显影观察及其联合超声靶向微泡破裂对兔VX2肝癌的作用
目的利用超声检查监测肿瘤情况,并探讨载多西紫杉醇脂质微泡体内的显影效果。研究载多西紫杉醇脂质微泡联合UTMD介导药物靶向释放对兔VX2肝癌的治疗效果。方法90只新西兰大白兔,采用瘤块种植法于肝左叶内植入VX2肿瘤组织块,建立兔肝癌模型。用2D、CDFI及微泡造影观察肿瘤的大小、回声及血流灌注,并和CT检查结果相比较,从而评估超声在兔VX2肝癌模型监测中的应用价值。病理解剖验证VX2瘤株在兔肝脏的成瘤情况。实验动物随机分成6组,每组15只,包括空白对照组(C)、载药微泡组(LMLD)、单纯药物组(Doc)、药物+超声组(Doc+US)、单纯微泡+超声组(PLM+US)、载药微泡+超声组(LMLD+ US)。治疗前后用超声和CT观察肿瘤并测量其大小,计算其体积和肿瘤抑制率;免疫组化法检测肿瘤组织CD34、MMP2及PCNA的表达;TUNEL法检测肿瘤细胞凋亡;原位杂交法检测MMP2和Caspase3 mRNA表达水平;比较各组动物的存活时间和转移情况。
结果采用瘤块种植法成功建立了兔肝VX2肝癌模型,成瘤率为100%。超声能较准确地测量肿瘤的大小,能实时、准确地监测肝内肿瘤的回声变化及肿瘤血流情况,超声与CT测量的肿瘤大小无显著性差异(P>0.05);静脉注射载药微泡后,兔肝实质见良好、持续的增强显影,肝癌可见典型的“快进快出”显影表现。LMLD+ US组的肿瘤抑制率明显高于其他各组。治疗后肿瘤内部多呈混合回声,CDFI显示肿瘤的血流明显减少。LMLD+ US组的凋亡指数(AI)显著高于其它各组(P<0.01);LMLD+ US组的肿瘤增殖受到明显抑制,其增殖指数明显低于其它各组(P<0.01);LMLD+ US组MMP2蛋白及mRNA表达水平明显低于其它各组,而Caspase3 mRNA表达明显高于其它各组;LMLD+ US组的动物存活时间明显长于其它组。各组动物均见有腹腔转移,但LMLD+US组的远处转移最少。结论瘤块种植法建立兔VX2肝癌模型操作较简单、成瘤率高。载药微泡对肝实质及肿瘤具有良好的显影效果,超声检查能无创、方便、较准确地监测肝脏VX2肿瘤的大小、形态等;超声检查为利用VX2肝癌模型评价载多西紫杉醇脂质微泡联合UTMD的疗效及实时监控下的治疗提供了有效的监测方法。载多西紫杉醇脂质微泡联合UTMD能抑制兔VX2肝癌的增殖、促进其凋亡,对VX2肝癌具有显著的抗肿瘤作用,为寻求一种有效、安全、靶向性高的肿瘤治疗手段提供了新的思路,为研究一种基于超声微泡的靶向定位控释技术提供了依据。
Hepatocellular carcinoma (HCC) is the sixth most common tumor worldwide, and the incidence and mortality in China are highest around the world, for these reasons, research on liver carcinoma treatment is important and essential. At present, surgical resection is the main treatment method for it, but because of the low rate of surgical resection and the high recurrence rate, chemotherapy is usually the main available treatment method, however, most of chemotherapy drugs are cytotoxic, lacking of targeting to cancer cells, and causing severely side effects to body. Therefore, it is imminent to explore a kind of effective, safe, high-targeting and low toxicity treatment method. Recently, ultrasound microbubbles as a kind of drug or gene delivery vehicles are paid an ever increasing attention. After intravenous injection, microbubbles can arrive at the target tissues through blood circulation, ultrasound can be used to monitor the appearance of microbubbles in target organs and certain intensity of ultrasound could make microbubbles rupture, and then, the drug could be released locally in target tissues and absorbed more by target cells, by this way, the treatment effects could be improved and the side effects on other tissues could be decreased. Docetaxel is an extensively used chemotherapy drug in clinical trials, which has been evaluated as one of the better effective chemotherapy drug, however, water solubility of docetaxel is so poor that it is solubilized in polysorbate 80 (Tween 80) and alcohol for clinical use, which usually causes adverse events such as haemolysis and anaphylaxis, therefore, clinical application of docetaxel is inconvenient and limited. Current studies showed loading taxane drugs into lipid materials could lower toxic effects, improve the tolerance of patients, and the same antitumor activity as injection could be kept. In this study, lipid ultrasoud microbubbles was used as a drug vehicle, and UTMD was applied to release the drug locally in target tissues. Lipid ultrasoud microbubbles loaded docetaxel were prepared, and the properties of which were determined. We explored the effect of LMLD combined with UTMD on liver tumor and related mechanisms in vitro and in vivo level. This research may give a new strategy for composing pharmaceutical dosage form, and drug-loaded microbubbles combined with UTMD may provide a novel thought for tumor targeted-treatment. The study consisted of the following three parts:
PART I The preparation and general properties of lipid ultrasoud microbubbles loaded docetaxel
Objective To prepare a kind of lipid ultrasoud microbubbles loaded docetaxel, study its physical properties, and evaluate as a new ultrasound contrast agent for chemotherapeutic drug delivery. Methods The LMLD were made using mechanic vibration technique. Properties were studied contained concentration, size distribution, zeta potential, drug entrapment efficiency and drug-loading amount. Primary stability experiments were performed including comparison of the properties of lipid ultrasoud microbubbles loaded docetaxel under different storage condition, before and after sterilizing with 60Coγirradiation. Drug released with ultrasound was observed.Results LMLD had a concentration of approximately 2.2×109~3.2×109/ml, a mean size of 623.1nm and a zeta potential of–(3.1±0.9)mV. Size distribution was 473.4~706.6nm. Drug entrapment efficiency was more than 70% and drug-loading amount was more than 17.5%. There was no significant difference in properties between before and after sterilizing with 60Co irradiation, properties were changed a little after being stored at 4℃and -20℃for 7 days. Certain intensity of ultrasound energy can rupture microbubbles and release docetaxel. Conclusions In this study, we successfully prepared lipid ultrasoud microbubbles loaded docetaxel, and this kind of microbubbles had high drug entrapment efficiency, the size was well-distributed and it was stable,docetaxel could be released from microbubbles by ultrasound irradiation.
PART II Effect of lipid ultrasoud microbubbles loaded docetaxel combined with ultrasound-targeted microbubble destruction on SMMC-7721 hepatoma carcinoma cells
Objective To study the cultivation parameters and suitable docetaxel treatment concentration on hepatoma carcinoma cell strain SMMC-7721 in vitro. To investigate the effects of lipid ultrasoud microbubbles loaded docetaxel combined with ultrasound-targeted microbubble destruction on cells proliferation, ultrastructure, cell cycle distribution, apoptosis and expression of apoptosis-related proteins and genes. Methods SMMC-7721cells were cultured in vitro,cells growth characteristics were determined by morphologic observation and cell counting.The suitable treatment concentration was decided according to the proliferating inhibiton rate of SMMC-7721 cells in different dose groups, the proliferating inhibiton rate was detected by MTT assay. SMMC-7721 cells were divided into 6 groups:blank control group (C),docetaxel group(Doc),lipid microbubbles loaded docetaxel group(LMLD), docetaxel+ultrasound group(Doc+US), pure lipid microbubbles+ultrasound group(PLM+US) and lipid microbubbles loaded docetaxel+ultrasound group(LMLD+US). Proliferating inhibition rates at different time point were detected with MTT assay. Ultrastructural changes were observed by transmission electron microscope(TEM). Cell cycle distribution was observed by flow cytometry(FCM). Cell apoptosis was detected with Annexin V-FITC technology. Protein and mRNA expression of Caspase3, Bcl-2, Bax were separately detected by Western Blotting and real-time PCR. Results During the course of cultivation,SMMC-7721cells were inoculated with a concentration of 1×105/ml. Certain concentration of docetaxel could inhibit the growth of SMMC-7721 cells, the inhibition effects were positively related with the concentration of docetaxel and time, which showed an obvious dose-time-response relation. Compared with other groups, the proliferating inhibition effect of LMLD+US group was strongest and the apoptotic rate was highest; percentage of cells blocked in G2/M phase was highest in LMLD+US group. Many apoptosis bodies were observed in LMLD+US group, early stage apoptosis cells could be seen in Doc+US group. The expression of Caspase3 and Bax in LMLD+US group was significantly higher than other groups at protein and mRNA level, the Bcl-2 expression in LMLD+US group was lowest among all groups. Conclusions Docetaxel could inhibit the growth of SMMC-7721 cells in vitro, 1×10-9 mol/L was chosen as the suitable concentration in following experiments. Lipid microbubbles loaded docetaxel combined with ultrasound-targeted microbubble destruction could obviously inhibit the proliferation and induce apoptosis of SMMC-7721 cells,and also blocked SMMC-7721 cells in G2/M phase;the possible mechanisms of which may be lie in influencing the expression of apoptosis-related proteins and genes.
PART III Tumor imaging with lipid microbubbles loaded docetaxel and effect of lipid microbubbles loaded docetaxel combined with ultrasound-targeted microbubble destruction on rabbit VX2 liver tumor
Objective To evaluate the ultrasound imaging quality of tumor with LMLD; to explore the antitumor effect of LMLD combined with UTMD on rabbit VX2 liver tumor. Methods VX2 liver tumor models were established in 90 rabbits. Size, echo and blood flow of tumors were detected by 2D, CDFI and CEUS, the axes values detected by ultrasound were compared with CT measurements. The successful establishment of VX2 tumor was testified by pathologic biopsy. 90 rabbits were randomly divided into 6 groups: blank control group (C),docetaxel group(Doc),lipid microbubbles loaded docetaxel group(LMLD), docetaxel+ultrasound group(Doc+US), pure lipid microbubbles+ultrasound group(PLM+US) and lipid microbubbles loaded docetaxel+ultrasound group(LMLD+US). Tumor sizes were observed by ultrasound and CT before and after treatment; tumor volume(TV) and the tumor inhibition rate(TIR) were calculated and compared. Apoptosis was detected by TUNEL. Protein expression of CD34, PCNA and MMP2 was detected by immunohistochemistry(IHC). mRNA expression of Caspase3 and MMP2 was detected by hybridization histochemistry (ISH). Tumor metastasis rate and survival time of animals were compared. Results The rabbit VX2 liver tumor models were successfully established with tumor block transplanting method, and the successful rate was 100%. Ultrasound could detect the tumors accurately and provide real-time monitoring, there was no significant difference between the axes measured by ultrasound and CT(P>0.05). Liver imaging of rabbits could be enhanced obviously and persistently,“fast in and out”phenomenon was typical of VX2 liver tumor. TIR and apoptotic index (AI) of LMLD+US group were highest among all groups(P<0.01) and proliferating labelling index (LI) was lowes(tP<0.01).MVD of LMLD+US group was lowest among all groups(P<0.01).Protein and mRNA expression of MMP2 in LMLD+US group were lowest(P<0.01), and Caspase3 mRNA expression in LMLD+US group was highest among all groups(P<0.01).Abdominal cavity metastasis was found in all groups, extensive metastasis rate was lowest in LMLD+US group, and survival time of animals in LMLD+US group was longest. Conclusions Tumor block transplanting method is a simple way to establish rabbit VX2 liver tumor models with high successful rate. Ultrasonography is a non-invasive, convenient and accurate method to monitor the tumor size,shape,and blood flow in tumors, which provides an effective monitoring method for estimating the treatment effect of real-time monitoring treatments. LMLD combined with UTMD could inhibit the growth of rabbit VX2 liver tumor by inhibiting proliferation and promoting apoptosis, which may give a novel strategy for exploring an effective, safe and high-targeting way for cancer therapy and provide the foundation for study on a kind of ultrasound microbubbles-based locally targeted-releasing technology.
引文
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[8] Chen JH, Li YC, Huang YS, et al. Induction of VX2 carcinoma in rabbit liver: comparison of two inoculation methods[J]. Lab Anim. 2004, 38(1):79-84.
[9] Lin WY, Chen J, Lin Y, et al. Implantation of VX2 carcinoma into the liver of rabbits: a comparison of three direct-injection methods[J] . J Vet Med Sci. 2004, 64(7): 649-652.
[10] Czekierdowski A, Czekierdowska S, Czuba B, et al. Microvessel density assessment in benign and malignant endometrial changes[J] .J Physiol Pharmacol. 2008, 59 (Suppl 4): 45-51.
[11] Nanashima A, Nakayama T, Sumida Y, et al. Relationship between microvessel count and post-hepatectomy survival in patients with hepatocellular carcinoma[J]. World J Gastroenterol. 2008, 14(31): 4915-4922.
[12] Hwang SH, Rait A, Pirollo KF, et al. Tumor-targeting nanodelivery enhances the anticancer activity of a novel quinazolinone analogue[J] . Mol Cancer Ther. 2008, 7(3): 559-568.
[13] Condorelli G, Roncarati R, Ross J Jr, et al. Heart-targeted overexpression of caspase3 in mice increases infarct size and depresses cardiac function[J] . Proc Natl Acad Sci USA. 2001, 98(17): 9977-9982.
[14] Dean RA, Butler GS, Hamma-Kourbali Y, et al. Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis[J] . Mol Cell Biol. 2007, 27(24): 8454-8465.
[15]Li X, Wang Z, Ran H, et al. Experimental research on therapeutic angiogenesis induced by hepatocyte growth factor directed by ultrasound-targeted microbubble destruction in rats[J] . J Ultrasound Med. 2008, 27(3): 453-460.
[16] Vancraevnest D, Havaux X, Pouleur AC, et al. Myocardial delivery of colloid nanoparticles using ultrasound-targeted microbubble destruction[J] . Eur Heart J. 2006, 27(2): 237-245.
[17] Chen S, Ding JH, Bekeredjian R, et al. Efficient gene delivery to pancreatic islets with ultrasonic microbubble destruction technology[J] .Proc Natl Acad Sci USA. 2006, 103(22): 8469-8474.
[18] Xing W, Gang WZ, Yong Z, et al. Treatment of Xenografted Ovarian Carcinoma Using Paclitaxel-loaded Ultrasound Microbubbles[J] . Acad Radiol. 2008, 15(12): 1574-1579.
[1] Hitoshi Maruyama, Masaaki Ebara. Recent applications of ultrasound: diagnosis and treatment of hepatocellular carcinoma[J] . Int J Clin Oncol. 2006, 11:258–267.
[2] Yiyao Liu,Hirokazu Miyoshi,Michihiro Nakamura. Encapsulated ultrasound microbubbles: Therapeutic application in drug/gene delivery[J] . Journal of Controlled Release. 2006, 114:89-99.
[3]Klibanov AL.Microbubble contrast agents:Targeted ultrasound imaging andultrasound-assisted drug-delivery applications[J] .Invest Radiol. 2006, 41(3):354-362.
[4]卞爱娜,高云华,谭开彬,等.人肝癌靶向脂质体微泡造影剂的鉴定及免疫学性质研究[J] .中华超声影像学杂志. 2004, 13:696-699.
[5] Barbarese E,Ho SY,Darrigo JS,et a1,Internalization of microbubbles by tumor-cells in-vivo and in-vitro[J] . J Neuro Oncology. 1995, 26:25-34.
[6] Ellegala DB,Leong-Poi H,Carpenter JE,et a1.Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted toαvβ3[J] .Circulation. 2003,108:336-341.
[7] Leong-Poi H,Christiansen J,Klibanvo AL,et a1.Noninvasive assessment of angiogenesis by ultrasound and microbubbles targeted toαv-Integrins[J] .Circulation. 2003, 107:455-460.
[8]叶永强,刘政,付赤学,等.靶向肿瘤血管αvβ3整合素的脂质声学造影剂体外研究[J] .临床超声医学杂志. 2005, 7:88-91.
[9] Christopher J. Harvey,Adrian K.P. Lim,Martin J.K. Blomley,et al. Detection of an occult hepatocellular carcinoma using ultrasound with liver-specific microbubbles[J] . Eur Radiol. 2002, 12:S70-S73.
[10] Gregory E.R. Weller,Michael K.K. Wong, Ruth A. Modzelewski,et al. Ultrasonic Imaging of Tumor Angiogenesis Using Contrast Microbubbles Targeted via the Tumor-Binding Peptide Arginine-Arginine-Leucine[J]. Cancer Res. 2005, 65:533-539.
[11] Mattrey RF,Aguirre DA. Advances in contrast media research[J] .Academic Radiology. 2003, 10: l450-1460.
[12]谭开彬,高云华.纳米级微泡:一种新的超声造影剂[J] .中国医学影像技术. 2005, 21:516-518.
[13] Oeffinger BE,Wheatley MA.Development and characterization of a nanoscale contrast agent[J] .Ultrasonics. 2004, 42:343-347.
[14] Price RJ,Skyba DM,Kaul S,et al. Delivery of Colloidal Particles and Red Blood Cells to Tissue Through Microvessel Ruptures Created by Targeted Microbubble Destruction With Ultrasound[J] .Circulation. 1998, 98:1264-1267.
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[16]张群霞,王志刚,冉海涛,等.超声微泡促进肿瘤细胞报告基因转染[J] .临床超声医学杂志. 2005, 7:78-80.
[17] Haag P,Frauscher F,Gradl J ,et al. Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide targeting the human androgen receptor into prostate tumours[J] . J Steroid Biochem Mol Biol. 2006, 102:103-113.
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[19] Hayashida K,Ooi J,Omagari K,et al.Percutaneous ethanol injection therapy by ethanol mixed with CO2 microbubble for hepatocellular carcinoma[J] .Nippon Shokakibyo Gakkai Zasshi. 1997, 94:730-738.
[20]陈宝安,姜藻,董伟民,等.低功率超声空化效应二氧化碳微泡剂治疗淋巴瘤初探[J] .中华内科杂志. 2004, 43:697-698.
[21] Korpanty G, Carbon JG et al. Monitoring response to anticancer therapy by targeting microbubbles to tumor vasculature[J] .Clin Cancer Res. 2007, 13:323-330.
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[1]戚跃勇,邹利光,魏泓.兔VX2肝癌模型在TACE中的研究进展.中国比较医学杂志[J] . 2004,14(1):61-65.
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[4] Weidner N. Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors [J]. Breast Cancer Res Treat. 1995, 36(2): 169-180.
[5]张洪新,王执民,曹伟,等.兔Vx-2移植性肝癌模型的建立及其影像学表现[J] .介入放射学杂志.2002,11(3):193-196.
[6]王荞,王佚,王少春,等.兔肝VX2肿瘤模型的建立及超声影像监测的研究[J].重庆医科大学学报.2003,28(5):585-611.
[7]吴文娟,崔慧先,李海涛,等.兔VX2肝癌模型的建立及超声观察[J].中华超声影像学杂志.2005,14(2):147-150.
[8] Chen JH, Li YC, Huang YS, et al. Induction of VX2 carcinoma in rabbit liver: comparison of two inoculation methods[J]. Lab Anim. 2004, 38(1):79-84.
[9] Lin WY, Chen J, Lin Y, et al. Implantation of VX2 carcinoma into the liver of rabbits: a comparison of three direct-injection methods[J] . J Vet Med Sci. 2004, 64(7): 649-652.
[10] Czekierdowski A, Czekierdowska S, Czuba B, et al. Microvessel density assessment in benign and malignant endometrial changes[J] .J Physiol Pharmacol. 2008, 59 (Suppl 4): 45-51.
[11] Nanashima A, Nakayama T, Sumida Y, et al. Relationship between microvessel count and post-hepatectomy survival in patients with hepatocellular carcinoma[J]. World J Gastroenterol. 2008, 14(31): 4915-4922.
[12] Hwang SH, Rait A, Pirollo KF, et al. Tumor-targeting nanodelivery enhances the anticancer activity of a novel quinazolinone analogue[J] . Mol Cancer Ther. 2008, 7(3): 559-568.
[13] Condorelli G, Roncarati R, Ross J Jr, et al. Heart-targeted overexpression of caspase3 in mice increases infarct size and depresses cardiac function[J] . Proc Natl Acad Sci USA. 2001, 98(17): 9977-9982.
[14] Dean RA, Butler GS, Hamma-Kourbali Y, et al. Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis[J] . Mol Cell Biol. 2007, 27(24): 8454-8465.
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[16] Vancraevnest D, Havaux X, Pouleur AC, et al. Myocardial delivery of colloid nanoparticles using ultrasound-targeted microbubble destruction[J] . Eur Heart J. 2006, 27(2): 237-245.
[17] Chen S, Ding JH, Bekeredjian R, et al. Efficient gene delivery to pancreatic islets with ultrasonic microbubble destruction technology[J] .Proc Natl Acad Sci USA. 2006, 103(22): 8469-8474.
[18] Xing W, Gang WZ, Yong Z, et al. Treatment of Xenografted Ovarian Carcinoma Using Paclitaxel-loaded Ultrasound Microbubbles[J] . Acad Radiol. 2008, 15(12): 1574-1579.
[1] Hitoshi Maruyama, Masaaki Ebara. Recent applications of ultrasound: diagnosis and treatment of hepatocellular carcinoma[J] . Int J Clin Oncol. 2006, 11:258–267.
[2] Yiyao Liu,Hirokazu Miyoshi,Michihiro Nakamura. Encapsulated ultrasound microbubbles: Therapeutic application in drug/gene delivery[J] . Journal of Controlled Release. 2006, 114:89-99.
[3]Klibanov AL.Microbubble contrast agents:Targeted ultrasound imaging andultrasound-assisted drug-delivery applications[J] .Invest Radiol. 2006, 41(3):354-362.
[4]卞爱娜,高云华,谭开彬,等.人肝癌靶向脂质体微泡造影剂的鉴定及免疫学性质研究[J] .中华超声影像学杂志. 2004, 13:696-699.
[5] Barbarese E,Ho SY,Darrigo JS,et a1,Internalization of microbubbles by tumor-cells in-vivo and in-vitro[J] . J Neuro Oncology. 1995, 26:25-34.
[6] Ellegala DB,Leong-Poi H,Carpenter JE,et a1.Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted toαvβ3[J] .Circulation. 2003,108:336-341.
[7] Leong-Poi H,Christiansen J,Klibanvo AL,et a1.Noninvasive assessment of angiogenesis by ultrasound and microbubbles targeted toαv-Integrins[J] .Circulation. 2003, 107:455-460.
[8]叶永强,刘政,付赤学,等.靶向肿瘤血管αvβ3整合素的脂质声学造影剂体外研究[J] .临床超声医学杂志. 2005, 7:88-91.
[9] Christopher J. Harvey,Adrian K.P. Lim,Martin J.K. Blomley,et al. Detection of an occult hepatocellular carcinoma using ultrasound with liver-specific microbubbles[J] . Eur Radiol. 2002, 12:S70-S73.
[10] Gregory E.R. Weller,Michael K.K. Wong, Ruth A. Modzelewski,et al. Ultrasonic Imaging of Tumor Angiogenesis Using Contrast Microbubbles Targeted via the Tumor-Binding Peptide Arginine-Arginine-Leucine[J]. Cancer Res. 2005, 65:533-539.
[11] Mattrey RF,Aguirre DA. Advances in contrast media research[J] .Academic Radiology. 2003, 10: l450-1460.
[12]谭开彬,高云华.纳米级微泡:一种新的超声造影剂[J] .中国医学影像技术. 2005, 21:516-518.
[13] Oeffinger BE,Wheatley MA.Development and characterization of a nanoscale contrast agent[J] .Ultrasonics. 2004, 42:343-347.
[14] Price RJ,Skyba DM,Kaul S,et al. Delivery of Colloidal Particles and Red Blood Cells to Tissue Through Microvessel Ruptures Created by Targeted Microbubble Destruction With Ultrasound[J] .Circulation. 1998, 98:1264-1267.
[15] Lanza GM,Yu X,Winter PM,et al.Targeted antiproliferative drug delivery to vascular smooth muscle cells with a magnetic resonance imaging nanoparticle contrastagent:implications for rational therapy of restenosis[J] . Circulation. 2002, 106: 2842– 2847.
[16]张群霞,王志刚,冉海涛,等.超声微泡促进肿瘤细胞报告基因转染[J] .临床超声医学杂志. 2005, 7:78-80.
[17] Haag P,Frauscher F,Gradl J ,et al. Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide targeting the human androgen receptor into prostate tumours[J] . J Steroid Biochem Mol Biol. 2006, 102:103-113.
[18] Aaron F H Lum, Mark A Borden, Paul A Dayton,et al. Ultrasound Radiation Force Enables Targeted Deposition of Model Drug Carriers loaded on Microbubbles[J] .J Control Release. 2006, 111: 128–134.
[19] Hayashida K,Ooi J,Omagari K,et al.Percutaneous ethanol injection therapy by ethanol mixed with CO2 microbubble for hepatocellular carcinoma[J] .Nippon Shokakibyo Gakkai Zasshi. 1997, 94:730-738.
[20]陈宝安,姜藻,董伟民,等.低功率超声空化效应二氧化碳微泡剂治疗淋巴瘤初探[J] .中华内科杂志. 2004, 43:697-698.
[21] Korpanty G, Carbon JG et al. Monitoring response to anticancer therapy by targeting microbubbles to tumor vasculature[J] .Clin Cancer Res. 2007, 13:323-330.