Experimental Study On The Antitumor Effect Of Lipid Ultrasound Microbubbles Loaded Docetaxel Combined With Ultrasound-targeted Microbubble Destruction On Liver Tumor

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 docetaxelObjective 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 cellsObjective 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 tumorObjective 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.