Antitumor Efficacy And Mechanisms Of Dr5-targeted Docetaxel-loaded Lipid Microbubbles Combined With Ultrasound Targeted Microbubble Destruction On Liver Cancer

Liver cancer is one of the common worldwide malignant tumors withhigh morbidity and mortality, and global cancer statistics in the year2011showed that almost half of world's newly diagnosed and death cases ofliver cancer were reported in China. In recent years, with the continuousimprovements of our medical technology and economic levels, the rates ofsurgical treatment of liver cancer have gradually increased, while on theother hand, multimodal therapy including chemotherapy could prolongpatients' survival time and therefore plays an important role in patients withrecurrented or metastasized liver cancer. But traditional methods ofchemotherapy always result in poor targeting ability and efficacy, as well asside effects and lack of patient tolerance, and therefore there remains anurgent need to develop new drug delivery systems with improved targetingability and antitumor effect as well as alleviated side effects.With the constant developments of ultrasound molecular imaging technology in recent years, ultrasound-targeted molecular probes combinedwith ultrasound targeted microbubble destruction (UTMD) provide us anew method of noninvasive tumor-targeted drug delivery withcontrol-releasing ability, which may help us to improve the targeting abilityand antitumor efficacy of chemotherapeutic drugs for the treatment ofrecurrented liver cancer.Anti-death receptor5monoclonal antibody (DR5mAb) canspecifically bind to TRAIL-R2(death receptor5,DR5) which has elevatedexpressions on the surface of liver cancer cell, and then induce apoptosis inliver cancer cells through intracellular apoptotic pathways, however,DR5mAb would not cause hepatocyte apoptosis in normal liver tissuesbecause of their low expression of DR5, and therefore DR5mAb could beused as an ideal targeting material for the treatment of liver cancer withlittle damage in normal liver tissue.Docetaxel (DTX) is one promising antitumor drug of the taxane class,and plenty of researches have demonstrated that DTX has a more powerfulantitumor effect than paclitaxel. DTX could promote microtubulepolymerization and enhance microtubule stability in cell cycles, and recentstudies have demonstrated that lipid-coated docetaxel combined withUTMD could achieve effective improvements of docetaxel solubility andsignificant inhibition of liver cancer. Therefore, in this study, DR5mAbwere binded to DTX-loaded microbubbles via biotin–avidin technology, and then physical properties as well as targeting ability of microbubbleswere detected. Effect and mechanisms of DR5-targeted microbubblescombined with UTMD on liver cancer were investigated throughcytological experiments and animal models, which may provide newexperimental evidence for targeting treatment of liver cancer and futherresearch on ultrasound molecular imaging.The main subject of this study includes the following three parts:PARTⅠPREPARATION AND TARGETING ABILITY OFDR5-TARGETED DOCETAXEL-LOADED LIPIDMICROBUBBLES IN VITROObjective To develop a novel DR5-targeted and docetaxel-loadedmicrobubble and investigate its characterizations and specific targetingfunction in vitro.Methods DR5-targeted docetaxel-loaded lipid microbubbles(DTDLLM) were constructed by the avidin-biotin interaction which bindedbiotinylated DR5mAb to the surfaces of DLLM prepared by mechanicalvibration. The immunofluorescent assay was used to test the combinationbetween DR5mAb and DLLM. Microbubble stability was preliminarilyevaluated. The physical properties were determined and microbubblecomposition was optimized. Compared with DLLM as control, themorphology and targeting ability of DTDLLM to HepG2cells wereinvestigated in vitro. Results DTDLLM prepared in this study have similar particle sizewhich was1232nm on average, and were well dispersed with theconcentration of3.1×109/ml. The average drug entrapment efficiency anddrug-loaded amount was73.5%and25.3%, respectively. We also observedthat green fluorescents were obviously expressed around the surface ofDTDLLM, and targeted microbubbles tightly conjugated with HepG2cellsin DTDLLM group while few microbubble was observed binding to HepG2cells in DLLM group.Conclusions DTDLLM can be successfully constructed andspecifically attached to HepG2cells in vitro. DTDLLM,as a novel contrastagent with good properties and superior targeting ability, may become oneof the effectively targeting drug deliveries for ultrasound molecularimaging and HCC targeting therapy.PARTⅡ EFFIECT AND MECHANISMS OF DR5-TARGETEDDOCETAXEL-LOADED LIPID MICROBUBBLES COMBINEDWITH ULTRASOUND TARGETED MICROBUBBLEDESTRUCTION ON HEPG2CELLS IN VITROObjective To study the proliferation-inhibited and apoptosis-inducedeffects of DR5-targeted docetaxel-loaded lipid microbubbles (DTDLLM)combined with ultrasound targeted microbubble destruction on HepG2cellsin vitro, and investigate mRNA and protein expression levels of Caspase-8,Bcl-2and DR5. Methods Human hepatoma cell line HepG2were cultured and dividedinto nine groups, including the control group (Con), docetaxel group(DTX), docetaxel combined with ultrasound group(DTX+US), puremicrobubbles group(MB), pure microbubbles group combined withultrasound group(MB+US), docetaxel-loaded lipid microbubbles group(DLLM), docetaxel-loaded lipid microbubbles combined with ultrasoundgroup (DLLM+US), DR5-targeted docetaxel-loaded lipid microbubblesgroup (DTDLLM), DR5-targeted docetaxel-loaded lipid microbubblescombined with ultrasound group (DTDLLM+US). The CCK-8and TUNELassay were used to detect the proliferation-inhibited and apoptosis-inducedeffects, retrospectively, and then cell cycles were analyzed by flowcytometry. The mRNA and protein expression levels of Caspase-8, Bcl-2and DR5were tested by RT-PCR and Western Blot.Results Of all the groups, DTDLLM+US group demonstrates thestrongest proliferation-inhibited effect with an obvious time-dose-responserelationship (P<0.01), and the strongest apoptosis-induced effect (P<0.01)as well as an increased G2/M phase arrest showed by flow cytometrydetection of HepG2cell cycle (P<0.01). The mRNA and protein expressionof Caspase-8and DR5in DTDLLM+US group were significantlyup-regulated (P<0.01), and those of Bcl-2were conspicuouslydown-regulated (P <0.01) when compared to other groups.Conclusions DTDLLM combined with UTMD can significantly inhibit the proliferation of HepG2cells, and increase G2/M phase arrest aswell as apoptosis-induced effect, which resulted from regulated mRNA andprotein expressions of apoptosis-related factors including Caspase-8, DR5and Bcl-2.PART Ⅲ EFFIECT AND MECHANISMS OF DR5-TARGETEDDOCETAXEL-LOADED LIPID MICROBUBBLES COMBINEDWITH ULTRASOUND TARGETED MICROBUBBLEDESTRUCTION ON LIVER CANCER IN NUDE MICEObjective To investigate the antitumor effect and mechanisms ofDR5-targeted docetaxel-loaded lipid microbubbles (DTDLLM) on livercancer in nude mice.Methods Subcutaneous hepatoma was established in nude mice andthen randomly divided into five groups, including the control group (Con),docetaxel group(DTX), docetaxel combined with ultrasound group(DTX+US), docetaxel-loaded lipid microbubbles combined with ultrasoundgroup (DLLM+US), DR5-targeted docetaxel-loaded lipid microbubblescombined with ultrasound group (DTDLLM+US). Tumor growth curvewere drew, and tumor inhibition rate (TIR) were calculated. The TUNELassay was used to detect apoptosis-induced effects, and expression ofMMP-2and VEGF were detected by immunohistochemistry (IHC). ThemRNA and protein expression levels of Caspase-8, Bcl-2and DR5weretested by RT-PCR and Western Blot methods. Results Compared with other groups, DTDLLM+US demonstrates thehighest TIR (P<0.01) and AI (P<0.01) as well as the lowest expression ofMMP-2(P<0.01) and VEGF (P<0.01). The mRNA and protein expressionsof Caspase-8and DR5in DTDLLM+US group were significantlyup-regulated (P<0.01), and those of Bcl-2were conspicuouslydown-regulated (P <0.01) when compared to other groups.Conclusions DTDLLM+US can significantly inhibit the growth ofliver cancer in nude mice, and the stronger antitumor effect than those innon-targeted DLLM and DTX groups resulted from the stronger inhibitionin tumor angiogenesis and invasion, as well as down-regulated expressionsof Bcl-2and up-regulated expressions of Caspase-8and DR5. DTDLLMcombined with UTMD demonstrates a better targeting ability and strongerantitumor effect than non-targeted docetaxel-loaded microbubbles and pureDTX, and therefore provide a novel, efficient and targeting strategy forliver cancer therapy and ultrasound molecular imaging.