Targeted Release And Treatment By 10-hcpt-loaded Microbubbles In Mice With H22 Tumor With Ultrasound Irradiation

10-HCPT (10-hydroxycamptothecin) is a kind of alkaloids and which is extracted from the camptotheca acuminata, it's a sort of chemotherapy drug commonly used in clinic. It has strong antitumor activity against a wide range of tumors with a mechanism of targeting the nuclear enzyme topoisomerase I, and 10-HCPT has no cross-resistance with the commonly used anticancer drugs. The active site of 10-HCPT'molecular structure is theα-hydroxy lactone ring. But the 10-HCPT itself does not dissolve in water, the clinic use alkaline solution to dissolve it. By this way the structure ofα-hydroxy lactone ring opens, that will not only reduce the effect, but also lead to serious side effects, as hypolekocytosis, thrombocytopenia, hemorrhagic enterocolitis and so on.In order to increase efficacy while reducing side effects, new dosage forms for the 10-HCPT research has attracted wide interest.Lipid ultrasound microbubbles are formed of phospholipid bilayer enwraping fluorocarbon gases. Except as an ultrasound contrast agent, it has also been used to carry genes or drugs in targeted therapy in recent years. The study aims to prepare a new lipid ultrasound microbubble containing HCPT(HLM) ,then to observe targeted release of 10-HCPT in H22 liver tumor in mice by HLM with ultrasound targeted microbubble destruction (UTMD) technique and evaluate it's anticancer effect .Therefore the study includes three parts:PARTⅠPreparation and Identification of HLMObjective To prepare the 10-HCPT loaded lipid ultrasound microbubbles and to study its characteristics then evaluate it as an ultrasound contrast agent.Methods The 10-HCPT was dissolve in lipid thoroughly by sonic boom and film drying method ,then the mechanical vibration method was used to prepare HLM. The concentration, particle size of HLM was measured by the DFY ultrasound image analysis software, then combined optical microscope to calculate concentration of microbubble. Drug-loading capacity and drug encapsulation of HLM was established by the high performance liquid chromatography (HPLC). Subcutaneous tumor imaging in mice and destroyed by ultrasound were observed by ultrasonic inspection.Results The drug-loaded microbubbles were round and uniform. The diameter range of HLM was 0.63~1.93μm,the average particle size of HLM was 1.32±0.18μm, the diluted concentration was about 1.97×109/ml. HLM could enhance the subcutaneous tumor imaging in mice and could be destroyed by ultrasound irradiation.Conclusion The basic physical properties of HLM were similar to that of the common lipid microbubble. The HLM had higher entrapment efficiency and drug-loading amounts established by HPLC. HLM was effective for the enhancement of subcutaneous tumor imaging in mice and also could be destroyed by ultrasound irradiation.PARTⅡTargeted Release of 10-HCPT in H22 Transplantation Tumor in Mice by HLM with UTMDObjective To establish H22 tumor-bearing mice model and targeted release 10-HCPT in H22 transplantation tumor by HLM with ultrasound targeted microbubble destruction (UTMD) technique.Methods H22 cells were cultured until the cells growth to the logarithmic growth phase. Collected cells by centrifugation, re-suspended with PBS then inoculated to each mouse subcutaneously. 42 tumor-bearing mice were collected for the experiment. There were 12 tumor-bearing mice in group A for observation of The distribution of fluorescently-labeled HLM in tumor.There were 30 tumor-bearing mice in groupB for detection of drug concentration in tissues.Mice in groupA were divided into fluorescently-labeled HLM group and ultrasound + fluorescently-labeled HLM group . Mice were sacrificed after 1h to take their tumor of each mouse to made cryostat section and the cryostat sections were observed by microscope. Mice in group B were randomly divided into five groups: ultrasound+10-HCPT injection group, ultrasound+10-HCPT injection + blank microbubbles group, HLM group, ultrasound +HLM group and saline control group. Mice were sacrificed after 1h to take their heart, liver, spleen, lung, kidney and tumor of each mouse to measure the drug concentrations in each tissue.Results Tumor biopsy showed fluorescein distribution in tumor of ultrasound + HLM group was significantly more than that of HLM group. The highest drug concentration in tumor was ultrasound +HLM group and the drug concentration in other tissues of mice treated with HLM were higher than mice treated with 10-HCPT injection(P<0.05).Conclusion HLM could release drug in target organ by UTMD technique to elevate drug concentration in tumor and HLM could prolong the circulation time of HCPT in vivo.PARTⅢAnticancer Effect of HLM with UTMD in Mice with H22 Transplantation TumorObjective To evaluate the antitcancer effects of HLM with ultrasound targeted microbubble destruction (UTMD) technique in mice with H22 transplantation tumor. Methods Tumor-bearing mice were randomly divided into five groups: ultrasound+10-HCPT injection group, ultrasound+ blank microbubbles +10-HCPT injection group, HLM group, ultrasound +HLM group and control group.Each of the mice was administered respectively quaque die alterna all quintic.Tumor volume of each mouse was evaluated by sonogram, then draw the growth curve chart and calculated the tumor inhibition rate. The tumor of each mouse was harvested to detect the apoptosis by TUNEL method and detect the hyperplasy by PCNA immunohistochemistry.Results The tumor inhibition rate was the highest in ultrasound +HLM group while the tumor growth rate was the lowest in ultrasound +HLM group(P<0.05). The result of TUNEL indicated the apoptosis rate of each treatment group was higher than that of the control group,and the apoptosis rate of ultrasound+HLM group was the highest(P<0.05).The result of PCNA immunohistochemistry indicated the multiplication rate of each treatment group was lower than that of the control group,and the multiplication rate of ultrasound+HLM group was the lowest(P<0.05).Conclusion Ultrasound irradiation mediate HLM destruction can significantly enhance the tumor inhibition effect of 10-HCPT in the H22 transplantation tumor. This technique is expected to be adopted as a novel tool for liver cancer chemotherapy.