Experimental Study Of Targeted Nanometer Cationic Lipid Microbubbles Loaded HSV-TK Gene Can Synergistic Treatment Residual Liver Cancer After Hifu Incomplete Ablation

Primary liver cancer is one of the most common malignant tumor,In our country, the incidence and mortality rate ranked first in the world. At present, the treatment of liver cancer with multiple choice. The surgical operation is still the main treatment for early liver cancer, but there is a high rate of recurrence after operation, and the majority of patients have been in advanced stage or end-stage,lost the chance of operation. At present, in our country, patients with advanced hepatocellular carcinoma treated with radiofrequency ablation and transcatheter arterial embolization and chemotherapy is the main way. But the existence of serious complications and bring much pain to patients. In recent years, with the continuous development of science and technology, High intensity focused ultrasound (HIFU) as a new treatment method has been widely used in clinical, achieved good results in the treatment of many tumors and non neoplastic diseases. The principle of treatment of HIFU is mainly through the thermal effect of ultrasound to kill lesions. However, in clinical treatment of tumor, the tumors mostly located in deep tissue or organ, Ultrasonic wave through the organization will cause the sound energy decay, at the same time the tumor region rich in blood supply, energy will decrease with the blood flow. These factors have restricted the thoroughness treatment,of HIFU,reducing the treatment effect of HIFU, Resulting in tumor tissues can not be completely destroyed, increases the risk of tumor recurrence. Research shows that once tumor recurrence, the invasive ability increase significantly than the original. Therefore, how to improve the effect of treatment of HIFU, reduced tumor residue or recurrence become current research hot spot. With the progress of the study,the occurrence and progress of tumor associated with gene mutation or deletion. Therefore, the majority of scholars believe that gene therapy is the most promising treatment. Suicide gene therapy is the most effective schemes in cancer gene therapy,HSV-TK/GCV system is a kind of suicide gene, HSV-TK does not have the function of killing, after transfected into cells non-toxic GCV can be transformed into toxic substances to kill cells. How to put the HSV-TK plasmid is efficient and safe, noninvasive, nontoxic and transfected into the cells to become the bottleneck of its development.In recent years, targeted ultrasound microbubbles is widely applied in gene carrier, The microbubbles are injected into the body, through a specificity combination of microbubble surface ligands, antibodies and receptor of tumor tissue, thus in the target region clustering, use of a certain intensity ultrasound from the body surface irradiation make the micro bubble burst, In order to release the loaded purpose gene, At the same time, microbubble destruction generated cavitation effect and acoustic hole effect, can promote the cell membrane permeability increased, to facilitate gene transfection.Therefore, in this study, we prepared the nano cationic microbubble, the expression of hepatoma specific GPC3 antibody was constructed targeting cationic microbubble, Then the HSV-TK plasmid connected with targeting cationic microbubble,construction of nano targeting cationic microbubble loaded HSV-TK gene. Detect of its physical properties, to explore the ability of its target and treatment effect of HSV-TK/GCV system for tumor in vivo and in vitro. This study includes the following three parts:THE FIRST PART PREPARATION OF NANO TARGETED MICROBUBBLES LOADED GENETHE FIRST SECTION PREPARATION AND CHARACTERIZATION OF NANO MICROBUBBLEOBJECTIVE:To construct different types of ultrasound microbubbles for loaded the gene of interest.METHOD:Through the mechanical vibration method to construct different lipid microbubbles, The cationic microbubble with DC-cholesterol in the membrane material (DC-cholesterol) to the surface of the microbubble with positive charge, in order to construct cationic microbubble (CMB), To observe the morphology and uniformity of microbubble under light microscope, detect surface potential and particle size of microbubbles.RESULT:The successful construction of all kinds of micro bubble, Light microscope:the microbubble distribution, particle size uniform, Common microbubble surface charge of-2.38±0.56 mV, a weak negative charge, The surface potential of cationic microbubble was26.44±2.13 mV, Ordinary micro bubble diameter was 512.57±25.18nm,Cationic microbubble diameter was 502.63±21.26nm.CONCLUSION:Two kinds of micro bubble interaction, potential have significant difference (P< 0.01), size comparison, the difference was not statistically significant (P> 0.05).CONSTRUCTION OF TARGETED MICROBUBBLES AND PERFORMANCE TESTINGOBJECTIVE:Connect the GPC3 antibody and the HSV-TK plasmid with microbubbles,to construct targeted microbubbles loaded gene.METHOD:The prepared cationic connected the GPC3 antibody with microbubbles via "avidin biotin method", At the same time, cationic microbubble connect with isotype control antibody; The successful construction of the common microbubbles, cationic microbubble, targeting cationic microbubble (5×108) and plasmid (HSV-TK) were co incubated with, use of electrostatic adsorption principle so that the two fully integrated, then, the centrifugal and wash, to detect different types of microbubble loaded ability of plasmid was determined by the amount of unbound plasmid, Laser scanning confocal microscopy detection of plasmids and antibodies and microbubble connections.RESULT:Successful construction of target cationic microbubble loaded gene and contrast antibody cationic microbubble. Microbubble size uniformity under confocal laser microscope, the connection is successful, the dispersion is better, observed under the laser scanning confocal microscope: GPC3 antibody and microbubble connected showed green fluorescence, the HSV-TK plasmid with microbubbles with red fluorescence, the two overlapping showed yellow fluorescence in situ, show that successfully construted targeted microbubbles loaded gene. Ability of common microbubbles, cationic microbubble and cationic targeted microbubbles loaded HSV-TK plasmid were 0.36±0.03 μg,17.91±1.15μg�'�17.26±2.01μg, two group of cationic microbubble loaded plasmid had no statistical significance (P> 0.05), cationic microbubble and common microbubbles comparison, the difference was significant (P< 0.01).CONCLUSION:Nanometer microbubble, HSV-TK plasmid, GPC3 antibody can be successfully connected via "biotin avidin system", successfully constructed nano targeted microbubble loaded gene, Cationic microbubble loaded genetic ability is significantly higher than common microbubbles, Cationic microbubbles attached to the antibody does not affect its loaded capacity.THE SECOND PART EXPERIMENTAL STUDY OF COMMON MICROBUBBLES, CATIONIC MICROBUBBLE, TARGETING CATIONIC MICROBUBBLE TRANSFECTED HEPG-2 CELLSTHE FIRST SECTION DETECT THE ABILITY OF TARGETED MICROBUBBLE LOADED GENE TO FIND TARGET IN VITRO AND IN VIVOOBJECTIVE:To detect the ability of common microbubbles, cationic microbubble and targeting cationic microbubble to find HepG-2 cells in vitro and in vivo.METHOD:(1) The use of immunohistochemistry to confirmed that HepG-2 cell membrane expression of GPC3 antigen. (2) the HepG-2 cells were cultured by using self-designed glass plate, the cells adhered to the wall, then the cells were adherent glass plate and incubated with microbubbles fully, The glass plate flipped, Observation the number of microbubbles combined with HepG-2 cells under inverted microscope, to detect ability of different types microbubbles to find target in vitro. (3) Subcutaneous injection of HepG-2 cells in nude mice, The establishment of tumor model of human hepatocellular carcinoma in nude mice planting, Immunohistochemistry demonstrated that GPC3 expressed on tumor cell surface. Different types of micro bubble into nude mice by tail vein injection, to detect ability of microbubbles targeting find tumor cells in vivo by ultrasound imaging. In the experiment, the control antibody cationic microbubbles as cationic targeted microbubbles in control group, Competitive inhibition assay was used to confirm the specificity of targeting cationic microbubble binding with target cell.RESULT:(1) Immunohistochemistry confirmed that HepG-2 cell surface expression of GPC3 antigen; (2) Targeting test, in addition to the common microbubble, cationic microbubble, targeting cationic microbubble, control antibody cationic microbubble can bind with HepG-2 cell, who can express GPC3 antigen, while the number of cationic targeted microbubbles targeting with HepG-2 cell is the highest (P< 0.01); Pretreatment with GPC3 antibody incubation, number of targeting cationic microbubble combined with HepG-2 cells decreased significantly (P< 0.01); But the cells with isotype control antibody were incubated, the number of targeting cationic microbubble combined with HepG-2 cells and had no significant effect (P> 0.05). (3) In vivo experiment, ultrasound imaging confirmed:in addition to targeting cationic microbubble, common microbubbles, cationic microbubble and control antibody cationic microbubble can not bind with tumor cells who express GPC3 antibody; In ultrasound imaging of contrast model and traditional grey model, Compared with the control group, the common microbubbles, cationic microbubble with cationic targeted microbubble injection, the tumor showed echo enhancement; and echo in cationic microbubble group and targeting cationic microbubble group is stronger than in common microbubbles group, The strongest echo in targeting cationic microbubble group. In vivo competitive inhibition experiment:Through the tail vein of nude mice were injected with GPC3 antibody, the number of targeting cationic microbubble bind with targeting tumor cells decreased significantly (P< 0.001).CONCLUSION:(1) Cationic microbubble, targeting cationic microbubble and control antibody cationic microbubble can bind with HepG-2 cell, But only targeting cationic microbubble targeting bind with HepG-2 cell, while the cationic microbubble and control antibody cationic microbubbles combined with HepG-2 cells nonspecific, the combination of the advantages no longer exist in vivo; (2) Ultrasound imaging confirmed the targeting cationic microbubbles has good targeting effect in vivo, Fully confirmed the targeting cationic microbubble in vitro and in vivo, has good targeting ability.THE SECOND SECTION EXPERIMENTAL STUDY OF COMMON MICROBUBBLES, CATIONIC MICROBUBBLE AND TARGETING CATIONIC MICROBUBBLE LOADED HSV-TK PLASMID WAS TRANSFECTED INTO THE HEPG-2 CELLS UNDER THE EFFECT OF ULTRASONICOBJECTIVE:To investigate transfection efficiency of common microbubbles, cationic microbubble and targeting cationic microbubble loaded HSV-TK plasmid was transfected into HepG-2 cells under the action of ultrasound, and the effect on cell proliferation inhibition rate, tumor cell invasion.METHOD:The experiment is divided into control group, common microbubbles group, cationic microbubble group, targeting cationic group, different types of micro bubble connected with HSV-TK plasmid, was transfected into HepG-2 cells under the action of Ultrasound (1 MHz,1 W/cm2,50% duty cycle and (DC),30 seconds)o The efficiency of transfection was detected after transfection, cell proliferation inhibition rate was detected by MTT, qPCR and Western were used to detect the TK, VEGF, Caspase-3 mRNA and protein expression; Through the HepG-2 cell invasion in vitro to evalue treatment of HSV-TK plasmid in different microbubble mediated transfection.RESULT:(1) 24 hours after transfection and transfection efficiency of each group were 0.31±0.05%,2.67±0.27%,21.38±2.04%,34.39±2.93%, with statistically significant difference between the groups (P< 0.01). (2) 24 hours after transfection, the proliferation inhibition rate of cell was detected by MTT, were 8.25±0.93%,10.01±0.85%,42.36±3.87%,58.17±5.08%, targeting cationic microbubble group cell proliferation activity was decreased, the difference between groups was statistically significant (P< 0.01). (3) QPCR and Western were detected in the cationic microbubble and targeting cationic microbubble group are TK and Caspase-3 mRNA and protein expression increased, while the expression of VEGF mRNA and protein decreased, with a significant difference between the two groups (P< 0.05). (4) In tumor cell invasion assay, the migration of tumor cells was 61.25±4.29、 66.53±5.25、63.86±4.98、34.59±2.94、24.91±2.25, the difference between the two groups was significant (P<0.01).CONCLUSION:Cationic microbubble and targeting cationic microbubble group compared with control group and common microbubbles group, transfection efficiency was increased, the targeting cationic microbubble group has higher transfection efficiency and the corresponding biological effects, can significantly inhibit the proliferation of tumor cells and tumor cell invasion.THE THIRD PART C TRANSFECTED OF RESIDUAL CANCER WITHCOMMON MICROBUBBLES, AND CATIONIC MICROBUBBLE TARGETING CATIONICMICROBUBBLES LOADED WITH HSV-TK PLASMID AFTER HIFU INCOMPLETE ABLATION OF LIVER CANCEROBJECTIVE:To investigate the effect of common microbubbles, cationic microbubbleand targeting cationic microbubble loaded with HSV-TK plasmid ontransfecting into HepG-2 planting tumor after HIFU incomplete ablation under the function of ultrasonic; to observe the effects of different types of micro bubble n tumor growth and treatment effect.METHOD:(1) Tumor model in nude mice was established by subcutaneous injection of HepG-2 cells in the left hind limb suspension.14 days after modeling, thenude mice model was established successfully, and HIFU treatment of incompleteablation was conducted to construct residual cancer model. (2) This experiment was randomly divided into 4 groups, control group (respectively via tail vein injection of normal saline), common microbubbles group (intravenous injection of common microbubbles), cationic microbubble group (intravenous injection of cationic microbubble) and targeting cationic microbubble group (intravenous injection of microbubbles targeting cationic). (3) Each group of microbubbles was connected with HSV-TK plasmid. In vivo imaging scanning was performed after transfection, and the image analysis software was used to evaluate the effect of targeting. (4) After the success of the targeting, all groups were transfected into tumor cells under the action of ultrasound (1 MHz,2 W/cm2, and 50% DC,30 seconds), to intraperitoneal injection of GCV after transfection. (5) The expression of TK, PCNA, CD31 and VEGF in tumor tissue was detected by immunohistochemistry; the formation of microvessel in tumor was analyzed through the calculation on expression of CD31; the expression of TK, VEGF and caspase3mRNA was detected by q-PCR; the cell apoptosis was detected by TUNEL; the survival time of tumor bearing nude mice was recorded.RESULT:(1) HE staining of the residual cancer cells showed that HIFU incomplete ablation model was constructed successfully. After injecting microbubble. the residual cancer tissue showed green fluorescence by using in vivo imaging, which demonstrated that HSV-TK plasmid was successfully transfected into the cells. (2) Immunohistochemistry and q-PCR results showed that except the control group, K protein was expressed in each group, and the cationic targeted microbubbles group showed the highest expression level, which confirmed again that the HSV-TK gene was successfully transfected into tumor cells; (3) Immunohistochemistry and TUNEL results showed that in targeting cationic microbubble group,the tumor proliferation was significantly inhibited (P<0.01), apoptosis index was significantly increased (P<0.01), angiogenesis was inhibited (P<0.05), and the survival time was prolonged.CONCLUSION:(1) HSV-TK plasmid targeting cationic microbubble can accurately target tumor cells, and combined with ultrasound, it can promote the release of transfected plasmid, improve the transfection efficiency, in order to achieve the effect of the targeting treatment of residual tumor. (2) The HSV-TK/GCV system can inhibit proliferation, angiogenesis and tumor invasion of the tumor, increase apoptosis of tumor cells and also prolong the survival time enhance the therapeutic effect ofHIFUsignificantly.