The Experimental Study On Microbubble Contrast Agent Imaging Containing Nitrous Oxide And Synergism Of Hifu To Treat Osteosarcoma

PART I PREPARATION AND PERFORMANCE TESTINGOF MICROBUBBLE CONTRAST AGENT CONTAININGNITROUS OXIDEObjective To prepare ultrasonic lipid microbubble contrast agentloaded with nitrous oxide (N2O-LM), choose lipid as the shell material,C3F8and nitrous oxide N2O as the core, and detect the basic characteristicsand development effect in vitro and in vivo of the lipid microbubblecontrast agent, providing basis for follow-up study in vivo and in vitro.Methods （1）Prepare Lipid microbubbles loaded with nitrous oxidewith the mechanical oscillation method, with DSPC, DPPE liposomes asthe shell materials, C3F8and nitrous oxide(N2O) as the cores.（2）Observethe morphology after contrast agents redissolving with water, detectaverage particle sizes, count the concentrations. Place redissolvedmicrobubble contrast agent at room temperature in6h,3d,1d,1w, observethe changes of basic characteristics.（3）Use the Malvin laser detector todetect particle sizes of microbubble in different time (1d,3d,7d), detectthe lipid microbubbles potential with ZETA surface potential detection, atthe same time store each take part in4℃refrigerator, measure the particlesizes, potentials again after1week.（4）Observe the shapes, sizes, concentrations, Zeta potential changes of N2O-LM after60Co irradiation.（5）Gel imagings in vitro, preparate gel imaging model in vitro, observethe effect of microbubble in vitro enhancing ultrasound imaging,investigate its feasibility as a contrast imaging agent, observe the effect ofmicrobubbles on rat osteosarcoma imagings in vivo experiments,understand the abilities and principles of microbubble imagings.Results Lipid microbubbles loaded with nitrous oxide (N2O-LM)seemed to be white curd observed by naked eyes with this preparatingmethod, were packed in penicillin bottle, very thick on surface, opaque;observing under the light microscope, microbubbles were gathered innumber, good shapes, round, uniform size distribution, no obviousaggregation. Microbubble concentration was about (3.35±0.31)×109/ml,average particle diameter was (1105.7±121.2) nm, the Zeta potential was(-18.7±2.65) mV. At7day of observation, no significant changes of thephysicochemical properties was found (p>0.05). After contrast agentsdissolved, N2O-LM were at high concentrations in1day. Contrast agentshad been placed for3days, dissolved after1week, seemed white emulsion,and its basic characteristics had no obvious changes. After N2O-LM wereirradiated by60Co, sizes、Zeta potentials、concentrations of microbubbleshad no significant changes (p>0.05). Ultrasound imagings in vitro showedthat microbubbles were shaped high echo, and echo intensities decreased asthe concentrations decreased.Conclusions Successfully prepared shell materials with DSPC, DPPE,perfluoropropane (C3F8) and nitrous oxide (N2O) as the cores of lipidmicrobubble contrast agents. Microbubbles shaped very regular, spherical,stable properties, high concentrations, uniform sizes and the grain sizes,without obvious change of the physicochemical properties within7days.N2O-LM has the ability to enhance ultrasound imaging in vitro, has the potential being the ultrasound contrast agent. In vivo experiments,microbubbles could effectively enhance the contrast signal intensity ofrabbit liver imagings, worthy of further study. PART II THE RESEARCH ON N2O-LM COMBINEDWITH FOCUS ULTRASOUND TO BIOLOGICALEFFECTS OF RAT OSTEOSARCOMA CELL UMR-106Objective To investigate the mechanism of biological effect of N2O-LM combined with focus ultrasound effecting on rat osteosarcoma cellsUMR-106.Methods Use the method of MTT to test the effect on the survivalrates of rat osteosarcoma cells UMR-106in different ultrasound irradiatingtime (10s,20s,30s,60s,90s), to determine the optimum irradiating time,ultrasound irradiating parameters (ultrasonic frequency of10.50MHz,sound intensity:0.5W/cm2). UMR-106rat osteosarcoma cells suspensionswere divide into: control group、only N2O、only focus ultrasound、focusultrasound+C3F8-LM、focus ultrasound+N2O-LM, MTT method was usedto detect the survival rates of the cells, annexin V/PI double staining byflow cytometry was performed to measure the way of death, observe howmuch of each group of living cells in the crystal violet staining experiment.The focus ultrasound parameters (ultrasonic frequency of10.50MHz,sound intensity:0.5W/cm2, irradiation time:20seconds), detect survivalrates of UMR-106rat osteosarcoma cells in12h、24h、36h、48h by MTTmethod. Detect cells mortality by flow cytometry, cells apoptosis byhoechst33258staining of nuclei. flow cytometry and Hoechst33258nuclear staining were used to observe changes in nuclear morphology andcell apotosis。Results Focus ultrasound irradiation could significantly reduce thesurvival rates of UMR-106rat osteosarcoma cells (P <0.05) for30s、60sand90s, without significantly reducing the survival rate（sP＞0.05）for20s. Simple nitrous oxide had no significant effect on UMR-106cells survivalrates (P＞0.05). When cells were expose at the irradiation for20s, themicrobubbles was50ul, cells survival rate of N2O-LM combined focusultrasound group decreased significantly (P <0.05), and annexin V/PIdouble staining and transmissing electronic microscope showed thatUMR-106cells suffered from necrosis and apoptosis. Throughtransmission electron microscope, can see changes: vacuoles in cytoplasmof rat osteosarcoma cells having formed, UMR-106rat osteosarcoma cellmicrovilli， mitochondria， endoplasmic reticulum swelling，intracytopl-asmic vacuoles with different sizes forming，mitochondrialmedullary changing，intranuclear pseudo inclusion bodies，gap around thenucleus increasing，rupture of the cell membranes、cytoplasm loss, nuclearfragmentation、dissolved etc.MTT detection method showed that focus ultrasound+N2O-LM cellssurvival rates were93.18%(10s),80.28%(20s),55.38%（60s）70.68%(30s),and40.01%(90s), with a statistically significant differences(P<0.05), focus ultrasound+N2O-LM group comparing with the controlgroup、 simple N2O group、 simple focus ultrasound group、 focusultrasound+C3F8-LM group at the same time. Detcted by flow cytometry,focus ultrasound+N2O-LM group compared with other groups, withsignificant differences (P<0.05). hoechst33258cells nuclear stainingshowed necrosis and apoptosis、nuclear condensation、flocculent change、nuclear margination.Conclusions focus ultrasound+N2O-LM had a significant killingeffect on rat osteosarcoma cells UMR-106cultured in vitro, couldsignificantly inhibit the proliferation of osteosarcoma cells in UMR-106rats. N2O may be a potential gas sound sensitive agent, which may provide a new method for the treatment of osteosarcoma. PART III THE EXPERIMENTAL STUDY ONN2O-LM ENHANCING HIFU TO ABLATE BOVINELIVER IN VITROObjective Observe the effect of N2O-LM combined with HIFUablating bovine livers in vitro, verify the feasibility that it was used asHIFU synergist. Explore the optimal doses and the best irradiationparameters for HIFU efficiency, to the next step provide experimental basisfor synergism HIFU ablating tumors in vivo, and explore the applicationvalues of N2O-LM as synergist of HIFU.Methods Get fresh bovine livers in vitro. After injecting with differentproportions (proportions of PBS with N2O-LM wrer5:1,10:1,20:1) ofN2O-LM microbubble contrast agents in the local, give the different outputacoustic power, HIFU irradiation in different time, evaluate ablating effectof N2O-LM combined with HIFU by calculating the gray value changes ofthe irradiation zone and volumes of coagulative necrosis, At the same time,contrast with groups which were respectively injected with PBS solutionand C3F8-LM. After HIFU irradiated, observe changes of target gray valuesand volumes of coagulative necrosis, investigate synergistic effect in vitroof N2O-LM emulsion.Results After the liver bovines were injected in the local withmicrobubbles loaded nitrous oxide and irradiated by HIFU, there wereobvious effect of ablation. At the same time, gray changes in values of theirradiation zone and volumes of coagulative necrosis increasedsignificantly contrasting with the groups of injection of PBS solution、C3F8-LM (P<0.05).The N2O-LM could reduce the HIFU ablating time, reduce the HIFUablating power, enhance the HIFU ablating effect to bovine livers.Conclusions N2O-LM can effectively enhance the biological effects of HIFU ablating bovine livers, it is a good HIFU synergist. PART IV THE EXPERIMENTAL STUDY OF N2O-LMPOTENTIATING HIFU TO ABLATE RATOSTEOSARCOMAObjective To observe the inhibition effect of N2O-LM combined withHIFU ablation of SD rat osteosarcoma cells UMR-106transplanted tumors,discuss the application values of HIFU synergist.Methods furtherly establish thirty SD transplanted tumor models ofUMR-106rat osteosarcoma cells, in14days after tumor inoculation, anddivide randomly into group A (blank control group)、group B (simple N2Ogroup)、group C(simple HIFU group)、groupD(HIFU+C3F8-LM group)、group E(HIFU+N2O-LM group). Group A was blank control group, noradiation, no injection of microbubbles also without nitrous oxide; group Bgot intratumoral injected with1ml nitrous oxide dissolved in saline beforeirradiation; group C carried out a simple HIFU irradiation; group D gotintratumoral injection with1ml C3F8-LM emulsion diluted10times; groupE was injected with1ml N2O-LM emulsion diluted10times, with HIFUirradiation after injection, radiating acoustic power180W, irradiation time5s. After HIFU irradiation, observe changes of target gray values andvolumes of coagulative necrosis, collect Samples and stain HE, detectproliferating cell nuclear antigen (PCNA) and observe by transmissionelectron microscope. observe the tumor volume changes after treatments,observe the inhibitory effect of N2O-LM on rat osteosarcoma cells tumorUMR-106solid. After tumor tissues were embedded in paraffin、sliced、stained by HE, observe the morphologic changes of tumor tissues, getimmunohistochemical staining, analyse expression of PCNA、TUNNEL byIPP image, and explore its antitumor mechanism of inhibition. Results In the experiments of HIFU ablating rat osteosarcomas,combined with N2O-LM，gray value changes and tissue ablation zone ofcoagulative necrosis volumes increased significantly (P<0.05), andosteosarcoma cells PCNA expressions were significantly reduced (P<0.05).Under light microscope after HE staining, observe the cells structures thatthe irradiation zones were destroyed, the necrosis area and surroundingtissue boundaries were clear. Under electron microscope, observe thatcancer cell structures were not clear, cell membranes and nuclearmembranes were discontinuous, chromatin pyknosis, fragmentation withmass. Comparing the foucus ultrasound+N2O-LM group with the othercontrol groups, tumor volumes and weights reduced, and the differenceswere statistically significant (P＜0.05). Tumor tissues PCNA expressionsdecreased, the differences were statistically significant (P <0.05).Conclusions N2O-LM can effectively enhance the biological effect ofHIFU ablation of SD rat osteosarcomas, is a good HIFU synergist. There isa significant inhibition effect on UMR-106rat osteosarcoma cellstransplantation tumor proliferation. hope to open up a new path for clinicaltreatment of osteosarcoma.