Combination Of Ultrasound Contrast Agent And Diagnostic Ultrasound In High Mechanical Index For Synergistic Microwave Ablation Of Tumors

PART I PREPARATION AND CHARACTERIZATION OF PERFLUOROCARBON ULTRASOUND CONTRAST AGENTObjective To prepare the ultrasound contrast agent(UCA) emulsions with lipid mixture as a shell and Octafluoropropane(OFP) as a core, and further characterize their physical and chemical properties and evaluate their biocompatibilities.Methods The UCA emulsions were prepared by a rotary evaporation-mechanical oscillations method. And their basic physical and chemical properties were determined, including surface morphology, mean size and potential distribution, concentration and stability. Additionally, Conventional culture of human hepatoma cell line SMMC-7721 was processed for cell activity assay to observe the biological activity of UCA after incubation with tumor cells.Results The UCA emulsions exhibited a perfect spherical morphology and a smooth surface. They were good dispersion without obvious adhesion and aggregation phenomenon. The mean size, potential, and concentration of UCA were 1.18 ± 0.33 μm, 0.08 ± 7.45 m V, and(2.31 ± 0.24 × 108) m L-1. In the CCK8 assay, no significant effect on the tumor cells could be found after incubation with UCA at different concentration, demonstrating the high biocompatibility of UCA.Conclusions The perfluorocarbon UCA with stable physical and chemical properties and high biocompatibility was successfully fabricated by the rotary evaporation- mechanical oscillations method, which would take a basic role for the further experiment ex vivo and in vivo.PART II COMBINATION OF ULTRASOUND CONTRAST AGENT AND DIAGNOSTIC ULTRASOUND IN HIGH MECHANICAL INDEX FOR AUGMETATION OF MICROWAVE ABLATION EFFICIENCYObjective To investigate the feasibility of augmenting the efficacy of microwave ablation(MWA) of normal bovine liver specimens ex vivo and healthy rabbit livers in vivo using UCA with high MI diagnostic ultrasound, and explore its preliminary mechanisms.Methods There were five groups in this study. Group I, MWA only; Group II, MWA only in the presence of UCA(1 m L at a concentration of 0.5 mg/m L); Group III, MWA plus simultaneous US; Group IV, MWA plus simultaneous US in the presence of UCA(1 m L at a concentration of 0.5 mg/m L); Group V, MWA plus simultaneous US in the presence of PBS(1 m L). During the MWA, the ultrasonography in general B-Mode and contrast enhanced ultrasonography(CEUS) mode were collected at 1 min of ablation in bovine liver specimens ex vivo and 30 s of ablation in rabbit livers in vivo, and the temperature ex vivo and in vivo in the real time at the ablation zone were measured. After MW ablation, the tissue volume(mm3) of coagulation necrosis in the bovine liver specimens ex vivo and rabbit livers in vivo was calculated and compared among different groups.In the in vivo study, the ablated tissue from 5 mm along the horizontal axis area of the MW radiation source was harvested in each group to stain with hematoxylin and eosin(H&E) and nicotinamide adenine dinucleotide hydrogen(NADH) for pathological examination. Additionally, 1 mm3 tissue here was excised to observe the ultra-structure changes of the targeted region by transmission electron microscopy(TEM).Results The comparison among groups under the same ablative parameters in terms of the necrosis volume, temperature in the ablated zone, and the gray variance in ultrasonography follows: No statistical differences(P > 0.05) were found between MWA group and MWA + UCA group, between MWA + US group and MWA + US + PBS group. However, there was a significant difference between MWA + US group and MWA group(P < 0.05). Furthermore, another significant difference can be found between MWA + US + UCA group and MWA + US group(P < 0.05).On microscopic examination of tissues in healthy rabbit livers experiments, the normal morphology and structure of cells were disappeared, only remnants of the nuclear fragmentation remained in the MWA + US + UCA group. And in the MWA and MWA + UCA groups, both mitochondria structure and nuclear membranes were intact. By contrast, both MWA + US and MWA + US + PBS groups displayed some degree of tissue damage including interstitial hyperemia, most cell membranes and nuclear membranes interrupted or disintegrated, but some cell morphologies still existed.Conclusions The combinatorial strategy using high MI ultrasound and UCA can elevate the temperature of target zone, expand ablation sphere, and augment the MWA efficiency. The underlying mechanisms were related to the cavitation effect, the changed local environment, and the target energy deposition etc., owning to the exposure of UCA under the high MI ultrasound.PART III COMBINATION OF ULTRASOUND CONTRAST AGENT AND DIAGNOSTIC ULTRASOUND IN HIGH MECHANICAL INDEX FOR ENHANCED INHIBITION OF RESIDUAL TUMORES AFTER MICROWAVE ABLATION Objective To explore the high MI ultrasound combined with UCA for inhibiting proliferation and promoting apoptosis of residual tumors after MWA through the establishment of tumor-bearing rabbit liver VX2 tumor model. The long-term effects of the synergism on tumor-bearing rabbits were further observed for safety assessment..Methods The MWA therapy experiment was made(20 W, 1 min) among different groups according to the aforementioned methods after establishment of VX2 tumor-bearing rabbit model. After ablation, the tumor tissues from 5 mm along the horizontal axis area of the MW radiation source were harvested for H&E and NADH staining and TEM examination. The tumor tissues situated in the peripheral regions of MWA were harvested on the first day, 3 days, 7 days after ablation for immunohistochemistry TUNEL, PCNA staining to assess apoptotic and proliferous properties of residuals.In the additional 40 tumor-bearing rabbits(n = 8 per group), tumor sizes were identified through ultrasound imaging. At two time points 1 day before and the 9th day after ablation, blood specimens were drawn from ear veins for the blood biochemistry assay. Continuous weighing of each group was done at once per three days, from the beginning of the operative day to the 9th day after MWA. Simultaneously, all rabbits were weighed for analysis at the 10 th day, 20 th day, 30 th day, and 40 th day, respectively. All rabbits were breed and observed to natural death. And the survival analysis was performed at last.Results Findings of H&E and NADH staining and TEM examination in each group were consistent with healthy rabbit liver experiments in vivo. Immunohistochemistry showed that: On the first day after ablation, the apoptosis index(AI) and proliferation index(PI) in each group were around 10% and 70%, and no significant difference(P > 0.05) was found among groups. On the third day after ablation, the AI steadily increased and the PI steadily descended. Compared with other groups, however, the MWA + US + UCA group has the highest AI and the lowest PI(P < 0.05). On the seventh day, both AI and PI in the MWA and MWA + UCA groups were no significant differences(P > 0.05) between the third day and the seventh day, but those were statistically significant(P < 0.05) in the other three groups. And the MWA + US + UCA group has the highest AI and the lowest PI, compared with other four groups(P < 0.05).The tumor size comparison among groups after ablation follows: No statistical differences(P > 0.05) were found between MWA group and MWA + UCA group, between MWA + US group and MWA + US + PBS group. However, there was a significant difference between MWA + US group and MWA group(P < 0.05). Furthermore, another significant difference can be found between MWA + US + UCA group and MWA + US group(P < 0.05). There were no significant difference(P > 0.05) among groups in the blood biochemistry assay between the first day and the 9th day. And no significant difference(P > 0.05) can be found in the weight among groups during the perioperative period. After the perioperative period, the weight comparison among groups were that: there were no statistical differences(P > 0.05) between MWA and MWA + UCA groups, and between MWA + US and MWA + US + PBS groups. However, The MWA group weighed less than MWA + US group(P < 0.05). And the weight in the MWA + US + UCA group was the most than the other groups(P < 0.05). On the survival analysis, the VX2 rabbits in the MWA + US + UCA group lived longer than others(P <0.05).Conclusions The High MI diagnostic ultrasound combined with UCA can promote the residual apoptosis and inhibit the tumor growth after MWA. In the long-term follow-up period, this combination strategy can delay disease progression and improve prognosis without obvious complications for the potential clinical applications.