Application Of A Phase-change Type Nano-size Ultrasound Contrast Agent To Enhance The Efficacy Of HIFU Ablation

Objective To prepare lipids wrapped phase-change type liquidperfluorocarbon nanoparticles (abbreviation as Pct-PFCNP) emulsioncontrast agent with shell of lipids and kernel of PFH. The basiccharacteristics of the emulsion and the possibility of its phase transitionpromoted by any factors were studied in vitro.Methods The study of this part was divided into two sections. Insection one, the two-step emulsification was used to prepare lipids wrappedPCT-PFCNP emulsion. The appearance, size, potential, concentration,stability testing of the emulsion were investigated to sum up the factors forbetter preparation. In section two, temperature increasing, diagnosticultrasound, low frequency therapeutic ultrasound, high-intensity focusedultrasound excitation was used respectively to promote the phase-change of Pct-PFCNP in vitro. Ultrasound and optical microscopy were used toobserve the phase-change. By this means, the feasibility and the favorableconditions were concluded.Results In section one, the stable lipids wrapped Pct-PFCNP emulsionwith uniform particle size, evenly distribution was manufactured by thetwo-step emulsification method. The ratio of.1:8between the PFH and thelipid suspension, and the vibroacoustic time of90second could prepare themost stable Pct-PFCNP emulsion with minimum size. In section two, whenthe temperature rises above70℃,or low frequency treatment ultrasoundexcitation or high-intensity focused ultrasound excitation can promotePct-PFCNP undergo a phase transition and produce microbubbles toenhance ultrasound imaging. No phase transition of Pct-PFCNP wasobserved after diagnostic ultrasound excitation with various mechanicalindexes.Conclusions The lipids wrapped Pct-PFCNP emulsions could beprepared by a relatively simple process. Temperature rising and highsound-pressure ultrasond excitation could promote the phase transition ofPct-PFCNP and produce microbubbles, which has promising prospect inmolecular imaging. Objective To investigate the feasibility of enhancing the efficacy ofHIFU ablation of normal livers and rabbit liver VX2tumors in vitro and invivo with self-made Pct-PFCNP emulsion. The mechanism of the enhancedefficacy was explored preliminary.Methods The study of this part was divided into two sections. Insection one, firstly, the time, squares, values of the phase-changed areas onscreen, and the necrosis anatomicalnecrosis volumes of bovine livers werecompared between each groups to investigate the role of Pct-PFCNPemulsion in HIFU after different power of HIFU ablation. The differentconcentration of Pct-PFCNP emulsion injected group was as theexperimental group. The lipids suspension injected group was as thecontrol group, and the PBS injected group as the blank control group. Thenthe raw dose range and PCT-PFCNP concetration of HIFU ablation in thenext study was filtered. Secondly, the normal livers of rabbit underwentHIFU ablation with different power and different time. The Pct-PFCNPinjected group was as experimental group. The PBS injected group as the blank control group. The gray scale change and the anatomicalnecrosiswere compared between each group to investigate the role of Pct-PFCNPemulsion in HIFU ablation in vivo, and then filtered the more appropriatedosage for study of Pct-PFCNP as synergist for HIFU ablation in vivo.Lastly, a study had been carried out to compare the effect of injection timeof Pct-PFCNP emulsion at24h,48h and30s before HIFU ablation on itsefficacy.In section two, rabbits with VX2tumor in liver were select to undergoHIFU ablation of tumor with dosage of150W,5s. The gray scale changes,the anatomicalnecrosis in target regions, the ultrastructural changes,apoptotic index and expressing rate of PCNA out of target areas werecompared to examine the role of Pct-PFCNP in HIFU ablation of tumor.Results In section one, in vitro experiments, There were significantlydifferences in time, size and value of gray scale change andanatomicalnecrosis between Pct-PFCNP group and lipid suspension groupand the blank control group(P<0.05). There were significantly differencesin time of gray scale change in dosage of120W and150W between thelatter two groups.(P<0.05), and there was no difference in otherparameters between the two groups(P>0.05). There were significantlydifferences in size and value of gray scale change and anatomicalnecrosisbetween5times and10times diluted Pct-PFCNP groups and10times and20times diluted groups(P<0.05)There was no difference in all parameters between the5times diluted group and10times diluted grou(pP>0.05), andthere was significant difference in all parameters between the20timesdiluted group and40times diluted group(P<0.05). In vivo ablation ofnormal liver experiments, there weresignificant differences in allparameters between the Pct-PFCNP group and blank control group invarious dosages of HIFU (P <0.05). As the energy increased and the timeincreased, the anatomicalnecrosis of livers increased. The dosage of150W,5s was suitable for the synergist study of HIFU ablation of rabbitliver.There were significant difference in gray-scale changes and necroticvolumes between the ablaiont day injection group, the24h before injectiongroup and the48h before injection group(P <0.05). There was nosignificant difference between the former two groups in all parameters(P>0.05).In section two, there were significant differences in size and value ofgray scale change and anatomicalnecrosis between the Pct-PFCNP groupand the blank control group (P <0.05). Three days later, the ultrastructuralchanges were more significant in the Pct-PFCNP group. There weresignificant differences in apoptotic index and expressing rate of PCNA outof target regions between the Pct-PFCNP group and blank control group (P<0.05)Conclusions The preliminary study confirmed the self-madePct-PFCNP emulsion can shorten HIFU ablation time, reducing the HIFU ablation power, thus enhance the therapeutic effect of HIFU ablation oflesions in vivo and in vitro. The main mechanism was: under the effect ofHIFU, Pct-PFCNP underwent a phase transition and produce microbubbles,which could increase the ultrasonic cavitation, the target energy depositionand heating up. The sonochemical promoting non-target lesion necrosisalso played an important role in the enhanced efficacy. Objective To prepare the FR targeted Pct-PFCNP emulsion and studyits targeting performance in vitro and its role in HIFU ablation of ovariancancer xenograft tumors in nude mice.Methods The study of this part was divided into two sections. Insection one, DSPE—PEG (2000) Folate with other lipids were dissolved inchloroform. Rotary evaporation was used to remove the organic solventand obtain the lipids film. After re-hydration,1:8ratio of PFH was added inand underwent two-step emulsion to prepared the FR targeted Pct-PFCNP emulsion, the trarget performance of which was verified in human ovariancarcinoma cells line SKOV3, non-targeting Pct-PFCNP group and freefolic acid intervention group were used as the control group. In section two,the model of tumor of human ovarian cancer in nude mice was establishedby subcutaneous injection of tumor cells. The FR targeted Pct-PFCNPemulsion was injected by tail vein injection to investigate its role in HIFUablation of tumor, and the non-targeting Pct-PFCNP emulsion injectedgroup as the control group. The gray scale change area and value on screen,the anatomicalnecrosis and ultrastructure differences in tumor werecompared between the two groups after HIFU ablation.Results In section one, the FR targeted Pct-PFCNP emulsion wassuccessfully prepared. A great amount of Pct-PFCNP adhered andaggregated firmly to the SKOV3cells, and some Pct-PFCNP were in thecells observed by light microscopy. Observed by fluorescence microscopywith blue light excitation, lots of green fluorescence seen Pct-PFCNP weresurrounding the orange-red fluorescence seen cells in the targeting group.No obvious binding trend to the cells was observed in the two controlgroups. Confocal laser scanning microscope showed that a lot of greenPct-PFCNP adhered to red cells in targeting group too. In section two, indosage of120W,5s, obvious white necrosis with clear boundaries tosurrounding TTC dyed red non-necrosis tissue was seened after HIFUablation in the Pct-PFCNP group. No obvious necrosis was seen in3 tumors in the control group, there were significant differences in gray scalechange area, value and the anatomical necrosis between the two groups (P<0.05). Targeting Pct-PFCNP group of, necrosis was larger areas ofnecrosis and less island remnants cells can be seen in FR targetedPct-PFCNP group by HE staining. More serious necrosis sign like not clearoutline of cells and nuclear membrane rupture were seen in most of tumorsin the FR targeted Pct-PFCNP group than the control group.Conclusions The performance of the FR targeted Pct-PFCNP wasgood, which could used in HIFU ablation of tumor. The enhanced effectwas more obvious than the non-targeted group. This would provide aeconomical, efficient and useful molecular probes for future targeteddiagnosis and treatment of tumor.