Low Intensity Focused Ultrasound Combined With Adriamycin Loaded Novel Microbubbles For VX2Tumor Therapy

PART Ⅰ PREPARATION AND CHARACTERIZATION OFADM-NMCsObjective To prepare ADM-NMCs, and to investigate their generalproperties.Methods ADM-NPs was prepared by double emulsion technique byintroducing ADM in the water phase. NH2-MB was prepared by mechanicvibration technique. ADM-NMCs were prepared by covalent reaction ofterminal carboxyl of ADM-NPs (activated by EDC/sulfo-NHS) and aminoof NH2-MB. These contrast agents were characterized by several analyticaltools. The morphology was observed using the light microscope. The meandiameter and zeta potential were measured by dynamic light scatteringtechnique. Drug encapsulation efficiency and loading capacity wereanalyzed using high performance liquild chromatography system. ADMrelease behavior of ADM-NMCs with/without ultrasound was investigated.Results The size distribution of ADM-NPs was uniform, and the averagediameter was249.8nm, the average zeta potential was-0.82mv. The averaged encapsulation efficiency and loading capacity were86.1%and7.8%, respectively. The average size of NH2-MB was1032nm, the averagezeta potential was0.7mv. The average size of ADM-NMCs was1721nm.The ADM-NMCs contained an average of0.28ng ADM of per MB and therelease ADM was higher with low intensity focused ultrasound irradiationcompared to without ultrasound irradiation.Conclusions In this study, we successfully prepared ADM-NMCs, thedistribution of size was uniform, and the ADM-NMCs had high drugloading capacity. ADM could be released using low intensity focusedultrasound irradiation, we expect that the ADM-NMCs could release drugfor local tumor therapy under real-time ultrasound guidance. PART Ⅱ EVALUATION OF MICROBUBBLES DESTRUCTIONUSING LIFUObjective To explore the ability of LIFU destruction microbubbleslocally in vitro. To explore the ability of LIFU destruction ADM-NMCsand release drug more accurately in vivo.Methods Lipid microbubbles prepared by mechanical vibration werediluted and placed into latex water bags (home-made). The same intensity LIFU and non-focused ultrasound transducer were placed perpendicular tothe surface of the water bag for10s respectively. The range ofmicrobubbles destruction was observed using Two-dimensional ultrasoundimaging mode. A total of twenty healthy rabbits were chosen andADM-NMCs were injected via ear vein, and then liver was irradiated withLIFU for60s under B-mode ultrasound guidance. Four liver tissuses atdifferent distances from the focal point were harvested1h later and theADM level were determined by RP-HPLC.Results Lipid microbubbles presented high uniform ultrasound signalintensity prior to destruction. We can see wide range of microbubblesdestruction presented a nonuniform decreased ultrasound signal in responseto non-focused ultrasound triggering. However, LIFU resulted in a circularregion of decreased signal intensity perpendicular to the directionpropagation of ultrasound waves and a fusiform region of decreased signalintensity along direction of propagation of the ultrasound waves. The rangeof circular region of decreased signal intensity increased along with theacoustic power of LIFU. ADM levels in the target area were significantlyhigher than the surrounding areas after LIFU irradiation to ADM-NMCsand the ADM levels increased depending on the acoustic power of LIFU.Conclusions LIFU has the ability of targeted destruction microbubblesaccurately and release drug in situ locally. These findings recommendLIFU could target destruction drug loaded microbubbles and release drug in situ for targeted tumor therapy. PART Ⅲ ENHANCED ULTRASOUND IMAGING OF ADM-NMCsAND ANTITUMOR EFFECT COMBINED WITH LIFU ON VX2LIVERCANCER IN RABBITSObjective To explore the efficacy of ADM-NMCs enhanced ultrasoundimaging in vitro and VX2liver tumor in rabbits. To explore the antitumoreffect of LIFU targeted destruction ADM-NMCs and release drug inrabbits.Methods VX2liver cancer models were established in sixty-four rabbitsby implantation VX2tumor tissues into the left lobe of liver. Two weeksafter implantation, ten rabbits were screened using an ultrasonic diagnosticsystem under fundamental wave condition with B-mode and divided intotwo groups randomly for ultrasound imaging.1ml ADM-NMCs solutionwas injected via ear vein. Pure lipid microbubbles were used control. Theremaining fifty-four tumor-burdened rabbits were divided into six groupsrandomly:(1) control group;(2) ADM-NPs combined with LIFU group(LIFU+ADM-NPs);(3)MBs combined with LIFU group (MBs+LIFU);(4)the mixture of ADM-NPs and microbubbles combined with LIFU group (ADM-NPs+MBs+LIFU);(5) ADM-NMCs combined with non-focusedultrasound group (ADM-NMCs+Non-FUS);(6) ADM-NMCs combinedwith LIFU group (ADM-NMCs+LIFU). In the control group,4ml salinewas injected through marginal ear veins; in the ADM-NPs+LIFU group,0.8ml ADM-NPs was diluted into4ml and injected, followed by exposureto LIFU; in the MBs+LIFU group,1.2ml of pure lipid microbubbles werediluted into4ml and injected, followed by exposure to LIFU; in theADM-NPs+MBs+LIFU group,1.2ml of pure lipid microbubbles weremixed with0.8ml of ADM-NPs and the final volume was diluted into4mland injected, followed by LIFU irradiation; in the ADM-NMCs+Non-FUSgroup,1.2ml ADM-NMCs diluted into4ml and injected, and then wasexposed to Non-FUS; in the ADM-NMCs+LIFU group,1.2mlADM-NMCs diluted into4ml and injected, and then was exposed to LIFU.Tumor volume of each group was monitored by diagnostic ultrasoundsystem before and after treatment and the longest axis and shortest axiswere measured compared to each other. Three rabbits of each group weresacrificed and tumor masses were harvested after the last treatment. Thetumor tissues were washed in saline and placed in polyoxymethylene forproliferating cell nuclear antigen (PCNA) detection byimmunohistochemical examinations and apoptosis by terminaldeoxyuridine nick end labeling. The remaining tumor-bearing rabbits fromeach group were monitored until their cachexia death and the median survival time was recorded to compare the antitumor effects.Results Both ADM-NMCs and pure lipid microbubbles producedenhanced ultrasound imaging on VX2liver tumor, typical “fast in and out”phenomena could be found. It is noteworthy that the peak intensity time,peak height and the clearance time of ADM-NMCs was similar to that ofpure lipid microbubbles. The results of immunohistochemical examinationsshowed that the PCNA expression and proliferation index in ADM-NMCs+LIFU group was obviously lower than that in other groups（P<0.05）, theapoptotic cells and apoptosis index was the highest among these group（P<0.05）. The volume inhibition rate of the ADM-NMCs wassignificantly higher than all the other groups. LIFU combined theADM-NMCs treatment increased the median survival to71days.Conclusions ADM-NMCs could enhance ultrasound imaging, whichindicated that ADM-NMCs could be used for visual monitoring destructionand drug release. The results showed that the combination of LIFU andADM-NMCs could inhibit the proliferation and accelerate the cellapoptosis of VX2tumor. This may provide a novel safe and non-invasivestrategy for clinical tumor therapy.