Studies On The Enhanced Treatment Of Rabbit VX2Tumors By Thermal Ablation And Ultrasound Mediating Targeted Release Of Vinorelbine Long-circulating Thermosensitive Liposomes

Part One Establishment and biological evaluation of rabbitVX2muscle tumor modelsObjectives: Rabbit VX2tumor cell lines have been widely used in tumor modelsestablished in rabbit livers, kidneys, bones and brains and other parts. While vaccination inestablishing tumor models within the muscles is often used for the transmission of tumorstrains, it is rarely applicable to present experiments. Establish rabbit VX2muscle tumormodels and observation of their biological characteristics. Methods:①Experimental materials:30purebred New Zealand white rabbits, two or three months old,1.5to2.0kg.②Experimental processes: with tissue blocks embedded, VX2tumor tissues weretransplanted in30New Zealand white rabbits’ thigh vastus lateralis muscles to establishmuscle tumor models.③Evaluation: A group of15randomly selected tumor-bearingrabbits were under Ultrasound examination after the7,10,14,21,26d tumor inoculationrespectively; calculate the tumor volume (V)=0.5×ab2, wherein a, b represents the longdiameter and short diameter of tumors respectively. Tumor growth curves were drawnwith regard to the average TV. Calculate the tumor growth rate (TGR) according to theformula TGR=(a2b2-a1b1)/a1b1×100%. As for another group of15tumor-bearingrabbits, three tumor-bearing rabbits were randomly killed at10,15,20,25,30d; theirtumors were stripped of and the tumor volumes measured and compared with ultrasoundmeasurements. Pathologically examine the tumors’ histological characteristics. Results:30rabbits were under the final analysis.①The rate of establishing rabbit VX2muscletumor models reached up to100%. After vaccination for10-14d, the tumors grew rapidlyat the rate of66.9%; for14to21d, the tumors grew at the rate of97.2%.②On7th daysafter vaccination, by ultrasound, the small isoechoic nodules were found visible in someexperimental rabbits’ muscle tissues. On the14th days low echo or isoechoic nodules werefound visible in the rabbits’ muscle tissues with clear boundary and average echoes. ColorDoppler showed, inside the tumors, blood flow signals in the form of sparse rods and, anamount of blood flow signals can be seen surrounding around. On the21st days a smallpart of the tumor echoes was uneven and, there were small pieces of echo-free zones andcalcification pots scattered in the hyperechoic zones; as the growth of tumors, tumornecrosis increased.③Pathological observation: with low magnification microscope, tumorcells were discovered in the nested distribution crossed with pieces of necrosis distribution.Tumor cells in the necrosis distribution were in low density. With high-poweredmicroscope, tumor cell volumes increased in irregular shape, with irregular arrangement.And nuclei were large and deeply stained; cytoplasm less, mitotic more and, nuclearcondensation and fragmentation were present in the necrotic region.④The tumors werestripped of, tumor volumes measured and compared with ultrasound measurements; the differences were not statistically significant.Conclusion:This model can be transplanted at a high rate, with short modeling time, rapid tumorgrowth and sufficient blood supply. It is an ideal experimental model used to study tumorlocal treatment.Part two Studies on the treatment of rabbit VX2tumors bylow-power RF technique mediating targeted release ofvinorelbine long-circulating thermosensitive liposomesObjectives: Use in rabbit VX2tumor therapy targeted release of thermosensitiveliposomes with portable chemotherapy drugs through precise positioning and thermaleffects of RF technology. Methods: Establish VX2tumor models in the thigh muscles ofNew Zealand white rabbits by embedding tissue blocks. When the tumors grew to0.8to1.0cm,24tumor-bearing rabbits were randomly divided into4groups according to tumorsizes. Control group (CON): normal saline was intravenously injected. Low-frequency RFgroup (RF): Output power,1W,30min, target temperature of42~43℃. Vinorelbinetartrate long cycle thermosensitive liposomes (LCTSL):1.8mg/kg vinorelbine tartratelong-circulating thermosensitive liposomes were given. Vinorelbine tartrate long cyclethermosensitive liposomes combined with low-frequency RF (LCTSL+RF): power,1W,time30min. Before the low-frequency RF, the1.8mg/kg vinorelbine long cyclethermosensitive liposomes were given. Treatment was carried out on the1st,4th, and7thdays respectively, in the total of3times. Observe the tumor sizes without a stop underUltrasonic before and after treatment; Distribution within the internal tissues afterintravenous vinorelbine injection was determined by HPLC. After8d treatment, westarted extraction of VX2venous blood followed by blood routine examination before therabbits were killed, and then we obtained different data on white blood cells and plateletsof the different experimental groups. The samples were obtained for HE staining and detecting protein expression through Western blotting. Results: All rabbits werewell-tolerated in treatment, which completed the experiment. The inhibition rate ofLCTSL+RF group was70.3%, significantly higher than the RF and LCTSL group; thedifference was statistically significant (P <0.05). There was no significant ablation effectin Low-frequency RF group. With histopathological examination, less obvious tumornecrosis was revealed under a low frequency RF. But in TSL+RF group tumor cell injuryand necrosis were significantly found visible. And compared with the LCTSL group,LCTSL+RF group presented itself in the highest concentration of tumor tissue vinorelbine,24h later after administration. And the concentrations of vinorelbine in hearts and lungswere the lowest. The detection of tumor cell apoptosis: The LCTSL group and LCTSL+RF group could be seen to significantly promote tumor cells apoptosis and inhibit tumorcells proliferation. Compared with the LCTSL group, the rate of tumor cells apoptosis wasmore significant than that of the LCTSL+RF group.Conclusion:Low-power RF targeted release of the vinorelbine hot long circulating thermosensitiveliposomes was discovered to have an obvious effect on inhibiting rabbit VX2tumors, withvery good drug targeting and the lowest cardiopulmonary side effects. Thusradiofrequency ablation treatment can be applied, which is of great significance forreducing tumor recurrence after radiofrequency ablation.Part three Anti-tumor experimental study on HIFU targetedrelease of vinorelbine long-circulating thermosensitiveliposomesObjectives: Study the anti-tumor efficiency of HIFU targeted release of vinorelbinelong-circulating thermosensitive liposomes, with HIFU as targeted release of heat.Methods: Establish VX2tumor models in New Zealand white rabbits’ thigh muscles with tissue blocks embedded. Until the tumors grew to0.8～1.0cm,24tumor-bearing rabbitswere divided randomly into4groups according to tumor sizes: control group (CON),HIFU group, V-LCTL group, HIFU+V-LCTL group. Treatment was carried out every twodays or on the1st,3rd, and5th days respectively. Western blotting were be used fordetection of protein expression in the tumors, which would then obtained for pathologicalHE staining. And tumor pathomorphological changes would be observed. After treatment,observe tumor volumes and weight changes and calculate inhibitory rates. After8dtreatment, we started extraction of VX2venous blood followed by blood routineexamination before the rabbits were killed, and then we obtained different data on whiteblood cells and platelets of the different experimental groups. Results: Tumor growthcurves for tumor-bearing rabbits in each group: Control group presented itself with thefastest tumor growth; the tumor volumes of HIFU group and control group wereequivalent when growing. The tumor growth was significantly inhibited in HIFU+VBLgroup and HIFU+LCTSL group. And the tumor volumes were significantly reduced ascompared to the other groups. Compared with HIFU+VBL group, the tumor volumes inHIFU+LCTSL group were also more significantly reduced. The rate of tumor inhibitionwas71.0%in LCTSL+HIFU group and significantly higher than HIFU group and HIFU+VBL group (0.8%and48.9%, P <0.05). In TV-LCTL group and HIFU+the V-LCTLgroup caspase-3active fragments and bax expression levels were significantly increasedwhile bcl-2expression levels were significantly decreased. The pathological resultsshowed that, in control group and HIFU group, tumor cells, big, with abundant cytoplasmand complete intratumoral microvasculars, were closely arranged in nested shape. Thecells of HIFU+LCTSL group were obviously condensed smaller, accompanied withnuclear condensation, fragmentation and dissolution. The capillary wall structures becamefuzzy with eosinophilic staining increasing and a large number of red blood cells werefocused in endovascular.Conclusion: Chemotherapeutic drugs-carrying thermosensitive liposomes were combinedwith HIFU technique in our study. By HIFU targeted release, with highly concentration ofchemotherapeutic drugs-carrying thermosensitive liposomes in local tumors, chemotherapeutic drugs can be used to enhance the tumor ablation through HIFU. Theranges of HIFU thermal ablation were enlarged as well as its adaptation and the systemictoxicity of anti-tumor chemotherapy drugs was relieved. This study provides a new way ofseeking an effective, safe and highly targeting tumor treatment.