| Background:With the wide application of ultrasound contrast agent (UCA), increasing attention had been paid to the bioeffects of ultrasound. Ultrasound-combined microbubble has shown its new prospect in clinical therapy. It has been applied to increase gene transfection efficiency, enhance therapeutic effect of anti-tumor drugs, etc, which are generally accepted by most of researchers to be the induction of acoustic cavitation. Our preliminary study showed that, compared with simple carboplatin by intratumoral injection, it was better to employ carboplatin-loaded polylactic-co-glycolic acid microspheres (CBP-PLGA Ms, abbreviate CBPMs) to treat tumors in animal models, and there were less side effects as well as longer life span. But the pathology showed that the neoplastic cells were not completely destroyed in situ. So it is urgent to find a new to completely destroy neoplastic cells in situ as well as effectively prevent the metastasis and recurrence of tumors. Based on the presumption, we assumed that , after we inject the combination of CBPMs and contrast agent intratumoraly the acoustic cavitation would destroy partial neoplastic cells, and then increase the intracellular amount of chemo and enhance the bioavailability of chemo, consequently, augment the local therapeutic effect.Objective:1. To explore the bioeffects of intratumoraly injected microbubbles induced by ultrasound from the point of pathology2. To investigate the synergistic effect of intratumoraly injected microbubble and CBPMs in local chemotherapy.Methods:1. Twenty-four SD rats were randomly divided into two groups, twelve in each group. The subcutaneous transplantation tumor models were prepared by directly injection of enriched cancerous ascites or by the implantation of tumor tissue. The positive rate of inoculation, tumor size, growth rate were observed by ultrasound volume probe. The vascularization index (VI) of two groups was subjected to three-dimensional power Doppler angiography (3D-PDA) examinations.2. Twelve rats were randomly divided into three groups after twenty-four Walker-256 carcinomas were subcutaneousely implant into the right and left hind legs. In group A, after intratumoral injection of microbubbles, ultrasound with the 1MHz frequency, and the intensity of 2.0W/cm2 was applied on the tumors of the rats; In group B, the intensity and time of ultrasound irridiation were the same as in group A; In group C, the same amount of microbubbles as in group A were injected into the tumor. One hour after the treatment, histopathology damage was observed under light microscope and the edge tumor was harvested for electron microscope examination by means of Lanthanum (La) tracer.3. The Walker-256 tumor-bearing rats were divided into control group, microbubbles+ ultrasound group (MB+US), carboplatin microspheres group (CBPMs), carboplatin microspheres+ultrasound group (CBPMs+US), carboplatin microspheres+ microbubbles +ultrasound group (CBPMs+MB+US), eight in each group. Two-dimensional ultrasonography was applied to survey the tumor size. Three-dimensional power Doppler angiography was performed pre-and post-treatmently to evaluate the change of vascular perfusion in tumors and the numbers of vessels in tumors so as to evaluate the therapeutic effect of the treatment. The tumor was excised and the pathologic characterization was observed to evaluate the intensity and extent of the necrosis of tumor after eighteen days.Results:1. The positive rate of inoculation in the injection group was higher than that in the implantation group, which were 100% vs. 75% respectively (P<0.05). And the growth rate was faster in the injection group than in the implantation group. The time of peak amplitude of VI of the injection group and the implantation group were 12 days and 16 days respectively.2. In group A, many coagulated necrotic areas were spotted in the tumors under light microscope, and La particles were seen in the cells and vascular endothelial cellular space as well as intercellular space under electronic microscope;there were no necrotic cancer cells seen under light microscope in group B and C, and under electronic microscope little La particles were seen in the cells in group B, and La particle appeared only in the intercellular space in group C.3. The sequences of tumor volume and VI from small to big were CBPMs+MB+US, CBPMs+US, CBPMs, MB+US, and control group. There was no significant difference in the tumor volume between group CBPMs+US and CBPMs, and there was no significant difference in the VI between group CBPMs+US and CBPM s either. The histopathology showed that there were severe coagulation necrosis in group CBPMs+MB+US, a few scatter neoplastic cells and inflammatory cell were found in CBPMs+US and CBPM group, while there were a lot of active neoplastic cells in MB+US and control groupConclusion:1. Both the injection and the implantation technique can prepare subcutaneous transplantation tumor model in rats, the positive rate of inoculation of the injection group was higher than that of the implantation group. Three-dimensional sonography was useful in the evaluation of subcutaneous tumors.2. Ultrasound-mediated intratumoral injection of microbubbles and destruction could enhance the permeability of tumor cell membrane and could cause coagulate necrosis areas in the tumors.3. Tumor growth inhibition was most significant in CBPMs+MB+US Group. The method significantly inhibited the growth of subcutaneous tumors with the intensive and complete necrosis of neoplastic tissues, and disappearing of neoplastic vessels. The CBPMs+MB+US way gain a bright future in the treatment of cancer.
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