| Research background:The intracranial tumor (including primary and secondary) has been a serious threat to human life and health. Due to its special anatomical position and endogenous susceptibility of the radiation therapy, as for most of the tumors which can not receive surgery or can not be completely removed by the surgery, the effect of chemotherapy will decide the prognosis of patients to a large extent. Unfortunately, the traditional chemotherapy way has little effect for the treatment of intracranial tumors because of the blood brain-barrier (blood brain-barrier, BBB). Research shows that, although the BBB of necrotic tissue in the intracranial tumor has been damaged, but that on the edge of the tumor is still integrated. The existence of BBB has not only lowered the drug's utilization rate and increased the toxic and side effects, but also limited the utilization of 95% of the drug in the central nervous system. The existing methods of reversible opening of the BBB as well as others bypassing the BBB, such as partly chemotherapy for interstitial substance and so on, can not significantly raise the survival rate of cancer patients.In recent years, the ultrasound interventional therapeutic development which is mediated by acoustic contrast agent (microbubble contrast agent or "micro-bubble" for short) has provided a new thought for the chemotherapy for brain tumor. Acoustic contrast agent is a suspension with a high concentration of micro-bubble, and it has two features in the interventional therapy:⑴it is a good carriers for drugs or genes.⑵If it is given a certain power of the ultrasound wave irradiation (including diagnostic ultrasound of high mechanical index), the micro-bubble will break down and release the carrying drugs or genes targeting to the target as well as promote their infiltration. By using this character of the micro-bubble, some achievements have been made in BBB-related research. Recently, JAMESd abroad used the focusing ultrasound with the 1.5 MHz frequency, 0.8 ~ 2.7 MPa sound pressure and the 20% dutyfactor to radiate the vole cornu ammonis per cutem, and the BBB opening has been tested by using nuclear magnetic resonance testing. Stephen M reported that the focusing ultrasound can realize highly selective and non-invasive blood brain-barrier opening. Under the condition that the acoustic contrast agent exists, the ultrasonic energy for opening up the blood brain-barrier has decreased significantly, which can facilitate the purpose to selectively open the blood brain-barrier by lower energy ultrasound without damaging the normal brain tissue. Acoustic contrast agent's intervention has significantly increased the number of cavitation nuclei and reduced the ultrasonic cavitation threshold, making the sound hole effect come true with less power diagnostic ultrasound, which can just be achieved by treatment ultrasound in the past. In prophase, this laboratory has performed the brain ultrasound visualization by the rat cranial cerebral using the diagnostic ultrasound with 1.7 MHz/3.3 MHz frequency. It has been found that when certain doses of bubbles are used and the mechanical index is more than 0.8, or when the mechanical index of the ultrasound launching is 1.3 and the microbubble dose is over 0.05 ml / kg, it can be observed that the selective opening of the BBB in rats emerges. It features that Evans blue which has combined with hemoglobin deposits in the brain tissue in the ultrasound convergence region and even in the brain cells through the BBB, and with the changing dose of the contrast agent or the increasing machinical index, the amount of seepage will increase. But the changes of cell morphology as well as the rat nerve behavior can not found. Since the human skull is significantly thicker than the rat skull, making the attenuation of ultrasound more obvious, it is still not sure that whether the diagnostic ultrasound attenuated by the human skull can open the BBB or not. Even if it can effectively open the BBB, whether this method is safe or not? Whether it will initiate the delayed effect or not? Whether it will promote the micro-bubble carrying drug to targeting release or not? Whether it will be effective with ultrasound involved in brain tumor chemotherapy? All of these questions need in-depth study.Research purpose:On the basis of prophase results that "the diagnosis ultrasound with high mechanical index united with microbubble can selectively open the blood-brain barrier", we simulate the condition of human skull acoustic window and establish the VX2 brain tumor model as well as discuss the feasibility and effectiveness of paclitaxel microvesicle crossing BBB targeting to the brain tumor. We will explore a new treatment method and mechanism for brain tumors.Main research contents:1. To discuss the feasibility of microbubble destruction of people skull specimens in different age groups radiated by diagnosis ultrasound with different instruments and different energy.2. To discuss the feasibility, safety and mechanism of diagnosis ultrasound united with micro-bubble opening the BBB by human skull specimens, and determine the parameters of the following experiment promoting drug mediated by the microbubble to cross BBB and transport into the brain tumor.3. Improve the preparative method of the microbubble carrying paclitaxel anti-cancer drug, and perform the testing for the microbubble carrying paclitaxel; research the targeting and distribution of paclitaxel microbubbles in the human body to provide experimental basis for ultrasound-mediated microbubble carrying paclitaxel targeting to the brain tumors.4. Research the feasibility and the initial effect of the ultrasound-mediated microbubble carrying paclitaxel targeting to the brain tumors.Research method and technical lines:1. We choose different types of ultrasonoscope, under the condition of largest output sound pressure and lowest ultrasound frequency, to find out the feasibility of ultrasonic wave passing the acoustic window of the children, the young and the elderly people's skull specimens to damage the microbubbles so as to determine the ultrasound instruments and parameters for the follow-up research.2. First, we open the skull window in the experimental rabbit, and put the young people's temporal bone specimens in the window, and then simulate the diagnostic ultrasound radiation mediated by the microbubble through the human skull acoustic window for 10min. Using EB as the labelled compound of the increasing permeability of the blood-brain barrier, the quantitatively analysis of EB effusion in the targeting region is carried out to infer whether the BBB open or not and its opening extent. We use the maximum sound pressure (MI reached the maximum), under the premise of the lowest frequency, to determine the dose of microbubbles and ultrasound launch mode for safely and effectively promoting the release of anticancer drugs targeting to the brain tissue. 3. To discuss using the high solubility of paclitaxel in triacetin to improve the preparation method of microbubble carrying paclitaxel, and to optimize the preparation method of microbubble by detecting the encapsulation efficiency and drug loading dosage.4. Build the rabbit VX2 brain tumor model. Based on the research of the biology characteristics of the tumor model and the ultrasound visualization, EB perfusion, and other experiments, we will further study the possibility of ultrasound stimulating the release of microbubbles carrying paclitaxel targeting to the tumor area and evaluate the preliminary efficacy.The main findings and conclusions:1. When the ultrasound intensity of the ultrasound diagnostic instrument is about MI> 0.99, the ultrasound can damage the microbubble after attenuating the temporal and occipital bone specimens of all ages. By ultrasound diagnosis of radiation of parietal bone specimens of all ages, the bubble can not be significantly damaged. As a result, it is possible that after attenuated by the skull specimens irradiance, the ultrasound diagnosis wave mediated by microbubble can open BBB and promote the transit of paclitaxel crossing BBB.2. We radiate the experiment rabbits through the simulated human skull (bone specimens) with the ultrasound diagnostic wave of the frequency of 1.75MHz/3.5MHz,MI1.9 for 10min. The microbubble dose is 0.5ml/kg. After the deliquation, we inject it by the intravenous injection of rabbit ear edge even. By using the blasting imaging model, we found that: under the same conditions, the EB leaking by intermittent triggered is more clearly than the radiation way. Four hours later, there is no cell morphology change by the organization cytological examination, no delay effect after 4w, and there is no significant nerve cell apoptosis.3. Due to the high solubility of paclitaxel in the triacetin, we have improved the preparative method of microbubble carrying paclitaxel, and it has got higher encapsulation efficiency and drug loading than that of the lyopyilization preparative method.4. Both the microbubble combined with ultrasound plus paclitaxel group and the ultrasound united with microbubble carrying paclitaxel group were able to mediate the release of paclitaxel to target to the brain tissue, but the concentration of the latter group in the brain tissue was significantly higher than the former one, and the difference between the two groups is very significant, P <0.01. In addition, as for the microbubble combined with ultrasound plus paclitaxel group, the concentration of free paclitaxel in the blood was significantly lower than the control group, and the difference was significant, P <0.01. The tumor inhibition rate of the ultrasound united with microbubble carrying paclitaxel group was significantly higher than the control groups, P<0.05. Furthermore ,the apoptotic index tumor cell was highest of all the exprienmt groups.
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