| Background:Breast cancer is an epithelial malignant tumor originated from the ductal and lobular.Infiltrating ductal carcinoma is the most common type of breast cancer in women. Mass inbreast area, nipple retraction or collapse, orange peel skin and lymph node enlargement aremain clinical manifestations. There are three major metastatic pathways of malignant cancer,including the hematogenous metastasis, lymphatic metastasis and direct invasion.Lymphatic system is the major passage of tumor metastasis and the systemic metastasis toimportant organs may involve lung, brain and bone. They will directly endanger people’slife.There are several methods for breast cancer treatment, including surgery, radiationtherapy, chemotherapy and targeted drug therapy, etc. Targeted drug treatments forlymphatic metastasis include anti-lymphogenesis and regional lymphoid tissue targetedchemotherapy.In the research of therapeutic ultrasound technology, microbubble ultrasound contrastagent is widely used as a drug or gene carrier. Microbubble, as an effective cavitationnucleus, is able to induce acoustic cavitation at low acoustic intensity. When cavitationoccurs, microbubble implodes and produces shock waves, micro-jets, which could enhancethe local release and penetration of therapeutic drugs. Cavitation related sonoporationincreases the tissue permeability which may help the local drug delivery. For lymphaticsystem, conventional intravenous injection does not reach the effective drug concentration.Our previous research confirmed that subcutaneously injection of microbubble contrastagent surrounding tumors can enhance the development of the drainage of axillary lymphnodes. Therefore, we hypothesize that percutaneous injection of fluorescent microbubble combined with ultrasound excitation can enhance the sonorelease in the draining lymphnodes of tumor. We hope that this targeted chemotherapy may be useful in the future.Objective:1. To prepare DiO-labeled microbubbles and to investigate their physicochemicalproperty, fluorescent labeling conditions and the development effects in rabbit liver.2. To observe the sonorelease of fluresence microbubbles to the lymph nodes of VX2breast cancer by ultrasound excitation. This method is potential to be a new, less invasiveand targeted chemotherapy for translymatic metastasis.Materials and Methods1. Materials:1,2-Distearoyl-sn-glycero-3-phosphocholine(DSPC),1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol sodium salt(DPPG), Polyethylene glycol-4000(PEG-4000), Glycerol,50%Glucose, Proplylene glycol,3,3dioetadeeyloxaearboeyanineperehlorate (DiO)(BiYunTian biotechn-ology research institute), dimethyl sulfoxide (DMSO),perfluoropropane (Physical and chemical engineering institute of nuclear industry, Tianjing,China).Therapeutic ultrasound device: A therapeutic ultrasound device named by CZ-960digital ultrasonic cavitation device was applied to generate pulsed therapeutic ultrasound(TUS), The transducer was operated at frequency of831kHz with peak negative pressureof2.9MPa. The actual working duty cycle was approximately5.95%.Diagnostic ultrasound instrument: A Acuson S2000ultrasound system equipped with a9L4high-frequency probe, eligible to perform contrast enhanced ultrasound (CEUS) withlow mechanic index.2. Methods:The DiO-labeled microbubbles (DiO-MB) were prepared with a freeze-dried powderand mixed Dimethyl sulfoxide (DMSO) dissolved DiO. Then we replace the air in bottleswith perfluoropropane. Finally the DiO-labeled microbubbles were made by high speedmechanical agitation. The size and distribution of DiO-MB were determined by a laserparticle sizer. The bubble fluorescent loading was approximately estimated by a laserconfocal microscopy. In this study, DiO-MB was intravenously injected to six healthy New Zealand whiterabbits weighted between2.0and2.5kilograms. Then contrast enhanced ultrasound wasperformed to detect liver enhancement. Fluorescent microscopy was used to observe thedeposition of green fluorescein in the frozen section of liver tissues.VX2breast tumor model was produced by injecting live tumor tissues to twentyfemale rabbits. Then the animals were divided into two groups: the DiO-MB group and thesonorelease group combining DiO-MB and ultrasound cavitation. In sonorelease group,1.0ml DiO-MB was injected subcutaneously around breast tumor and it was followed by1minhand massage at the injection site. Once the lymph node was contrast enhanced, it wasexposed to intermittent therapeutic ultrasound treatment for5mins in total. In DiO-MBgroup, the same injection without ultrasound irradiation was performed. The laser confocalmicroscope was used to observe the deposition of green fluorescein in the frozen section oflymph node. Integrated optical density (IOD), fluorescent area and average optical density(AOD) were analyzed.Result:1. The DiO-MB was round in shape with uniform size. The mean concentration andparticle size of SFLM was (4-7×109)/mL and (429±119) nm respectively. Confocal laserscanning microscopy showed that green apertures were surrounded the microbubble shell.The rabbit hepatic angiography ultrasound image shows continuous enhancement. Lessfluorescein distribution area were detected in the rabbit liver under fluorescencemicroscopy.2. Compared with DiO-MB group, the fluorescent area, IOD (P<0.01) and AOD oflymph node were higher in the sonorelease group (P<0.05). A high correlation between thevisual score of the fluorescence intensity and the visual score of the CEUS of the lymphnode were demonstrated with a spearman rank correlation analysis (rs=0.439, P>0.05).Conclusions:1. The particle sizes of DiO-labeled microbubbles is uniform and this enable it passthrough the pulmonary circulation. The contrast effect of DiO-MB in rabbit liver wassignificant.2. DiO-MB can not only enter lymph vessel around breast cancer, but also can bedelivered to the regional lymph nodes with a high fluorescent concentration by using pulse therapeutic ultrasound. |
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