| BackgroundThere are several kinds of tumor treatment, mainly including surgery, radiotherapy, chemotherapy, interventional therapy, high intensity focused ultrasound therapy, radio frequency ablation and laser treatment, etc. Chemotherapy is one of the important methods of traditional treatment, and it has the following unique advantages:①It Acts on the split phase cells, including cancer stem cells, tumor primary lesion and distant metastases lesions.②It can reduce the body’s immune suppression. However, Chemotherapy curative effect is restricted by some factors:①Antitumor drugs are difficult to achieve effective concentration in tumor tissues.②Systemic adverse reactions caused by intravenous administration limits the dose of drug.③The half-life of antineoplastic drugs is generally short, and tumor tissues are difficult to steadily maintain the effective drug concentration. Therefore, the key of chemotherapy is to improve the local drug concentration in tumor tissues.Lots of experimental studies show that ultrasound-activated microbubbles "cavitation effect" can improve the drug concentrations within the tumor cells and inhibit the growth of tumor tissues. Microbubbles activated by ultrasonic occurs oscillation, contraction, expansion and a series of dynamic processes, namely "cavitation effect". The micro beam or the shock wave arised by "cavitation effect" can make the cell membranes around bubbles form micropores, namely "sonoporation ", the molecules outside the membranes can enter into the cells."sonoporation" is divided into "reversible sonoporation" and "irreversible sonoporation".It has been reported that the effect of tumor chemotherapy can be enhanced by therapy ultrasound-activated microbubbles. Many scholars explore parameters optimization problem including therapy ultrasound intensity, pulse mode, the duty cycle, frequency, irradiation time and so on. They assess the influence of micron grade microbubbles, liquid-gas phase change microbubbles, drug-loading microbubbles and the microbubbles with both diagnosis and treatment function on curative effect. Beyond that, They observe the curative effect of different tumor cells and transplant tumor models.Also, it has been reported that two-dimensional diagnosis ultrasound-activated microbubbles, under appropriate mechanical index (MI) and irradiation time, can promote cell membranes to form micropores. It can increase the permeability of microvessels, result in spillover of red blood cells, and facilitate bone marrow mesenchymal stem cell to across the capillary wall and induce microvasculars rebirth of the myocardial infarction site.Howere, an experimental study about the real-time three-dimensional ultrasound-activated microbubbles has been rarely reported.In vitro studies, microbubbles are mixed with tumor cell suspension directly, microbubbles close to the cells. In vivo experiments, the contrast agent is given via venous injection. Microbubbles flow in capillaries, and are subjected to the influence of pulmonary circulation. The pre-existing literature has confirmed that therapy ultrasound-activated intratumoral injection microbubbles can increase the number of lanthanum particles within the tumor cells. However, according to the transmission electron microscopy, the number of intracellular lanthanum particles is only2.In terms of clinical ultrasonic equipment, the intensity of the ultrasonic field is generally adjusted with the mechanical index (MI).The MI depends on the maximum value of peak negative pressure and the centre frequency of the ultrasound field. For MI<0.3, the intensity of sound field is considered to be low. For0.3<MI<0.7, there is a possibility of minor damage to neonatal lung or intestine. For MI>0.7, there is a risk of cavitation if an ultrasound contrast agent containing gas microspheres is being used, and there is a theoretical risk of cavitation without the presence of ultrasound contrast agents. The risk increases with MI values above this threshold. In commercial scanners, the MI has been limited to1.9for medical imaging.Contrast agent microbubbles—octafluoropropane-albumin microbubbles are that perfluorinated propane gases are wrapped in human blood albumin to form microspheres suspension of average diameter2.5～4.0μm by acoustic vibration. It is impossible that perfluorinated propane gases may diffuse from bubbles into blood for they are of little solubility in blood, thus perfluorinated propane gases in bubbles can circulate steadily through all parts of the body. However, when octafluoropropane-albumin microbubbles are injected by virtue of ear marginal vein of a rabbit, they could run through lung. According to Henry’s law, there are a lot of gases escape from bubbles in the pulmonary capillary into atmosphere. The rest of octafluoropropane-albumin microbubbles can stride lung and enter left heart, flow through microcirculations of all organs, and realize ultrasound contrast of target region.Octafluoropropane-albumin microbubbles can not pass through microvascular walls, because they are micron-size. It is in microvasculars where ultrasonic act on microbubbles, bringing about damage to vascular endothelial directly. VX2neoplasm is malignant papilloma which Shope virus induced the skin of a rabbit to form into. After being72times cultured, it is formally established VX2, and it is a kind of transplantable tumor strains. VX2tumor can be administered and grow steadily within any internal organ, bone and soft tissue of a rabbit due to no reject reaction. Comparison with inoculation in sites such as the liver, kidney, bone,and lung of a rabbit tumor model, the muscle of a rabbit tumor model has its unique advatages. One is that the location of xenograft tumor is superficial, and the formed tumor can be found earlier by touching the surface of the skin. Also, the formed tumor can be used in the early study of cancer treatment and its evaluation of curative effect. However, other parts of inoculation tumor are very deep, and their observation are often with the aid of imaging technologies, vulnerable to the constraints of time and space, and found relatively delayed. The other is that the time of metastasis is late, and the metastasis location is single. After being inoculated over25days, a rabbit can be found small lung metastases, or only pulmonary metastases until a natural death. On the contrary, other parts of the xenograft tumor metastases are earlier in lung, liver, mediastinal lymph nodes, greater omentum and so on. Therefore, the intramuscular VX2xenograft tumor model is an ideal animal model which is used for the evaluation of local curative effect and local treatment effects on lung metastasis.When the tumor tissue become greater than2mm3or the number of cancer cells is over107, they need new vessles to provide enough oxygen and nutrients for their growth continously. As long as the volume of tumor reaches1～2mm3, if there were not enough new vessles produced in neoplasm, the tumor tissues would maintain dormancy or become vestigial. Once new vessles grow into tumor tissues, the neoplasm will grow rapidly and uncontrollably for there is sufficient blood supplied from new vessels. Doxorubicin (Dox) is a kind of anthracyclines antineoplastic and characterized by wide anti-tumor spectrum,and it aims at hypoxia cells in particular. In general, it is used in the form of hydrochloride complex in clinic, being orange crystal and soluble in water. Generally speaking, Dox is injected by intravenous or artery for its oral malabsorption and it enter cells with the help of active transportation. Dox can stop the replication of DNA, and influence the transcription of RNA. A lot of studies show that Dox maintain active during the whole cell cycle, including cell interphase. Dox has the fluorescence property exclusively, which could be used to detect the concentration of drug in the cell easily.The objective of this study is to observe whether real-time three-dimensional ultrasound-activated microbubbles can enhance the chemotherapy effect of xenograft tumor or not, and microbubbles are given by intratumoral injection. Real-time three-dimensional ultrasound can not only display the tumor but also irradiate the tumor in deep part. VX2xenograft tumor model is established in this experiment. The tumor within the muscle layer is irradiated by real-time three-dimensional ultrasound combined with intratumoral injection microbubbles. It is expected to provide the basis for an experimental study about the chemotherapy effect strengthened on tumor in the deep part by real-time three-dimensional ultrasound-activated intratumoral injection microbubbles.ObjectiveTo investigate the possibility of application of real-time three-dimensional ultrasound to strengthen chemotherapy effect on VX2xenograft tumor.Materials and methods1. MaterialsInstruments and reagents:①Ultrasonic irradiation apparatus:Diagnostic ultrasound3V-D probe of ViVi E9(GE company manufacture),1.5～4.0MHz. Ultrasonic diagnostic instrument:Philips iU22coclor Doppler imaging, L9-3high frequency line array probe,4.0～9.0MHz, equipped with contrast enhanced ultrasonography (CEUS) with low MI.②Contrast agent microbubbles: Octafluoropropane-albumin microbubbles (made by Nanfang Hospital affiliated Nanfang Medical University). Its film-forming materials and core gases are human blood albumin and perfluoropropane respectively. It is dissolved in3ml saline and made into suspension in order to be used in experiments. The bubbles size ranges form2.5μm to4.0μm, and the concentration of the suspension is0.8～2.2×109/ml. The volume of bubbles injected in tumor directly is equal to half of the volume of the tumor.③Doxorubicin Hydrochloride for injection (Dox, Shenzhen Wan Le Pharmaceutical Limited Company),10mg of one bottle, each bottle of Dox dissolved in5ml saline, and1.2ml/kg of Dox in saline is given by ear marginal vein of each rabbit.Other reagent:Sumianxin Ⅱ for injection, Atropine,3%Pentobarbital, Heparin.Experiment animals:29healthy New Zealand white rabbits which were male or female and2.0kg～2.5kg body weight, and another2rabbits for passage. All rabbits were provided by experimental animal center of Nanfang Medical University. The frozen storage VX2tumor strains were supplied by experimental animal center of Sun Yat-sen University.2. VX2xenograft tumor modelThe two tubes of frozen storage VX2tumor strains are conventionally recovered, and made into2ml of tissue suspension respectively.1ml of tissue suspension is inoculated into one place of muscle layer in the right hind limb of each rabbit which is used for continuous passage. In the14th day, a New Zealand white rabbit bearing VX2xenograft tumor was anesthetized with0.35ml/kg Sumianxin Ⅱ and0.25ml/kg Atropine through intramuscular injection, and established intravenous access for injecting3%pentobarbital. The rabbit was maintained lateral position. The position located tumor tissue and its1.5cm around were cleaned and sterilized. The tumor tissue was taken out under aseptic condition, and was removed the connective tissue and necrotic tissue quickly. The retained part was put in sterile sodium chloride, sheared into1mm3of small pieces approximately, and transplanted into one site in muscular layer of the right hind limbs of29healthy New Zealand white rabbits.3. Experimental procedureUltrasound diagnostic instrument monitored growth situation of tumor. In the11th day,24healthy New Zealand white rabbits bearing VX2xenograft tumor survived, and the volume of the tumor ranged from0.2cm3to0.9cm3. All the rabbits were used for experiment. Concrete steps as follows:①All the rabbits were divided into4groups randomly. Group A:blank control, not was given any treatment. Group B:Dox was injected through ear marginal vein merely. Group C:Microbubbles were injected into the tumor directly, and activated by real-time three-dimensional ultrasound. Group D:After Microbubbles being injected into tumor directly and Dox being given by ear marginal vein of a rabbit, real-time three-dimensional ultrasound irradiated the tumor.②Ultrasonic diagnostic instrument orientated tumor.③Output intensity of Ultrasonic irradiation apparatus measured by mechanical index (MI) was1.3,1.5～4.0MHz, and the irradiation time was20min. Microbubbles were injected into the tumor directly. Dox was given by ear marginal vein of a rabbit. Real-time three-dimensional ultrasound irradiated the tumor immediately. The tumor was treated in the1st,3rd,5th,8th day.④In the10th day, the tumor tissue was acquired for HE to observe the pathological histology of the tumor. Before each treatment and the Pathological materials, the diagnostic ultrasonic instrument detected the bigggst longitudinal section of the tumor, and measured the biggest suprainferior diameter (a) and the biggest anteroposterior diameter (b) of the xenograft tumor. In the a section perpendicular to the maximum longitudinal section, diagnostic ultrasonic instrument detected the biggest transverse diameter(c) of the xenograft tumor. Calculation of the tumor volume, the relative growth rate and the total growth rate of the xenograft tumor.4groups were conducted orderly.4. The volume, relative growth rate and the total growth rate of the tumorTumor volume(V)=Π/6×a×b×c(cm3)Tumor volume relative growth rate=(Vn-V1)/(Vn’-V1’)×100%,(Vn, V1) and (Vn’,V1’) were the tumor volume of test group and control measured respectively, start of the treatment at n day and1st day.Tumor volume total growth rate=(V10-V1)/(V10’-V1’)×100%5. Statistical analysisThe data was performed using SPSS13.0software. The tumor volumes as outcome measured of chemotherapy effect on VX2tumor were expressed as Mean±SD, the comparisons of the tumor volume relative growth rate and total growth rate among4groups at the same time were subjected to One-Way ANOVA. A p value<0.05was deemed statistically significant.Results①HE pathological histology:Group D majored in coagulation necrosis. There were a wide range of cells pyknosis in group C. The necrotic cells were distributed among the nonpolar tumor cells which arranged disorderly in Group B. Different degree of cells necrosis were in invasive growth cancer cells in Group A.②uring the treatment, the inhibition of tmor growth achieved in group B and D, and inhibition effect was more apparent in group D. The growth of tumors were promoted in group C. The tumor tissues of group A continue to grow.ConclusionsThe obvious inhibition effect is supported by microbubbles injected in tumor directly activated by real-time three-dimensional ultrasound combination with Dox injected through vein. |
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