Preparation Of Lipid Microbubbles Loaded Hematoporphyrin For Treatment Of Rabbit Liver VX2 Tumor

Hepatic cell carcinoma (HCC) is a common malignance of digestive system. Currently the preferred treatment of HCC is operation, but the low rate of surgical resection and the high rate of recurrence, chemotherapy is often the main alternative treatment method. Most chemotherapy drugs are cytotoxic drugs, lack of specificity and targeting to the cancer cells, have side effect to body. So a simple, safe, effective and reusable local chemotherapy is a hot and difficult research. Sonodynamic therapy (SDT) is a promising new modality for cancer treatment based on the synergistic effect on cell killing by the combination of sonosensitizer and ultrasound. Ultrasound waves could be precisely focused on the target volume, which made it possible to control the generation of active radicals in a definite area. This indicated that SDT has the potential for targeted therapy. However, the tissue distribution of sonosensitizers is a problem. Recent studies have shown that ultrasound-targeted microbubble destruction (UTMD) is a new noninvasive targeted drug delivery technology. Microbubbles carrying the therapeutic compound may be targeted to specific tissues through the use of sonic energy, which is directed to the target area and causes the microbubbles to rupture and release the therapeutic compound. UTMD provides a new way to overcome the shortage of SDT. Now UTMD and SDT are made tremendous progress, their basis of biological effects are the cavitation effects, so we have assumed that incorporation of porphyrin into lipid ultrasound microbubbles bilayer should provide an effective drug delivery system for cancer therapy.Therefore, the aim of the study was prepared a new lipid microbubble loaded-hematoporphyrin (LMLH) and detected its size, concentration, stability, encapsulation efficiency and ultrasound imaging in vivo and therapeutic effect on rabbit liver VX2 tumor, to provide reliable experimental basis for ultrasound-mediated target therapy. The study included the following two parts:PARTⅠ: PREPARATION AND CHARACTERISTICS OF LIPID MICROBUBBLES LOADED HEMATOPORPHYRINObjective To prepare a kind of LMLH and to study its characteristics and to evaluate as a new ultrasound contrast agent for chemotherapeutic drug delivery. Methods LMLH was prepared by mechanical vibration, the appearance, concentration, mean diameter, Zeta potential and encapsulation efficiency were detected before and after irradiance of 60CoγRay, and the enhancement of ultrasound imaging in vivo was assessed. Results The mean diameter range of the self-made LMLH was 1.5-5.3μm, the mean diameter was 2.8±0.4μm, the concentration was (3.24±0.49)×109/ml, Zeta potential was ?(15.4±2.4)mV, the drug encapsulation efficiency was 30.25%. After sterilization with irradiance of 60CoγRay, and the mean diameter and encapsulation efficiency were 2.2±0.3μm, 27.39±2.91%, respectively, no significant difference was observed. The liver imaging of the rabbit could be enhanced obviously and persistently. Conclusion LMLH prepared with mechanical vibration had high encapsulation efficiency with well distributed particle size, effective for the enhancement of ultrasound imaging in vivo. Based on this phenomenon, to achieve real-time monitoring of targeted drug delivery in vivo may be expected for future.PARTⅡ: EXPERIMENTAL STUDY OF ULTRASOUND COMBINED WITH MICROBUBBLE LOADED HEMATOPORPHYRIN TREATMENT ON RABBIT LIVER VX2 TUMORSECTIONⅠESTABLISHMENT OF RABBIT LIVER VX2 TUMOR MODEL AND MONITORED BY ULTRASOUNDObjective To explore a method of high successful rate to establish the rabbit liver VX2 tumor model, and to monitor the tumor with ultrasonography.Methods Tumor block transplanting method was used to make the rabbit liver VX2 tumor model, and the tumor in rabbit′s liver was confirmed by pathology. Ultrasonography including 2DUS, CDFI and CEUS was applied to detect the size, echo and blood flow of tumors, and to find out the characteristics of the rabbit liver VX2 tumor. Results The rabbit liver VX2 tumor model was made successfully with tumor block transplanting method,and the successful rate was 93.33%. Ultrasonography could measure the size of tumor exactly,and could monitor the changes of the echoes and tumor blood vessels accuratly at the real-time.The tumors showed typical enhancement patterns of"quick wash-in and quick wash-out".Conclusion Tumor block transplanting method is a simple method to make rabbit liver VX2 tumor model with high successful rate. Ultrasonography is an accurate, convenient and non-invasive method to monitor the tumor and blood vessels in tumors, the results provide experimental evidence for evaluating the efficacy of new techniques and therapeutics for rabbit liver VX2 tumor.SECTIONⅡEFFECT OF ULTRASOUND COMBINED WITH MICROBUBBLE LOADED HEMATOPORPHYRIN TREATMENT ON RABBIT LIVER VX2 TUMORObjective To explore the anti-tumor effects of LMLH on rabbit liver VX2 tumor using the technique of ultrasound mediated target drug dilivery. Methods Thirty-five New-Zealand rabbits with liver VX2 tumor were randomly divided into seven groups:①ultrasound+LMLH group (US+LMLH),②HP and ultrasound group(HP+US),③HP and lipid microbubble group(HP+MB),④LMLH alone group(LMLH),⑤HP alone group(HP),⑥US alone group(US) and⑦NS alone as control group(C). Ultrasound targeted irradiation was applied to the tumors after injecting LMLH through the ear-vein of rabbit in US+LMLH group.After treatment, all the rabbits were sacrificed and the tumors were harvested to measure the diameter.The tumor growth inhibiting rate was calculated and the expressions of CD34, PCNA in the tumor tissues were detected by immunohistochemistry, the tumor cell apoptosis was detected by TUNEL assay. Results The echoes of tumors in US+LMLH group were mixing echo,the results of CDFI and CEUS showed the blood vesseles were obviously decreased, the tumor growth inhibiting rate was higher than orther groups. In metabasis, there was few metabasis in US+LMLH group. Ultramicrostructure showed cellular membrane was broken, mitochondria was obviously swell. In US+LMLH group, MVD and PCNA were the lowest but AI was the highest, there were significant difference between US+LMLH group and others groups (P<0.05).Conclusion HP can be efficiently released and activated by ultrasound. This technique enhanced the drug delivery efficiency at the tumor site and inhibitted the tumor growth significantly in vivo, which could provide a novel strategy for chemotherapy of tumor.