Optimization For Formulation Of Hematoporphyrin-loaded PLGA Ultrasound Contrast Agents And Application In Sonodynamic Therapy For H22 Tumor-bearing Mice

PARTⅠOPTIMIZATION FOR FORMULATION OF HEMATOPORPHYRIN-LOADED PLGA ULTRASOUND MICROBUBBLE CONTRAST AGENTS AND DETECTION OF THEIR PROPERTIESObjective:To optimize the formulation of a new kind of ultrasound contrast agents carrying the sensitizer of hematoporphyrin with PLGA for material. Then the basic physical properties, drug releasing behavior in vitro, ultrasound imaging in vitro and in vivo and the skin phototoxicity of the contrast agents were detected.Methods:1. The techniques of double emulsion, mechanical oscillation, vacuum freeze-drying were applied to produce hematoporphyrin-loaded PLGA ultrasound microbubbles, which were optimized through orthogonal test using encapsulation efficiency for the main detected index. 2. Morphology and drug distribution of HP-PLGA microbubbles were observed through light microscope(LM), fluorescence microscope(FM) and scanning electron microscope(SEM). The mean size,Zeta potential and the properties of releasing behavior in vitro were detected by Malvern laser particle size analysis apparatus, Malvern surface potential measuring apparatus and high performance liquid chromatography. Then ultrasound imaging in vitro and in vivo and the skin phototoxicity of HP-PLGA contrast agents were investigated, respectively.Results:1. Through orthogonal test, the optimization parameters were picked out as 10 mg/ml for concentration of HP, 40 mg for amount of PLGA, and 1/5 for volume ratio of water inside to dichloromethane.2. The optimized HP-PLGA contrast agents were spheric with the me- an size of 602.3 nm and Zeta potential of–(17.1±1.6) mV. The drug loading and encapsulation efficiency of HP-PLGA were (2.15±0.15) % and (63.5±2.6) %, respectively. And the releasing behavior of HP-PLGA contrast agents in vitro was that after an obvious release of about 35.1 % in former 24 h, there were 86.5 % HP-PLGA released within 14 days. The ultrasound image of HP-PLGA could be enhanced in vitro and in vivo. Compared with HP, HP-PLGA contrast agents could significantly reduce the phototoxicity side-effect of hematoporphyrin.Conclusions: The self-made HP-PLGA ultrasound microbubble could meet the basic requirements for an ideal drug carrier, which might be a useful tool for delivering sensitizer in sonodynamic therapy on tumor.PARTⅡSONODYNAMIC THERAPY FOR H22 TUMOR-BEARING MICE WITH HEMATOPORPHYRIN-LOADED PLGA ULTRASOUND CONTRAST AGENTSObjective:To study the effect of sonodynamic therapy on H22 tumor-bearing mice using ultrasound combined with hematoporphyrin-loaded PLGA ultrasound contrast agents.Methods:Sixty H22 tumor-bearing mice were divided into A and B groups randomly. Each group was divided again into five groups for different treatment: control group(C); group of ultrasonic irradiation alone(US); group of ultrasound combined with microbubbles without hema- toporphyrin(US+MB); group of ultrasound combined with hematopor- phyrin(US+HP); group of ultrasound combined with microbubbles containing hematoporphyrin(US+HP-PLGA). In group A, the growth curve of H22 tumor was depicted within 15 days after treatment; The inhibition rate of tumor quality on the 15th day was investigated; The index about apoptosis and proliferation on tumor of each group were detected by TUNEL and immunohistochemical method. In group B, survival days of 30 H22 tumor-bearing mice receiving different treatment were recorded.Results:Compared with other groups, the tumors of US+HP-PLGA group grew the most slowly. The inhibition rate of tumor quality (53.86 %), the apoptosis index (52.82 %) and the mean survival days (47.83 days) of US+HP-PLGA group were significantly higher and longer than the other four groups (P <0.05), while the proliferation index (37.83 %) was significantly lower than other groups (P <0.05). There was no statistic difference among US group, US+MB group and C group.Conclusions:Ultrasound combined with hematoporphyrin-loaded PLGA ultrasound contrast agents could inhibit growth and promote the apoptosis of H22 tumor, and prolong the survival days of H22 tumor-bearing mice.