| PARTⅠ PREPARATION AND CHARACTERIZATION OF HEMATOPORPHYRIN MONOMETHYL ETHER-LOADED POLYMER SYNERGISTObjective To prepare a Hemato Porphyrin Monomethyl Ether(HMME)-loaded poly(lactic-co-glycolic acid)(PLGA) microcapsules(HMME/PLGA), investigate their properties of physics, optics and so on. After characterization, the cell-killing and cell proliferation-inhibiting effects of HMME/PLGA microcapsules on ovarian cancer SKOV3 cells were assessed.Methods PLGA microcapsules encapsulating HMME were prepared by the double emulsion evaporation method. The surface morphology and structure, the mean size and zeta potential distribution, the encapsulation efficiency and drug-loading efficiency were assessed using several analytical methods. The effects of different ultrasound irradiation time(10,30, 60, 90 s) on the survival rate of ovarian cancer SKOV3 cells was detected by CCK8 method. The experiment was divided into five groups: the control group(Group I), the group subjected to US(US; Group II), the group treated with PLGA subjected to US(PLGA+US; Group III), the group treated with 20 μg/ml HMME subjected to US(HMME+US; Group IV), and the group treated with HMME(20 μg/ml)/PLGA microcapsules subjected to US(HMME/PLGA+US; Group V). Treatment parameters were(the intensity was 0.50 w/cm2 and the frequency was 1 MHz).Survival rate of ovarian cancer SKOV3 cells at 12, 24, 36, 48 h were detected based on results from the above experiments.Results HMME/PLGA microcapsules became pink emulsion, had better dispersion. Light microscope(LM) and scanning electron microscope(SEM) imaging indicated that the microcapsules exhibited a smooth and uniform spherical morphology. Transmission electron microscope(TEM) indicated the presence of HMME nanoparticles in the shells of these microcapsules, as a large number of black particles embedded in the spherical shell. The HMME/PLGA microcapsules exhibited strong red florescence as detected with confocal laser scanning microscopy(CLSM). Laser Particle Size Analyzer System showed that, the average diameter of the HMME/PLGA microcapsules was 357±0.72 nm(PDI=0.932), and the microcapsule surface was negatively charged(zeta potential:-7.89 m V). UV-Vis spectrophotometry showed that the HMME/PLGA microcapsules covered a wide absorption wavelength range in the UV visible region, with a strong absorption band at 418 nm and four absorption peaks between 500-700 nm(at 515, 549, 590, and 648 nm, respectively).The encapsulation efficiency of the HMME/PLGA microcapsules was 58.33±0.95%, and the drug-loading efficiency was 4.73±0.15%. HMME/PLGA microcapsules in cell toxicity-proliferation test showed that Group V( US+HMME/PLGA+cell) report the largest reduction for cell viability(P<0.05) and the slowest cell proliferation(P<0.05).Results HMME/PLGA microcapsules have been successfully synthesized. The microcapsules have good spherical morphology, uniform size, high encapsulation efficiency. They have excellent optical absorption and remarkably killed and inhibited the SKOV3 cell proliferation, which make them a potential multifunctional contrast agent for HIFU diagnosis and treatment.PART Ⅱ HEMATOPORPHYRIN MONOMETHYL ETHER-LOADED POLYMER SYNERGIST FOR ENHANCED US AND PA IMAGINGObjective To observe the effect of HMME/PLGA for enhanced US and PA imaging in vitro, investigating the feasibility of these microcapsules as a dual-modality imaging agents. To observe the influence of these agents on nude mice ovarian cancer PA imaging in vivo, to investigate the enhanced capacity and the principle of HMME/PLGA in PA imaging.Methods Imaging was assessed with the gel mold. The holes were filled with PBS, PLGA, HMME, and HMME/PLGA microcapsules with different HMME concentrations(0.25, 0.50, and 1.00 mg/ml) to obtain the US imaging results of these samples. All images were acquired with the conventional B mode, with the same instrument parameters. For the in vitro PA imaging, PBS, PLGA, HMME, and HMME/PLGA microcapsules were subjected to the laser exposure at 690 nm for about 3 min, and the PA intensity variation was observed. In vivo US and PA dual-modality imaging was performed about one month after cell inoculation, totally 10 mice intravenously injected with 0.2 ml HMME/PLGA microcapsule solution(the HMME concentration was 1.50 mg/ml), followed by the laser exposure at 690 nm. PA images and US images for tumors were recorded, understanding the relationship between ultrasound/ photoacoustic signal intensity and time of ovarian cancer.Results At the same concentration, the HMME/PLGA had the strongest echo intensity, and PBS had the weakest. The PLGA and HMME did not exhibit significant difference. Meanwhile, the US signals were increased obviously with the increasing HMME concentrations. The PA signal of HMME/PLGA was stronger than HMME, while PBS and PLGA almost had no PA signals. An obvious linear correlation was observed between the HMME/PLGA microcapsule concentration and PA signal. Especially, the maximum concentration was associated with the strongest PA signal. The PA signal intensity was gradually increased at the tumor sites for all the time points, while there was very little change in the US signal in the tumor region in vivo.PART Ⅲ HEMATOPORPHYRIN MONOMETHYL ETHER-LOADED POLYMER SYNERGIST FOR HIGH INTENSITY FOCUSED ULTRASOUND CANCER ABLATIONObjective To investigate the efficacy of HMME/PLGA combined with HIFU in bovine liver tissue ablation, and to investigate the influence of HMME/PLGA combined with HIFU in nude mice with detectable ovarian cancer.Methods This part was divided into two parts. The experiment in vitro was assessed with the common fresh bovine liver. The sample was divided into the following groups: PBS(Group I), PLGA(Group II), HMME(Group III), and HMME/PLGA microcapsule(Group IV) groups(CHMME was 0.25, 0.50, 1.00 mg/ml, respectively). HIFU ablation was performed with single irradiation, with output acoustic powers of 120, 150, and 180 w, respectively. For each output acoustic power, the treatment duration was set as 3, 5, and 10 s, respectively. After irradiation, the gray scale values, coagulative necrosis volume(V) and energy efficiency factor(EEF) were calculated. In vivo 50 nude mice bearing xenograft tumors were randomly divided into the control(Group I), PBS(Group II), PLGA(Group III), HMME(Group IV), and HMME/PLGA microcapsule(Group V) groups. Group I only received HIFU ablation. For Groups II- V, the nude mice received injection of 200 μl PBS, PLGA, HMME, and HMME/PLGA microcapsule(CHMME=1.50 mg/ml), respectively, at the tumor sites. For all the five groups, each tumor was destroyed by one single exposure, with the acoustic power at 120 w and exposure duration for 5 s. After HIFU ablation, the gray-scale value change, the volume of coagulative necrosis and the EEF was calculated, HE staining, TEM and immunohistochemical examination of PCNA and TUNEL were performed to detect the structure changes of the targeted tissue caused by HIFU ablation.Results The gray scale variation value and coagulative necrosis volume in the target area of the HMME/PLGA group(Group IV) increased greater than all the other groups, while the EEF value was much smaller in vitro. In vivo, Group V(HMME/PLGA+HIFU) presented the greatest gray scale change and coagulative necrosis volume, while the EEF value was much smaller too. Microscopic examination displayed, the ultrastructural changes were significant of Group V, the expression of PCNA and TUNEL were significantly different with other gyoups. |
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