| PARTⅠ Preparation and detection of folate-targeted oxygen and paclitaxel loaded lipid microbubblesObjective: To prepare a novel folate-targeted oxygen and paclitaxel loaded Lipid microbubbles(TOPLMBs) and to evaluate its characteristics.Methods: TOPLMBs and no-targeted oxygen and paclitaxel loaded lipid microbubbles(OPLMBs) were prepared by mechanical vibration method. Stability of TOPLMBs and OPLMBs was preliminarily evaluated under 4℃and 37℃ after prepared. The particle size distribution and zeta potential were identified by a Malvern Zeasizer Nano ZS unit. The ability of oxygen release was tested by a dissolved oxygen instrument in the presence and absence of US. The PTX loading rate was monitored by HPLC. lipid microbubbles(MBs) coated with folate was detected by immunofluorescent staining, and the binding efficiency of the ligands was quantified via flow cytometry.Results: Stability of TOPLMBs and OPLMBs changed with time and temperature change, the aggregation phenomenon appeared in both microbubbles at 4 ℃ for 14 d,and the size of both microbubbles became larger at 37℃ for 2h. The synthesized TOPLMBs had an average size distribution of(1.81±0.04)mm,a mean zeta potential of-(11.75±1.56)mv, a drug entrapment efficiency of(86.10±3.47)%, and a drug-loading of(24.60±1.23)%. In comparision, the OPLMBs had an average size distribution of(1.78±0.05)mm, a mean zeta potential of-(14.93±0.50)m V, a drug entrapment efficiency of(93.51±4.74)%, and a drug-loading rate of(26.72±2.31)%, respectively. Both the two microphones were able to release oxygen in the presence of ultrasound exposure(US). TOPLMBs showed positive in immunofluorescent staining assay, the ligands binding efficiency of TOPLMBs was(96.29±0.67)%, and it was much higher than that of OPLMBs(P<0.05).Conclusion: TOPLMBs were successly synthesized with highly floated ligands attached on, and they own perfect oxygen and drug loading ability, may become a new type targeted drug carriers that can be used for ultrasonic controlled release.PARTⅡ The targeting ability of TOPLMBs to SKOV3 human ovarian cancer cells and macrophages in vitroObjective: To identify the expression of folate receptor in SKOV3 human ovarian cancer cells and macrophages, and to investigate the targeting ability of TOPLMBs in vitro.Method: SKOV3 human ovarian cancer cells and thioglycollate-el icited macrophages extracted by peritoneal lavage were cultured in vitro, the presence of folate receptors on SKOV3 human ovarian cancer cells and macrophages was identified by fluorescent immunocytochemistry. According to the different incubation conditions, cells were divided into 3 groups: group a(OPLMBs); group b(TOPLMBs+ free folic acid); groups c(TOPLMBs), the targeting ability of TOPLMBs for SKOV3 cells and macrophages in vitro was observed by bright field microscope.Result: Fluorescence analyses revealed that the folate receptors were distributed all over the cell surface of SKOV3 cells and macrophages. In the targeting test in vitro,a number of TOPLMBs were bounded around SKOV3 cells tightly, but the conjugation were inhibited after free folic acid added in, there was always no conjugation in OPLMBs group; for macrophages, a large number of TOPLMBs were uptake and internalized by macrophages in group c, and few microbubbles internalized by macrophages in the group a and the group b.Conclusion: Folate receptors are highly expressed in human ovarian cancer cells and macrophages; and TOPLMBS could be bind well to SKOV3 cells and macrophages in vitro.PARTⅢ The effection and mechanism of ultrasound mediated folate-targeted oxygen and paclitaxel loaded lipid microbubbles on the proliferation and apoptosis of SKOV3 ovarian cancer cellsObjective: To investigate the effection and mechanism of ultrasound mediated TOPLMBs on the proliferation and apoptosis of hypoxia SKOV3 ovarian cancer cells.Method: Cobalt chloride-induced SKOV3 hypoxia ovarian cancer cells were established in vitro, then cells were divided into seven groups and treated as follows:(a) applying RPMI1640 only(i.e.,control);(b) applying PTX only(i.e., PTX);(c) applying PTX followed by ultrasound destruction(i.e.,PTX +US);(d) applying OPLMBs only(i.e., OPLMBs);(e) applying OPLMBs followed by ultrasound destruction(i.e., OPLMBs+US);(f) applying TOPLMBs only(i.e., TOPLMBs);(g)applying TOPLMBs followed by ultrasound destruction(i.e., TOPLMBs+US). PTX was administered at a dose of 6mg/ml in each group, and ultrasound pulses was held in a fixed position,immersed 2 mm above the cell suspension and applied an watt density of 0.5 W/cm 2 for 15 s after PTX, OPLMBs or TOPLMBs were added to the cell medium for 30 min of incuburation. The ratio of proliferation inhibition of cells was detected by MTT at 24 h,48h,72 h after treatment. The cell apoptosis was determined by flow cytometry 24 h after different treatment. Gene transcription levels of hypoxia-inducible transcription factors 1α(HIF-1α)and Multi drug resistant gene(MDR-1) were evaluated using quantitative reverse transcription-po lymerase chain reaction(RT-PCR),and the expression of HIF-1α and p-gp protein were determined by western blot.Result: A significant anti-proliferative activities and apoptosis effects were observed in group c,e and g, among those three groups,group g demonstrated anti-proliferative activities of(64.61±12.53)%,(79.34±7.39)% and(89.83±5.61)% at 24 hour, 48 hour, and 72 hour after the treatment, respectively. The cell apoptosis ratio at 24 hour after the treatment is(49.20±7.18)%, which is significantly higher than other treatment groups(P<0.05). The results of RT-PCR showed that the gene transcription levels of HIF-1α and MDR-1was significantly lower in group b,c,e,and g, the gene expression of HIF-1α and MDR-1 in group e and g was significant than other groups(P<0.05), but there was no significant differences between group e and g. The Protein expression of HIF-1α and MDR-1 were significantly lower in group e and g in comparison with other groups, and group g was significantly higher than other groups(P<0.05).Conclusion: Ultrasound mediated TOPLMBs destruction has significant effects on SKOV3 hypoxia ovarian cancer cells proliferation and apoptosis, the mechanism may be related to the down-regulation of HIF-1α and MDR-1expression.PARTⅣ The cytotoxicity of ultrasound mediated folate-targeted oxygen and paclitaxel loaded lipid microbubbles on the macrophagesObjective: To investigate the cytotoxicity of ultrasound mediated TOPLMBs on the macrophages.Method: Thioglycollate-elicited macrophages extracted by peritoneal lavage were cultured divided into seven groups:(a) applying RPMI1640 only(i.e.,control);(b) applying PTX only(i.e., PTX);(c) applying PTX followed by ultrasound destruction(i.e.,PTX +US);(d) applying OPLMBs only(i.e., OPLMBs);(e) applying OPLMBs followed by ultrasound destruction(i.e., OPLMBs+US);(f) applying TOPLMBs only(i.e., TOPLMBs);(g)applying TOPLMBs followed by ultrasound destruction(i.e., TOPLMBs+US). The viability of cells in each group was detected by MTT at 24 h,48h,72 h after treatment, the apoptosis rate of cells was determined by flow cytometry.Result: The treatment of TOPLMBs+US had significant effects on the cytotoxicity of macrophages, with the viability of cells at 24 h, 48 h, 72 h was respectively(49.72±4.60)%,(44.99±7.19)%、(32.17±6.73)%,which was the lower than other groups(P<0.05), the apoptosis rate of cells in this group was(48.36±9. 39)%, which was significantly higher than other groups(P<0.05).Conclusion: Ultrasound mediated TOPLMBs destructon has cytotoxicity on macrophages in vitro. |
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