Ultrasound-mediated Destruction Of Oxygen And Paclitaxel Loaded Dual-targeting Microbubbles For Intraperitoneal Treatment Of Ovarian Cancer Xenografts

Part one: Preparation of oxygen and paclitaxel loaded folate dual-targeting microbubbles and observation of the targeting ability of oxygen and paclitaxel loaded folate dual-targeting microbubbles in vivoSection one The synthesis of oxygen and paclitaxel loaded folate dual-targeting microbubbles and the study of targeting ability in vivoObjectives: To synthesize oxygen and paclitaxel loaded folate dual-targeting microbubbles and evaluate the targeting ability of oxygen and paclitaxel loaded folate dual-targeting microbubbles in vivo.Methods: The human epithelial ovarian carcinoma models were established by intraperitoneal injection of the SKOV3 cells in nude mice.The growth behavior of mice and the expression of folate receptors were detected by immuneohistochemistry.The oxygen and paclitaxel loadedfolate dual-targeting microbubbles(TOPLMBs)and the oxygen and paclitaxel loaded microbubbles(OPLMBs)were synthesized by mechanic vibration technique,and both TOPLMBs and OPLMBs were labeled by Di I.Tumor-bearing mice were randomly divided into 3 groups: TOPLMBs group,OPLMBs group and TOPLMBs after folic acid blocking group.Cells within the ascites fluid and tumor tissue were collected from sacrificed mice 30 min,24h and 48 h after injection with Di I-labeled OPLMBs or Di I-labeled TOPLMBs;Free folic acid was used to block FRs and then Di I-labeled TOPLMBs were intraperitoneal injected and cells within the ascites fluid and tumor tissue were collected from sacrificed mice 30 min post-injection.The distribution of fluorescence in ascites cells and tumor tissues was imaged by a confocal microscopy.The mice were divided into two groups(TOPLMBs group and OPLMBs group)randomly.The animal model was prepared and handled as described above.Ascites fluid was collected from the sacrificed mice and the uptake of SKOV3 cells and tumor-associated macrophages(TAMs)were detected by flow cytometry analysis afterward.Results: The success ratio of establishing the human ovarian carcinoma intraperitoneal models were 100%.The latency period were8.2±0.84 days.The expression of folate receptors(FRs)were positive in the tumor tissue.Confocal microscopy of slices: TOPLMBs group exhibits stronger red fluorescence emission than OPLMBs group at 30 min,24h and 48 h.TOPLMBs group exhibits strongest red fluorescence emission at 30 min.No obvious red fluorescence was observed in TOPLMBs plus folate blocking group at 30 min,and the fluorescence was similar to OPLMBs group.Flow cytometry: TOPLMBs uptaken by SKOV3 cells was three times than OPLMBs(P<0.05),while the uptake of TOPLMBs by TAMs was five times more than that of OPLMBs(P<0.05).Conclusion: The human ovarian carcinoma intraperitoneal models of SKOV3 are established successfully.TOPLMBs have the dual-targeting capability of both tumor cells and TAMs and TOPLMBs can penetrate into the tumor tissue.TOPLMBs have a better targeting ability in vivo.Section two The analysis of drug delivery by oxygen and paclitaxel loaded folate dual-targeting microbubbles and biodistribution In vivoObjects: To explore the biodistribution of oxygen and paclitaxel loaded folate dual-targeting microbubbles In vivo and to determine the time point for ultrasound mediation.Methods: Tumor-bearing mice were randomly divided into 3 groups:(a)PTX,(b)OPLMBs,(c)TOPLMBs.All the mice received the same single dose of PTX(20mg/kg)through the intraperitoneal administration.Each animal was sacrificed 30 min after injection and the tissue samples(i.e.,ascites fluid,lymph node,blood,tumor node)were collected.The concentration of PTX in various tissue were detected by HPLC.Results: The PTX concentration of tumor tissue in the TOPLMBs group,OPLMBs group and PTX group were(51.63 ± 6.29 mg/m L),(20.56 ± 5.39 mg/m L)and(3.59 ± 0.81 mg/m L),respectively.The PTX concentration of lymph nodes in the TOPLMBs group,OPLMBs group and PTX group were(3.27 ± 0.76 mg/m L),(1.20 ± 0.12 mg/m L)and(0.75 ± 0.09 mg/m L),respectively.The PTX concentration of blood in the TOPLMBs group,OPLMBs group and PTX group were(0.56 ± 0.10 mg/m L),(1.19 ± 0.12 mg/m L)and(3.11 ± 0.10 mg/m L),respectively.The PTX concentration of Ascites in the TOPLMBs group,OPLMBs group and PTX group were(3.25 ± 0.18 mg/m L),(6.51 ± 0.13 mg/m L)and(8.96 ± 0.23 mg/m L),respectively.The TOPLMBs group showed a significantly higher PTX concentration in the tumor tissue and lymph nodes(P<0.05).The PTX concentration of TOPLMBs group in blood and ascites were significantly lower than in other groups(P<0.05).Conclusion: These results implied that the TOPLMBs could easily delivery PTX into the tumor tissue and lymph nodes.Only few PTX were detected in blood and ascites by TOPLMBs administration,which may promote the antitumor efficiency and reduce the nonspecific side effects.Part two: Ultrasound-mediated destruction of oxygen and paclitaxel loaded dual-targeting microbubbles for intraperitoneal treatment of ovarian cancer xenograftsObjects: To explore the antitumor efficiency and the mechanism of TOPLMBs mediated with ultrasound on intraperitoneal ovarian cancer xenografts.Methods: 56 Tumor-bearing mice were randomly divided into seven treatment groups:(a)PBS,(b)PTX,(c)PTX+US,(d)OPLMBs,(e)OPLMBs+US,(f)TOPLMBs,(g)TOPLMBs+US,eight mice in each group.Three mice in each group were randomly sacrificed 24 h after the last treatment and the tumor tissue and the ascites fluid were harvested.The tumor apoptosis was measured by Td T mediated UTP nick end labeling(TUNEL)method.The apoptosis of macrophages in the tumor were detected by immunohistochemical staining of CD68;The tumor microvascular density(MVD)and the expression of vascular endothelial growth factor(VEGF)were measured by immunohistochemical technique.The apoptosis of ascites cells with FRs positive were detected by flow cytometry analysis.The survival time of other mice were recorded.Results: The corresponding median survival times in group(a)to group(g)were 31,37,36,31,41,32,and 52 days,respectively.The treatment group TOPLMBs+US,OPLMBs+US,PTX and PTX+US increased the median survival time compared with PBS group(P<0.05).The TOPLMBs+US group showed a superior significantly longer median survival time than other treatments(P<0.05).The apoptosis of tumor cells and TAMs: The apoptosis of group PTX,PTX+US,OPLMBs+US and TOPLMBs+US were higher compared with PBS group(P<0.05).The TOPLMBs+US group showed a significantly highest apoptosis than other treatments(P<0.05).The expression of VEGF and MVD: The group PTX,PTX+US,OPLMBs+US and TOPLMBs+US showed lower expression of VEGF and MVD compared with group PBS(P<0.05).VEGF and MVD in group TOPLMBs+US was significantly lowest than those in the other treatment groups(P<0.05).The FRs positive cells apoptosis ratios in group(a)to group(g)were(5.84 ± 0.28)%,(18.72 ± 3.44)%,(18.46 ± 0.80)%,(7.63 ± 0.90)%,(37.88 ± 8.04)%,(7.34 ± 1.08)% and(86.62 ± 2.38)%,respectively.Compared with the other treatment groups,group TOPLMBs+US(g)had the highest apoptosis efficiency than other groups(P < 0.05).Conclusion: TOPLMBs combined with US synergistically enhanced the survival time by killing ovarian cancer cells and TAMs,improving the hypoxia environment and inhibiting the angiogenesis.