Ultrasound Assisted Temperature-sensitive Liposome-microbubble IR780-loaded Complex For Targeted Thermo-chemotherapy In Breast Cancer

ObjectiveTo develope a multi-functional laser-responsive liposome-microbubble complex via conjugating cRGD targeted DOX temperature-sensitive liposome with IR780-loaded microbubble.,and explore the anti-tumor efficacy for human breast cancer MCF-7 cell,both in vitro and in vivo,using complex with different treatment protocols.MethodscRGD targeted DOX temperature-sensitive liposome-IR780-loaded microbubble complex was prepared via biotin-avidin system.The characterization of complex was investigated for drug loading,size and morphology using UV-vis spectrophotometry,dynamic laser light scattering(DLS),Laser scanning confocal microscope and transmission electron microscope(TEM).In particular,the anti-tumor efficacy of the complex with/without US/laser irradiation in MCF-7 cells was detected using the MTT assay.MCF-7 tumor-bearing nude mice were divided randomly into six treatment groups(each n=6):Control,free DOX,cRGD targeted DOX temperature-sensitive liposome with HT,complex with US,complex with laser,as well as complex with US and laser.At the 15 day after the first treatment,anti-tumor therapeutic efficacy(volume and weight of tumors)of different treatment groups was analyzed.Furthermore,tumors of six groups were collected and analyzed by immunohistochemical analyses of Ki67 and CD34.The targeted imaging capacity of complex was analyzed using contrast-enhanced US imaging.The biodistribution of therapeutic agent with/without US/laser irradiation was acquired via in vivo fluorescence imaging system.ResultsThe liposomes with green fluorescence conjugating onto the surface of a microbubble were confirmed via confocal microscopy.Corresponding DLS revealed its homogeneous size distribution(average diameter:1436.00±283.40 nm).The drug loading content of complex was 10.5 μg/108 MBs for IR-780 and 21.3μg/108 MBs for DOX.The cell viability of MCF-7 treated with the complex with US and laser assisted,compared with complex alone,significantly reduced(p<0.05).At the end of anti-tumor experiment,the tumor volume of tumor-bearing nude mice treated with complex plus US and laser irradiation showed an obvious decrease compared with free DOX group and RTSL with HT group(p<0.05).After administration of complex,the video intensity localized in the tumor region from tumor-bearing nude mice was obviously higher than that of blank MBs via contrast-enhanced US imaging.Stronger in vivo fluorescence intensity was detected at the tumor site after administration of complex combined with localized US,compared with free IR780 and complex alone groups,at the initial 0.5 h after injection(p<0.001).For immunohistochemical analysis,compared to the other groups,the lowest rate of Ki-67 staining cells was detected in the tumor tissues of mice treated with complex plus US and laser irradiation(p<0.001),and lower MVD per field compared to free DOX group(p<0.05).ConclusionWith the assistance of localized US and laser irradiation,successfully prepared cRGD targeted temperature-sensitive liposome-IR780-loaded microbubble complex displayed excellent antitumor activity both in vitro and in vivo.The mechanisms which are considered to be responsible for enhanced therapeutic efficacy are the ultrasound-mediated early extravasation of nanomedicine into the tumor and followed by laser-triggered ultrafast DOX release from RTSL.And then,this therapeutic strategy could improve DOX bioavailability to tumor cells,and exert significant antitumor efficacy through inhibiting cell proliferation and antagonizing angiogenesis.In addition,this multifunctional complex displayed the potential for theragnosis of tumor with excellent ultrasound molecular targeting imaging and NIR fluorescence imaging abilities.