Targeted And Phase-shifted Lipid-coated Contrast Agents Loaded With Hcpt And Perfluorocarbon For Multi-modality Image And Therapy

PART I PREPARATION AND CHARACTERIZATION OF FA/Fe3O4/PFP/HCPT@LIPID CONTRAST AGENTSObjectives To prepare folate-modified phase-shifted and HCPT-loaded nanodroplets(FA/Fe304/PFP/HCPT@ Lipid)and investigate their properties of stability,phase transitional characteristics,drug loading,drug release and targeting efficiency in vitro and in vivo.Methods The FA/Fe3O4/PFP/HCPT@ Lipid were prepared through the rotary evaporation and sonication process.Size,morphology,mean particle size,zeta potential and Fe loading were all characterized.And mean particle size were determined at 0.5 h,1 h,2 h,6 h,12 h,24 h,36 h,48 h,60 h,72 h and 96 h after preparation.Phase transition of nanodroplets at different temperature(40?>45?>50? and 55?)were observed with optical microscopy.Drug loading and drug release were determined with Reverse-phase high-performance liquid chromatographic(HPLC)system.Targeting efficiency of FA/Fe3O4/PFP/HCPT@ Lipid was validated with cell and mice experiment.Results The prepared FA/Fe3O4/PFP/HCPT@ Lipid nanoemulsion was brown.Nanodroplets showed spherical or dotted morphology under optical microscope and fluorescence microscope.Fe3O4 nanoparticles were located into the shell of FA/Fe3O4/PFP/HCPT@Lipid under TEM.The mean diameter of prepared nanodroplets was 321±67 nm and the average surface zeta potential was-50 mV.The corresponding Fe concentrations of the FA/Fe3O4/PFP/HCPT@Lipid with different concentrations(5 ?g mL-1,10 ?g mL-1,20 ?g mL-1,40 ?g mL-1,80 ?g mL-1,160 ?g mL-1,320 ?g mL-4,640 ?g mL-1,1280 ?g mL-1,2560 ?g mL-1),as obtained using atomic absorption spectrometry,were 0.43±0.08,0.71±0.09,1.63±0.31,3.52±1.21,7.60±1.35,15.05±3.16,29.12±4.05,53.32±5.25,117.65±6.36 and 236.37±11.97 ?g mL-1,respectively.The mean particle size of FA/Fe3O4/PFP/HCPT@ Lipid nanoemulsions did not vary significantly within 48 h at a storage temperature of 4?.Two days later,the nanodroplets would enlarge slightly.At 40? the nanodroplets showed no obvious changes,and almost no MBs were observed under the microscope.Many nanodroplets enlarged to form MBs when the temperature rose to 45?.Almost all nanodroplets enlarged/fused and abundant MBs formed when the temperature reached 50?.Only a few larger bubbles could be observed at 55?.The drug encapsulation efficiency and drug loading content were 60.5%and 8.3%,respectively.FA/Fe3O4/PFP/HCPT?Lipid nanoemulsions slowly released HCPT and LIFU accelerated drug release.Conclusion The successfully fabricated FA/Fe3O4/PFP/HCPT@Lipid nanoemulsions were normal in shape,uniform and stable.The nanodroplets would change into microbubbles through liquid to gas transition.LIFU sonication would accelerated drug release from FA/Fe3O4/PFP/HCPT @ Lipid.PART ? CONTRAST AGENTS OF FA/Fe3O4/PFP/HCPT@LIPID FOR ENHANCED US,PA AND MR IMAGINGObjectives Study on the effect of FA/Fe3O4/PFP/HCPT@Lipid for enhanced US,PA and MR imaging in vitro and in vivo,to investigate the feasibility of the nanodroplets as a multi-modality imaging agents.Methods This section was divided into three parts.First,FA/Fe3O4/PFP/HCPT@Lipid nanoemulsions were heated to different temperatures,and the ultrasound imaging of nanoemulsions as well as optical microscopy of nanodroplets were observed.In the acoustic droplet vaporization(ADV)experiments LIFU instrument was used with different acoustic intensity(0.8W/cm2?1.6W/cm2?2.4 W/cm2?3.2 W/cm2)and duration time(1min?2min?3min?4min).Ultrasound imaging of the nanoemulsions were obtained.Second,Fe3O4 solution,FA/Fe304/PFP/HCPT@ Lipid nanoemulsions and FA/PFP/HCPT@ Lipid nanoemulsions were prepared for photoacoustic(PA)imaging.FA/Fe3O4/PFP/HCPT@ Lipid nanoemulsions with different concentrations were prepared for PA imaging.SKOV3 tumor cells were incubated with FA/Fe3O4/PFP/HCPT@ Lipid nanoemulsions for different duration time for PA imaging.In vivo PA imaging experiment,mice were received FA/Fe304/PFP/HCPT@Lipid injection,Fe304/PFP/HCPT@Lipid injection and FA/PFP/HCPT@ Lipid injection via tail vein.The PA imaging was conducted before and 0.5 h,1 h,12 h after nanoemulsions injection.Thirdly,FA/Fe3O4/PFP/HCPT@Lipid nanoemulsions with different concentrations were prepared for MR imaging.In vivo MR imaging experiment,mice were received FA/Fe3O4/PFP/HCPT@ Lipid injection,Fe3O4/PFP/HCPT@Lipid injection and FA/PFP/HCPT@Lipid injection via tail vein.The MR imaging was conducted before and 0.5 h,1 h,12 h after nanoemulsions injection.Results First,nanoemulsions showed nearly no contrast-enhanced signals at 40?.Moderate,strong,and minimal contrast enhancement was evident at 45 ? and 50?,and 55?,respectively.Most gas bubbles were observed under optical microscope at 45? and 50?.After LIFU sonication with acoustic intensity of 2.4 W/cm2 and duration time of 2 min,nanoemulsions showed obvious ADV in vitro,and in vivo obvious ADV required LIFU sonication with acoustic intensity of 3.2 W/cm2 and duration time of 2 min.Second,Fe3O4 solution and FA/Fe3O4/PFP/HCPT@Lipid nanoemulsions showed obvious PA signal,while FA/PFP/HCPT@ Lipid nanoemulsions showed nearly no PA signal.For FA/Fe3O4/PFP/HCPT@Lipid nanoemulsions,the higher concentration of Fe3O4 was,the more obvious PA signals were.The in vivo PA experiment revealed that in group with FA/Fe3O4/PFP/HCPT@Lipid injection the tumor displayed PA signals during observation time and PA signals was obvious at 1 h post injection,and the PA signal intensity was obviously higher than the other two groups(P<0.05).Thirdly,in vitro MRI experiments revealed that FA/Fe3O4/PFP/HCPT@ Lipid negatively enhanced the T2*-weighted MR images.The MRI signal intensity decreased with increasing iron concentration.The in vivo MRI experiment revealed that in group with FA/Fe3O4/PFP/HCPT@Lipid injection the tumor displayed MR signals during observation time and PA signals was obvious at 1 h post injection,and the MR signal intensity was obviously higher than the other two groups(P<0.05).Conclusion FA/Fe3O4/PFP/HCPT@Lipid could enhance US,PA and MR imaging in vitro and in vivo and were provided with the feasibility to be multi-modality imaging contrast agents.PART III FA/Fe3O4/PFP/HCPT@LIPID FOR TARGETED THERAPY OF OVARY CANCERObjectives To observe the in vivo distribution of FA/Fe3O4/PFP/HCPT @ Lipid and HCPT,and the therapeutic effect to SKOV3 ovary cancer.Methods Thirty tumor-bearing mice were divided randomly into targeted group and non-targeted group.DiI-dyed FA/Fe304/PFP/HCPT @ Lipid/Fe304/PFP/HCPT @ Lipid nanoemulsions were injected into the targeted group/non-targeted group through tail vein.Mice were sacrificed at 0.5,1,6,12 and 24 h after nanoemulsions injection and mice tumor were excised for ultrathin section.All the sections were observed with a fluorescence microscope to make sure the distribution of nanodroplets at different time point.Fifty nude mice were divided randomly into two groups and received HCPT injection and FA/Fe3O4/PFP/HCPT@Lipid injection through tail vein respectively.Blood were taken by removing mice eyeball at 0.5,1,6,12 and 24 h post-injection.Mice tissues including liver,spleen,heart,lung,kidney and tumor were excised at 0.5,1,6,12 and 24 h post-injection for RP-HPLC to determine drug concentration in tissue and tumor.Furthermore,drug concentration in tumor was determined after LIFU sonication.Thirty mice were randomly grouped(5 per group)three weeks after tumor inoculation.The experimental groups received HCPT injection,Fe3O4/PFP/HCPT@Lipid injection,FA/Fe3O4/PFP?Lipid injection plus LIFU,FA/Fe304/PFP/HCPT @ Lipid inJection and FA/Fe3O4/PFP/HCPT@Lipid injection plus LIFU exposure once every two days for 14 consecutive days,respectively.The control group was treated with saline.Mice tumors were measured to calculate the tumor growth curve and tumor inhibition rate.Immunohistochemical staining examination,proliferating cell nuclear antigen(PCNA)and TdT-mediated dUTP nick end labeling(TUNEL),were performed to acquire information on the proliferation and apoptosis of tumor tissue of each group.Results Red fluorescence representing FA/Fe3O4/PFP/HCPT@ Lipid was present in ultrathin section in the whole observation time,and it was obvious at 1 h in the targeted group.But no red fluorescence was found in ultrathin section of non-targeted group.The group treated with FA/Fe3O4/PFP/HCPT@ Lipid exhibited a higher plasma HCPT concentration than the group with HCPT injection at all the tested time points.HCPT concentration of tumor in the group admistered with FA/Fe3O4/PFP/HCPT@Lipid was significantly higher than that of the group with free HCPT injection at every time point(P<0.05).HCPT concentration in the tumor receiving FA/Fe3O4/PFP/HCPT@Lipid injection plus LIFU sonication was significantly higher than that receiving FA/Fe304/PFP/HCPT@Lipid injection only(P<0.05).For the mice administered with FA/Fe3O4/PFP/HCPT@Lipid+LIFU,the tumor growth curve was the lowest and tumor inhibition rate was the highest compared with all the other groups.The PI for the tumor of the FA/Fe3O4/PFP/HCPT@Lipid plus LIFU exposure group was significantly lower than those of any other groups(p<0.05).The AI for the tumor in the group of FA/Fe3O4/PFP/HCPT@ Lipid plus LIFU exposure was significantly higher than those of the other groups(p<0.05).Conclusion FA/Fe3O4/PFP/HCPT @ Lipid could prolong the circulation time of HCPT in vivo.After targeting to tumor cells,FA/Fe3O4/PFP/HCPT@Lipid,in comparison with LIFU,had satisfactory therapeutic effect to SKOV3 tumor.It's promising for FA/Fe3O4/PFP/HCPT@ Lipid compared with LIFU to open new ways for tumor therapy.