Targeted Multifunction Ct Contrast Agent For CT Molecular Imaging And Therapy Of Ovarian Cancer

PART Ⅰ PREPARATION AND CHARACTERIZATIONOF TARGETED PERFLUORONOCYLBROMIDENANOPARTICLESObjective To prepare a novel folate-receptor targetedperfluoronoctylbromide nanoparitcles contrast agent, evaluating thecharacterization and in vitro targetability by several analytical tools, anddiscussing the potentiality for in vivo CT enhancement by in vitro CTexperiment.Methods The FR-TPNP contrast agent with different mass ratio ofPFOB/PLGA were prepared by two-step emulsion technique, and thesurface of the nanoparticles was modified by Fol-PEG-COOH.The sizedistribution and surface potential was measured by Laser Particle SizeAnalyzer System.The emulsification of contrast agent in different massratio were observed under the light microscope（LM).The morphology andstructure were observed by scanning electron microscope (SEM) andtransmission electron microscope (TEM). The targetablity of FR-TPNP to SKOV3cell was observed by confocal laser scanning microscope (CLSM),and the mean CT value of FR-TPNP contrast agent was acquired by CTscanner.Results The size distribution of the FR-TPNP contrast agent with massratio was found to be about300~560nm and the surface potential wasnegative.The size of FR-TPNP was increased with the higher level of massratio. The nanoparticles was emulsified well in all mass ratio of LMimaging except the1:1.The contrast agent was spherical, uniform and welldispersed according to the SEM imaging, and a typical core-shell structurewith PFOB encapsulated in the central which demonstrated by TEM.A lotof FR-TPNP were attached to the SKOV3cell in FR-targeted group, whichwere much less in control group and folic acid intervention group.The CTvalue of all FR-TPNP contrast agent was rang from200-800HU.Conclusions In this study, a novel FR-TPNP contrast agent wasprepared by a relatively simple method.The FR-TPNP contrast agent washighly effective targeting in vitro and with strong CT enhancedability.Neverthenes, the mass ratio of PFOB/PLGA was1:2maybe a betterchoice for synthesis the FR-TPNP contrast agent.Above all, this targetednanoemulsion contrast agent has broad application prospects in CTmolecular imaging. PART Ⅱ FR-TPNP CONTRAST AGENT FOR IN VIVO CTENHANCMENT AND TARGETED CONTRAST IMAGINGOF OVARIAN CANCERObjective1. To explore the in vivo circulation time, safety and enhanced efficacyof FR-TPNP contrast agent in mice.2. To evaluate the in vivo targeted efficiency of FR-TPNP contrastagent to ovarian cancer xenografts in nude mice.Methods1.Twelve healthy SD rats were randomly divided into three groups(n=4).Respectively, FR-TPNP group (intravenous FR-TPNP contrast agentvia tail vein,100mg/ml,2ml), Iohexol group (intravenous Iohexol via tailvein,350mgI/ml,2ml), control group (intravenous saline via tail vein,0.9g/ml,2ml). The CT contrast imagings of three groups were acquired atbefore and5min,1h,3h,6h,12h,24h,48h after injection.The enhancedeffects were evaluated by using the image quantitative softwares. The CTvalue of aorta, liver, spleen, kidneys and bladder in three groups weremeasurement by workstation ADW4.3. The mice was observed1monthafter injection of FR-TPNP contrast agent.2.The human ovarian cancer cells SKOV3were first cultured, then, themodels of xenografts of human ovarian cancer in nude mice on back wasestablished by subcutaneous injection of tumor cells.10nude mice with ovarian cancer xenografts beard were randomly divided into two groups(n=5), respectively, FR-targeted group (intravenous FR-TPNP contrastagent via tail vein,50mg/ml,0.5ml), control group (intravenous PNPcontrast agent via tail vein,50mg/ml,0.5ml).The CT contrast imaging oftumor, liver, spleen, kidneys were acquired at before and timed intervalsafter injection.The enhanced imaging was evaluated by using the imagequantitative softwares and the CT values were measured to evaluate the invivo targeted efficiency of FR-TPNP contrast agent.Result1.In the in vivo CT experiment of mice, the FR-TPNP contrast agentperformed a much longer time in blood circulation than Iohexol did, about3h. Furthermore, the enhancement of liver and spleen was last for48h inFR-TPNP group. However, the enhanced effect of liver and spleen wasminimized3h after injection in Iohexol group.There were no obviouscontrast-enhanced effects was observed in control group.The mean CT valueof aorta in FR-TPNP group was lower than Iohexol group between5min to2h after injection, but the mean CT value of liver and spleen in FR-TPNPgroup was much higher than Iohexol group from6h to48h after injection.2.Not any abnormalities about the mice were found during thefollow-up period.3.In the in vivo targeted experiment, the tumor performed a significantcontrast-enhanced effect6h after injection in FR-targeted group, the contrast effect was peak12h after injection, and last to48h after injection.The meanCT value of tumors were higher than that of liver, spleen and kidneys after6h.In control group, the mean CT value of tumors were increased about30HU2h after injection, but decreased at12h after injection. Furthermore,the mean CT value of tumors was lower than liver and spleen.Conclusion FR-TPNP contrast agent could be used as a long timecirculation, high targeted efficiency, effective contrast and safe nanosizedCT contrast agent for enhanced CT imaging in vivo. PART III FOLATE-RECEPTOR TARGETEDMULTIFUNCTIONAL NANOPARTICLES FOR THERAPYOF OVARIAN CANCERObjective To prepared a novel folate-receptor targeted multifunctionalnanoparticles（FR-TMNP）contrast agents for therapy of ovarian cancerxenografts.Methods1.The FR-TMNP contrast agent was prepared by two-step emulsiontechnique.10mg paclitaxel was first dissolved in CH2Cl2together withPLGA and Fol-PEG-COOH.Then the PFOB was added dropwise in themass ratio of1:2. The process of synthesis was the same as partⅠ. Theaverage size and surface potential were acquired by Laser Particle SizeAnalyzer System. Paclitaxel encapsulation efficiency, drug loading amountand in vitro drug release were determined by High Performance LiquidChromatography(HPLC).The SKOV3cells were first seeded in96platformand were divided into four groups, respectively, targeted therapy group(adding FR-TMNP contrast agent); non-targeted therapy groups (addingPNP contrast agent with paclitaxel loaded); free paclitaxel group (addingpaclitaxel solution) and the control group (adding FR-TPNP contrast agent),then the cells were kept incubated for24h.MTT assay was used to detect theviability of the cells.2.Twenty nude mice beard ovarian cancer xenografts were randomly divided into four groups as sectionⅠ. All the mice were injected withdifferent contrast agents and paclitaxel solution via tail vein (twice per week,two weeks). The volume of tumor were measured by vernier caliper everytwo days after the last injection. One month later, the tumors were takenaway from nude mice, and the tumor tissue were stained byimmunohistochemical.The apoptosis and tumor inhibition rate wereobserved by TUNEL, PCNA. The micro vessel density(MVD) werecounted.Result1.The paclitaxel was successfully encapsulated in FR-TMNP contrastagent. The mean size was402±27.12nm, surface potential was-19.2±1.7mV.The encapsulation efficiency of paclitaxel was70±24.78%, drug loadingwas7±2.48%.The PTX was released slowly from the FR-TMNP, about35%of drug loading amount.2.Immunohistochemistry showed that the apoptosis index (AI) oftargeted therapy group was significantly higher than other groups, the tumorproliferation index (PI) was significantly inhibited in targeted therapy group,tumor proliferation index was significantly lower than the other groups.MVD was lowest among all groups.Conclusion FR-TMNP was high drug loading and the PTX releasedfrom the FR-TPNP well in vitro.The tumor growing was effectivelycontrolled by the FR-TPNP.Above all, the FR-TPNP contrast agent was a simple synthesis, high efficient multifunctional molecular probe for targetedcancer imaging and therapy.