The Experiment Study Of Ultrasound/CT Dualmodality Targeted Molecular Imaging And Assessment Of Liver Fibrosis

PART I PREPARATION AND CHARACTEZATION OF ULTRASOUND/CT DUAL-MODAL NANOPARTICLES CONTRAST AGENT OF LIQUID FLUOROCARBONObjective To prepare dual-modality nanoparticles(abbreviation as PLGA-PFOB NPs)contrast agent with shell of poly(lactic-co-glycolic acid)(PLGA)and core of perfluorooctyl bromide(PFOB).The basic characteristics and dual-modal imaging capability of nanoparticles,as well as their bio-safety,were studied in vitro.Methods The study of this part was divided into two sections.In the first section,the two-step emulsification was used to prepare PLGA-PEGCOOH wrapped PLGA-PFOB NPs suspensions.The morphology,particle size,zeta potential and stability testing of the NPs were investigated using several analytical methods to sum up an optimizing prepation condition.In the second section,the function of PLGA-PFOB NPs as dual-mode contrast agents for US and CT were imaged in vitro with a commercial US imaging system and Spiral CT scanner.The influence of PLGA-PFOB NPs intracellular uptake on the cell viability was estimated using CCK8 assay.The samples of different concentrations of PLGA-PFOB NPs emulsion were injected into normal rat through caudal vein to investigate the effect of these NPs on rat hepatic and renal function at different time(1d?3d?7d?14d).Results In the first section,PLGA-PFOB NPs suspensions were successfully prepared and milky white in color.Light microscope(LM)and scanning electron microscopy(SEM)images showed that most PLGAPFOB NPs were spherical with smooth surfaces and small spheres distributed evenly in the suspension.They appeared as spherical particles with a core-shell structure which showed by images of transmission electron microscope(TEM)and confocal laser scanning microscopy(CLSM).The proporation of adding 60?L of PFOB solution to 0.1g of PLGA-PEG-COOH and the vibroacoustic time of 4min as well as the evaporation time of more than 6h could prepare the most stable PLGA-PFOB NPs suspensions with minimum size.The mean diameter and zeta potential were 255.3 nm and-16.4 m V,respectively.In the second section,the PLGA-PFOB NPs suspension appear hyperechogenicity in US imaging with non-linear mode in vitro,the echo intensity(EI)increases in a concentration-dependent fashion.The PLGA-PFOB NPs suspension presented obvious high-density shadow in CT imaging,and the CT values were elevated with the increased concentration.The cell viability was not affected after PLGA-PFOB NPs suspensions incubate with BRL-3A cell by CCK8 assay.And the PLGAPFOB NPs suspension had no significant effect on the hepatic and renal function after injection into normal rat.Conclusion PLGA-PFOB NPs have been successfully synthesized;these NPs have good spherical morphology,uniform size and good biosafety.They have excellent acoustic characteristic and radiopaque properties,which enable them have a potential candidate for US and CT imaging.PART II THE EXPERIMENT STUDY OF PREPARATION AND CELL-TARGETING ABILITY OF CYCLIC ARGININE-GLYCINE-ASPARTIC ACID OCTAPEPTIDE FUNCTIONALIZED NANOPARTICLES CONTRAST AGENT OF LIQUID FLUOROCARBON IN VITROObjective To synthesize a cyclic RGD octapeptide by solid-phase synthsis method.The sequence of cyclic RGD(C*GRGDSPC)was selected based on its cell adhesion mediated by activated HSCs,and a subset were labeled with fluorescence isothiocyanate(FITC)for fluorescence detection.To prepare a new cyclic RGD octapeptide functionized nanoparticles for US/CT dual-modal imaging and to test their physical properties,biosafety and targeting ability in vitro.Methods The study of this part was divided into two sections.In the first section,the cyclic RGD octapeptide was solid-phase synthesized by N-tertbutyloxycarboryl(Boc)method and its side chain was labeled by FITC.The purity and molecular weight of the FITC-c RGD were determined using HPLC and MALDI-TOF-MS.The cyclic RGD octapeptide immobilization on PLGA-PFOB NPs was completed through the amide condensation reaction.The conjugation efficiency of cyclic RGD octapeptide to PLGAPFOB NPs was detected by CLSM and FCM.In the second section,rat stellate cell line(HSC-T6)was cultivated and its activated phenotype was determinded by immunofluorescence staining.The targeting effect of c RGDPLGA-PFOB NPs to activated HSCs was examine by CLSM and FCM.The influence of c RGD-PLGA-PFOB NPs intracellular uptake on the cell viability was estimated using CCK8 assay.The c RGD-PLGA-PFOB NPs suspensions of different concentrations were injected into normal rat through caudal vein to investigate the effect of these NPs on rat hepatic and renal function at different time(1d?3d?7d?14d).In order to test the chronic biosafety of c RGD-PLGA-PFOB NPs,the heart,liver,spleen,lungs,and kidney of health rats were collected after injection of c RGD-PLGA-PFOB NPs for 30 days and subjected to hematoxylin and eosin(H&E)staining.Results In the first section,the purity of c RGD and c RGD-FITC were 99.42% and 97.6%,respectively.The molecular weight of of c RGD and c RGD-FITC were 792.5 and 1293.2,respectively.The size and distribution of c RGD-PLGA-PFOB NPs were uniformed.The particle size and zata potential were approximately 273.7 nm and-10.7 m V.The conjection rate of c RGD octapeptide to PLGA-PFOB NPs was approximately 99.73%.In the second section,after being passage cultivated,the HSCs were immunofluorescently stained for ?-SMA and Desmin positively.FCM showed that intracellular fluorescence intensity of c RGD-PLGA-PFOB NPs in HSCs was higher than that of PLGA-PFOB NPs.CLSM iamges showed that c RGD-PLGA-PFOB NPs were aggregated to HSC-T6 cells much more than to BRL-3A,while fewer PLGA-PFOB NPs were aggregated to HSCT6.The c RGD-PLGA-PFOB NPs suspension didn't affect the cell viability of BRL-3A cells in vitro and the hepatic and renal function in vivo under the certain concentration.In addition,H&E staining of the heart,liver,spleen,lungs,and kidney revealed no observable changes between the c RGDPLGA-PFOB NPs injection group and control group.Conclusion The cyclic RGD octapeptide and FITC-labeld cyclic RGD octapeptide were successfully synthesized by solid-phase synthesis mothod.A novel targeted nanoparticle as a dual-modal contrast agent for US/CT imaging(c RGD-PLGA-PFOB nanoparticle)was prepared successfully,which engineer PLGA-PFOB nanoparticle by conjugation with c RGD through the amide condensation reaction.The c RGD-PLGA-PFOB NPs has good biological safety and exhibited good targeting effect to a HSCs,which laid a solid foundation for subsequent studies.PART III ESTABLISMENT OF RAT HEPATIC FIBROSIS MODEL AND DETECTION OF INTEGRIN ALPHA V BETA 3 EXPRESSION IN FIBROTIC LIVER OF RATSObjective To establish a rat hepatic fibrosis model and analyse pathological changes of different degree of fibrosis liver.To detect the expression of ?-smooth muscle actin(?-SMA)and integrin ?v?3 which related to activated hepatic stellate cells,as well as TGF-?1 which related to macrophages,during hepatic fibrogenesis.And to analyse the correlation between expression of ?-SMA,integrin ?v?3 and TGF-?1 in fibrogenic liver and degree of liver fibrosis.Methods Sprague Dawley rats rats were injected CCl4 solution(40% in olive oil,the first dosage 0.5ml/100 g,the others 0.3ml/100g)subcutaneously in back twice a week for either 3,6,or 9 weeks,to induce fibrosis at different stages.The control group received only normal saline.The pathological changes and percentage of collagen fibers in each group were observed and compared by hematoxylin–eosin(H&E)and Masson staining.The expression of integrin ?v?3 and ?-SMA in ?-SMA were detected by immunofluorescent staining.The expression of ?-SMA,integrin ?v,integrin ?3 and TGF-?1 in each group were detected by western blotting.Results Hematoxylin-eosin staining showed that the degree of pathological changes of fibrosis gradually increases with longer induction time.Masson staining showed a gradual increase in the percentage of collagen fiber area as the induction time prolonged.Immunofluorescence staining showed that the expression of integrin ?v?3 and ?-SMA gradually increased with the severity of fibrosis,and the difference between the groups was statistically significant.Western blotting analysis showed that the expression levels of ?v,?3,?-SMA and TGF-?1 in liver fibrosis were significantly higher than those in the normal control group,and gradually increased as the degree of fibrosis worsened.Conclusion We successfully established the rat liver fibrosis model.The expression of integrin ?v?3,?-SMA and TGF-?1 in fibrotic liver gradually increased with the severity of hepatic fibrosis.PART IV ULTRASOUND/CT DUAL-MODALITY TARGETED LIQUID FLUOROCARBON NANOPARTICLES CONTRAST AGENT FOR ENHANCED IMAGING AND ASSESSMENT OF LIVER FIBROSISObjective To observe the characteristics of enhanced ultrasound imaging and CT imaging of liver in normal rats within 48 hours after injection of PLGA-PFOB NPs contrast agent.And to select the observation and comparison time points of the contrast-enhanced imaging of liver fibrosis according to the changes of ultrasound echo intensity and CT values.Methods Normal rats were injected with contrast agent of PLGA-PFOB NPs through the caudal vein.The ultrasound enhanced diagnostic imaging instrument and spiral CT machine were used to dynamically observe the enhanced liver imaging and collect images.The DFY ultrasound image quantitative analysis software and CT image analysis software were respectively used to detect the echogenicity values and CT values before and 48 h after injection.The observation and comparison time points were selected according to the image intensity variation of contrast enhaced imaging effect of c RGD-PLGA-PFOB NPs on hepatic fibrosis.All contrast enhancement progress of fibrogenic liver within the selected time point was observed dynamically,and all liver images were captured and stored.The echo intensity values and CT values within the region of interest(ROI)in the liver parenchyma were quantitatively analysed by an Ultrasonic Quantitative Analysis Diagnostic System and a CT image analysis software,respectively.Results After intravenous injection of PLGA-PFOB NPs,in US imaging,the inferior vena cava presented immediate enhancement,and large vessels within the liver and liver parenchyma were subsequently enhanced.The EI in the liver peaked at 5 min,but contrast enhancement persisted for ~6 h.After that,the liver EI decreased significantly and reduced to the baseline value at 12 h.In CT contrast enhanced imaging,liver parenchyma were enhanced immediately at 5min after intravenous injection of PLGAPFOB NPs,and contrast enhancement was persisted for 12 h.After that,the CT values decreased significantly and reduced to the baseline value at 48 h.In subsequent enhanced imaging of hepatic fibrosis experiments,US and CT images were only acquired and analyzed before injection and within 6 h after injection.Rats in the fibrosis group(3 weeks,6 weeks,and 9 weeks after CCl4 injection of the c RGD-PLGA-PFOB NPs)had significantly higher EI in the liver at 6 h than those injected with PLGA-PFOB NPs(p < 0.001).After c RGD-PLGA-PFOB NP injection,the EI for rats with advanced liver fibrosis(9-week group)was significantly higher than that of rats with mild liver fibrosis(3-and 6-week groups)or normal liver(0-week group).After PLGA-PFOB NP injection,the EI of the 9-week group was significantly higher than the 0-,3-,and 6-week groups(p< 0.05).However,no changes in EI were found among the 0-,3-,and 6-week groups after PLGA-PFOB NP injection.In CT enhanced imaging experiment of liver fibrosis groups,the CT values in liver fibrosis group(3 weeks,6 weeks,and 9 weeks after CCl4 injection)were was significantly higher than that of rats with normal liver at 6 h after c RGD-PLGA-PFOB NP injection(P<0.05),and CT values were found significantly difference among the 3-,6-,and 9-week groups,respectively.Rats in the fibrosis group(3 weeks,6 weeks,and 9 weeks after CCl4 injection of the c RGD-PLGA-PFOB NPs)had significantly CT values in the liver at 6 h than those injected with PLGA-PFOB NPs(P<0.05).Conclusions The prepared c RGD-PLGA-PFOB NPs are successfully acted as dual-modal contrast agents for US and CT imaging in vivo.The c RGD-PLGA-PFOB NPs showed stronger contrast-enhanced imaging capability of fibrogenic liver in rats than of normal rats.The liver echo intensity and CT values were increased gradually with the development of hepatic fibrosis.Targeted ultrasound/CT dual-modality nanoparticles contrast agent for molecular imaging could potentially be a noninvasive method to assess liver fibrosis degree.