Preparation Of Multifunctional Ultrasound Ontrast Agents For Us, Mr, And Ct Imaging

PART Ⅰ PREPARATION AND CHARACTERIZATIONOF A MULTIFUNCTIONAL ULTRASOUND CONTRASTAGENTObjective To prepare a novel multifunctional ultrasound contrastagent Fe3O4-PFOB nanoemulsions, and to investigate its potential forultrasonography (US), magnetic resonance (MR), and computedtomography (CT) imaging in vitro.Methods The multi-functional ultrasound contrast agentFe3O4-PFOB nanoemulsions was prepared by introducingsuperparamagnetic iron oxide Fe3O4nanoparticles into the shell ofperfluorooctylbromide (PFOB) nanoemulsions through the hydration andsonication process. Several analytical tools were employed to characterizethe contrast agent. The morphology was observed under the lightmicroscope (LM), atomic force microscope (AFM) and transmissionelectron microscope (TEM) respectively. The mean size and sizedistribution were measured by a laser instrument. The concentration of Fe3O4loading in the contrast agent was detected by an atomic absorptionspectrophotometer (AAS). The magnetization properties were detected byusing a vibrating smaple magnetometer (VSM). The efficacy of the contrastagent for US, MR and CT imaging was evaluated by in vitro experiments.Results Fe3O4-PFOB contrast agent with a typical core-shellstructure was spherical, uniform and well dispersed. It was demonstrated byTEM that Fe3O4nanoparticles were successfully encapsulated into the shellof the contrast agent. The iron concentration loading in the contrast agentwas about62.5μg/ml. The size of the contrast agent was found to be about200~250nm and the size distribution was nearly symmetric. Fe3O4-PFOBcontrast agent could preserve the superparamagnetic properties of theencapsulated Fe3O4material, and its magnetic susceptibility effect wasassociated with the concentration of Fe3O4in the shell. The in vitroimaging showed that Fe3O4-PFOB nanoemulsions could not only producehigher echo intensity than non-Fe3O4-loaded PFOB nanoemulsions, butalso posses the magnetic suscpetibility and radiopacity for MR and CTimaging.Conclusion The newly developed Fe3O4-PFOB may have thepotential to serve as a multifunctional ultrasound contrast agent forenhanced US, MR, and CT imaging. PART Ⅱ MULTIFUNCTIONAL ULTRASOUNDCONTRAST AGENT FOR ENHANCED US, MR, AND CTIMAGINGObjective1. To explore the in vitro macrophages imaging after Fe3O4-PFOBloading.2. To evaluate the efficacy and safety of Fe3O4-PFOB for enhanced US,MR, and CT imaging in rat's liver.Methods1. Mouse mononuclear macrophages RAW264.7were incubated for3h with different concentrations of Fe3O4-PFOB nanoemulsions. Prussianblue staining was performed in order to observe the nanoemulsions uptake.MTT assay was used to detect the viability of the cells. The imaging effectsof macrophages loading with or without Fe3O4-PFOB were determined byin vitro US, MR and CT imaging.2. Twenty rats were randomized into two groups (ten rats each group).One group was injected with Fe3O4-PFOB nanoemulsions, and anothergroup was injected with PFOB nanoemulsions. US, MR, and CT imaging ofrat's liver were performed before and at different time after injection of thecontrast agents. The contrast-enhancement effects were evaluated by usingthe image quantitative softwares. At2h after the contrast agent administration, rat's liver tissues were excised for HE and Prussian bluestaining.3. The changes of vital signs of rats were observed in one month afterthe invo imaging. Additionally, rat's blood was obtained before and at7,14,30days after Fe3O4-PFOB injection for analysis of the liver and kidneyfunctions.Results1. Prussian blue staining allowed direct visualization of Fe3O4-PFOBnanoemulsions. A dose-dependent nanoemulsions uptake by macrophagescan be observed after3h of incubation. MTT assay showed Fe3O4-PFOB atthe desired concentrasions did not affect the cells viability. Macrophagesloading with Fe3O4-PFOB exhibited an obvious contrast enhancement inUS, MR and CT imaging compared with the control cells.2. In the in vivo US experiments, compared with PFOBnanoemulsions,Fe3O4-PFOB produced better contrast enhancement of rats'liver, and had longer enhancement duration time. In MR imaging, a negtiveenhancement was observed in Fe3O4-PFOB group, while no effectiveenhancement was observed in PFOB group. The shortest T2*time of theliver was achieved at2h after Fe3O4-PFOB injection. The highest PSIL(percentage of signal intensity loss) was shown by GRE-T2*WI comparedwith other used sequences. In CT imaging, the density enhancement of livercould be observed in both Fe3O4-PFOB and PFOB group. There was no significant difference in CT values between the two groups. Prussian bluestaining showed that Fe3O4-PFOB were mainly present in the liver sinusoid.3. No any abnormalities about the rats were found during thefollow-up period. No marked changes in the liver and kidney functionswere detected at different time after Fe3O4-PFOB administration.Conclusion1. Fe3O4-PFOB can be taken up by macrophages in vitro and itsimaging characteristics could not be affected after the uptake.2. Fe3O4-PFOB could be used as a safe and effective multifunctionalultrasound contrast agent for enhanced US, MR, and CT imaging in vivo. PART Ⅲ EXPERIMENTAL STUDY ON VALUE OFMULTIFUNCTIONAL ULTRASOUND CONTRASTAGENT IN DIAGNOSIS OF LOCAL HEPATIC LESIONSObjective1. To explore the efficacy of Fe3O4-PFOB-enhanced CT imaging inthe detection of VX2hepatic tumor in rabbits.2. To explore the efficacy of Fe3O4-PFOB-enhanced US, MR, and CTimaging in characterization and detection of hepatocellular carcinoma(HCC) and regenerative nodules (RNs) in rats, and to investigate thecorrelation between the image enhancement and the histopathologicchanges of local hepatic lesions.Methods1. Models of rabbit hepatic tumor were induced through VX2tumorblock implantation. Plain and Fe3O4-PFOB enhanced CT imaging wereperformed for all rabbits bearing VX2tumor in the liver. CT values in theregion of interest within the liver were measured before and afterFe3O4-PFOB administration. The ratio of CT values of the hepaticparenchyma to that of the tumor was calculated. At24h after injection ofFe3O4-PFOB, the tumorous and normal liver tissues were excised for thefrozen-section examination,HE and Prussian blue staining and were thenobserved under the light microscope. 2. Models of DEN induced hepatocellular carcinoma and cirrhosis ofrats were established.50SD rats were randomly divided into two groups:Experimental Group (40rats) and Control Group (10rats). The rats inExperimental Group were given DEN solution by gavage once a day (5times a week for14weeks). The rats in Control Group were given theequivalent normal saline. From week14to week22, rats of the two groupswere randomly chosen and examined by using Fe3O4-PFOB for enhanecdUS, MR, and CT imaging. The nodules diameter>3mm and normal livertissues were excised for the histologic examination to distinguish HCCfrom RNs. Kupffer cells were detected immunohistochemically usingCD68monoclonal antibodies. The Kupffer-cell amount in the tumoroustissue relative to that in the normal liver tissue (Kupffer-cell ratio) wascalculated. The relative image intensity of HCC and RNs were calculatedby comparing the intensity of the lesions with that of the background liver.The relative intensity changes on Fe3O4-PFOB-enhanced images(Fe3O4-PFOB-enhanced ratio) were evaluated. The relationship betweenFe3O4-PFOB-enhanced ratio and Kupffer-cell ratio of the liver lesions wasquantitatively annalyzed. The sensitivity, specificity, and accuracy incharacterization of malignant and benign lesions using single imagingmodality and combined imaging modalities were evaluated respectively.Results1. After Fe3O4-PFOB administration, the density of the hepatic parenchyma was enhanced as a function of time, while the density of tumordid not show any enhancement. The ratio of CT values in the parenchyma tothat in the tumor was significantly increased as compared with pre-injectionof Fe3O4-PFOB (P<0.05). Both frozen-section examination and Plussionblue staining showed much more contrast agents in the liver tissue than inthe tumorous tissue.2. Models of hepatocellular carcinoma and cirrhosis were successfullyinduced in23experimental rats.57nodules including HCC (n=38) andRNs (n=19) were studied. In Fe3O4-PFOB-enhanced US, MR, and CTimaging, the performance of HCC and RNs was different. Contrastenhancement can be observed in RNs under three modalities while noobvious change were found in HCC after Fe3O4-PFOB administration.Immunostaining examination showed that there were much more Kupffercells in RNs than that in HCC. There was significant difference inKupffer-cell ratio between HCC and RNs (P<0.05). Fe3O4-PFOB-enhancedratio was correlated with kupffer-cell ratio of the liver lesions. Thesensitivity, specificity, and accuracy in characterization of malignant andbenign lesions using single imaging modality could be improved by usingthe combined imaging modalities.Conclusion1. Fe3O4-PFOB could markedly increase the contrast differencebetween tumor and normal hepatic parenchyma,which is useful for the detection of hepatic tumor.2. Fe3O4-PFOB-enhanced imaging could indirectly reflect the numberof Kupffer cells, which is useful for the differential diagnosis of hepaticmalignant and benign lesions.3. Fe3O4-PFOB enhanced multimodality imaging could yieldcomplimentary information for the detection of liver lesions, whih mayprovide a valuable method for the characterization of nodular lesionsassociated with the cirrhotic liver.