Folate-targeted Perfluorohexane Nanoparticles Carrying Bismuth Sulfide Foruse In US/CT Dual Mode Imaging And Synergistic High-intensity Focused Ultrasound Ablation Of Cervical Cancer

PART Ⅰ PREPARATION,CHARACTERIZATION AND PHASE TRANSITION RESEARCH OF FOLATE-TARGETED PERFLUOROHEXANE NANOPARTICLES CARRYING BISMUTH SULFIDEObjectivesTo prepare the folate targeted perfluorohexane lipid nanoparticles carrying bismuth sulfide(FLBS-PFH-NPs)and investigate their basic characteristics,phase-transition property,cell targeted property in vitro,biosafety and bio-distribution of nanoparticles.MethodsIn this part,film-hydration + emulsification was used to prepare FLBS-PFH-NPs contrast agent.The morphological structure,the zeta and size potential,the element content,the concentration of Bi loading in FLBS-PFH-NPs were detected using several analytical methods.In vitro temperature increasing and high-intensity focused ultrasound excitation was applied respectively to induce the phase-transition of FLBS-PFH-NPs.Optical microscopy and ultrasound were used to observe the phase-change.The DiI labeled FLBS-PFH-NPs were incubated with human cervical cancer Hela cells and human hepatic L02 cells,then the targeted ability of these nanoparticles to cells was observed by confocal laser-scanning microscope(CLSM),comparing to the non targeted group(NLBS-PFH-NPs).Different concentrations of FLBS-PFH-NPs were prepared and incubated with L02 cells,then we applied the MTT assay to test the viability of the cells.Then max concentration of FLBS-PFH-NPs and PBS were used to incubated with L02 cells for 24 h.Next,the cell cycles and apoptosis were tested by applying flow cytometry.What is more,rat’s blood was gained before and at 7,14,30 days after injection of FLBS-PFH-NPs by vein for analysis of the heart,liver and kidney functions.To obtain the circulation time of FLBS-PFH-NPs,the New Zealand Rabbits’ blood sample was gathered at different interval time(2 min,5 min,10 min,30 min,1 h,2 h,4 h and 24 h)after injection of FLBS-PFH-NPs.The Bi concentration was measured using ICP after digestion using HClO4/HNO3 solution.Meanwhile,for an in vivo distribution experiment,the tumor-bearing nude mice was injected of FLBS-PFH-NPs via tail vein.The mice was put to execution at 3 hours after administration.The organs containing heart,liver,spleen,lung,kidney,and tumor were collected and the Bi concentrations were measured by using ICP after digestion by HC104/HNO3 solution.ResultsFLBS-PFH-NPs were brown emulsion.Imaging of the FLBS-PFH-NPs with LM and TEM showed perfect spherical morphologies with high dispersion.Bi2S3 nanoparticles were randomly distributed in the lipid shell,as determined by transmission electron microscopy(TEM).The FLBS-PFH-NPs emitted fairly strong red fluorescence during fluorescence microscope observations,which showed that FLBS-PFH-NPs were equipped with fluorescence imaging capability.The FLBS-PFH-NPs were uniform in size with a mean diameter of 458.5 nm.The average surface electric potential was measured to be-(15.5 ± 5.8)mV by dynamic light scattering(DLS).The peaks for Bi and S element were detected in the EDS Spectrum.The reflection peaks of FLBS-PFH-NPs in XRD patterns were well consistent to the Bi2S3 nanoparticles.Both of EDS and XRD demonstrated that the Bi2S3 nanoparticles were successfully inserted into the lipid droplets.The corresponding concentrations of the Bi encapsulated in the FLBS-PFH-NPs after the addition of different volumes of Bi2S3(200,400,600,800,and 1000 μL,as measured by ICP-OES,were 1.0,2.0,3.0,4.0,and 5.0 mg/mL.when the temperature rises above 64 ℃,or high-intensity focused ultrasound excitation can promote FLBS-PFH-NPs undergo a phase transition and become microbubbles to enhance ultrasound imaging.Many punctate particles emitting red fluorescence were observed around the cytoplasm of Hela cells,which emitted green fluorescence;fewer of these red particles were present in the non-targeted group.Simultaneously,we measured the targeting connection rate using flow cytometry.The connection rate gradually increased with the increases in the concentration of nanoparticles.Few red particles were observed around the L02 cells both in targeted and non-targeted groups.The L02 cells proliferation activity was not affected after incubation by MTT results.The flow cytometry results showed that the percentage of apoptotic cells after incubation with FLBS-PFH-NPs was approximately 7.88%,which was similar to the PBS control group(7.48%).The cell cycles were normal in FLBS-PFH-NPs and PBS groups with use of flow cytometry.No significant changes in the heart,liver and kidney functions were detected at different time after FLBS-PFH-NPs administration.The distribution of Bi in nude mice further proved that the nanoparticles of tumor tissue in targeted group(FLBS-PFH-NPs)were obviously more than non-targeted group(NLBS-PFH-NPs).The half circulation time of FLBS-PFH-NPs was calculated to be 2.2 h.ConclusionFLBS-PFH-NPs have been successfully synthesized;these lipid nanoparticles have good spherical morphology,good dispersion,uniform size,stable properties and good biosafety.The contrast agents have good phase-transition and targeted abilities.It is laying the foundation for the future researches.PART II FOLATE-TARGETED PERFLUOROHEXANE NANOPARTICLES CARRYING BISMUTH SULFIDE FOR ENHANCED US AND CT IMAGINGObjectivesTo observe the effect of FLBS-PFH-NPs for enhanced US and CT imaging in vitro,to investigate the feasibility of the lipid nanoparticles as a dual-modality contrast agent;to evaluate the imaging effects of FLBS-PFH-NPs for enhanced US and CT imaging in rabbits’s liver;meanwhile,to observe the imaging effects of FLBS-PFH-NPs for improving targeted imaging in nude mice with cervical cancer;to investigate the enhanced capacity and the principle of FLBS-PFH-NPs in dual-modality imaging.MethodsThis studying part was divided into three sections.Saline solution,L-PFH-NPs,NLBS-PFH-NPs and FLBS-PFH-NPs at different Bi concentrations were applied to perform US and CT imaging using imaging model in vitro.Twelve rabbits were randomly divided into four equal groups with the injection of saline,L-PFH-NPs,NLBS-PFH-NPs,and FLBS-PFH-NPs 1 ml/kg via ear vein respectively.The liver of the rabbits were subjected to HIFU ablation(acoustic power:70 W,exposure duration:2 s)after injection.US images were acquired before and immediately after phase transition.DFY Image Quantitative Analysis software was used to analyze changes in echo intensities and gray values.Similarly,different contrast agents were intravenously injected.Liver CT imaging was performed before injection and at 30 min,1 h and 2 h after injection.The CT values of liver tissues in the ROI were measured using image analysis software.Nude mice(n = 12)bearing cervical cancer were divided into four equal groups and intravenously injection of saline solution,L-PFH-NPs,NLBS-PFH-NPs and FLBS-PFH-NPs,respectively.Then,US and CT images of tumors were acquired before and at 3 h after administration.The echo intensity and CT density were detected as well.ResultsIn vitro,FLBS-PFH-NPs,NLBS-PFH-NPs,and L-PFH-NPs displayed punctiform,tiny,and homogeneous hyperechogenicities,whereas the saline group was anechoic.The echo intensities were gradually enhanced with increases in the Bi2S3 concentrations.The CT densities of both the NLBS-PFH-NPs and FLBS-PFH-NPs groups were higher than those of the saline and L-PFH-NP groups.The CT value of the FLBS-PFH-NPs clearly increased linearly with increases in Bi2S3 concentration.The echo intensities and gray value of the liver tissues in US images were obviously improved in the L-PFH-NPs,NLBS-PFH-NPs,and FLBS-PFH-NPs groups compared to the saline group and that before injection(P<0.05).After administration for 30 min,1 h or 2 h,both the NLBS-PFH-NPs and FLBS-PFH-NPs generated substantial contrast enhancement in the liver during CT imaging compared to the enhancement observed for the same nanoparticles pre-injection,and those injected with saline or L-PFH-NPs.The corresponding CT values of the NLBS-PFH-NPs and FLBS-PFH-NPs groups were higher than the values obtained for the same nanoparticles pre-injection and for the groups injected with saline and L-PFH-NPs.In vivo targeting imaging,the ultrasound echo intensity in tumor tissue of folate-targeted group was obviously higher than that in the non-targeted groups.In vivo tumor-targeted CT imaging,the tumors from the targeted group could be readily recognized in CT images,in contrast to the non-enhanced tumors from the non-targeted group.The CT value of tumor in the targeted group was obviously higher than that in the non-targeted groups.ConclusionThe FLBS-PFH-NPs are successfully acted as dual-modality biological imaging contrast agents for US and CT imaging in vitro and vivo.The FLBS-PFH-NPs are one kind of safe and efficient multi-functional contrast agents,which have a potential application value in the detection of carcinoma.PART III FOLATE-TARGETED PERFLUOROHEXANE NANOPARTICLES CARRYING BISMUTH SULFIDE FOR HIGH INTENSITY FOCUSED ULTRASOUND CANCER ABLATIONObjectivesTo evaluate the effects of FLBS-PFH-NPs combined with HIFU in bovine liver tissue ablation,and to study the optimal irradiation conditions.Next,to investigate the influence of FLBS-PFH-NPs combined with HIFU ablation in nude mice bearing cervical cancer,and to observe the application value of FLBS-PFH-NPs acted as HIFU synergistic agents.MethodsThe study of this part was divided into two sections.In section one HIFU ablation of bovine in vitro:saline,L-PFH-NPs,NLBS-PFH-NPs and FLBS-PFH-NPs(1 mL)were vertically injected into the fresh bovine liver tissue,respectively.The HIFU ablation parameters were as follows:exposure duration of 2 s;and acoustic power levels of 90,120,150,and 180 W.After HIFU ablation,the gray-scale value change,echo intensity change and the volume of coagulated tissue irradiated by HIFU were calculated to assess the HIFU synergistic ablation effects.In section two HIFU ablation nude mice bearing cervical cancer:Twelve nude mice were randomly divided into four equal groups,①HIFU+NACL,②HIFU+L-PFH-NPs,③HIFU+NLBS-PFH-NPs,④HIFU+FLBS-PFH-NPs,subsequently,different contrast agents were injected into the nude mice via the tail vein.Tumor tissues were exposed to HIFU ablation with the following parameters:acoustic power,120 W;exposure duration,2 s.After HIFU ablation,the gray-scale value change,the anatomical necrosis and the volume of coagulative necrosis were calculated,HE staining,TEM and immunohistochemical examination of PCNA and TUNEL were performed to detect the structure changes of the targeted tissue caused by HIFU ablation.ResultsThe gray-scale value change,echo intensity change and the mean coagulative necrosis volumes of liver tissues caused by HIFU in the groups that received the FLBS-PFH-NPs and NLBS-PFH-NPs treatments were higher than the volumes in the groups that received saline and L-PFH-NPs(P<0.05).Moreover,the ablated volume of bovine liver after L-PFH-NPs injection was larger than that observed for the group injected with saline(P<0.05).In contrast,no obvious differences were observed in tissues treated with the FLBS-PFH-NPs and NLBS-PFH-NPs under the same conditions(P>0.05).Concurrently,an increasing trend was found with regards to the coagulative necrosis volumes,gray values,and acoustic intensities when the HIFU acoustic power was gradually increased.In nude mice bearing cervical cancer,obvious white necrosis with clear boundaries to surrounding TTC dyed red non-necrosis tissue was seen after HIFU ablation in targeted group(FLBS-PFH-NPs).No obvious necrosis was seen in the non-targeted groups(saline,L-PFH-NPs and NLBS-PFH-NPs),there were significant differences in gray scale change value and the coagulative necrosis volume between the targeted and non-targeted groups(P<0.05).The coagulated necrotic tissue exhibited typical damage consisting of lysed cell membranes and nuclear fragments,which were accompanied by a considerable amount of evenly stained red substances in targeted group by HE staining.Ultrastructural examinations reconfirmed the irreversible cellular damage in the targeted regions,the cell and nuclear membranes ruptured,and the nuclei and organelles disappeared.Additionally,the positive index(PI)of PCNA in the folate-targeted group(FLBS-PFH-NPs)was lower than that of the other three non-targeted groups(saline,L-PFH-NPs and NLBS-PFH-NPs,P<0.05).We concluded that the tumor cell apoptotic index(AI)was higher in the folate-targeted group than in the other three non-targeted groups(P<0.05).ConclusionThe performance of the folate targeted FLBS-PFH-NPs was good,which could act as HIFU synergistic agents.The FLBS-PFH-NPs can effectively enhanced the biomedical effect of HIFU ablation both on bovine liver tissue and on nude mice bearing cervical cancer.The enhanced effect was more obvious than the non-targeted group.This would lay a foundation for future targeted diagnosis and treatment of tumor.