Hollow Mesoporous Prussian Blue Nanoparticles For Multi-modality Imaging And Hifu/chemotherapy Synergistic Therapy

PART 1 THE PREPARATION,CHARACTERIZATION AND DRUG RELEASE RESEARCH OF HOLLOW MESOPOROUS PRUSSIAN BLUE NANOPARTICLESObject To prepare perfluorohexane and doxorubicin-loaded hollow mesoporous Prussian Blue nanoparticles as stable multifunctional contrast agents(HMPBs-DOX/PFH),and detecting its physical properties,chemical properties,optical properties,stability,performance of drug release and phase transition.Methods HMPBs and HMPBs-DOX were prepared by selective etching method and drug adsorption method,and their morphological structure and chemical composition were investigated.Then,HMPBs-DOX/PFH was fabricated by vacuum perfusion method,and its particle size,potential,drug loading efficiency rate and optical absorption property were studied.The effect of phase-transition of multifunctional nanoparticles was detected by optical microscope.The drug release rate of HMPBs-DOX/PFH at different p H values(4.6,6.0,7.4)with or without HIFU exposure was investigated.Then,the drug release rate of HMPBs-DOX/PFH in the tissue with or without HIFU exposure was studied.Finally,HPLC was used to detect the thermal stability of DOX.The HMPBs-DOX/PFH was incubated into the MDA-MB-231 cells.Then,the cell phagocytosis was studied by CLSM.The cell activity of HMPBs was detected with different concentrations(400,200,100,50,25 mg/ml).Results HMPBs had uniform size and good monodispersion,showing a cube-like morphology with a hollow mesoporous structure,which were detected by transmission electron microscope(TEM).The corresponding EDS mapping exhibited that multifunctional nanoparticles were constituted of carbon,nitrogen and iron,which further indicated that HMPBs have been successfully synthesized by Prussian Blue(PB).The average diameter of HMPBs-DOX/PFH was(269 ± 37)nm,and zeta potential was(-33.7 ± 5.4)Mv measured by the Malvern Laser Particle Size Analyzer System.It was found that HMPBs-DOX/PFH had high drug loading efficiency detected by ultraviolet-visible-near infrared spectrophotometer(Uv-vis-NIR).The absorption peaks of HMPBs and HMPBs-DOX/PFH were both at 710 nm.When the temperature rose to 63 ?,HMPBs-DOX/PFH could generate microbubble to enhance ultrasound imaging.In vitro drug release showed that the drug release rate of HMPBs-DOX/PFH was significantly higher than that of other groups in the acid environment after HIFU exposure.HMPBs-DOX/PFH combined with HIFU showed a significant increased fluorescence signal in liver tissue.The high stability of DOX was detected by HPLC.Cell phagocytic experiments showed that HMPBs-DOX/PFH could be taken by MDA-MB-231 cells.MTT results showed HMPBs had good cell activity even at high concentration.Conclusion HMPBs-DOX/PFH have been successfully prepared to encapsulate DOX and PFH.These nanoparticles have uniform size,single composition,high drug-loading,good stability and high biocompatibility.The nanoparticles showed good phase transformation ability,optical absorption and special chemical structure to enhance ultrasound imaging,as well as the potential of photoacoustic imaging,magnetic resonance imaging.PART 2 HOLLOW MESOPOROUS PRUSSIAN BLUE NANOPARTICELS FOR ENHANCING US,PA AND MRI IMAGINGObject To investigate the efficacy of HMPBs-DOX/PFH for enhancing the ultrasound imaging,photoacoustic imaging and magnetic resonance imaging.Method First,saline,HMPBs-DOX,PFH and HMPBs-DOX/PFH were performed the effect of enhancing US and PA imaging by using agar in vitro model before and after HIFU exposure.The US and PA images data were analyzed by imaging system.Then,the magnetic resonance imaging of HMPBs-DOX/PFH with different concentrations were performed and analyzed.In vivo experiment was divided into two parts.(1)The rabbits bearing breast cancer were established and intratumorly injected by saline and HMPBs-DOX/PFH.The ultrasound imaging and photoacoustic imaging were obtained before and after HIFU exposure.The corresponding ultrasound and photoacoustic images data was analyzed by imaging system.(2)The rabbits bearing liver cancer were injected by saline and HMPBs-DOX/PFH,respectively.MR imaging was performed before and after the injection,the signal intensity(SI)of ROI was measured before and after enhancement.Results In vitro,HMPBs-DOX and HMPBs-DOX/PFH showed high signal before and after HIFU excitation in PA imaging,while saline and PFH had no difference before and after HIFU excitation.HMPBs-DOX/PFH could obviously enhanced ultrasound signal after HIFU exposure.Although the echo intensity of pure PFH slightly increased in B-mode after HIFU exposure,the contrast-mode has no obvious difference.Saline,HMPBs-DOX also had no obvious difference in ultrasound imaging before and after HIFU exposure.In the T2*-weighted MR images,HMPBs-DOX/PFH showed negative enhanced imaging.With the increasing concentration,the transverse relaxation time of MR imaging decreased significantly.In vivo PA imaging,rabbits bearing breast cancer showed that the signal intensity of tumor site increased significantly after injecting HMPBs-DOX/PFH.Then,the echo intensity of tumor site after injecting HMPBs-DOX/PFH and saline in B-mode and contrast-mode imaging had no obvious difference before HIFU exposure.While after HIFU exposure,HMPBs-DOX/PFH exhibited obviously higher echo intensity than that of saline group in tumor injection site.In vivo MR imaging,the signal intensity of ROI in rabbits bearing liver cancer decreased significantly compared with saline group after local injection of HMPBs-DOX/PFH.Conclusion HMPBs-DOX/PFH are safe and stable multifunctional contrast agents to effectively enhance US,PA and MR imaging in vitro and in vivo,which has application capacity in the diagnosis and treatment of tumors.PART 3 HOLLOW MESOPOROUS PRUSSIAN BLUE NANOPARTICLES FOR ENHANCING HIFU/ CHEMOTHERAPY SYNERGISTIC THERAPYObject To study the cell viability of HMPBs-DOX/PFH combined with HIFU.To verify the efficacy of HMPBs-DOX/PFH for enhancing HIFU ablation in ex-vivo experiment,and to investigate the optimized irradiation intensity.To investigate the efficacy of HIFU combined with chemotherapy in the treatment of rabbits bearing breast cancer and liver cancer.Methods In vitro treatment experiment was divided into two parts.The treatment of HMPBs-DOX/PFH combined with HIFU on MDA-MB-231 cells in vitro was studied.The cells were divided two groups,and one group was added PBS,HMPBs,DOX,HMPBs-PFH,HMPBs-DOX and HMPBs-DOX/PFH,respectively.The other group was exposed by HIFU after incubating with aforementioned agents.After treatment in each group,the cell apoptosis was tested by flow cytometry and CLSM.Ex-vivo HIFU treatment: the fresh degassed bovine liver was injected into 200 ?l different agents(saline,HMPBs,DOX,HMPBs-DOX,HMPBs-PFH,HMPBs-DOX/PFH)and exposed by different HIFU intensity(120 W,150 W,180 W)for 5 s.Then,B-mode gray values of ROI and the volume of coagulated necrosis before and after HIFU exposure were measured.In vivo treatment is divided into two parts: Firstly,rabbits bearing breast tumor were randomly divided into eight groups,and percutaneously injected into saline,DOX,HMPBs-PFH,HMPBs-DOX/PFH at 3,6,9 and 12 dots.Then,5-7 groups were also injected with saline,HMPBs-PFH and HMPBs-DOX/PFH,and exposed by HIFU at the same power intensity.Eighth group was injected into saline and exposed by HIFU at 400 W for 5 s.After treatment of each group,the tumor relative volume and immunohistochemical detection were performed.Secondly,rabbits bearing liver tumor were randomly divided into five groups,and injected into saline,HMPBs,DOX,HMPBs-DOX,HMPBs-DOX/PFH.Then,these rabbits were exposed by HIFU at the same power intensity.After treatment of each group,the tumor relative volume and immunohistochemical detection were performed.Results In vitro treatment experiment,compared with other groups,HMPBs-DOX/PFH+HIFU group presented the strongest apoptosis of MDA-MB-231 cells.The change of gray-scale value and the volume of coagulated bovine liver tissue exposed by HIFU in HMPBs-PFH group and HMPBs-DOX/PFH group were significantly higher than other groups.After 15 days of treatment,the tumor growth rate in HMPBs-DOX/PFH+HIFU group was significantly slower than other groups.After immunohistochemical staining,the apoptosis positive index of TUNEL in HMPBs-DOX/PFH+HIFU group was significantly higher than that of other groups,while the tumor proliferation positive index of PCNA was significantly lower than that of other control groups.After seven days of treatment,the tumor growth rate in HMPBs-DOX/PFH+HIFU group was significantly slower than other groups.After immunohistochemical staining,the tumor proliferation positive index of PCNA was significantly lower than that of other control groups.Conclusion HMPBs-DOX/PFH can effectively enhance the synergistic effect of HIFU and chemotherapy.At the same time,combined with the characteristics of multimodality imaging,it is further demonstrated that HMPBs-DOX/PFH has extensive research prospects in the field of diagnosis and treatment integration.