The Basic Research Of Fibrin Targeted Phase-Transitional Mutimodel Nanoparticles For Thrombus Imaging And Thrombolysis

PART ? PREPARATION AND CHARACTERIZATION OF FIBRIN TARGETED PHASE-TRANSITIONAL MULTIMODAL NANOPARTICLESPurpose Preparation of a safe phase-transitional fibrin targeted nanoparticles and verification of its basic physical and chemical properties and stability.Methods This nanoparticles containing perfluorohexane?PFH?,Fe₃O₄ and CREKA polypeptides,were prepared using polylactic acid hydroxyacetic acid?PLGA?as the carrier by modified double emulsification and volatilization method.Malvern particle size tester to prove its size and stability;Atomic absorption spectra were used to measure its capacity of iron carrying.Transmission electron microscopy?TEM?and elemental mapping were used to analyze its structure and properties.Fourier infrared spectrometer?FTIR?was used to prove the formation of amide bond;The amount of CREKA was analyzed by flow cytometry semiquantitatively,and the successful connection between CREKA and PLGA was confirmed by enzyme hydrolysis experiment and fluorescence microscope.Results Fe₃O₄-PLGA-PFH-CREKA nanoparticles were successfully prepared with a particle size of 311.3±4.3 nm and a polydispersion coefficient of 0.094±0.048.No significant change in particle size was observed during the 9 days.TEM and element mapping confirmed that Fe₃O₄ and PFH had been successfully encapsulated.FTIR results showed that the amide bond was generated,and the flow cytometry showed that the carrying rate of the nanoparticle of CREKA-FITC was 98.37%.Fluorescence coincidence of the two channels could be observed under the fluorescence microscope.Conclusions Fe₃O₄-PLGA-PFH-CREKA nanoparticles were successfully prepared by the double-emulsion volatilization method.The nanoparticles had good particle size,dispersibility and uniform size,and were successfully loaded with Fe₃O₄,PFH and CREKA,laying a foundation for subsequent phase-transitional thrombolysis therapy and multimodal imaging.PART ? FIBRIN TARGETED PHASE-TRANSITIONAL MULTIMODAL NANOPARTICLES FOR IMAGING AND THROMBOLYSIS EVALUATION IN VITROPurpose To verify the capability of multimodal imaging of the targeted fibrin nanoparticles in vitro and to evaluate the thrombolytic effect of the targeted phase-transitional nanoparticles in vitro.Methods The ability of the phase-transitional nanoparticles to target thrombi was verified by frozen section in vitro.The Fe₃O₄-PLGA-PFHCREKA nanoparticles were diluted to a concentration gradient of 0-2.6286 m M according to the concentration of Fe₃O₄,and MR images were collected.Additionally,nanoparticles with a concentration of Fe₃O₄ gradient of 0.1643-2.6286 m M were prepared for photoacoustic imaging?PA?by Vevo LAZR.Low-intensity focused ultrasound?LIFU?irradiated the phase-transitional nanoparticles outside the gel mold,observed the process of acoustic phase change in vitro,measured the temperature,and observed the characteristics of the phase change of nanoparticles.Meanwhile,an in vitro thrombus model was established,and the phase-transitional nanoparticle were irradiated with LIFU at different acoustic power densities to achieve thrombolysis.Results More Fe₃O₄-PLGA/Di I-PFH-CREKA NPs were significantly adhered to thrombus than Fe₃O₄-PLGA/Di I-PFH NPs.Magnetic resonance imaging?MR?in vitro indicated that the relaxation rate of the nanoparticles was 50.98 m M-1s-1.Photoacoustic imaging?PA?in vitro indicated that 680 nm was the optimal excitation wavelength for the nanoparticles.After 30 min LIFU irradiation,B-mode and CEUS contrast increased by 12 times and 6.6 times respectively,and the particle size was expanded from nanometers to microns,causing 8°C temperature rise.Under low acoustic power density irradiation,the thrombolysis rate of the phase-transitional thrombolysis group was 63.29±1.92 %,and the final thrombus weight was 0.112±0.01 g,which was better than that of the other three groups.Conclusions Such targeted phase-transitional nanoparticles have outstanding targeting ability against thrombus in vitro,as well as the ability of MR and PA imaging,and can change from liquid to gaseous state under LIFU irradiation.This process also endows them the ability of phasetransitional thrombolysis,and its thrombolytic effect is preliminarily verified in vitro.PART ? FIBRIN TARGETED PHASE-TRANSITIONAL MULTIMODAL NANOPARTICLES FOR IMAGING, THROMBOLYSIS EVALUATION IN VIVO AND ITS SAFETYPurpose To verify the capability of multimodal imaging of the phasevariant multimodal molecule,and the thrombolysis process was monitored in real time to evaluate the effect of phase-transitional thrombolysis,as well as the safety of the nanoparticle and LIFU.Methods A rat model of abdominal aortic thrombosis was established to evaluate targeting ability in vivo.Targeted phase-transitional nanoparticles,nontargeted phase-transitional nanoparticles,and loading double-distilled water targeted nanoparticles were injected into the rat tail vein,and the rats were irradiated with low acoustic power density of LIFU and injected with normal saline as the sham operation group.Then,US,PA and CEUS images were collected after modeling,immediately after injection,15 min and 60 min after injection,respectively.The efficacy of thrombolysis were evaluated by ELISA and immunohistochemistry before and after modeling,15 min and 60 min after injection.Near infrared imaging,pathological sections and blood biochemical tests were also used to evaluate the safety of the phase-transitional nanoparticles and LIFU irradiated with different acoustic power densities.Results Fe₃O₄-PLGA/Di I-PFH-CREKA NPs can specifically bind to fibrin in thrombus.The CEUS,US and PA imaging could be collected after the nanoparticles were injected in vivo for 15 min,and the thrombolysis effect could be preliminarily monitored.After 60 min,FDPs and D-dimer were increased?P < 0.05?,PAI-1 was more expressed in the sham group and only a small amount in the targeted phase-transitional thrombolysis group.LIFU irradiation with medium and low acoustic power density could keep security in vivo.Conclusions Fe₃O₄-PLGA/Di I-PFH-CREKA NPs can monitor the thrombolysis process in vivo through PA,US and CEUS,and its thrombolytic mechanism may mainly activate the fibrinolytic system rather than inhibit platelet aggregation.Irradiation with LIFU at medium and low acoustic power densities does not cause thermal or mechanical damage and can induce nanoparticle phase transition.