Thrombin-resposive Phase Transiton Liposomes For The Detection And Treatment Of Venous Thrombosis

PART1 PREPARATION AND CHARCTERIZATION OF THROMBIN-RESPOSIVE PHASE TRANSITON DUAL-TARGETED MULTIMODAL LIPOSOMESObjective To prepare a safe dual-targeted phase transition multi-functional nano-agents of thrombi and verify its basic physical and chemical properties and stability.Method PFP-ACPP-FTP-DIR liposomes were prepared using thin-film dispersion and ultrasound emulsification.The particle size and zeta potential of the liposomes were measured by Malvern dynamic light scattering analyzer.The structure and properties were analyzed by Transmission electron microscopy.The connection amount of ACPP-FTP peptide was measured through flow cytometry and verified under the confocal laser scanning microscope.The stability of the liposomes was observed in the PBS solution.HPLC was used to validate the thrombin-directed cleavage of ACPP peptide.Results The mean particle size for the PFP-ACPP-FTP liposomes was 383.3 nm,PDI was 0.100,the zeta potential was 8.39 m V.Confocal imaging demonstrated that the DIR and FITC fluorescence was overlaid,further confirming that the ACPP and FTP peptides were loaded on the liposome surface.Meanwhile,the labeling efficiency of the PFP-ACPP-FTP liposomes was 77.48%,confirmed using flow cytometry.The results of the HPLC confirmed the successful cleavage of ACPP peptide into CPP+ and polyanionic domains in the presence of thrombin.No significant change in size was observed in the stability test of the liposomes.Conclusions PFP-ACPP-FTP-DIR liposomes were successfully prepared.The structure of liposomes was spherical in morphology,no significant change was observed in particle size,the results of flow cytometry and two peptides were successfully loaded onto the liposome surface.PART? THROMBIN-RESPOSIVE PHASE TRANSITON DUAL-TARGETED MULTIMODAL LIPOSOMES FOR IMAGING AND THROMBOLYSIS EVALUATION IN VITRONObjective To verify the capability of the dual imaging of the thrombin-responsive phase transition dual-targeted multimodal liposomes in vitro and to evaluate the target effect and the thrombolytic effect in vitro.Method In vitro experiments,to evaluate the capacity of PFP-ACPP-FTP-DIR liposomes as a contrast agent for photoacoustic imaging,we observed the liposome solutions at different concentration of DIR.To evaluate the process of nano-agents vaporization,ultrasound images of the microemulsion in agar gel phantom were obtained by ultrasound using B-mode and contrast-enhanced ultrasound mode(CEUS)before and after LIFU irradiation,the morphological changes were observed under light microscope as well.Meanwhile,an in vitro thrombus model was established,and incubated with the phase-transitional nanoparticle were irradiated with LIFU to achieve thrombolysis.The treated thrombosis model was made into frozen slices and the target aggregation effect of the liposomes were observed under a confocal microscope.Results The dual-targeted phase transition liposomes worked as a suitable photoacoustic contrast agent,750 nm was the excellent excitation wavelength,where maximum photoacoustic absorbance occured.During the process of LIFU irradiation at 7 W cm-2,the particle size of the PFP liposomes expanded from nanometers to micron,at the same time,after 6 minutes of LIFU insonification,B-mode and CEUS contrast increased by25 times and 19 times respectively.In vitro thrombosis experiment,the thrombolysis rate of the dual-targeted group was 47.8±9.1%,which was significantly higher than the single-targeted group or the control group.Conclusions This thrombin-responsive phase transition dual-targeted liposomes showed great target thrombotic ability in vitro experiments,as well as the capability of PA and US imaging under LIFU irradiation.The process endows them the capability of phase transitional thrombolysis,which laid a good foundation for subsequent in vivo experiments.PART? THROMBIN-RESPOSIVE PHASE TRANSITON DUAL-TARGETED MULTIMODAL LIPOSOMES FOR IMAGING AND THROMBOLYSIS EVALUATION IN VIVO AND ITS SAFTYObjective To verify the capability of the dual imaging of the thrombin-responsive phase transition dual-targeted multimodal liposomes in vivo and to evaluate the target effect and the thrombolytic effect in vivo.Method A rat model of inferior vena cava was established to evaluate the targeting ability in vivo.The liposomes were injected into the rat tail vein,and then LIFU performed on the thrombus area at a power of 7 W/cm2 for 15 min.Ultrasound and photoacoustic imaging was performed before liposome injection,and at 2,5,10 and 15 minutes of LIFU insonification.After LIFU treatment for 5 min,the animals were sacrificed and the thrombi sectioned and stained for H&E,masson and reticular fibers.The toxicity of the dual-targeting liposome was investigated after liposome injection and LIFU treatment.The animals were sacrificed,and the parenchymal organs were sectioned and with H&E stained.Blood samples were also acquired for blood biochemical examination.Results The in vivo experiment demonstrated the superior targeting efficiency and full-thickness penetration of the PFP-ACPP-FTP-DIR liposome formulation.And the ACPP-FTP-PFP liposome can be used as ultrasound imaging contrast agents and PA imaging agents for real-time monitoring of the thrombolytic process caused by the LIFU stimulated vaporization.In the section of vascular thrombosis after 5 minutes of treatment,the better thrombosis effect of the dual-targeted group was visible.Pathological sections and blood biochemical tests were used to evaluate the safety of the liposomes.Conclusions We have developed a non-toxic,thrombin-responsive,full-thickness infiltration and pharmaceutical-free nanoplatform for the purpose of combating DVT under imaging guidance.Phase change molecular probe for the treatment of thrombosis and the dual-targeting peptides ensures deep penetration into the thrombus.Once activated with low intensity focused ultrasound,the liposomes vaporize into microbubbles,damaging the thrombus and reducing its size,without damaging the surrounding vasculature.Treatment can be visualized with ultrasound and photoacoustic imaging,enabling real time thrombolytic monitoring and confirming that the agents hit their target.This treatment is non-invasive and highly localized,minimizing side effects compared to long term systemic drug therapies that are commonly used.