Platelet Membrane Nanobubble Delivery System And Its Application In Ischemic Stroke Diagnosis And Therapy

In recent years,inspired by the structure and function of natural cells,the design strategies of biomimetic nanomaterials,especially drug delivery systems based on cell membranes,had been extensively studied due to their ability to actively target diseases and interact with complex in vivo microenvironment with high specificity and selectivity.Platelets(PLTs),as one of the important visible components of human blood,play an important role in early identification of vascular damage,maintenance of vascular integrity and thrombosis.They are also important potential targets for the early diagnosis and treatment of vascular injury and thrombosis.Therefore,various drug delivery systems based on platelet membrane can be used for the diagnosis and treatment of vascular injury,thrombosis,tumor and many other diseases.In this dissertation,platelet membranes were used to construct platelet membrane reassembled biomimetic nanobubbles and nitric oxide prodrug delivery system.Based on the advantages of natural platelet membrane,the in vitro and in vivo targeting ability to the damaged blood vessels has been systematically investigated.Furthermore,the capability of multimodality imaging enhancement and vascular recanalization intervention of the fabricated biomimetic delivery system in the early stage of ischemic stroke have been evaluated.The specific research content includes the following parts:(1)Fabrication of the platelet membrane reassembled nanobubble delivery systemFresh PLTs were collected and platelet membranes vesicles(PMVs)were extracted by a repeated freeze-thawing process.An ultrasonic cavitation method was established to construct nanobubbles of platelet membrane-coated sulfur hexafluoride(SF6)gas(PNBs).Morphological characterization showed that PNBs have an intact bubble structure with a hydrodynamic size distribution of 131.43 ±19.84 nm and good in vitro stability.Moreover,the species and quantity of platelet membrane proteins and phospholipids were characterized by SDS-PAGE electrophoresis,LC-MS/MS mass spectrometry and high-performance liquid chromatography(HPLC),respectively.The results showed that among a total of 2069 platelet proteins identified.The 1510 species were retained in PNBs,and the proportion of proteins localized on the platelet membrane was 74.42 %.The lipid composition was also consistent with platelets.All of the retained components have ensured that the PNBs have inherited the function of natural platelets.(2)Study on the applications of PNBs in the early diagnosis of ischemic strokeThe biological effects of PNBs were further evaluated at the cellular and animal levels.At the cellular level,THP-1 phagocytes were used to interact with PNBs.It is verified that PNBs have good biocompatibility and immune escape functions.At the animal level,the mouse cortical ischemic stroke model was established and the Di R-labeled PNBs were injected intravenously.The nearinfrared fluorescence imaging(NIRFI)analysis showed that PNBs were able to rapidly target the ischemic lesions of stroke,and the fluorescence signal intensity of lesions in the administration groups was 2.24 times higher than that of the blank control groups.Further,the multimodal optical imaging platform(MIP)and full-field laser perfusion imager(FLPI)characterization of brain microvascular networks and blood flow showed that PNBs could target and adhere to the damaged blood vessels in the stroke lesions,and can prevent further expansion of the ischemic area.Finally,the small animal ultrasound imaging system(USI)was used to evaluate the ultrasound imaging enhanced effect of PNBs.It was proved that PNBs can realize the targeted diagnosis of damaged blood vessels in the early stage of ischemic stroke with the optical imaging and ultrasonic contrast imaging.(3)Fabrication of the magnetic platelet membrane nitric oxide prodrug delivery systemTo achieve targeted delivery of therapeutic gas molecules and revascularization in the early stages of ischemic stroke,platelet membrane magnetic nanocarriers(PAMNs)for targeted delivery of nitric oxide(NO)donor and in situ generation of NO nanobubbles were constructed.The purified PMVs were resuspended in a mixed solution of NO donor L-arginine and ?-Fe2O3 magnetic nanoparticles followed by sonication and extrusion.Morphological characterization revealed that PAMNs have a hydrodynamic size distribution of 215.50 ± 8.05 nm with good morphology and in vitro stability.PAMNs also have good superparamagnetic and in vitro magnetic resonance T2 imaging enhance effects due to the encapsulation of magnetic nanoparticles.(4)Study on the application of PAMNs in the early of ischemic strokeThe biological effects of PAMNs were further evaluated at the cellular and animal levels.At the cellular level,the effects of PAMNs on cell viability were first determined to assess the biosafety of PAMNs.Subsequently,the in vitro production of NO after incubation of PAMNs with b End.3endothelial cells was observed by fluorescence microscopy and quantitative analysis was performed.Finally,platelet aggregation assay was performed to evaluate the effect of PAMNs on platelet aggregation.It was found that PAMNs can significantly reduce platelet aggregation rate induced by three clotting agents(thrombin,adenosine diphosphate(ADP),and arachidonic acid(AA)),which proved that PAMNs can inhibit the formation of thrombus.In the in vivo evaluation,after the occurrence of stroke,the NIRFI characterization revealed that under the guidance of an external magnetic field,Di R-labeled PAMNs can be rapidly targeted to the lesion site within 0.5 h due to the encapsulation of magnetic nanoparticles.PAMNs can realize in vivo T2 imaging enhancement.Finally,MIP and FLPI were used to characterize the vascular morphology and blood flow in the ischemic lesion It was found that with the targeted accumulation of PAMNs and the release of L-arginine,significant vasodilation and blood flow recovery was observed due to the in situ generation of NO.It is proved that PAMNs can achieve the integration of targeted diagnosis and treatment in the early stage of stroke.