Mechanism Of Cadmium-induced Blood-brain Barrier Damage In Zebrafish And Construction Of Thrombolytic Biomimetic Nano-drug Carriers

Petroleum is an important strategic energy source for the sustainable development of the national economy,which not only promotes national economic development,but also stabilizes national economic growth.However,with the development of the petroleum industry,the problem of petroleum pollution has become increasingly prominent.In petroleum pollutants,heavy metal ions will cause immeasurable damage to human beings and nature after entering the environment.Among them,the heavy metal cadmium（Cd）mainly exists in the form of free and easily bioavailable Cd²⁺,which is transmitted through the food chain and enriched in various tissues and organs of the human body,seriously endangering human health.Studies have shown that Cd²⁺accumulated in the blood increases the risk of neurological diseases such as Parkinson’s and Alzheimer’s disease.Since the blood-brain barrier（BBB）is a natural barrier separating blood and brain nerve tissue,the mechanism of how Cd²⁺breaks through the BBB and produces neurotoxicity remains unclear.In addition,Cd²⁺in the blood increases the risk of cardiovascular disease.Thrombosis is the main cause of cardiovascular disease.How to safely and efficiently reduce blood clots is a key link in relieving the process of cardiovascular disease.In view of the above two problems,this paper used the zebrafish blood brain barrier model to study the effects of different degrees of Cd²⁺exposure on BBB function and its toxicological mechanism;and cooperate with the design strategy of biomimetic and controllable drug release to construct a biomimetic nano drug carrier with targeted thrombolysis function.The main research contents of this paper are as follows:（1）Toxicity evaluation of heavy metal Cd²⁺on zebrafish BBB function.Transgenic zebrafish models were utilized,combined with the in vivo imaging technology of confocal laser scanning microscope,different concentrations of Cd²⁺solutions were used to conduct exposure zebrafish embryo experiments,zebrafish DAPI fluorescence staining and human brain microvascular endothelial cells（HBMECs）transepithelial electrical resistance experiments.The experimental results showed that:Cd²⁺had concentration-dependent embryotoxicity and developmental toxicity in zebrafish.Cd²⁺exposure at 200-500μM caused cerebral hemorrhage and retinal hemorrhage in zebrafish.Cd²⁺exposure increased BBB permeability,leaded to BBB dysfunction.（2）Discussion on the mechanism of heavy metal Cd²⁺-induced BBB dysfunction.Combined with molecular detection methods such as immunofluorescence staining,co-immunoprecipitation and western blotting,the effects and toxicological mechanism of Cd²⁺exposure on BBB related tight junction proteins,adhesion connexins,cytoskeleton,reactive oxygen species and protein tyrosine phosphatase were studied through transgenic zebrafish in vivo and HBMECs in vitro.The results showed that Cd²⁺exposure interfered with the formation of tight junction between zebrafish BBB vascular endothelial cells and the morphology of cytoskeleton.And Cd²⁺could destroy the adhesion junction and tight junction between cells by changed the distribution of connexin between HBMECs.Cd²⁺also induced increased levels of reactive oxygen species in zebrafish and HBMECs to mediate the inhibition of protein tyrosine phosphatase activity.Furthermore,the addition of reactive oxygen species inhibitor diphenyleneiodonium chloride could eliminate Cd²⁺-induced protein tyrosine phosphatase activity inhibition,connexin degradation and effectively alleviate Cd²⁺-induced cerebral hemorrhage and BBB injury.It was further confirmed that Cd²⁺-induced intracerebral hemorrhage was dependent on reactive oxygen species-mediated disruption of adhesive junctions and tight junctions,leading to BBB dysfunction.（3）Construction and characterization of thrombolytic biomimetic nano-drug carriers.Since the toxic effect of heavy metal Cd²⁺increases the risk of people suffering from cardiovascular disease,in order to improve the ability to treat the induced cardiovascular disease,a biomimetic nano drug carrier for the treatment of thrombosis disease was constructed.Poly（lactic-co-glycolic acid）was selected as nanocarrier material,loaded with urokinase with thrombolytic effect and perfluoro-n-pentane with phase change effect,and urokinase-loaded phase-change nanomedicines（PPUNPs）were prepared by a double-emulsion solvent evaporation method.The red blood cell membrane was extracted by hypotonic hemolysis,and RGD was modified to provide targeting effect on the red blood cell membrane.Furthermore,the RGD-targeted modified red blood cell membrane was coated on the surface of PPUNPs by the cell membrane coating technology,and the thrombolytic biomimetic nano drug carrier（RBC-RGD@PPUNPs）was prepared.The prepared PPUNPs and RBC-RGD@PPUNPs were characterized by morphology,particle size,potential,encapsulation efficiency,drug loading rate,drug release in vitro in response to ultrasound,and protein.The experimental results showed that PPUNPs were spherical,had good dispersibility.PPUNPs encapsulation efficiency and drug loading rate were 87.1%and 6.14%.In response to ultrasound,PPUNPs could rapidly release urokinase to realize the drug-loading system of urokinase drugs.RBC-RGD@PPUNPs had a"core-shell"structure with uniform particle size and good dispersion,with a particle size of 275.4±12.13 nm,which was negatively charged;and could well retain the composition of the original red blood cell membrane protein.（4）In vitro functional evaluation of thrombolytic biomimetic nano-drug carriers.In order to test the safety,targeting,and thrombolytic effects of the constructed RBC-RGD@PPUNPs,in vitro hemolysis experiments,endothelial cell and smooth muscle cytotoxicity experiments,macrophage phagocytosis and endothelial cell targeting experiments,and in vitro thrombolysis experiments were carried out.The experimental results showed that:RBC-RGD@PPUNPs had good blood compatibility;no obvious toxicity to endothelial cells and smooth muscle cells.Due to the RGD-modified red blood cells membrane coated,RBC-RGD@Di DNPs could effectively inhibit the phagocytosis of macrophages and had the potential to actively target thrombus.In addition,RBC-RGD@PPUNPs showed better thrombolytic function in vitro.Based on the above research,this paper uses a multi-strain transgenic zebrafish model,combined with confocal laser scanning microscope in vivo imaging technology,to demonstrate the toxicological effects of Cd²⁺-induced cerebral hemorrhage and BBB injury in vivo and in vitro.Using molecular detection methods such as immunoblotting and co-immunoprecipitation,the molecular mechanism of Cd²⁺-induced BBB dysfunction was elucidated.In addition,for the vascular diseases caused by Cd²⁺,a thrombolytic biomimetic nano drug carrier with biomimetic nano drug properties,good blood compatibility and cytocompatibility,inhibition of macrophage phagocytosis,active targeting potential and thrombolytic effect was developed and prepared.These studied provide new etiological evidence for Cd²⁺-induced neurological diseases;provide a new animal model for the toxicity study of environmental pollutants on BBB;and provide more efficient and safe treatment strategies for cerebrovascular diseases caused by thrombosis.