Sodium Nitroprusside-doped Mesoporous Prussian Blue Nanoparticles Improved Pulmonary Hypertension In Rats By Eliminating Superoxide Anion And Slowly Releasing Nitric Oxide

[Background]Pulmonary hypertension（PH）is a progressive disease.The combined effects of endothelial dysfunction,increased contractility of pulmonary arterioles,proliferation and remodeling of endothelial cells and smooth muscle cells,and in situ thrombosis increase pulmonary vascular resistance（PVR）,then induce the generation of PH.The continuous rise of PVR leads to a decrease in cardiac output.It eventually results in right heart failure and then death.As a critical vascular relaxation factor,nitric oxide（NO）regulates smooth muscle relaxation,inhibits the proliferation of vascular smooth muscle cells,and reduces platelet aggregation and intravascular thrombosis.NO participates in the whole process of the occurrence and development of PH and improves PH.Superoxide anion(O₂^·-)production is increased in PH,which decouples endothelial nitric oxide synthase（e NOS）and reduces NO production.In addition,O₂^·-reacts with NO to form a peroxynitrite anion（ONOO^-）,which increases NO consumption.Prussian blue（PB）,also known as iron ferrocyanide（Fe₄[Fe（CN）₆]₃）,is a coordination compound that has been used in various fields,including magnetism,optics,electrochemistry and biomedicine.FDA approved PB as an antidote for thallium in 2010,and it has a high binding affinity for cyanide ions,indicating that PB has good biosafety.Mesoporous Prussian blue（m-PB）nanoparticles can eliminate O₂^·-,and NO donor sodium nitroprusside（SNP）and m-PB synthesis raw material potassium ferricyanide have similar chemical structure,so under acidic conditions,sodium nitroprusside-doped mesoporous Prussian blue nanoparticles（m-PB-SNP）can be obtained by inserting the added SNP molecules into the growing PB nano frame.The nitro group of m-PB-SNP is connected to Fe³⁺through a coordination bond,and there will be weak dissociation in the solution,thus slowly releasing NO.[Objective]To investigate whether m-PB-SNP can improve pulmonary hypertension in rats by eliminating O₂^·-and slowly releasing NO.[Method]1.Synthesis and detection of m-PB-SNP:SNP was reacted with potassium ferricyanide（K₃[Fe（CN）₆]）at a ratio of 9:1 at 80℃for 12 h,and m-PB-SNP was collected by centrifugation.The composition of m-PB-SNP was analyzed by Ultraviolet spectrophotometry（UV-Vis）,Fourier transform infrared spectroscopy（FT-IR）,X-ray photoelectron spectroscopy（XPS）and X-ray diffraction（XRD）.The particle size and morphology of m-PB-SNP were observed by transmission electron microscopy（TEM）to verify whether the synthesis of m-PB-SNP is successful.O₂^·-elimination ability of m-PB-SNP was detected by UV-Vis,and NO release of m-PB-SNP was detected by Griess assay kit in vitro,confirming the feasibility of the experimental idea.2.Effect of m-PB-SNP on systemic circulation blood pressure of normal rats:SD male rats were randomly divided into 7 groups（n=6）:the saline group;the SNP（9 mg/kg）group;the m-PB-SNP（10 mg/kg）group;the SNP（6.3 mg/kg）group;the m-PB-SNP（7 mg/kg）group;the SNP（4.5 mg/kg）group;the m-PB-SNP（5 mg/kg）group;non-invasive caudal arterial manometry was used to measure the blood pressure of normal rats at different time points,including before drug administration and 5 min,15 min,30 min,1 h,1.5 h,2 h,4 h,8 h,12 h,1 d,2 d,3 d,4 d,5 d and6 d after drug administration.3.Therapeutic effect of m-PB-SNP on rats with pulmonary hypertension:SD male rats were randomly divided into 5 groups:the control group（n=8）;the PH group（n=12）;the PH plus m-PB-SNP（10mg/kg）group（n=10）;the PH plus m-PB-SNP（7 mg/kg）group（n=10）;the PH plus m-PB-SNP（5 mg/kg）group（n=10）.The PH model was induced by a single intraperitoneal injection of monocrotaline（MCT）at60 mg/kg.The corresponding preparation was injected into the tail vein after 14 days of the adminstration of MCT,once every 6 days.The rats were weighed 28 days after the adminstration of MCT,the function of heart and lung was measured by small animal echocardiography under anaesthesia condition;right ventricular systolic pressure（RVSP）was detected by cannulation of the jugular vein and the right heart.After the rats were sacrificed,the right ventricle（RV）,left ventricle and interventricular septum（LV+S）were dissected and weighed,and the tibial length was dissected and measured.The pulmonary vascular remodelling was observed through lung Hematoxylin-eosin（HE）staining.The expression of PCNA,a pulmonary proliferative protein was detected by Western blot.The anti-O₂^·-detection kit detects the anti-O₂^·-activity of proteins in pulmonary arteries.4.m-PB-SNP toxicological experiment:SD male rats were randomly divided into 2 groups（n=6）:the control group;the normal plus m-PB-SNP（7 mg/kg）group.After 14 days of regular feeding,corresponding preparations were injected into the tail vein once every 6days.The rats were sacrificed after 14 days,and the heart,liver,spleen,lung,kidney and brain were collected for HE staining to observe the histological changes.Blood was collected for a routine blood test,and plasma was collected for liver and renal function tests.[Results]1.m-PB-SNP was successfully synthesized,with a particle size of about 200 nm and a cubic shape.Both m-PB and m-PB-SNP concentration-dependently eliminated O₂^·-at p H=7.4 and p H=6.5.m-PB-SNP released NO slowly and continuously in 37℃water bath.2.The results of blood pressure monitoring of rats by non-invasive caudal arterial manometry showed that the systolic blood pressure and diastolic blood pressure of rats after 5mg/kg and 7mg/kg m-PB-SNP administration were slightly decreased compared with those before drug administration,but there was no significant difference.However,rats’systolic blood pressure and diastolic blood pressure after 10 mg/kg m-PB-SNP administration were significantly decreased compared with those before drug administration.The blood pressure of rats returned to normal condition 6 days after the adminstration.The rats’systolic blood pressure and diastolic blood pressure decreased sharply 5 minutes after injection of 4.5 mg/kg,6.3 mg/kg and 9 mg/kg SNP into the tail vein,presenting severe hypotension symptoms and death occurred in 6.3 mg/kg and 9 mg/kg SNP groups.3.Compared with the control group,the survival rate,body weight,right ventricular remodelling,systolic function and overall cardiac function of rats in the PH group were markedly decreased,RVSP was significantly increased,the expression of PCNA was increased,and the anti-O₂^·-activity was decreased;m-PB-SNP improved the survival rate,increased body weight,inhibited right ventricular remodeling,ameliorated the overall cardiac function,reduced RVSP,and also down-regulated the expression of PCNA and enhanced the anti-O₂^·-activity of PH rats.The m-PB-SNP（7 mg/kg）group had the best improvement effect.4.Compared with the control group,the histomorphology of the heart,liver,spleen,lung,kidney and brain of the normal rats treated with7 mg/kg m-PB-SNP showed no significant changes,and the values of blood routine and liver and kidney tests were all within the normal range,which verified the relative safety of the drug at this dose.[Conclusion]m-PB-SNP improved MCT-induced pulmonary hypertension in rats by eliminating superoxide anion and slowly releasing nitric oxide.