Ameliorative Effect Of Prussian Blue/ceria Co-modified Nanoparticles On Hypoxia-induced Pulmonary Hypertension In Rats And The Underlying Mechanisms

BackgroundPulmonary hypertension（PH）is a progressive disease caused by a variety of factors,including alveolar damage,pulmonary intravascular thromboembolism,and lumen stenosis caused by vascular wall thickening.Hypoxia lesions,as one of the common inducers of PH,play an important role in its occurrence and development.Pulmonary vascular remodeling caused by the imbalance of reactive oxygen species（ROS）in the body caused by hypoxia has been confirmed to be a decisive factor in the pathogenesis of hypoxia-induced PH.At present,there is no report on drugs that directly improve the hypoxia microenvironment of the body to treat PH.Although drugs targeting the hypoxia/ROS pathway to treat PH can improve the quality of life of patients to a certain extent,they are largely limited by the pharmacokinetics and route of administration.The morbidity and mortality of PH still face great challenges,and it is urgent to develop new PH-targeted agents that can improve the hypoxia microenvironment and remove excess ROS.The properties of nanomaterials（including size,shape,uniformity,modifiability,etc.）make them biocompatibility,high targeting and low toxicity,and have great application potential in the biomedical field.Prussian blue（PB）is a newly developed biomimetic enzyme in recent years,which can interact with H₂O₂to continuously generate O₂,but the accompanying production of a large amount of hydroxy radical（·OH）under acidic conditions limits its application in extra-tumor fields.Ceria（CeO₂）is the most used inorganic nanomedicine in current antioxidant therapy,which can rapidly and efficiently convert·OH to O₂.In addition,PB and CeO₂possess both superoxide dismutase（SOD）and catalase（CAT）mimetic activities.Therefore,Prussian blue/ceria co-modified nanomaterials are expected to synergistically improve hypoxia-induced PH by promoting O₂generation and ROS scavenging,providing new ideas for the treatment of PH.MethodsSynthesis and Characterization of Nanomaterials:Using melanin（PDA）as a carrier,Prussian blue（PB）and ceria（CeO₂）were co-modified to construct Prussian blue/ceria co-modified nanomaterials（PDA-PB-CeO₂）.Transmission electron microscopy（TEM）was used to detect the size and morphology of nanomaterials;X-ray Powder Diffraction（XRD）was used to detect composition of nanomaterials;X-ray Photoelectron Spectroscopy（XPS）was used to analyze valence State of PDA-PB-CeO₂;Inductively coupled plasma（ICP）was used to analyze elemental content of PDA-PB-CeO₂.Determination of Reactive Oxygen Species（ROS）Scavenging and O₂Generation ability of Nanomaterials in Vitro:Nitro blue tetrazole（NBT）method was used to detect SOD-like activity;3,3’,5,5’-Tetramethylbenzidine（TMB）method was used to detect·OH generate ability;Griess assay kit was used to detect NO scavenging ability;Pyrogallol red method was used to detect·ONOO^-scavenging ability;Dissolved oxygen meter was used to detect O₂generation ability.Biological distribution of nanomaterials in lung tissue:Male SD rats were randomly divided into normoxia+PDA-PB-CeO₂group（21%O₂）and hypoxia+PDA-PB-CeO₂group（10%O₂）.On the 14th day of hypoxia,PDA-PB-CeO₂was injected into the tail vein,and the Ce content in the lung tissue of the rats was detected on the 1 d,3 d,5 d and 7 d after the administration.Animal p H experiment:Male SD rats were randomly divided into normoxia group,hypoxia group,hypoxia+PDA group,hypoxia+PDA-PB group,and hypoxia+PDA-PB-CeO₂group.The rats were administered by tail vein injection on the 14 d and 21 d after modeling.After 28 days,the hemodynamics and right ventricular remodeling indexes was detected;Small animal echocardiography was used to detect heart and lung function;HE staining was conducted to observe pulmonary vascular remodeling;O₂·^-detection kit and·OH detection kit was used to detect the anti-superoxide anion activity and anti-hydroxyl free radical activity of nanomaterials in pulmonary artery.Toxicology Testing:Male SD rats were randomly divided into control group and PDA-PB-CeO₂group.After 14 days of administration by tail vein injection,the heart,liver,spleen,lung and kidney were taken for HE staining;The blood was collected for routine blood test;The plasma was collected for liver and kidney function test.Result1.PDA-PB-CeO₂was a spherical particle with a particle size of about 200 nm;XRD,XPS and ICP results confirmed the successful construction of the indicated nanomaterials from the aspects of composition,element valence and element content.2.PDA,PDA-PB,PDA-PB-CeO₂all had strong SOD-like activity;PB exhibited CAT-like activity in an alkaline environment,reacting with H₂O₂to generate O₂,and exhibited peroxidase（POD）-like activity in an acidic environment,reacting with H₂O₂to generate·OH;CeO₂effectively converted·OH generated by PB into O₂,turning harm into profit,increasing the amount of O₂generation,and reducing toxic and side effects;PDA-PB-CeO₂had a strong scavenging effect on·ONOO^-,but had no scavenging effect on NO.3.Compared with the normal control group,the Ce content in the hypoxia-induced rat lung tissue was significantly increased,indicating that PDA-PB-CeO₂was accumulated in the lung tissue of hypoxia-induced PH rats.4.Under hypoxia environment for 28 days,the PH rat model was successfully established;PDA-PB-CeO₂had a significant therapeutic effect on hypoxia-induced PH in rats,with the best effect at a dose of 7.5mg/kg;At the same dose,PDA-PB also improved the hypoxia-induced PH in rats to a certain extent,and the efficacy of PDA-PB-CeO₂was significantly better than that of PDA-PB.5.At the dose of 7.5 mg/kg,compared with the normal control group,the PDA-PB-CeO₂group showed no significant changes in the morphology of the heart,liver,spleen,lung and kidney,blood routine indexes,and liver and kidney function indexes,which confirmed the relative safety of PDA-PB-CeO₂.Conclusion1.PDA-PB-CeO₂nanomaterials was accumulated in the lungs of hypoxia-induced PH rats and had an improved lung targeting.2.PDA-PB-CeO₂can promoted O₂generation,ROS scavenging,and played a synergistic role in improving hypoxia-induced PH in rats.