Ameliorative Effect Of Nanoparticles Encapsulated With HIF-1 Dimerization Inhibitor Acriflavine On Hypoxia-Induced Pulmonary Hypertension In Rats And The Underlying Mechanisms

BackgroundPulmonary hypertension（PH）is a serious pulmonary vascular disease.It is characterized by hyperproliferation of vascular cells,increased deposition of extracellular matrix,and accumulation of inflammatory cells within the pulmonary vessel wall,which together lead to increased pulmonary vascular resistance and increased right ventricular afterload.This eventually leads to right heart failure and death.At present,the conventional treatment methods for PH include oxygen inhalation,anticoagulation,diuresis,cardiotonic,pulmonary artery dilatation etc.,but the long-term quality of life of patients cannot be effectively improved.Therefore,it is of great significance to explore the pathological mechanism of PH and find more effective therapeutic drugs.Hypoxia-inducible factor-1（HIF-1）and its signaling pathway play an important role in hypoxia-induced PH.HIF-1 is a specific transcription factor that is activated under hypoxia.HIF-1 is involved in many important physiological processes,such as cardiovascular formation,cartilage development,neuroembryogenesis,and tumor development,and is closely related to various human pathological processes.Given the importance of HIF-1,the development of HIF-1 inhibitors has received increasing attention.Acriflavine（ACF）prevents the dimerization of HIF-1αand HIF-1βby binding to the PAS-B domain,thereby inhibiting the DNA-binding and transcriptional activity of HIF-1.It has a better effect on tumor growth and angiogenesis.Due to the short half-life of ACF in vivo and the metabolic elimination is fast,this study used hollow mesoporous silica nanoparticles（HMONs）to encapsulate acriflavine to increase its circulation time in vivo,and to explore its therapeutic effect on hypoxic PH.Methods1.Chemical synthesis experiment:（1）Synthesis of hollow mesoporous silica nanoparticles（HMONs）:CTAC aqueous solution（2 g,10 wt%）and TEA solution（0.08 g,10 wt%）were mixed.After 10 min of magnetic stirring at 95℃,1 m L of TEOS was added dropwise;1 h later,a mixture of 1 m L of TEOS and 0.6 m L of BTDS was added,reacted for 4 h,centrifuged and washed with ethanol for several times.The solution was refluxed with hydrochloric acid/ethanol（50 m L,10%v/v）for 12 h at78℃,repeated 3-5 times,and the product was washed 3 times with ultrapure water and stored in 20 m L of ultrapure water.Finally,5 m L of sample was placed in 100 m L ultrapure water diluted in water,2 m L of ammonia water was added.The etching process was continued at 95℃ for 3 h to obtain the final HMONs.（2）Drug loading:After HMONs-PEG was obtained by modifying m PEG-Saline on the surface of HMONs,100mg of ACF and 20 mg of HMONs-PEG were added to 10 m L of ultrapure water and stirred overnight to obtain the final product ACF-HMONs-PEG.2.Characterization of nanoparticles:（1）Transmission electron microscopy（TEM）to observe the morphology of nanoparticles;（2）X-ray photoelectron spectroscopy（XPS）for component identification;（3）Identification of ACF loading and determination of drug loading by ultraviolet spectrophotometry（UV-Vis）;（4）Dialysis method to measure the release curve of ACF-HMONs-PEG in vitro.3.Animal experiments:Hypoxia-induced PH rat model was constructed,and SD male rats were randomly divided into normoxia group,hypoxia group,hypoxia+free ACF group,hypoxia+empty HMONs-PEG group,hypoxia+ACF-HMONs-ACF group.Hypoxia（10%O₂）was used to induce PH model for 4 weeks.Body weight,hemodynamic indexes,right heart hypertrophy index and tibial length were measured,and ultrasound were used to detect pulmonary artery and cardiac function.HE staining was used to observe pulmonary vascular remodeling.The HIF-1αand HIF-1βdimerization in pulmonary artery was detected by co-immunoprecipitation.The expression of PCNA in the pulmonary artery was detected by Western blot.4.Toxicological experiment:SD male rats were randomly divided into normoxia group and normoxia+ACF-HMONs-PEG group.After 14 days of the drug administration,the heart,liver,spleen,lung and kidney were taken for HE staining to observe the drug distribution.Blood plasma was collected to test liver function（alanine aminotransferase/aspartate aminotransferase,bilirubin）and renal function（serum creatinine,blood urea nitrogen and blood uric acid）;whole blood was collected to measure the number of cells,hemoglobin and platelets in the blood.Results1.HMONs had a hollow mesoporous structure.The size of HMONs was about 50 nm.2.Compared with the unloaded HMONs-PEG nanoparticles,the final product ACF-HMONs-PEG had a new N element spectrum.After the peak processing of the N element,it was found that the N element existed in the form of N-C,N-H,and N⁺-C bonds.It was proved that ACF was successfully loaded into the nanoparticles.3.The remaining content of ACF in the supernatant of ACF-HMONs-PEG was measured,and the loading rate of ACF was calculated to be about 33%.4.ACF-HMONs-PEG could biodegrade in PBS solution（p H=7.4）.The drug release rate in PBS solution containing 10 mM GSH（pH=7.4）was significantly faster than that in pure PBS solution,about 5 day to reach the maximum release.5.Hypoxia（10%O₂,28 d）induced the occurrence of PH in rats.The expression of PCNA protein in the pulmonary artery of the hypoxia group was significantly increased,and the expression of PCNA protein was significantly decreased after the administration of 30 mg/kg ACF-HMONs-PEG.There was no significant difference in PCNA protein expression between free ACF and HMONs-PEG groups compared with hypoxia group.ACF-HMONs-PEG at the dose of 30 mg/kg inhibited the dimerization of HIF-1αand HIF1βin pulmonary artery and effectively improved hypoxia-induced PH in rats.6.On the 14th day after administration of 30 mg/kg ACF-HMONs-PEG in the tail vein of rats,there were no significant differences in the morphology of major organs,liver and kidney indexes,and serum biochemical indexes in each group compared with the experimental group.It confirmed that 30 mg/kg ACF-HMONs-PEG had no obvious toxicity and was relatively safe after the administration.Conclusion1.ACF-HMONs-PEG had a hollow mesoporous structure and could release ACF slowly in vitro.2.ACF-HMONs-PEG at the dose of 30 mg/kg effectively improved hypoxia-induced PH in rats by inhibiting the dimerization of HIF-1αand HIF-1β.