Perfluorocarbon-based H₂O₂ Responsive Nanocarrier For Reversing Hypoxia In Ischemic Kidney

In recent years,the number of patients with end-stage renal disease has surged all around the world,making it a major risk factor for global public health.Since the first successful kidney transplant operation in the 1950s,kidney transplantation has gradually become the best treatment option for patients with end-stage renal disease.Currently,Donation after Cardiac Death（DCD）is the most widely used source of donor kidneys in clinical.However,in the process of DCD surgery,the donor organ will undergo a warm ischemic process due to cardiac death,resulting in insufficient oxygen supply and overexpression of reactive oxygen species,which will seriously damage kidney tissues and ultimately reduce the quality of donor organs.Also,it will affect the survival rate after transplantation and increase the risk of postoperative complications.Therefore,how to deliver oxygen to the kidney during the ischemic process of DCD surgery to reverse renal hypoxia and simultaneously remove ROS to protect the kidney has become a key scientific problem in the field of organ transplantation.Based on the above issues,this study designed a perfluorocarbon-based H₂O₂-responsive nanosystem,M-PHNs,which utilizes the reactive consumption of Mn O₂and H₂O₂（consumed excessively in ischemic tissue）.The oxygen generation ability of H₂O₂ with the superior oxygen-bearing and oxygen-releasing properties of perfluorocarbon make M-PHNs become a“reservoir”of oxygen.We hope that M-PHNs can be served as an integrated oxygen self-supply nanoplatform with“oxygen production-oxygen storage-oxygen release”in one system.This thesis will be studied from the following three parts.Part I:Preparation and characterization of M-PHNs nanoparticles.Oxygen supply nanoparticles（M-PHNs）containing Mn O₂ and PFC were prepared by in-situ bio-mineralization and ultrasonic emulsification method.The hydrated particle size and surface potential of M-PHNs was measured by a potential and particle size analyzer,and then its morphology was observed by transmission electron microscopy（TEM）.Next,the existence of the characteristic absorption peak of Mn O₂ was checked in the full-wavelength ultraviolet absorption spectrum of M-PHNs.Similarly,the use of gas chromatography to characterize the characteristic peaks of FDC and FMCP,respectively,proved the successful loading of Mn O₂ and perfluorocarbon in M-PHNs nanoparticles.The storage stability and physiological environment stability of M-PHNs nanoparticles was investigated by monitoring the particle size changes at different time points.The results in this section indicate the successful preparation of M-PHNs nanoparticles and M-PHNs have a suitable particle size and excellent stability,which can be used in subsequent in vivo and in vitro experiments.Part II:Study on the oxygen generation ability of M-PHNs nanoparticles in vitro.After adding H₂O₂,the reactivity of M-PHNs nanoparticles at different reaction time points and the residual amount of H₂O₂ were compared to prove the reactivity of M-PHNs with H₂O₂.We use dissolved oxygen microelectrode to investigate the oxygen generation ability of M-PHNs nanoparticles in vitro and oxygen could release for a long period of time.Finally,HK-2 cell model was established to verify that M-PHNs nanoparticles significantly improved the viability of cells under hypoxic environment,and downregulated HIF-1αexpression in hypoxic HK-2 cells.Part III:The hypoxia reversion ability of M-PHNs nanoparticles in vivo and protect the kidney from ischemic injury.Establish a mouse kidney ischemia model,use dissolved oxygen microelectrode to determine the oxygen production of M-PHNs nanoparticles in ischemic kidney tissue homogenate.HIF-1αimmunohistochemical analysis of kidney slices proved that M-PHNs nanoparticles can downregulate the expression of HIF-1αin ischemic kidney.Next,the protective effect of M-PHNs nanoparticles on ischemic kidney was proved by measuring the change of anti-apoptotic protein Bcl-2 expression in the ischemic kidney,the expression of antioxidant indexes SOD,MDA,and histopathological examination of H&E staining.The results showed that M-PHNs can reverse the hypoxic microenvironment caused by renal ischemia and protect the kidney from ischemic injury.M-PHNs nanoparticles,as an integrated oxygen self-supplying nanoplatform of"oxygen production-oxygen storage-oxygen release",can not only effectively deliver oxygen in vivo and in vitro to reverse renal hypoxia,but also can eliminate excessive production of H₂O₂ during renal ischemia,which can alleviate the ROS damage to the ischemic kidney.These characteristics make M-PHNs have great clinical potential,or we can hope that M-PHNs may improve the quality of donor organs during the actual DCD kidney transplantation.