PET Molecular Imaging Of Prussian Blue Nanozymes For Treatment Of Cerebral Ischemia

Ischemic stroke is one of the leading causes of mortality worldwide,accounting for 5.2%of deaths globally.It generally refers to a transient or permanent reduction of cerebral blood flow caused by local blockage of the cerebral artery via a thrombus or an embolus.The transient blood flow reduction could trigger ischemia/reperfusion injury,which initiates overwhelming production of reactive oxygen and/or nitrogen species(RONS,i.e.,ROS and/or RNS).Overproduction of RONS is known as a key factor in exacerbating brain damage.RONS-targeting scavenger that can block excessive production of RONS is considered as an ideal approach to protecting neurons against RONS-induced impairments and further promoting therapeutic outcomes for ischemic stroke.Currently,to protect ischemia-injured neurons,special attention has been devoted to developing feasible nanomaterials with enzyme-like activity(nanozymes)which present stable enzyme-like activity,robust anti-oxidative property,great physiological stability,and have a low synthetic cost.Prussian blue which is an FDA-approved antidote for cesium and thallium intoxication presents excellent biosafety.Prussian blue-based nanoparticles hold various advantages including flexible molecular structures,adjustable physical and chemical properties,porosity,and a low density.Over the past decade,they have attracted extensive interest in biomedical field and been developed as sophisticated multimodal imaging agents,as a drug delivery system,and as photothermal therapeutic agents,etc..Recently,PB nanoparticles have been found to be efficient RONS scavengers due to their superoxide dismutase-,catalase-,and peroxidase-like activities.The great RONS-scavenging capability of PB nanoparticles indicates their potential for driving neuroprotection against RONS-related disorders like ischemic stroke,which,however,has not yet been explored.Positron emission tomography(PET)is an emerging non-invasive molecular imaging modality,which is capable of in vivo visualizing the biological processes at cellular and molecular levels.PET imaging with 18F-fluorodeoxyglucose(18F-FDG)can in vivo monitor the glucose metabolic changes and reflect the cell viability.It has been widely used for the diagnosis and evaluation of therapeutic outcomes of various neurological diseases.In the present study,we aimed to construct novel hollow Prussian blue nanozymes(HPBZs)via developing a new,green,and convenient systhetic method,to evaluate the neuroprotective effect of HPBZs against hypoxia/ischemia,and more importantly,to explore the potential mechanisms underlying the neuroprotection of HPBZs both in vitro(in hypoxia cell model)and in vivo(in a rat model of ischemic stroke).Meanwhile,we performed 18F-FDG PET imaging to in vivo assess the neuroprotective effect of HPBZs against cerebral ischemia in a rat model of ischemic stroke,providing the evidence for further clinical translation of HPBZs.Part 1 Synthesis,Characterization,and Performance of HPBZsWe developed a facile Bi3+-assistant template-free synthetic strategy to construct HPBZs with multienzyme-like activities.This novel template-free synthetic strategy possesses several advantages,including easy bulk preparation and no need for any post-treatment or complex preparation processes.The prepared HPBZs with a size of approximately 65 possess a cavity inside and demonstrate good physiological stability with stable hydrodynamic diameters in a physiological environment.Furthermore,owing to the variable valence state and extraordinary redox capability,HPBZs with multienzyme-like activity could not only convert harmful ROS into harmless molecules(i.e.,H2O and O2),but also scavenged detrimental RNS.The unique hollow structure endows HPBZs with a large specific surface area to counteract the RONS and greatly improves the RONS scavenging capacity of HPBZs.These results indicated the potential of HPBZs for further in vitro and in vivo applications.Part 2 HPBZs Drive Neuroprotection in In Vitro ModelsWe investigated whether HPBZs could drive neuroprotection against hypoxia in vitro and further explored the potential mechanisms underlying the neuroprotective effect of HPBZs.First,we established an in vitro cell model of oxidative stress,and found that HPBZs could drive neuroprotection against oxidative damage;Later,we established CoCl2-induced in vitro hypoxia cell model and found that HPBZs could drive neuroprotection against hypoxia by reducing the generation of RONS and regulating the expression of pro-apoptotic p53 and anti-apoptotic Bcl-2 proteins;In addition,we discovered that HPBZs could also exert anti-inflammation effects in vitro through reducing the expression of inflammation mediators including cyclooxygenase-2 and inducible nitric oxide synthase and suppressing the release of inflammation factor IL-1(3.Taken together,HPBZs might drive cytoprotection in vitro through scavenging RONS,counteracting cell apoptosis,and suppressing inflammation response.Part 3 HPBZs Drive Neuroprotection against Cerebral Ischemia in a Rat Model of Ischemic StrokeConsidering the anti-oxidative,anti-apoptotic,and anti-inflammatory activities of HPBZs in vitro,we therefore further investigated their disease-modifying effects in a rat model of transient ischemic stroke induced via a 90-min middle cerebral artery occlusion surgery(MCAO).HPBZs(40 ?g/mL,10 ?L)were intracerebroventricularly administered into the brains of rats at 2 d before the MCAO surgery or at 1,4,or 8 h after the MCAO surgery.The results demonstrated that HPBZs administered 2 d before or 1 h after the MCAO surgery significantly reduced cerebral infarct volume,increased cerebral glucose metabolism,and attenuated neurofunctional behavior deficits.Further biological results indicated that HPBZs might drive neuroprotection via anti-oxidative,anti-inflammatory,and anti-apoptotic activities,thereby contributing to the reduced vulnerability of brain to ischemic injury.Preliminary evaluation of the in vivo safety of HPBZs including 18F-FDG PET imaging,neurological tests,and 2,3,5-triphenyltetrazolium chloride staining indicated that HPBZs might not induce brain tissue damage or neurological behavioral impairments in vivo.