Functionalized Nanoparticles With Monocyte Membranes And Rapamycin Achieve Synergistic Chemoimmunotherapy For Reperfusion-Induced Injury In Ischemic Stroke

Ischemic stroke is a serious acute neurological disease,one of the main causes of disability and death,and brings a serious burden to the society.Reperfusion after ischemia is an effective way to treat brain injury after stroke.However,ischemia/reperfusion(I/R)can also cause damage to the brain,nerve cells and even the whole body due to restoration of blood flow and intense inflammation.At present,clinical drugs can only partially relieve the symptoms of stroke patients,and there is an extreme lack of drugs with specific target and high specificity.Therefore,it is an urgent need to develop innovative and effective therapeutic approaches for I/R.Based on I/R with a strong inflammation,this research uses poly lactic-co-glycolic acid copolymer(PLGA)copolymer as a nanocarriers loaded with rapamycin(RAP)drugs to prepare nano-drug RNPs.Because monocyte membrane(Mc M)has the escape function of immune clearance and can specifically bind to the inflammatory endothelial cell,the monocyte membrane(Mc M)is coated on the surface of RNPs to obtain biomimetic nanoparticles Mc M/RNPs.We proposed that Mc M/RNPs are a kind of nanoparticles with the “shield-sword” function.On the one hand,Mc M/RNPs can pass through inflammatory endothelial cells to reach the injury site,and then release RAP drugs to inhibit the proliferation of inflammatory cells,which is similar to a “sword”.On the other hand,Mc M/RNPs can actively target the inflammatory endothelium,and inhibit the adhesion of monocytes to endothelial cells,which is similar to a “shield”.Therefore,Mc M/RNPs reduced local inflammation,relieved and improve I/R damage,and significantly improved neurological score by simultaneously blocking the infiltration of monocytes and inhibiting the proliferation of microglia.Moreover,in vivo experiments showed that Mc M/RNPs had good therapeutic safety.Therefore,Mc M/RNPs can be used as a potential,effective and safe nanomedicine for the treatment of I/R injury.Specifically,the main research contents and conclusions of this paper are as follows:(1)Mc M/RNPs are successfully prepared.Nano-precipitation method was used to prepare RNPs.The hydrodynamic diameter,surface zeta potential,morphology,drug loading and encapsulation efficiency of RNPs were characterized.The results showed that RNPs are uniform nano-scale size,with uniform particle size and good dispersion.Moreover,RNPs could significantly increase the solubilization of RAP.The biomimetic nanoparticle Mc M/RNPs were prepared by co-extrusion method.The Mc M/RNPs were characterized by transmission electron microscopy(TEM)and Western Blot(WB).Moreover,Mc M/RNPs have the ability of sustaining drug release.(2)The biomimetic nanoparticles Mc M/RNPs can block the adhesion of monocytes to the inflammatory endothelium in vitro.Hemolysis experiments showed that Mc M/RNPs have good blood compatibility.The macrophage phagocytosis experiment proved that Mc M/RNPs has the ability to inhibit the phagocytosis of macrophages.Mc M and Mc M/RNPs can block the adhesion of monocytes on inflamed endothelium.Transwell experiment showed that Mc M/RNPs can penetrate the endothelial layer.In short,Mc M/RNPs have excellent blood compatibility,can escape the phagocytosis of macrophages,block the adhesion of monocytes on endothelium,and pass through the endothelial layer.(3)Mc M/RNPs can effectively treat I/R in the SD rat model.In vivo experiments showed that the Mc M/RNPs injected through the tail vein have a longer circulation time in the blood than RNPs,indicating that the monocyte membrane functionalized nanoparticles can evade its elimination by the immune system in vivo.Fluorescence imaging results of main organs in ex-vivo showed that the Mc M/RNPs mostly accumulated in the brain of the I/R model,especially in the right brain of the ischemic area than that the left brain,which indicated that Mc M/RNPs can identify the I/R lesion area and gather in the ischemic area.It is administered once 12 hours after I/R,TTC staining on 7 days showed that the ischemic area of Mc M/RNPs was significantly reduced.The infarction rate of the Mc M/RNPs group was only 6.92%,which significantly improved neurological behavior.At the cell level,neurons were labeled with Neu N and astrocytes with GFAP.Immunofluorescence staining shows that Mc M/RNPs were beneficial to the recovery of the number of neurons and astrocytes.Mc M/RNPs does not cause inflammation in vivo,In addition,Mc M/RNPs have very low side effects and good biocompatibility.(4)The mechanism of Mc M/RNPs in I/R therapy may depend on inhibiting inflammatory cells and reducing oxidative stress.To verify the effect of the“shield-sword” function of Mc M/RNPs,the brain tissues treated for 1 day with drugs were analyzed by immunofluorescence staining.It was found that the CD11 B and CD68 positive cell in the Mc M/RNPs treatment group decreased significantly at the injured site,which indicated that Mc M/RNPs can effectively inhibit local inflammation.At the same time,Mc M/RNPs can significantly enhance the activity of SOD and reduce the MDA in the serum,which reduce the concentration of oxygen free radicals and effectively prevent oxidative damage.Meanwhile,Mc M/RNPs significantly inhibits the proliferation of microglia and the recruit of macrophages from the blood.In addition,immunofluorescence confirmed that Mc M/RNPs can also inhibit cell apoptosis in the early stage.Therefore,Mc M/RNPs may treat I/R by inhibiting local inflammation and reducing oxidative stress.In summary,this study has prepared a novel drug delivery system Mc M/RNPs with a functionalized monocyte membrane,which can realize the immune-chemotherapeutic drug synergistic treatment of ischemic stroke.In vitro experiments showed that Mc M/RNPs has a low macrophage phagocytosis rate,can pass through the inflammatory endothelium and can also prevent monocytes adhering to inflammatory endothelium cells.In vivo experiments showed that Mc M/RNPs can pass through the inflammatory endothelium,then enter the I/R injured area.In addition,Mc M/RNPs can inhibit inflammation and improve I/R injury,significantly improve neurological scores and reduce infarct area in vivo.Therefore,Mc M/RNPs can be used as a potential nanomedicine in clinical treatment of I/R injury.