Preparation And In Vitro Evaluation Of ANG-PEG-2ME2 Micelles For Mitigating Cerebral Ischemia/Reperfusion Injury And Peg-Rapa Micelles For Inducing Immunotolerance

Polymer-drug conjugated micelles（PDCM）is a nanoscale drug delivery system composed of amphiphilic polymer-drug conjugates formed by self-assembly in aqueous solution.PDCM as delivery vehicles of hydrophobic drugs have many advantages,such as increasing the solubility and stability of hydrophobic drugs,improving drug loading capacity,prolonging circulation time in vivo,increasing intracellular uptake and enhancing the therapeutic efficacy of drugs,etc.Compared with the nano-based drug delivery system in which the drugs are physically entrapped,PDCM show no initial burst release and exhibit a much slower drug release or a triggered drug release property due to the requirement for covalent bonds cleavage.In addition,a variety of polymers containing functional groups can also be used to prepare multi-functional PDCM,improving the active targeting effect and achieving drug accumulation at the targeting sites.Polyethylene glycol（PEG）is a polymer that has been approved by the U.S.FDA for clinical application.It is widely used due to its strong hydrophilicity and excellent biocompatibility.Chemically,the technique of connecting a therapeutic drug molecule with one or more PEG molecules to form a PEG-drug molecule conjugate is also known as PEGylation technology.In this paper,2-methoxyestradiol（2ME2）and rapamycin（RAPA）were used as model drugs,and PEG was used as a hydrophilic material.Two PDCM drug delivery systems were constructed and evaluated.In the first part of this paper,we fabricated and evaluated free 2ME2-loaded PEGylated 2ME2 micelles for mitigating cerebral ischemia/reperfusion（I/R）injury.Ischemic stroke is one of the leading causes of death and disability worldwide,but reperfusion caused by the restoration of blood oxygen supply to the brain tissue will cause secondary damage to the cerebral ischemic area,that is,cerebral I/R injury.Cerebral I/R injury is a complex pathophysiological process,which can cause a lower level of ATP production,electrolyte imbalance,Ca²⁺overload,mitochondrial damage and ROS retention,resulting in cell apoptosis,autophagy and necrosis.2ME2 is a kind of microtubules and HIF-1αblocker,which can inhibit the overexpression of HIF-1α,thereby inhibiting the activation of p53 signal pathway and autophagic cell death,and is a promising drug for the treatment of cerebral I/R injury.However,2ME2 has low bioavailability due to its poor water solubility,strong liver first-pass effect and short half-life,which have limited its efficacy as a neuroprotective agent.In order to increase its therapeutic efficacy,we planned to fabricate brain-targeted 2ME2-loaded micelles（ANG-PEG-2ME2/2ME2）and evaluate its neuroprotective effect.Firstly,we synthesized PEGylated 2ME2 conjugate（m PEG-2ME2）and angiopep-2 capped PEGylated 2ME2 conjugate（ANG-PEG-2ME2）.Then,we prepared common drug-loaded micelles（m PEG-2ME2/2ME2）and brain-targeted drug-loaded micelles（ANG-PEG-2ME2/2ME2）by emulsion-solvent evaporation method.The active targeting effect and the neuroprotective effect were evaluated at the cellular level:（1）Intracellular uptake experiments were performed using brain capillary endothelial cells（BCECs）,and the results showed that angiopep-2 modified micelles had higher intracellular uptake rate;（2）PC12 cells with neuronal characteristics were used to construct oxygen-glucose deprivation/reoxygenation（OGD/R）models with different severity of injury to simulate I/R injury.The results showed that for mild and moderate injury models（OGD 0.5 h/R 24 h and OGD 4 h/R 24 h）,different 2ME2 preparations can both exert neuroprotective effects;Compared with free 2ME2,its drug-loaded micelles could significantly improve the survival rates of PC12 cells,inhibited the generation of reactive oxygen species（ROS）and cell apoptosis,and could effectively reduce the damage caused by OGD/R.In the second part of this paper,we fabricated and evaluated free RAPA-loaded PEGylated RAPA micelles for inducing immunotolerance.In recent years,biologic drugs have become an important driving force for the development of the global pharmaceutical industry.However,almost all biologic drugs have immunogenicity of eliciting antidrug antibodies（ADAs）,which can adversely affect the efficacy of drugs,cause hypersensitivity reactions and cross-reaction with endogenous proteins or even deprive patients of life-sustaining therapies.RAPA is a macrolide immunosuppressant and is hardly soluble in water.It has been reported that PLGA nanoparticles loaded with RAPA（PLGA/RAPA）could prevent the production of ADAs of biologic drugs,and it has also been proved that the mechanism was immunotolerance rather than immunosuppression.This nanoparticle is actually a drug delivery system for RAPA,and its induction of immunotolerance should be related to its immune targeted delivery and drug release behavior.In order to explore the factors that affect the immunotolerance induced by RAPA nanoparticles,we planned to prepare another nano-based drug delivery system which was different from PLGA/RAPA in the aspect of drug release behavior——RAPA-loaded PEGylated RAPA micelles（m PEG-RAPA/RAPA）.Firstly,we prepared PLGA/RAPA and m PEG-RAPA/RAPA micelles using emulsion-solvent evaporation method.The particle size,particle size distribution,encapsulation efficiency,and drug loading efficiency of these two RAPA-loaded nanoparticles were characterized.In vitro drug release experiments showed that m PEG-RAPA/RAPA exhibited slower release rate.Studies on the effects of RAPA to dendritic cells（DCs）on the expression of surface molecules and secretion of cytokines showed that compared with mature DCs stimulated by lipopolysaccharide（LPS）,after treating with free RAPA and its drug-loaded nanoparticles,the expression of MCH classⅡmolecules on the surface of DCs increased,the expression of CD40 and CD86decreased,and the secretion of transforming growth factor-β1（TGF-β1）increased.These results showed that RAPA can inhibit the immune function of DCs,but the efficacy of free RAPA,PLGA/RAPA and m PEG-RAPA/RAPA was not significantly different.This mainly could be attributed to the time-dependent internalization manner of nanoparticles and time-dependent intracellular release manner of entrapped RAPA.Therefore,in an in vitro setting（the drug treatment time has not been investigated）,the different intracellular uptake and drug release behavior characteristics of different nanoparticles will lead to a lack of differentiation among DCs surface molecule expression,cell molecule secretion and immune function.