Study On The Targeted Treatment Of Multiple Sclerosis Via Neutrophil Membrane Biomimetic Nanomedicine

Objective:Neutrophil membrane biomimetic nanomedicine(TFMN)was designed and synthesized,and the targeted therapeutic effect of TFMN on multiple sclerosis(MS)was investigated by in vitro and in vivo pharmacodynamic experiments.Methods:1.Synthesis and characterization of TFMN(1)Synthesis of TFMN:Neutrophil membrane(NM)was extracted from peripheral blood of mice following a previously published protocol,supramolecular nanoparticles(TA-F68,TF)was synthesized from tannic acid(TA)and Poloxamer 188(Poloxamer 188,F68)supramolecular nanoparticles(TA-F68,TF)are fabricated by tannic acid and Pluronic F-127 by self-assembly,and TF and methylprednisolone(MPS)were encapsulated in NM through aqueous filters with 800 and 400 nm pore sizes in turn.Inside,a neutrophil biomimetic nanomedicine(TA-F68-MPS-NM,TFMN)was constructed.The erythrocyte membrane(RM)biomimetic nanomedicine(TA-F68-MPS-RM,TFMR)was prepared by the same method as a control.(2)Characterization of TFMN:the particle size and morphology were observed by laser nanoparticle size analyzer(DLS)and transmission electron microscope(TEM);drug loading rate and encapsulation rate were determined by high performance liquid chromatography(HPLC);to the level of neutrophil membrane proteins on TFMN were analyzed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE);DLSwere used to assess the stability of TFMN;enzyme-linked immunosorbent assay(ELISA)to detect the in vitro capacity of perforin adsorption of TFMN;simulated in vivo microenvironment to investigate the drug release of TFMN;the ability of TFMN to scavenge reactive oxygen species(ROS)were detected by electron spin resonance(ESR).2.Inflammatory targeting of TFMN and its protective effect on neuron damage in vitro(1)Study on inflammatory targeting of TFMN in vitro:BBB model was established in vitro,and the BBB permeability of TFMN was evaluated by Transwell test.(2)Cytotoxicity test of TFMN:TFMN with different concentrations(0,20,50,75,100,150,20μg/mL)was incubated with PC 12 for 24 hours by the cytotechnology reagent(CCK-8)method,and then determined by a microplate reader.The absorbance value of PC 12 cells at 450nm was used to calculate the survival rate.The cytotoxicity of TF was detected by the same method.(3)Study on the protective effect of TFMN on neuron injury in vitro:Rat adrenal pheochromocytoma cell(PC 12)in vitro oxidative stress injury model was established,and TF(100 μg/mL)or TFMN(50μg/mL)were administered for 10 h,the viability of PC 12 cells was detected by CCK-8 method;cell apoptosis was observed by Calcein/PI cell viability and cytotoxicity detection kit and fluorescence microscope;the ability of scavenging ROS of TFMN was detected by reactive oxygen species detection kit.3.CNS inflammatory targeting of TFMN and study on the treatment of experimental autoimmune encephalitis(1)CNS inflammation targeting test of TFMN:preparation the TFMN@DiO and TFMR@DiO.Female C57BL/6 mice were used to replicate the experimental autoimmune encyclopedia(EAE)model,and the model animals were randomly divided into TFMN@DiO group(2mg/kg)and TFMR@DiO group(2mg/kg).The corresponding nanoparticles were administered by tail vein injection once only,the administration volume was 10 mL/kg.In vivo imaging was performed on mice in each group at 1,2,4,8,and 12 h after iv to investigate the CNS inflammation targeting of TFMN.(2)TFMN treatment of EAE C57BL/6 mice were randomly divided into TFMN(neutrophil membrane biomimetic nanomedicine)group(2mg/kg),TF(TA-F68)group(14.8mg/kg),MPS(methylprednisolone)group(2mg/kg),TFM(TA-F68-MPS)group(2mg/kg),model group,control group,20 mice in each group.Except for the animals in the control group,which were not treated with modeling,the other groups of mice replicated the EAE model.Tail vein injection,except the model and control group were given equal volume of normal saline,the other groups of mice were given the corresponding preparation of normal saline solution,10mL/kg,once per day,for 14 days consecutively.The detection indicators:neurological function score,serum IFN-γ and IL-17 content,Th1,Th17,Treg ratio in CNS,ROS content in CNS,positive area of myelin in corpus callosum,number of neuronal apoptosis,neutrophils cellular infiltration,brain and spinal cord tissue,heart,liver,spleen,lung,kidney pathology.Results:1.Synthesis and characterization of TFMN The neutrophil membrane biomimetic nanomedicine TFMN with a "core-shell" structure was successfully prepared,with particle size about 220nm and potential about-48.2 mV;the maximum drug loading rate and encapsulation rate can reach to 31.22±2.24(%)and 22.57±1.01(%),respectively;TFMN retained NM protein during the preparation process,and good stability of TFMN was proved at room temperature;TFMN can adsorb perforin,and in the presence of perforin,it can quickly release the encapsulated MPS;TFMN can effectively scavenge O2·-、·OH、H2O2 free radicals in the system.2.Inflammatory targeting of TFMN and its protective effect on neuron damage in vitro By the "recruitment" of inflammatory factors IL-17 and TNF-α in vitro,strong inflammatory targeting and BBB penetration effect of TFMN was detected,less side effects and higher biological safety was proved.In vitro studies have shown that TFMN can Significantly increased the survival rate of oxidative stress injury cells and inhibited their apoptosis,and the mechanism may be related to the effect of TFMN in scavenging ROS in the inflammatory microenvironment in vitro.3.Study on the CNS inflammatory targeting of TFMN and its treatment of experimental autoimmune encephalitis The CNS inflammation targeting of TFMN was detected under the action of inflammatory cytokines and chemokines in vivo,TFMN can significantly reduce the neurological function scores of EAE mice,and reduce the content of IL-17 and IFN-γ in the serum,at the same time,the proportion of Treg cells was increased and Thl and Th17 was reduced significantly in the CNS.TFMN can ROS in the inflammatory microenvironment was effectively scavenged,the positive area of myelin in mice was significantly increase.TFMN could inhibit the apoptosis of CNS cells,reduce the infiltration of neutrophil,and significantly improve the lesions of the brain and spinal cord in EAE mice.TFMN has good biological safety.Conclusion:1、A neutrophil biomimetic nanomedicine,TFMN,loading with MPS for targeted therapy of MS was designed and prepared.2、TFMN could significantly regulate the inflammatory microenvironment,improve the oxidative stress caused by ROS,and inhibit the infiltration of inflammatory cells,thereby playing a role in neuroprotection.