Preparation Of Mitochondria-targeted Cyclosporin A Nanomicelle And Its Therapeutic Effect On Alzheimer’s Disease In Mice

Background:Alzheimer’s disease（AD）,a neurodegenerative disease characterized by memory deficits and cognitive impairment,is the leading cause of death in elderly patients with dementia.The signature pathological changes of AD are the deposition ofβ-amyloid protein（Aβ）outside neurons and neurofibrillary tangles caused by hyperphosphorylation of Tau protein inside neurons.The toxicity of Aβincreases rapidly after the formation of insoluble oligomers,and excessive deposition of Aβexhibits a dual neurotoxic effect.On the one hand,Aβoligomers can induce microglia to transform into pro-inflammatory type of M1,which continuously secrete inflammatory factors and produce a large amount of reactive oxygen species（ROS）to damage neurons.On the other hand,Aβoligomers can induce the irreversible opening of mitochondrial permeability transition pore（m PTP）in neurons,resulting in mitochondrial dysfunction.Mitochondrial dysfunction includes the decrease of mitochondrial membrane potential and ATP synthesis,the increase of mitochondrial superoxide and ROS levels,the release of cytochrome c.All of these injuries lead to neuronal apoptosis and further progression of neurodegeneration.Therefore,more and more attention has been paid to neuronal mitochondrial dysfunction,neuroinflammation and oxidative stress caused by the imbalance of microglia polarization.Clinical studies have shown that the incidence of AD in organ transplant patients receiving long-term cyclosporine A（Cs A）treatment is significantly lower than that in the general population.A further literature review revealed that Cyclosporin A（Cs A）can effectively protect neurons from Aβ-induced injures by inhibiting the abnormal sustained opening of m PTP,restoring and maintaining mitochondrial homeostasis,and promoting the growth of damaged neuron synapses.Therefore,Cs A has the potential to treat AD.However,Cs A has poor water solubility and non-selective distribution in vivo.Hence,how to transport Cs A into the injured mitochondria of neurons in the brain is the key to its application in the treatment of AD.The water-soluble cationic peptide SS-31 can be adsorbed to anion sites on the surface of cerebral vascular endothelial cells through electrostatic interaction,and then cross the blood-brain barrier（BBB）.At the same time,SS-31 can selectively enrich in mitochondrial matrix and inhibit cardiolipin oxidation,which plays the role of stabilizing mitochondrial membrane and exerting antioxidant function.Our previous study found that thioketal（TK）can specifically break down and decompose in the presence of ROS.The lipophilic Cs A and hydrophilic SS-31 were linked together by TK to synthesize Cs A-TK-SS-31,and Cs A-TK-SS-31 can self-assembly in water to form nanomicelles（CTS micelles）.SS-31 is able to drive Cs A across BBB and targets to mitochondria of neurons and microglia in the brain.Objective:The preparation of ROS-responsive CTS micelles can explore a new method for the treatment of AD by restoring the mitochondrial function of damaged neurons and activated microglia,inhibiting apoptosis of damaged neurons,promoting the transformation of activated microglia into M2 type,improving the inflammatory microenvironment of neurons,restoring damaged neuronal function,and ultimately improving memory and cognitive ability.Methods:1.Cs A-TK-SS-31（CTS）were synthesized from Cs A,TK and SS-31.The structure of CTS was identified by nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.2.CTS micelles were prepared by self-assembly method in water.The particle size,zeta potential,stability,drug release and hemolytic characteristics of CTS micelles were characterized by laser particle size analyzer,high performance liquid chromatography and high resolution mass spectrometry.3.The CCK-8 was used to investigate the effect of CTS micelles on the activity of mouse BV2 cells,human SH-SY5Y cells and mouse N2a cells.4.A model of Aβ_1-42-induced BV2 cells polarization was established,and the effect of CTS micelles on the morphology of BV2 cells damaged by Aβ_1-42 was observed by inverted microscopy.Flow cytometry and ELISA were used to determine the effects of CTS micelles on ROS levels,inflammatory factors（IL-1β,TNF-α）and anti-inflammatory factors（IL-10）in BV2 cells damaged by Aβ_1-42.5.Two models of Aβ_1-42-induced SH-SY5Y and N2a cells damage were established,and the effect of CTS micelles on the morphology of neurons damaged by Aβ_1-42 was observed by inverted microscope.Flow cytometry was used to detect the effects of CTS micelles on the level of ROS and mitochondrial superoxide,the potential of mitochondrial membrane,the opening of m PTP and the apoptosis of neurons damaged by Aβ_1-42.Western blot was used to detect the effect of CTS micelles on the expression of mitochondrial dynamics-related proteins and apoptosis-related proteins in neurons damaged by Aβ_1-42.6.BV2 cells,SH-SY5Y cells and N2a cells were co-cultured,and laser screening confocal microscopy（LSCM）was used to observe the uptake of FITC-labeled CTS micelles（FITC@CTS micelles）by three kinds of cells.Flow cytometry was used to detect the uptake of FITC@CTS micelles by three kinds of cells under normal condition and Aβ_1-42induced injury.7.The in vitro blood-brain barrier model was established,and the cumulative transport rate of FITC@CTS micelles across the in vitro blood-brain barrier was observed by LSCM.After FITC@CTS micelles across the in vitro blood-brain barrier,the mitochondrial colocalization of FITC@CTS in BV2 cells,SH-SY5Y cells and N2a cells co-cultured in the lower chamber was also observed by LSCM.8.APP/PS1 transgenic mice with five familial AD gene mutations（5×FAD mice）were selected to study AD disease.The distribution of Cy5-labeled CTS micelles in vivo and brain tissues of 5×FAD mice was observed by in vivo imaging system.9.The effects of CTS micelles on spatial learning,memory ability and cognitive index of 5×FAD mice were investigated by open field experiment,new object experiment and morris water maze experiment.10.H&E staining,Nishi staining and Neu N staining were used to observe the effects of CTS micelles on the morphology,number and function of neurons in hippocampus and the cortex of 5×FAD mice.11.Transmission electron microscopy（TEM）,ATP kit and western blot were used to detect the effects of CTS micelles on the morphology,energy supply function and expression of mitochondrial division and fusion-related proteins in hippocampal and cortical neurons of 5×FAD mice.12.Thioflavin S staining and ELISA were used to detect the effect of CTS micelles on the number of Aβplaques,soluble and insoluble Aβ1-42 levels in the hippocampus and cortex of 5×FAD mice.13.Flow cytometry,DHE staining,ELISA and western blot were used to investigate the effects of CTS micelles on the phenotype of microglia in the brain,the contents of ROS,IL-1β,TNF-αand IL-10 and the expression of apoptosis-related proteins in the hippocampus and cortex of 5×FAD mice.14.H&E staining,automatic biochemical analyzer and blood routine analyzer were used to detect the influence of CTS micelles on serum alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,urea nitrogen（BUN）,creatinine（CREA）and immune cells in peripheral blood of normal mice.Results:1.The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry determined that the precise molecular weight of CTS was consistent with the theoretical value,and nuclear magnetic resonance hydrogen spectroscopy further confirmed the molecular structure of CTS.2.CTS can self-assemble in water to form CTS micelles with a critical micelle concentration of 1.4μM.TEM showed that the CTS micelles were spherical and relatively uniform in size distribution.The average particle size was 102.6±5.1 nm,the potential was 21.5±4.3 Mv.The average particle size remained almost unchanged within 5 days,and the poly-dispersion indices were all less than 0.3,indicating good stability of CTS micelles.CTS micelles exhibited ROS responsive drug release properties.When the concentration of CTS micelles was below 50μM,the hemolysis rate was within the safe range.3.The micellar concentration of CTS at 50μM and below had no effect on the activity of BV2,SH-SY5Y and N2a cells.4.CTS micelles could restore the morphology of BV2 cells injured by Aβ_1-42,reduce the levels of ROS and inflammatory cytokines IL-1βand TNF-α,and increase the levels of anti-inflammatory cytokine IL-10.5.CTS micelles could restore the morphology of SH-SY5Y cells and N2a cells injured by Aβ_1-42,reduce the ROS and mitochondrial superoxide levels in SH-SY5Y cells and N2a cells,inhibit the abnormal opening of m PTP,and restore the mitochondrial membrane potential and the expression levels of mitochondrial division and fusion related proteins.Ultimately,the apoptosis of neurons was reduced by CTS micelles.6.FITC@CTS micelles could be effectively taken up by BV2 cells,SH-SY5Y cells and N2a cells in a time-dependent manner.Compared with normal BV2 cells,BV2cells injured by Aβ_1-42 showed a significantly higher uptake of FITC@CTS micelles.micelles Compared with normal SH-SY5Y cells and N2a cells,there was no significant difference in uptake of FITC@CTS micelles by SH-SY5Y cells and N2a cells injured by Aβ_1-42,suggesting that injury of Aβ_1-42 did not affect uptake of FITC@CTS micelles by neuros.7.CTS micelles could cross the blood-brain barrier in vitro,and the cumulative transport rate increased in a time-dependent manner.After crossing the blood-brain barrier in vitro,CTS micelles could target mitochondria of BV2 cells,SH-SY5Y cells and N2a cells co-cultured in the lower chamber.8.CTS micelles could effectively accumulate in the brain of 5×FAD mice,and reached the peak value at 6 h after administration.CTS micelles could enter into neurons in the hippocampus and cortex.9.Open field experiment,new object experiment and morris water maze experiment showed that CTS micelles could systematically restore the impaired spatial learning and memory ability of 5×FAD mice,improve the cognitive index,but did not affect their spontaneous activities.10.CTS micelles could restore the normal morphology of neurons in the hippocampus and cortex of 5×FAD mice,increase the number of Nissl bodies,the expression level of Neu N,and subsequently improved the function and state of neurons.11.CTS micelles could restore the mitochondrial morphology of neurons in the hippocampus and cortex of 5×FAD mice,reduce the expressions of mitochondrial division related proteins DRP1 and FIS1,increase the expressions of mitochondrial fusion related proteins OPA1,FMN1 and FMN2,improve the level of ATP synthesis,subsequently restored normal mitochondrial division and fusion dynamic balance and energy supply function.12.CTS micelles could effectively reduce Aβload by reducing the number of Aβplaques,soluble and insoluble Aβ_1-42 levels in hippocampus and cortex of 5×FAD mice13.CTS micelles could decrease the polarization ratio of M1 microglia and increase the polarization ratio of M2 microglia in the brain of 5×FAD mice.CTS micelles also decreased the content of ROS,IL-1β,TNF-αand the expression of Cleaved caspase-3,Caspase-3,Bax,Cytochrome c protein,increased the content of anti-inflammatory factor IL-10 and Bcl-2 protein in the hippocampus and cortex.14.CTS micelles had no significant effects on ALT,AST,BUN,CREA in serum and immune cells in peripheral blood of normal mice at therapeutic doses,showing good biocompatibility and safety in vivo.Conclusions:CTS micelles exhibited mitochondrial targeting and ROS responsive drug release properties.CTS micelles could effectively accumulate in the brain of 5×FAD mice,and restore the morphology of damaged mitochondria,increase ATP synthesis,improve mitochondrial dysfunction,reduce neuron apoptosis,degeneration and loss,and restore the function of injured neurons by reducing the expression of mitochondrial fission protein and increasing the expression of fusion protein.In addition,CTS micelles could also improve the inflammatory microenvironment in the brain by reducing the number of Aβplaques,reducing the level of soluble and insoluble Aβ1-42,reducing the polarization of microglia to M1,increasing the proportion of M2 microglia,reducing the secretion of ROS and inflammatory factors IL-1βand TNF-α.In conclusion,CTS micelles had the potential to treat AD by improving the inflammatory microenvironment where neurons are located,restoring the function of injured neurons,and improving the memory and cognitive ability of 5×FAD mice.