Design And Evaluation Of The Biomimetic Engineered Nanodelivery Systems Stepwise Targeting Brain Neuronal Mitochondria

Recent studies have shown that mitochondrial oxidative stress is closely associated with the clinical development of AD symptoms and might be a potential therapeutic target,especially due to the failures of phase III clinical trials of conventional amyloid-??A??targeted therapy.However,the efficient intravenous delivery of therapeutic agents to neuronal mitochondria in the brain remains a major challenge for drug delivery due to the complicated physiological and pathological environment.Currently,biomimetic nanotechnology represents a promising approach to deliver therapeutic agents in neuronal mitochondria.Herein,we devised and tested a strategy for functional antioxidant delivery to neuronal mitochondria by loading antioxidants into red blood cell?RBC?membrane-coated human serum albumin nanoparticles bearing T807 and TPP molecules attached to the RBC membrane surface?T807/TPP-RBC-NPs?.Drug content and entrapment efficiency of biological nanoparticles were determined by high performance liquid chromatography and microcolumn centrifugation in vitro.Through dialysis,lipid extrusion and single factor analysis combined with central composite design,the average particle size of CUR-loaded-RBC-NP was 102.62±0.59nm,the encapsulation efficiency was 88.43±1.25%and the drug loading was 4.87±1.04%.The two chemical chains including DSPE-PEG3400-T807 and DSPE-PEG2000-TPP were prepared by chemical synthesis,separation and purification,and the structural identification of the chemical chains were determined by nuclear magnetic resonance spectroscopy and laser-assisted analysis-time-of-flight mass spectrometry,respectively.The optimal modification ratio of the two chemical chains were determined through BBB model and mitochondrial extraction experiments in vitro.The stability experiment showed that the biological nano-carriers were stable within 72 h in p H7.4 PBS?containing10%fetal bovine serum?.The average particle size of the T807/TPP-RBC-NPs was112.13±0.74 nm,the PDI was 0.087±0.012,and the average Zeta potential was-39.3±0.9 m V.The results showed that compared with the free CUR,the prepared biological nano-carrier groups release drugs slowly and continuously within 48 hours.In order to evaluate the safety and therapeutic effect ability of drug-loaded biological nano-carriers,the MTT assay,macrophage uptake,CD47 protein determination and pharmacokinetics and other experiments were conducted in vitro an in vivo.The results show that the T807/TPP-RBC-NPs are stabilized and promote sustained drug release,providing improved biocompatibility and long-term circulation.To explore whether these ligand-modified formulations targeted the mitochondria after successful internalization,the nano-carriers were labeled with coumarin 6,the mitochondrial colocalization,mitochondrial uptake and BBB permeability studies were conducted,respectively.The results show that T807/TPP-RBC-NPs demonstrated that the dual-modified biomimetic formulations could not only penetrate the BBB but also target the nerve cells and further localize in the mitochondria under the synergistic effect of T807 and TPP.In order to prove that T807/TPP-RBC-NPs can reach the sufficient concentration to treat mitochondrial damage,we then studied the antioxidant effect of drug-loaded biological nano-carriers in vitro by loading the model drug CUR on nanoparticles.We found that compared with CUR,T807/TPP-RBC-NP loaded on CUR significantly reduced the level of mitochondria ROS of AD model cells that induced by OA,regulation of antioxidant enzymes and oxides?SOD,MDA,?-GT,H₂O₂?and finally inhibited the apoptosis of neuron-like cells.The in vivo pharmacodynamic results which injected by tail vein showed that the drug-loaded biological nano-carrier?T807/TPP-RBC-NPs?significantly improved the cognitive impairment of AD model mice,regulate the contents of antioxidant enzymes and oxides in hippocampus,weaken neuronal damage.The research data of this work collectively suggest that T807/TPP-RBC-NPs might provide a promising intravenous mitochondria-targeted delivery platform for AD treatment.