Designed Fabrication Of Acid-Responsive Mitochondrial Targeting Nanoformulation For Asthma Treatment

Asthma is a serious disease that affects millions of people worldwide and inhaled corticosteroids are applied as the first-line strategy for controlling asthma.However,there are still a part of asthma patients poorly controlled with traditional inhaled corticosteroids.ABT-199,as a selected inhibitor of Bcl-2 protein,can inhibit the expression of’ Bcl-2 in mitoclhondria of inammatory cells and induce the apoptosis However,ABT-199 is hydrophobic and without targeting property to mitochondria which significantly influence the therapeutic efficiency.Therefore,it is necessary to develop new drug delivery system to enhance the efficiency of ABT-199.The development of nanocarriers and targeting nano-delivery provide novel strategy for efficient treatment of astluna.Considering the acidic microenvironment in the lesion of inflanmmation associated disease and the lysosome is acidic inside,we designed acid-responsive nanocarrier for f-urther drug loading.The acid-responsive amphiphilic polymer was synthesized and spontaneously formed micelle with hydrophobic ABT-199 in aqueous solution.The asthma disease model was chosen for further investigation of the therapeutic efficiency of Nf-ABT-199.Firstly,the amphiphilic NH2-PEG with high biocompatibility was chosen as the initiator of ring-opening polymerization with R-amino-N-carbon acid anhydride and imidazole was f’unctioned as the acid-sensitive group through an aminolysis reaction.Finally,the acid-responsive polymer was obtained.The nuclear magnetic resonance spectroscopy(NMR)was applied for the characteristic analysis of acid-responsive polymer.The pH sensitive property of’ polymer was characterized by ultraviolet spectrophotometer(UV-vis).When the pH was lower than the pKa,the protonation of acid-responsive ionizable groups would lead the increase of surface zeta potential which would enhance endocytosis and further mitochondria-targeting with electrostatic interaction.In this paper,we synthesized acid-sensitive nanoformulated Bcl-2 inhibitor(Nf-ABT-199).Nf-ABT-199 can efficiently deliver ABT-199 to the inflammatory cells and then target to mitochondria.And the nanocarrier was synthesized with the same method as the control.Then the measuring method of ABT-199 was set up by ultraviolet spectrophotometer and the drug loading capacity was measured.The zeta potential change of Nf-ABT-199 with pH was characterized by zeta potential analyzer.The morphology and size of Nf-ABT-199 was characterized by transmission electron microscopy(TEM)and dynamic light scattering(DLS).The results showed that Nf-ABT-199 was pH responsive.In low pH condition,the surface zeta potential of Nf-ABT-199 increased with positive electricity as well as the size swell.Furthermore,the endosomal escape of Nf-ABT-199 was investigated in inflammatory cells and the result confirmed endosomal escape behavior of Nf-ABT-199.Upon the uptake of Nf-ABT-199,it underwent a charge enhancement in the acidic environment of endo/lysosomes and induced a proton sponge effect,leading to endosomal escape of Nf-ABT-199.Finally,Nf-ABT-199 targeted to mitochondria with electrostatic interaction.The mitochondria targeting ability of Nf-ABT-199 was also investigated.Compared with the insensitive nanoformulated ABT-199(is-ABT-199),as the result showed,Nf-ABT-199 can targeted and accumulated in mitochondria.And the concerntration of ABT-199 in mitochondrias after cell’s incubation with different incubation was compared which further confirmed the same result.The chicken ovalbumin induced allergic airway inflammation models were applied.The mice were intratracheally(i.t.)administered with ABT-199,is-Nf=ABT-199 and Nf-ABT-199.The number of inflammatory cells,inflanmmatory cytokine levels in the airway,the inflamiatoly cell Irecruitment,mucus production in lung tissue through section-staining and the inflammatory cell apoptosis conditions were investigated to assess the efficacy of Nf-ABT-199.The results showed that:1.Nf=ABT-199 can sp read into deeper lung tissue and showed superior efficicency in small airways;2.Nf-ABT-199 can reduce the number of inflammatory cells,inflammatory cytokine levels,and inhibit peribronchial inflammatory cell recrLuitment.mucus production and airway hyperresponsiveness.And the nanocarrier didn’t influence the inflammatoly condition;3.Nf-ABT-199 can activate caspase-3 pathway and promote inflzammatory cell apoptosis.Furthermore.the biocompatibility of Nf-ABT-199 and nanocarrier was investigated.The toxicity was detected by CCK-8 assay and the apoptosis level of HBE was analyzed by fluorescence activated cell sorting(FACS).The damage to the airway epithelium was detected through the evaluation of E-Cadherin.The weight change of mice and the levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST)and actate dehydrogenasealso(LDH)were detected.The results showed that:1.Nf-ABT-199 or nanocarrier didn’t make obvious influence on cell viability;2.Nf-ABT-199 or nanocarrier cause no damage to E-Cadherin and airway epithelium;3.Nf-ABT-199 or nanocarrier didn’t make obvious liver injury nor influence the body weight.In coinclusion,the ABT-199 loaded micelles(Nf-ABT-199)were prepared with acid-responsive mitochondria targeting nanocarrier.Nf-ABT-199 was with nano size and improved the water solubility of drugs and facilitated the drug delivery.Moreover,Nf-ABT-199 can deeply spread into lung tissue and accumulated in inflaimmatory position.And with the acid-responsive ability,when Nf-ABT-199 was ingested by inflammatory cells,the charge switched due to the acidic environment of endo/lysosomes.Then Nf-ABT-199 can target to mitochondria with electrostatic interaction.The enhanced drug delivery finally led to the inhibition of Bcl-2 and resulted in the apoptosis of inflammatory cells.In addition,Nf-ABT-199 and nanocarrier possess good biocompatibility.The pH responsive mitochondria-targeting nano-delivery can provide a new strategy for future design and development of organelle targeting drug.