Studies On The Therapeutic Effects And Mechanisms Of Multifunctional Nanotherapies Based On Bioactive Carriers In The Treatment Of Neutrophilic Asthma

There is no effective treatment for moderate to severe asthma with neutrophilic infiltration due to glucocorticoid resistance and lack of precise targeted drugs.Studies have shown that the pathogenesis of neutrophilic asthma is closely related to the imbalance of oxidative stress caused by excessive reactive oxygen species（ROS）.Improving the efficiency of lung targeting,scavenging ROS,reducing neutrophil inflammation and regulating immune homeostasis have attracted much attention as new strategies for the treatment of asthma.In view of the above problems,the development of multifunctional bioactive nanodrug delivery systems with ROS scavenging,lung targeting,high mucus penetration,immune regulation,bronchodilation,safety and efficiency may be a new way to tre at neutrophilic asthma.To test this hypothesis,we first prepared ROS scavenging nanoparticles PTC NP by covalently binding the ROS-reactive units phenylboronic acid pinacol ester（PBAP）and Tempol（Tpl）to aβ-cyclodextrin（β-CD）skeleton,to verify the effectiveness of oxidation-sensitive group bonded skeleton molecule in the treatment of asthma.However,the nanoparticles showed a certain degree of uncertainty in structure and exhibited poor solubility.Therefore,we further optimized the delivery system on this basis and used hexachlorocyclotriphosphazene（HCCP）as a skeleton to co-bond PBAP and Tpl to obtain biomaterials with precisely controllable structure,multi-solvent solubility and good biocompatibility,by which a series of multifunctional bioac tive nano micelles were constructed.The screened nano micelles PP₄T₁ NM exhibited the best neutrophil asthma targeting therapeutic effect and showed strong mucus penetration.After loading with the clinical bronchodilator indacaterol（IND）,the anti-asthma efficacy was enhanced by synergistic effects.Finally,the mechanism was explored.Methods1.Synthesis and characterization of ROS scavenging nanoparticles PTC NPROS scavenging material PTC was obtained by covalent binding of PBAP and Tpl onβ-CD skeleton.The structure was characterized by FT-IR and ¹H NMR.ROS scavenging nanoparticles（PTC NP）were synthesized by nanoprecipitation self-assembly method.The morphology,particle size and potential of PTC NP were explored.2.Evaluation of the efficacy of PTC NP in the treatment of neutrophilic asthmaOVA/LPS induced neutrophilic asthma model was established in mice,and PTC NP was treated by aerosol inhalation.The proportion of neutrophils in bronchoalveolar lavage fluid（BALF）,oxidative stress indicators,pro-inflammatory factors,and the level of Ig E in blood were detected.H&E staining,PAS staining,Masson staining,N-cadherin staining,andα-SMA staining in lung tissue were observed.Respiratory resistance was measured using a small animal spirometer.3.Synthesis and characterization of multifunctional bioactive nano micellesThe amphiphilic polymer PP_xT_y was constructed by covalently binding PEG,PBAP,and Tpl to the HCCP backbone,and the structure was characterized by FT-IR and ¹H NMR.Micelle PP₃T₂ NM,PP₄T₁ NM,and PP₅T₀ NM were prepared,the particle size and potential were measured,and the morphology was observed by transmission electron microscopy（TEM）.4.Targeting of PP₄T₁ NM to OVA/LPS-induced asthmaThe distance PP₄T₁ NM penetrated the artificial mucus was measured at different time points.In vivo imaging was used to determine the fluorescence intensity of Cy5/PP₄T₁ NM retained in the bottom gelatin after penetration of artificial mucus.Confocal laser scanning microscopy（CLSM）was used to observe the Cy5/PP₄T₁ NM in the Transwell system after penetrating the artificial mucus and entering the bottom A549.Mice were aerosolized with Cy5/PP₄T₁ NM and lung fluorescence intensity was measured by in vivo imaging.Lung tissues were sectioned and CLSM was used to observe the penetration of Cy5/PP₄T₁ NM into the airway through mucus.Cy5/PP₄T₁ NM was inhaled by aerosol or injected into the tail vein of asthmatic mice.The targeting ability of PP₄T₁ NM to the lungs was examined by in vivo imaging,and its distribution in lung tissue or BALF cells was analyzed by flow cytometry.5.Evaluation of the therapeutic effect of different nano-micelles on asthmaAsthmatic mice inhaled PP₃T₂ NM,PP₄T₁ NM,and PP₅T₀ NM,respectively,and the efficacy of the three was compared as described in Method 2 of this abstract.6.In vitro biological effects of PP₄T₁ NMAfter A549 or HBSMCs were incubated with different concentrations of Cy5/PP₄T₁NM for different times,the uptake of PP₄T₁ NM by cells was examined by CLSM and flow cytometry.A549 or HBSMCs were treated with PP₄T₁ NM and then stimulated with PMA.Intracellular ROS levels were investigated using CLSM and flow cytometry.A549was treated with PP₄T₁ NM and then stimulated with LPS.The levels of tumor necrosis factor-α（TNF-α）and interleukin-1β（IL-1β）in the supernatant were detected.A549 was treated with PP₄T₁ NM,followed by H₂O₂ to induce apoptosis.Flow cytometry was used to analyze the apoptosis rate.7.Evaluation of the efficacy of PP₄T₁ NM in asthmaOVA/LPS-induced asthmatic mice were aerosolized with different concentrations of PP₄T₁ NM or budesonide（BDN）.Efficacy was compared as described in Method 2 of this abstract.A house dust mite（HDM）induced asthma model was established,and the efficacy of aerosolized PP₄T₁ NM was explored by the same method.8.Synthesis,characterization and in vitro biological effects of drug-loaded micelles IND/PP₄T₁ NMIND/PP₄T₁ NM was synthesized by micellar encapsulation of IND.The drug loading and encapsulation efficiency of the micelles were measured,the particle size and potential were detected,and the morphology was observed.The drug release properties of IND/PP₄T₁ NM were investigated.The intracellular free calcium fluorescence intensity of HBSMCs and collagen contraction assay were used to explore the anti-contraction ability of the drug-loaded micelles.9.Evaluation of the efficacy of IND/PP₄T₁ NM in the treatment of asthmaAerosolized IND/PP₄T₁ NM was inhaled in asthmatic mice.Efficacy was evaluated as described in Method 2 of the abstract.10.Mechanism of PP₄T₁ NM in the treatment of asthmaPP₄T₁ NM was incubated with neutrophils and stimulated with PMA,and the content of neutrophil cytoplasts was examined by flow cytometry and CLSM.After asthmatic mice were treated with aerosolized PP₄T₁ NM,flow cytometry and CLSM were used to measure the content of neutrophil cytoplasts in BALF.Neutrophil cytoplasts were incubated with dendritic cells（DCs）for 24 h,then na?ve CD4⁺T cells were co-cultured for 3 days,and the proportion of Th17 cells was determined.Activated DCs added with PP₄T₁ NM were co-cultured with na?ve CD4⁺T cells for 3 days to determine Treg cell content.11.Safety evaluation of PP₄T₁ NM and IND/PP₄T₁ NM.After incubation with A549 and HBSMCs with different concentrations of PP ₄T₁NM and IND/PP₄T₁ NM,cell viability was measured by CCK-8.PP₄T₁ NM was inhaled by aerosol.15 days later,blood samples were collected for routine blood test,liver and kidney function analysis,and organ index was calculated after weighing heart,liver,spleen,lung and kidney.H&E sections of each organ were observed under the microscope.The levels of oxidative stress and inflammatory factors were measured after lung tissue grinding.After incubation of PP₄T₁ NM with H₂O₂,neutrophils,and lung homogenates from asthmatic mice,the supernatants were coll ected for NMR or LC-MS.Results1.The synthesis of PTC was confirmed by FT-IR and ¹H NMR.PTC NP had a spherical structure with regular morphology and uniform size.PTC NP c ould anti-inflammatory,anti-oxidation,anti-airway remodeling,reduce airway resistance and improve lung function.2.The synthesis of PP_xT_y was confirmed by ¹H NMR and FT-IR.The synthesized nano micelles had a particle size of 35-50 nm,a slightly positive potential,and a spherical structure with regular morphology and uniform size.3.PP₄T₁ NM had the longest penetration distance in artificial mucus.In vivo imaging showed that Cy5/PP₄T₁ NM fluorescence intensity was the highest in gelatin.Transwell experiment showed that the fluorescence intensity of Cy5/PP ₄T₁ NM group in A549 cells was higher.After aerosol inhalation,the fluorescence intensity of PP ₄T₁ NM group was the highest.Lung sections showed that Cy5/PP₄T₁ NM penetrated airway mucus better and distributed more widely.Cy5/PP₄T₁ NM was efficiently targeted to the lung and mainly distributed in DC,neutrophils and epithelial cells.4.After inhalation of different nano micelles,PP₄T₁ NM showed more significant anti-inflammatory,anti-oxidative and anti-airway remodeling effects.5.PP₄T₁ NM could be effectively taken up by related cells,and could reduce intracellular ROS,TNF-αand IL-1βlevels,and reduce cell apoptosis rate.6.In OVA/LPS or HDM induced asthma models,10 mg/kg PP₄T₁ NM showed better anti-inflammatory,anti-oxidation,anti-airway remodeling,and has better efficacy than BDN.7.IND/PP₄T₁ NM was synthesized,and the drug loading,encapsulation efficiency,size and potential were measured,and the morphology was observed.It ha d a good ROS response drug release performance,which could reduce the intracellular calcium fluorescence intensity of HBSMCs and inhibit collagen gel contraction.IND/PP₄T₁ NM not only had all the efficacy of PP₄T₁ NM,but also dilated the bronchus.8.Neutrophil cytoplasts can stimulate the maturation of DCs,which in turn induces the differentiation of naive CD4⁺T cells into Th17.PP₄T₁ NM could significantly inhibited the generation of neutrophil cytoplast and reduced Th17 cells in vitro and in vivo.PP₄T₁ NM could also regulate DCs to promote Treg cell differentiation.9.PP₄T₁ NM had no obvious cytotoxicity and did not affect the physiological indexes of mice.It could be hydrolyzed to HMP,pinacol borate and PEG under different conditions.Conclusions1.It was demonstrated that the ROS scavenging group bonded to the skeleton molecule had a good therapeutic effect in OVA/LPS-induced asthma.2.After bonding HCCP,a series of amphiphilic polymers PP _xT_y with precise and controllable structure were constructed,and the micelle PP₃T₂ NM,PP₄T₁ NM and PP₅T₀NM were synthesized.PP₄T₁ NM was selected for the prevention and treatment of asthma by comparing its efficacy in vivo.3.PP₄T₁ NM has a strong mucus penetration,could efficiently target the lung and mainly distribute in DCs,neutrophils and epithelial cells.4.PP₄T₁ NM could be efficiently uptaken by A549 and HBSMCs in vitro and exhibited good anti-inflammatory,anti-oxidative and anti-apoptotic abilities.In asthma models,it showed the most significant effects of anti-inflammation,anti-oxidation and improvement of airway remodeling.5.The efficacy of IND/PP₄T₁ NM was significantly enhanced:it not only showed anti-inflammation,anti-oxidation and anti-airway remodeling,but also dilated bronchi and improved lung function.6.The mechanism of PP₄T₁ NM against neutrophil asthma showed that in the neutrophil-neutrophil cytoplast-DC-Treg/Th17 pathway,it could inhibit the formation of neutrophil cytoplasts by scavenging ROS and thus reduced the content of Th17 cells,and it could induce the differentiation of Treg cells by acting on DCs,and finally achieve d the function of regulating the balance of Treg/Th17 cells to maintain imm une homeostasis.7.In vitro and in vivo safety experiments showed that PP₄T₁ NM had good biological safety.In conclusion,this study demonstrated that nano-drug delivery systems constructed with ROS scavenging unit bonded backbone molecules are a reliable strategy to combat neutrophil asthma by reducing oxidative stress,inflammatory response,and neutrophil cytoplasts content and thereby maintaining immune homeostasis.In addition,given the important pathological roles of neutrophils,neutrophil cytoplasts,and DCs,the neutrophil-neutrophil cytoplasm-DC-Treg/Th17 pathway may be a new target for the treatment of neutrophil asthma and other lung diseases,and is expected to provide potential new drugs.