Study On The Therapeutic Effects And Mechanisms Of Multiple Pharmacologically Active Nanoparticles Based On Luminol-conjugated Cyclodextrin For Targeted Treatment Of Neutrophil Asthma

Asthma is a respiratory disease,especially neutrophils-mediated severe asthma is an important cause of death and morbidity worldwide.Neutrophil asthma（NA）is closely related to severe asthma,but there is still a lack of safe and effective treatment.Studies have shown that a potent neutrophil chemokine IL-8 produced by damaged airway epithelial cells can induce neutrophil recruitment to the inflamed lung,and neutrophils adhereed to lung epithelial cells can further induce IL-8 released.Activated neutrophils secrete excessive neutrophil extracellular traps（NETs）that can cause lung tissue damage,airway epithelial cell necrosis and smooth muscle cell proliferation,resulting in airway inflammation,airway remodeling and airway hyperresponsiveness.In addition,neutrophils can induce monocyte/macrophage adhesion,recruitment and activation,which can further aggravate NA inflammatory responses.Therefore,the development of novel anti-inflammatory nanomedicines that can simultaneously target and regulate multiple lung pathological cells and inhibit the formation of NETs may provide a new strategy for the effective treatment of NA.Therefore,in this project we successfully constructed nanoparticles LaCD NP with mutiple pharmacological activities and neutrophil membrane-modified LaCD NP（NM-LaCD NP）based on luminol-conjugatedα-cyclodextrin,which achieve an effective treatment for NA.Methods1.Synthesis and characterization of LaCD NP with multiple pharmacological activitiesAfter activated by CDI,α-CD was bonded with luminol to form LaCD.Subsequently,LaCD was characterized by infrared spectroscopy,¹H NMR and UV-Vis spectroscopy.NPs based on LaCD were prepared by a nanoprecipitation method.In brief,LaCD was dissolved in DMF,into which deionized water was slowly added.Then,LaCD NP was collected and characterized by particle size analyzer,TEM,SEM and AFM.2.The targeting and therapeutic effects of LaCD NP in neutrophil asthmaTo exam the targeting ability in NA lung and the distribution of LaCD NP in lung tissue cells,Cy7.5/LaCD NP was intravenously administered through the tail vein or Cy5/LaCD NP was given by aerosol inhalation.In order to further investigate the therapeutic effect of LaCD NP,LaCD NP was administrated by either i.v.or inhalation.The bronchoalveolar lavage fluid（BALF）was collected to detect the neutrophil content,the levels of MPO,H₂O₂ and inflammatory factors TNF-α,IL-1βand IL-17.The levels of allergy-related antibody Ig E in peripheral blood of mice were determined.Lung tissue was taken for immunofluorescence staining,H&E staining,PAS staining,α-SMA staining and Masson staining.The respiratory resistance of mice was assessed by a small animal invasive pulmonary function instrument.3.Preparation of neutrophil membrane-modified LaCD NPPeritoneal neutrophils were collected from mice and neutrophil membrane vesicles（NMVs）were then obtainedby sonicated and frozen.Finally,neutrophil membrane-functionalized LaCD NP（NM-LaCD NP）was produced by mixing LaCD NP with NMVs through a sonication-extrusion method and then characterized.4.The targeting and therapeutic effects of NM-LaCD NP in neutrophil asthmaCy5/LaCD NP and Cy5/NM-LaCD NP were administrated by i.v.,then the targeting ability of different nanoparticles in the lung of neutrophil asthma was analysed by in vivo imaging system.In order to further investigate the therapeutic effect of NM-LaCD NP.LaCD NP and NM-LaCD NP were administrated by i.v.After the treatment,the BALF of the mice was extracted,subsquently the content of neutrophils and the levels of inflammatory factors were detected.Lung tissue was taken for H&E staining,PAS staining,α-SMA staining and Masson staining.5.Preliminary mechanism study of LaCD NP in the treatment of neutrophil asthma（1）Biological effects of LaCD NP in vitroAfter Cy5/LaCD NP with different doses were incubated with peritoneal neutrophils,peritoneal macrophages and A549 epithelial cells for different times,the phagocytic behavior was analysed by confocal laser microscopy and flow cytometry.Different concentrations of LaCD NP were co-incubated with PMA-stimulated neutrophils,and ELISA kits were used to determine the contents of MPO,TNF-αand IL-1β.Different concentrations of LaCD NP were co-incubated with macrophage-stimulated neutrophils to explore the effect of LaCD NP on the migration of neutrophils by transwell assay.（2）LaCD NP inhibited neutrophil extracellular traps（NETs）formation in vitroThe differential genes（DEGs）of neutrophil asthma mice compared with the normal group or the treatment group of LaCD NP were analyzed,which provide theoretical support for screening intervention targets.Peritoneal neutrophils were seeded and different doses of LaCD NP were added.After stimulation with PMA,the levels of major components of NETs（ds DNA,NE,MPO and Cit H3）were determined by fluorescence method and ELISA kits.The effect of LaCD NP on inhibiting the formation of NETs was observed by laser confocal microscopy in vitro.After i.v.administration of different concentrations LaCD NP in NA mice,lung tissues were extracted.The expression of Cit H3 was observed by laser confocal microscopy.（3）LaCD NP inhibited NLRP3 inflammasome activation in neutrophilsPeritoneal neutrophils were seeded and different doses of LaCD NP were added.After stimulation with PMA or LPS/ATP,the levels of NLRP3 inflammasome components NLRP3,ASC,pro-caspase-1 and IL-1βwere determined by WB and ELISA kits.After i.v.administration of different concentrations of LaCD NP,lung tissues were extracted.The levels of NLRP3inflammasome components NLRP3,ASC and pro-caspase-1 were determined by WB.（4）LaCD NP regulated lung epithelial cells and bronchial smooth muscle cellsDifferent concentrations of LaCD NP were pre-incubated with LPS-stimulated A549 lung epithelial cells,subsequently,IL-8 levels of the supernatant were detected by ELISA kit.The effect of LaCD NP on neutrophil migration activated by A549 lung epithelial cells was investigated by transwell assay.The effect of NETs with different treatments on the migration of HBSMCs was investigated by transwell experiment.NETs treated with different concentrations of LaCD NP were co-incubated with A549 lung epithelial cells,and the cell viability was detected by CCK8 kit and flow cytometry.NETs treated with different concentrations of LaCD NP were co-incubated with HBSMCs,and cell viability was detected by CCK8 kit.6.Preliminary biosafety evaluations of LaCD NPDifferent concentrations of LaCD NP were incubated with neutrophils,A549 lung epithelial cells and HBSMCs for different times,and cell viabilities were detected by CCK8 kits.LaCD NP were administered by i.v.injection at days 1 to 3（1 g/kg）or by inhalation on days 1 to 5（50 mg/kg）in BALB/c mice.14 days after different treatments,possible toxicity of LaCD NP was examed in vivo.Results1.Both FT-IR,¹H NMR,and UV-Vis spectrometry revealed successful synthesis of LaCD.LaCD nanoparticles（LaCD NPs）were then prepared by nanoprecipitation method.LaCD NP displayed well-defined spherical morphology with the mean diameter of about 226 nm andζ-potential of-11 m V.2.We found that LaCD NP can efficiently be accumulated in the lung of NA mice after either i.v.administration or inhalation,and mainly localize in neutrophils,macrophages,and lung epithelial cells.LaCD NP can significantly reduce the neutrophil content and the levels of relevant inflammatory factors in NA mice.H&E,PAS,α-SMA,and Masson staining of lung tissue further confirmed that LaCD NP can effectively inhibit neutrophil-mediated pulmonary inflammatory response,reduce mucus secretion,and relieve high airway resistance,thereby reducing the severity of allergic asthma.3.Neutrophil cell membrane-modified LaCD NP（NM-LaCD NP）was prepared successfully and showed a uniform spherical core-shell structure.Compared with uncoated LaCD NP,the mean diameter of NM-LaCD NP increased by approximately 20 nm.Theζ-potential value of NM-LaCD NP was reduced,which was comparable to that of NM.Moreover,Coomassie blue staining indicated the presence of neutrophil membrane-associated proteins on NM-LaCD NP.4.Compared with uncoated LaCD NP,NM-LaCD NP exhibited better lung targeting performance in NA mice.NM-LaCD NP could effectively reduce the content of neutrophils and the levels of related inflammatory factors of BALF in neutrophil asthma.Meanwhile,lung tissue stainings also confirmed that NM-LaCD NP had better effects.5.LaCD NP were efficiently phagocyted by neutrophils,macrophages,and A549 lung epithelial cells in vitro.LaCD NP could inhibit the migration of neutrophils and reduce neutrophil-mediated inflammatory responses.6.RNA-sequencing analysis in NA mice showed that among these differentially expressed genes（DEGs）,the Mpo gene is one of most significantly varied genes after treatment with LaCD NP.LaCD NP effectively inhibited PMA-mediated NETs in neutrophils in vitro,as indicated by the remarkably decreased Cit H3 expression and NETs degree.In vivo,LaCD NP significantly reduced the formation of NETs of lung in NA mice.7.Western blotting analyses revealed significantly increased expressions of NLRP3,ASC,and pro-caspase-1 by NETs-induced neutrophils.Correspondingly,significantly increased IL-1βlevel by NETs-stimulated neutrophils was detected.By contrast,treatment with LaCD NP attenuated the formation and activation of the NLRP3 inflammasome in NETs-stimulated neutrophils.Moreover,LaCD NP could directly inhibit the formation and activation of the NLRP3 inflammasome induced with LPS and ATP in neutrophils.NLRP3 inflammasome activation in lung tissues from asthmatic mice was notably inhibited after treatment with LaCD NP in vivo.8.LaCD NP effectively reduced the expression of IL-8 by LPS-stimulated pulmonary epithelial cells.Correspondingly,LaCD NP was able to significantly suppress the migration of neutrophils induced by LPS-activated epithelial cells.On the other hand,LaCD NP effectively protected pulmonary epithelial cells from NETs-induced death and significantly inhibited migration and proliferation of HBSMCs.9.Regardless of neutrophils,A549 lung epithelial cells,or HBSMCs,high cell viabilities were found after incubation with various doses of LaCD NP.Further,acute toxicity of LaCD NP after either i.v.or inhalation administration showed that the weight gain,organ index,blood routine,biochemical indexes,and major organs revealed no change in mice treated with LaCD NP.After inhalation delivery,we found no infiltration of inflammatory cells.Conclusions1.The LaCD with multiple pharmacological activities was successfully constructed fromα-CD and luminol,and the multiple pharmacologically active nanoparticle LaCD NP was prepared.2.LaCD NP can be efficiently taken up by neutrophils,macrophages and lung epithelial cells in lung tissue of NA mice by i.v.administration or inhalation.In NA mice,LaCD NP were administrated by either i.v.or inhalation showed excellent therapeutic effects by effectively inhibiting neutrophil-mediated pulmonary inflammatory response,reducing mucus secretion,relieving high airway resistance,and reducing severity of allergic asthma.3.The neutrophil membrane-modified nanoparticle（NM-LaCD NP）was successfully prepared by nanomimetic technology.The lung targeting ability and efficacy of LaCD NP in NA mice was further potentiated by surface functionalization with the neutrophil membrane.4.LaCD NP can significantly inhibit the formation of NETs in vitro and in vivo,and further inhibit the activation of neutrophil NLRP3 inflammasome mediated by NETs.In addition,LaCD NP could inhibit activated neutrophil-induced macrophage migration and macrophage inflammatory response.Meanwhile,LaCD NP could inhibit the secretion of IL-8 from lung epithelial cells,reduce neutrophil migration,and inhibit NETs-mediated epithelial cell necrosis and SMC migration and proliferation.5.We found that LaCD NP exhibited good safety performance after either i.v.administration or inhalation in vivo or vitro.Taken together,this research showed that LaCD NP played an important role in inhibiting neutrophil migration and NETs formation,which could further inhibit neutrophil NLRP3inflammasome activation,macrophage inflammatory response,as well as airway epithelial cell death and smooth muscle cell proliferation.Therefore,nanotherapy based on LaCD NP that could simultaneously regulate multiple pathological cells was expected to be used for the precise treatment of neutrophil asthma,and it could also provide new strategies and methods for the clinical treatment of other diseases related to neutrophil inflammation.