Study On The Effect And Mechanism Of ROS-scavenging Nanoparticles In The Treatment Of Neutrophilic Asthma

Asthma,one of the most common lung diseases,remains a serious global healthy problem.Approximately 3%-10%of asthma patient have severe asthma,mainly infiltration with neutrophils.Currently,the clinical drugs for asthma therapy have poor effects on neutrophilic asthma,and most of them have drug resistance or adverse effects.Oxidative stress imbalance plays a vital role in the pathological process of neutrophilic asthma,and the oxidative respiratory burst of neutrophils leads to increased reactive oxygen species（ROS）.Recent studies have revealed that targeting neutrophil-related mediators or inflammatory was a new strategy for asthma treatment.Neutrophils capture and kill pathogens by releasing neutrophil extracellular traps（NETs）,which were assembled from ds DNA,proteases,and histone.However,the excessive NETs formation was the pathogenesis of many inflammatory and autoimmune diseases.Therefore,there is still an urgent need for effective and safe therapies which can target pulmonary neutrophils against neutrophilic asthma.Here we hypothesized that site-specific inhibition of NETosis in the lungs could effectively suppress local inflammation,reverse airway remodelling,and improve pulmonary function,thereby alleviating neutrophilic asthma in mice,which was achieved by selectively attenuating oxidative stress.Herein,we constructed a ROS scavenging nanomaterial（i.e.,TPCD）from Tempol（Tpl）and phenylboronic acid pinacol ester（PBAP）,and then developed nanotherapy for site-specific delivery to the lungs in mice with neutrophilic asthma.In vitro and in vivo experiments were conducted to investigate the mechanism of this nanotherapy in the treatment of neutrophilic asthma,preliminary studies shown that the suppressed NETs formation might afforded beneficial effects on the balance of T helper 17（Th17）and regulatory T（Treg）cells by reducing NET trapping of na（?）ve T cells.Methods1.Construction and characterization of ROS-scavenging materials and nanoparticlesActivated with CDI,Tpl was covalently bonded withβ-CD in the presence of DMAP to form TCD.Subsequently,PBAP activated by CDI was covalently combined with TCD under the catalysis of DMAP to obtain TPCD.Fourier transform infrared（FT-IR）spectra and ¹H NMR spectra were used to characterize TPCD.Reactive oxygen scavenging nanoparticles（defined as TPCN）were prepared by modified nanoprecipitation self-assembly method.Dissolve lecithin and DSPE-m PEG2000 in water as the aqueous phase.Under vigorous stirring,add the methanol solution of TPCD into the aqueous phase to self-assemble to obtain TPCN.Transmission electron microscopy（TEM）and scanning electron microscopy（SEM）were carried out to observed the appearance of TPCN,and Malvern Zetasizer Nano ZS instrument was used to detect the size distribution and surface potential of TPCN.2.Evaluation of targeted therapeutic effect of TPCN in neutrophilic asthmaA neutrophilic asthma model was established by inducing with LPS and OVA.In vivo imaging system was employed to investigate the targeting ability of TPCN in the lungs of neutrophilic asthma by intravenous injection and aerosol inhalation of Cy7.5/TPCN.Using Cy5/TPCN,the distribution of TPCN in pulmonary leukocytes was analyzed after intravenous injection and aerosol inhalation by flow cytometry.Mice with neutrophilic asthma were treated with different drugs by intravenous（i.v.）injection and inhalation.After treatment,bronchoalveolar lavage fluid（BALF）was collected to detect the content of neutrophils and analyze the levels of TNF-α,H₂O₂,MPO,IL-17 and KC.Furthermore,immunofluorescence was employed to analysis the counts of neutrophil and the levels of MPO in lung tissues.In a separate study,lung tissues were performed with H&E and PAS staining.Invasive measurement of airway hyperresponsiveness was performed using a computer-controlled small animal ventilator system.3.Treatment of asthma in mice by a mitochondrial-targeting TPCN nanotherapyCy5/TTPCN modified with different proportions of stearyl triphenylphosphine（defined as STPP）were prepared by nanopreciptation self-assembly method,and the size distribution of different nanoparticles was measured by Nano ZS instrument.After the tail injection of Cy5/TTPCN modified different proportions of STPP,in vivo imaging system was used to explore the optimal ratio of STPP to achieve the best targeting in the lungs of neutrophilic asthma.After Cy5/TTPCN was co-incubated with A549 cells and neutrophils,mitochondria of the cells were labeled with Mitotracker Green,and the co-localization of the nanoparticles and mitochondria was observed by Confocal laser scanning microscopy（CLSM）.Mice with neutrophilic asthma were treated with TTPCN and TPCN according to the intravenous administration schedule.After the experiment,the counts of neutrophils and the levels of H₂O₂,MPO,KC,TNF-α,and IL-1βin BALF were measured to evaluated the efficacy of TTPCN.Observe the effect of TTPCN on alleviating airway stricture,inhibiting mucus secretion and inflammatory cell infiltration by H&E staining and PAS staining of the lungs.4.In vitro and in vivo mechanistic studies4.1 Study on the biological effects of TPCN in vitroAfter different concentrations of Cy5/TPCN were incubated with human epithelial cell line（A549 cells）or mouse peritoneal neutrophils for various periods of time,the cellular uptake behavior of TPCN was observed by flow cytometry and CLSM.The antioxidant effect of TPCN on the production of ROS in neutrophils stimulated by phorbol 12-myristate 13-acetate（PMA）was investigated by flow cytometry and CLSM,and the antioxidant capacity of TTPCN was further explored.ELISA was used to analyze the anti-inflammatory effects of TPCN on PMA-stimulated neutrophils to secrete TNF-α,KC,and IL-1β.The effect of TPCN on neutrophils migration was investigated by Transwell assay.4.2 Regulation of TPCN on pulmonary transcriptome genes in neutrophilic asthmaThe transcriptome differential genes of neutrophilic asthma compared with the normal group and TPCN treatment group were analyzed by RNA sequencing,and the results were verified by q RT-PCR,so as to provide genetic guidance for further exploring the anti-asthma mechanism of TPCN.4.3 In vitro inhibition of the formation of neutrophil extracellular traps（NETs）by TPCNAfter incubation with different concentrations of TPCN,neutrophils were stimulated to NETs formation by PMA.The levels of the main components（ds DNA,NE,MPO,and cit H3）of NETs were determined by fluorescence assay,ELISA and WB.The inhibitory effect of TPCN on NETs formation in vitro was further observed by CLSM.4.4 Analysis of NETs related biomarkers in lungs of asthmatic mice after treatment with TPCNQuant-i T Pico Green ds DNA reagent and mouse elastase ELISA kit were used to measure the levels of ds DNA and elastase in the acellular fraction of BALF according to the manufacturer’s protocols,respectively.Furthermore,the relative levels of cit H3 and elastase were explored with WB and CLSM.4.5 TPCN regulated immune imbalance in neutrophilic asthmaMice with neutrophilic asthma were treated with TPCN by i.v.injection.After single cell suspensions were prepared from lung or spleen tissues,flow cytometry was employed to investigate Treg cells and Th17 cells that were stained with the corresponding antibodies.4.6 TPCN regulated the differentiation of Treg cells by inhibiting the NETs formationUnder Treg cells differentiation conditions,the isolated na（?）ve CD4⁺T cells were co-incubated with freshly isolated peritoneal neutrophils that were stimulated with or without PMA,the effect of NETs on Treg cells differentiation was analyzed by flow cytometry.To test whether TPCN can influence Treg cell differentiation by inhibiting NETs formation,neutrophils were preincubated with or without TPCN and then stimulated with PMA before addition of na（?）ve CD4⁺T cells.The differentiation of Treg cells were detected by flow cytometry.After preincubated with or without TPCN,mouse peritoneal neutrophils were stimulated with PMA,then supernatants were eliminated and na（?）ve CD4⁺T cells were added.After incubation,cells were fixed with glutaraldehyde and dehydrated with ethanol and isopropanol,followed by SEM observation.5.Biosafety evaluation of nanoparticlesA549 cells were cultured with the gradient concentrations of TPCN or TTPCN for various durations to test the cell cytotoxicity.BALB/C mice were administered with TPCN by inhalation at a theoretical dose of 50mg/kg or 100 mg/kg for 7 days,and mice were weighed every other day.One week later,mice were euthanized and peripheral blood was collected for hematological analysis.Major organs were weighed and performed with H&E staining.In addition,the levels of H₂O₂,MPO,IL-1β,and TNF-αin pulmonary tissues were analyzed by ELISA after lung homogenization.TPCN were co-incubated with PMA-stimulated neutrophils or lung homogenate from mice with neutrophilic asthma,and the hydrolysates were detected by ¹H NRM and MADIF-TOF.Results1.By covalently conjugating Tpl and PBAP ontoβ-CD,a bioactive material（i.e.,TPCD）which can eliminate ROS was successfully synthesized.Calculation according to the1H NMR spectrum revealed 2 Tempol and 5 PBAP units in each TPCD.TPCD nanoparticles（defined as TPCN）that were produced by a nanoprecipitation/self-assembly method displayed well-defined spherical shape,with a narrow size distribution and a mean diameter of 80 nm,and a negative Zate-potential of-19 m V.2.TPCN could be effectively accumulated in the lungs of mice with neutrophilic asthma and specifically ingested by neutrophils after both i.v.injection and inhalation.TPCN notably decreased the ROS and MPO levels,and neutrophils counts in BALF collected from diseased mice.Moreover,TPCN effectively reduced typical pro-inflammatory cytokines,including TNF-α,IL-1β,and IL-17.In addition,TPCN could attenuate inflammatory cell infiltration,pulmonary edema,mucous secretion,and airway resistance.3.Using Cy5/TTPCN,confocal microscopic observation demonstrated excellent mitochondrial targeting capability of TTPCN in neutrophils and A549 cells.Meanwhile,TTPCN showed significantly higher pulmonary accumulation than TPCN after i.v.delivery in mice with neutrophilic asthma.Compared to TPCN,TTPCN more effectively inhibited the expression levels of ROS,TNF-α,IL-1β,KC,and MPO as well as the neutrophil count in BALF.Inspection on histological sections stained with H&E or PAS also revealed desirable efficacy of TTPCN.4.In vitro,microscopic observation and flow cytometric quantification showed effective endocytosis of TPCN by neutrophils and A549 cells,in both time-and dose-dependent manners.TPCN significantly inhibited PMA-induced ROS generation and suppressed the overexpression of typical pro-inflammatory cytokines in neutrophils.In addition,migration of neutrophils was remarkably attenuated after TPCN treatment.Of note,TTPCN more effectively reduced ROS production in neutrophils.5.RNA-seq showed the expression of neutrophil-associated genes Elane and Mpo were significantly increased in asthmatic mice than those healthy mice,which were recovered to the normal levels after TPCN therapy.q RT-PCR analysis confirmed these results.6.In vitro,expression levels of the characteristic components of NETs,such as extracellular double-stranded DNA（ds DNA）,NE,MPO,and cit H3,significantly increased in PMA-activated neutrophils,which were remarkably reduced by TPCN in a dose-response pattern.Further,CLSM observation showed that the release of extracellular ds DNA and cit H3that appeared as fibrous strands.By contrast,TPCN treatment significantly reduced the degree of NETosis and cit H3 area.7.In vivo,mice with neutrophilic asthma showed significantly higher levels of ds DNA and NE in BALF as well as NE and cit H3 in lungs,compared to normal mice.However,treatment with TPCN at either 0.1 or 1 mg/kg remarkably reduced expressions of ds DNA,NE,and cit H3 in BALF and lungs.Compared with those of healthy mice,the proportion of Treg cells in asthmatic mice significantly decreased,while an opposite change was observed for Th17 cells,resulting in a remarkably decreased ratio of Treg/Th17 cells.By contrast,treatment with TPCN significantly increased Treg cells and decreased Th17 cells,and therefore effectively reversed the proportion of Treg/Th17 cells.8.In vitro,NETs notably suppressed the differentiation of na（?）ve T cells into Treg cells,whereas normal neutrophils showed no significant effects.Treatment with TPCN significantly restored Treg cells differentiation from na（?）ve T cells in the presence of NETs.Further SEM observation revealed that na（?）ve T cells were covered by NETs,but TPCN decreased NETs coating on T cells.9.In vitro safety experiments shown that even high doses of TPCN or TTPCN had no significant cytotoxicity on A549 cells.After mice were inhaled TPCN at 50 or 100 mg/kg for7 consecutive days,there were no change in body weight gain and organ indices of major organs.H&E staining revealed no discernible destruction of tissue microstructure or infiltration of inflammatory cell in TPCN groups.Moreover,TPCN treatment did not lead to significant changes of H₂O₂,MPO,TNF-α,and IL-1βin lung tissues.Likewise,there were no abnormal changes in typical hematological parameters and biomarkers relevant to liver/kidney functions in TPCN-treated mice.In neutrophils and mouse lung homogenates,hydrolysis and/or metabolism of TPCN producedβ-CD and other water-soluble molecules.Conclusions1.The ROS-scavenging material TPCD was successfully constructed fromβ-CD,Tpl and PBAP,and the ROS-scavenging nanoparticles TPCN were prepared.2.TPCN could site-specifically distribute in pulmonary neutrophils of mice with neutrophilic asthma after i.v.injection and inhalation.Treatment with TPCN via either i.v.injection or inhalation in asthmatic mice afforded beneficial therapeutic effects,as manifested by significantly decreased oxidative stress,attenuated inflammatory reactions,inhibited mucus hypersecretion,suppressed airway remodeling,and improved pulmonary function.3.Surface decoration with a mitochondrial targeting unit could simultaneously enhance pulmonary delivery efficiency and in vivo efficacy of TPCN after i.v.administration.4.TPCN could markedly inhibit oxidative stress,inflammatory response,and NETs formation in neutrophils after efficient cellular internalization.Further,TPCN exhibited a good anti-asthmatic effect by inhibiting NETs formation,regulating Treg/Th17 cells balance,and maintaining immune homeostasis.In addition,TPCN could rescue NET-attenuated Treg cell differentiation from na（?）ve T cells,probably by decreasing NETs coating on T cells and inhibiting detrimental effects of the DNA/protein components of NETs on T cells.5.In vivo studies revealed good safety of TPCN after inhalation at a dose that is100-fold higher than those examined in therapeutic studies.In summary,our findings demonstrated nanotherapy-mediated site-specific attenuation of oxidative stress and inflammatory responses in neutrophils is a promising strategy for effective treatment of neutrophilic asthma,by precisely inhibiting NETosis and promoting immune homeostasis.Our results also suggested that the ROS-NETs-Treg/Th17 axis may be potentially new therapeutic targets for severe asthma and other neutrophilic inflammation-associated noninfectious and infectious lung diseases such as adult respiratory distress syndrome,chronic obstructive pulmonary disease,cystic fibrosis,and COVID-19,in view of the important pathological effects of neutrophils.