Study On Construction Of A Neutrophil Extracellular Trap Inhibiting Nanotherapy And Its Efficacies And Mechanisms For Targeted Treatment Of Abdominal Aortic Aneurysm

Abdominal aortic aneurysm（AAA）,characterized by a permanent dilatation of abdominal aortas which defined as 1.5 times larger than the normal diameter,remains most commonly diagnosed aneurysm in clinical practice.Currently,open surgical repair and endovascular aneurysm repair（EVAR）are the only two effective clinical intervention for AAA patients to prevent death caused by rupture.However,high risk of surgery and 6%post-operative mortality reduce compliance of AAA patients and 90%of AAA patients are not eligible for surgical and endovascular repair.So far,most pharmacological treatment cannot provide beneficial effects.Consequently,developing novel and effective therapies represents an attractive alternative.In view of the pivotal role of neutrophilic inflammation in the pathogenesis of AAA,herein we propose a new paradigm for targeted therapy of AAA by site-specifically regulating neutrophil extracellular traps（NETs）.A nanotherapy based on a bioactive oligosaccharide material was first engineered.After efficient accumulation in the aneurysmal aorta and localization in pathologically relevant inflammatory cells via passive targeting in rats with Ca Cl₂-induced AAA,nanoparticles significantly alleviated AAA progression,as implicated by the decreased aortic expansion,suppressed elastin degradation,inhibited calcification,and improved structural integrity of the abdominal aorta.By functionalizing nanoparticle with a calcification-targeting moiety,in vivo aneurysmal targeting capability of nanoparticle was considerably enhanced,thereby affording significantly potentiated therapeutic outcomes in AAA rats.Mechanistically,nanoparticles can effectively inhibit neutrophil-mediated inflammatory responses in the aneurysmal aorta.In particular,nanoparticles potently attenuated NETs,thereby suppressing NETs-mediated pro-inflammatory events and NETs-associated negative effects responsible for AAA progression.Nanoparticles also displayed good safety in preliminary studies.Methods1.Inflammatory cell infiltration during AAA developmentThe sections of infrarenal abdominal aortas isolated at different days after Ca Cl₂treatment were stained with hematoxylin and eosin（H&E）,elastin van Gieson,Alizarin Red,anti-MPO antibody or anti-CD68 antibody to evaluate the relationship between morphology changes and inflammatory cell infiltration.2.Synthesis and characterization of a bioactive material based onα-CDA bioactive material（defined as LaCD）was synthesized by covalently conjugating luminol ontoα-CD and characterized by FT-IR,UV-Vis and ¹H NMR.3.Fabrication and characterization of NETs-inhibiting nanoparticles by nanoprecipitationLaCDnanoparticle（LaCDNP）was prepared by a nanoprecipitation method.Briefly,LaCDwas dissolved in DMF,into which deionized water was added slowly.Based on the similar procedures,LaCDNP labeled with Cy3 or Cy5 were fabricated.Mean sizes,size distribution profiles,andζ-potential values of NPs were measured by a Malvern Zetasizer Nano ZS instrument.The morphology of LaCDNP was observed by transmission electron microscopy（TEM）and scanning electron microscopy（SEM）.4.In vivo aneurysm targeting capability and biodistribution of LaCDNP in ratsCy5/LaCDNP was administrated by intravenous injection in AAA rats.At 8 h after injection,rats were euthanized and the whole aortas were collected.Ex vivo imaging was conducted with an IVIS Spectrum living imaging system.In a separate experiment,the sections of aortas were stained with anti-MPO antibody,anti-CD68 antibody,anti-α-smooth muscle actin（α-SMA）antibody,and anti-CD31 antibody.Images of abdominal aortas were acquired by fluorescence microscopy after nuclei were stained with DAPI.5.In vivo therapeutic effects of different nanotherapies in rats with AAAAfter establishment of AAA,the rats were randomly divided into five groups.Different doses of LaCDNP were administrated via i.v.injection twice a week at day 2 after Ca Cl₂application in the nanotherapy groups.At day 23 after different treatments,the abdominal aorta diameter were evaluated by ultrasonography and digital photos.Subsequently,sections of abdominal aortas were stained with H&E,elastin van Gieson,or Alizarin Red to evaluate vascular morphology,elastic fibers and calcium deposition,respectively.For immunohistochemistry analysis,the sections were incubated with anti-CD68antibody,anti-CD31 antibody,anti-MMP-2 antibody,anti-MMP-9 antibody.The levels of MMP-2 and MMP-9 were examined by Western blot analysis.Besides,the levels of tumor necrosis factor（TNF）-α,interleukin（IL）-1β,monocyte chemoattractant protein（MCP）-1,and C-X-C motif chemokine ligand 1（CXCL1）were quantified by corresponding ELISA kits.Calcium contents and hydrogen peroxide levels in the aortas were evaluated by the corresponding kits.6.Synthesis and characterization of ALN-functionalized LaCDNPsAlendronate-functionalized DSPE-PEG（defined as DSPE-PEG-ALN）was synthesized by co-dissolving DSPE-PEG-Maleimide and ALN.To determine the ALN content in DSPE-PEG-ALN,a previously reported method was used by quantifying the formation of chromophoric complex between ALN and Fe3+in perchloric acid solution.Then,to fabricate ALN-decorated LaCDNPs（defined as ALaCDNPs）,the mixtures of DSPE-PEG-ALN and DSPE-PEG were co-dissolved in deionized water.Then,lecithin dispersed in ethanol was added into the aqueous solution.Subsequently,the water phase was dropped slowly into DMF containing LaCD.The resulting ALaCDNPs were also collected after centrifugation.In another separate study,characterization of ALaCDNP was conducted in the similar procedures described above.7.In vitro calcification-targeting and in vivo AAA targeting capability of ALaCDNPTo examine calcification-targeting capability of ALaCDNP in vitro,hydroxyapatite microparticles were incubated with Cy3/LaCDNP or Cy3/ALaCDNP.Fluorescence images were captured by an inverted fluorescence microscope.Additionally,to examine active targeting capacity of ALaCDNPs,at 8 h after i.v.administration of Cy5/ALaCDNPs in AAA rats,ex vivo fluorescence images were acquired and quantitative analysis was conducted.8.In vivo therapeutic effects of ALaCDNP in AAA ratsFollowing the similar procedure mentioned above,therapeutic effects of LaCDNP and ALaCDNP at 50 mg/kg were compared.Additionally,ALaCDNP was i.v.administrated twice a week at day 8 after AAA induction by Ca Cl₂.After treatment for five times,different evaluations were performed.9.Mechanism studies in vitro and in vivo（1）Biological effects of LaCDNP in vitroNeutrophils were incubated with Cy5/LaCDNP for predetermined time periods,followed by confocal laser scanning microscopy（CLSM）observation.Similarly,dose-dependent cellular uptake profiles were examined after cells were incubated with varied doses of Cy5/LaCDNP.Fluorescence intensities were determined via flow cytometry for quantitive analysis.A Transwell assay was used to examine the anti-migration activity of LaCDNP.3-μm pore polycarbonate inserts were planted onto a 24-well plate to evaluate migration of neutrophils.In addition,8-μm pore polycarbonate inserts were placed onto a 12-well plate with or without the neutrophil suspension to evaluate the migration of macrophages induced by neutrophils.Images of migrated cells were obtained and counted by optimal microscopy.（2）RNA-sequencing analysis of aortic tissuesAfter different treatments,the abdominal aortas were collected from rats.The total RNA was extracted from the aorta using Trizol according to the manual instruction.Essentially,analysis of differential expression was conducted by the DESeq2.The significant levels of terms and pathways were corrected by Q value with a rigorous threshold（Q value≤0.05）.（3）Analysis of the MPO expression by activated neutrophils and in aortic tissuesMPO levels excreted by neutrophils after stimulation with PMA and treatment with different formulations were quantified by ELISA.Also,the contents of MPO in the aortic tissue homogenates were detected.The sections of aortas were stained with anti-MPO antibody and anti-CD177 antibody.Immunofluorescence images were captured by a confocal microscope.（4）Analysis of the NETs formation after treatment with LaCDNPThe abdominal aortas were collected from AAA rats after different treatments.Then,aortic sections were stained with anti-CitH3 antibody and anti-neutrophil elastase（NE）antibody.Fluorescent images were captured after nuclei were counterstained with DAPI.In addition,neutrophils were incubated with LaCDNP and then stimulated by PMA for 4h.After that,neutrophils were stained with anti-CitH3 antibody.CLSM images were acquired after nucleic acids were stained with SYTOX green.The levels of double-stranded DNA（dsDNA）and NE in different groups were measured by ELISA.（5）Effects of LaCDNP on NETs-induced VSMCs apoptosisNETs were collected after neutrophils were incubated with various doses of LaCDNP and then induced by PMA.Then,VSMCs were incubated with NETs obtained from different groups.VSMCs apoptosis was assayed by using FITC annexin V apoptosis detection kit with propidium iodide.Finally,the collected VSMCs were analyzed by flow cytometry.10 Preliminary biosafety evaluation of nanoparticlesVSMCs were incubated with different doses of LaCDNP for 6 or 12 h,the cell viability was examined by CCK-8 assay.Then,rats were i.v.administered with LaCDNP or ALaCDNP at 500 mg/kg,respectively.Then,rats were weighed at predetermined time.All rats were euthanized at day 14.Blood samples were acquired for hematological analysis.The organ index was measured as the ratio of organ weight to the body weight.In addition,histopathological sections of major organs were stained with H&E.Results1.Inspection on aortic sections indicated that notable pathological changes in the aortas could be found at day 4.Besides,immunofluorescence analysis revealed that macrophages showed delayed infiltration as compared to neutrophils,which affirmed that neutrophils may serve as an initiator to mediate the AAA development by inducing the recruitment of macrophages,thereby representing a key therapeutic target.2.Luminol-conjugatedα-cyclodextrin（α-CD）（LaCD）was synthesized and characterized by different spectroscopy measurements.According to ¹H NMR spectrum,approximately 1-2luminol units were conjugated onto eachα-CD molecule.LaCDNP could be easily produced by nanoprecipitation method.Observation by TEM and SEM indicated that LaCDNP displayed well-defined spherical shape,with relatively narrow size distribution.The mean diameter of LaCDNP was 221 nm,while theζ-potential was-17.8±1.2 m V.3.Ex vivo imaging showed notable fluorescence at the injury site of abdominal aortas isolated from AAA rats,compared to the normal aorta.Further,immunofluorescence analysis showed considerable co-localization of Cy5/LaCDNP with MPO⁺neutrophils and CD68⁺macrophages.These results demonstrated that LaCDNP delivered by i.v.injection can efficiently accumulate in the aneurysmal aorta and localize in pathologically relevant cells,particularly neutrophils.4.Subsequently,direct observation and transabdominal ultrasound imaging of the isolated abdominal aortas revealed enlarged aortas in the model group.These abnormalities were notably mitigated after therapy with LaCDNP.Further,histological and immunohistochemistry analysis of aortic sections showed significant therapeutic effects after treatment with LaCDNP.Additionally,the levels of representative pro-inflammatory cytokines and chemokines could be significantly decreased,which indicated anti-inflammatory effects of LaCDNP.Collectively,these results demonstrated that treatment with LaCDNP could efficaciously delay the development.5.ALN-conjugated DSPE-PEG was synthesized and verified by ¹H NMR spectroscopy and complexation tests.ALN-decorated LaCDNP（ALaCDNP）was prepared by a nanoprecipitation/self-assembly method.ALaCDNPs displayed spherical morphology,with narrow size distribution profiles as well as comparable average diameters and negativeζ-potential values.6.After incubation with Cy3/ALaCDNP,the whole microparticles showed strong fluorescence signals,which suggested that ALaCDNP can effectively bind to calcium-enriched hydroxyapatite.Further,in vivo targeting effects of ALaCDNP were evaluated.Ex vivo imaging revealed a significantly enhanced accumulation of ALaCDNPs in isolated abdominal aortas,as compared to LaCDNP without ALN coating.Moreover,the anti-aneurysmal activity of LaCDNP was significantly potentiated by decorating with ALN according to the therapeutic effect evaluations.7.RNA-sequencing analysis of abdominal aortas from AAA rats showed that LaCDNP remarkably reduced the abnormal expression of the Mpo gene.Both confocal microscopy observation and flow cytometry quantification clearly showed time-dependent and dose-dependent internalization of Cy5/LaCDNP in neutrophils.Further,Transwell assay suggested that LaCDNP could significantly inhibit the migration of neutrophils and macrophages induced by neutrophils.Moreover,LaCDNP effectively inhibited the MPO expression in PMA-stimulated neutrophils and aneurysmal aortas of AAA rats.These results substantiated that the nanotherapy LaCDNP can alleviate the AAA development by attenuating neutrophil-mediated inflammatory responses.8.At day 4 after adventitial Ca Cl₂ application in rats,immunofluorescence analysis of abdominal aortic cryosections revealed the notable expression of citrullinated histone H3（CitH3）and neutrophil elastase（NE）,two typical biomarkers of NETs.In vitro studies showed that LaCDNP effectively reduced the release of CitH3,double-stranded（dsDNA）,and NE by PMA-activated neutrophils.Moreover,therapy with LaCDNP significantly decreased the NETs formation in the abdominal aorta.In addition,NETs released by activated neutrophils significantly increased apoptosis of VSMCs.By contrast,when neutrophils were pre-treated with LaCDNP,VSMCs apoptosis was significantly decreased.Agreeing with this finding,immunohistochemistry analysis of the abdominal aortic sections showed notably inhibited loss of VSMCs and significantly reduced cell apoptosis in LaCDNP groups.9.After incubation with different doses of LaCDNP for 6 or 12 h,relatively high cell viability was detected.Even 5 mg/m L LaCDNP did not cause discernible hemolysis.Additionally,all rats treated with either LaCDNP or ALaCDNP with a single i.v.injection at500 mg/kg exhibited good biosafety in acute toxicity tests.Conclusion1.We have successfully synthesized a neutrophil extracellular trap inhibiting nanotherapy LaCDNP based on a bioactive cyclodextrin material,for precisely regulating neutrophilic inflammation for effective treatment of AAA.Taking advantage of the passive targeting effect,LaCDNP can efficiently accumulate at the aneurysmal site and efficaciously delay the development of Ca Cl₂-induced AAA in rats.2.By functionalizing LaCDNP with a calcification-targeting moiety ALN,the obtained nanotherapy ALaCDNP showed significantly enhanced accumulation in aortic aneurysms,thereby offering notably potentiated therapeutic effects on attenuating AAA development,as compared to LaCDNP.3.Mechanistically,LaCDNP can be efficiently endocytosed by neutrophils as well as inhibit neutrophil recruitment and activation.RNA-seq implied that activated neutrophils and enhanced MPO expression play a central role in therapeutic effects of LaCDNP.Consistently,we found that LaCDNP can efficaciously inhibit the NETs formation and suppressing NETs-mediated apoptosis of VSMCs.Consequently,our nanotherapies can effectively attenuate AAA progression by site-specifically inhibiting neutrophilic inflammation,in particular,the NETs-amplified inflammatory response and relevant negative effects.4.Preliminary safety evaluations suggested that LaCDNP and ALaCDNP exhibited good biosafety for i.v.administration at the examined dose.