| Background:Cardiovascular diseases(CVDs)remain the leading cause of morbidity and mortality worldwide.It has been estimated that CVDs may result in about 17.9 million deaths each year,accounting for?31%of all global deaths.Vascular inflammation is closely related to the pathogenesis of a diverse group of CVDs,such as atherosclerosis,myocardial infarction,restenosis,intracranial and aortic aneurysms,stroke,and peripheral artery disease.By regulating different molecular and cellular processes involved in the inflammatory response,a large number of therapeutics have been investigated to prevent and treat CVDs.Despite major achievements in preclinical studies,desirable efficacy of most examined anti-inflammatory agents has not been fully demonstrated in clinical practice.To a large degree,this may be associated with inefficient delivery of therapeutic molecules to the site of vascular inflammation,resulting from their nonspecific distribution and rapid elimination from the circulation.Even for locally absorbed drugs in the inflamed blood vessels,their retention time is very short due to uncontrolled diffusion.Recently,nanoparticle(NP)-based targeting has been considered as a promising strategy for site-specific delivery of different therapeutic and imaging agents to detect or treat vascular inflammation.In particular,a broad spectrum of NPs have been engineered as targeting earriers for treatment of atherosclerosis,myocardial infarction,heart failure,ischemia-reperfusion injury,critical limb ischemia,restenosis,abdominal aortic aneurysm,and ischemic stroke.In these cases,polymeric and lipid NPs,liposomes,recombinant high-density lipoproteins,cell-derived vesicles,inorganic and metal NPs,and hybrid NPs have been used for delivery of therapeutic agents varying from small-molecule drugs,peptides/proteins,to nucleic acids,for the management of CVDs associated with vascular inflammation.In addition to passive targeting via the damaged endothelial cell layer or the enhanced permeability and retention effect,vascular targeting efficiency of NPs can be further increased by modulating their physical properties(such as geometry and flexibility),decorating with molecular moieties,or functionalizing with specific cell membranes.On the other hand,the compositions of NPs can be designed and tailored to release cargo molecules in response to abnormally changed biochemical cues at the inflammatory sites of blood vessels.However,translation of these vascular targeting nanotherapies remains challenging.Whereas vascular accumulation of NPs can be enhanced by directly manipulating epitope-specific affinity,targeting efficiency post decoration with molecular moieties alone is still very limited.For nano therapies derived from the cell membrane-based biomimetic strategy,the relatively complicated formulation procedures and undefined components may hinder their following large-scale production and clinical studies.Also,uncontrolled release at the vascular sites of interest need to be further improved.Increasing evidence has demonstrated that NPs responsive to dual or multiple stimuli can more precisely control their cargo release profiles at the site of action,thereby affording considerably potentiated efficacies.In this aspect,NPs sensitive to multiple biochemical signals or biochemical/physical signals have been extensively examined for targeted treatment of diverse diseases,such as cancers,diabetes,and inflammatory diseases.Nevertheless,bench-to-bedside translation of these responsive nanotherapies is not straightforward,largely resulting from more complex pharmaceutical development,especially in regard to poor reproducibility and quality control.Moreover,in vivo safety is another important issue that should be fully addressed for most responsive nanocarriers currently developed for vascular targeting,particularly for the treatment of chronic diseases,since the majority of intravenously injected NPs will be cleared by the mononuclear phagocyte system and accumulate in organs such as liver and spleen.Consequently,there is a crucial need to develop more effective and safe NPs with desirable targeting capacity and well-controlled drug release performance in response to biochemical signals relevant to vascular inflammation.By facile chemical functionalization of cyclodextrins,our group has recently developed a series of responsive materials with highly tailorable hydrolysis behaviors under acidic and oxidative conditions.Both in vitro cell culture experiments and in vivo evaluations in different animal models demonstrated that NPs based on these materials display good safety profile.Herein we hypothesize that pH and reactive oxygen species(ROS)dual-responsive NPs engineered by integrating pH-and ROS-responsive cyclodextrin materials can serve as an effective and safe nanoplatform for therapeutic delivery to sites of vascular inflammation,in view of the presence of acidosis and oxidative stress at inflammatory sites.Furthermore,vascular targeting and in vivo efficacies of pH/ROS dual-responsive nanotherapies can be additionally enhanced by combination with a molecular targeting strategy.In an animal model of vascular inflammation in rats subjected to balloon injury in carotid arteries,therapeutic advantages of the dual-responsive nanotherapy were first affirmed,by comparison with non-responsive and pH-or ROS-responsive nanotherapies,using rapamycin as a model drug.Then we demonstrated in vivo targeting and therapeutic effects of the dual-responsive,active targeting nano therapy.Methods:1.Synthesis of carrier materials based on ?-cyclodextrin1.1 Synthesis of a pH-responsive materialAcetalation of p-CD was performed at room temperature in the presence of excess amount of MP,using PTS as a catalyst.Briefly,4 g p-CD was dissolved in 80mL anhydric DMSO,then 16 mL MP and 64 mg PTS were added.After 3 h,approximately 1 mL of triethylamine was added to terminate the reaction.Finally,the acetal products were collected by centrifugation after precipitation with water,and washed with deionized water four times.The residual water was removed by lyophilization to give rise to white powder.1.2 Synthesis of a ROS-responsive material5.55 g PBAP was dissolved in 36 mL of anhydrous dichloromethane,and then 7.65 g CDI was added.After reaction for 30 min,40mL DCM was added again and washed with 30ml deionized water for three times.The organic phase was further washed with saturated NaCl solution,dried with anhydrous NaZSO4 for 0.5 h,and vacuum concentrated to obtain CDI activated PBAP.Subsequently,250 mg ?-CD and 1.52 g CDI-activated PBAP were dissolved in 20 mL of anhydrous DMSO,Then 0.8 g DMAP was added.The resulting mixture was stirred magnetically overnight at 20?.The final product was precipitated from 80 mL of deionized water,collected by centrifugation,rinse thoroughly with deionized water,and collected after lyophilization.The obtained carrier material was characterized by 1H NMR spectroscopy and FT-IR spectroscopy.2.Preparation of different nanoparticlesRAP-loaded nanoparticles(NPs)based on different materials were prepared by Nanoprecipitation/self-assembly method.Specifically,50 mg carrier material and 10 mg RAP were co-dissolved in 2 mL of organic solvent to obtain an organic phase.To obtain an aqueous phase,9 mg DSPE-PEG2000 and 6 mg lecithin were first dispersed in 0.6 mL anhydrous ethanol,and 10 mL deionized water was added.followed by heating at 65 C for 1 h.Then,the organic phase was slowly added to the pre-heated aqueous phase solution drop by drop under magnetic agitation.After vortexing for 3 min,the mixture was cooled to room temperature,incubated for 2 h,and then dialyzed against deionized water at 25 C for 24 h.Finally,the solidified NPs were harvested by lyophilization.For PLGA and ACD NPs,acetonitrile was used,while solvent mixture of methanol and acetonitrile at 1:1(v/v)was employed to prepare the organic phase containing OCD or ACD/OCD blends at various weight ratios(varying from 80:20,60:40,50:50,40:60,to 20:80).Through similar procedures,blank NPs and Cy5-or Cy7.5-labeled NPs were prepared.3.Characterization of nanoparticlesThe nanoparticle size,size distribution and zeta-potential of different nanoparticles in aqueous solution were detected by a Malvern Zetasizer.The shape of nanoparticles was observed by TEM and SEM.For RAP-loaded NPs,the drug concentration was measured by HPLC.the contents of Cy5 or Cy 7.5 were determined by fluorescence spectroscopy.4.Hydrolysis of in vitro nanoparticles in different solutionsHydrolysis profiles of different NPs were separately earried out in 0.01 M PBS at pH 5,pH 6,or pH 7.4,with or without 1 mM HZO2.After incubation at 37 C for varied time periods,transmittance of different NP-containing solutions was determined at 500 nm by UV-Visible spectroscopy.The degree of hydrolysis was calculated based on the transmittance values.5.In vitro release testsTo test pH/ROS dual-responsive drug release profiles,5 mg RAP-containmg NPs freshly prepared was incubated at 37 C in 8 mL of 0.01 M PBS at pH 5,pH 6,or pH 7.4,with or without 1 1M H2O2,with shaking at 125 rpm.At a specific time interval,the aqueous solution containing the blank nanoparticles was centrifuged at 19118 9,and 4 mL releasing medium was taken out.Meanwhile,the corresponding fresh medium of the same volume was supplemented.The concentration of RAP was quantified as aforementioned.6.Studies on cellular uptake profiles in rat VSMCsMOYAS cells were seed to 12-well plates at 2×105 cells per well in 1 mL of growth medium.After 24 h,the culture medium was removed and 1 mL of fresh medium containing Cy5-labeled NPs at 20 ?g/mL was added,followed by incubation at 37? for various periods of time.Before observation,the late endosomes and lysosomes were stained with lysosome probes(50 nM,incubation for 2h),and the nuclei were stained with DAPI.Fluorescence images were obtained by CLSM.Likewise,dose-dependent cellular internalization behaviors were examined after incubation for 6 h.For quantification of internalized NPs by flow cytometry,MOVAS cells were seeded in 12-well plates at a density of 2 7×105 cells per well.After 24 h,the culture medium was switched to 1 mL of fresh medium containing Cy5-labeled NPs with a concentration of 20 ?g/mL and incubated for different periods.Then,the cells were digested,centrifuged and collected,fluorescence intensity was measured by FACS.Dose-dependent internalization profiles were detected in a similar manner,with incubation time of 6 h.7.In vitro anti-proliferative and anti-migration activity of different nanotherapies against rVSMCs in ratsMOVAS cells were seeded in a 96-well microplate at 5× 103 cells per well in DMEM and incubated at 37"C.After 24 h of incubation,the medium was changed to fresh culture medium containing 0.5%FBS and 20 ng/mL PDGF-BB,concomitant with the addition of various RAP formulations containing 1 ?M of RAP.The normal control group was treated with growth medium only,and the positive control group was treated with PDGF-BB alone.After incubation for 24 hours,cell viability was measured by CCK-8 assay.A Transwell experiment of VSMCs was carried out to evaluate the anti-migration activity of different nanotherapies by using a modified Boyden chamber using the Costar Transwell apparatus with 8.0-?m pore size.Briefly,MOVAS cells were seeded in 12-well plates at a density of 2 × 105 cells/well and allowed to adhere overnight.The culture medium was then switched to DMEM containing 0.5%FBS,into which PDGF-BB was added at 20 ng/mL.Cells were incubated with various RAP formulations containing 1 ?M of RAP for 6 hours.In the positive control group,cells were induced with PDGF-BB alone,while PDGF-BB and RAP were not added in the normal control group.Subsequently,each well was washed,digested with trypsin,resuspended with 0.2ml growth medium containing 0.5%fetal bovine serum,and plated to the upper chamber.The lower chamber was filled with 0.6 mL of medium containing 0.5%FBS and 20 ng/mL PDGF-BB.After 8 h,all non-migrated cells were gently removed from the upper face of the Transwell membrane with a cotton swab.Migrated cells were fixed in 4%paraformaldehyde and stained with 0.1%crystal violet.Finally,the migrated cells were quantified by counting the number of stained cells with a Nikon microscope.8.Cell cycle assayCell cycle assay was performed as described previously.MOVAS cells were seeded into 6-well plates at a density 5 ×105 cells per well and treated with various RAP formulations containing 1 ?M of RAP for 24 h.In the positive group,cells were only stimulated by PDGF-BB,while in the normal group,cells were treated with culture medium.The cells were harvested at 24 h after different treatments and fixed by 70%ethanol,followed by incubation overnight at 4?.After through washing,cells were treated with ribonuclease(RNase)(0.5 mg/mL)at 37OC for 30 min,and stained with propidium iodide(50 ?g/mL)at 4? for 30 min in the dark.The cells were analyzed by flow cytometry to determine the proportion of cells within the Gl,S,and G2/M phases.9.Western blot analysisMOVAS cells in the normal,model,RAP/PLGA NP,RAP/ACD NP,RAP/OCD NP,and RAP/AOCD NP groups were treated with medium alone,PDGF-BB,PDGF-BB plus RAP/PLGA NP,PDGF-BB plus RAP/ACD NP,PDGF-BB plus RAP/OCD NP,and PDGF-BB plus RAP/AOCD NP,respectively.The cells were harvested after at 24 h after different treatments to detect the levels of cell cycle-related proteins by Western blotting.To this end,cells were lysed in lysis buffer.The lysates were centrifuged at 12,000 g for 20 min at 4?.The supernatant was collected and protein concentrations were determined using a Bicinchoninic acid(BCA)assay kit.Then samples containing 50 ?g proteins were separated on 10%sodium dodecyl sulfonate(SDS)-polyacrylamide gels and were transferred electrophoretically on nitrocellulose membranes.After blocking nonspecific binding with 5%skim milk,the membranes were incubated with the following primary antibodies at 4“C overnight:(1)Cyclin D1 antibody,dilution 1:200;(2)p27kipl antibody,dilution 1:500;(3)?-actin antibody,dilution 1:500.Subsequently,the membranes were hybridized at 37? for 2 h with horseradish peroxidase-conjugated secondary antibody at a 1:2000 dilution.After washing three times,specific bands were visualized by fluorography using an enhanced chemiluminescence kit.The relative densities were quantified using the Quantity One analysis system.10.Cytotoxicity of various nanoparticlesMOVAS cells were seeded in a 96-well plate and grown for 24 h.The culture medium was removed and the cells were treated with 100 ?L of fresh medium containing differentconcentrations of blank NPs.After 24 h,cell viability was measured by CCK-8 assay.11.Establishment of a carotid balloon injury model in ratsMale Sprague-Dawley rats(weighing 400-450g)were administered with aspirin by oral gavage and heparin by i.v.injection before surgery.Immediately,pentobarbital at 0.7 mg/kg were intraperitoneally injected for anesthesia.Subsequently,the left external carotid arteries were exposed,and the endothelium of common carotid arteries was denuded by intraluminal passage of a 2-French arterial embolectomy catheter,which was passed to the proximal common carotid artery and then withdrawn.This procedure was repeated three times.All procedures were carried out by the same operator.Then,Endothelial injury of the carotid artery was assessed by staining with Evans blue.12.Evaluation of acidic inflammatory microenvironment and oxidative stress in the injured carotid arteriespH changes at the injured sites of carotid arteries were qualitatively evaluated by using Using an intracellular ratiometric pH indicator BCECF-AM.Similarly,the carotid artery tissues were collected at various time points after balloon injury in the carotid artery.Then the carotid arteries were frozen in Tissue-Tek O.C.T.Compound.The frozen arteries were cut into 8-?m sections.Subsequently,the sections were separately incubated with 10 uM DCFDA or 5 ?M DHE.The stained sections were observed by fluorescence microscopy.Also,the levels of hydrogen peroxide(H2O2)and superoxide dismutase(SOD)activity were determined using the corresponding diagnostic reagent kits.The levels of malondialdehyde(MDA)and myeloperoxidase(MPO)were separately detected with the related ELISA kits.Also,the levels of tumor necrosis factor(TNF)-? and interleukin(IL)-1? were separately measured by ELISA kits.13.Fabrication of RAP-loaded and collagen ? targeting nanoparticlesCollagen IV-targeting peptide(KLWVLPKGGGC-Am)was reduced using Bond-breaker TCEP solution,Neutral pH in PBS containing 5 mmol/L EDTA at a disulfide/TCEP molar ratio of 1:1.Then KLWVLPKGGGC-conjugated DSPE-PEG was synthesized in 4%ethanol at a peptide/DSPE-PEG-maleimide molar ratio of 5:4,under magnetic stirring at room temperature for 4 h.The free peptide was removed by dialysis overnight.The above mentioned method was then adopted to prepare RAP-loaded and collagen IV-targeting NPs based on a blend of at an OCD/ACD weight ratio of 80:20,with a DSPE-PEG-peptide/DSPE-PE molar ratio of 1:9.14.Collagen binding by targeting nanoparticlesType ? collagen was dissolved in 0.25%acetic acid at a concentration of 0.5 mg/mL.Then 100 ?L of the collagen solution was added into a 96-well plate and incubated overnight at 4 C.For the binding study,collagen-coated or non-coated plates were first blocked with 50%calf serum for 1 h and then incubated with 100 ?L of Cy7.5/TAOCD NP(0.2 mg/mL)in 25%calf serum.After 1 h of incubation,the microplates were washed with PBS containing 0.05%Tween 20 three times.Subsequently,fluorescence images were taken by an IVIS Spectrum system.15.Evaluation of targeting ability of AOCD NP to the injured carotid arteryAfter the establishment of balloon injury model of carotid artery in SD rats,cy7.5 labeled nanoparticles were injected into tail vein immediately.After 8 h,the whole carotid artery tissues were harvested for in vivo imaging and the fluorescence intensity of carotid artery tissue was analyzed.In a separate study,carotid artery injury rats were injected with Cy7.5/AOCD NP via the tail vein,and carotid artery tissue was collected at specific time points,followed by in vitro imaging and quantitative analysis of tissue fluorescence intensity.16.Specific binding of targeting nanoparticles to the injured carotid arteryFollowing carotid artery balloon injury in rats,Cy5/TAOCD NP was i.v.injected at 25?g/kg.After 0.5 h,carotid artery tissues were harvested and frozen in Tissue-Tek O.C.T.Compound.The frozen arterial tissues were then cut into 5-?m sections and placed on glass slides.After nuclei were stained with DAPI,the sections were observed by CLSM.17.Effect of different nanotherapies on balloon injury of carotid artery in SD ratsThirty male SD rats were randomly divided into 6 groups(n=5),including the normal group(sham operation group),the model group and the groups separately administered with RAP/ACD NP,RAP/AOCD NP,RAP/OCD NP or RAP/PLGA NP.Except rats in the sham operation group,rat carotid artery balloon injury was induced in other rats according to the above described procedures.After endovascular angioplasty,different RAP formulations were given at lmg/kg immediately after surgery and respectively on day 4,day 8 and day 12 via the tail vein.In a separate study,RAP/AOCD NP or RAP/TAOCD NP were i.v.administered at 1 mg/kg of RAP at days 0,5,and 10 after carotid balloon injury was induced in rats.On day 14 after angioplasty,rats were euthanized.The carotid arteries and major organs were excised for further histological and immunohistochemical studies.18.Acute toxicity evaluation of nanoparticles and Chronic toxicity evaluation of the dual-responsive RAP nanotherapy in SD ratsTwelve male SD rats(180-250 g)were randomly divided into 4 groups(n=3).In AOCD NP groups,rats were treated with 1 mL of saline containing different doses of AOCD NP(at 250,500,or 1000 mg/kg)by intravenous injection through the tail vein.Rats(control group)were administered with 1ml saline via tail vein.For all animals,signs of toxicity and their behaviors were observed for about 14 days.The body weight was checked at defined time points.After 14 days,the animals were euthanized.Blood samples were collected for hematological and biochemical tests.Meanwhile,the main organs were collected and weighed.The organ index was calculated,tissue sections were prepared and H&E staining was performed.To assess the long-term toxicity of the dual-responsive nanotherapy in rats,healthy male SD rats(180-250 g)were randomly divided into four groups.The vehicle group was treated with AOCD NP at 11.5 mg/kg(corresponding to the nanotherapy dose at 1 mg/kg),while two groups were separately administered with RAP/AOCD NP at 1 or 3 mg/kg by i.v.injection 2 times per week for consecutive 12 weeks.In the normal control group,rats were i.v.administered with saline.All animals were observed daily for mortality,general appearance,and behavioral abnormality.Food and water consumption as well as body weight were recorded weekly.At week 12,rats were euthanized.Blood samples were collected for analysis of representative hematological and biochemical parameters.The major organs were excised and weighed for calculation of the organ index.Furthermore,H&E stained histological sections were prepared for the collected organs.In addition,immunohistochemistry analysis of splenic sections was performed after staining with anti-rat CD3 and anti-rat CD 19 antibody.19.Quantification of T and B cells in the spleen of mice after long-term treatment with RAP/AOCD NPTo further evaluate the effects of RAP/AOCD NP treatment on the adaptive immune system,24 male C57BL/6J mice(18-22 g)were randomly divided into four groups.Mice in the normal control were administered saline,while the vehicle group was treated with AOCD NP at 1 mg/kg by i.v.injection.Mice in the other two groups were separately administered with RAP/AOCD NP at 1 or 3 mg/kg by i.v.injection 2 times per week for consecutive 12 weeks.During treatment,mice were monitored for any changes in the general physical conditions,such as appearance,behaviors,and mortality.At week 12,mice were euthanized.The isolated splenic tissues were cut into pieces,which were thoroughly grinded in the presence of sterile PBS.Then cells were collected by centrifugation in the lymphocyte separation medium.After incubation with a mixture of anti-CD3-FITC and anti-CD19-PE at 4OC for 0.5 h,the cells were washed twice and resuspended with PBS.Subsequently,flow cytometry was conducted to quantify the percentage of T and B cells.Results:1.Preparation of pH/ROS dual-responsive nanoparticlesA pH-responsive material(ACD)was synthesized by acetalation of ?-cyclodextrin(?-CD).Calculation based on the NMR spectrum of ACD showed that the molar ratio of cyclic acetal to linear acetal was approximately 0.62,acetalation degree of?90%.In addition,an ROS-responsive material(OCD)was obtained by chemical bond interaction between beta CD and 4-PBAP compound,IH NMR spectrum confirmed that approximately 7 PBAP units in each ?-CD molecule.Different NPs were prepared by a modified nanoprecipitation and self-assembly method,pH/ROS dual-responsive NPs were fabricated by the combination of ACD and OCD at different weight ratios.NPs based on ACD/OCD at the weight ratios of 100:0,80:20,60:40,50:50,40:60,20:80,and 0:100,abbreviated as ACD NP,AOCD8020 NP,AOCD6040 NP,AOCD5050 NP,AOCD4060 NP,AOCD2080 NP,and OCD NP,respectively.TEM showed that the nanoparticles were spherical.All the prepared NPs exhibited negative Zeta-potential,with the mean hydrodynamic diameter varied from 121±2 to 186±4 nm.Relatively narrow size distribution was found for different NPs.ACD and OCD exhibited good blend compatibility.2.Dual-responsive hydrolysis profiles of different nanoparticlespH/ROS dual-responsive performance of NPs were based on ACD and OCD,in vitro hydrolysis tests were conducted in PBS with 1 mM H2O2 and at various pH values.For ACD NP,notably rapid hydrolysis was detected at either pH 5 or pH 6,while the presence of H2O2 showed negligible effects.Independent of pH,H2O2 dramatically accelerated the hydrolysis of OCD NP.By contrast,OCD NP showed comparable hydrolysis profiles at pH 5,6,or 7.4.As for NPs derived from ACD and OCD,their hydrolysis behaviors were dependent on both pH and H2O2,they were separately tested in different buffers.These data revealed pH/ROS dual-responsive capacity for ACD/OCD-based NPs.more desirable dual-responsive character was observed for AOCD2080 NP and AOCD8020 NP at 1 mM H2O2 and pH 5,6,or 7.4.Moreover,in the presence of HZOZ,AOCD2080 NP displayed relatively excellent hydrolysis behaviors at varied pH values,therefore it was employed for further experiments.A pH/ROS dual-responsive nanoplatform was successfully established by simply using composites based on a pH-responsive material ACD and a ROS-labile material OCD.Of note,the exact sensitivity of resulting nanovehicles can be easily modulated by changing the weight ratios of ACD/OCD.3.Preparation and characterization of pH/ROS dual-responsive nanotherapiesRAP nanotherapies were prepared based on different responsive NPs.Also,a non-responsive nanotherapy was fabricated for comparison studies,by using PLGA.Similar to the corresponding blank NPs,all these four nanotherapies exhibited negative Zeta-potential,with the mean hydrodynamic diameter varying from 121±2 to 179±6 nm.For RAP/PLGA NP,RAP/ACD NP,RAP/OCD NP,and RAP/AOCD NP,the RAP loading content was 4.5±0.4%,9.4±0.2%,8.2±0.1%,and 8.7±0.45,respectively.In vitro release studies were performed to examine responsive release behaviors of different nanotherapies.For RAP/AOCD NP,RAP release was considerably accelerated in PBS at either pH 5 or pH 6,as compared to that at pH 7.4.Regardless of varied pH values,the presence of 1 mM H2O2 dramatically enhanced drug release rate.Accordingly,RAP/AOCD NP showed desirable pH/ROS dual-responsive drug release capacity,which is well consistent with the dual-responsive hydrolysis profiles of the corresponding nanocarrier AOCD2080 NP.The single-responsive drug release profiles of RAP/ACD NP and RAP/OCD NP agree with the hydrolysis performance of corresponding nanovehicles.Consequently,a pH/ROS dual-responsive RAP nanotherapy can be facilely and successfully developed simply by using nanocomposites of pH-responsive and ROS-responsrve materials.4.In vitro cellular uptake of nanoparticlesAfter 1 h of incubation with Cy5-labeled dual-responsive NPs(Cy5/AOCD NP),fluorescence signals in VSMCs were notably increased with increase in the dose of Cy5/AOCD NP,indicating dose-dependent internalization.Consistent with the observation by confocal microscopy,quantification by fluorescence-activated cell sorting(FACS)via flow cytometry further confirmed effective cellular uptake of Cy5/AOCD NP by VSMCs,in a dose-response pattern.At the same dose of Cy5/AOCD NP,distribution of Cy5 fluorescence was increased in VSMCs with prolonged incubation.Moreover,staining of late endosomes and lysosomes by LysoTracker revealed endolysosomal trafficking of internalized Cy5/AOCD NP in VSMCs.Likewise,FACS analysis indicated that endocytosis of Cy5/AOCDNP was enhanced with increased incubation time.These results demonstrated that rat VSMCs could efficiently internalize the pH/ROS dual-responsive NPs based on ACD and OCD.In subcellular organelles with acidic and oxidative microenvironment,the dual-responsive NPs were able to more rapidly release the loaded drug molecules,compared to either pH-or ROS-responsive control.5.In vitro biological activities of the dual-responsive RAP nanotherapyThe cytotoxicity of various blank NPs were evaluated in VSMCs.different responsive NPs showed low cytotoxicity comparable to PLGA NP.Further,cytotoxicity of RAP nanotherapies had low cytotoxicity at relatively low doses.Pretreatment with RAP and RAP nanotherapies for 24 h dramatically inhibited VSMCs migration.Of note,all nanotherapies showed notably stronger anti-migration effects,compared to free RAP.Moreover,responsive nano therapies suppressed VSMCs migration to a much more significant degree than the non-responsive nanotherapy RAP/PLGA NP.Importantly,the most potent activity was achieved by the dual-responsive RAP/AOCD NP.Treatment with various nanotherapies at the same dose of RAP much more effectively inhibited VSMCs proliferation,particularly in the case of responsive nanotherapies.Of note,the best anti-proliferation effect was achieved by the dual-responsive nanotherapy RAP/AOCD NP that exhibited significant difference compared to the single-responsive counterparts.The effects of different nanotherapies on cell cycle progression indicated that RAP nanotherapies attenuated VSMCs proliferation mainly by inhibiting the Gl/S transition.The mechanism responsible for cell cycle arrest at G1 phase by RAP nanotherapies,suggest that the dual-responsive RAP nanotherapy significantly arrested the G1 phase by up-regulating p27Kipl and down-regulating cyclin D1 in VSMCs.6.In vivo targeted treatment of restenosis by the dual-responsive RAP nanotherapy in ratsA rat model of vascular inflammatory disease was established by balloon-induced carotid artery injury,which was confirmed by staining of a typical artery with Evans blue.The pH changes in the carotid arteries were evaluated by using a pH-sensitive fluorescent probe BCECF-AM.Whereas the normal artery displayed remarkable fluorescence,the arterial tissues isolated at days 1,7,and 14 after injury showed remarkably weak fluorescent signals,indicating the presence of relatively acidic microenvironment in injured arteries.Cryosections of the rat carotid arteries were stained with dihydroethidium(DHE)and DCFHDA fluorescent probe,compared to the normal artery,the carotid artery collected at day 1 after balloon injury showed slightly high fluorescence intensity.At days 7 and 14 post injury,notably strong fluorescence was observed.These results revealed the existence of oxidative stress in the injured sites of arteries,largely resulting from the progression of inflammation.Targeting capability of the dual-responsive NPs at the injured carotid showed the examined NPs could target the carotid artery with endothelial injury,quantitative analysis revealed no significant differences between different groups treated with either non-responsrve or responsive NPs.Treatment with RAP nanotherapies showed varied degrees of benefits,the lumen area of carotid arteries significantly increased,while the intimal area and proliferation index were remarkable decreased after intervention with RAP nanotherapies,no significant changes were found in the medial area.In all cases,the most desirable outcome was obtained by the dual-responsive nanotherapy.After treatment with different RAP nanotherapies,Immunohistochemistry analysis exhibited that the total number of ?-SMA positive cells was considerably decreased,although they still existed in the intima.In addition,PCNA were notably reduced in all nanotherapy groups.In both cases,much better effects were found in the RAP/AOCD NP group.Similarly,Immunohistochemical staining of MMP-2 was also consistent with the previous results.Moreover,dramatically low expressions of 8-OHdG were detected in arteries from rats administered with responsive nanotherapies.quantitative analysis also revealed significantly reduced levels of typical oxidative mediators,including HZOZ,MDA,and MPO in arterial tissues,after treatment with the dual-responsive nanotherapy.On the other hand,the activity of SOD was significantly reduced in injured carotid arteries,it was efficiently rescued by treatment with RAP/AOCD NP.In addition,the expressions of typical pro-inflammatory cytokines tumor necrosis factor(TNF-?)and interleukin(IL)-lp in the injured tissues were significantly reduced by treatment with RAP/AOCD NP,as compared to the model group treated with saline.7.Preparation and characterization of a pH/ROS-dual responsive,type ? collagen targeting nanotherapyThe peptide KLWVLPKGGGC was conjugated with DSPE-PEG via thiol-maleimide click chemistry.The Col-? targeting,pH/ROS dual-responsive NPs(TAOCD NP)were prepared by the similar nanoprecipitation/self-assembly procedures.TEM and SEM transmission electron microscope(TEM)and scanning electron microscope(SEM)showed that TAOCD NP was spherical shape,with relatively narrow size distribution.The average hydrodynamic diameter was 131 nm,while Zeta-potential was-27.2±0.3 mV.RAP can be packaged into TAOCD NP,giving rise to a targeting,dual-responsive nanotherapy RAP/TAOCD NP,with spherical shape and narrow size distribution as well as effici... |
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