| Background Coronary arterial disease(CAD)remains the leading cause of death globally,resulting in 8.14 million deaths in 2013.In most cases,CAD is caused by atherosclerosis,a chronic process characterized by inflammation of blood vessels due to a variety of etiologies.Percutaneous coronary interventions(PCI)including balloon dilation,excisional atherectomy,and endoluminal stenting are frequently used nonsurgical techniques for treating CAD such as acute myocardial infarction and acute coronary syndrome.Unfortunately,regardless of revascularization procedures,thrombosis and/or neointimal hyperplasia generally occurs after initial successful angioplasty,which in turn leads to restenosis,an arterial reobstruction.It has been found that restenosis is responsible for the 30-40% long-term failure rate after coronary revascularization.Inflammation plays a critical role in vascular response to the arterial injury during PCI.Immediately after PCI,endothelial denudation and platelet deposition occur at the injury site.In addition to thrombus formation,activated platelets recruit circulating leukocytes via platelet receptors.Subsequently,the recruited leukocytes roll along the injured surface and release several proinflammatory cytokines such as interleukin-6 and tumor necrosis factor-?.This in conjunction with factors released from platelets and endothelial cells,such as basic fibroblast growth factor and platelet-derived growth factor(PDGF),induce migration and proliferation of vascular smooth muscle cells(VSMCs),sequentially enhance extracellular matrix synthesis,and ultimately result in neointima formation.Based on these pathophysiological features,various therapeutics including anticoagulant,anti-inflammatory,and anti-proliferative agents have been investigated to treat restenosis.However,for these drugs delivered by systemic administration,only limited efficacy was observed in clinical trials,despite their success in animal studies.This undesirable therapeutic effect has been considered to be mainly caused by rapid drug clearance from the vessel,poor drug accumulation at the site of arterial injury,and inefficiency in maintaining therapeutics in adequate levels for appropriate periods of time as well as relatively low doses administered due to systemic toxicity.Consequently,new delivery strategies and novel therapeutics are necessary to circumvent these drawbacks.Recently,nanomedicinal approaches have been demonstrated promising for diagnosis and therapy of inflammatory and cardiovascular diseases.As well documented,overproduced reactive oxygen species(ROS)play a significant role in the pathogenesis of restenosis.Substantially increased ROS levels were found in coronary arteries following balloon injury,which was positively correlated with the development of restenosis.On the other hand,mildly acidic microenvironments have been observed at inflamed sites.Based on these issues,herein we hypothesize nanomedicines that are triggerable by inflammatory microenvironments and can preferentially release their cargo molecules at the diseased site,may function as effective therapeutics for targeted treatment of restenosis.To test our assumption,either pH-or ROS-responsive nanotherapies were developed.Both in vitro evaluations and in vivo studies were performed to interrogate therapeutic benefits of responsive nanomedicines,with rapamycin as a candidate drug.Methods1.Synthesis of inflammatory microenvironment-responsive materials1.1 Synthesis of a pH-responsive material of acetalated ?-cyclodextrinAcetalated ?-cyclodextrin(Ac-bCD)was synthesized by acetalation of ?-cyclodextrin(?-CD)in the presence of excess amount of 2-ethoxypropene(EP),using p-toluene sulfonate(PTS)as a catalyst.In brief,4.5 mL of EP and 160 mg PTS were added into 8 mL of anhydrous DMSO containing 1 g ?-CD.After 3 h,the reaction was terminated by triethylamine.The product was collected by precipitation from deionized water and centrifugation at 14000 revolutions per minute(rpm).After thorough rinsing with deionized water,the sample was lyophilized to give a white powder.1.2 Synthesis of a ROS-responsive material from ?-CD4-(Hydroxymethyl)benzeneboronic acid pinacol ester(PBAP,2 g)was dissolved in 12 m L dry dichloromethane(DCM),into which 2.8 g 1,1'-Carbonyldiimmidazole(CDI)was added.After 30 min of reaction,14 mL of DCM was added into the mixture,and then washed with 10 mL of deionized water three times.The organic phase was further washed with saturated sodium chloride solution,dried over sodium sulphate,and concentrated to obtain CDI-activated PBAP.ROS-responsive ?-CD(Ox-bCD)was then synthesized by conjugating CDI-activated PBAP units onto ?-CD.To this end,250 mg ?-CD was dissolved in 20 mL of anhydrous DMSO,into which 0.8 g 4-dimethylaminopyridine(DMAP)was added,followed by addition of 1.5 g CDI-activated PBAP.Thus obtained mixture was magnetically stirred at room temperature(RT)for 12 h.The final product was obtained by precipitation from water,and collected by centrifugation.After thorough rinsing with deionized water,the sample was lyophilized to give a white powder.The obtained material was characterized by 1H Nuclear Magnetic Resonance(NMR)spectroscopy(Agilent 600MHz)and Fourier transform infrared spectroscopy(FT-IR)(S100,PerkinElmer).2.Preparation of various nanoparticlesA modified nanoprecipitation/self-assembly method was employed to prepare different nanoparticles(NPs).Briefly,50 mg carrier material and 10 mg rapamycin(RAP)was first dissolved in 2.0 mL of acetonitrile(methanol was used as the solvent in the case of Ox-b CD).Lecithin(4 mg)and DSPE-PEG(6 mg)were dispersed in 0.4 m L of ethanol,into which 10 m L of deionized water was added.Thus obtained aqueous dispersion was heated to 65°C for 0.5 h.Subsequently,the organic solution containing a carrier material and RAP was added into the preheated lipid aqueous solution dropwise under gentle stirring,followed by vortexing for 3 min.After 2 h of incubation,solidified RAP-loaded NPs were harvested by centrifugation at 16000 rpm and rinsing with deionized water three times.Through similar procedures,blank NPs and NPs containing either Cy5 or Cy7.5 were fabricated.3.Characterization of nanoparticlesThe size,size distribution,and zeta-potential of various NPs in aqueous solution were measured using a Malvern Zetasizer Nano ZS instrument.The morphology of NPs was characterized by transmission electron microscopy(TEM).For RAP-loaded NPs,the drug loading content was quantified by high performance liquid chromatography(HPLC)at 278 nm.On the other hand,the content of Cy5 or Cy7.5 was determined by fluorescence spectroscopy.4.Hydrolysis of nanoparticlesFor hydrolysis of Ac-bCD NP,it was separately performed in PBS buffers at pH 5.0 or 7.4.In the case of Ox-bCD NP,hydrolysis was conducted in PBS buffers containing different concentrations of hydrogen peroxide.Quantitative tests were conducted by measuring the absorbance of NPs-containing aqueous solution at 500 nm at various time points.Similar procedures were utilized to examine hydrolysis of poly(lactide-co-glycolide)(PLGA)NP under various conditions.5.In vitro release testsTo examine the effect of pH values on the release profiles of RAP-loaded Ac-b CD NP,in vitro drug release tests were carried out under mildly acidic(p H 5.0)or physiological(pH 7.4)conditions.At various time points,the aqueous solutions containing NPs were centrifuged at 16000 rpm and 4.0 mL of supernatant was withdrawn,and the same volume of fresh medium was replenished.According to similar protocols,in vitro release experiments of RAP/Ox-bCD NP in the presence of various concentrations of hydrogen peroxide were implemented.Also,similar methods were followed to study release behaviors of RAP/PLGA NP.RAP concentrations in the supernatant were determined by HPLC.6.In vitro cellular uptakeRat primary vascular smooth muscle cells(rVSMCs)were isolated from Sprague-Dawley(SD)rat thoracic aorta by explant method and cultured in Dulbecco modified eagle medium(DMEM)containing 10% fetal calf serum(FBS)at 37°C in a humidified atmosphere containing 5% CO2.rVSMCs at passage 3-6 were seeded in 6-well plates at a density of 3×105 cells per well.After 24 h,the culture medium was replaced with fresh medium containing Cy5-labeled NPs and incubated for various durations.Before observation,late endosomes/lysosomes were stained with LysoTracker Green,while nuclei were stained with 4,6-diamidino-2-phenylindole(DAPI).Fluorescence images were acquired by confocal laser scanning microscopy(CLSM).Similarly,dose-dependent uptake was examined after incubation for 6 h.Further quantification of cellular uptake of NPs was conducted by flow cytometry.To this end,r VSMCs were seeded in 12-well plates at a density of 2 × 105 cells per well.After 24 h,the culture medium was replaced with fresh medium containing Cy5-labeled NPs(20 ?g/mL)and incubated for various periods of time,then the cells were digested and fluorescence intensity was determined via fluorescence activated cell sorting(FACS).Through similar procedures,dose-dependent internalization profiles were examined after 6 h of incubation.7.In vitro anti-migration activity of nanotherapies in primary r VSMCsA transwell assay was performed to investigate anti-proliferation activity of different nanotherapies.Specifically,rVSMCs were seeded in 12-well plates allowed to adhere overnight.After the culture medium was replaced with DMEM growth medium containing 0.5% FBS,platelet-derived growth factor-BB(PDGF-BB)at 20 ng/m L was added.Cells were treated with different RAP formulations at 1 ?M of RAP for 6 h.In the normal control group,no PDGF-BB and RAP were added,while cells were stimulated with PDGF-BB alone in the positive control group.Then each well was washed,trypsinized,resuspended in 0.5 mL growth medium containing 0.5% FBS,and plated onto 8-?m pore polycarbonate inserts in a 12-well plate with 1.5 mL medium containing 0.5% FBS and 20 ng/mL PDGF-BB.Cells were allowed to migrate for 8 h,and cells on the upper side of each insert were gently removed with a cotton swab.Cells on the lower side of each insert were then fixed and stained with 0.3% crystal violet.After staining,5 images of migrated cells per high-power field(HPF,10 × 10)were taken and counted with a Nikon microscope.8.In vitro anti-proliferative activity of nanotherapies in rVSMCsrVSMCs were cultured in a 96-well plate for 24 h.After the medium was changed to one containing 0.5% FBS,PDGF-BB at the dose of 20 ng/m L was added,concomitant with the addition of various RAP formulations at 1 ?M of RAP.Cells in the normal control group were treated with growth medium alone,while only PDGF-BB was added in the PDGF-BB group.After 24 h of incubation,the viability of r VSMCs was quantified by a cell counting kit-8(CCK-8).9.Cytotoxicity of various nanoparticlesTo evaluate cytotoxicity of different NPs,r VSMCs were seeded in 96-well plates.After 24 h,the culture medium was replaced by 100 ?L of fresh medium containing gradient concentrations of blank NPs and incubated for 24 h.The cell viability was quantified by CCK-8 assay.10.Acute toxicity evaluation on nanoparticlesTwelve male Sprague Dawley rats were randomly assigned into 4 groups(n = 3 per group).PLGA NP,Ac-b CD NP,or Ox-b CD NP in 1 mL of saline was administered via intravenous(i.v.)injection at 500 mg/kg.In the control group,rats were i.v.injected with 1 m L of saline.Post administration,rats were weighed at defined time points and their behaviors were monitored for any signs of illness each day.After 2 weeks,animals were euthanized.Blood samples were collected for hematological analysis.Major organs were harvested and weighed.Organ index was calculated as the ratio of organ weight to the body weight of each rat.In addition,histopathological sections of harvested organs were prepared and stained with hematoxylin-eosin(H&E).11.Establishment of an experimental restenosis model in rats and ROS detectionThe rat carotid artery balloon injury model was prepared.Briefly,male SD rats(480-500 g)were anesthetized.Then the left common carotid artery was exposed through a midline cervical incision.The catheter was advanced from carotid bifurcation to just under the proximal edge of the omohyoid muscle three times with a 2 Fogarty embolectomy catheter(Model 120602F;Edwards Lifesciences)at the moderate resistance in a rotating fashion.All procedures were performed by the same operator.Immediately after angioplasty,the carotid artery was isolated and stained by Evans Blue to confirm the formation of endothelial injury.After balloon-induced rat carotid artery injury model was established,dihydroethidium(DHE)was used to evaluate oxidative stress in injured carotid arteries.Also the level of hydrogen peroxide(H2O2)and malondialdehyde(MDA)in injured carotid arteries were determined with chemical kits.12.In vivo targeting of the injured carotid arteryImmediately after the SD rat carotid artery balloon injury model was prepared,Cy7.5-labeled NPs were i.v.administered.After 8 h,whole carotid artery tissues were harvested and imaged simultaneously using in vivo imaging system(IVIS)Spectrum.Fluorescence intensity of carotid artery was then analyzed.In a separate study,the carotid artery tissue was collected at defined time points after i.v.administration of Cy7.5/Ac-bCD NP in rats with injured carotid artery,followed by ex vivo imaging and quantitative analysis of fluorescence intensity.13.Treatment of rat carotid artery balloon injury by different nanotherapiesThirty SD rats were divided into six groups(n=5),including a sham group,a saline-treated group(control group),a free RAP-treated group,and groups separately administered with RAP/PLGA NP,RAP/Ac-bCD NP,or RAP/Ox-bCD NP.After angioplasty,different RAP formulations were i.v.administered at 1 mg/kg of RAP at day 0,3,7,and 11.The vessels were harvested 14 days after injury.The main organs were harvested,weighed,and fixed in 4% paraformaldehyde(PFA).Pathological sections were prepared and stained with H&E and immunohistochemistry analysis.Results1.Preparation of inflammation-responsive nanoplatformsAc-bCD was synthesized by a one-pot acetalation strategy that can be easily scaled up.Characterization by FT-IR and 1H NMR spectroscopy indicated successful synthesis of Ac-b CD.On the other hand,a ROS-responsive material Ox-b CD was produced by chemical functionalization of ?-CD with an oxidation-labile group of PBAP.Calculation based on the 1H NMR spectrum revealed 7 PBAP units were conjugated onto each ?-CD molecule.In addition to these responsive materials,PLGA was used as a non-responsive material.Both responsive and non-responsive NPs were fabricated by a modified nanoprecipitation/ self-assembly method.Regardless of different materials,well-defined spherical NPs were obtained as illustrated by TEM images.Measurement by dynamic light scattering showed the average diameter was about 200 nm.2.Hydrolysis and in vitro release profiles of various nanoparticlesSlow hydrolytic profiles were observed for PLGA NP at either pH 5.0 or pH 7.4.Similarly,the presence of hydrogen peroxide did not affect the hydrolysis of PLGA NP at p H 7.4.These results agree with the fact that PLGA is a non-responsive polymer.By contrast,p H-dependent hydrolysis could be clearly observed for Ac-b CD NP.Accordingly,Ac-bCD NP exhibited good pH-sensitivity.For Ox-b CD NP,the rate and degree of hydrolysis of Ox-b CD NP was positively correlated with the concentration of hydrogen peroxide.Consequently,Ox-b CD NP displayed well-defined oxidation-sensitivity.Consistent with non-responsive hydrolysis behaviors,both p H and hydrogen peroxide had no remarkable effects on in vitro drug release profiles of RAP/PLGA NP.For RAP/Ac-b CD NP,significantly faster release was observed at p H 5.0 as compared to that at p H 7.4.In the case of RAP/Ox-b CD NP,we found notably accelerated drug release in the presence of hydrogen peroxide.These in vitro release profiles of responsive nanotherapeutics are consistent with the hydrolysis profiles of corresponding blank NPs.3.Cellular uptake of various nanoparticles by r VSMCsObservation by fluorescence microscopy showed significant internalization of Cy5/Ox-bCD NP.Moreover,localization of Cy5/Ox-b CD NP in rVSMCs was remarkably increased with prolonged incubation.Also,we could find enhanced intracellular fluorescence signals as the dose of Cy5/Ox-b CD NP was increased.Furthermore,staining of endolysosomes by Lyso Tracker Green revealed that endocytosed Cy5/Ox-b CD NP was transported in rVSMCs via the endolysosomal pathway.FACS quantification analysis revealed that internalized Cy5/Ox-b CD NP was increased in a dose and time-dependent manner.Also,r VSMCs could effectively endocytose Cy5-labled PLGA NP or Ac-bCD NP in both time and dose dependent manners.Moreover,intracellular trafficking of Cy5/PLGA NP or Cy5/Ac-b CD NP was also implemented through the endolysosomal compartments.Consequently,these findings demonstrated that primary r VSMCs could efficiently internalize various NPs of interest.4.In vitro anti-migration and anti-proliferative activities of RAP nanotherapiesAnti-migration and anti-proliferative activities of RAP could be strikingly increased by packaging into NPs.In addition,either p H-or ROS-responsive nanovehicles more effectively potentiated RAP activity as compared to the non-responsive nanocarrier.5.Enhanced oxidative stress in the injured carotid artery in ratsEvans blue staining showed that the endothelium of carotid artery was successfully denuded.DHE staining revealed that O2-was dramatically increased,aggravating with the progression of inflammation in the injured vasculature.Significantly higher contents of hydrogen peroxide and MDA could be detected in the injured carotid artery.Accordingly,these results clearly demonstrated the presence of oxidative stress in the carotid artery after endothelial injury was induced by the balloon procedure.6.Targeting vascular injury in an experimental model of restenosis in ratsThree different NPs can be accumulated at the injury site of the carotid artery.By contrast,extremely weak fluorescence intensity was observed in the right artery without endothelial injury.What's more,accumulated NP could be maintained at the injured site of carotid artery for relatively long time.7.Targeted treatment of restenosis by different RAP nanotherapiesTreatment with free RAP decreased intimal thickness,and more significant therapeutic effect was realized after intervention with RAP/PLGA NP.Treatment with either p H-responsive RAP/Ac-b CD NP or ROS-responsive RAP/Ox-bCD NP more prominently attenuated restenosis.Three nanotherapies notably reduced intimal ?-SMA positive cells and PCNA positive cells,in which responsive nanomedicines showed the most desirable efficacy.Likewise,consistent results were obtained by immunohistochemistry staining of MMP-9.All these results substantiated that proliferation of rVSMCs at the injured sites were remarkably inhibited by responsive nanotherapies,well agreeing with the finding on in vitro anti-migration and anti-proliferation studies.8.Toxicology evaluationThree different NPs displayed slight cytotoxic effects in r VSMCs.In vivo toxicology evaluation showed that no significant differences were found between the NP-treated groups and the saline control.Rats treated with different NPs displayed normal food and water intake,and no abnormal behaviors appeared.All the treated rats showed the comparable organ index for major organs.Also,no distinct variations were examined in typical hematological parameters and clinical chemistry tests of liver and kidney functions biomarkers.H&E stained histopathological sections indicated no distinguishable injuries or pathological changes in major organs of NP-administered rats.Conclusions1.Both pH-and ROS-responsive nanoplatforms were successfully fabricated using cyclodextrin-derived functional materials that could be synthesized by an easily translatable approach.2.Preliminary in vitro and in vivo evaluations demonstrated that these responsive nanovehicles displayed good safety profile.3.The newly engineered NPs could effectively package RAP,and thus obtained RAP nanotherapies were capable of inhibiting migration and proliferation of r VSMCs.4.Effective localization of NPs at the injured vasculature was achieved by passive targeting.5.Furthermore,RAP nanomedicines responsive to the inflammatory microenvironment could notably attenuate restenosis in the carotid artery of rats,which was more potent than the non-responsive counterpart.Accordingly,responsive nanotherapies hold great potential for the management of vascular restenosis by targeting the inflammatory microenvironment with mild acidity and high ROS.Although in-depth studies are necessary to elucidate exact mechanisms underlying their efficacy,this study represents the first investigation that demonstrates therapeutic advantages of responsive NPs for prevention and therapy of arterial restenosis,which also provides new insight into the design of novel therapeutics for other inflammatory diseases. |
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