| Heart failure(HF)is a serious clinical and public health problem,with a considerable increase in the incidence and prevalence over time.The activation of renin-angiotensin-aldosterone(RAAS)systems and the sympathetic nervous plays a key role in HF.In addition,activation of both correlates with ROS production.In view of the crucial role of myocardial oxidative stress in the development and progression of heart failure,different antioxidant strategies have been investigated in both preclinical and clinical studies for the treatment of heart failure,by regulating mitochondrial ROS generation,scavenging excessive ROS,or attenuating oxidative damage.Unfortunately,most current strategies have failed to provide beneficial clinical outcomes.To a large degree,this can be attributed to low accumulation and short retention time of therapeutic molecules in the heart,adverse effects due to nonspecific drug distribution and long-term or overdose treatments,and/or limited antioxidative activity of available drugs.Accordingly,both new therapies and novel delivery approaches are necessary for the treatment of heart failure.The advantages of inhalation delivery include easy handling,self-administration,good compliance and adherence,rapid absorption/action,low metabolism,and reduced risk of systemic adverse effects.Inhalation delivery has become a method of noninvasive systemic delivery of drugs through the lungs.Here we hypothesize that nanoparticles deposited in the lungs can be gradually and continuously transported to the heart by the pulmonary circulation.As a proof of concept,considering the importance of oxidative stress in heart failure,a nanotherapy based on antioxidative nanoparticles was employed in this study.We found time-dependent translocation of the inhaled nanotherapy via the lung epithelial cells and endothelial cells,with subsequent heart accumulation through the pulmonary circulation in mice,thereby affording good therapeutic effects in a mouse model of heart failure.Surface decoration of the bioactive nanotherapy with a mitochondrial targeting moiety notably enhanced its heart targeting efficiency.The bioactive targeting nanotherapy was loaded with anti-inflammatory peptide Ac2-26,resulting in better therapeutic benefit.Methods1.Synthesis and characterization of a reactive oxygen species(ROS)-scavenging material based on?-CDTPCD was synthesized by conjugating Tempol and PBAP unit onto?-CD and characterized by 1H NMR and FT-IR.2.Synthesis and characterization of stearyl-TPPStearyl bromide and triphenylphosphine were dissolved in acetonitrile.The resulting oil was washed with hexanes and diethyl ether and dried to obtained Stearyl-TPP(i.e.,STPP).The obtained materials of STPP were characterized by 1H NMR spectroscopy.3.Preparation of TPCD nanoparticles(TPCD NP)We added DSPE-m PEG2000 and lecithin to ethanol,and then added to deionized water.The final liquid was stirred.Then TPCD dissolved in methanol was added to the aqueous phase and was stirred for 2 h at room temperature.Finally,TPCD NP was obtained by removing organic solvent and excess distilled water via vacuum rotary evaporation.4.Preparation of TPP-decorated TPCD nanoparticles(TTPCD NP)We added DSPE-m PEG2000,STPP and lecithin to ethanol,followed by deionized water.TPCD is then added to methanol,which is then added to the aqueous phase.Methanol was removed under reduced pressure to obtain TTPCD NP.5.Preparation and characterization of Ac2-26-loaded TPCD NPWe put DSPE-m PEG2000 and lecithin in ethanol and then added deionized water.TPCD was then dissolved in methanol,into which Ac2-26 dissolved in DMSO was added.The liquid was added to the aqueous phase and was stirred.Finally,Ac2-26-loaded TPCD NP(defined as ATPCD NP)was obtained by removing organic solvent and excess distilled water via vacuum rotary evaporation.6.Characterization of various nanoparticlesWe used Malvin Zetasizer to measure size distribution profiles,zeta-potential values and means sizes of NPs.We used TEM and SEM to observe the morphology of NPs.7.Determination of ROS-scavenging capabilityFresh solution of DPPH·was incubated in methanol solutions containing different concentrations of TPCD NP for 30 min in dark.Absorbance was then recorded by UV-visible spectroscopy and eliminated DPPH·was calculated.The superoxide anion was detected with the superoxide anion detection kit.The concentration of H2O2 was determined by using a hydrogen peroxide detection kit.8.Studies on cellular uptake profilesH9C2 cells and Cy5-labeled TPCD NP(Cy5/TPCD NP)were incubation for different time periods,then 4%paraformaldehyde was added,washed once with PBS.Finally,DAPI working solution was added,washed with PBS for five times,stored in dark,and confocal laser photography was taken.In addition,cell uptake was observed after 2 h ours incubation with different concentrations of TPCD NP.In separate experiments,cellular uptake and mitochondrial localization of Cy5/TPCD NP or Cy5/TTPCD NP in H9C2 cells were observed by CLSM.After incubation for different times,cells were stained with Mito-tracker Green FM and followed by CLSM observation.A549 cells,HUVECs,RAW264.7 cells,and H9C2 cells were cultured in 12-well plates.Then H9C2 cells were treated with or without DOX,while A549 cells,HUVECs,and RAW264.7 cells were incubated without DOX.After cells and Cy5/TPCD NP were incubation for different times,the cells were collected for flow cytometry(FACS)analysis.Similarly,cell uptake was studied after incubation with different doses of Cy5/TPCD NP for 2 h.Furthermore,cellular uptake of Cy5/TPCD NP or Cy5/TTPCD NP in H9C2 cells were compared by FACS analysis.In this case,H9C2 cells were treated with 2?M DOX.After incubation for different times,cells were then collected for FACS analysis.9.Intracellular ROS generation in H9C2 cellsAfter cells were pretreated with different doses of TPCD NP,they were stimulated with DOX.Subsequently,cells were treated with DCFH-DA.The cells were washed with PBS and observed by CLSM.In a separate study and following the similar procedures,cells were collected and analyzed by FACS.10.Quantification of MDA,c Tn I,and LDHCells were pretreated with nanoparticles and then stimulated with DOX.Subsequently,cell culture supernatant was collected and centrifuged.The levels of c Tn I and LDH were detected by c Tn I and LDH kits,respectively.Meanwhile,cells were collected and subjected to be frozen and thawed five times before they lyse completely.Then the supernatant was collected into the EP tube.The level of MDA was detected by an MDA kit following the manufacturer's instructions,and the total protein level was quantified with the BCA kit.11.In vitro cytotoxicity of various nanoparticlesWe cultured A549 cells,HUVECs,H9C2 cells and RAW264.7 macrophages in 96-well plates.Cells were co-incubated with different concentrations of NPs at different times periods.The cell viability was quantified by CCK-8 assay.12.In vivo biodistribution after inhalation of nanoparticlesMale C57BL/6 mice were exposed to a halation chamber and nebulized with Cy7.5-labeled NPs(Cy7.5/TPCD NP).At different time points after inhalation,heart and other major organs were collected.We used the IVIS Spectrum living imaging system to observe the fluorescence of major organs and analyze the fluorescence intensities of these major organs.To estimate the exact dose after inhalation,intratracheal nebulization and inhalation were compared.IMice in the intratracheal nebulization group were administered by inhalation via intratracheal nebulization of(Cy7.5/TPCD NP).Mice in the inhalation group were exposed to the halation chamber and nebulized with Cy7.5-labeled NPs.After the experiment,lungs were collected for ex vivo imaging,and fluorescence intensities were calculated by the Living Image software.13.Intrapulmonary distribution of nanoparticlesMice were exposed to Cy5/TPCD NP by inhalation.After 24 h,lungs were collected,fixed with 4%paraformaldehyde,and embedded in paraffin.The lung sections were incubated with antibodies to Ep CAM,CD31,and CD68.After staining with DAPI,lu ng slices were visualized by CLSM.In addition,Cy5/TPCD NP in the heart was analyzed by immunofluorescence after 60 h of inhalation.In separate experiments,the cellular distribution of Cy5/TPCD NP in pulmonary tissues was analyzed by FACS.Briefly,mice were nebulized with Cy5/TPCD NP.After 24h,and lungs were minced and incubated DMEM containing Liborase TM and DNase I.We used red blood cell lysis buffer to destroy the RBC.Then the antibodies to CD45 R,CD326,CD31,F4/80 and CD11b were mixed in EP tubes and co-incubated with cells for a certain period of time,followed by analysis via FACS.14.Blood cell distribution of nanoparticles after inhalationMice were nebulized with Cy5/TPCD NP.At 12 h after inhalation,whole blood samples were collected.Then cell suspensions were stained with different antibodies,followed by analysis via FACS.15.Treatment of DOX-induced cardiac dysfunction by different nanotherapies in miceMice in DOX and DOX+TPCD NP groups were intraperitoneally administrated with DOX and all mice were given DOX only once.Mice in the DOX+TPCD NP groups were nebulized with TPCD NP(4,10,or 25 mg/kg)every day for 7 days beginning from 2 days before DOX injection.We collect hearts,tibia,and other major organs.We weighed all the organs and measured the length of the tibia with a caliper.Blood samples were collected and analyzed for blood routine and biochemical analyses.To compare the therapeutic effects of DEX and TPCD NP,mice were subjected to a single intraperitoneal injection of DEX(200 mg/kg)at 2 h before administration of DOX or nebulization with TPCD NP(25 mg/kg)2 days before administration of DOX.Mice were euthanized after 7 days.At the end of the experiment,we collected the tibia and the heart.We weighed the heart and measured the length of the tibia with a caliper.The ratio of organ weight to tibial length was analyzed.In a separate study,mice were nebulized with TPCD NP or TTPCD NP(at 25 mg/kg)2days before administration of DOX.After 7 days,we collected the tibia and the heart.We weighed the heart and measured the length of the tibia with a caliper.The ratio of organ weight to tibial length was analyzed.Furthermore,mice were nebulized with Ac2-26(14.75?g/kg),TTPCD NP or ATTPCD NP(at 25 mg/kg)2 days before administration of DOX.After 7 days,hearts and tibia were harvested to calculate the ratio of heart weight to the tibia length.16.EchocardiographyMice were anesthetized with 1-1.5%isoflurane and the mice were fixed on the operating table with thermal insulation function.Cardiac function was measured by small animal ultrasound system and and the images were collected and saved.Finally,we calculated and analyzed LVEF and LVFS to assess cardiac function.17.Measurements of serum levels of c Tn I,LDH,CK-MB,and CKWe got whole blood from mice by removing their eyeballs and then putting it in an EP tube for a certain amount of time.The blood is centrifuged and the upper fluid is collected.The levels of c Tn I,LDH,CK-MB,and CK were measured by ELISA assay.18.Evaluation of oxidative stressDHE staining was used to detect superoxide anion generation.Fresh heart slices were incubated with DHE at 37°C in dark for 30 min.We used fluorescence microscope to observe the fluorescence of heart sections.The images were then captured and saved.Finally,the fluorescence intensity of the slices was calculated and analyzed.The MDA and H2O2 levels in the myocardium were measured to further assess oxidative stress.19.Histological analysisWe put the heart in 4%paraformaldehyde for a certain period of time.Paraffin sections were then made and we assess the heart with H&E staining.20.Acute toxicity evaluation of TPCD NP in miceThe experiment consisted of three groups:control group,62.5 mg/kg TPCD NP group(TPCD NP-L)and 125 mg/kg TPCD NP group(TPCD NP-H).There were five mice in each group.After seven days,blood samples were collected and analyzed for route blood tests and biochemical analyses.The main organs were collected.We weigh the dry w eight and wet weight of the lung tissue respectively.We obtained the ratio of dry weight to wet weight,and the ratio reflected the state of the lung tissue.In a separate study,lavage fluid was collected.The TNF-?and IL-1?levels were measured by ELISA assay.Results1.Preparation and characterization of a bioactive nanoparticle-derived nanotherapyAn antioxidative and anti-inflammatory material(defined as TPCD)was synthesized by conjugating Tempol and PBAP unit onto?-CD.The structure of TPCD was characterized.According to the results of 1H NMR spectrum,approximately 2 Tempol and5 PBAP were conjugated onto each?-CD molecule of TPCD.TPCD NP were produced by a modified nanoprecipitation/self-assembly method.Observation by TEM and SEM revealed well-defined spherical shape of TPCD NP.The mean diameter was101 nm,while?-potential was-29.4±1.7 m V.With an increase in TPCD NP concentration,the amount of scavenged H2O2 increased,with a nearly liner profile.Similarly,TPCD NP showed dose-dependent elimination of superoxide anion.In addition,both time and dose dependent scavenging profiles of DPPH radical were observed.2.In vitro biological activities of TPCD NPCLSM and FACS showed that H9C2 cells could effectively internalize Cy5/TPCD NP,and the uptake of Cy5/TPCD NP increased with time and concentration.CLSM and FACS indicated efficient internalization of Cy5/TPCD NP by H9C2 cells and the internalization of Cy5/TPCD NP increased with time and concentration.Moreover,the results showed that the uptake of Cy5/TPCD NP in DOX-stimulated H9C2 cells were significantly higher than those of cells without DOX pretreatment,regardless of different incubation time periods.We also examined cellular uptake in other cells related to the absorption and translocation of inhaled TPCD NP,including A549 cells,HUVECs,and RAW264.7 cells.All these tested cells showed effective uptake of Cy5/TPCD NP in time-and dose-dependent manners.Pretreatment with TPCD NP dose-dependently reduced ROS levels in H9C2 cells treated with DOX.Treatment with this nanotherapy also significantly inhibited the production of MDA.Moreover,pretreatment with TPCD NP effectively reduced excretion of c Tn I and LDH by H9C2 cells.3.Heart targeting capability and mechanistic studies of inhaled TPCD NPPulmonary deposition of TPCD NP was largely completed within 12 h.Correspondingly,fluorescence intensities in the heart increased significantly with time from12 to 60 h after inhalation.The heart accumulation of Cy5/TPCD was further affirmed by confocal microscopic observation of the heart cryosection.whereas the lung accumulation of Cy7.5/TPCD NP considerably decreased at 60 h after inhalation,the heart distribution remained increased at the examined time points from 60 to 120 h.These results indicated that inhaled Cy7.5/TPCD NP can be gradually absorbed and translocated to other major organs.Immunofluorescence analysis revealed the co-localization of Cy5/TPCD NP with Ep CAM+epithelial cells,CD31+endothelial cells,and CD68+macrophages of lungs after inhalation.The distribution of Cy5/TPCD NP in distinct pulmonary cell types of the lung tissue was further quantified by FACS.It was found that 29.7±5.6%lung endothelial cells were Cy5-positive,while 25.6±1.9%epithelial cells contained Cy5/TPCD NP.By contrast,only a very low proportion of lung macrophages(6.9±1.4%)were Cy5-positive.Consequently,absorption and translocation of Cy5/TPCD NP are mainly mediated by epithelial and endothelial cells.Since previous studies reported that inhaled nanoparticles can be transported into the blood circulation through the pulmonary lymphatic system,Blood was collected from mice after nanoparticle inhalation,and the amounts of nanoparticles in white blood cells was measured and counted by FACS.Although we found the localization of Cy5/TPCD NP in different blood cells including lymphocytes,neutrophils,Ly6Clow monocytes,Ly6Chighmonocytes,dendritic cells,and macrophages,the proportion of Cy5-positive cells for each cell type was less than 5%.Accordingly,lymphatic transport may not be the main route for the heart accumulation of Cy5/TPCD NP.4.Targeted treatment of DOX-induced cardiac dysfunction by inhaled TPCD NP in miceBased on the above results,we examined in vivo efficacies of TPCD NP.Treatment with the nanotherapy TPCD NP notably increased both heart weight and HW/TL,alleviated cardiac dysfunction and reduced LVEF and LVFS caused by DOX.TPCD NP significantly reduced the levels of markers related to oxidative stress,such as MDA and H2O2,and typical biomarkers of cardiac injury,such as c Tn I and CK-MB.Moreover,treatment with TPCD NP could also reverse DOX-induced reduction in the ratios of organ weight to tibia length for other major organs.Likewise,TPCD NP therapy enhanced the number of WBC and PLTs as well as reduced serum levels of ALT and ASTNotably,the lung accumulation efficiency of inhaled Cy7.5/TPCD NP was estimated by comparing fluorescence intensities with those of Cy7.5/TPCD NP subjected to direct intratracheal administration.The result indicated that approximately 4%TPCD NP was deposited in the lung after inhalation.This suggested that the inhalation doses of 4,10,and25 mg/kg TPCD NP are actually 0.16,0.4,and 1 mg/kg TPCD NP,respectively.5.Comparison of in vivo efficacy of TPCD NP with dexrazoxane(DEX)in mice wit h DOX-induced cardiac dysfunctionTreatment either DEX or TPCD NP significantly increased the heart weight and HW/TL ratio as well as remarkably improved heart dysfunction by increasing the DOX-induced abnormal reduction in LVEF and LVFS.Correspondingly,the levels of MDA and H2O2 in the myocardium and serum levels of c Tn I and CK were considerably decreased after treatment with DEX or TPCD NP,and more beneficial outcomes were achieved by TPCD NP.6.Engineering of a TPCD NP-derived mitochondrial-targeting nanotherapy for heart failureTPCD NP was functionalized with STPP.TTPCD NP was also formulated by the aforementioned self-assembly/nanoprecipitation method,using TPCD,lecithin,DSPE-m PEG2000,and STPP.The results show that the particle size distribution of TTPCD NP is uniform.Similar to TPCD NP,Cy5/TTPCD NP also showed time-dependent cellular uptake in H9C2 cells.Nevertheless,quantification via FACS indicated that TTPCD NP was more effectively internalized by H9C2 cardiomyocytes.Although both Cy5/TPCD NP and Cy5/TTPCD NP showed mitochondrial-targeting capacity,quantitative analysis revealed that the co-localization ratios of Cy5/TTPCD NP were significantly higher than those of Cy5/TPCD NP at the examined time points.These results demonstrated th at both cellular uptake and mitochondrial-targeting capability of TPCD NP can be effectively enhanced after decoration with STPP,thereby affording a more efficient nanotherapy TTPCD NP.In line with this finding,TTPCD NP more effectively protected H9C2 c ells from DOX-induced oxidative stress injury,compared to TPCD NP,as implicated by notably decreased levels of cellular MDA and expression of c Tnl and LDH.7.In vivo heart targeting and therapeutic effects of TTPCD NP in miceInhaled Cy7.5/TTPCD NP exhibited a significantly higher heart accumulation than that of Cy7.5/TPCD NP.However,comparable fluorescence intensities in the lung were found after inhalation of Cy7.5/TPCD NP or Cy7.5/TTPCD NP.We then compared in vivo efficacies of two nanotherapies TPCD NP and TTPCD NP in mice with DOX-induced cardiac dysfunction.Consistent with enhanced heart targeting,inhalation of TTPCD NP more effectively inhibited the heart weight loss and reduction of the HW/TL ratio,in comparison to TPCD NP.Likewise,TTPCD NP prominently alleviated cardiac dysfunction and improved LVEF and LVFS.Moreover,the lowest levels of MDA and H2O2 in the myocardium as well as c Tn I and CK in serum were detected in the TTPCD NP group.8.Evaluation of therapeutic effects of ATTPCD NP in miceAc2-26 peptide was encapsulated into an TPCD NP or TTPCD NP,using a nanoprecipitation/self-assembly method,and this Ac2-26-containing,TPCD-or TTPCD-derived nanotherapy were defined as ATPCD NP or ATTPCD NP.Observation via TEM and SEM indicated that ATPCD NP and ATTPCD NP displayed a spherical shape.The results show that the particle size distribution of TTPCD NP is uniform.The drug loading content of Ac2-26 was 0.59?g/mg.ATPCD NP displayed a rapid hydrolysis and release profile profile in 0.01 M PBS(p H 7.4)containing 1.0 m M of H2O2.ATPCD NP or ATTPCD NP more effectively protected H9C2 cells from DOX-induced oxidative stress injury,compared to Ac2-26,as implicated by notably decreased levels of cellular MDA and expression of c Tnl and LDH.Inhalation of ATPCD NP or ATTPCD NP more effectively inhibited the heart weight loss and reduction of the HW/TL ratio,in comparison to Ac2-26.Likewise,ATPCD NP or ATTPCD NP prominently alleviated cardiac dysfunction and improved LVEF and LVFS.Moreover,the lowest levels of MDA,H2O2,TNF-?,and IL-1?in the myocardium as well as c Tn I and CK in serum were detected in the ATPCD NP or ATTPCD NP group.9.Safety studies on TPCD NPAfter incubation with different doses of TPCD NP,high cell viability was found for H9C2 cells,A549 cells,HUVECs,and RAW264.7 macrophages.Even at the highest dose of 1000?g/m L,cell viability was still above 80%in most cases.Since TPCD NP is expected to be delivered via inhalation,its possible side effects after inhalation at low(62.5 mg/kg/day)and high(125 mg/kg/day)doses were tested in mice.During 7 days of continuous inhalation,no significant changes were found for the body weight,the major organ index,and lung wet/dry weight ratios.Moreover,no significant changes were detected for two typical proinflammatory cytokine of TNF-?and IL-?in bronchoalveolar lavage fluid.Likewise,TPCD NP-treated mice exhibited no significant abnormal changes in the levels of representative hematological parameters and biomarkers relevant to hepatic and kidney functions.Further,assessment of H&E sections revealed no discernable injuries in the trachea and other major organs.Conclusions1.An antioxidative and anti-inflammatory material TPCD was synthesized by conjugating Tempol and PBAP unit onto?-CD.TPCD NP effectively alleviated oxidative stress and myocardial cell injury by efficient internalization in H9C2 cardiomyocytes and scavenging overproduced reactive oxygen species.2.Inhaled nanoparticles can accumulate in the heart by transport across the lung epithelial and endothelial barriers through the pulmonary circulation,mainly via the transcellular and/or paracellular pathways.3.Inhaled TPCD NP notably inhibited myocardial dysfunction induced by doxorubicin in mice,showing significantly higher potency than that a clinically used drug dexrazoxane.4.By decoration with a mitochondrial-targeting moiety,the heart targeting capability,cellular uptake efficiency,and mitochondrial distribution capacity of TTPC D NP were considerably enhanced,thereby leading to notably potentiated therapeutic effects.the mitochondrial-targeting nanotherapy TTPCD NP showed considerably potentiated therapeutic effects in amelioration of DOX-induced cardiac dysfunction.Ac2-26-loaded ATTPCD NP is more effective against the treatment of DOX-induced heart failure in mice.5.TPCD NP displayed good safety profile after inhalation at the dose at least 5-fold higher than those used in therapeutic studies.Consequently,inhalation delivery of nanoparticles can serve as an effective,safe,and noninvasive regimen for targeted treatment of heart diseases.TPCD-based nanotherapies are promising for the treatment of heart failure and other acute/chronic heart diseases associated with oxidative stress. |
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