• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Liposomal codelivery of inflammation inhibitor and collagen protector to the plaque for effective anti-atherosclerosis

    2023-03-14 06:52:14XiaotongLiJiayiGuQingqingXiaoYingLiuPingZhouLifangFanXiulianZhangXiangLuJunWuZhngxiaLiuWi
    Chinese Chemical Letters 2023年1期

    Xiaotong Li,Jiayi Gu,Qingqing Xiao,Ying Liu,Ping Zhou,Lifang Fan,Xiulian Zhang,Xiang Lu,g,Jun Wu,Zhngxia Liu,**,Wi H

    a School of Pharmacy,China Pharmaceutical University,Nanjing 211198,China

    b Department of Geriatrics,The Second Affiliated Hospital,Nanjing Medical University,Nanjing 210011,China

    c Key Laboratory for Aging & Disease,Nanjing Medical University,Nanjing 210011,China

    d Jiangsu Aosaikang Pharmaceutical Co.,Ltd.,Nanjing 211112,China

    e Department of Respiratory Disease,Baoshan Branch,Shuguang Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200120,China

    f Department of Geriatric Cardiology,Jiangsu Provincial Key Laboratory of Geriatrics,The First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China

    g Department of Geriatrics,The Affiliated Sir Run Run Hospital of Nanjing Medical University,Nanjing 211166,China

    Keywords:Atherosclerosis Plaque Inflammation Collagen Macrophages Co-delivery Liposomes

    ABSTRACT Plaque plays a central role in atherosclerosis (AS) progression,whereas inflammation and destruction of the plaque microenvironment contribute to plaque advancement.As a result,a therapy regime,which combines anti-inflammation and inhibition-degradation of plaque matrix,appears to be a promising strategy to combat AS.Herein,we report a pH-sensitive liposome co-loading with the anti-inflammatory agent(oridonin,ORD) and plaque-collagen protector (marimastat) for anti-AS therapy.ORD was first conjugated with hyaluronic acid (HA) to target the inflammation contributor,pro-inflammatory macrophages.Then,the conjugate assembled onto the MATT-loaded liposomes.The co-loaded system (~150 nm) significantly improved pharmacokinetics over the liposomes without anchoring the conjugate and accumulated effectively in the plaque.The preparation administration allowed efficient anti-AS activities in high-fat diet(HFD)-Apoe-/- mice by decreasing the pro-inflammatory cytokine expression in the serum,lessening the lesion area,alleviating the plaque collagen degradation,promoting macrophage polarization from phenotypic M1 to M2,reducing T helper (Th) 17 cells (Th17)/T regulatory cells (Tregs) and Th1/Th2 ratio, etc.Furthermore,the serum determination in AS patients demonstrated high expression of the inflammatory cytokines,indicating our finding may offer a potential guideline for clinical practice.

    Atherosclerosis (AS) is a chronic inflammatory disease characterized by plaque buildup at the arterial subendothelial space primarily owing to the deposition of activated cells such as macrophages and endothelial cells,lipid and extracellular matrixlike collagen,and glycosaminoglycan [1–4].Clinically,the rupture of the plaque frequently leads to severe cardiovascular events,such as acute myocardial infarction (AMI),stroke,and peripheral arterial disease [5],accounting for 30% of cardiac death worldwide[6].Statin therapy for lipid-lowering is the most commonly used for AS treatment [7,8].However,this regimen has a modest effect on plaque progression [9],and inversely,may increase the risk of coronary heart disease [10].

    Constant inflammation reaction is a vital determinant of plaque development [11].Whereas macrophages,the most abundant leukocytes in the plaque,significantly contribute to inflammation responseviathe production of pro-inflammatory cytokines [12].Unfortunately,single anti-inflammation therapy remains not reduce AS patient mortality [13].Furthermore,the degradation of extracellular plaque-matrix,which disrupts the plaque microenvironment,is another essential factor in promoting plaque advancement[14,15].Collagen constitutes a central portion of the plaque matrix and forms a structural framework under the fibrous cap [16].Viaproviding strength and integrity for the fibrous cap,collagen can protect the plaque from breakup [17].Accordingly,a combined regimen to inhibit decomposition of plaque collagen and suppress inflammationviatargeting plaque macrophages appears to be a promising approach for plaque stabilization.

    Fig.1.Preparation and characterization.(A) Iillustration of the co-loaded pHSLs preparation.(B) Influence of HA-ORD/lipid mass ratio on the pHSLs diameter.(C) FRET between FITC-HA-ORD/Rho-pHSLs with different FITC and Rho mass ratios (excitation wavelength: 430 nm).The HA-ORD was conjugated with FITC,and Rho was loaded in the pHSLs cores.(D) TEM examination.

    Liposomes have been widely used as drug carriers to improve drug delivery due to their various benefits.These include increasing stability through encapsulation,increasing drug efficacy and therapeutic index,improving pharmacokinetic effects (i.e.,decrease elimination and increase circulation lifetime),and reducing the toxicity of encapsulated agents [18,19].This study developed a pH-sensitive liposomal system to co-deliver an anti-inflammatory drug (oridonin,ORD) and a plaque-collagen protector (marimastat,MATT) to the plaque.Briefly,ORD is an anti-inflammatory compound with poor water solubility [20].To improve its solubility and target macrophages,ORD was modified with hyaluronic acid (HA).The HA in the conjugate (HA-ORD) could target cell CD44 receptors [21].MATT,a water-soluble compound,is an effective protector of collagen [22],and MATT could inhibit plaque-collagen degradation by matrix metalloproteinases (MMPs) [23].The pH-sensitive co-delivery liposomes,termed co-loaded pHSLs,were prepared by encapsulating the collagen protector into the aqueous cores and anchoring the conjugate onto the liposomes (Fig.1A).The plaque has an over-metabolism and the resultant acidic microenvironment[24].We proposed that the co-loaded pHSLs could release the two therapeutics in response to the acidic microenvironment after accumulating in the plaque,thereby achieving efficient anti-AS therapy.To obtain proof of concept,we conducted various experimentsin vitroandin vivo.Apoe-/-mice fed with a high-fat diet (HFD)were utilized as a model in the anti-AS studyin vivo.

    The pH-sensitive co-delivery liposomes (co-loaded pHSLs) were prepared by assembling the conjugate HA-ORD onto the MATTloaded pH-sensitive liposomes (MATT-pHSLs) (Fig.1A).First,the targeted conjugate hyaluronic acid-oridonin (HA-ORD) was synthesizedviacondensation reaction.The synthesis of HA-ORD was characterized by1H and13C NMR spectra.Consequently,approximately 70% of fed ORD was linked to HA.Five ORD molecules were involved in one conjugate.MATT-pHSLs were prepared by the film dispersion method and had a diameter of 100–120 nm.Then,the co-loaded pHSLs were prepared by anchoring the hydrophobic section of conjugate onto the liposomesviahydrophobic interactions under ultrasonic conditions.As shown in Fig.1B,the increased proportion of HA-ORD/lipid (w/w) increased the particle size of co-loaded pHSLs from 90 nm to 130 nm.To verify the conjugate loading onto the liposomes,we performed the FRET test by labeling the conjugate with the fluorescence donor (FITC) and encapsulating the fluorescence acceptor (Rho) in the cores of liposomes.As a result of an increase in the mass ratio of FITC/Rho,the maximum intensity of the fluorescence acceptor (Rho) at 575 nm rises gradually,whereas the maximal fluorescence intensity of the donor (FITC) at 515 nm declines (Fig.1C).The results demonstrated the happening of the FRET effect.FRET is always utilized to explore the interaction between two materials by tagging fluorescent donors and acceptors to them [25–27].These results implied the co-assembly of the conjugate and the liposomes.TEM examination displayed the co-loaded pHSLs have a spherical morphology with a 150 nm diameter (Fig.1D).The incubation in 10% serum for 6 h imposed little change on the particle size of MATT-pHSLs and co-loaded pHSLs and,therefore,indicated potential stability in the blood after intravenous injection (Fig.S1 in Supporting information).In addition,little FRET effect between the 6 h incubation liposomes and 0 h incubation samples,implying the two payloads were loaded in the liposomes after serum incubation (Fig.S2 in Supporting information).HPLC assay demonstrated that total drugloading in the co-loaded pHSLs was 8.27% ± 0.19%,wherein the MATT loading was 2.35% ± 0.20% with an encapsulation efficiency of 72.78% ± 6.27%,and while the ORD loading was 6.06%,respectively.After 24 h incubation in 10% serum,the remaining drugs(%) were 98.68% MATT and 99.85% ORD in the co-loaded pHSLs compared with the initial drug loading,demonstrating little drug leakage.The release study depicted that the release of MATT and HA-ORD conjugate from the liposomes at pH 6.0 in first 1 h period was significantly greater than that at pH 7.4 (Fig.S3 in Supporting information,P<0.05),thereby verified the pH-sensitive characteristics of the liposomes.

    To study anti-ASin vitro,we performed various experiments,including M1-like macrophage-targeting,uptake of ox-LDL in macrophages and inflammation inhibition.The accumulation of pro-inflammatory macrophages (M1-like phenotype) promotes the AS pathological process [28].We expected that delivering the targeted anti-inflammatory agent,HA-ORD,to the M1-like macrophages could retard plaque progression through regression of ox-LDL uptake and inflammation activity.First,the targeted uptake of HA-ORD by M1-like macrophages induced by LPS was assayed.The CD44 expression on the M1-like macrophages was verified by confocal imaging after staining with the FITC-CD44 antibody(Fig.S4 in Supporting information).To study the conjugate target ability to the CD44 receptor,we saturated the receptor on the M1-like macrophages by incubation with a high HA-concentration.As shown in Figs.S5A and B (Supporting information),the Rho-HA-ORD uptake in the HA-saturated cells declined significantly(P<0.01).Furthermore,the uptake in the M1-like macrophages was significantly higher than that in nonpolarized RAW264.7(Figs.S5C and D in Supporting information).In addition,the M1-like macrophages uptake was time-related and reached maximal internalization at 2 h post-incubation (Fig.S6 in Supporting information).Overall,the data implied that the conjugate (HA-ORD)could target M1-like macrophages.

    The excessive influx of ox-LDL facilitates the formation of the foam cells and then induces the development of plaques [29].Accordingly,the inhibition of ox-LDL uptake in macrophages functionally impedes AS progress and promotes plaque stability [30,31].As depicted in Figs.S7A and B (Supporting information),the M1-macrophages (LPS-induced RAW264.7) possess improved ability to take up ox-LDL over the nonpolarized RAW264.7 without LPStreatment (Figs.S7A and B,Fig.S8 in Supporting information).The treatment with co-loaded pHSLs restrained the ox-LDL influx with higher efficacy than the incubation with other formulations(Fig.S7B,P<0.001).Furthermore,the anti-inflammatory study indicated that HA-ORD-containing formulations,HA-ORD and coloaded pHSLs,reduced the production of inflammatory cytokines,IL-6 and TNF-α(Figs.S7C and D in Supporting information),while co-loaded pHSLs allowed enhanced anti-inflammation effect.These results demonstrated that co-loaded pHSLs could alleviate ASin vitro.

    Next,we investigated anti-AS efficacyin vivoin theApoe-/-mice with HFD feedviaassaying plaque volume and blood lipid level.After 4 weeks of HFD feeding,theApoe-/-mice were intravenously administrated with different formulations every five days for 8 weeks (Fig.S9A in Supporting information).We first stained descending aorta with oil-red-O (ORO) to assay the lesion area after treatment.As displayed in Figs.S9B and C (Supporting information),combination treatment with the physical mixture or the co-loaded pHSLs reduced the lesion area in the descending aorta compared with the single treatment of free MATT or HA-ORD(P<0.01).Significantly,the dosing of co-loaded pHSLs at low and high doses rendered 0.5 (P<0.05) and 1-fold decrease (P<0.01)in the lesion area over the single administration.Next,we determined the plaque area by staining the aortic sinus cryosection with H&E and ORO (Figs.S9D and E in Supporting information).As showed in H&E and ORO staining,the lesions were inhibited after therapy.The plaque area in the group treated with the co-loaded pHSLs was reduced compared with the groups dosed with the PBS,free HA-ORD,or physical mixture of MATT and HA-ORD (Fig.S9D).The semi-quantitative analysis indicated that the plaque area in groups dosed with PBS,free MATT,free HA-ORD,physical mixture,co-loaded pHSLs at low and high doses was 32.34% ± 1.96%,31.77% ± 0.63%,26.10% ± 3.30%,25.20% ± 2.40%,17.33% ± 4.72%and 12.34% ± 2.11%,respectively (Fig.S9E).The treatment with the co-loaded pHSLs enabled a 1–1.5-fold plaque-area decrease over the PBS treatment.In addition,the mouse weight usually increased during the treatment duration.As a result,no apparent pathologic change after treatment was observed by H&E staining,indicating the safety of these formulations (Figs.S9F and S10 in Supporting information).Overall,the results demonstrated that the therapy with the co-loaded pHSLs could impede plaque advancement effectively.

    Blood lipid is a significant factor contributing to AS progression,including low-density lipoproteins (LDL),cholesterol,high-density lipoproteins (HDL) cholesterol,and other components [32].As reported,the HDL protected the HFD diet animals from AS and,inversely,the LDL is a kind of “bad” cholesterol that its accumulation accelerates the development of AS [33,34].First,we determined the blood lipid levels after dosing.The treatment with the co-loaded pHSLs lowered the serum LDL and total cholesterol(TC) compared with PBS-treatment (P<0.05),accompanied with a modest effect on triglyceride (TG) and HDL (Figs.S11A–D in Supporting information).Next,we calculated the plasma TG/HDL ratio and atherogenic index (common logarithm of TC/HDL ratio).The treatment with co-loaded pHSLs reduced the ratio of TG/HDL and the atherogenic index compared with PBS treatment (Figs.S11E and F in Supporting information,P<0.05).The increase in the TG/HDL ratio and atherogenic index indicates AS exacerbation and a growing risk of cardiovascular events [35].As a result,the coloaded pHSLs administration is promising to attenuate AS by lowering the lipid levels.

    To investigate the contributors to the effective anti-AS effi-cacy,we next studied the pharmacokinetics of co-loaded pHSLs and explored their targetability to the active axis of plaquemicroenvironment components (macrophages and collagen)-inflammatory factors.

    Blood circulation is an essential factor that governs the accumulation of drug carriers in the target site of interest [36,37].And the fluorescence dye was widely used to quantify the pharmacokinetics of carriers,especially the environment-responsive dye,such as aggregation-caused quenching (ACQ) probe [38,39].Furthermore,previous reports demonstrated that the carrier circulation performance obtained by determining the drug was generally similar to fluorescence-dye determination [40].As depicted in Fig.2A,the DiR labeled co-loaded pHSLs displayed significantly higher plasma concentrations at each time point post-injection than DiR-pHSLs without anchoring of HA-ORD.Next,the pharmacokinetic parameters were calculated based plasma concentration(Table S1 in Supporting information).The co-loaded pHSLs demonstrated prolongedt1/2of blood circulation and mean residence time (P<0.05),increased AUC (P<0.05),and lowered clearance rate (CL,P<0.05) in contrast to pHSLs.Therefore,the data indicated that the conjugate (HA-ORD) anchoring enabled prolonged blood circulation time and improved pharmacokinetics.The improved performancein vivoof co-loaded pHSLs may be because the surface HA could offer an extra energy barrier for nanoparticles by presenting bound water molecules and compromising the absorption of opsonin [41,42].

    Then,we first studied the plaque-targeted ability of CF-labeled co-loaded pHSLs in theApoe-/-HFD model.As well known,numerous M1-like macrophages inhabit inside the plaque [43].Accordingly,the plaque targeting was characterized by co-localizing the CF-labeled pHSLs with the M1-like macrophages marked by Cy3 for CD68.As shown in Fig.2B,profound yellow fluorescent spots were exhibited in the group dosed with the CF-labeled pHSLs or co-loaded pHSLs; in contrast,slight yellow fluorescence was observed in the group treated with free dye.Importantly,coloaded pHSLs have more profound co-localization compared with CF-labeled pHSLs.The results demonstrated that the co-loaded pHSLs with HA-ORD anchoring effectively accumulated at the plaque and located inside the lesion area.

    Collagen,a significant component of the plaque extracellular matrix,is critical to stabilizing the plaque and preventing rupture [44].The higher the collagen content,the more stable the plaque [45].Herein,the collagen was identified by Masson staining.As shown in the Masson staining (Fig.2C),the positive collagen stained in blue was demonstrated in the groups treated with MATT-loading preparations,especially with co-loaded pHSLs at low or high doses.Quantified assay indicated that the treatment with co-loaded pHSLs allowed a 1–1.5-fold increase of collagen expression compared to PBS treatment (Fig.2D,P<0.01).Furthermore,co-loaded pHSLs (high dose) demonstrated elevated collagen expression compared to the physical mixture (P<0.05).These results verified that the co-loaded pHSLs could alleviate the plaquecollagen degradation and,therefore,is promising to protect the plaque from disruption.

    To study macrophage polarization switch,we first assayed the polarization pattern of macrophages in the plaque inApoe-/-HFD model after treatment.As depicted in the result of confocal imaging (Fig.3A),a large amount of red fluorescent spots (marking CD68+) and slight green fluorescence (marking CD206+) appeared in the PBS group.Conversely,we observed fewer red fluorescent spots and considerable green fluorescence in the groups administered with HA-ORD loading preparations.Notably,coloaded pHSLs demonstrated elevated green fluorescence intensity compared to the physical mixture (PM) and other preparations.Additional quantified assay indicated that the ratio of CD68+/CD206+macrophages from the groups administered with co-loaded pHSLs at the low or high dose displayed approximately 80% and 40% reduction compared with the PBS- and PMtreated groups,respectively (Fig.3B,P<0.001 andP<0.05).To confirm the phenotype switch of macrophages,we also assayed the M1/M2-like subset in the liver after treatment.The administration of co-loaded pHSLs did not affect the total number of liver macrophages as compared with PBS (Figs.S12A and B in Supporting information),whereas the administration resulted in the expansion of CD206+ macrophages and reduction of CD68+phenotype (Figs.S12C and D in Supporting information,P<0.05).

    Fig.2.Pharmacokinetics,plaque targeting and collagen in the lesion area.(A) Pharmacokinetics of pHSLs and co-loaded pHSLs.DiR labeled formulations were intravenously injected at a DiR dose of 5 mg/kg,according to the body weight.The fluorescence intensity was detected by a microplate reader at 748 nm (excitation) and 780 nm(emission). n=5.(B) Targeting the plaque in Apoe-/- HFD mice.The tricuspid ring of the mouse heart was isolated for sectioning after 2 h injection with CF-labeled formulations and the control (free CF) via the tail vein at a CF dose of 2.5 mg/kg,according to the body weight.The plaque was marked by labeling the CD68 on macrophages with Cy3 (red,5 μg/mL).Scale bar: 50 μm.(C) Masson staining of the descending aortas after administration.The collagen fibers were stained to blue and muscle fibers to red.The plaque was stained to red.The dosage of ORD and MATT in the pHSLs was 5/5 mg/kg and 5/10 mg/kg for low dose and high dose,respectively.(D) Semi-quantitative of collagen in the plaque calculated by ImageJ.pHSLs herein were the co-loaded pHSLs.Scale bar: 200 μm. n=3,*P<0.05,**P<0.01,compared with saline; #P<0.05,compared with free MATT; ns,not significant.

    Fig.3.Targeting macrophages and ameliorating inflammation.(A) Analysis of macrophages by immunofluorescence staining and (B) semi-quantitative of the M1/M2 ratio by ImageJ.M1 macrophages (CD68) were stained to red with Cy3-labeled second antibody,and M2 macrophages (CD206) were stained to green with FITC-labeled second antibody.Scale bar: 20 μm.(C–E) The inflammatory cytokines expression in the serum.IL-6 and TNF-α were measured using the Bio-Plex? multiplex array systems.IL-22 was determined with ELISA kits.The dosage for ORD and MATT was 5 mg/kg.The low dose and high dose indicate the different dosages of co-loaded pHSLs,5/5 mg/kg and 5/10 mg/kg for ORD and MATT. n=3,*P<0.05,**P<0.01,***P<0.001,compared with saline; #P<0.05,##P<0.01,compared with free HA-ORD,free MATT or physical mixture; ns,not significant.

    In addition,we detected the immune cells,T regulatory cells(Tregs) and T-lymphocyte helper (Th) cells,in the serum,owing to that Tregs could inhibit inflammation and AS progression,while the increased ratio of Th17/Tregs and Th1/Th2 always exacerbate inflammation and atherogenesis [46–48].As displayed in Figs.S13A–D (Supporting information),dosing the co-loaded pHSLs at low or high dose demonstrated limited influence on the Treg percent and,while reducing the ratio of Th17/Tregs (P<0.05)and Th1/Th2 (P<0.01) compared with PBS administration.Overall,the data revealed the treatment with co-loaded pHSLs regressed macrophage polarization switch towards pro-inflammatory phenotype in the plaque and livers and decreased the ratio of Th1/Tregs and Th1/Th2,which could benefit the plaque stabilization [49].

    Finally,the inflammatory serum cytokines were measured after treatment.The pro-inflammatory factors,IL-6,IL-22,and TNFα,were significantly downregulated after treatment with HA-ORD loading preparations,including free HA-ORD,PM,and co-loaded pHSLs,compared with PBS treatment (Figs.3C–E,P<0.001).Especially,the dosing of co-loaded pHSLs at a high dose reduced the levels of IL-6 and IL-22 by 3- and 1.5-fold,respectively,over PM treatment (Figs.3C–E).The data implied that the treatment with co-loaded pHSLs could lessen the inflammation.

    To confirm the present results that might provide potential guidelines for the clinical treatment,we further tested the serum cytokines in patients with acute myocardial infarction (AMI)caused by the rupture of AS plaque [50] and healthy volunteers without cardiovascular disease.The population baseline table is shown in Table S2 (Supporting information).As displayed in Fig.S14 (Supporting information),elevated inflammatory cytokines,IL-6,IL-22,and TNF-α,were demonstrated in the patients compared with the healthy volunteers (P<0.001).The data indicated that our results could be translated into the clinic practices of AS therapy.

    Taken together,we proved that the co-loaded pHSLs had extended blood circulation time,effectively accumulated in the plaque,facilitated macrophage polarization into M2-like phenotype,and rendered inhibition of both inflammation and collagen degradation (Fig.S15 in Supporting information).Furthermore,their administration to HFD-Apoe-/-mice reduced the lesion area,elevated the percent of Tregs,decreased the Th1/Th2 ratio.Targeted co-delivery of inflammation inhibitor and collagen protector to the plaque represents a promising approach for anti-AS therapy.

    The anchoring strategy developed here acts as a promising approach to load a polymer-based conjugate/prodrug in liposomes to improve delivery.The conjugate HA-ORD with a molecular weight of over 10,000 Da is challenging to be loaded in liposome core or lipid bilayer.Rather than loading the conjugate at the two locations,we loaded it on liposomes by the anchoring strategy that the hydrophobic section in conjugate inserted into the liposome’s lipid membrane.Furthermore,the anchoring improved the pharmacokinetic performance of the pHSLs.In addition,the anchoring facilitated plaque accumulation of the co-loaded pHSLs because HA on the co-loaded pHSLs could target the CD44 receptors that are overexpressed on the cells inhabiting the plaques.The CD44 receptors in the plaque have>10-fold increase over that in healthy vascular tissue [51].Nonetheless,the plaque targeting-efficacy of drug delivery systems is correlated with plaque advancement [52].Additional study is needed to clarify the targetability of the co-loaded pHSLs to the early and advanced plaque.

    The features,including thin fibrous cap,inflammation,accumulation of inflammatory cells,and raised blood lipid levels are closely linked to the rupture of the plaque [53].Significantly,the loss of fibrillar collagen is a direct cause to make the fibrous cap become thinning [54].In this study,we found that the targeting co-delivery of the collagen protector MATT and antiinflammatory HA-ORD conjugate using pHSLs to the plaque relieved the degradation of plaque collagen,reduced the accumulation of M1-like macrophages,alleviated inflammation,and declined the blood level.These indicators confirmed that the coloaded pHSLs could stabilize the rupture-prone plaque in HFDApoe-/-mice.Furthermore,we found that the plaque was regressed,as evidenced by dosing the co-loaded pHSLs to HFDApoe-/-mice reduced the plaque area by 1–1.5-fold compared to the PBS treatment.The plaque regression may be ascribed to the lowering of blood lipids,LDL and TC,and reduced residence of proinflammatory macrophages after administration of co-loaded pHSLs [55].

    IL-6 plays a leading role in the inflammatory progressions underlying cardiovascular diseases [28,56],and meanwhile,TNF-αfacilitates the development of plaque at all stages [12,57].Herein,we demonstrated that treatment with the co-loaded pHSLs significantly reduced the IL-6,IL-22,and TNF-αin HFD-Apoe-/-mice(Figs.3C–E).The data is consistent with the serum determination from the AS patients,in which the two indicators markedly upregulated,as displayed in Fig.S14.As a result,our data may inspire the clinic treatment of AS,which is combinatorial use of antiinflammatory drugs and collagen protectors to improve AS therapy.

    In summary,the co-loaded pHSLs allowed effective anti-AS therapy by targeted co-delivery of the anti-inflammatory (HA-ORD)and collagen protector (MATT) to the plaque.Moreover,the targeted co-delivery suppressed plaque regression.Besides promoting macrophage polarization into M2-like phenotype and alleviating inflammation and plaque-collagen degradation,we confirmed that the co-delivery could lower the blood lipid level and modulate the immune-cell ratio of Th17/Tregs and Th1/Th2 as well.We believe the current liposomal strategy is promising to target the AS plaque.Targeted co-delivery of anti-inflammatory drugs and collagen protectors to the plaque represents an efficient regimen to treat AS.The finding may provide a potential guideline for the clinic practices of AS treatment.

    MATT utilized in the study was a broad-spectrum MMP inhibitor and was not approved for clinical use.However,Periostat,an MMP inhibitor,has been permitted clinically [58].Accordingly,the combinatorial use of an anti-inflammatory drug and Periostat has the potential as an AS therapy regimen.

    Declaration of competing interest

    The authors have no conflicts of interest to declare.

    Acknowledgments

    This study was supported by the National Natural Science Foundation of China (Nos.81872823,82073782,81970374),the Shanghai Science and Technology Committee (No.19430741500).We are thankful for the guidance and help provided by Lab Center,the Second Affiliated Hospital of Nanjing Medical University.

    Supplementary materials

    Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.cclet.2022.04.081.

    在线视频色国产色| 十八禁网站免费在线| 日韩欧美免费精品| 无人区码免费观看不卡| 亚洲精品国产色婷婷电影| 一区二区三区国产精品乱码| 亚洲欧美精品综合一区二区三区| 国产成人系列免费观看| 日本a在线网址| 99久久国产精品久久久| 美女福利国产在线| av中文乱码字幕在线| 女性生殖器流出的白浆| 中文字幕人妻熟女乱码| 免费一级毛片在线播放高清视频 | 亚洲成人久久性| 国产免费av片在线观看野外av| а√天堂www在线а√下载| 精品无人区乱码1区二区| 人人妻人人添人人爽欧美一区卜| 麻豆成人av在线观看| 午夜福利欧美成人| 精品福利永久在线观看| 国产成人啪精品午夜网站| 国产亚洲精品综合一区在线观看 | 97超级碰碰碰精品色视频在线观看| 免费观看精品视频网站| 天天躁夜夜躁狠狠躁躁| 久久久精品欧美日韩精品| 亚洲欧美激情在线| 精品乱码久久久久久99久播| 午夜精品在线福利| 窝窝影院91人妻| 激情视频va一区二区三区| 亚洲国产看品久久| av超薄肉色丝袜交足视频| 精品午夜福利视频在线观看一区| 亚洲国产精品合色在线| 精品人妻在线不人妻| 高清黄色对白视频在线免费看| 中文字幕色久视频| 曰老女人黄片| 久久天堂一区二区三区四区| 欧美成狂野欧美在线观看| 国产aⅴ精品一区二区三区波| 女性被躁到高潮视频| 在线视频色国产色| 国产精品 国内视频| av网站在线播放免费| 精品国产国语对白av| 日本免费一区二区三区高清不卡 | 国产av又大| 久久人妻av系列| 午夜91福利影院| 欧美亚洲日本最大视频资源| 日本五十路高清| 嫩草影视91久久| 亚洲伊人色综图| 男人舔女人下体高潮全视频| 精品久久久久久,| 美女午夜性视频免费| 日韩欧美一区视频在线观看| 宅男免费午夜| 女生性感内裤真人,穿戴方法视频| 久久中文字幕一级| 日韩大码丰满熟妇| 热99re8久久精品国产| 成熟少妇高潮喷水视频| 黑人欧美特级aaaaaa片| av电影中文网址| 一区二区三区激情视频| 日韩精品免费视频一区二区三区| 桃红色精品国产亚洲av| 国产日韩一区二区三区精品不卡| 12—13女人毛片做爰片一| 亚洲精品美女久久久久99蜜臀| 欧美成狂野欧美在线观看| 久久人妻av系列| 精品国产亚洲在线| 久久精品人人爽人人爽视色| 中出人妻视频一区二区| 757午夜福利合集在线观看| 亚洲第一av免费看| 国产黄色免费在线视频| 久久青草综合色| 每晚都被弄得嗷嗷叫到高潮| 叶爱在线成人免费视频播放| 精品久久久久久成人av| 女同久久另类99精品国产91| 亚洲精品国产精品久久久不卡| 在线观看日韩欧美| 精品高清国产在线一区| 搡老岳熟女国产| 亚洲欧美一区二区三区黑人| 国产高清国产精品国产三级| 欧美日韩黄片免| 免费高清视频大片| 亚洲一卡2卡3卡4卡5卡精品中文| 黄色怎么调成土黄色| 亚洲国产欧美日韩在线播放| av在线天堂中文字幕 | 欧美日韩亚洲高清精品| 免费观看人在逋| 亚洲第一欧美日韩一区二区三区| 又紧又爽又黄一区二区| 国产精品一区二区三区四区久久 | 国产精品日韩av在线免费观看 | 精品高清国产在线一区| av在线播放免费不卡| 电影成人av| 久久人妻av系列| 欧美日韩亚洲高清精品| 日日干狠狠操夜夜爽| √禁漫天堂资源中文www| 九色亚洲精品在线播放| 国产精品九九99| 人人妻人人澡人人看| 99久久99久久久精品蜜桃| 亚洲美女黄片视频| 超色免费av| 午夜免费鲁丝| 久久久久亚洲av毛片大全| 日韩欧美免费精品| 两个人看的免费小视频| 12—13女人毛片做爰片一| 嫩草影院精品99| 久久精品国产99精品国产亚洲性色 | 亚洲一区高清亚洲精品| 亚洲成人免费电影在线观看| 午夜福利免费观看在线| 操美女的视频在线观看| 国产精品免费视频内射| 久久香蕉国产精品| 欧洲精品卡2卡3卡4卡5卡区| 大型黄色视频在线免费观看| 欧美在线一区亚洲| 免费在线观看日本一区| 久久人人精品亚洲av| 免费搜索国产男女视频| 国产精品电影一区二区三区| 久久国产精品男人的天堂亚洲| 最近最新免费中文字幕在线| 久久热在线av| 自线自在国产av| 性色av乱码一区二区三区2| 亚洲国产中文字幕在线视频| 午夜两性在线视频| 露出奶头的视频| 少妇的丰满在线观看| 欧美不卡视频在线免费观看 | 免费在线观看日本一区| 久久狼人影院| 女警被强在线播放| 97人妻天天添夜夜摸| 国产精品一区二区三区四区久久 | av网站免费在线观看视频| 伦理电影免费视频| 日韩免费av在线播放| 国产精品av久久久久免费| 成人三级做爰电影| 国产无遮挡羞羞视频在线观看| 国产精品爽爽va在线观看网站 | 大型av网站在线播放| 夫妻午夜视频| 亚洲少妇的诱惑av| 超色免费av| 成人精品一区二区免费| 天天影视国产精品| 久久久国产成人免费| 91在线观看av| 国产成年人精品一区二区 | 美女大奶头视频| 久久天躁狠狠躁夜夜2o2o| 国产成人av激情在线播放| 女人高潮潮喷娇喘18禁视频| 超色免费av| 色播在线永久视频| 欧美黑人精品巨大| 99久久国产精品久久久| 日韩精品中文字幕看吧| 五月开心婷婷网| av网站在线播放免费| 国产无遮挡羞羞视频在线观看| 女性生殖器流出的白浆| 精品久久久久久成人av| av天堂在线播放| 69av精品久久久久久| 精品国产乱子伦一区二区三区| 神马国产精品三级电影在线观看 | 女同久久另类99精品国产91| 欧美日韩乱码在线| 国产精品一区二区在线不卡| 麻豆成人av在线观看| 精品人妻1区二区| 国产精品98久久久久久宅男小说| 亚洲国产毛片av蜜桃av| 久久香蕉国产精品| 亚洲精华国产精华精| www.www免费av| 99精品欧美一区二区三区四区| 久久久久久久久久久久大奶| 国产aⅴ精品一区二区三区波| 一进一出抽搐gif免费好疼 | 中文字幕色久视频| 亚洲av成人一区二区三| 国产高清视频在线播放一区| 国产黄a三级三级三级人| 在线观看日韩欧美| 免费一级毛片在线播放高清视频 | 天天影视国产精品| 日本a在线网址| 成熟少妇高潮喷水视频| 亚洲午夜精品一区,二区,三区| 男人舔女人下体高潮全视频| 交换朋友夫妻互换小说| 欧洲精品卡2卡3卡4卡5卡区| 国产精品免费视频内射| 免费av毛片视频| 久久久久国内视频| 国产精品永久免费网站| 亚洲欧美精品综合一区二区三区| 国产免费av片在线观看野外av| 久久久久久久久久久久大奶| 精品一区二区三区av网在线观看| 亚洲av成人不卡在线观看播放网| 国产亚洲精品久久久久5区| 精品一区二区三区av网在线观看| 麻豆久久精品国产亚洲av | 91老司机精品| 国产一区二区三区视频了| 美女高潮喷水抽搐中文字幕| 91国产中文字幕| 黄色a级毛片大全视频| 99久久久亚洲精品蜜臀av| 亚洲精品美女久久av网站| 69精品国产乱码久久久| 亚洲精品av麻豆狂野| 国产欧美日韩一区二区三区在线| 国内毛片毛片毛片毛片毛片| av片东京热男人的天堂| 久久国产亚洲av麻豆专区| 亚洲 国产 在线| 极品教师在线免费播放| 精品日产1卡2卡| 亚洲精品一卡2卡三卡4卡5卡| 午夜免费观看网址| 国产精品成人在线| 精品久久久久久久久久免费视频 | 美女高潮喷水抽搐中文字幕| 制服诱惑二区| av国产精品久久久久影院| 午夜精品国产一区二区电影| 成人免费观看视频高清| 日本wwww免费看| 中文字幕精品免费在线观看视频| 天天躁狠狠躁夜夜躁狠狠躁| 久久精品国产亚洲av高清一级| 欧美日韩亚洲国产一区二区在线观看| 一边摸一边抽搐一进一出视频| 黄色女人牲交| 中国美女看黄片| 精品国产国语对白av| 国产精品久久久人人做人人爽| 黄色片一级片一级黄色片| 亚洲精品一卡2卡三卡4卡5卡| 99国产综合亚洲精品| 国产日韩一区二区三区精品不卡| 两个人免费观看高清视频| x7x7x7水蜜桃| 欧美一区二区精品小视频在线| a级毛片在线看网站| 美女福利国产在线| 国产午夜精品久久久久久| svipshipincom国产片| 久久久久亚洲av毛片大全| 亚洲五月婷婷丁香| 不卡av一区二区三区| 亚洲,欧美精品.| 性少妇av在线| 91字幕亚洲| 91国产中文字幕| 欧美不卡视频在线免费观看 | 欧美日韩瑟瑟在线播放| 午夜亚洲福利在线播放| 午夜福利影视在线免费观看| 天天添夜夜摸| 黄色毛片三级朝国网站| 99国产精品免费福利视频| 成人18禁在线播放| 又黄又爽又免费观看的视频| 99国产综合亚洲精品| 丁香六月欧美| 免费高清视频大片| 91老司机精品| 欧美黑人欧美精品刺激| 国产高清国产精品国产三级| 另类亚洲欧美激情| 高清在线国产一区| 国产高清激情床上av| 狂野欧美激情性xxxx| 乱人伦中国视频| 久久香蕉激情| 身体一侧抽搐| 亚洲五月婷婷丁香| 亚洲 欧美 日韩 在线 免费| 一区二区三区精品91| 水蜜桃什么品种好| 电影成人av| 欧美国产精品va在线观看不卡| 视频区欧美日本亚洲| 亚洲在线自拍视频| 免费在线观看完整版高清| 亚洲avbb在线观看| 丁香欧美五月| 老司机福利观看| 久久国产乱子伦精品免费另类| 久99久视频精品免费| 曰老女人黄片| 人成视频在线观看免费观看| 激情在线观看视频在线高清| 精品福利永久在线观看| 国产乱人伦免费视频| 亚洲七黄色美女视频| 午夜福利影视在线免费观看| 少妇裸体淫交视频免费看高清 | 91麻豆精品激情在线观看国产 | 国产91精品成人一区二区三区| www国产在线视频色| 婷婷精品国产亚洲av在线| 成人手机av| 国产视频一区二区在线看| 老鸭窝网址在线观看| 亚洲一区中文字幕在线| 一个人观看的视频www高清免费观看 | 亚洲免费av在线视频| 久久婷婷成人综合色麻豆| 中文字幕另类日韩欧美亚洲嫩草| 亚洲第一欧美日韩一区二区三区| 在线观看舔阴道视频| 巨乳人妻的诱惑在线观看| 亚洲精品中文字幕一二三四区| 亚洲欧洲精品一区二区精品久久久| 免费在线观看影片大全网站| 国产亚洲精品久久久久5区| 伦理电影免费视频| 自线自在国产av| 夜夜爽天天搞| 黄色怎么调成土黄色| 国产又爽黄色视频| 男人操女人黄网站| 久久精品亚洲熟妇少妇任你| 天堂动漫精品| 大码成人一级视频| 麻豆久久精品国产亚洲av | 欧美性长视频在线观看| www.999成人在线观看| 亚洲成国产人片在线观看| 亚洲熟女毛片儿| 极品教师在线免费播放| 夜夜夜夜夜久久久久| 成人特级黄色片久久久久久久| av在线天堂中文字幕 | 久久精品亚洲熟妇少妇任你| 亚洲男人天堂网一区| 久久久久久亚洲精品国产蜜桃av| 国产视频一区二区在线看| 极品教师在线免费播放| 91大片在线观看| 在线视频色国产色| 成人黄色视频免费在线看| 日韩精品中文字幕看吧| 美女高潮到喷水免费观看| √禁漫天堂资源中文www| 国产深夜福利视频在线观看| av超薄肉色丝袜交足视频| 久久久久久久久中文| 18禁国产床啪视频网站| 首页视频小说图片口味搜索| 日韩成人在线观看一区二区三区| 亚洲第一欧美日韩一区二区三区| 午夜a级毛片| av网站在线播放免费| 国产精品香港三级国产av潘金莲| 国内久久婷婷六月综合欲色啪| 一区二区日韩欧美中文字幕| 91大片在线观看| 美女高潮喷水抽搐中文字幕| 中文字幕av电影在线播放| 美女高潮到喷水免费观看| 精品高清国产在线一区| 91av网站免费观看| 成年版毛片免费区| 桃红色精品国产亚洲av| 一进一出抽搐gif免费好疼 | 日韩欧美一区视频在线观看| 久久影院123| 深夜精品福利| 一级毛片高清免费大全| 中文字幕另类日韩欧美亚洲嫩草| 激情在线观看视频在线高清| 十八禁人妻一区二区| 一边摸一边做爽爽视频免费| 久9热在线精品视频| 999久久久国产精品视频| 18禁黄网站禁片午夜丰满| 亚洲伊人色综图| 久久久久久亚洲精品国产蜜桃av| 一a级毛片在线观看| √禁漫天堂资源中文www| 免费av毛片视频| 国产在线精品亚洲第一网站| 国产精品日韩av在线免费观看 | 国产野战对白在线观看| 高清av免费在线| 国产免费av片在线观看野外av| 最新美女视频免费是黄的| 亚洲熟妇中文字幕五十中出 | 男女下面插进去视频免费观看| 水蜜桃什么品种好| 两性夫妻黄色片| 欧美亚洲日本最大视频资源| 很黄的视频免费| 亚洲欧美日韩无卡精品| 丰满人妻熟妇乱又伦精品不卡| 亚洲激情在线av| 午夜免费鲁丝| 日韩视频一区二区在线观看| 亚洲一区中文字幕在线| 一区二区三区精品91| 成人av一区二区三区在线看| 亚洲欧美激情综合另类| 日本欧美视频一区| 中文字幕最新亚洲高清| 老汉色av国产亚洲站长工具| 韩国av一区二区三区四区| 亚洲七黄色美女视频| 黄色成人免费大全| 欧美乱妇无乱码| 亚洲 国产 在线| 国产精品亚洲av一区麻豆| 男女下面进入的视频免费午夜 | 一边摸一边抽搐一进一小说| 麻豆久久精品国产亚洲av | 亚洲片人在线观看| 国产成人av激情在线播放| 久久香蕉国产精品| 日韩国内少妇激情av| 久久天躁狠狠躁夜夜2o2o| 国产无遮挡羞羞视频在线观看| 波多野结衣高清无吗| 久99久视频精品免费| 美女扒开内裤让男人捅视频| 欧美午夜高清在线| 国产1区2区3区精品| 欧美日韩亚洲综合一区二区三区_| 欧美 亚洲 国产 日韩一| 一个人观看的视频www高清免费观看 | 91大片在线观看| 日韩三级视频一区二区三区| 高清欧美精品videossex| 色精品久久人妻99蜜桃| 亚洲人成网站在线播放欧美日韩| 黄片大片在线免费观看| 国产精品影院久久| 亚洲男人的天堂狠狠| 国产成年人精品一区二区 | 夜夜爽天天搞| 免费在线观看影片大全网站| 在线av久久热| 日韩欧美一区视频在线观看| 精品国内亚洲2022精品成人| 免费女性裸体啪啪无遮挡网站| 亚洲专区国产一区二区| 欧美日韩一级在线毛片| 亚洲第一av免费看| 国产在线精品亚洲第一网站| 免费高清视频大片| 好看av亚洲va欧美ⅴa在| 国产成人精品无人区| 女同久久另类99精品国产91| 99久久国产精品久久久| 男人舔女人下体高潮全视频| 亚洲情色 制服丝袜| 午夜福利免费观看在线| 亚洲色图av天堂| 后天国语完整版免费观看| 乱人伦中国视频| 午夜久久久在线观看| 美女高潮喷水抽搐中文字幕| 亚洲av片天天在线观看| av超薄肉色丝袜交足视频| 少妇粗大呻吟视频| 欧美成人免费av一区二区三区| 99热只有精品国产| 国产精品美女特级片免费视频播放器 | 午夜成年电影在线免费观看| 亚洲精品一区av在线观看| 1024视频免费在线观看| 色婷婷久久久亚洲欧美| 国产精品自产拍在线观看55亚洲| 一级片'在线观看视频| 黄色丝袜av网址大全| 波多野结衣高清无吗| 成人手机av| 亚洲人成网站在线播放欧美日韩| 香蕉国产在线看| 国产亚洲精品第一综合不卡| 国产aⅴ精品一区二区三区波| 在线免费观看的www视频| 亚洲国产欧美网| 亚洲aⅴ乱码一区二区在线播放 | 亚洲黑人精品在线| 国产高清视频在线播放一区| av网站在线播放免费| 亚洲熟妇中文字幕五十中出 | 超色免费av| 人人妻,人人澡人人爽秒播| 日韩一卡2卡3卡4卡2021年| 黄色女人牲交| 精品卡一卡二卡四卡免费| 中亚洲国语对白在线视频| 欧美一区二区精品小视频在线| 婷婷丁香在线五月| 亚洲熟妇熟女久久| 激情在线观看视频在线高清| 免费在线观看黄色视频的| 久久国产精品男人的天堂亚洲| 我的亚洲天堂| 最近最新中文字幕大全免费视频| 老汉色∧v一级毛片| 欧美黄色淫秽网站| 欧美激情 高清一区二区三区| 咕卡用的链子| a在线观看视频网站| 涩涩av久久男人的天堂| 国产高清国产精品国产三级| 欧美日韩亚洲综合一区二区三区_| 国产亚洲精品久久久久久毛片| 操美女的视频在线观看| 久久精品国产清高在天天线| 国产亚洲精品一区二区www| 男人的好看免费观看在线视频 | 亚洲欧洲精品一区二区精品久久久| 久久久国产精品麻豆| 亚洲专区国产一区二区| 日本撒尿小便嘘嘘汇集6| 国产精品久久久人人做人人爽| 在线国产一区二区在线| 十八禁人妻一区二区| 村上凉子中文字幕在线| 国产精品一区二区免费欧美| 51午夜福利影视在线观看| 丝袜在线中文字幕| 亚洲男人的天堂狠狠| av有码第一页| 777久久人妻少妇嫩草av网站| 午夜老司机福利片| 国产蜜桃级精品一区二区三区| netflix在线观看网站| 亚洲人成电影观看| 亚洲欧美日韩无卡精品| 欧美最黄视频在线播放免费 | 韩国av一区二区三区四区| 日韩人妻精品一区2区三区| 欧美日韩亚洲高清精品| √禁漫天堂资源中文www| 亚洲人成77777在线视频| 侵犯人妻中文字幕一二三四区| 精品无人区乱码1区二区| a级片在线免费高清观看视频| 韩国av一区二区三区四区| 青草久久国产| 一a级毛片在线观看| 国产成人av激情在线播放| 大型av网站在线播放| 日韩免费av在线播放| 男女高潮啪啪啪动态图| av在线天堂中文字幕 | 久久亚洲真实| 日本免费一区二区三区高清不卡 | 欧美人与性动交α欧美软件| 国产av又大| 一区福利在线观看| 久久久久久久久中文| 黄片大片在线免费观看| 啦啦啦免费观看视频1| 怎么达到女性高潮| 亚洲精华国产精华精| 国产三级在线视频| 丰满迷人的少妇在线观看| 满18在线观看网站| 日本免费一区二区三区高清不卡 | 黑人巨大精品欧美一区二区mp4| 97超级碰碰碰精品色视频在线观看| 欧美激情 高清一区二区三区| 欧美精品啪啪一区二区三区| 无人区码免费观看不卡| 日韩欧美三级三区| 国产精品乱码一区二三区的特点 | 在线国产一区二区在线| 91国产中文字幕| 在线观看一区二区三区| 91在线观看av| 一区在线观看完整版| 日本 av在线| 午夜福利影视在线免费观看| 欧美日韩视频精品一区| 男女做爰动态图高潮gif福利片 | 国产精品亚洲一级av第二区| 欧美中文日本在线观看视频| 天堂√8在线中文| 亚洲欧美日韩另类电影网站| 老司机午夜福利在线观看视频| 久久久国产精品麻豆| 热re99久久精品国产66热6|