• <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 ?

    A hybrid nano-assembly with synergistically promoting photothermal and catalytic radical activity for antibacterial therapy

    2022-11-05 06:47:44YunxiWngChungxinZhngHuiZhngLihengFengLibingLiu
    Chinese Chemical Letters 2022年10期

    Yunxi Wng,Chungxin Zhng,Hui Zhng,Liheng Feng,*,Libing Liu

    a School of Chemistry and Chemical Engineering,Shanxi University,Taiyuan 030006,China

    b Department of Nutrition and Health,China Agricultural University,Beijing 100193,China

    c Beijing National Laboratory for Molecular Sciences,Institute of Chemistry,Chinese Academy of Sciences,Beijing 100190,China

    Keywords:Conjugated oligomer Nano-assembly Fenton reaction Photothermal therapy Bacterial infections

    ABSTRACT It is of great significance to develop effective antibacterial agents and methods to combat drug resistant bacterial infections due to its increasing threaten to human health and the ineffectiveness of antibiotics.Herein,a multifunctional hybrid nano-assembly (M1-Fe NPs) based on conjugated oligomer and ferrous ion was engineered with favorable bactericidal activity for synergetic antibacterial therapy.The chelation of ferrous ion not only enhances the photothermal conversion efficiency of M1 but also endows the nano-assembly with catalytic capability of transferring H2O2 into stronger oxidant hydroxyl radicals(·OH).Meanwhile,the generated heat can further promote the Fenton reaction activity.By generating cytotoxic heat and oxidative ·OH,M1-Fe NPs can effectively kill Staphylococcus aureus in vitro and in vivo with the aid of low dosage of H2O2.The work provides a new multifunctional platform for combinational drug resistant antibacterial therapy and even antitumor therapy.

    Although the generation of various antibiotics,the drug resistant bacterial infections still threaten the health of human being over the world [1-4].It is emergent to develop effective antibacterial agents and antibacterial strategies to resist the evolution of bacteria and eliminate the damage from bacterial infections.By now,different kinds of materials have been developed to antibacterial agents relied on diverse antibacterial mechanisms [5-10].Agbased materials can kill bacteria by inhibiting replication through the binding of Ag ions with ATP synthetic enzymes [11].Cationic polymers,quaternary ammonium salts,and antibacterial peptides can perturb and disrupt the cell membrane of bacteria to induce the death of bacteria [12-15].Conjugated polymer [16-20],metalorganic frameworks [21-23],black phosphorus [24,25],copper sulfide [26,27],Au-[28-30]and carbon-based materials [31,32]can generate reactive oxygen species (ROS) or heat under light irradiation to oxidize or damage phospholipids,proteins and even DNA to kill bacteria,which is involved in photodynamic therapy (PDT)and photothermal therapy (PTT).In addition,hydrogels are also developed as multifunctional anti-infective materials by combining above mentioned antibacterial mechanism with ability of tissue repair [33].However,there are still some disadvantages in current antibacterial agents for practical antibacterial therapy,such as low antibacterial ability and long-term cytotoxicity,and it remains a challenge to develop highly effective antibacterial agents and strategies with low biological toxicity and little possibility to trigger drug resistance.

    PDT and PTT are considered to be effective antibacterial therapies with characteristics of broad-spectrum antibacterial activity,spatial and temporal control and low side effects [34].The ROS and hyperthermia generated by photosensitizers and photothermal agents can kill all type of bacteria,including antibiotic resistant strains,with little chance to induce drug resistance.However,these two therapies have respective shortcomings,such as the limited damage to bacteria due to the oxygen-dependent antibacterial activity and the unnecessary damage to normal cells due to the excessive ROS and temperature [35,36].To address the issues,combined therapy of PDT and PTT is increasingly developed to maximize the advantages of two therapies and make up for the shortages of each other.Hydroxyl radical (·OH) is one kind of ROS and has stronger oxidation ability than other ROS,such as H2O2and1O2[37,38].Moreover,·OH can be generated by decomposition of H2O2with the aid of ferrous ion (Fe2+),which termed as Fenton reaction [39],without dependence of oxygen.It shows remarkable advantage over oxygen-dependent traditional PDT.Hence,Fenton reaction has attracted broad interest of researchers for development of chemodynamic therapy (CDT).Increasing works focus on the selection of nanomaterials and strategy to enhance the efficiency of CDT,offering important findings and rational instructions to the development of antitumor therapy [39,40].As most CDT agents are inorganic materials,organic materials also begin to be applied.Although the combined PTT and CDT for cancer treatment has been intensively investigated,for the antibacterial therapy combined two therapies remains rare,especially that mediated by organic materials.Therefore,it is imperative to develop organic materials with photothermal and catalytic Fenton reaction activity to realize synergistic treatment of bacterial infections.

    In this work,to effectively eradicate bacteria and lower the damage to normal tissue,we proposed a new antibacterial strategy of photothermal and chemodynamic synergistic promotion.Accordingly,we developed a hybrid nano-assembly with photothermal and catalytic Fenton reaction activity for synergetic antibacterial therapy (Scheme 1).The nano-assembly,termed as M1-Fe NPs,is comprised of ferrous ion-chelated conjugated oligomer M1 and amphiphilic polymer PLGA-PEG.M1 is designed to chelate with Fe2+offering catalytic activity of Fenton reaction and also act as photothermal agent.Furthermore,the chelation with Fe2+can in turn enhance the photothermal effect of M1.Hence,in the presence of H2O2and light irradiation (660 nm),M1-Fe NPs can catalyze decomposition of H2O2to generate stronger oxidizer·OH to damage bacteria.Meanwhile,the heat generated by M1 can further kill bacteria and promote the generation of·OH by accelerating Fenton reaction.TakingStaphylococcus aureus(S.aureus)as a typical representative,the synergistic damage of heat and·OH can effectively kill it by destroying membrane,proteins,enzyme and DNA.Besides,under light irradiation,M1-Fe NPs successfully treatS.aureus-infected wound of mice at the aid of low dosage of H2O2.Therefore,this work provides a hybrid nanoassembly based on conjugated oligmer to achieve highly effective and synergistic antibacterial PTT and CDT,and it also offers a novel multi-functional antibacterial platform for effective anti-infective therapy.

    Scheme 1.Schematic illustration of designed multifunctional hybrid nano-assembly M1-Fe NPs for highly effective and synergetic antibacterial therapy.

    Conjugated oligomer M1 is a kind of photothemal agent and can be prepared to nanoparticle M1-NPs,which has been reported by our previous literature [41].Its synthetic route is shown as Fig.S1 (Supporting information).However,it is necessary to develop new method to improve the photothmal property for better antibacterial application.Since the molecular structure of M1 contains heteroatoms such as sulfur and nitrogen,it is promise to have good chelation between M1 and Fe2+.The chelation probably changes the molecular configuration and light absorption ability of M1.And Fe2+is considered to have catalytic Fenton reaction activity that is heat-related and benefits antibacterial application.Therefore,Fe2+is designed to chelate with M1 to change the photothermal property and endow M1 with catalytic activity of Fenton reaction at the same time for obtaining enhanced antibacterial capability.

    M1 was cheated with Fe2+and then mixed with PLGA-PEG to prepare nanoparticles noted as M1-Fe NPs by nanoprecipitation method.The optical properties of chelated compound (M1-Fe) and nanoparticles were next investigated.As shown in Fig.S2 (Supporting information),the maximum absorption peak of oligomer M1 had an obvious blue-shift from 642 nm to 610 nm after chelated with Fe2+.Even processed to nanoparticles,the obvious blue-shift from 670 nm to 655 nm in the absorption spectra was still found(Fig.1a).It indicates that the chelation between M1 and Fe2+exist and is stable.Besides,the formation of nanoparticles made absorption spectrum broader (extending to 900 nm),especially better absorption at 660 nm,which provided excellent conditions for photothermal therapy.At an excitation wavelength of 650 nm,almost no fluorescence peak was observed in the fluorescent spectrum (ranging from 700 nm to 900 nm) of M1-Fe NPs (Fig.S3 in Supporting information),which further facilitated the conversion from light to heat.The average hydrodynamic diameter of M1-Fe NPs was measured to be ~60 nm by using dynamic light scattering(DLS) and was shown in Fig.1b.To further understand the morphology of M1-Fe NPs,scanning electron microscopy (SEM) was employed.As shown in Fig.S4 (Supporting information),M1-Fe NPs showed a uniformly spherical shape.

    Next,to verify the catalytic activity of M1-Fe NPs for catalyzing decomposition of H2O2,aminophenylfluorescein (APF) was used to detected the production of·OH by measuring the fluorescence of oxidized APF [42].Considering the acidic microenvironment at the site of infection,the detection was performed at a pH of 6.5.As shown in Fig.1c,compared with M1 NPs,M1-Fe NPs can catalyze H2O2to generate more·OH with stronger fluorescent intensity.After light irradiation,the amount of generated·OH was a little more,which might because M1-Fe NPs generate heat to accelerate the heat-related catalytic reaction.With the pH at 7.4,the generated·OH was obviously lower than that of 6.5 (Fig.1d).It indicates the introduction of Fe2+endows the nanoparticles with catalytic property and the catalytic activity is potential to be better in bacterial microenvironment under light irradiation.

    The ability of converting light to heat is important for photothermal antibacterial application,therefore,we investigated the photothermal property of M1-Fe NPs,and a 660 nm laser was used to irradiate nanoparticle solution.At a power density of 1.0 W/cm2,the temperature of M1-Fe NPs dispersion with different concentrations increased to varying degrees and the increase was positively correlated with nanoparticles concentrations and irradiation time(Fig.2a).After an irradiation of 8 min,the temperature of M1-Fe NPs was up to to 55 °C at a low concentration of 10.0 μg/mL,which indicated M1-Fe NPs had good photothermal capability.The corresponding thermal images that visualized the temperature changes of solutions are displayed as Fig.2b.In addition,the increase of temperature was also positively correlated with power density of light.As shown in Fig.S5 (Supporting information),the temperature of M1-Fe NPs (10.0 μg/mL) increased to 60 °C at an irradiated power density of 1.25 W/cm2.To further verify the boost effect of Fe2+on the photothermal property of M1 NPs,we compared the temperature changes of two nanoparticles under the same condition.As shown in Fig.2c,the temperature of M1-Fe NPs was higher than that of M1 NPs about 4 °C.It proved that the chelation indeed enhanced the photothermal conversion capability of M1 NPs.After five heating-cooling cycles,the temperature profiles of M1-Fe NPs were little changed,indicating the good photothermal stability (Fig.2d).All above mentioned results illustrate that fabricated M1-Fe NPs are potential to be applied in the photothermal antibacterial application.

    Fig.2.(a) Temperature changes and (b) infrared thermal images of M1-Fe NPs with different concentrations under light irradiation (660 nm,1.0 W/cm2).(c) Temperature profiles of M1 NPs and M1-Fe NPs under the same experimental condition (660 nm,1.0 W/cm2),respectively.(d) Temperature curve of M1-Fe NPs for 5 cycles of on/off light irradiation (660 nm laser,1 W/cm2).

    After verifying the photothermal and catalytic property of M1-Fe NPs,we next examined its antibacterial activity against planktonic bacteria.S.aureusis chosen as the model bacteria for it is a common pathogen and has better heat resistance [43].H2O2is employed as a source of·OH.Considering that the microenvironment of bacterial infected site was acidic,we performed the antibacterial experiment at a pH of 6.5 to simulate the infected environment,and set pH of 7.4 as contrast condition.As shown in Fig.3a,bacterial viability was decreased with the increase of M1-Fe NPs concentration,proving efficient and synergistic dual treatment mode.The corresponding bacterial plates are displayed as Fig.3b and Fig.S6 (Supporting information).In contrast,even treated with the highest concentration of M1-Fe NPs,about 70% of bacteria were still alive at pH of 7.4 and 6.5 without light irradiation.It indicates that·OH generated by decomposition of H2O2is not enough to eradicate bacteria.As the light introduced,the bacterial activity decreased obviously,no matter pH was 7.4 or 6.5.It verifies that heat plays an important role in killing bacteria.The decrease of bacterial activity was more remarkable at pH of 6.5,stating that the Fenton reaction was favored by the heat and acidic condition.These results illustrate that M1-Fe NPs are potential to effectively kill bacteria at infected site by generating heat and catalyzing H2O2.

    Fig.3.(a) Antibacterial activity of M1-Fe NPs with the aid of H2O2 under different concentration and pH conditions.(b) Corresponding bacterial plate images at killing concentration (10.0 μg/mL).(c) Antibacterial activity of different components at pH of 6.5 in dark or under irradiation and corresponding (d) bacterial plate images.(e)Fluorescence staining and (f) SEM images of S.aureus without and with treatment of M1-Fe NPs+H2O2 in dark or under irradiation.The scale bars are 20 μm in (e)and 1 μm in (f).Data are presented as mean ± SD (n=3).The light is a 660 nm laser (1.0 W/cm2).The concentration of M1-Fe NPs is 10.0 μg/mL.The concentration of H2O2 is 1 mmol/L.

    To figure out the synergistic antibacterial effect of heat and·OH,S.aureuswas treated with H2O2,Fe2+,H2O2+Fe2+,M1 NPs,M1-Fe NPs,and M1-Fe NPs+H2O2,respectively.The concentration of M1-Fe NPs is 10.0 μg/mL.As shown in Fig.3c,under light irradiation,the·OH generated from catalytic decomposition of 1 mmol/L H2O2by individual Fe2+could only kill 30% of bacteria.The heat generated from M1-Fe NPs under the same light irradiation damaged 50% of bacteria.Without light irradiation,the·OH generated from catalytic decomposition of H2O2by M1-Fe NPs might inhibit 34%of bacteria.In contrast,95% of bacteria could be killed by M1-Fe NPs and H2O2under the same light irradiation.The corresponding bacterial plates are displayed in Fig.3d and Fig.S7 (Supporting information).The result proves that the heat and·OH synergistically kill bacteria to obtain the best antibacterial effect in M1-Fe NPs-mediated antibacterial therapy.

    Further,the antibacterial mechanism of M1-Fe NPs with aid of H2O2was investigated.After different treatments,S.aureuswas stained by SYTO9 and PI to observe the state of bacteria through confocal laser scanning microscopy (CLSM).As shown in Fig.3e,bacteria treated with M1-Fe NPs and H2O2without light irradiation was mostly stained by SYTO9 showing green fluorescence.It indicates that most bacteria have intact membrane and kept alive.While,the bacteria was almost all stained by PI showing red fluorescence when light was performed.It demonstrates that the membranes of bacteria are ruptured and bacteria are dead.To further observe the morphology of bacteria after different treatments,SEM was employed to capture the images of bacteria.As depicted in Fig.S8 (Supporting information),the surface of bacteria treated with Fe2+and H2O2had a little collapsed,which might result from the oxidative damage of·OH that generated by Fe2+-catalyzed decomposition of H2O2.For bacteria treated with M1 NPs and M1-Fe NPs without light irradiation had little morphology change compared to that of blank group.However,upon light irradiation,the surface of some bacteria was fused,which might result from the heat produced by nanoparticles.Only the bacteria treated with M1-Fe NPs and H2O2under light irradiation appeared obvious wrinkled and fused morphology (Fig.3f).It indicates that the heat and·OH generated by M1-Fe NPs and H2O2synergistically break the membrane structure and other components of bacteria to kill them.

    Fig.4.(a) Cell viability of EA.hy926 and 293T after treatment of M1-Fe NPs with different concentrations.(b) Infrared thermal images of infected wound of mice after different treatments (660 nm,1.0 W/cm2).(c) Photographs of S.aureus-infected wounds of mice after 10-day treatment by various therapies.

    After understanding the antibacterial effects of M1-Fe NPs and H2O2,we next investigated their anti-infective ability.Firstly,the cytotoxicity of M1-Fe NPs was determined by standard MTT method.As shown in Fig.4a,M1-Fe NPs had little effect on the growth of two human cells EA.hy926 and 293T even at a concentration of 20.0 μg/mL,implying the low cytotoxicity and application possibility of M1-Fe NPs for anti-infective treatment.ThenS.aureus-infected mice modes were established to perform the antiinfective experiment.All animal procedures were performed according to the relevant laws and guidelines approved by the Animal Care and Use Committee of Shanxi University.Besides the treatment of M1-Fe NPs+H2O2,other treatments including saline,H2O2+Fe2+,M1 NPs,and M1-Fe NPs were performed as control treatments.The concentration of H2O2was 1.0 mmol/L,a low concentration for antibacterial application [38,44].Each treatment involved two groups: dark and light.The light group employed a 660 nm laser and the power density was 1.0 W/cm2plus an irradiation time of 8 min.As displayed in Fig.4b,after laser irradiation,the temperature of wound treated with M1-Fe NPs+H2O2increased to 55 °C,which was obviously higher than that of saline treated (43°C).Such locally high temperature of 55 °C can kill most bacteria and trigger little damage to normal tissue.Hence,the wound area of mice in M1-Fe NPs+H2O2plus light group decreases about 60%on the fourth day,which decreased the most in all groups (Fig.S9 in Supporting information).And after a 10-day treatment,the wound treated with M1-Fe NPs+H2O2under light irradiation had the best healing effect compared to other treatments (Fig.4c).It confirms that the synergistic PTT/CDT based on M1-Fe NPs and H2O2can effectively treatS.aureus-infected wound and get the best therapeutic effect than any other single therapy.All of the results demonstrated that M1-Fe NPs-mediated synergistic antibacterial therapy had a great potential for highly effective and safe treatment of bacterial infection.

    In summary,we have fabricated a multifunctional hybrid nanoassembly (M1-Fe NPs) based on conjugated oligomer M1 and Fe2+for synergetic PTT/CDT antibacterial therapy.The introduction of Fe2+not only increased the photothermal conversion property (increased 4 °C at 10.0 μg/mL),but also endowed the nanoparticles with catalytic capability of transferring H2O2into stronger oxidant·,OH.Moreover,the generated heat could further accelerate the catalytic decomposition of H2O2to kill more bacteria.Hence,upon light irradiation,the M1-Fe NPs could efficiently killS.aureuswith the aid of low dosage H2O2,which antibacterial effect was better than any other monotherapy.Finally,they also treated theS.aureus-infected wound of mice.This study offers a novel drug resistant antibacterial platform for effective,safe and synergistic anti-infective therapy.

    Declaration of competing interest

    The authors report no declarations of interest.

    Acknowledgments

    We are grateful to the National Natural Science Foundation of China (Nos.21977065,22177065 and 21807067),SanJin Scholars Support Plan under Special Funding (No.2017-06),Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (Nos.201802106,2019L0022),Supported by the Fund for Shanxi “1331” Project (1331),and the Program for Introducing Overseas High-level Talents of Shanxi (Hundred Talents Plan).

    Supplementary materials

    Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.cclet.2022.03.076.

    俄罗斯特黄特色一大片| 99久久成人亚洲精品观看| 亚洲av熟女| 午夜a级毛片| 9191精品国产免费久久| 午夜激情福利司机影院| 色精品久久人妻99蜜桃| 丰满人妻一区二区三区视频av | 色综合站精品国产| 一进一出抽搐gif免费好疼| 成人一区二区视频在线观看| 精品无人区乱码1区二区| 久久久精品欧美日韩精品| 国产乱人视频| 午夜福利高清视频| 国产亚洲精品一区二区www| 欧美最黄视频在线播放免费| 99精品在免费线老司机午夜| 国产综合懂色| 国产亚洲欧美98| 久久九九热精品免费| 老鸭窝网址在线观看| 亚洲熟妇熟女久久| 1024香蕉在线观看| 午夜福利18| 亚洲成人免费电影在线观看| 老司机深夜福利视频在线观看| 黑人欧美特级aaaaaa片| 亚洲成a人片在线一区二区| 一个人免费在线观看电影 | 丝袜人妻中文字幕| 国产精品一区二区三区四区久久| 国产高潮美女av| 在线国产一区二区在线| 伦理电影免费视频| 亚洲人与动物交配视频| 亚洲黑人精品在线| 久久精品综合一区二区三区| 欧美在线黄色| 成人av一区二区三区在线看| 国产亚洲精品综合一区在线观看| 一卡2卡三卡四卡精品乱码亚洲| 真人一进一出gif抽搐免费| 舔av片在线| 国产伦精品一区二区三区视频9 | 欧美中文日本在线观看视频| 久久天躁狠狠躁夜夜2o2o| 亚洲熟妇中文字幕五十中出| 免费av毛片视频| 欧美激情在线99| 午夜免费成人在线视频| 在线a可以看的网站| 国产亚洲精品av在线| 91九色精品人成在线观看| 国产1区2区3区精品| 欧美大码av| av片东京热男人的天堂| 日本 欧美在线| 国产亚洲欧美在线一区二区| 深夜精品福利| 男人舔女人下体高潮全视频| 亚洲精品在线观看二区| 99久久久亚洲精品蜜臀av| 精品国产超薄肉色丝袜足j| 久久国产精品影院| 在线十欧美十亚洲十日本专区| 欧美一级a爱片免费观看看| 国产精品久久视频播放| 国产高清有码在线观看视频| 久久久国产成人免费| 精品久久久久久久毛片微露脸| 99re在线观看精品视频| 精品人妻1区二区| 在线国产一区二区在线| 日韩大尺度精品在线看网址| 香蕉国产在线看| 天天躁日日操中文字幕| 亚洲熟妇熟女久久| 老汉色av国产亚洲站长工具| 99re在线观看精品视频| 国产av在哪里看| 日本 av在线| 九色成人免费人妻av| 亚洲狠狠婷婷综合久久图片| 国产高清有码在线观看视频| 国产亚洲av嫩草精品影院| 好男人在线观看高清免费视频| 久久久色成人| 午夜激情欧美在线| 精品一区二区三区四区五区乱码| 毛片女人毛片| 女警被强在线播放| 日本撒尿小便嘘嘘汇集6| 成人亚洲精品av一区二区| 法律面前人人平等表现在哪些方面| 色尼玛亚洲综合影院| 一区二区三区国产精品乱码| 国产午夜精品论理片| 91九色精品人成在线观看| 岛国在线观看网站| 国产精品99久久久久久久久| netflix在线观看网站| 看免费av毛片| 国产伦精品一区二区三区四那| 中文字幕最新亚洲高清| 女人被狂操c到高潮| 国产麻豆成人av免费视频| 国产淫片久久久久久久久 | 久久精品影院6| 精品久久久久久,| 久久久久久人人人人人| 免费av不卡在线播放| 欧美日韩综合久久久久久 | 99久久精品国产亚洲精品| 成人av在线播放网站| 国产麻豆成人av免费视频| 后天国语完整版免费观看| 日韩有码中文字幕| 欧美丝袜亚洲另类 | 精品一区二区三区视频在线观看免费| 又紧又爽又黄一区二区| 动漫黄色视频在线观看| 精品不卡国产一区二区三区| 国产综合懂色| 99久久成人亚洲精品观看| 日日夜夜操网爽| 一区二区三区国产精品乱码| 国产激情欧美一区二区| 激情在线观看视频在线高清| 成人特级黄色片久久久久久久| 午夜福利在线观看免费完整高清在 | 亚洲国产欧洲综合997久久,| 久久精品国产清高在天天线| 久久亚洲精品不卡| 91麻豆精品激情在线观看国产| 成年女人永久免费观看视频| 日本免费a在线| 午夜福利免费观看在线| a级毛片在线看网站| 这个男人来自地球电影免费观看| 好男人在线观看高清免费视频| 老司机午夜十八禁免费视频| 欧美日韩亚洲国产一区二区在线观看| 免费看日本二区| 国产高清videossex| 国产激情欧美一区二区| 国产亚洲精品av在线| 又紧又爽又黄一区二区| 999久久久精品免费观看国产| 日韩人妻高清精品专区| 天天躁狠狠躁夜夜躁狠狠躁| 99re在线观看精品视频| 在线观看一区二区三区| 在线免费观看的www视频| 亚洲熟妇熟女久久| 99国产综合亚洲精品| 夜夜爽天天搞| 亚洲人成电影免费在线| 特大巨黑吊av在线直播| 91老司机精品| 黑人操中国人逼视频| 天天一区二区日本电影三级| 熟女电影av网| 国产极品精品免费视频能看的| 在线观看舔阴道视频| 黄色片一级片一级黄色片| 亚洲色图 男人天堂 中文字幕| 后天国语完整版免费观看| 性欧美人与动物交配| 黑人欧美特级aaaaaa片| 亚洲av成人一区二区三| 91九色精品人成在线观看| 成人国产综合亚洲| 欧美成人一区二区免费高清观看 | 国产综合懂色| 蜜桃久久精品国产亚洲av| 亚洲在线观看片| 亚洲va日本ⅴa欧美va伊人久久| 日本成人三级电影网站| 18禁观看日本| 人人妻人人澡欧美一区二区| 中文字幕人成人乱码亚洲影| 国产成年人精品一区二区| 天堂√8在线中文| 99国产精品一区二区蜜桃av| 色精品久久人妻99蜜桃| 18美女黄网站色大片免费观看| 热99re8久久精品国产| 叶爱在线成人免费视频播放| 岛国视频午夜一区免费看| 国产精品爽爽va在线观看网站| 天天添夜夜摸| 午夜福利在线观看吧| 在线免费观看不下载黄p国产 | 99精品久久久久人妻精品| 欧美一级毛片孕妇| 国产探花在线观看一区二区| 亚洲av成人精品一区久久| 搞女人的毛片| www.自偷自拍.com| 亚洲国产看品久久| 亚洲av电影在线进入| 最近最新中文字幕大全免费视频| 欧美日韩福利视频一区二区| 一本精品99久久精品77| 一级毛片高清免费大全| 亚洲av成人一区二区三| 在线观看66精品国产| 国产精品综合久久久久久久免费| 男人舔女人的私密视频| 丁香欧美五月| 最新中文字幕久久久久 | 亚洲精品美女久久av网站| 听说在线观看完整版免费高清| 国产亚洲av嫩草精品影院| 蜜桃久久精品国产亚洲av| 毛片女人毛片| 99在线视频只有这里精品首页| 成人三级做爰电影| 99国产精品99久久久久| 特级一级黄色大片| 午夜视频精品福利| av欧美777| 国产欧美日韩一区二区三| 久久久久久国产a免费观看| 国产精品久久久久久人妻精品电影| 亚洲五月婷婷丁香| 狂野欧美白嫩少妇大欣赏| 老司机午夜福利在线观看视频| 免费观看精品视频网站| 老司机深夜福利视频在线观看| 黑人巨大精品欧美一区二区mp4| 午夜激情欧美在线| 中文字幕精品亚洲无线码一区| 日韩欧美在线二视频| 男女那种视频在线观看| 夜夜躁狠狠躁天天躁| 国产亚洲精品综合一区在线观看| 黄色 视频免费看| 国产91精品成人一区二区三区| a级毛片a级免费在线| 最近最新免费中文字幕在线| 毛片女人毛片| av女优亚洲男人天堂 | 亚洲一区二区三区不卡视频| 757午夜福利合集在线观看| 熟女电影av网| 国产成人系列免费观看| 午夜免费观看网址| 午夜福利欧美成人| 亚洲自拍偷在线| 国产精品九九99| 99热6这里只有精品| 精品久久蜜臀av无| 久久伊人香网站| 成人一区二区视频在线观看| 一本综合久久免费| 亚洲成人免费电影在线观看| 美女午夜性视频免费| 国产精品久久久久久精品电影| 国产午夜精品久久久久久| 舔av片在线| 成人无遮挡网站| 香蕉国产在线看| 精品无人区乱码1区二区| 午夜成年电影在线免费观看| 两性午夜刺激爽爽歪歪视频在线观看| 99视频精品全部免费 在线 | 精品久久久久久久末码| 后天国语完整版免费观看| 国产单亲对白刺激| 特级一级黄色大片| 欧美三级亚洲精品| 亚洲精品在线观看二区| 久久天堂一区二区三区四区| 嫩草影院精品99| 久久天躁狠狠躁夜夜2o2o| 在线免费观看不下载黄p国产 | 亚洲人成电影免费在线| 欧美中文日本在线观看视频| 男女那种视频在线观看| 好看av亚洲va欧美ⅴa在| 夜夜看夜夜爽夜夜摸| 午夜福利在线在线| 日韩欧美三级三区| 一个人免费在线观看电影 | 免费在线观看视频国产中文字幕亚洲| 欧美日韩综合久久久久久 | 日日夜夜操网爽| 在线观看免费视频日本深夜| 波多野结衣高清无吗| 在线永久观看黄色视频| 国产精品精品国产色婷婷| 这个男人来自地球电影免费观看| av片东京热男人的天堂| 国产成人精品久久二区二区免费| 每晚都被弄得嗷嗷叫到高潮| 男女做爰动态图高潮gif福利片| 精品免费久久久久久久清纯| 非洲黑人性xxxx精品又粗又长| 国产成人av激情在线播放| 亚洲欧美日韩东京热| 亚洲美女黄片视频| 又紧又爽又黄一区二区| 精品福利观看| 在线永久观看黄色视频| 国产亚洲av嫩草精品影院| 精品一区二区三区av网在线观看| 97碰自拍视频| 一个人免费在线观看的高清视频| 亚洲国产欧洲综合997久久,| 欧美性猛交╳xxx乱大交人| 国产精华一区二区三区| 法律面前人人平等表现在哪些方面| 成年版毛片免费区| 亚洲国产看品久久| 叶爱在线成人免费视频播放| 亚洲成a人片在线一区二区| 人人妻人人看人人澡| 日本成人三级电影网站| 国产成人系列免费观看| 91av网站免费观看| 无人区码免费观看不卡| 久久久久久久久免费视频了| 婷婷亚洲欧美| 欧美av亚洲av综合av国产av| 色综合站精品国产| 黄色片一级片一级黄色片| 一区二区三区国产精品乱码| 两个人的视频大全免费| 久久精品国产清高在天天线| 国产精品 国内视频| 狂野欧美激情性xxxx| 99久久精品一区二区三区| 日本黄大片高清| 精品乱码久久久久久99久播| cao死你这个sao货| 丁香六月欧美| 国产一区二区三区视频了| 国产91精品成人一区二区三区| 日韩av在线大香蕉| 老司机福利观看| 国产精品乱码一区二三区的特点| 欧美+亚洲+日韩+国产| www.999成人在线观看| 十八禁人妻一区二区| 日本三级黄在线观看| 老司机深夜福利视频在线观看| 久久久国产欧美日韩av| 亚洲七黄色美女视频| 亚洲第一欧美日韩一区二区三区| 国产亚洲精品综合一区在线观看| 日韩免费av在线播放| АⅤ资源中文在线天堂| 1024手机看黄色片| 亚洲在线观看片| av在线蜜桃| 国产高清视频在线观看网站| 国产成人影院久久av| 人妻久久中文字幕网| 韩国av一区二区三区四区| 亚洲专区国产一区二区| 全区人妻精品视频| 国产免费av片在线观看野外av| 九九热线精品视视频播放| 日本撒尿小便嘘嘘汇集6| 国产精品乱码一区二三区的特点| 久久久久亚洲av毛片大全| 久久久久久国产a免费观看| 精品日产1卡2卡| 亚洲 欧美 日韩 在线 免费| 露出奶头的视频| 国产成人啪精品午夜网站| 99久久99久久久精品蜜桃| 男女下面进入的视频免费午夜| 久久午夜亚洲精品久久| 淫妇啪啪啪对白视频| 嫩草影视91久久| 色播亚洲综合网| 91老司机精品| 无遮挡黄片免费观看| 黄片大片在线免费观看| 国产伦精品一区二区三区四那| 两个人的视频大全免费| 国产一区二区在线av高清观看| 精品熟女少妇八av免费久了| 色综合婷婷激情| av中文乱码字幕在线| 亚洲五月天丁香| 久久久精品大字幕| av在线蜜桃| 亚洲无线在线观看| 国产成人av教育| 亚洲国产日韩欧美精品在线观看 | netflix在线观看网站| 波多野结衣高清无吗| 视频区欧美日本亚洲| 国产视频内射| 亚洲最大成人中文| 国产高清激情床上av| 麻豆国产av国片精品| 男女那种视频在线观看| 国内精品美女久久久久久| 亚洲精品美女久久久久99蜜臀| 真人一进一出gif抽搐免费| 少妇的逼水好多| 少妇人妻一区二区三区视频| 制服丝袜大香蕉在线| 亚洲成a人片在线一区二区| 欧美乱码精品一区二区三区| 欧美日本亚洲视频在线播放| 国内毛片毛片毛片毛片毛片| 久久久国产成人精品二区| 亚洲国产精品合色在线| 一区福利在线观看| 国产欧美日韩一区二区精品| 免费无遮挡裸体视频| 91麻豆精品激情在线观看国产| 国产精华一区二区三区| 亚洲欧洲精品一区二区精品久久久| 亚洲人成网站在线播放欧美日韩| 男人舔女人下体高潮全视频| 亚洲成人精品中文字幕电影| 日韩欧美一区二区三区在线观看| 亚洲五月天丁香| 亚洲av成人精品一区久久| ponron亚洲| 一二三四在线观看免费中文在| 在线免费观看的www视频| 久9热在线精品视频| 丁香六月欧美| 色在线成人网| 国产精品久久视频播放| 久久久久久久久免费视频了| 12—13女人毛片做爰片一| or卡值多少钱| 老司机午夜福利在线观看视频| 国语自产精品视频在线第100页| 国产精品久久久久久精品电影| 真人一进一出gif抽搐免费| 午夜日韩欧美国产| 美女大奶头视频| 久久精品91无色码中文字幕| 怎么达到女性高潮| 国产男靠女视频免费网站| 国产成人精品久久二区二区91| 午夜久久久久精精品| 一区二区三区高清视频在线| 国内毛片毛片毛片毛片毛片| 国产久久久一区二区三区| 麻豆久久精品国产亚洲av| 久久中文字幕人妻熟女| 99久久综合精品五月天人人| 午夜两性在线视频| 午夜福利免费观看在线| 99久久综合精品五月天人人| 国产亚洲av高清不卡| 成人特级av手机在线观看| 中出人妻视频一区二区| 日韩中文字幕欧美一区二区| 国产一区二区三区在线臀色熟女| 亚洲男人的天堂狠狠| 悠悠久久av| 亚洲av熟女| 精品国产乱子伦一区二区三区| 亚洲真实伦在线观看| 中亚洲国语对白在线视频| 美女被艹到高潮喷水动态| 97人妻精品一区二区三区麻豆| 欧美xxxx黑人xx丫x性爽| 成人一区二区视频在线观看| 91av网一区二区| 午夜精品在线福利| 久久精品亚洲精品国产色婷小说| 午夜免费观看网址| 午夜激情欧美在线| 国内精品一区二区在线观看| 久久精品影院6| 婷婷丁香在线五月| 人妻夜夜爽99麻豆av| 黄频高清免费视频| 亚洲人成网站高清观看| 人人妻,人人澡人人爽秒播| 男女视频在线观看网站免费| 美女被艹到高潮喷水动态| 中文字幕人成人乱码亚洲影| 久久久色成人| 成人高潮视频无遮挡免费网站| 88av欧美| 国产精品亚洲av一区麻豆| 精品电影一区二区在线| 色精品久久人妻99蜜桃| 精品久久久久久久人妻蜜臀av| 日韩成人在线观看一区二区三区| 国产精华一区二区三区| 成人永久免费在线观看视频| 欧美黑人欧美精品刺激| 女人被狂操c到高潮| 日本黄色视频三级网站网址| 国产黄色小视频在线观看| 淫妇啪啪啪对白视频| 18禁黄网站禁片免费观看直播| 精品一区二区三区视频在线 | 夜夜夜夜夜久久久久| 一区二区三区激情视频| 狠狠狠狠99中文字幕| 亚洲成av人片在线播放无| 久久精品影院6| 观看免费一级毛片| 婷婷六月久久综合丁香| 久久久久国产一级毛片高清牌| 婷婷精品国产亚洲av在线| 精品一区二区三区视频在线 | 国产视频一区二区在线看| 69av精品久久久久久| 无限看片的www在线观看| a级毛片a级免费在线| 成人一区二区视频在线观看| 黄片小视频在线播放| 日本在线视频免费播放| 97碰自拍视频| 免费人成视频x8x8入口观看| 国产精品一区二区免费欧美| 久久久久亚洲av毛片大全| cao死你这个sao货| 中文字幕久久专区| 亚洲国产精品sss在线观看| 丝袜人妻中文字幕| 麻豆一二三区av精品| 日本 av在线| 国产精品亚洲av一区麻豆| 久久久久久人人人人人| 成人无遮挡网站| 欧美乱色亚洲激情| 九色成人免费人妻av| 丰满人妻熟妇乱又伦精品不卡| 2021天堂中文幕一二区在线观| 久久午夜亚洲精品久久| 精品福利观看| 听说在线观看完整版免费高清| 91老司机精品| 国内揄拍国产精品人妻在线| 很黄的视频免费| 亚洲国产欧美网| 男女床上黄色一级片免费看| 欧美另类亚洲清纯唯美| 亚洲精品在线观看二区| 好男人在线观看高清免费视频| 国产乱人伦免费视频| 国产精品女同一区二区软件 | 一区二区三区高清视频在线| 99国产精品一区二区蜜桃av| 日韩欧美在线乱码| 十八禁网站免费在线| 久久午夜综合久久蜜桃| av国产免费在线观看| 很黄的视频免费| 成人av一区二区三区在线看| 麻豆av在线久日| 婷婷亚洲欧美| 免费观看人在逋| 9191精品国产免费久久| 19禁男女啪啪无遮挡网站| 国产又黄又爽又无遮挡在线| 热99在线观看视频| 欧美日韩亚洲国产一区二区在线观看| 亚洲乱码一区二区免费版| 亚洲国产色片| 国产一级毛片七仙女欲春2| 国产精品久久视频播放| 久久久久国产精品人妻aⅴ院| 日本免费a在线| 国产熟女xx| 90打野战视频偷拍视频| 看免费av毛片| 亚洲av免费在线观看| 成人18禁在线播放| 国产主播在线观看一区二区| 久久久久国产一级毛片高清牌| 国产三级在线视频| 国产麻豆成人av免费视频| 日韩欧美 国产精品| 国产淫片久久久久久久久 | 久久人人精品亚洲av| 国产麻豆成人av免费视频| 一区二区三区高清视频在线| 久久久色成人| 国产午夜福利久久久久久| 18禁裸乳无遮挡免费网站照片| 精品福利观看| 夜夜爽天天搞| 国产精品一区二区三区四区免费观看 | 一夜夜www| 免费在线观看视频国产中文字幕亚洲| 国产精品女同一区二区软件 | 久久人妻av系列| 亚洲激情在线av| 国产综合懂色| 欧美黑人欧美精品刺激| 久久久久久久久免费视频了| 亚洲av美国av| 国产又黄又爽又无遮挡在线| 久久天堂一区二区三区四区| 黄色丝袜av网址大全| 久久精品国产亚洲av香蕉五月| 久久久久精品国产欧美久久久| 国产蜜桃级精品一区二区三区| 精品久久蜜臀av无| av国产免费在线观看| 亚洲欧美精品综合久久99| 一级a爱片免费观看的视频| 免费看日本二区| 2021天堂中文幕一二区在线观| 两人在一起打扑克的视频| 欧美在线黄色|