• <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 ?

    Reducing systemic absorption and macrophages clearance of genistein by lipid-coated nanocrystals for pulmonary delivery

    2023-03-14 06:52:16YunHeChngLiuRunHnYingminLingJuithChoiWoMkYinghoZhuHifengLiYingZheng
    Chinese Chemical Letters 2023年1期

    Yun He,Chng Liu,Run Hn,Yingmin Ling,Juith Choi Wo Mk,Yingho Zhu,Hifeng Li,Ying Zheng,*

    a State Key Laboratory of Quality Research in Chinese Medicine,Institute of Chinese Medical Sciences,University of Macau,Macau,China

    b Department of Pharmacy,Xuzhou Medical University,Xuzhou 221004,China

    c LKS Faculty of Medicine,Department of Medicine,The University of Hong Kong,Hong Kong,China

    d Institute of Applied Physics and Materials Engineering,University of Macau,Macau,China

    Keywords:Pulmonary delivery Lipid-coated nanocrystals Cell uptake Transport Systemic absorption Macrophage clearances

    ABSTRACT Pulmonary delivery is an effective drug delivery strategy for the treatment of local respiratory diseases.However,the rapid systemic absorption through the lung due to the thin barrier and persistent lung clearances influence the drug retention in the lung.In this study,we designed a lipid-coated genistein nanocrystals (Lipo-NCs) formulation to achieve enhanced efficiency of local pulmonary delivery.The Lipo-NCs were fabricated by modifying genistein nanocrystals (NCs) with phospholipid membrane through thin film hydration following the homogenization method.The prepared Lipo-NCs exhibited a decreased drug release rate compared with the naked NCs.Our results demonstrated that intracellular uptake and transcellular transport of NCs by the Calu-3 epithelial layer were reduced after lipid coating.Furthermore,the macrophages clearance was also impeded by this Lipo-NCs formulation. In vivo lung retention and distribution revealed that more genistein was retained in the lung after intratracheal administration of Lipo-NCs.The pharmacokinetic study displayed that the AUC(0-t) values of Lipo-NCs were 1.59-fold lesser than those of the NCs group,indicating a reduced systemic absorption.In conclusion,this research indicated that Lipo-NCs could be a suitable formulation for reducing systemic absorption and macrophages clearance,and thus enhancing drug concentration in lung by pulmonary delivery.

    In recent years,lung diseases including pneumonia,asthma,chronic obstructive pulmonary disease (COPD) and cystic fibrosis have become a big threat to human health.According to the statistics,over 300 million adults and children suffered from asthma worldwide [1],and COPD was evaluated to be the third leading cause of death in 2030 [2].Pulmonary delivery of therapeutic drugs for these lung diseases is a promising strategy since it allows targeted drug to be delivered to the lung with a relatively high local concentration and low systemic exposure compared with the oral or parenteral routes [3–5].

    Currently,the commercial inhalable drug products for the treatment of lung diseases are mainly fast-release formulations.Despite great success in commercial products,these formulations have faced the dilemma of reaching the peak concentration of the drug immediately after administration and the subsequent rapid decline in drug concentrations [5],leading to undesirable systemic side effects and increased administration frequency.This phenomenon was mainly attributed to the systemic absorption of drugs.Because the lung has a large alveolar surface area (about 100 m2),and the lung epithelial barrier is quite thin,the free drug molecules released from deposited particles are easily transported across the lung epithelium and to be absorbed into the bloodstream [6].While this property facilitates systemic delivery,it is not beneficial for local delivery of drugs that need to be retained in the lung.Besides,the lung clearances,including mucociliary clearance and macrophages clearance,could also reduce the available drugs [2,7].Many microparticles designed with suitable particle size for deposition were easily cleared by macrophage phagocytosis [7].These clearance and systemic absorption processes ultimately result in decreased retention of drugs in the lung.In this context,drugloaded particles with sustained-release property and prolonged retention ability are desirable for the treatment of local lung diseases by pulmonary delivery.

    Fig.1.(A) Size distribution of GES NCs and Lipo-NCs; (B) TEM images of NCs and Lipo-NCs; (C) representative CLSM images of Lipo-NCs; the NCs was stained with coumarin 6 (green),the lipid membrane was stained with DiD (red),and the fluorescent points showed good colocalization in the merged Lipo-NCs group (the scale bar is 20 μm,the scale bar of the enlarged image on the right corner is 0.5 μm); (D)Pearson’s colocalization value of the physical mixture of NCs and lipid membrane and Lipo-NCs calculated by ImageJ software (n=6,**P<0.01); (E) in vitro drug release profile of GES formulations (n=3).

    In recent years,the utilization of nano-drug systems for pulmonary delivery has attracted increasing attention because their small size could help avoid macrophages phagocytosis [8,9].Nanocrystals (NCs),the submicron drug particle dispersions system [10],are considered as a promising drug platform for pulmonary delivery due to their high drug loading capacity and low excipient addition property [11,12].For pulmonary delivery,NCs could improve the homogeneity of hydrophobic drugs in nebulized aerosols and exhibited well aerodynamic performance for deposition [13–15].However,the NCs dissolved very rapidly,which resulted in a fast systemic absorption in the lung [16].Amikacin liposome inhalation (ALIS; Arikayce?) with well biocompatibility and biosafety has been approved for use as part of a combination antibacterial drug regimen in the USA.More importantly,the phospholipid membrane enfolded vesicle displayed a declined drug release rate [17].Inspired by these properties,we considered combining the two nano-drug systems to obtain a novel lipid-coated nanocrystal drug delivery system with sustained-release property and high drug loading capacity.Furthermore,we would like to investigate how the release property influences the lung epithelial uptake and transport,and find out whether this formulation reduces lung clearance.

    In this study,we fabricated a lipid-coated nanocrystal formulation to reduce lung clearance and prolong local retention by pulmonary delivery.The hydrophobic drug genistein (GES),which was an isoflavonoid compound derived from soy,was used as the model drug for nanocrystal preparation [18,19].It exhibited anti-inflammatory and antioxidant activities for the treatment of pulmonary diseases such as acute lung injury [20] and COPD [21].The lipid-coated NCs were prepared by coating the phospholipids on the surface of genistein NCs cores by the thin film hydration following the homogenization method.The systemic absorption and clearance of this nanocarrier were then assessed by thein vitroCalu-3 cell uptake,transport and macrophages phagocytosis studies.After that,the drug retention and biodistributionin vivowere also studied.The systemic absorption of GES was assessed by measuring the drug concentration in plasma after intratracheal administration.

    The GES NCs were prepared by ball wet milling method.The parameters of the mill machine including rotation speed and milling time were adjusted to obtain GES NCs.After the preparation of NCs,the lipid membrane was formed and coated on the surface of NCs.In this lipid membrane,lecithin and cholesterol were used as the main lipid materials.TPGS was added to improve the stability of lipid coated NCs.Stearamine were incorporated to obtain a positive charged lipid membrane,so that the lipid membrane could successfully coat on the surface of NCs which possessed a negative charge.Size distributions of GES NCs and Lipo-NCs were shown in Fig.1A.The prepared GES NCs was at the size of 289.7±21.3 nm with the zeta potential of -30.4±8.1 mV.After coating with the lipid membrane,Lipo-NCs were 206.2±15.3 nm with the zeta potential of 21.1±4.1 mV.The size,polydispersity index (PDI) and zeta potential values were listed in Table S1 (Supporting information).The size reduction of Lipo-NCs was probably due to the high-pressure homogenization process.The stability of NCs and Lipo-NCs in cell culture mediums were shown in Fig.S1(Supporting information).

    Transmission electron microscope (TEM) images revealed that NC had a rod-like shape.Whereas,the Lipo-NC had a more spherical shape with a core-shell structure (Fig.1B).As shown in the Lipo-NCs group,the black core possibly referred to the NC,the grey shell referred to the phospholipids on the surface of NC.The core-shell structure of Lipo-NCs was further validated by the double fluorescence labelling method by using confocal laser scanning microscopy (CLSM,Leica TCS SP8,German).According to Fig.1C,the green and red fluorescence showed well colocalization in the Lipo-NCs group.Pearson’sR-value of the Lipo-NCs group was 0.62,which was significantly higher than that of the physical mixture group 0.15 (P<0.01) (Fig.1D).This indicated that the phospholipids were successfully coated on the NCs.

    Thein vitrorelease study of GES NCs,Lipo-NCs and coarse crystals was carried out in a dialysis bag in PBS 7.4 medium,and the release profile was shown in Fig.1E.According to the result,the GES coarse crystals displayed the slowest drug release rate.There was only about 37% of drugs released within 6 h.For the NCs group,the drug release rate was significantly increased with nearly 100% GES dissolving within 6 h,compared with the group of the coarse crystal.This enhancement was attributed to the reduced particle size of crystals and increased total surface area [11].After lipid coating on NCs in the Lipo-NCs group,the drug release rate decreased.About 78% of the drug was released from Lipo-NCs after 6 h.This indicated that coating phospholipid membrane on the surface of NCs could impede the release of drug.

    Since systemic absorption of the drug from the lung to blood circulation is a major lung clearance pathway that influences local drug concentration,reducing systemic absorption is a useful strategy to increase drug retention in the lung.In this study,the systemic absorption process was elucidated by lung epithelial cells uptake and transport studies,and Calu-3 cells were used as the cell model because the characteristics of these cells were similar with bronchiolar epithelium [22].The viability of cells treated with GES formulations with different concentrations was assessed by MTT method (Fig.2A).The NCs group showed no obvious cytotoxicity.However,for the Lipo-NCs group,the cell viability decreased at the concentration of 200 μg/mL.It has been reported that the integrity and viability of Calu-3 cells decreased when particles with positive charge were used in the cell layers without mucus protection.However,when there was a mucus blanket lying on the Calu-3 cells,the integrity and viability remained [23].Therefore,the decreased cell viability in this study was possible due to the positive charge of Lipo-NCs.In contrast,under thein vivocondition,the integrity and viability of lung epithelial cells would not be influenced due to the presence of the mucus on the airways of the lung.In further cell studies,a concentration of GES at 50 μg/mL was chosen to ensure the cell viability.

    Fig.2.(A) Cell viability of Calu-3 upon exposure to GES formulations at different drug concentrations; (B) CLSM images of Calu-3 cells after incubation with GES NCs and Lipo-NCs at different time points (the blue fluorescence represented nucleus; the green fluorescence was C6 labelled GES formulations; the scale bar is 50 μm); (C) mean fluorescence intensity of cells determined by flow cytometry after incubation (n=3).

    The cell uptake studies were conducted by adding C6 labelled GES formulations in Calu-3 cells and incubating them for different time lengths (the concentration of C6 in GES formulations was about 2.5 μg/mL).The images acquired by CLSM were shown in Fig.2B.Both GES formulations exhibited a time-dependant uptake manner that the fluorescence increased with incubation time.The Calu-3 cells treated with the NCs group showed a brighter green fluorescence at every time point when compared with the Lipo-NCs.This indicated that coating phospholipid on NCs could decrease the cellular uptake amount.Quantitative analysis of the intracellular fluorescence intensity by flow cytometry also showed that the uptake of Lipo-NCs by Calu-3 was lower than NCs at all tested time points which was consistent with the CLSM result(Fig.2C).Previously,researchers found that nanoparticles with positive surface charge were more easily internalized by Calu-3 cells compared with neutral and negative charged nanoparticles[23].However,in this study,the cell uptake of positive charged Lipo-NCs was less than the negative charged NCs.To further explore the underlying mechanism,we calculated the ratio of drug release rate in NCs and Lipo-NCs at 40 min (0.5 h points were lacked in release profile),1 h and 2 h points.The result showed that the release rate of NCs were 1.23-fold,1.41-fold and 1.33-fold greater than Lipo-NCs,respectively.The intracellular fluorescence intensity determined at 0.5 h,1 h,and 2 h showed that the uptake of NCs were 1.51-fold,1.44-fold and 1.30-fold higher than Lipo-NCs,respectively.This result showed that the drug release rate and cellular uptake had a good correlation.The decreased cellular uptake of Lipo-NCs group was possibly due to the decreased drug release rate.

    The penetration of GES formulations across lung epithelium was then investigated by using the Calu-3 cell layer by the air-liquid interface culturing (ALC) method.This culturing method formed a pseudostratified Calu-3 columnar epithelium which was similar to thein vivobronchiolar epithelium because tight junctions were formed,and mucus was secreted on the cell surface [24,25].The schema of NC formulations transport across the Calu-3 cell layer was depicted in Fig.3A.After 2 h exposure,results showed that a lower amount of GES transported across the Calu-3 layer at every time point in the Lipo-NCs group compared with that of the NCs group (Fig.3B).And the calculatedPappvalue of Lipo-NCs ((2.32±0.31)×10-6cm/s) was also significantly smaller than that of NCs ((3.46±0.48)×10-6cm/s) (P<0.05) (Fig.3C).The GES coarse crystals group had the lowest drug transport amount and thePappvalue ((1.67±0.16)×10-6cm/s) because of its slow dissolution rate.Then,the fluorescence of the Calu-3 layer after transport of GES formulations was observed by 3D andz-stack confocal models (Figs.3D and E).It is worth mentioning that the Calu-3 cells cultured by the ALC method formed a pseudostratified layer(Fig.3E).The cells incubated with Lipo-NCs had a weaker fluorescence than naked NCs.Images taken by using thez-stack scanning model (Fig.3E) also exhibited that the green fluorescence of Lipo-NCs in the basolateral side of the Calu-3 cell layer was weaker than that of the NCs group.These results indicated that after lipid coating,the transport of GES had been impeded,fewer drugs were transported across the lung epithelium.According to the result of Calu-3 uptake study (Fig.2),the uptake amount of Lipo-NCs was lower than NCs because of the slower drug release rate.The impeded transport of Lipo-NCs could attribute to less drug accumulation within the cells.Another reason that was responsible for the transport was the mucus secreted on the surface of Calu-3 layer.It was reported that the respiratory mucus had a mesh-like structure with negative charge.The positive-charged Lipo-NCs could interact with the mucus layer resulting in reduced contact with cells and decreased transport.This feature of Lipo-NCs could help increase the local retention of the drug in the lung.

    Macrophage phagocytosis is another important lung clearance pathway [26].To evaluate whether lipid coating could help avoid macrophages clearance,the phagocytosis of GES formulations by RAW 264.7 cells was conducted.The CLSM images at 0.5 h and 2 h were shown in Fig.4.The fluorescence intensity of RAW 264.7 cells in both NCs and Lipo-NCs groups increased with the incubation time.Whereas the macrophage phagocytosis of Lipo-NCs decreased when compared with naked NCs.According to the particle size of NCs and Lipo-NCs in DMEM (Fig.S1),the particle size kept stable within 2 h in both groups.But the size of Lipo-NCs was smaller than NCs.It was reported that the macrophages preferred to phagocytize particles with larger size [3].Therefore,the reduced phagocytosis effect of Lipo-NCs was possibly due to the smaller particle size in the culture medium.We further investigated the integrity of Lipo-NCs after phagocytosis by RAW 264.7 cells.The C6 labelled NCs and DiD labelled lipid showed well colocalization which demonstrated that the particles were phagocytized integrally.This study indicated that the Lipo-NCs formulation could decrease macrophages phagocytosis clearance in the lung and further contribute to prolong drug retention.

    Fig.3.(A) A schema of NCs transport across Calu-3 cell layer; (B) transport amount of GES across Calu-3 cell layer at different time points; (C) Papp values of GES formulations after transport across Calu-3 cell layer (n=3,*P<0.05,**P<0.01); (D) 3D images of Calu-3 cell layer after transport of GES formulations; (E) z-stack confocal images of the Calu-3 cell layer with the polyester membrane after transport of GES formulations for 2 h (the scale bar in xy plane was 50 μm).

    Fig.4.(A) CLSM images of RAW 264.7 cells after phagocytosis of NCs and Lipo-NCs at 0.5 h and 2 h (the blue fluorescence represented nucleus; the green fluorescence was C6 labelled GES formulations; the scale bar is 20 μm); (B) integrity of Lipo-NCs after phagocytosis by RAW 264.7 cells (the green fluorescence was C6 labelled GES NCs,and the red fluorescence was DiD labelled lipid membrane).

    The retention of GES in the lung after intratracheal administration of C6 labelled GES formulations were observed byin vivoImaging System (IVIS,Lumina XR III) (Fig.5A).In this study,all relevant animal experiments were approved by the Animal Ethics Committee of the University of Macau.The result showed that the fluorescence in the lungs was brighter after 0.5 h administration than at 2 h.The decreased fluorescence was attributed to the lung clearance and systemic absorption.By comparing the fluorescence between the two GES formulations,the fluorescence of the Lipo-NCs group was brighter than the NCs group.It is important that these results were consistent with ourin vitrorelease and transport studies,indicating that Lipo-NCs would obtain a slower drug release rate,a decreased Calu-3 cell layer transport amount and a higher retention in the lung after lipid coating.All above results suggested that the less retention of NCs mainly attributed to their fast systemic absorption.Then,the distribution of GES formulations within the lung was observed by tissue sections using CLSM visually.The fluorescence intensity shared a similar trend with the IVIS images.In the airway of the lung,a lot of Lipo-NCs particle fluorescence could be found in the lumen after 0.5 h administration (Fig.5B),while only a few particles could be observed in NCs.In the parenchyma region,many bright spots could also be found in Lipo-NCs and NCs groups.After 2 h administration,the fluorescence of both GES groups became weaker.Some particles of Lipo-NCs still remained in the interval space of cilium and parenchyma of the lung.But the bright spots could hardly be observed in the NCs group at 2 h.

    The GES retention in the lung after intratracheal administration was then quantitatively detected by LC-MS/MS.According to the results in Fig.6A,the GES concentration in lung tissue of the Lipo-NCs group was higher than the NCs group without lipid coating at 0.5 h point.This result coincided with the tendency of fluorescence observation in Fig.6 that the lipid coating on NCs could increase the retention of GES in the lung.After 2 h administration,the concentration of GES in the lung was further decreased in both groups.The decline of Lipo-NCs group was even faster than NCs from 0.5 h to 2 h.The reason was possibly that NCs dissolved quickly after deposition,the dissolved drug was then rapidly absorbed from the lung epithelium into the blood circulation leading to a fast decline of drug within 0.5 h.For the Lipo-NCs group,because of the slower drug release,the decline was gentler within 0.5 h.However,the unreleased Lipo-NCs particles in lung would undergo the lung clearance processes including mucociliary movement clearance,macrophages clearance and translocation from the lung to lymphatic system.These clearances preferred to clear particles rather than dissolved drug,therefore resulting in a faster decline of Lipo-NCs from 0.5 h to 2 h.

    Fig.5.(A) Ex vivo imaging of the whole lungs after intratracheal administration of GES formulations at the different time points (0.5 and 2 h); (B) histological examination of GES formulations distribution in the airway and parenchyma regions of the lung after intratracheal 0.5 h (upper panel) and 2 h (lower panel) administration (the scale bar is 50 μm).

    Fig.6.(A) Lung concentration of GES after intratracheal administration of NC formulations (n=3); (B) plasma concentration of GES after intratracheal administration of NC formulations at a dose of 5 mg/kg (n=4).

    Table 1 Pharmacokinetic parameters of GES after intratracheal administration of formulations to rats (n=4).

    The pharmacokinetic profiles of GES were indicative of the combined effect of several influence factors including dissolution,clearance and absorption in the lung.The plasma concentration of GES over time and pharmacokinetic parameters were shown in Fig.6B and Table 1,respectively.From the profiles,both NCs and Lipo-NCs groups reached the peak concentration at 5 min.Then the plasma concentration declined with time.The rapid pulmonary absorption could be attributed to the large surface area of lung alveoli and the thin anatomical barriers that limit the access to the blood [27].It was reported that the pulmonary absorption of lipophilic small molecules (logP >0) was in approximately 1 min [28].The GES peak concentration of the NCs group was 1.70±0.41 μg/mL which was 1.59-fold higher than that of the Lipo-NCs group.The calculated AUC(0-t)value of the NCs group was 1.48-fold greater than Lipo-NCs.This result indicated that the GES in the NCs group was more easily absorbed into the blood circulation.However,the statistical analysis of AUC(0-t)between NCs and Lipo-NCs groups was not significant (P >0.05) possibly because of the large difference between individual animals.Since the absorption of lipophilic GES free drugs happened within a minute,the rate-limiting step of absorption should be the drug release process.Therefore,the main reason leading to less absorption of GES from lung to blood in Lipo-NCs was attributed to the slower drug release rate.On the other hand,less systemic absorption facilitates more accumulation of drugs in the lung.

    In this study,lipid coated GES NCs were fabricated to enhance the drug accumulation and retention in the lung after pulmonary delivery.These Lipo-NCs showed a slower drug release ratein vitrowhen compared with the naked NCs.The Calu-3 cell uptake and transport studies demonstrated that Lipo-NCs formulation could decrease the transport of GES from apical side to basolateral side of the lung epithelium.And this lipid-coated formulation could also reduce macrophage clearance.Thein vivoretention and distribution studies verified that Lipo-NCs enhanced the drug concentration and residence in the lung by reducing the systemic absorption and macrophages phagocytosis.

    Declaration of competing interest

    The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

    Acknowledgments

    This work was supported by the Multi-Year Research Grants from the University of Macau (No.MYRG2019–00032-ICMS),Natural Science Foundation of Jiangsu Province (No.BK20210912),the startup grant of Xuzhou Medical University (D2021004),and Macau Science and Technology Development Fund (No.0017/2019/AKP).

    Supplementary materials

    Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.cclet.2022.04.082.

    18禁动态无遮挡网站| 亚洲精品日本国产第一区| 亚洲精品乱久久久久久| 欧美日韩成人在线一区二区| 免费大片黄手机在线观看| 国产一区二区三区综合在线观看 | 成人亚洲欧美一区二区av| 亚洲在久久综合| 国产又色又爽无遮挡免| 美女中出高潮动态图| 免费少妇av软件| 国产深夜福利视频在线观看| 久久99热6这里只有精品| 日韩 亚洲 欧美在线| 久久精品国产综合久久久 | 又黄又爽又刺激的免费视频.| 丰满饥渴人妻一区二区三| 久久久久精品性色| 春色校园在线视频观看| 午夜福利影视在线免费观看| 丝袜美足系列| 视频中文字幕在线观看| 久久影院123| 国产亚洲欧美精品永久| 99久久中文字幕三级久久日本| 国产极品粉嫩免费观看在线| 观看av在线不卡| 国产精品成人在线| 亚洲美女黄色视频免费看| 满18在线观看网站| 亚洲成色77777| 免费看光身美女| 人人妻人人爽人人添夜夜欢视频| 久久精品aⅴ一区二区三区四区 | 在线观看国产h片| 日韩制服骚丝袜av| 免费观看a级毛片全部| av在线观看视频网站免费| 欧美精品人与动牲交sv欧美| 狠狠婷婷综合久久久久久88av| 99国产精品免费福利视频| 欧美+日韩+精品| 久久久欧美国产精品| 国产色婷婷99| 成人无遮挡网站| 人人澡人人妻人| 亚洲成国产人片在线观看| 搡女人真爽免费视频火全软件| av播播在线观看一区| 2022亚洲国产成人精品| 永久免费av网站大全| 性高湖久久久久久久久免费观看| 久久亚洲国产成人精品v| 亚洲国产精品999| 久久狼人影院| 赤兔流量卡办理| 18+在线观看网站| 国产色婷婷99| 精品亚洲成a人片在线观看| 中文字幕制服av| 亚洲成人一二三区av| 欧美xxxx性猛交bbbb| 亚洲欧美中文字幕日韩二区| 热99国产精品久久久久久7| 丝袜美足系列| 我要看黄色一级片免费的| a级毛片黄视频| 水蜜桃什么品种好| 又黄又粗又硬又大视频| av卡一久久| av在线播放精品| 亚洲,一卡二卡三卡| 一二三四中文在线观看免费高清| 男女高潮啪啪啪动态图| 一级a做视频免费观看| 男女边吃奶边做爰视频| 欧美国产精品一级二级三级| 男女国产视频网站| 看免费av毛片| 热re99久久国产66热| 午夜老司机福利剧场| 色婷婷久久久亚洲欧美| 五月天丁香电影| 99热6这里只有精品| 亚洲av综合色区一区| 丝瓜视频免费看黄片| 国产在线一区二区三区精| 男人舔女人的私密视频| 亚洲伊人色综图| 国产成人精品一,二区| 成人午夜精彩视频在线观看| 秋霞伦理黄片| 精品人妻在线不人妻| 国产av一区二区精品久久| 色5月婷婷丁香| 免费在线观看完整版高清| 国产白丝娇喘喷水9色精品| 国产av精品麻豆| 日韩av在线免费看完整版不卡| 香蕉国产在线看| 日韩精品有码人妻一区| 美女国产视频在线观看| 大码成人一级视频| 欧美少妇被猛烈插入视频| 亚洲情色 制服丝袜| 国产成人av激情在线播放| 久久国产亚洲av麻豆专区| 午夜福利视频精品| 国产在视频线精品| 亚洲欧美中文字幕日韩二区| 国产成人aa在线观看| 国产成人欧美| 狠狠精品人妻久久久久久综合| 在线亚洲精品国产二区图片欧美| 伊人久久国产一区二区| 国产免费又黄又爽又色| 午夜免费男女啪啪视频观看| 91精品三级在线观看| 欧美激情国产日韩精品一区| 亚洲成av片中文字幕在线观看 | 成人二区视频| 久久精品国产鲁丝片午夜精品| 亚洲三级黄色毛片| 亚洲,欧美,日韩| 亚洲中文av在线| 欧美日韩综合久久久久久| 免费观看av网站的网址| 国产成人精品无人区| 一级,二级,三级黄色视频| 国产精品成人在线| 国产精品秋霞免费鲁丝片| 国产精品国产三级国产av玫瑰| 高清av免费在线| 国国产精品蜜臀av免费| 日本av手机在线免费观看| 亚洲国产最新在线播放| 亚洲四区av| a级毛色黄片| 久久精品国产亚洲av天美| 国产精品久久久av美女十八| 日韩精品免费视频一区二区三区 | 久久久亚洲精品成人影院| 日产精品乱码卡一卡2卡三| 久久精品国产自在天天线| 亚洲国产欧美在线一区| 久久ye,这里只有精品| 日韩欧美精品免费久久| 侵犯人妻中文字幕一二三四区| 国产精品一二三区在线看| 精品99又大又爽又粗少妇毛片| 久久久a久久爽久久v久久| 精品一品国产午夜福利视频| 亚洲在久久综合| 久久久久久久久久人人人人人人| 欧美精品一区二区大全| 日本av免费视频播放| 日韩制服骚丝袜av| 夫妻午夜视频| 国产成人免费无遮挡视频| 亚洲国产色片| 99热这里只有是精品在线观看| 亚洲av在线观看美女高潮| 久久久精品免费免费高清| 亚洲精品美女久久久久99蜜臀 | 亚洲经典国产精华液单| 国产福利在线免费观看视频| 国产一区二区三区av在线| 视频区图区小说| 国产国拍精品亚洲av在线观看| 免费大片18禁| 国产亚洲精品久久久com| 亚洲五月色婷婷综合| 高清毛片免费看| 宅男免费午夜| 美女脱内裤让男人舔精品视频| 亚洲精品自拍成人| 又大又黄又爽视频免费| 久久久国产一区二区| 久久青草综合色| 国产在视频线精品| 国产亚洲一区二区精品| 亚洲精品国产av蜜桃| 韩国精品一区二区三区 | freevideosex欧美| 精品国产乱码久久久久久小说| 啦啦啦在线观看免费高清www| 人人妻人人添人人爽欧美一区卜| 国产亚洲精品久久久com| 亚洲精品456在线播放app| 免费少妇av软件| 高清视频免费观看一区二区| 香蕉丝袜av| 亚洲美女黄色视频免费看| 国产成人精品婷婷| 久久青草综合色| av女优亚洲男人天堂| 晚上一个人看的免费电影| 久久国产精品男人的天堂亚洲 | 少妇的逼好多水| 国产成人a∨麻豆精品| 少妇人妻精品综合一区二区| 成年av动漫网址| 国产成人午夜福利电影在线观看| 成人亚洲欧美一区二区av| 男女边摸边吃奶| 久久这里只有精品19| 国产亚洲午夜精品一区二区久久| 免费观看无遮挡的男女| 亚洲四区av| 午夜精品国产一区二区电影| 国产淫语在线视频| 九草在线视频观看| 99视频精品全部免费 在线| 1024视频免费在线观看| av免费在线看不卡| 青青草视频在线视频观看| 美国免费a级毛片| 99热6这里只有精品| 涩涩av久久男人的天堂| 伦精品一区二区三区| 边亲边吃奶的免费视频| 亚洲美女黄色视频免费看| 国产国拍精品亚洲av在线观看| 国产成人免费无遮挡视频| 51国产日韩欧美| 97在线人人人人妻| 久久鲁丝午夜福利片| 国产精品久久久久久久电影| 国产精品无大码| 高清av免费在线| 中文字幕精品免费在线观看视频 | 亚洲av中文av极速乱| 黑人高潮一二区| 黄色毛片三级朝国网站| 麻豆精品久久久久久蜜桃| 精品一区二区三卡| 成年人免费黄色播放视频| 日韩av不卡免费在线播放| 一级毛片 在线播放| 最近中文字幕高清免费大全6| 久久久久精品性色| 久久精品国产鲁丝片午夜精品| kizo精华| 美女大奶头黄色视频| 亚洲精品乱久久久久久| 国产免费一区二区三区四区乱码| 成人二区视频| a级片在线免费高清观看视频| 亚洲欧美精品自产自拍| 日本wwww免费看| 久久热在线av| 欧美日韩av久久| 色94色欧美一区二区| 成人毛片a级毛片在线播放| 十分钟在线观看高清视频www| 日日爽夜夜爽网站| 97精品久久久久久久久久精品| 国产不卡av网站在线观看| 在线天堂最新版资源| 99久久中文字幕三级久久日本| 日日爽夜夜爽网站| 黄色 视频免费看| 国产不卡av网站在线观看| 成人毛片a级毛片在线播放| 国产一区亚洲一区在线观看| 亚洲国产最新在线播放| 亚洲精品国产av蜜桃| 国产精品人妻久久久久久| 天天操日日干夜夜撸| 中国三级夫妇交换| 丝袜美足系列| 狠狠精品人妻久久久久久综合| 欧美激情国产日韩精品一区| 1024视频免费在线观看| 国产亚洲欧美精品永久| 美国免费a级毛片| 国产亚洲av片在线观看秒播厂| 国产成人精品在线电影| 日本午夜av视频| 最近中文字幕高清免费大全6| 天堂俺去俺来也www色官网| 久久婷婷青草| 久久99一区二区三区| 亚洲综合色网址| 五月天丁香电影| 国产精品久久久av美女十八| 精品国产露脸久久av麻豆| 精品一区二区三区四区五区乱码 | 精品国产一区二区三区四区第35| 又黄又粗又硬又大视频| 国产亚洲欧美精品永久| 成人手机av| 母亲3免费完整高清在线观看 | 成人综合一区亚洲| 高清视频免费观看一区二区| 制服丝袜香蕉在线| av片东京热男人的天堂| 国产1区2区3区精品| 婷婷色av中文字幕| 久久久精品免费免费高清| 人成视频在线观看免费观看| 日本猛色少妇xxxxx猛交久久| 美女国产高潮福利片在线看| av在线老鸭窝| www.av在线官网国产| 欧美精品一区二区免费开放| 激情五月婷婷亚洲| 精品一区二区三区视频在线| 精品亚洲成国产av| 免费高清在线观看日韩| 国产熟女午夜一区二区三区| 欧美性感艳星| 男人爽女人下面视频在线观看| 亚洲国产毛片av蜜桃av| 老司机影院毛片| 精品一区二区三区四区五区乱码 | 一级,二级,三级黄色视频| 自拍欧美九色日韩亚洲蝌蚪91| 欧美日韩视频高清一区二区三区二| 欧美日韩av久久| 国产一区有黄有色的免费视频| 久久国产精品大桥未久av| 亚洲美女视频黄频| 国产精品久久久久久av不卡| 建设人人有责人人尽责人人享有的| 久久精品熟女亚洲av麻豆精品| 熟女电影av网| 亚洲av男天堂| 热99国产精品久久久久久7| 乱码一卡2卡4卡精品| 久久女婷五月综合色啪小说| 69精品国产乱码久久久| 青青草视频在线视频观看| 在现免费观看毛片| 国产成人免费无遮挡视频| 日韩精品免费视频一区二区三区 | 久久久久精品久久久久真实原创| 午夜福利视频精品| 亚洲精品一区蜜桃| 精品熟女少妇av免费看| 精品午夜福利在线看| 国产亚洲精品久久久com| 成年动漫av网址| 亚洲av欧美aⅴ国产| 国产精品人妻久久久影院| 国产精品嫩草影院av在线观看| 男女无遮挡免费网站观看| 国产免费视频播放在线视频| 久久人人97超碰香蕉20202| 亚洲经典国产精华液单| 日本vs欧美在线观看视频| 日韩 亚洲 欧美在线| 18+在线观看网站| 大话2 男鬼变身卡| 免费av中文字幕在线| 少妇人妻精品综合一区二区| 国产毛片在线视频| 亚洲天堂av无毛| 欧美日韩国产mv在线观看视频| 极品少妇高潮喷水抽搐| 在线看a的网站| 男女高潮啪啪啪动态图| 韩国高清视频一区二区三区| 亚洲久久久国产精品| 波多野结衣一区麻豆| 男男h啪啪无遮挡| 国产高清三级在线| 成人毛片a级毛片在线播放| 9色porny在线观看| 久久久久久久国产电影| 成人亚洲精品一区在线观看| 国产一区亚洲一区在线观看| 韩国av在线不卡| 亚洲久久久国产精品| 国产麻豆69| 国产精品久久久久久精品电影小说| 少妇的逼水好多| 午夜av观看不卡| 在线观看免费高清a一片| 国产高清三级在线| 亚洲精品视频女| av在线播放精品| 在线观看人妻少妇| 亚洲激情五月婷婷啪啪| 国精品久久久久久国模美| 午夜影院在线不卡| 伊人亚洲综合成人网| 国产成人免费无遮挡视频| 在线观看人妻少妇| 日韩一本色道免费dvd| 久久97久久精品| 国产日韩欧美亚洲二区| 国产精品 国内视频| av天堂久久9| 国产精品久久久久久精品古装| 老司机影院毛片| 国产激情久久老熟女| 婷婷成人精品国产| av.在线天堂| 青春草亚洲视频在线观看| 日韩制服丝袜自拍偷拍| 大话2 男鬼变身卡| 久久综合国产亚洲精品| 国产在线一区二区三区精| 纵有疾风起免费观看全集完整版| 久久影院123| 999精品在线视频| 美女脱内裤让男人舔精品视频| 国产男女内射视频| 久久人人爽人人片av| 国产毛片在线视频| 日韩不卡一区二区三区视频在线| 日韩欧美精品免费久久| 成人午夜精彩视频在线观看| 日本与韩国留学比较| 热re99久久国产66热| 日本欧美视频一区| 2022亚洲国产成人精品| 观看美女的网站| 亚洲图色成人| 男女边摸边吃奶| 国产1区2区3区精品| 国产又爽黄色视频| 亚洲熟女精品中文字幕| 国产精品久久久久久av不卡| 青春草视频在线免费观看| 91午夜精品亚洲一区二区三区| 国产综合精华液| 伊人久久国产一区二区| 肉色欧美久久久久久久蜜桃| 日本猛色少妇xxxxx猛交久久| 晚上一个人看的免费电影| 狠狠婷婷综合久久久久久88av| 丰满乱子伦码专区| 纯流量卡能插随身wifi吗| 亚洲欧美一区二区三区黑人 | 亚洲内射少妇av| 黄色 视频免费看| 久久99一区二区三区| 亚洲色图综合在线观看| 男女无遮挡免费网站观看| 亚洲天堂av无毛| 老司机亚洲免费影院| 久久国产精品大桥未久av| 久久午夜综合久久蜜桃| 亚洲少妇的诱惑av| 国产极品天堂在线| 欧美 亚洲 国产 日韩一| 91精品三级在线观看| 免费不卡的大黄色大毛片视频在线观看| 丰满饥渴人妻一区二区三| 人人妻人人澡人人爽人人夜夜| 一级a做视频免费观看| 乱人伦中国视频| 一区二区三区精品91| 色婷婷久久久亚洲欧美| 日本黄色日本黄色录像| 啦啦啦在线观看免费高清www| 九九爱精品视频在线观看| 免费高清在线观看视频在线观看| 一本久久精品| 色5月婷婷丁香| 国产成人精品婷婷| 天天躁夜夜躁狠狠躁躁| 欧美精品人与动牲交sv欧美| 美女内射精品一级片tv| 久久久久久久久久成人| 久久97久久精品| 欧美日韩亚洲高清精品| 天天躁夜夜躁狠狠久久av| 王馨瑶露胸无遮挡在线观看| 免费看不卡的av| 亚洲一级一片aⅴ在线观看| 999精品在线视频| 午夜福利网站1000一区二区三区| 在线观看免费高清a一片| 午夜免费男女啪啪视频观看| 菩萨蛮人人尽说江南好唐韦庄| 欧美成人午夜精品| 男女免费视频国产| 卡戴珊不雅视频在线播放| 成人午夜精彩视频在线观看| 中国三级夫妇交换| 中文字幕制服av| 免费人妻精品一区二区三区视频| 日本欧美国产在线视频| 大香蕉久久成人网| 日本欧美国产在线视频| 欧美最新免费一区二区三区| 久久ye,这里只有精品| 国产精品久久久av美女十八| 国产一区二区在线观看日韩| 亚洲成人一二三区av| 国产免费福利视频在线观看| 卡戴珊不雅视频在线播放| kizo精华| 一边亲一边摸免费视频| 男女午夜视频在线观看 | 国产黄色视频一区二区在线观看| 免费高清在线观看日韩| 美女xxoo啪啪120秒动态图| 国产一区有黄有色的免费视频| 久久久久精品久久久久真实原创| 三级国产精品片| 日本-黄色视频高清免费观看| 一二三四中文在线观看免费高清| 尾随美女入室| 黑人猛操日本美女一级片| 又黄又爽又刺激的免费视频.| 街头女战士在线观看网站| 丝袜脚勾引网站| 日本-黄色视频高清免费观看| 亚洲高清免费不卡视频| 免费看光身美女| 精品第一国产精品| 国产av一区二区精品久久| 国产精品人妻久久久影院| 高清在线视频一区二区三区| 美女主播在线视频| 侵犯人妻中文字幕一二三四区| 伊人亚洲综合成人网| 亚洲综合精品二区| 亚洲成色77777| 精品少妇久久久久久888优播| 日韩成人伦理影院| 日本猛色少妇xxxxx猛交久久| 久久狼人影院| 国产一区亚洲一区在线观看| 91成人精品电影| 菩萨蛮人人尽说江南好唐韦庄| 久久久久久久国产电影| 欧美人与性动交α欧美精品济南到 | 久久精品aⅴ一区二区三区四区 | 免费女性裸体啪啪无遮挡网站| 国产免费视频播放在线视频| 看免费成人av毛片| 人人妻人人添人人爽欧美一区卜| 午夜福利,免费看| 日本与韩国留学比较| 中文字幕精品免费在线观看视频 | 欧美日韩视频高清一区二区三区二| 国产精品国产av在线观看| 久久久国产一区二区| 亚洲欧美色中文字幕在线| 免费不卡的大黄色大毛片视频在线观看| 精品亚洲乱码少妇综合久久| 如日韩欧美国产精品一区二区三区| 99热全是精品| 2022亚洲国产成人精品| 麻豆乱淫一区二区| 亚洲精品自拍成人| 一二三四在线观看免费中文在 | 午夜激情久久久久久久| 少妇精品久久久久久久| 少妇的丰满在线观看| 国产高清三级在线| 亚洲精品色激情综合| 久久精品国产鲁丝片午夜精品| 亚洲国产av影院在线观看| 在线亚洲精品国产二区图片欧美| 久久精品久久久久久噜噜老黄| 在线亚洲精品国产二区图片欧美| 欧美日韩精品成人综合77777| 999精品在线视频| 亚洲天堂av无毛| 如何舔出高潮| 免费观看a级毛片全部| 久热久热在线精品观看| 久久99蜜桃精品久久| 久久精品久久久久久久性| 两性夫妻黄色片 | 尾随美女入室| 观看美女的网站| 精品酒店卫生间| 美女福利国产在线| 久久国产精品大桥未久av| 亚洲精品国产av成人精品| 精品国产露脸久久av麻豆| 中文字幕精品免费在线观看视频 | 亚洲婷婷狠狠爱综合网| 色94色欧美一区二区| 18禁在线无遮挡免费观看视频| 亚洲av电影在线进入| 99久久精品国产国产毛片| 日本黄大片高清| a 毛片基地| 国产永久视频网站| 丰满饥渴人妻一区二区三| 成人黄色视频免费在线看| 香蕉精品网在线| 少妇人妻 视频| 乱人伦中国视频| 一本—道久久a久久精品蜜桃钙片| 丁香六月天网| 大码成人一级视频| 日韩欧美一区视频在线观看| 久久久精品94久久精品| 欧美激情 高清一区二区三区| 成人漫画全彩无遮挡| www.av在线官网国产| 精品卡一卡二卡四卡免费| 国产av码专区亚洲av| 亚洲国产精品成人久久小说| 18在线观看网站| 美女视频免费永久观看网站| 亚洲欧美成人综合另类久久久| 亚洲少妇的诱惑av| 在线观看三级黄色| 久久这里有精品视频免费| 成人漫画全彩无遮挡| 黄色一级大片看看| 咕卡用的链子| 永久免费av网站大全| 欧美成人午夜精品| 五月伊人婷婷丁香|