• <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 ?

    Engineering J-aggregates for NIR-induced meso-CF3-BODIPY nanoparticles by activated apoptosis mechanism in photothermal therapy

    2023-10-14 03:02:12ChujingYeShnZhngDongxingZhngYueShenZhnWngHunWngJunyiRenXinDongJingJinjunDuRongShngGuilingWng
    Chinese Chemical Letters 2023年9期

    Chujing Ye, Shn Zhng, Dongxing Zhng, Yue Shen, Zhn Wng, Hun Wng,Junyi Ren, Xin-Dong Jing,?, Jinjun Du, Rong Shng, Guiling Wng,?

    a Liaoning & Shenyang Key Laboratory of Functional Dye and Pigment, Shenyang University of Chemical Technology, Shenyang 110142, China

    b Department of Cell Biology, China Medical University, Shenyang 110122, China

    c State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 110624, China

    d Department of Chemistry, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 7398526, Japan

    Keywords:NIR dye J-aggregate CF3-BODIPY Photothermal therapy Cell apoptosis

    ABSTRACT Forming J-aggregates by organic monomer is a fascinating strategy to urge spectroscopic redshift with respect to that of the monomer.Herein, we designed 1,7-diphenyl-substituted meso-CF3-BDP monomer confirmed by X-ray crystallographic analysis.The low-barrier rotation of the -CF3 group in meso-CF3-BDP 1 significantly enhances the non-radiative efficiency, and the photothermal conversion efficiency (PCE) of the self-assembled nanoparticles (1-NPs: λabs=746 nm) by J-aggregates was 82%.1-NPs could effectively block cell cycle progression, inhibit cancer cell proliferation and trigger cell apoptosis under low power laser irradiation (0.2 W/cm2).This study proposes an alternate molecular design platform by J-aggregates to promote PCE through the insertion of rotating segment and trigger the cancer cells apoptosis in photothermal therapy at low power laser density.

    Cancer phototherapy refers to the utilization of photon-energy to implement the tumor ablation, mainly involving photodynamic therapy (PDT) and photothermal therapy (PTT), which had been emerged as cancer treatment approach following surgery,chemotherapy and radiotherapy.Compared with other cancer therapies, phototherapy holds great promise for precisely navigating at the lesion site for diagnostic therapy, non-tissue invasiveness, high treatment efficiency and anti-drug resistance [1-6].Strong absorption of the near-infrared (NIR) photon with high penetration of tissue, and efficient conversion to heat energy through non-radiative decay are critical factors for constructing photothermal agents(PTAs) [7-13].Compared with the molecular engineering strategy of extendingπ-πconjugated structure or inserting electrondonating/withdrawing groups, theJ-aggregate by organic monomer endowed it attractively optical properties, such as spectroscopic bathochromic shift, high photobleaching resistance, strong lightharvesting feature [14-18].J-aggregates demand slip-stacked alignment (θ< 54.7°), but currently there are few reports aboutJ-aggregates of cyanine, chlorophyll, perylenediimide, squaranine dye and borondipyrromethene (BODIPY or BDP) [19-23].Owing to the excellent spectral characters of BODIPY, such as high molar extinction coefficients, outstanding photostability and easy modification, it is urgent to conduct a thorough analysis for the crystal aggregation structure of BODIPY, and explore light-induced application, especially in the field of biomedical therapeutics [24-27].

    In contrast with PDT, PTT is not restricted by the hypoxic of the tumor microenvironment.Whereas, PTT usually undergoes the necrosis, which may impair the treatment outcomes by triggering pro-inflammatory responses and promoting tumor growth [8].By molecular design and photoexcitation condition, PTT can also be modulated to induce apoptosis rather than necrosis, which is significative since apoptosis prevented an inflammatory response.Above all, PTT is an efficient, non-invasive treatment method that overcomes hypoxia restriction and inflammation [28].The relaxed molecules in the lowest vibrational level of the excited state can undergo one or more of the three paths, that is, non-radiative transition, radiative transition (fluorescence emission) and intersystem crossing (ISC), to return to the ground state.In this regard, three pathways compete with each other, and it is pivotal to effectively inhibit the other two processes for improving non-radiative relaxation, which is conducive for PTT.In short, integrating high photothermal conversion efficiency (PCE), deep tissue penetration and excellent photostability for the ideal PTAs are vital [29-33].

    To enhance PCE, researchers constructed various structural BODIPYs, which are often involved in intramolecular charge transfer (ICT), photoinduced electron transfer (PET), rotating segments and so forth.For instance, Maet al.showed a BODIPY-based PTA,enhanced phototherapeutic performance of which is resulted from the reduction of radiation transition by ICT [34].Based on PET to quench the fluorescence, Huanget al.reported dimethylaminosubstituted aza-BODIPY with a moderate PCE (η=35%) [35].Especially, the low-barrier rotation strategy of a bulky group (such as -CF3, -tBu) is employed to directly promote non-radiative decay.In 2017, our group prepared NIR-absorbingmeso-CF3-BODIPYs by one-pot synthesis for the first time and reveal the property of non-fluorescent emission [36].In 2019, Xiet al.successfully discovered the highest PCE (η=88.3%) of thismeso-CF3-BODIPY[37].Very recently, our group successfully synthesized 1,7-di-tertbutyl-substituted aza-BODIPY for the first time [38].Although the low-barrier rotation of the distal -tBu groups in aza-BODIPY results in low quantum yield, the PCE (η=48%) is remarkably enhanced[38].Thereby, by restricting fluorescence and ISC, the enhancement of PCE could be achieved by high-efficiency non-radiative decay [39].Herein, to understand the influence of the -CF3rotation effect on non-radiation attenuation profoundly, 1,7-diphenylsubstitutedmeso-CF3-BODIPY (namelymeso-CF3-BDP) was designed (Fig.1a).The crystal structure showed obvious slip-stacked alignment (θ=24°), and the dye nanoparticles constituted by selfassembly emerged obvious bathochromic-shift (λabs=746 nm) due toJ-aggregates.In addition, the low-barrier rotation of the -CF3group can directly promote non-radiative decay.Self-assembledmeso-CF3-BDP 1 nanoparticles (namely 1-NPs) showed excellent PCE (η=82%), which is highly desirable for an effective and potential tumor PTA.Although the photothermal radiation with different photon intensity is acquainted by trigger cell death through either necrosis or apoptosis [40], PTT is usually engaged in necrosis mechanism.In contrast, PTT caused by apoptosis pathway is rarely reported [40,41].Furthermore, based on American National Standard for Safe Use of Lasers Outdoors, the maximum permissible exposure (MPE) for skin exposure is 0.2 W/cm2at the 635 nm laser.Hence, the safe PTT at low power laser density should be advocated and could be involved in the apoptosis mechanism.In this work, 1-NPs fabricated byJ-aggregates could induce the cancer cells death at low laser power density by triggering the apoptosis mechanism, which is fascinating since apoptosis discourages an inflammatory response (Fig.1b).As a result, this study proposes an alternate molecular design platform byJ-aggregates to enhance PCE through the insertion of rotating segment (-CF3) and trigger the cancer cells apoptosis in PTT under low power laser irradiation.

    Fig.2.(a) ORTEP drawing of BDPs 1-3 (CCDC: 2189483 for 1; 1547540 for 2 [36];2189484 for 3).The dihedral angles: C14-C9-C1-C8: 126.4(3)°, C31-C30-C23-C21:134.7(3)° for 1; C12-C11-C2-C1: 107.1(5)°, C29-C24-C7-C6: 125.2(6)° for 2; C29-C28-C15-C14: 137.6(3)°, C35-C34-C11-C12: 134.3(3)° for 3.(b) ESP distribution diagram of BDPs 1-3.

    Based on the synthetic method pioneered by our group [36],one-pot synthesis ofmeso-CF3-BDP 1 is achieved in 43% yields, as shown in Scheme S1 (Supporting information).In a sharp contrast,the contrastable dyemeso-H-BDP 3 (H-substitute atmeso-site)was also prepared (Scheme S1 and Figs.S1-S5 in Supporting information).Moreover, the solid state structures of BDPs 1-3 were confirmed by X-ray crystallographic analysis (Fig.2a).The sp3hybridized boron center inmeso-CF3-BDP 1 appeared as slightly distorted tetrahedron geometry with angles N1-B1-N2 of 108.15(19)°and F1-B1-F2 of 111.2(2)°, deviating from the ideal value of 109.5°In a stark comparison withmeso-H-BDP 3 (the dihedral angles of C29-C28-C15-C14: 137.6°; C35-C34-C11-C12: 134.3°), the dihedral angles of C14-C9-C1-C8 and C31-C30-C23-C21 inmeso-CF3-BDP 1 were small and measured to be 126.4° and 134.7°, respectively.Moreover, the smaller dihedral angles of C12-C11-C2-C1 and C29-C24-C7-C6 inmeso-CF3-BDP 2 (non-ring-fused configuration) were also observed to be 107.1° and 125.2°, respectively [36].Therefore, the 1,7-diphenyl torsion is mainly due to the steric hindrance from the introduction of themeso-CF3group,which meanwhile provides the enough space for the rotation of the -CF3group atmeso-site.Moreover, the electrostatic potential(ESP) maps for 1-3 in the gas phase were also investigated (Fig.2b).The negative charges (red color) were mainly concentrated on the fluorine atoms and oxygen atoms of BODIPY units, including the -CF3group.In contrast, the positive charges (blue color)were evenly distributed in the remaining positions.These results demonstrated the uneven charge distribution and the significant structural distortion of BODIPY, which is beneficial for the rotation energy-releasing of the -CF3group.

    Fig.3.(a) Normalized absorption spectra of BDPs 1 (red), 2 (green) and 3 (black) in CH2Cl2 at 298 K.(b, c) Emission changes of BDPs 1 and 3 in different concentrations of glycerol/methanol (v/v: 0:10; 1:9, 2:8, 3:7, 4:6 and 5:5) solution.(d) Energy levels of the S0 states of chemical bond for BDP 1 with the dihedral angle θ (Scheme S1).

    To gain insight into the photophysical properties ofmeso-CF3-BDPs, the absorption and emission spectra for BDPs 1-3 were measured and outlined in Fig.3a and Table S1 (Supporting information).Compared to the spectroscopic information for corresponding dyemeso-H-BDP 3 (λabs/λem=658/687 nm,φf=0.55),the introduction of the electron-withdrawing group (-CF3) leads to a remarkable bathochromic shift (74 nm) ofmeso-CF3-BDP 1(λabs=732 nm), the absorption maximum of which locates at the NIR region.However,meso-CF3-BDP 1 was astoundingly found to exhibit no fluorescence character.The lack of fluorescence signal indicates the excited state decays through non-radiative pathways and results in highly efficient PCE.In comparison withmeso-HBDP 3 (ε=140,000 L mol-1cm-1; FWHM: 36 nm),meso-CF3-BDP 1 has higher molar extinction coefficients (155,000 L mol-1cm-1)and wider full width at half maxima (FWHM: 52 nm) which is mainly caused by the drastic vibration of the -CF3fragment.Additionally, the band gaps (LUMO/HOMO) were calculated to be 2.07,2.23 and 2.30 eV for BDPs 1-3, respectively (Fig.S6 in Supporting information).All the theoretical calculation results well explained and supported the difference of absorption maxima.Furthermore, in order to reveal the obstruction of the rotating segment, the effect of viscosity on the fluorescence by using different concentrations of glycerol was further investigated (Figs.3b and c).Generally, the substituent rotation can be leastwise restricted in viscous media, and the corresponding fluorescence enhancement should be observed [42-45].Comparing to the remarkable fluorescence enhancement ofmeso-H-BDP 3, no obvious change in fluorescence intensity was observed formeso-CF3-BDP 1 in the mixture of glycerol and methanol in different proportions (Figs.3b and c).This was attributed to the “l(fā)ow-barrier” rotation of the -CF3group (Fig.S7 in Supporting information).Comparing tomeso-HBDP 3, the smaller dihedral angles inmeso-CF3-BDP 1 dodges the steric hindrance between the 1,7-diphenyl groups and themeso-CF3group to exactly provide the space for the low-barrier rotation of the -CF3group (Fig.2a).Moreover, we also calculated the rotated potential energy barrier of the -CF3group inmeso-CF3-BDP 1, as picked in Fig.3d.The energy maxima inmeso-CF3-BDP 1 are 26.3 kJ/mol, indicating the low-barrier rotation of the -CF3group in this molecule.As a result, the -CF3rotation inmeso-CF3-BDP 1 significantly increases the non-radiative efficiency.

    We further investigated singlet oxygen generation ofmeso-CF3-BDPs 1 and 2 to inspect the ISC process.By utilizing 1,3-diphenylisobenzofuran (DPBF), a singlet oxygen (1O2) indicator, the efficiency of1O2generation was evaluated by detecting the decrease of DPBF indicator absorbance at 416 nm [46,47].Based on the slope coefficient of the decay lines, the1O2yields ofmeso-CF3-BDPs 1 and 2 were so low and calculated to be 0 and 0.006 respectively (Fig.S8 in Supporting information), indicating that ISC is basically prohibited.

    Fig.4.Molecular packing diagram of (a) front view and (b) side view for meso-CF3-BDP 1.(c) Self-assembly of meso-CF3-BDP 1.(d) DLS and (e) TEM of 1-NPs in aqueous solution.(f) Photo of pure water.(g) Photo of 1-NPs in water.(h) Normalized absorption of 5 μmol/L meso-CF3-BDP 1 (blue curve) in CH2Cl2 and 20 μmol/L 1-NPs in water (red curve).

    Since we preliminarily probed the key data of fluorescence (φf=0) and1O2yield (φΔ=0) of this novel dyemeso-CF3-BDP 1, such information urges us to further explore the insight into the photothermal conversion capacity.To enhance the water solubility and biocompatibility ofmeso-CF3-BDP 1 for application in photoimaging and phototherapy in biological system,meso-CF3-BDP 1 and amphipathic polymer material 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were selfassembled into dye nanoparticles (abbreviated 1-NPs) [48-50].To confirm the molecular design concept, the molecular packing mode ofmeso-CF3-BDP 1viasingle-crystal structure analysis was firstly investigated (Fig.4a).In the single-crystal structure, the C-H…F hydrogen bond (2.677 ?A) between the -OMe group and the -BF2-group, and the C-H…F hydrogen bond (2.514 ?A) between the -Ph group and the -BF2- group dominate the molecular packing structure ofmeso-CF3-BDP 1 (Fig.4b), which facilitates theJaggregation packing mode.The slipping angle and the distance between each molecule are determined to be 24° and ~3.6 ?A, respectively (Fig.4b).Based on transmission electron microscopy (TEM)photograph, their sizes were less than 110 nm (Fig.4e).Moreover,dynamic light scattering (DLS) of 1-NPs showed a suitable hydrodynamic diameter (10-110 nm) in Fig.4d, and the average hydrodynamic diameter and the polydispersity index (PDI) were about 56.35 nm and 0.215.The prepared 1-NPs in aqueous solution are stable for two weeks (Figs.4f and g).Owing to theJ-aggregation effect (Fig.4c), the absorption maximum (λabs=746 nm) of 1-NPs in aqueous solution bathochromically shifted 14 nm and its absorption band covered the ranges of the NIR region (650-900 nm) and became wider [14,17], comparing to those (650-800 nm) of 1 in CH2Cl2(Fig.4h) [51].

    To discover the photothermal efficacy of hydrosoluble 1-NPs(Figs.S9 and S10 in Supporting information), the temperature elevation of the multiple concentrations ranging from 20 μmol/L to 80 μmol/L 1-NPs were recorded in the presence of 690 nm laser irradiation (0.6 W/cm2) (Fig.S9a).As revealed in Figs.S9a and b,80 μmol/L 1-NPs exhibited a intense photothermal conversion ability (ΔT=55.5 °C) upon photon-irradiation (0.6 W/cm2in 5 min),comparing to those (ΔT=27.5 °C for 20 μmol/L; ΔT=36.1 °C for 40 μmol/L) in the low concentration, suggesting that temperature augment is concentration dependent.Thus, we further discussed the temperature enhancement under different illumination of 80 μmol/L 1-NPs, and found that the stronger the radiation intensity, the higher temperature enhancement (ΔT=28.9 °C in 0.2 W/cm2; ΔT=39.4 °C in 0.4 W/cm2; ΔT=55.5 °C in 0.6 W/cm2)(Fig.S9c).Therefore, higher concentration and stronger laser radiation are feasible for photothermal conversion process.1-NPs showed an outstanding photothermal conversion during three heating-cooling cycles, approving the possibility of reuse (Fig.S9d).The PCE of 1-NPs was established by acquiring the temperature response of the heating and cooling curves (Fig.S9e), as revealed in Fig.S9f (τ=129 s).The PCE value (η) of 1-NPs was calculated to be 82%, which was much higher than that of the commercialized PTAs indocyanine green (ICG) NPs (η= 17.3%) [52,53], Au nanorods (η=21%) [54] and was inferior to the highest one (η= 88.3%) [37].

    To further explore the biological compatibility and potential inhibiting cancer cells effect of 1-NPs, the double-staining kit calcein AM (stains live cells with green fluorescence presented) and prodium iodide (PI, stains dead cells with red fluorescence presented) was applied to demonstrate the effectiveness of 1-NPs with low-power photon-irradiation on cancer cell viability.As displayed in column 4 of Fig.S11 (Supporting information), 1-NPs induced death of gastric cancer cells SGC-7901, exhibiting a significant red fluorescence, suggesting cell death state under laser treatment.In contrast, control group, sole laser-treated or sole 1-NPstreated groups had distinct green fluorescence, demonstrating no phototherapy effect for killing cancer cells.These results exhibited that cancer cells destroyed by 1-NPs with laser irradiation (690 nm,0.2 W/cm2) was observed on the premise of ensuring biosafety.

    To deeply research the triggering mechanism of 1-NPs under photo-mediated on cancer cell death, then, flow cytometry on SGC-7901 cells was performed.In comparison to the other groups, the cells treated with 1-NPs plus low power laser irradiation (690 nm,0.2 W/cm2) displayed a reduction in the stage of DNA synthesis phase (S phase), indicating that 1-NPs intercepted cancer cell proliferation, block cancer cell cycle progression caused by laser irradiation, as shown in Fig.5a [55-57].Meanwhile, Fig.5b evaluated that the percentage of apoptotic cells increased from 14.42% to 54.33% after treatment with 1-NPs imposed laser irradiation, cells treated with 1-NPs alone or light irradiation alone showed lower apoptosis rates, demonstrating the valid competence of 1-NPs to induce cancer cells apoptosis under light-responsive.The effect of 1-NPs with NIR laser irradiation on cycle and apoptosis related factors was further verified in SGC-7901 cancer cell by real-time polymerase chain reaction (RT-qPCR) and Western blot at both RNA and protein levels as shown in Figs.5c and d and Fig.S12 (Supporting information).Over expression of Cyclin D1 resulted in cell cycle disorder and uncontrolled cancer cell growth, the decreased expression level of Cyclin D1 indicated that treatment with 1-NPs plus 690 nm laser irradiation induced cancer cell cycle stagnation,and suppressed cancer cell proliferation [58].Meanwhile, Bcl-2 is a negative factor of cell apoptosis and Bax is a positive regulator of apoptosis [59,60].As shown in Figs.5c and d, executing lighttreated in the 1-NPs groups, the RNA and protein levels of Bax increased, while the RNA and protein levels of Bcl-2 decreased, indicating that the photothermal therapeutic effect of 1-NPs can trigger apoptosis in cancer cells.The above results are in high consistency with those of AM/PI co-stained experiments, indicating that 1-NPs upon low-power laser irradiation effectively restrains cell cycle progression, triggers cell apoptosis factors, and inhibits cancer cell proliferation.Thus, the design principle for 1-NPs obtained the probability of a NIR PTA for cancer treatment.

    Fig.5.(a) Cell cycle analysis using flow cytometry in SGC-7901 cells.NC: negative control.(b) Apoptosis analysis using flow cytometry in SGC-7901 cells.??P < 0.01,n=2. t-test.All data were shown as mean ± standard deviation (SD).UT: untreated.(c) mRNA expression levels related to the regulation of apoptosis (Bax and Bcl-2)was evaluated using RT-qPCR in SGC-7901 cells.(d) Protein expression levels related to the regulation of cell cycle (Cyclin D1) and cell apoptosis (Bax and Bcl-2)were evaluated by Western blot in SGC-7901 cells.Different treatments are including untreated, 20 μmol/L 1-NPs, laser (690 nm, 20 min), and BDP 1-NPs plus laser irradiation (0.2 W/cm2).

    In conclusion, one-pot synthesis of 1,7-diphenyl subsititutedmeso-CF3-BDP was achieved in 43% yields.The low-barrier rotation of the -CF3group inmeso-CF3-BDP remarkably increases the non-radiative efficiency, and the photothermal conversion effi-ciency of the self-assembled nanoparticles (1-NPs:λabs=746 nm)byJ-aggregates based on X-ray crystallographic analysis was 82%.1-NPs plus low power laser irradiation (0.2 W/cm2) could effectively block cell cycle progression, inhibit cancer cell proliferation and trigger cell apoptosis.Therefore, this study proposes an alternate molecular design platform byJ-aggregates to promote PCE through the introduction of rotating segment and trigger the cancer cells apoptosis in PTT at low power laser density.

    Declaration of competing interest

    The authors declare no conflict of interest.

    Acknowledgments

    This work was supported by the National Natural Science Foundation of China (Nos.22078201, U1908202), Natural Science Foundation of Liaoning (No.2021NLTS1206), Serving Local Project of Education Department of Liaoning Province (No.LZ2020005), Liaoning & Shenyang Key Laboratory of Functional Dye and Pigment (Nos.2021JH13/10200018, 21-104-0-23) and the Distinguished Professor Project Liaoning Province (No.20183532).

    Supplementary materials

    Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.108223.

    亚洲久久久国产精品| 少妇被粗大猛烈的视频| 日韩av免费高清视频| 黑人高潮一二区| 欧美日韩在线观看h| 大香蕉久久网| 亚洲av中文字字幕乱码综合| 久久久久网色| 一级毛片我不卡| 亚洲av电影在线观看一区二区三区| 亚洲精品乱久久久久久| 亚洲av福利一区| 丰满人妻一区二区三区视频av| 少妇人妻 视频| 26uuu在线亚洲综合色| 国产精品成人在线| 日韩人妻高清精品专区| 亚洲精品乱码久久久久久按摩| av在线观看视频网站免费| 亚洲av国产av综合av卡| 3wmmmm亚洲av在线观看| 男女下面进入的视频免费午夜| a级毛片免费高清观看在线播放| 国产在视频线精品| 蜜桃久久精品国产亚洲av| 久久亚洲国产成人精品v| 午夜福利网站1000一区二区三区| 黑人高潮一二区| 亚洲婷婷狠狠爱综合网| 97在线人人人人妻| 黑丝袜美女国产一区| 日本色播在线视频| 亚洲,一卡二卡三卡| 国产中年淑女户外野战色| 亚洲人成网站高清观看| 我的老师免费观看完整版| 国产 一区 欧美 日韩| 欧美+日韩+精品| 三级国产精品欧美在线观看| 在线观看三级黄色| 一级毛片我不卡| 蜜桃久久精品国产亚洲av| 一个人看视频在线观看www免费| 久久久亚洲精品成人影院| 黑人高潮一二区| 久久午夜福利片| 熟女人妻精品中文字幕| 国产又色又爽无遮挡免| 自拍偷自拍亚洲精品老妇| 中国三级夫妇交换| 男女国产视频网站| 大片电影免费在线观看免费| 色婷婷久久久亚洲欧美| 亚洲成人手机| 99久久综合免费| 亚洲欧美清纯卡通| 日韩免费高清中文字幕av| 2021少妇久久久久久久久久久| 国内精品宾馆在线| 欧美精品亚洲一区二区| 久久精品夜色国产| 亚洲av综合色区一区| 久久国产精品男人的天堂亚洲 | 最近的中文字幕免费完整| 少妇熟女欧美另类| 亚洲国产成人一精品久久久| 亚洲av在线观看美女高潮| 国产色婷婷99| 高清视频免费观看一区二区| 精品酒店卫生间| 肉色欧美久久久久久久蜜桃| 亚洲欧美精品专区久久| 亚洲综合色惰| 黄片wwwwww| 国产一区二区在线观看日韩| 久久精品国产自在天天线| 一区二区av电影网| 成人国产av品久久久| 身体一侧抽搐| 纵有疾风起免费观看全集完整版| 午夜福利在线在线| 亚洲国产av新网站| 激情五月婷婷亚洲| 国产精品嫩草影院av在线观看| videos熟女内射| 一个人看的www免费观看视频| 日韩伦理黄色片| 久久99热这里只频精品6学生| 久久99热6这里只有精品| 国产精品国产av在线观看| 国产精品一区二区性色av| 最近最新中文字幕免费大全7| 丰满少妇做爰视频| 国产高清三级在线| 91久久精品电影网| 亚洲av福利一区| 老司机影院成人| 免费少妇av软件| 一级毛片黄色毛片免费观看视频| 久久久国产一区二区| 久久99热这里只频精品6学生| 亚洲aⅴ乱码一区二区在线播放| 久久久久久久久久久丰满| av视频免费观看在线观看| 麻豆成人av视频| 日本黄大片高清| 男女边摸边吃奶| 日韩伦理黄色片| 在线观看国产h片| 最后的刺客免费高清国语| 人妻系列 视频| 另类亚洲欧美激情| 成人高潮视频无遮挡免费网站| 在线观看av片永久免费下载| 99视频精品全部免费 在线| 亚洲欧美精品自产自拍| 国产精品一区二区性色av| 日本与韩国留学比较| 草草在线视频免费看| 中文精品一卡2卡3卡4更新| 丰满迷人的少妇在线观看| 王馨瑶露胸无遮挡在线观看| 亚洲国产色片| 中文字幕制服av| 精品一区二区三卡| 欧美+日韩+精品| 成年美女黄网站色视频大全免费 | 久久精品国产a三级三级三级| h日本视频在线播放| 美女国产视频在线观看| 女性被躁到高潮视频| 大又大粗又爽又黄少妇毛片口| 成人毛片60女人毛片免费| 国产国拍精品亚洲av在线观看| 国产老妇伦熟女老妇高清| 秋霞伦理黄片| 国产精品福利在线免费观看| 国产免费视频播放在线视频| 久久99热6这里只有精品| 联通29元200g的流量卡| 韩国av在线不卡| 久久精品国产a三级三级三级| 小蜜桃在线观看免费完整版高清| 老司机影院毛片| 久久人妻熟女aⅴ| 成人免费观看视频高清| av福利片在线观看| 91狼人影院| 久久人妻熟女aⅴ| 五月伊人婷婷丁香| 一边亲一边摸免费视频| 一区二区三区免费毛片| 精品久久久精品久久久| 老司机影院成人| 日本黄色片子视频| av在线老鸭窝| 国产精品不卡视频一区二区| 欧美区成人在线视频| 久久99蜜桃精品久久| 中文精品一卡2卡3卡4更新| 女人十人毛片免费观看3o分钟| 亚洲国产精品成人久久小说| 国产深夜福利视频在线观看| 人人妻人人看人人澡| 国产av精品麻豆| 亚洲精品,欧美精品| 国产男人的电影天堂91| 久久久色成人| 亚洲久久久国产精品| 亚洲欧美成人综合另类久久久| 91精品国产国语对白视频| 秋霞在线观看毛片| 在线观看免费视频网站a站| 插逼视频在线观看| 国产极品天堂在线| 国产成人免费观看mmmm| 国产一区亚洲一区在线观看| 亚洲国产精品一区三区| 国模一区二区三区四区视频| 国产片特级美女逼逼视频| 亚洲欧美一区二区三区黑人 | 一级片'在线观看视频| 亚洲国产av新网站| 波野结衣二区三区在线| 色网站视频免费| av国产免费在线观看| 美女xxoo啪啪120秒动态图| 国语对白做爰xxxⅹ性视频网站| 国产人妻一区二区三区在| 成年av动漫网址| 国产精品人妻久久久久久| 国产黄片美女视频| 免费高清在线观看视频在线观看| 亚洲欧美成人综合另类久久久| 成人漫画全彩无遮挡| 日本一二三区视频观看| 91精品国产九色| a级一级毛片免费在线观看| 天堂中文最新版在线下载| 3wmmmm亚洲av在线观看| 99九九线精品视频在线观看视频| 日韩亚洲欧美综合| av在线app专区| 日韩国内少妇激情av| 日韩av在线免费看完整版不卡| 亚洲av男天堂| 久久99热这里只频精品6学生| 久久人人爽人人片av| 99热这里只有是精品50| 日韩国内少妇激情av| 久久99热这里只有精品18| 国产精品一区www在线观看| 18+在线观看网站| 视频区图区小说| 性色avwww在线观看| 午夜视频国产福利| 视频区图区小说| 在线免费十八禁| 免费看光身美女| 少妇人妻久久综合中文| 国产中年淑女户外野战色| 国产白丝娇喘喷水9色精品| 中文精品一卡2卡3卡4更新| 亚洲成人一二三区av| 大陆偷拍与自拍| 精品一区在线观看国产| 直男gayav资源| 日韩av不卡免费在线播放| 国产免费视频播放在线视频| 国产又色又爽无遮挡免| av又黄又爽大尺度在线免费看| 久久人人爽人人片av| 波野结衣二区三区在线| 视频中文字幕在线观看| 黄色欧美视频在线观看| 国产精品av视频在线免费观看| 在线 av 中文字幕| 久久久久国产网址| 街头女战士在线观看网站| 久久6这里有精品| 嘟嘟电影网在线观看| 蜜桃在线观看..| 日本wwww免费看| 国产精品一区www在线观看| 黄色配什么色好看| 国产高清国产精品国产三级 | 欧美日韩视频精品一区| 亚洲人与动物交配视频| 97超碰精品成人国产| 国产精品一区二区性色av| 多毛熟女@视频| 国产爽快片一区二区三区| 亚洲av欧美aⅴ国产| 国产黄片视频在线免费观看| 国产午夜精品一二区理论片| 18禁裸乳无遮挡动漫免费视频| 美女高潮的动态| 日本-黄色视频高清免费观看| 深爱激情五月婷婷| 久久婷婷青草| 99热国产这里只有精品6| 男人爽女人下面视频在线观看| 一个人看视频在线观看www免费| 亚洲自偷自拍三级| 蜜桃久久精品国产亚洲av| 亚洲精品日韩在线中文字幕| 亚洲精品久久久久久婷婷小说| 欧美成人一区二区免费高清观看| 十八禁网站网址无遮挡 | 天天躁日日操中文字幕| 欧美老熟妇乱子伦牲交| 国产男女内射视频| 最近最新中文字幕大全电影3| 国产一区二区三区av在线| 最近手机中文字幕大全| 国产精品一区二区在线观看99| 久久久久人妻精品一区果冻| 夜夜爽夜夜爽视频| 国产 一区精品| 黄色视频在线播放观看不卡| 99精国产麻豆久久婷婷| 国产亚洲最大av| 精品人妻一区二区三区麻豆| 少妇丰满av| 国产美女午夜福利| 国产精品.久久久| 国产精品爽爽va在线观看网站| 国产色爽女视频免费观看| 日本av免费视频播放| 日韩亚洲欧美综合| 亚洲av二区三区四区| 日韩强制内射视频| 男人添女人高潮全过程视频| 亚洲一级一片aⅴ在线观看| 如何舔出高潮| 精品人妻偷拍中文字幕| 一本一本综合久久| 欧美日韩综合久久久久久| 午夜免费鲁丝| 少妇的逼水好多| 色吧在线观看| 高清在线视频一区二区三区| 欧美日韩精品成人综合77777| 免费黄色在线免费观看| av.在线天堂| 国产高清有码在线观看视频| 亚洲久久久国产精品| 国产精品欧美亚洲77777| 又爽又黄a免费视频| av国产免费在线观看| 99热6这里只有精品| 大话2 男鬼变身卡| 97热精品久久久久久| 精品少妇黑人巨大在线播放| 五月玫瑰六月丁香| 尤物成人国产欧美一区二区三区| 久久国产乱子免费精品| 成年免费大片在线观看| 亚洲真实伦在线观看| 亚洲国产精品成人久久小说| 女人久久www免费人成看片| 国产亚洲91精品色在线| 欧美成人午夜免费资源| 亚洲一区二区三区欧美精品| 最近2019中文字幕mv第一页| 欧美精品人与动牲交sv欧美| 99久国产av精品国产电影| 人人妻人人澡人人爽人人夜夜| 人人妻人人添人人爽欧美一区卜 | 99久久中文字幕三级久久日本| 欧美 日韩 精品 国产| 国产精品国产三级专区第一集| 亚洲av免费高清在线观看| av天堂中文字幕网| 噜噜噜噜噜久久久久久91| 九九爱精品视频在线观看| 亚洲精品亚洲一区二区| 欧美xxxx性猛交bbbb| 夜夜看夜夜爽夜夜摸| 91在线精品国自产拍蜜月| 久久亚洲国产成人精品v| 黑人猛操日本美女一级片| 99热这里只有是精品50| 亚州av有码| 亚洲国产精品国产精品| 亚洲国产av新网站| 亚洲av在线观看美女高潮| 国产一区二区在线观看日韩| 亚洲av不卡在线观看| 亚洲av欧美aⅴ国产| 亚洲欧美日韩卡通动漫| 亚洲人成网站高清观看| 不卡视频在线观看欧美| 欧美高清成人免费视频www| 日韩精品有码人妻一区| 亚洲人与动物交配视频| 最近最新中文字幕大全电影3| 亚洲av国产av综合av卡| 春色校园在线视频观看| 91狼人影院| 国内揄拍国产精品人妻在线| 亚洲国产精品成人久久小说| 欧美+日韩+精品| 欧美精品国产亚洲| 久热久热在线精品观看| 欧美日韩综合久久久久久| 国产综合精华液| 欧美高清性xxxxhd video| 精品熟女少妇av免费看| 亚洲国产欧美人成| 欧美丝袜亚洲另类| 夫妻性生交免费视频一级片| 日本猛色少妇xxxxx猛交久久| 成年免费大片在线观看| 欧美日韩综合久久久久久| 亚洲精品国产成人久久av| 婷婷色av中文字幕| 国产精品久久久久久久电影| 偷拍熟女少妇极品色| 又爽又黄a免费视频| 亚洲av中文av极速乱| 王馨瑶露胸无遮挡在线观看| 国产伦精品一区二区三区四那| 美女主播在线视频| 亚洲国产色片| 内射极品少妇av片p| 国产精品人妻久久久影院| 亚洲精品中文字幕在线视频 | 大话2 男鬼变身卡| 亚洲欧美中文字幕日韩二区| 精品人妻偷拍中文字幕| 一级黄片播放器| 国产乱人视频| a级毛色黄片| 一区二区三区精品91| 亚洲精品日本国产第一区| 国产精品一区二区性色av| 国产淫片久久久久久久久| 交换朋友夫妻互换小说| 99热6这里只有精品| 亚洲av国产av综合av卡| 美女高潮的动态| 日本av免费视频播放| 一级av片app| 欧美精品一区二区免费开放| 中文字幕制服av| 99热国产这里只有精品6| 亚洲精品国产av蜜桃| 国产一区二区在线观看日韩| 亚洲av在线观看美女高潮| 美女国产视频在线观看| 日本欧美视频一区| 多毛熟女@视频| 另类亚洲欧美激情| 国产综合精华液| 内射极品少妇av片p| 久久精品久久精品一区二区三区| 女人久久www免费人成看片| 天天躁日日操中文字幕| 夜夜看夜夜爽夜夜摸| 99久国产av精品国产电影| 成人毛片60女人毛片免费| 国产 精品1| 91午夜精品亚洲一区二区三区| 人妻少妇偷人精品九色| 免费高清在线观看视频在线观看| 极品教师在线视频| 成人国产麻豆网| 久久精品国产鲁丝片午夜精品| 亚洲av.av天堂| 亚洲国产毛片av蜜桃av| 亚洲内射少妇av| 小蜜桃在线观看免费完整版高清| 欧美日韩视频高清一区二区三区二| 麻豆成人av视频| av在线播放精品| 岛国毛片在线播放| 国产免费一区二区三区四区乱码| 国产成人精品久久久久久| 99热网站在线观看| 一级爰片在线观看| 多毛熟女@视频| 午夜免费观看性视频| 婷婷色综合大香蕉| 午夜激情福利司机影院| 亚洲欧美一区二区三区黑人 | 男女啪啪激烈高潮av片| 亚洲婷婷狠狠爱综合网| 国产探花极品一区二区| xxx大片免费视频| 亚洲av在线观看美女高潮| 亚洲天堂av无毛| 简卡轻食公司| av.在线天堂| 97热精品久久久久久| 免费大片黄手机在线观看| 国产精品女同一区二区软件| h视频一区二区三区| 九九久久精品国产亚洲av麻豆| 少妇人妻精品综合一区二区| 最近中文字幕2019免费版| 亚洲精品国产成人久久av| tube8黄色片| 精华霜和精华液先用哪个| 久久精品国产亚洲网站| 我要看日韩黄色一级片| 欧美精品人与动牲交sv欧美| 18禁在线无遮挡免费观看视频| 亚洲在久久综合| 中国国产av一级| 精品久久久噜噜| 国产一区二区三区av在线| 免费黄色在线免费观看| 亚洲av日韩在线播放| 丰满少妇做爰视频| www.av在线官网国产| 国产精品人妻久久久影院| 新久久久久国产一级毛片| 国产伦精品一区二区三区视频9| 如何舔出高潮| 深夜a级毛片| 乱系列少妇在线播放| 我的老师免费观看完整版| 少妇猛男粗大的猛烈进出视频| 99国产精品免费福利视频| 五月玫瑰六月丁香| 高清在线视频一区二区三区| 精华霜和精华液先用哪个| 三级国产精品片| 天天躁夜夜躁狠狠久久av| 五月天丁香电影| 国产熟女欧美一区二区| 精品久久久久久久末码| 伦理电影大哥的女人| 亚洲国产精品一区三区| 99久久精品一区二区三区| 亚洲av.av天堂| 99久久人妻综合| 又大又黄又爽视频免费| 欧美成人精品欧美一级黄| 欧美精品一区二区免费开放| 久久精品国产亚洲网站| 天天躁夜夜躁狠狠久久av| av女优亚洲男人天堂| 天堂中文最新版在线下载| 亚洲精品国产av成人精品| 插逼视频在线观看| av不卡在线播放| 蜜臀久久99精品久久宅男| 晚上一个人看的免费电影| 久久99热这里只有精品18| 成年女人在线观看亚洲视频| 日本爱情动作片www.在线观看| 成人亚洲欧美一区二区av| 黄色视频在线播放观看不卡| 婷婷色综合大香蕉| 亚洲av成人精品一区久久| 久久ye,这里只有精品| 亚洲色图av天堂| 亚洲精品日本国产第一区| 国产午夜精品久久久久久一区二区三区| av线在线观看网站| 精品久久国产蜜桃| www.av在线官网国产| 美女内射精品一级片tv| 久久国产精品大桥未久av | av黄色大香蕉| 亚洲精品日韩av片在线观看| 天堂俺去俺来也www色官网| 午夜福利在线在线| 麻豆乱淫一区二区| 男人舔奶头视频| 国产老妇伦熟女老妇高清| 亚洲av欧美aⅴ国产| 亚洲人与动物交配视频| 国产午夜精品久久久久久一区二区三区| 国产69精品久久久久777片| 亚洲精品日韩av片在线观看| 狠狠精品人妻久久久久久综合| 久久久成人免费电影| 少妇精品久久久久久久| 我的女老师完整版在线观看| 久久久久久久久久久丰满| 亚洲欧洲国产日韩| 成年免费大片在线观看| 免费人妻精品一区二区三区视频| 三级国产精品片| 日本与韩国留学比较| 超碰97精品在线观看| 亚洲成人中文字幕在线播放| 少妇熟女欧美另类| 国产亚洲5aaaaa淫片| 欧美3d第一页| 五月伊人婷婷丁香| 九九爱精品视频在线观看| 少妇人妻久久综合中文| 乱码一卡2卡4卡精品| av又黄又爽大尺度在线免费看| 精品一区二区三卡| 97在线视频观看| 国产精品不卡视频一区二区| 青青草视频在线视频观看| 99国产精品免费福利视频| 亚洲色图综合在线观看| 亚洲最大成人中文| 国内精品宾馆在线| 天堂中文最新版在线下载| 久久久久精品性色| 联通29元200g的流量卡| 2022亚洲国产成人精品| 人妻少妇偷人精品九色| 亚洲激情五月婷婷啪啪| 熟女人妻精品中文字幕| 日本黄大片高清| 下体分泌物呈黄色| av网站免费在线观看视频| 97精品久久久久久久久久精品| 免费高清在线观看视频在线观看| 啦啦啦啦在线视频资源| 日本午夜av视频| 亚洲精品乱码久久久v下载方式| 国产成人91sexporn| 最近最新中文字幕大全电影3| 啦啦啦在线观看免费高清www| av视频免费观看在线观看| 国产精品女同一区二区软件| 久久久久国产网址| 大片免费播放器 马上看| 国产有黄有色有爽视频| 在线免费十八禁| 国产亚洲5aaaaa淫片| 欧美日韩亚洲高清精品| www.色视频.com| 黄色欧美视频在线观看| av一本久久久久| 亚洲国产精品999| 久久 成人 亚洲| 免费观看av网站的网址| 简卡轻食公司| 日本色播在线视频| 久热这里只有精品99| 欧美日韩国产mv在线观看视频 | 黄色视频在线播放观看不卡| 精品久久久久久电影网| kizo精华| 六月丁香七月| 两个人的视频大全免费| 国产黄色视频一区二区在线观看| 午夜福利网站1000一区二区三区| 黄片无遮挡物在线观看| 国产91av在线免费观看| 国产精品三级大全| 精品一品国产午夜福利视频| 国产精品欧美亚洲77777| 亚洲国产精品999|