共载盐酸阿霉素和水飞蓟宾口服肝靶向脂质体机制评价
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摘要
目的:研究基于胆酸转运体的DSPE-PEG-胆酸修饰脂质体体外细胞转运和摄取及其机制。方法:构建共载盐酸阿霉素(DOX)和水飞蓟宾(silybin)的DSPE-PEG-胆酸修饰脂质体(CA-LP-DOX/silybin),采用表面等离子体共振(surface plasmon resonance,SPR)技术考察CA-LP-DOX/silybin与细胞的相互作用;采用Caco-2细胞转运考察CA-LP-DOX/silybin经肠吸收机制;采用HepG2细胞摄取考察CA-LP-DOX/silybin经肝细胞摄取机制。结果:所建立的CA-LP-DOX/silybin与Caco-2细胞和HepG2细胞均可相互作用,且与HepG2细胞相互作用更强。与未修饰脂质体(LP-DOX/silybin)和游离药物(DOX/silybin)相比,其经Caco-2细胞转运和Hep G2细胞摄取均增强,其摄取量受温度、浓度和内吞抑制剂影响。结论:CA-LP-DOX/silybin能够增强DOX和silybin的细胞转运和摄取,其机制为胆酸转运体介导,且有网格蛋白和小窝蛋白参与的内吞过程。
Objective: To study the in vitro cell transport,uptake by HepG2 cells and uptake mechanism of DSPE-PEG-cholic acid modified liposome co-encapsulated silybin and doxorubicin hydrochloride(DOX). Methods:Preparing the DSPE-PEG2000-cholic acid modified liposomes co-encapsulated silybin and doxorubicin hydrochloride(CA-LP-DOX/silybin). Using surface plasmon resonance(SPR) technology to determine the interaction of CALP-DOX/silybin with HepG2 cells and Caco-2 cells. To study the gastrointestinal absorption mechanism of CA-LPDOX/silybin by in vitro Caco-2 cells transport experiment. To study hepatic uptake characteristics and mechanism of CA-LP-DOX/silybin by in vitro HepG2 cells uptake experiment. Results: The CA-LP-DOX/silybin built has good interaction with Caco-2 cells and HepG2 cells,and the interaction with HepG2 cells is stronger than Caco-2 cells.Compared with the unmodified liposomes(LP-DOX/silybin) and raw materials(DOX/silybin),the CA-LP-DOX/silybin's transport and uptake are both decreased across Caco-2 cells monolayer and by HepG2 cells in vitro. What's more,the uptake is affected by temperature,drug concentration and endocytosis inhibitors. Conclusion: CA-LPDOX/silybin can improve the transport and uptake of DOX and silybin,and the mechanism involves cholic acid receptor mediated reaction and caveolin and clathrin protein mediated endocytosis pathway.
Objective: To study the in vitro cell transport,uptake by HepG2 cells and uptake mechanism of DSPE-PEG-cholic acid modified liposome co-encapsulated silybin and doxorubicin hydrochloride(DOX). Methods:Preparing the DSPE-PEG2000-cholic acid modified liposomes co-encapsulated silybin and doxorubicin hydrochloride(CA-LP-DOX/silybin). Using surface plasmon resonance(SPR) technology to determine the interaction of CALP-DOX/silybin with HepG2 cells and Caco-2 cells. To study the gastrointestinal absorption mechanism of CA-LPDOX/silybin by in vitro Caco-2 cells transport experiment. To study hepatic uptake characteristics and mechanism of CA-LP-DOX/silybin by in vitro HepG2 cells uptake experiment. Results: The CA-LP-DOX/silybin built has good interaction with Caco-2 cells and HepG2 cells,and the interaction with HepG2 cells is stronger than Caco-2 cells.Compared with the unmodified liposomes(LP-DOX/silybin) and raw materials(DOX/silybin),the CA-LP-DOX/silybin's transport and uptake are both decreased across Caco-2 cells monolayer and by HepG2 cells in vitro. What's more,the uptake is affected by temperature,drug concentration and endocytosis inhibitors. Conclusion: CA-LPDOX/silybin can improve the transport and uptake of DOX and silybin,and the mechanism involves cholic acid receptor mediated reaction and caveolin and clathrin protein mediated endocytosis pathway.
引文
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[3] WANG S,ZHU X,WANG X,et al. Flavonoids from Malus hupehensis and their cardioprotective effects against doxorubicin-induced toxicity in H9c2 cells[J]. Phytochemistry,2013,87:119-125.
[4] WANG J,CHUNG MH,XUE B,et al. Estrogenic andantiestrogenic activities of phloridzin[J]. Biol Pharm Bull,2010a,33(4):592-597.
[5] HU NJ,IWATA S,CAMERON AD,et al. Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT[J].Nature,2011,478(7369):408-411.
[6] YAN H,ZHONG G,XU G,et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus[J]. e Life,2012,1:e00049.
[7] SEEGER C,MASON WS. Sodium-dependent taurocholic cotransporting polypeptide:a candidate receptor for human hepatitis B virus[J]. Gut,2013,62(8):1093-1095.
[8] RAIS R,FLETCHER S,POLLI JE. Synthesis and in vitro evaluation of gabapentin prodrugs that target the human apical sodiumdependent bile acid transporter(h ASBT)[J]. J Pharm Sci,2011,100(3):1184-1195.
[9] MAJER F,SALOMON JJ,SHARMA R,et al. New fluorescent bile acids:synthesis,chemical characterization,and disastereoselective uptake by Caco-2 cells of 3-deoxy 3-NBD-amino deoxycholic and ursodeoxycholic acid[J]. Bioorg Med Chem,2012,20(5):1767-1778.
[10] KOLHATKAR V,POLLI JE. Structural requirements of bile acid transporters:C-3 and C-7 modifications of steroidal hydroxyl groups[J]. Eur J Pharm Sci,2012,46(1-2):86-99.
[11] ZYBIN A,SHPACOVITCH V,SKOLNIK J,et al. Optimal conditions for SPR-imaging of nano-objects[J]. Sensor Actuat BChem,2017,239:338-342.
[12] KHURANA S,KING LR,MANISCHEWITZ J,et al. Novel antibody-independent receptor-binding SPR-based assay for rapid measurement of influenza vaccine potency[J]. Vaccine,2014,32(19):2188-2197.
[13] SITERS KE,SANDER SA,DEVLIN JR,et al. Bifunctional Zn(ii)complexes for recognition of non-canonical thymines in DNA bulges and G-quadruplexes[J]. Dalton Trans,2015,44(8):3708-3716.
[14] JUN E,KIM S,JONG-HO K,et al. Design of a multicomponent peptide-woven nanocomplex for delivery of siRNA[J]. PLoS One,2015,10(2):e0118310.
[15] LI Y,ZHU CY. Enhanced hepatic targeted delivery via oral administration using nanoliposomes functionalized with novel DSPEPEG-cholic acid conjugate[J]. RSC Advances, 2016, 6:28110-28120.
[16] LI Y,YANG DD,ZHU CY,et al. Novel DSPE-PEG-cholic acid-modified liposomes with hepatic targeting properties improve the anti-tumor efficacy of oral doxorubicin hydrochloride for liver tumor-bearing mice[J]. J Biomed Nanotechnol,2017,13:727-736.
[17] GREGORIDADIS G(ed). Liposomes Technology[M]. New York:Informa Healthcare,2007:341-372.
[18] COHEN AW,HNASKO R,SCHUBERT W,et al. Role of caveolae and caveolins in health and disease[J]. Physiol Rev,2004,84:1341-1379.
[19] CHARLTON SA,COYM JW. The use of methyl-beta-cyclodextrin to solubilize cholestesterol prior to coating onto a C18 stationary phase[J]. J Chromatogr A,2012,1266:69-75.
[20] SENGUPTA D. Cholesterol modulates the structure,bingding modes,and energetics of caveolin-membrane interactions[J]. J Phys Chem B,2012,116(50):14556-14564.