NGR和RGD配体修饰脂质体对人脐静脉内皮细胞靶向效果的比较

摘要/Abstract

摘要：

肿瘤新生血管内皮细胞特异性高表达一些能被某些配体识别的蛋白质。本文以阿霉素脂质体为载体, 在体外细胞水平上比较研究了多肽RGD和NGR对于人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVEC)的靶向效果。本研究制备了阿霉素立体稳定脂质体(doxorubicin-loaded sterically stabilized liposomes, SSL-DOX), RGD (arginine-glycine-aspartic acid) 或NGR(asparagine-glycine-arginine) 修饰的立体稳定脂质体 (RGD-SSL-DOX或NGR-SSL-DOX), 并对其粒径、Zeta电位、包封率和释放等性质进行了研究。通过针对HUVEC细胞的流式细胞术、激光共聚集显微技术以及SRB测定在体外水平上研究两个多肽对于肿瘤血管内皮细胞的靶向效果。本研究中制备的脂质体包封率达到98%以上, 粒径约65–75 nm, 表面带弱的负电荷。体外释放实验证明RGD或NGR的修饰没有改变脂质体的释放行为。流式细胞实验中, SSL-DOX, NGR-SSL-DOX, RGD-SSL-DOX和阿霉素溶液中阿霉素被HUVEC摄取的顺序为: 阿霉素溶液>RGD-SSL-DOX>NGR-SSL-DOX>SSL-DOX, 配体修饰脂质体中的阿霉素进入细胞后分布在细胞核和细胞质内, 而SSL-DOX内的药物仅分布在细胞核中。SRB实验中各制剂的细胞毒顺序与摄取实验中的顺序相同。脂质体的性质表征研究说明脂质体表面的配体修饰没有影响立体稳定脂质体的各项性质。由于配体介导的内吞作用, 相对未经修饰的脂质体, 配体修饰脂质体处理的HUVEC摄取的阿霉素有所增多, 并且RGD-SSL-DOX的靶向效果强于NGR-SSL-DOX。

关键词: NGR, RGD, 人脐静脉内皮细胞, 脂质体

Abstract:

Endothelial cells in the angiogenic vessels of solid tumors over-express several proteins, which could be recognized by some peptide ligands. In this study, the targeting properties of two peptides, RGD (arginine-glycine-aspartic acid) and NGR (asparagine-glycine-arginine), towards human umbilical vein endothelial cells (HUVEC) were compared in vitro using doxorubicin entrapped liposomes as vehicles. The doxorubicin-loaded sterically stabilized liposomes (SSL-DOX) and RGD or NGR modified liposomes (RGD-SSL-DOX or NGR-SSL-DOX) were prepared and characterized. The studied properties included particle size, zeta potential, encapsulation efficiency and in vitro release rate. Flow cytometry, confocal microscopy and SRB assay were used on HUVEC to assess the targeting effect of the two peptides towards endothelial cells of tumor vasculature. All of the liposomes prepared in this study were obtained with encapsulation efficiencies of above 98%, particle sizes of about 65–75 nm and slight negative surface charges. The in vitro release results demonstrated that the modification of RGD or NGR did not alter the release behaviors of liposomes. It was observed in flow cytometry that the uptake of doxorubicin by HUVEC from SSL-DOX, NGR-SSL-DOX, RGD-SSL-DOX and doxorubicin solution followed the order of doxorubicin solution>RGD-SSL-DOX >NGR-SSL-DOX>SSL-DOX, and the internalized doxorubicin distributed in both nuclei and cytoplasm for ligand modified SSL and only in nuclei for non-targeted SSL. The order of cytotoxicity in SRB assay was the same as that of the uptake study. The characterization study indicated that modifications did not significantly change the properties of the sterically stabilized liposomes. HUVEC treated with both modified liposomes showed higher uptake of doxorubicin as compared to those with SSL-DOX as a result of the receptor-mediated endocytosis. Moreover, RGD-SSL-DOX exhibited better targeting effect than NGR-SSL-DOX.

Key words: NGR, NGR, RGD, RGD, Human umbilical vein endothelial cells, Human umbilical vein endothelial cells, Liposomes, Liposomes

中图分类号:

R943.42

Supporting:

Foundation item: National Natural Science Foundation of China (Grant No. 30873170).

^*Corresponding author. Tel./fax: 86-10-82802683;

e-mail: xuanzhang@bjmu.edu.cn

陈晓梅, 王珣, 黄跃, 张烜^*, 张强. NGR和RGD配体修饰脂质体对人脐静脉内皮细胞靶向效果的比较[J]. .

Xiao-Mei Chen, Xun Wang, Yue Huang, Xuan Zhang^*, Qiang Zhang . A comparison study of the targeting properties of NGR-liposomes and RGD-liposomes towards human umbilical vein endothelial cells[J]. .

参考文献

[1] Folkman, J. N. Engl. J. Med. 1971, 285, 1182-1186.

[2] Holmgren, L.; O’Reilly, M.S.; Folkman, J. Nat. Med. 1995, 1, 149-153.

[3] Jain, R.K. Nat. Med. 2003, 9, 685-693.

[4] Brooks, P.C.; Montgomery, A.M.; Rosenfeld, M.; Reisfeld, R.A.; Hu, T.; Klier, G.; Cheresh, D.A. Cell. 1994, 79, 1157-1164.

[5] Brown, L.F.; Berse, B.; Jackman, R.W.; Tognazzi, K.; Manseau, E.J.; Dvorak, H.F.; Senger, D.R. Am. J. Pathol. 1993, 143, 1255-1262.

[6] Pasqualini, R.; Koivunen, E.; Kain, R.; Lahdenranta, J.; Sakamoto, M.; Stryhn, A.; Ashmun, R.A.; Shapiro, L.H.; Arap, W.; Ruoslahti, E. Cancer Res. 2000, 60, 722-727.

[7] Koivunen, E.; Arap, W.; Valtanen, H.; Rainisalo, A.; Medina, O.P.; Heikkila, P.; Kantor, C.; Gahmberg, C. G.; Salo, T.; Konttinen, Y.T.; Sorsa, T.; Ruoslahti, E.; Pasqualini, R. Nat. Biotechnol. 1999, 17, 768-774.

[8] Schlingemann, R.O.; Rietveld, F.J.; Kwaspen, F.; van de Kerkhof, P.C.; de Waal, R.M.; Ruiter, D.J. Am. J. Pathol. 1991, 138, 1335-1347.

[9] Dedhar, S. Cancer Metastasis Rev. 1995, 14, 165-172.

[10] Ruoslahti, E. Annu. Rev. Cell Dev. Biol. 1996, 12, 697-715.

[11] Curnis, F.; Arrigoni, G.; Sacchi, A.; Fischetti, L.; Arap, W.; Pasqualini, R.; Corti, A. Cancer Res. 2002, 62, 867-874.

[12] Hosseinkhani, H.; Tabata, Y. J. Control. Release. 2004, 97, 157-71.

[13] Xiong, X.B.; Huang, Y.; Lu, W.L.; Zhang, X.; Zhang, H.; Nagai, T.; Zhang, Q. J. Pharm. Sci. 2005, 94, 1782-1793.

[14] Hu, P.; Yan, J.; Sharifi, J.; Bai, T.; Khawli, L.A.; Epstein, A.L. Cancer Res. 2003, 63, 5046-5053.

[15] Curnis, F.; Gasparri, A.; Sacchi, A.; Longhi, R., Corti, A. Cancer Res. 2004, 64, 565-571.

[16] Mukhopadhyay, S.; Barnes, C.M.; Haskel, A.; Short, S.M.; Barnes, K.R.; Lippard, S.J. Bioconjug. Chem. 2008, 19, 39-49.

[17] Pastorino, F.; Brignole, C.; Marimpietri, D.; Cilli, M.; Gambini, C.; Ribatti, D.; Longhi, R.; Allen, T.M.; Corti, A.; Ponzoni, M. Cancer Res. 2003, 63, 7400-7409.

[18] Zarovni, N.; Monaco, L.; Corti, A. Hum. Gene Ther. 2004, 15, 373-382.

[19] Xiong, X.B.; Huang, Y.; Lu, W.L.; Zhang, X.; Zhang, H.; Nagai, T.; Zhang, Q. J. Control. Release. 2005, 107, 262-275.

[20] Jaffe, E.A.; Nachman, R.L.; Becker, C.G., Minick, C.R. J. Clin. Invest. 1973, 52, 2745-2756.

[21] Haran, G.; Cohen, R.; Bar, L.K.; Barenholz, Y. Biochim. Biophys. Acta. 1993, 1151, 201-215.

[22] Vichai, V.; Kirtikara, K. Nat. Protoc. 2006, 1, 1112-1116.

[23] Duzgunes, N.; Nir, S. Adv. Drug Deliv. Rev. 1999, 40, 3-18.

[24] Garde, S.V.; Forte, A.J.; Ge, M.; Lepekhin, E.A.; Panchal, C.J.; Rabbani, S.A.; Wu, J.J. Anti-cancer Drugs. 2007, 18, 1189-1200.

[25] Zhao, R.; Ma, X.; Xie, X.; Shen, G.X. Am. J. Physiol. Endocrinol. Metab. 2009. In print.

[26] Isberg, R.R.; Van Nhieu, G.T. Trends Cell Biol. 1995, 5, 120-124.

[27] Logan, D.; Abu-Ghazaleh, R.; Blakemore, W.; Curry, S.; Jackson, T.; King, A.; Lea, S.; Lewis, R.; Newman, J.; Parry, N. Rowlands, D.; Stuart, D.; Fry, E. Nature. 1993, 362, 566-568.

[28] Lee, R.J.; Low, P.S. Biochim. Biophys. Acta. 1995, 1233, 134-144.

[29] Sapra, P.; Allen, T.M. Cancer Res. 2002, 62, 7190-7194.

[30] Fukasawa, K.; Fujii, H.; Saitoh, Y.; Koizumi, K.; Aoznka, Y.; Sekine, K.; Yamada, M.; Saiki, I.; Nishikawa, K. Cancer Lett. 2006, 243, 135-143.