血管新生(Angiogenesis)在腫瘤細胞增生與實體腫瘤形成上扮演著不可或缺的角色。在腫瘤血管新生之過程中，內皮細胞形成新的微血管時，會表現相當量的αvβ3整合素，由於αvβ3整合素與具Arg-Gly-Asp (RGD)序列之胜□具有高度的專一結合性，因此，選擇αvβ3整合素作為腫瘤的生物性標記，開發標靶αvβ3整合素之治療劑乃是治療高血管新生腫瘤一新的策略。本研究旨在開發一包埋放射性藥物之標靶微脂體。此放射性標靶微脂體之建構係於微脂體表面耦合特定胺基酸序列Arg-Gly-Asp (RGD)，並同時於微脂體核內包埋Re-188放射性核種作為放射性治療藥物，以期導引正確腫瘤位置，以達到精確治療之目的。
方法︰首先將RGD胜□E[c(RGDyK)]2耦合於微脂體表面，同時將Re-188放射性核種以188Re-BMEDA形態包埋至微脂體，得到E[c(RGDyK)]2-188Re-liposome。另一方面，亦製備未具RGD胜□耦合之188Re-liposome。以具有大量αvβ3整合素表現之人類臍帶靜脈內皮細胞(Human umbilical vein endothelial cells)進行細胞攝取試驗，並以螢光顯微鏡觀察。進而以植有C26腫瘤小鼠，進行造影、生物分佈及藥物療效評估等試驗。
結果︰在細胞攝取試驗，靶向性微脂體E[c(RGDyK)]2-188Re-liposome於人類臍帶靜脈內皮細胞之攝取比非靶向性微脂體高出四倍之多，於螢光顯微鏡觀察下，靶向性微脂體之螢光強度亦明顯優於非靶向性微脂體。在活體外藥物穩定試驗顯示此靶向性微脂體在大鼠血清中，於72小時後仍具80%以上之穩定度。microSPECT/CT影像與生物分佈試驗顯示E[c(RGDyK)]2-188Re-liposome在植有C26腫瘤小鼠中，具有較高累積於網狀內皮系統(Reticuloendothelial system)，進而導致腫瘤部位累積量少於188Re-Liposome。但在療效評估試驗，E[c(RGDyK)]2-188Re-liposome與188Re-liposome對於抑制腫瘤生長並無顯著差異。
結論︰本研究證實靶向性微脂體E[c(RGDyK)]2-188Re-liposome於活體外細胞實驗上具有高親和力與高穩定度。在動物實驗部份，本研究發現此靶向性微脂體於動物活體內易被網狀內皮系統所辨識，因而降低腫瘤部位之累積。未來需進一步針對E[c(RGDyK)]2-188Re-liposome表面之修飾進行探討，以減少網狀內皮系統吸收，改善於腫瘤位置之累積，以期達到腫瘤抑制功效。

Angiogenesis is of a general property and is critical for tumor growth, where αvβ3 integrin is overexpressed on angiogenic endothelium in and around tumor tissue. αvβ3 integrin may therefore represent a possible target for drug delivery. In this study, a drug delivery system by a modified liposome specifically aiming at angiogenic tumor endothelial cells was developed. E[c(RGDyK)]2 peptide with affinity to this integrin was adopted to conjugate on the liposome surface and the 188Re radionuclide was simultaneously encapsulated in the liposome core. The resulted liposome, referred to E[c(RGDyK)]2-188Re-liposome, was investigated for its application potential in radionuclide therapy and diagnostic imaging for tumors.
Methods: In this study, an angiogenesis-targeting liposome system was constructed to have dimeric RGD peptide E[c(RGDyK)]2 conjugated on the liposome surface and a rhenium-188 radionuclide complex encapsulated inside the liposome core. The in vitro cellular uptake by radiotracing and fluorescence microscope imaging was studied using human umbilical vein endothelial cells with overexpression of αvβ3 integrin receptor. For animal trial, the main works comprised microSPECT/CT imaging and biodistribution study and investigation of antitumor efficacy of E[c(RGDyK)]2-188Re-liposomes in comparison with non E[c(RGDyK)]2 conjugated liposome using C26 murine colon tumor-bearing animal model.
Results: The in vitro stability study showed that E[c(RGDyK)]2-188Re-liposome was quite stable in rat plasma significantly for a long time, i.e., at least 72 h. The in vitro cell binding study demonstrated that the active targeting liposome surpassed non-targeted liposome in about four-fold higher of the cellular as observed from cellular uptake and cell staining. For in vivo studies, both of microSPECT/CT images and biodistribution studies indicated that the accumulation in the reticuloendothelial system for E[c(RGDyK)]2-188Re-liposome was apparently higher than the non-targeted liposome. The tumor uptake of E[c(RGDyK)]2-188Re-liposome was about six-fold lower than the non-targeted liposomes. Nevertheless, E[c(RGDyK)]2-188Re-liposome did inhibit the tumor growth as compared to untreated controls but no significant difference was observed in comparison with the non-targeted liposome.
Conclusion: This study demonstrated the high cell binding affinity and stability of the active targeting liposome in the cell experiments. It was revealed from this study that the decrease of 188Re radioactivity accumulation in the colon tumor was relevant to the RES system uptake of E[c(RGDyK)]2-188Re-liposome. Modification on the E[c(RGDyK)]2-188Re-liposome surface to enhance the tumor uptake would be the future work to be investigated.

1. Gimbrone, M.A., Jr., R.S. Cotran, S.B. Leapman, and J. Folkman, Tumor growth and neovascularization: an experimental model using the rabbit cornea. J Natl Cancer Inst, 1974. 52(2): p. 413-27.
2. Schaffner, P. and M.M. Dard, Structure and function of RGD peptides involved in bone biology. Cell Mol Life Sci, 2003. 60(1): p. 119-32.
3. Brooks, P.C., R.A. Clark, and D.A. Cheresh, Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science, 1994. 264(5158): p. 569-71.
4. Dunehoo, A.L., M. Anderson, S. Majumdar, N. Kobayashi, C. Berkland, and T.J. Siahaan, Cell adhesion molecules for targeted drug delivery. J Pharm Sci, 2006. 95(9): p. 1856-72.
5. Chen, X., M. Tohme, R. Park, Y. Hou, J.R. Bading, and P.S. Conti, Micro-PET imaging of alphavbeta3-integrin expression with 18F-labeled dimeric RGD peptide. Mol Imaging, 2004. 3(2): p. 96-104.
6. Liu, S., Radiolabeled cyclic RGD peptides as integrin alpha(v)beta(3)-targeted radiotracers: maximizing binding affinity via bivalency. Bioconjug Chem, 2009. 20(12): p. 2199-213.
7. Haubner, R., H.J. Wester, U. Reuning, R. Senekowitsch-Schmidtke, B. Diefenbach, H. Kessler, G. Stocklin, and M. Schwaiger, Radiolabeled alpha(v)beta3 integrin antagonists: a new class of tracers for tumor targeting. J Nucl Med, 1999. 40(6): p. 1061-71.
8. Torchilin, V.P., Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov, 2005. 4(2): p. 145-60.
9. Schiffelers, R.M., G.A. Koning, T.L. ten Hagen, M.H. Fens, A.J. Schraa, A.P. Janssen, R.J. Kok, G. Molema, and G. Storm, Anti-tumor efficacy of tumor vasculature-targeted liposomal doxorubicin. J Control Release, 2003. 91(1-2): p. 115-22.
10. Bao, A., B. Goins, R. Klipper, G. Negrete, and W.T. Phillips, 186Re-liposome labeling using 186Re-SNS/S complexes: in vitro stability, imaging, and biodistribution in rats. J Nucl Med, 2003. 44(12): p. 1992-9.
11. Chen, L.C., C.H. Chang, C.Y. Yu, Y.J. Chang, W.C. Hsu, C.L. Ho, C.H. Yeh, T.Y. Luo, T.W. Lee, and G. Ting, Biodistribution, pharmacokinetics and imaging of (188)Re-BMEDA-labeled pegylated liposomes after intraperitoneal injection in a C26 colon carcinoma ascites mouse model. Nucl Med Biol, 2007. 34(4): p. 415-23.
12. Bartlett, G.R., Phosphorus assay in column chromatography. J Biol Chem, 1959. 234(3): p. 466-8.
13. Ma, L., Y.L. Liu, Z.Z. Ma, H.L. Dou, J.H. Xu, J.C. Wang, X. Zhang, and Q. Zhang, Targeted treatment of choroidal neovascularization using integrin-mediated sterically stabilized liposomes loaded with combretastatin A4. J Ocul Pharmacol Ther, 2009. 25(3): p. 195-200.
14. Allen, T.M., P. Sapra, and E. Moase, Use of the post-insertion method for the formation of ligand-coupled liposomes. Cell Mol Biol Lett, 2002. 7(2): p. 217-9.
15. Enoch, H.G. and P. Strittmatter, Formation and properties of 1000-A-diameter, single-bilayer phospholipid vesicles. Proc Natl Acad Sci U S A, 1979. 76(1): p. 145-9.
16. Haran, G., R. Cohen, L.K. Bar, and Y. Barenholz, Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim Biophys Acta, 1993. 1151(2): p. 201-15.
17. Cressman, S., I. Dobson, J.B. Lee, Y.Y. Tam, and P.R. Cullis, Synthesis of a labeled RGD-lipid, its incorporation into liposomal nanoparticles, and their trafficking in cultured endothelial cells. Bioconjug Chem, 2009. 20(7): p. 1404-11.
18. Khandare, J.J., P. Chandna, Y. Wang, V.P. Pozharov, and T. Minko, Novel polymeric prodrug with multivalent components for cancer therapy. J Pharmacol Exp Ther, 2006. 317(3): p. 929-37.
19. Chang, Y.J., C.H. Chang, C.Y. Yu, T.J. Chang, L.C. Chen, M.H. Chen, T.W. Lee, and G. Ting, Therapeutic efficacy and microSPECT/CT imaging of 188Re-DXR-liposome in a C26 murine colon carcinoma solid tumor model. Nucl Med Biol, 2010. 37(1): p. 95-104.
20. Maddalena, M.E., J. Fox, J. Chen, W. Feng, A. Cagnolini, K.E. Linder, M.F. Tweedle, A.D. Nunn, and L.E. Lantry, 177Lu-AMBA biodistribution, radiotherapeutic efficacy, imaging, and autoradiography in prostate cancer models with low GRP-R expression. J Nucl Med, 2009. 50(12): p. 2017-24.
21. Chang, Y.J., C.H. Chang, T.J. Chang, C.Y. Yu, L.C. Chen, M.L. Jan, T.Y. Luo, T.W. Lee, and G. Ting, Biodistribution, pharmacokinetics and microSPECT/CT imaging of 188Re-bMEDA-liposome in a C26 murine colon carcinoma solid tumor animal model. Anticancer Res, 2007. 27(4B): p. 2217-25.
22. Lantry, L.E., E. Cappelletti, M.E. Maddalena, J.S. Fox, W. Feng, J. Chen, R. Thomas, S.M. Eaton, N.J. Bogdan, T. Arunachalam, J.C. Reubi, N. Raju, E.C. Metcalfe, L. Lattuada, K.E. Linder, R.E. Swenson, M.F. Tweedle, and A.D. Nunn, 177Lu-AMBA: Synthesis and characterization of a selective 177Lu-labeled GRP-R agonist for systemic radiotherapy of prostate cancer. J Nucl Med, 2006. 47(7): p. 1144-52.
23. ElBayoumi, T.A. and V.P. Torchilin, Tumor-targeted nanomedicines: enhanced antitumor efficacy in vivo of doxorubicin-loaded, long-circulating liposomes modified with cancer-specific monoclonal antibody. Clin Cancer Res, 2009. 15(6): p. 1973-80.
24. Holig, P., M. Bach, T. Volkel, T. Nahde, S. Hoffmann, R. Muller, and R.E. Kontermann, Novel RGD lipopeptides for the targeting of liposomes to integrin-expressing endothelial and melanoma cells. Protein Eng Des Sel, 2004. 17(5): p. 433-41.
25. Elbayoumi, T.A. and V.P. Torchilin, Tumor-specific anti-nucleosome antibody improves therapeutic efficacy of doxorubicin-loaded long-circulating liposomes against primary and metastatic tumor in mice. Mol Pharm, 2009. 6(1): p. 246-54.
26. Elbayoumi, T.A. and V.P. Torchilin, Enhanced accumulation of long-circulating liposomes modified with the nucleosome-specific monoclonal antibody 2C5 in various tumours in mice: gamma-imaging studies. Eur J Nucl Med Mol Imaging, 2006. 33(10): p. 1196-205.
27. Dubey, P.K., V. Mishra, S. Jain, S. Mahor, and S.P. Vyas, Liposomes modified with cyclic RGD peptide for tumor targeting. J Drug Target, 2004. 12(5): p. 257-64.
28. Park, J.H., G. von Maltzahn, L. Zhang, A.M. Derfus, D. Simberg, T.J. Harris, E. Ruoslahti, S.N. Bhatia, and M.J. Sailor, Systematic surface engineering of magnetic nanoworms for in vivo tumor targeting. Small, 2009. 5(6): p. 694-700.