Search tips
Search criteria 


Logo of aapspharmspringer.comThis journalToc AlertsSubmit OnlineOpen Choice
AAPS PharmSciTech. 2006 June; 7(2): E7–E16.
Published online 2006 April 7. doi:  10.1208/pt070232
PMCID: PMC2750290

A tumor vasculature targeted liposome delivery system for combretastatin A4: Design, characterization, and in vitro evaluation


The objective of this study was to develop an efficient tumor vasculature targeted liposome delivery system for combretastatin A4, a novel antivascular agent. Liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG), and DSPE-PEG-maleimide were prepared by the lipid film hydration and extrusion process. Cyclic RGD (Arg-Gly-Asp) peptides with affinity for αvβ3-integrins expressed on tumor vascular endothelial cells were coupled to the distal end of PEG on the liposomes sterically stabilized with PEG (long circulating liposomes, LCL). The liposome delivery system was characterized in terms of size, lamellarity, ligand density, drug loading, and leakage properties. Targeting nature of the delivery system was evaluated in vitro using cultured human umbilical vein endothelial cells (HUVEC). Electron microscopic observations of the formulations revealed presence of small unilamellar liposomes of ~120 nm in diameter. High performance liquid chromatography determination of ligand coupling to the liposome surface indicated that more than 99% of the RGD peptides were reacted with maleimide groups on the liposome surface. Up to 3 mg/mL of stable liposomal combretastatin A4 loading was achieved with ~80% of this being entrapped within the liposomes. In the in vitro cell culture studies, targeted liposomes showed significantly higher binding to their target cells than non-targeted liposomes, presumably through specific interaction of the RGD with its receptors on the cell surface. It was concluded that the targeting properties of the prepared delivery system would potentially improve the therapeutic benefits of combretastatin A4 compared with nontargeted liposomes or solution dosage forms.

Keywords: targeted liposome delivery system, combretastatin A4, tumor vasculature targeting, liposome characterization

Full Text

The Full Text of this article is available as a PDF (401K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
1. Lasic DD, Papahadjopoulos D. Medical Applications of Liposomes. Amsterdam, The Netherlands: Elsevier; 1998.
2. Forssen EA, Coulter DM, Proffitt RT. Selective in vivo localization of daunorubicin small unilamellar vesicles in solid tumors. Cancer Res. 1992;52:3255–3261. [PubMed]
3. Park JW, Hong K, Kirpotin DB, Meyer O, Papahadjopoulos D, Benz CC. Anti-HER2 immunoliposomes for targeted therapy of human tumors. Cancer Lett. 1997;118:153–160. doi: 10.1016/S0304-3835(97)00326-1. [PubMed] [Cross Ref]
4. Stephenson SM, Low PS, Lee RJ. Folate receptor-mediated targeting of liposomal drugs to cancer cells. Methods Enzymol. 2006;387:33–50. doi: 10.1016/S0076-6879(04)87003-4. [PubMed] [Cross Ref]
5. Dark GG, Hill SA, Prise VE, Tozer GM, Pettit GR, Chaplin DJ. Combretastatin A-4, an agent that displays potent and selective toxicity toward tumor vasculature. Cancer Res. 1997;57:1829–1834. [PubMed]
6. Young SL, Chaplin DJ. Combretastatin A4 phosphate: background and current clinical status. Expert Opin Investig Drugs. 2006;13:1171–1182. doi: 10.1517/13543784.13.9.1171. [PubMed] [Cross Ref]
7. Hynes RO. A reevaluation of integrins as regulators of angiogenesis. Nat Med. 2002;8:918–921. doi: 10.1038/nm0902-918. [PubMed] [Cross Ref]
8. Kumar CC, Armstrong L, Yin Z, et al. Targeting integrins alpha v beta 3 and alpha v beta 5 for blocking tumor-induced angiogenesis. Adv Exp Med Biol. 2000;476:169–180. [PubMed]
9. Haubner R, Gratias R, Diefenbach B, Goodman SL, Jonczyk A, Kessler H. Structural and functional aspects of RGD-containing cyclic pentapeptides as highly potent and selective integrin alpha v beta 3 antagonists. J Am Chem Soc. 1996;118:7461–7472. doi: 10.1021/ja9603721. [Cross Ref]
10. Pattillo CB, Sari-Sarraf F, Nallamothu R, Moore BM, Wood GC, Kiani MF. Targeting of the antivascular drug combretastatin to irradiated tumors results in tumor growth delay. Pharm Res. 2005;22:1117–1120. doi: 10.1007/s11095-005-5646-0. [PubMed] [Cross Ref]
11. Pettit GR, Singh SB, Boyd MR, et al. Antineoplastic agents. 291. Isolation and synthesis of combretastatins A-4, A-5, and A-6(1a) J Med Chem. 1995;38:1666–1672. doi: 10.1021/jm00010a011. [PubMed] [Cross Ref]
12. Hope MJ, Bally MB, Webb G, Cullis PR. Production of large unilamellar vesicles by a rapid extrusion procedure: characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim Biophys Acta. 1985;812:55–65. doi: 10.1016/0005-2736(85)90521-8. [PubMed] [Cross Ref]
13. New RRC, Black CDV, Parker RJ, Puri A, Scherphof GL. Liposomes in biological systems. In: New RRC, editor. Liposomes: A Practical Approach. Oxford, UK: IRL Press Ltd; 1993. pp. 221–252.
14. Prabhakarpandian B, Goetz DJ, Swerlick RA, Chen X, Kiani MF. Expression and functional significance of E-selectin on cultured endothelial cells in response to ionizing radiation. Microcirculation. 2001;8:355–364. doi: 10.1038/ [PubMed] [Cross Ref]
15. Gabizon A, Chemla M, Tzemach D, Horowitz AT, Goren D. Liposome longevity and stability in circulation: effects on the in vivo delivery to tumors and therapeutic efficacy of encapsulated anthracyclines. J Drug Target. 1996;3:391–398. doi: 10.3109/10611869608996830. [PubMed] [Cross Ref]
16. Kirby C, Clarke J, Gregoriadis G. Effect of the cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro. Biochem J. 1980;186:591–598. [PubMed]
17. Semple SC, Chonn A, Cullis PR. Influence of cholesterol on the association of plasma proteins with liposomes. Biochemistry. 1996;35:2521–2525. doi: 10.1021/bi950414i. [PubMed] [Cross Ref]
18. Torchilin VP, Omelyanenko VG, Papisov MI, et al. Poly(ethylene glycol) on the liposome surface: on the mechanism of polymer-coated liposome longevity. Biochim Biophys Acta. 1994;1195:11–20. doi: 10.1016/0005-2736(94)90003-5. [PubMed] [Cross Ref]
19. Hansen CB, Kao GY, Moase EH, Zalipsky S, Allen TM. Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures. Biochim Biophys Acta. 1995;1239:133–144. doi: 10.1016/0005-2736(95)00138-S. [PubMed] [Cross Ref]
20. Martin FJ, Papahadjopoulos D. Irreversible coupling of immunoglobulin fragments to preformed vesicles: an improved method for liposome targeting. J Biol Chem. 1982;257:286–288. [PubMed]
21. Allen TM, Brandeis E, Hansen CB, Kao GY, Zalipsky S. A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells. Biochim Biophys Acta. 1995;1237:99–108. doi: 10.1016/0005-2736(95)00085-H. [PubMed] [Cross Ref]
22. Maurer N, Fenske DB, Cullis PR. Developments in liposomal drug delivery systems. Expert Opin Biol Ther. 2001;1:923–947. doi: 10.1517/14712598.1.6.923. [PubMed] [Cross Ref]
23. Rouser G, Fkeischer S, Yamamoto A. Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 1970;5:494–496. doi: 10.1007/BF02531316. [PubMed] [Cross Ref]
24. Kawahara K, Sekiguchi A, Kiyoki E, et al. Effect of TRX-liposomes size on their prolonged circulation in rats. Chem Pharm Bull (Tokyo) 2003;51:336–338. doi: 10.1248/cpb.51.336. [PubMed] [Cross Ref]
25. Bredehorst R, Ligler FS, Kusterbeck AW, Chang EL, Gaber BP, Vogel CW. Effect of covalent attachment of immunoglobulin fragments on liposomal integrity. Biochemistry. 1986;25:5693–5698. doi: 10.1021/bi00367a052. [PubMed] [Cross Ref]
26. Spragg DD, Alford DR, Greferath R, et al. Immunotargeting of liposomes to activated vascular endothelial cells: a strategy for site-selective delivery in the cardiovascular system. Proc Natl Acad Sci USA. 1997;94:8795–8800. doi: 10.1073/pnas.94.16.8795. [PubMed] [Cross Ref]
27. Cheresh DA. Human endothelial cells synthesize and express an Arg-Gly-Asp-directed adhesion receptor involved in attachment to fibrinogen and von Willebrand factor. Proc Natl Acad Sci USA. 1987;84:6471–6475. doi: 10.1073/pnas.84.18.6471. [PubMed] [Cross Ref]
28. Czyz M, Cierniewski CS. Selective Sp1 and Sp3 binding is crucial for activity of the integrin alpha v promoter in cultured endothelial cells. Eur J Biochem. 1999;265:638–644. doi: 10.1046/j.1432-1327.1999.00754.x. [PubMed] [Cross Ref]
29. Koning GA, Morselt HW, Velinova MJ, et al. Selective transfer of a lipophilic prodrug of 5-fluorodeoxyuridine from immunoliposomes to colon cancer cells. Biochim Biophys Acta. 1999;1420:153–167. doi: 10.1016/S0005-2736(99)00091-7. [PubMed] [Cross Ref]
30. Goren D, Horowitz AT, Zalipsky S, Woodle MC, Yarden Y, Gabizon A. Targeting of stealth liposomes to erbB-2 (Her/2) receptor: in vitro and in vivo studies. Br J Cancer. 1996;74:1749–1756. [PMC free article] [PubMed]
31. Kirpotin D, Park JW, Hong K, et al. Sterically stabilized anti-HER2 immunoliposomes: design and targeting to human breast cancer cells in vitro. Biochemistry. 1997;36:66–75. doi: 10.1021/bi962148u. [PubMed] [Cross Ref]
32. Yuan F, Dellian M, Fukumura D, et al. Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size. Cancer Res. 1995;55:3752–3756. [PubMed]

Articles from AAPS PharmSciTech are provided here courtesy of American Association of Pharmaceutical Scientists