Liposomes pp 267-277 | Cite as

Conjugation of Ligands to the Surface of Preformed Liposomes by Click Chemistry

  • Benoît FrischEmail author
  • Fatouma Saïd Hassane
  • Francis Schuber
Part of the Methods in Molecular Biology book series (MIMB, volume 605)


Click chemistry represents a new bioconjugation strategy that can be used to conveniently attach various ligands to the surface of preformed liposomes. This efficient and chemoselective reaction involves a Cu(I)-catalyzed azide-alkyne cycloaddition, which can be performed under mild experimental conditions in aqueous media. Here, we describe the application of a model click reaction to the conjugation, in a single step of unprotected α-1-thiomannosyl ligands, functionalized with an azide group to liposomes containing a terminal alkyne-functionalized lipid anchor. Excellent coupling yields were obtained in the presence of bathophenanthrolinedisulphonate, a water soluble copper-ion chelator, acting as a catalyst. No vesicle leakage was triggered by this conjugation reaction and the coupled mannose ligands were exposed at the surface of the liposomes. The major limitation of Cu(I)-catalyzed click reactions is that this conjugation is restricted to liposomes made of saturated (phospho)lipids. Efficient copper-free azide-alkyne click reactions are, however, being developed, which should alleviate this constraint in the future.

Key words

Liposome Azide-alkyne cycloaddition Bioconjugation chemistry Click chemistry Mannose 


  1. 1.
    Torchilin VP (2006) Multifunctional nanocarriers. Adv Drug Deliv Rev 58:1532–1555CrossRefPubMedGoogle Scholar
  2. 2.
    Forssen E, Willis M (1998) Ligand-targeted liposomes. Adv Drug Deliv Rev 29:249–271CrossRefPubMedGoogle Scholar
  3. 3.
    Allen TM (2002) Ligand-targeted therapeutics in anticancer therapy. Nat Rev Cancer 2:750–763CrossRefPubMedGoogle Scholar
  4. 4.
    Sapra P, Allen TM (2003) Ligand-targeted liposomal anticancer drugs. Prog Lipid Res 42:439–462CrossRefPubMedGoogle Scholar
  5. 5.
    Schuber F (1995) Chemistry of ligand-coupling to liposomes. In: Philippot JR, Schuber F (eds) Liposomes as tools in basic research and industry. CRC Press, Boca Raton, pp 21–39Google Scholar
  6. 6.
    Nobs L, Buchegger F, Gurny R, Allemann E (2004) Current methods for attaching targeting ligands to liposomes and nanoparticles. J Pharm Sci 93:1980–1992CrossRefPubMedGoogle Scholar
  7. 7.
    Torchilin VP (2005) Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 4:145–160CrossRefPubMedGoogle Scholar
  8. 8.
    Schuber F, Said Hassane F, Frisch B (2007) Coupling of peptides to the surface of liposomes – Application to liposome-based synthetic vaccines. In: Gregoriadis G (ed) Liposome technology, 3rd edn. Informa Healthcare, New York, USA, pp 111–130Google Scholar
  9. 9.
    Barbet J, Machy P, Leserman LD (1981) Monoclonal antibody covalently coupled to liposomes: specific targeting to cells. J Supramol Struct Cell Biochem 16:243–258CrossRefPubMedGoogle Scholar
  10. 10.
    Martin FJ, Papahadjopoulos D (1982) Irreversible coupling of immunoglobulin fragments to preformed vesicles. J Biol Chem 257:286–288PubMedGoogle Scholar
  11. 11.
    Schelté P, Boeckler C, Frisch B, Schuber F (2000) Differential reactivity of maleimide and bromoacetyl functions with thiols: application to the preparation of liposomal diepitope constructs. Bioconj Chem 11:118–128CrossRefGoogle Scholar
  12. 12.
    Bourel-Bonnet L, Pecheur EI, Grandjean C, Blanpain A, Baust T, Melnyk O, Hoflack B, Gras-Masse H (2005) Anchorage of synthetic peptides onto liposomes via hydrazone and alpha-oxo hydrazone bonds. Preliminary functional investigations. Bioconj Chem 16:450–457CrossRefGoogle Scholar
  13. 13.
    Immordino ML, Dosio F, Cattel L (2006) Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. Int J Nanomedicine 1:297–315CrossRefPubMedGoogle Scholar
  14. 14.
    Zalipsky S, Mullah N, Harding JA, Gittelman J, Guo L, DeFrees SA (1997) Poly(ethylene glycol)-grafted liposomes with oligopeptide or oligosaccharide ligands appended to the termini of the polymer chains. Bioconj Chem 8:111–118CrossRefGoogle Scholar
  15. 15.
    Sudimack J, Lee RJ (2000) Targeted drug delivery via the folate receptor. Adv Drug Deliv Rev 41:147–162CrossRefPubMedGoogle Scholar
  16. 16.
    Kolb HC, Sharpless KB (2003) The growing impact of click chemistry on drug discovery. Drug Discov Today 8:1128–1137CrossRefPubMedGoogle Scholar
  17. 17.
    Lutz JF, Zarafshani Z (2008) Efficient construction of therapeutics, bioconjugates, biomaterials and bioactive surfaces using azide-alkyne “click” chemistry. Adv Drug Deliv Rev 60:958–970CrossRefPubMedGoogle Scholar
  18. 18.
    Hein CD, Liu XM, Wang D (2008) Click chemistry, a powerful tool for pharmaceutical sciences. Pharm ResGoogle Scholar
  19. 19.
    Moorhouse AD, Moses JE (2008) Click chemistry and medicinal chemistry: a case of “cyclo-addiction”. Chem Med Chem 3:715–723PubMedGoogle Scholar
  20. 20.
    Hassane FS, Frisch B, Schuber F (2006) Targeted liposomes: Convenient coupling of ligands to preformed vesicles using “click chemistry”. Bioconj Chem 17:849–854CrossRefGoogle Scholar
  21. 21.
    Cavalli S, Tipton AR, Overhand M, Kros A (2006) The chemical modification of liposome surfaces via a copper-mediated [3 + 2] azide-alkyne cycloaddition monitored by a colorimetric assay. Chem Commun (Camb):3193–3195Google Scholar
  22. 22.
    Sun EY, Josephson L, Weissleder R (2006) “Clickable” nanoparticles for targeted imaging. Mol Imaging 5:122–128PubMedGoogle Scholar
  23. 23.
    Espuelas S, Haller P, Schuber F, Frisch B (2003) Synthesis of an amphiphilic tetraantennary mannosyl conjugate and incorporation into liposome carriers. Bioorg Med Chem Lett 13:2557–2560CrossRefPubMedGoogle Scholar
  24. 24.
    Ponpipom MM, Bugianesi RL, Robbins JC, Doebber TW, Shen TY (1981) Cell-specific ligands for selective drug delivery to tissues and organs. J Med Chem 24:1388–1395CrossRefPubMedGoogle Scholar
  25. 25.
    Iyer SS, Anderson AS, Reed S, Swanson B, Schmidt JG (2004) Synthesis of orthogonal end functionalized oligoethylene glycols of defined lengths. Tetrahedron Lett 45:4285–4288CrossRefGoogle Scholar
  26. 26.
    Rouser G, Fleisher J, Yamamoto A (1970) Two dimensional thin layer chromatographic separation of plant lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5:494–496CrossRefPubMedGoogle Scholar
  27. 27.
    Monsigny M, Petit C, Roche AC (1988) Colorimetric determination of neutral sugars by a resorcinol sulfuric acid micromethod. Anal Biochem 175:525–530CrossRefPubMedGoogle Scholar
  28. 28.
    Engel A, Chatterjee SK, Alarifi A, Riemann D, Langner J, Nuhn P (2003) Influence of spacer length on interaction of mannosylated liposomes with human phagocytic cells. Pharm Res 20:51–57CrossRefPubMedGoogle Scholar
  29. 29.
    Lewis WG, Magallon FG, Fokin VV, Finn MG (2004) Discovery and characterization of catalysts for azide-alkyne cycloaddition by fluorescence quenching. J Am Chem Soc 126:9152–9153CrossRefPubMedGoogle Scholar
  30. 30.
    Gal S, Pinchuk I, Lichtenberg D (2003) Peroxid-ation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effects of surface charge on the oxidizability and on the potency of antioxidants. Chem Phys Lipids 126:95–110CrossRefPubMedGoogle Scholar
  31. 31.
    Lee LV, Mitchell ML, Huang S-J, Fokin VV, Sharpless KB, Wong C-H (2003) A potent and highly selective inhibitor of human a-1, 3-fucosyltransferase via click chemistry. J Am Chem Soc 125:9588–9589CrossRefPubMedGoogle Scholar
  32. 32.
    Baskin JM, Prescher JA, Laughlin ST, Agard NJ, Chang PV, Miller IA, Lo A, Codelli JA, Bertozzi CR (2007) Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci USA 104:16793–16797CrossRefPubMedGoogle Scholar
  33. 33.
    Ning X, Guo J, Wolfert MA, Boons GJ (2008) Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast Huisgen cycloadditions. Angew Chem Int Ed Engl 47:2253–2255CrossRefPubMedGoogle Scholar
  34. 34.
    Li Z, Seo TS, Ju J (2004) 1,3-Dipolar cycloaddition of azides with electron-deficient alkynes under mild condition in water. Tetrahedron Lett 45:3143–3146CrossRefGoogle Scholar
  35. 35.
    Tam A, Soellner MB, Raines RT (2007) Water-soluble phosphinothiols for traceless staudinger ligation and integration with expressed protein ligation. J Am Chem Soc 129:11421–11430CrossRefPubMedGoogle Scholar
  36. 36.
    Ryu EH, Zhao Y (2005) Efficient synthesis of water-soluble calixarenes using click chemistry. Org Lett 7:1035–1037CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Benoît Frisch
    • 1
    Email author
  • Fatouma Saïd Hassane
    • 1
  • Francis Schuber
    • 1
  1. 1.Départment de Chimie Bioorganique, Faculté de PharmacieInstitut Gilbert Lautriat, UMR 7175-LC1 CNRS-Université Louis PasteurStrasbourg-IllkirchFrance

Personalised recommendations