Studies on protein-liposome coupling using novel thiol-reactive coupling lipids: influence of spacer length and polarity.

To optimize the preparation of immunoliposomes, we investigated the coupling of thiolated IgG and BSA to liposomes using a novel group of coupling lipids. All lipids consist of cholesterol as membrane anchor and a thiol-reactive maleimide headgroup, linked by a spacer that differs in length and polarity (ethylene glycol, tetraethylene glycol, PEG 400, PEG 1000, dodecyl). In addition, lipids differ in the electrophilicity of the maleimide group (p- or m-maleimidobenzoic ester). In the case of BSA, coupling efficiency strongly depended on the electrophilicity of the maleimide group as well as on the spacer polarity: The less electrophilic meta constitution seems to be an advantage over the p-maleimidobenzoic ester, resulting in higher coupling efficiency. Polar spacers (tetraethylene glycol, 46%) achieved a higher coupling efficiency than a nonpolar spacer with approximately the same length (dodecyl, 15%).When liposomes containing coupling lipids with the spacers tetraethylene glycol, PEG 400, and PEG 1000 were linked to BSA, coupling efficiencies were in a medium range and similar (41-46%) but were lower for the short ethylene glycol spacer (30%). In contrast, for IgG coupling efficiencies correlated with increasing spacer length. Best results were obtained using coupling lipids with a long polar spacer (PEG 1000) (65%), whereas a coupling lipid bearing a short spacer (ethylene glycol) resulted in a low coupling efficiency of 12%.