During the past five decades, various types of chemistries have been used for conjugation of molecules such as antibodies to the surface of the liposomes. In general, the conjugation can be achieved through the N-terminus, the C-terminus or the available sulfur (e.g. Fab’ fraction or thiolated Ab). Not all chemistries have the same yield and efficiency of conjugation and often reproducing biocompatible batches can be a challenge. Coupling of sulfhydryl groups with maleimide groups has been the most widely used conjugation of antibodies to liposomes. Different lipids which are offered for thioether conjugation contain maleimide, aromatic maleimides such as N-[4-(p-maleimidophenyl)-butyryl] (MPB) or 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC) group. The maleimide function group of MCC which contains an aliphatic cyclohexane ring is more stable toward hydrolysis in aqueous reaction environments rather than the aromatic phenyl group of MPB. MPB and MCC lipids are non-PEGylated lipids and they have separate kits and protocols than PEGylated maleimide lipids.

One of the major problems of using maleimide chemistry for conjugation is the rapid hydrolysis of maleimide lipid. The rate of hydrolysis is much faster in alkaline pH and therefore controlling the pH throughout the entire process is necessary and it is recommended to use the pH of 7. Due to the hydrolysis of maleimide group, our kits are designed for post-insertion of ligand conjugated maleimide lipid into the preformed liposomes. After post conjugation the liposomes have to be used right away because hydrolysis may occur after sulfhydryl coupling to the maleimide as well. Another problem is the reactivity and oxygen sensitivity of sulfhydryl group on thiolated antibody or Fab’ fragment. Due to that the conjugation reaction should be done under argon or nitrogen using inflatable polyethylene glovebag chambers.

Thiolation which is adapted to the modification of all of the antibody functional groups, is relatively clean, fast, and efficient. However, different antibodies may be more sensitive to some procedures than others. Therefore, it is recommended to select the chemistry and site of modification depending on what procedures are compatible with the antibody.

ImmunoFluor™-Maleimide is a PEGylated product. For other sulfhydryl reactive (PEGylated and non-PEGyalated products) and also ImmunoFluor™ products suitable for other types conjugation methods see here.