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. The liposomes containing pyridyldithiopropionate (PDP) lipids are used to conjugate proteins, antibodies and other molecules containing the reactive moiety. PDP lipids are not as widely used as maleimide lipids, but they do have their own niche application. The PDP group contains disulfide, which can react with sulfhydryl or thiolated proteins/antibodies. Therefore, PDP-functionalized liposomes can be used in two ways: Method A. In this approach, the pyridyldithio group on the distal ends of the PEG chains contains PDP is forest reduced by a reducing agent (dithiothreitol, DTT). Maleimide-containing antibodies are then efficiently coupled to the surface of liposomes. The thiol-maleimide procedure is one of the most desirable reactions in bioconjugate chemistry due to its simplicity and high coupling efficiency in aqueous solution. The reaction, which is based on the stable thioether linkage between a thiol group (reduced form of PDP-liposome) and the corresponding maleimide group, occurs selectively and irreversibly at neutral pH (6.5-7.5), and the formed bonds are not cleaved by reducing agents. In addition, due to the presence of two different oxidation states of sulfur residues (oxidized and reduced states as a disulfide bond and sulfhydryl group, respectively) on the two conjugating components (i.e., the liposome and protein/antibody), the probability of the crosslinking of the homologous agents is low. Therefore, protein-protein and liposome-liposome crosslinking does not usually happen. Method B. Alternatively, the PDP group can participate in disulfide exchange reactions with thiols present on targeting proteins/antibodies. The coupling reaction is fast and conducted under mild conditions. However, the formed disulfide bonds have been reported to be less stable than thioether bonds. Moreover, even in an alkaline medium (pH 8.0), thiol groups are oxidized. The disulfide bond formed between the protein/antibody and liposomes can also be broken in the presence of a reducing agent and therefore, the conjugation reaction is reversible.

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. The liposomes containing pyridyldithiopropionate (PDP) lipids are used to conjugate proteins, antibodies and other molecules containing the reactive moiety. PDP lipids are not as widely used as maleimide lipids, but they do have their own niche application. The PDP group contains disulfide, which can react with sulfhydryl or thiolated proteins/antibodies. Therefore, PDP-functionalized liposomes can be used in two ways: Method A. In this approach, the pyridyldithio group on the distal ends of the PEG chains contains PDP is forest reduced by a reducing agent (dithiothreitol, DTT). Maleimide-containing antibodies are then efficiently coupled to the surface of liposomes. The thiol-maleimide procedure is one of the most desirable reactions in bioconjugate chemistry due to its simplicity and high coupling efficiency in aqueous solution. The reaction, which is based on the stable thioether linkage between a thiol group (reduced form of PDP-liposome) and the corresponding maleimide group, occurs selectively and irreversibly at neutral pH (6.5-7.5), and the formed bonds are not cleaved by reducing agents. In addition, due to the presence of two different oxidation states of sulfur residues (oxidized and reduced states as a disulfide bond and sulfhydryl group, respectively) on the two conjugating components (i.e., the liposome and protein/antibody), the probability of the crosslinking of the homologous agents is low. Therefore, protein-protein and liposome-liposome crosslinking does not usually happen. Method B. Alternatively, the PDP group can participate in disulfide exchange reactions with thiols present on targeting proteins/antibodies. The coupling reaction is fast and conducted under mild conditions. However, the formed disulfide bonds have been reported to be less stable than thioether bonds. Moreover, even in an alkaline medium (pH 8.0), thiol groups are oxidized. The disulfide bond formed between the protein/antibody and liposomes can also be broken in the presence of a reducing agent and therefore, the conjugation reaction is reversible.

During the past five decades, various types of chemistries have been used for conjugation of molecules such as antibodies, peptides, proteins or other reactive ligands to the surface of 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 antibodies). 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 must 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 glove bag chambers. Thiolation, which is adapted to the modification of all 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.

During the past five decades, various types of chemistries have been used for conjugation of molecules such as antibodies, peptides, proteins or other reactive ligands to the surface of 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 antibodies). 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 must 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 glove bag chambers. Thiolation, which is adapted to the modification of all 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.

Numerous techniques have been developed to prepare immunoliposomes based on the nucleophilic reactivity of free amine groups of proteins or peptides. However, the most common, versatile and straightforward activation chemistry for creating reactive acylating reagents and labeling peptides/proteins is to form NHS ester with primary amines. A single step nucleophilic substitution reaction between NHS ester derivative and alpha amines at the N terminal or the beta amines of lysine side chains leads to the formation of a stable amide bond. N-Hydroxysuccinimide (NHS) esters of DSPE-PEG-NHS liposomes react with the primary amine groups on the peptides, proteins, antibodies or other functional ligands for targeted drug delivery applications. The nucleophilic attack of the amine groups on the NHS-activated carbonyl group of the DSPE-PEG-NHS results in the elimination of the NHS group and formation of amide linkage. Immunodox-NHS is a PEGylated product. For other amine reactive (PEGylated and non-PEGyalated products) and also Immunodox products suitable for other types conjugation methods see here.