For most surface reactive liposome formulations, the reactive lipid is incorporated into the liposomes and the ligand, protein or peptide is conjugated directly to the liposomes that contain reactive lipids. However, this strategy might not work if the reactive lipid is prone to hydrolysis such as maleimide or NHS lipid. In order to get around this issue, the liposomes are made without the PEGylated (reactive and non-reactive) lipids. The scientist who is planning to use the kit will conjugate the ligand, protein or peptide to the reactive lipid. Next, the buffer is added to the lipid-conjugated ligand and micelle solution is formed. Another micelle solution is formed after the PEGylated non-reactive lipid is hydrated with the buffer. The two micelle solutions are mixed and incubated together at a temperature above the liquid-to-gel phase transition temperature of the HSPC (saturated matrix lipid). The PEGylated lipids in the micelles insert themselves with a high efficiency (above 80%) to the liposomes and form the immunoliposomes. Surface reactive doxorubicin liposomes (using various types of chemistries) suitable for conjugation of various antibodies, proteins, peptides and ligands are also available. For more information about surface reactive doxorubicin liposomes (Immundox) see here.

For most surface reactive liposome formulations, the reactive lipid is incorporated into the liposomes and the ligand, protein or peptide is conjugated directly to the liposomes that contain reactive lipids. However, this strategy might not work if the reactive lipid is prone to hydrolysis such as maleimide or NHS lipid. In order to get around this issue, the liposomes are made without the PEGylated (reactive and non-reactive) lipids. The scientist who is planning to use the kit will conjugate the ligand, protein or peptide to the reactive lipid. Next, the buffer is added to the lipid-conjugated ligand and micelle solution is formed. Another micelle solution is formed after the PEGylated non-reactive lipid is hydrated with the buffer. The two micelle solutions are mixed and incubated together at a temperature above the liquid-to-gel phase transition temperature of the HSPC (saturated matrix lipid). The PEGylated lipids in the micelles insert themselves with a high efficiency (above 80%) to the liposomes and form the immunoliposomes. Surface reactive doxorubicin liposomes (using various types of chemistries) suitable for conjugation of various antibodies, proteins, peptides and ligands are also available. For more information about surface reactive doxorubicin liposomes (Immundox) see here.

For most surface reactive liposome formulations, the reactive lipid is incorporated into the liposomes and the ligand, protein or peptide is conjugated directly to the liposomes that contain reactive lipids. However, this strategy might not work if the reactive lipid is prone to hydrolysis such as maleimide or NHS lipid. In order to get around this issue, the liposomes are made without the PEGylated (reactive and non-reactive) lipids. The scientist who is planning to use the kit will conjugate the ligand, protein or peptide to the reactive lipid. Next, the buffer is added to the lipid-conjugated ligand and micelle solution is formed. Another micelle solution is formed after the PEGylated non-reactive lipid is hydrated with the buffer. The two micelle solutions are mixed and incubated together at a temperature above the liquid-to-gel phase transition temperature of the HSPC (saturated matrix lipid). The PEGylated lipids in the micelles insert themselves with a high efficiency (above 80%) to the liposomes and form the immunoliposomes. Surface reactive doxorubicin liposomes (using various types of chemistries) suitable for conjugation of various antibodies, proteins, peptides and ligands are also available. For more information about surface reactive doxorubicin liposomes (Immundox) see here.

Numerous techniques have been developed to prepare immunoliposomes based on the nucleophilic reactivity of free amine groups of proteins or peptides. One of the most popular and commonly used methods is to covalently couple free carboxylic groups to primary amines through activation of the carboxyl groups with EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide). EDC, which is a so-called zero-length crosslinking agent, reacts with the carboxyl to form an amine reactive intermediate (O-acylisourea). The produced O-acylisourea can be easily displaced by nucleophilic attack from the primary amino groups in the reaction mixture. However, this intermediate is unstable and hydrolyzed in aqueous solutions. In order to prevent the intermediate hydrolysis, sulfo-NHS (N-hydroxysulfosuccinimide) is added to EDC to produce a significantly more stable and more soluble active intermediate (NHS ester). Consequently, the immunoliposomes are prepared by a two-step coupling procedure: first, activating the free carboxyl group of the linker lipid incorporated in the liposomes with EDC and sulfo-NHS, and then covalently conjugating the antibodies to the lipids through displacement of sulfo-NHS groups by antibody amines, as depicted below. EDC/sulfo-NHS coupling reactions are highly selective and highly efficient, and the biological activity of the protein or peptide is preserved.

Numerous techniques have been developed to prepare immunoliposomes based on the nucleophilic reactivity of free amine groups of proteins or peptides. One of the most popular and commonly used methods is to covalently couple free carboxylic groups to primary amines through activation of the carboxyl groups with EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide). EDC, which is a so-called zero-length crosslinking agent, reacts with the carboxyl to form an amine reactive intermediate (O-acylisourea). The produced O-acylisourea can be easily displaced by nucleophilic attack from the primary amino groups in the reaction mixture. However, this intermediate is unstable and hydrolyzed in aqueous solutions. In order to prevent the intermediate hydrolysis, sulfo-NHS (N-hydroxysulfosuccinimide) is added to EDC to produce a significantly more stable and more soluble active intermediate (NHS ester). Consequently, the immunoliposomes are prepared by a two-step coupling procedure: first, activating the free carboxyl group of the linker lipid incorporated in the liposomes with EDC and sulfo-NHS, and then covalently conjugating the antibodies to the lipids through displacement of sulfo-NHS groups by antibody amines, as depicted below. EDC/sulfo-NHS coupling reactions are highly selective and highly efficient, and the biological activity of the protein or peptide is preserved.