|Description||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.|
|Lipid composition||L-a-PC/Cholesterol/1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(dodecanyl) (69/30/1 molar ratio)|
|Liposome Size||100 nm|
|Lipid Mass Concentration||14.82 mg/mL|
|Lipid Molar Concentration||22.45 mM|
|Appearance||White translucent liquid made of nano size unilamellar liposomes. Usually due to the small size of liposomes no settling will occur in the bottom of the vial. The liposomes are packaged in an amber vial.|
|Buffer||Phosphate Buffered Saline, pH 7.4|
|Storage||Should always be stored at in the dark at 4 °C, except when brought to room temperature for brief periods prior to animal dosing. DO NOT FREEZE. If the suspension is frozen, the encapsulated drug can be released from the liposomes thus limiting its effectiveness. In addition, the size of the liposomes will also change upon freezing and thawing.|
|Technical Notes||EDC and sulfo-NHS should be prepared immediately before use and kept at room temperature.
The activation reaction with EDC and Sulfo-NHS is most efficient at pH 4.5-7.2, and EDC reactions are often performed in at pH 4.7-6.0. For this reason, we have formulated the liposomes in PBS buffer and adjusted the pH to 6.
Reaction of Sulfo-NHS-activated molecules with primary amines is most efficient at pH 7-8, and Sulfo-NHS-ester reactions are usually performed in phosphate-buffered saline (PBS) at pH 7.2-7.5.
Tris buffer should never be used in any step of the process since it contains amine.
If you are using a ligand or peptide that is hydrophobic then it is recommended to solubilize it in DMSO or DMF and then add the buffer to it. It is recommended not to use more than 5% volume of DMSO or DMF in the solution. DMF and DMSO are both compatible with liposomes and they are also miscible in water. Other organic solvent such as ethanol and chloroform are not compatible with liposomes and will cause the liposomes to lyse. If you end up using DMSO or DMF then after the conjugation reaction is done, you need to remove DMSO and DMF from the liposomes. In order to do that you need to use a dialysis cassette that is made from REGENERATED CELLULOSE MEMBRANE. NOTE: Not all membranes are compatible with DMF and DMSO. We recommend using a Slide-A-Lyzer™ MINI Dialysis Device with MWCO of 2K made from regenerated cellulose membrane manufactured by ThermoFisher. After DMSO or DMF is removed, you can use Float-A-Lyzer® dialysis device for the final step of cleaning up the prep.