Activation of PE Residues with Heterobifunctional Crosslinkers

The most common type of heterobifunctional reagent used for the activation of lipid components includes the amine- and sulfhydryl-reactive crosslinkers containing an N-hydroxysuccinim-ide (NHS) ester group on one end and either a maleimide, iodoacetyl, or pyridyl disulfide group on the other end (Chapter 5, Section 1). Principle reagents used to effect this activation process include SMCC (Chapter 5, Section 1.3), MBS (Chapter 5, Section 1.4), SMPB (Chapter 5, Section 1.6), SIAB (Chapter 5, Section 1.5), and SPDP (Chapter 5, Section 1.1). Other 

Activation of PE residues with these crosslinkers can proceed by one of two routes the purified PE phospholipid may be modified in organic solvent prior to incorporation into a liposome, or an intact liposome containing PE may be activated while suspended in aqueous solution. Most often, the PE derivative is prepared before the liposome is constructed. In this way, a stable, stock preparation of modified PE may be made and used in a number of different liposomal recipes to determine the best formulation for the intended application. However, it may be desirable to modify PE after formation of the liposomal structures to ensure that only the outer half of the lipid bilayer is altered. This may be particularly important if substances to be entrapped within the liposome are sensitive or react with the PE derivatives. 

For the activation of PE prior to liposome formation, it is best to employ a highly purified form of the molecule. While egg PE is abundantly available, it consists of a range of fatty acid derivatives—many of which are unsaturated—and is highly susceptible to oxidation. Synthetic PE, by contrast, can be obtained having a discrete fatty acid composition and is much more stable to oxidative degradation. 

Dissolve lOOpmol of PE in 5 ml of argon-purged, anhydrous methanol containing lOOpmol of triethylamine (TEA). Maintain the solution over an argon or nitrogen 

Add 50 mg of SMPB (Thermo Fisher) to the PE solution. Mix well to dissolve. 

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