p-nitrophenylcarbonyl-PEG-PE-liposomes: fast and simple attachment of specific ligands, including monoclonal antibodies, to distal ends of PEG chainsvia p-nitrophenylcarbonyl groups

We have attempted to simplify the procedure for coupling various ligands to distal ends of liposome-grafted polyethylene glycol (PEG) chains and to ma ke it applicable For single-step binding of a large variety of a primary am ino group-containing substances, including proteins and small molecules. Wi th this in mind, we have introduced a new amphiphilic PEG derivative, p-nit rophenylcarbonyl-PEG-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (pNP-PEG -DOPE), synthesized by reaction of DOPE with excess of bis(p-nitrophenylcar bonyl)-PEG in a chloroform/triethylamine mixture. pNP-PEG-DOPE readily inco rporates into liposomes via its PE residue, and easily binds primary amino group-containing ligands via its water-exposed pNP groups, forming stable a nd non-toxic urethane (carbamate) bonds. The reaction between the pNP group and the ligand amino group proceeds easily and quantitatively at pH around 8.0, and remaining free pNP groups are promptly eliminated by spontaneous hydrolysis. Therefore, pNP-PEG-DOPE could serve as a very convenient tool f or protein attachment to the distal ends of liposome-grafted PEG chains. To investigate the applicability of the suggested protocol for the preparatio n of long-circulating targeted liposomes, we have coupled several proteins, such as concanavalin A (ConA), wheat germ agglutinin (WGA), avidin, monocl onal antimyosin antibody 2G4 (mon2G4), and monoclonal antinucleosome antibo dy 2C5 (mon2C5) to PEG-liposomes via terminal pNP groups and studied whethe r the specific activity of these immobilized proteins is preserved. The met hod permits the binding of several dozens protein molecules per single 200 nm liposome. All bound proteins completely preserve their specific activity . Lectin-liposomes are agglutinated by the appropriate polyvalent substrate s (mannan for ConA-liposomes and glycophorin for WGA-liposomes); avidin-lip osomes specifically bind with biotin-agarose; antibody-liposomes demonstrat e high specific binding to the substrate monolayer both in the direct bindi ng assay and in ELISA. A comparison of the suggested method with the method of direct membrane incorporation was made. The effect of the concentration of liposome-grafted PEG on the preservation of specific protein activity i n different coupling protocols was also investigated. It was also shown tha t pNP-PEG-DOPE-liposomes with and without attached ligands demonstrate incr eased stability in mouse serum. (C) 2001 Elsevier Science B.V. All rights r eserved.