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

Abstract

We have attempted to simplify the procedure for coupling various ligands to distal ends of liposome-grafted polyethylene glycol (PEG) chains and to make it applicable for single-step binding of a large variety of a primary amino group-containing substances, including proteins and small molecules. With this in mind, we have introduced a new amphiphilic PEG derivative, p-nitrophenylcarbonyl-PEG-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (pNP-PEG-DOPE), synthesized by reaction of DOPE with excess of bis(p-nitrophenylcarbonyl)-PEG in a chloroform/triethylamine mixture. pNP-PEG-DOPE readily incorporates into liposomes via its PE residue, and easily binds primary amino group-containing ligands via its water-exposed pNP groups, forming stable and 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 for protein attachment to the distal ends of liposome-grafted PEG chains. To investigate the applicability of the suggested protocol for the preparation of long-circulating targeted liposomes, we have coupled several proteins, such as concanavalin A (ConA), wheat germ agglutinin (WGA), avidin, monoclonal antimyosin antibody 2G4 (mon2G4), and monoclonal antinucleosome antibody 2C5 (mon2C5) to PEG-liposomes via terminal pNP groups and studied whether the specific activity of these immobilized proteins is preserved. The method 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 substrates (mannan for ConA-liposomes and glycophorin for WGA-liposomes); avidin-liposomes specifically bind with biotin-agarose; antibody-liposomes demonstrate high specific binding to the substrate monolayer both in the direct binding 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 in different coupling protocols was also investigated. It was also shown that pNP-PEG-DOPE-liposomes with and without attached ligands demonstrate increased stability in mouse serum.

“SMART” Drug Delivery Systems: Double-Targeted pH-Responsive Pharmaceutical Nanocarriers

“…Monoclonal antibody (mAb) 2G4 was produced and purified in our laboratory. Also, pNP-PEG 3400 -PE was synthesized and purified according to an established method in our laboratory (27) Developing the method of coupling oxidized antibody to the PEG terminus through a hydrazone bond as suggested by Hansen et al (28), we devised our own scheme of conjugate reaction for synthesis of pH-cleavable PEG-PE. The reaction was performed in two steps: first, the activation of mPEG 2000 -CHO with PDPH, and second, the conjugation of 1,2-dipalmitoylsn-glycero-3-phosphothioethanol (sodium salt) (PE-SH) to activated mPEG-CHO.…”

Hydroxypropyl-β-Cyclodextrin Copolymers and Their Nanoparticles as Doxorubicin Delivery System

“…The reaction of HP-$-CD-PLA-pNP and DPPE as reported previously, 33 is performed with with slight modification. Briefly, 1.0 g of HP-$-CD-PLA-pNP was dissolved in 3 mL of chloroform.…”

“…Firstly, the inclusion complexes may be formed from the accessible residues of DOX with the hydrophobic cavity of HP-$-CD moiety, because such inclusion complexes were easily formed from small organic molecules with free HP-$-CD. 33 The cavity size of HP-$-CD is suitable to bind DOX. Therefore, the formation of inclusion complexes was quite possible.…”

Cell transfection in vitro and in vivo with nontoxic TAT peptide-liposome–DNA complexes

“…pNP-PEG-PE was synthesized according to a procedure published by us (45). Briefly, 0.1 mmol of PE was dispersed in 8 ml of chloroform supplemented with 2 ml of triethylamine.…”