No 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.

A series of dioleoyl N‐(monomethoxy polyethyleneglycol succinyl)phosphatidylethanolamine (PEG‐PE) of different polymer chain length was used in this study. Both the activity of PEG‐PE in prolonging the circulation time of liposomes and the relative steric barrier activity of amphipathic polymer, measured by a liposome agglutination assay, were found to be directly proportional to the chain length of PEG‐PE (PEG5000‐PE > PEG2000‐PE > PEG750‐PE). However, PEG5000‐PE caused a reduced target binding of immunoliposomes in mice due to its overly strong steric barrier activity. The best PEG‐PE species supporting the target binding of immunoliposomes was PEG2000‐PE, the activity of which was compatible to that of ganglioside GM1. However, GM1 only showed a weak steric barrier activity, suggesting a different mechanism for this glycolipid.

The less than optimal accumulation of immunoliposome-associated reagents at target sites has often been attributed to the rapid in vivo clearance of immunoliposomes from the blood. In an attempt to overcome the drawback of rapid clearance and use the targeting potential of immunoliposomes, we have prepared long-circulating, 111In-labeled immunoliposomes. Targeting properties and enhanced circulation times were demonstrated in a rabbit model of acute experimental myocardial infarct. The specificity of liposomes for newly exposed intracellular cardiac myosin at the necrotic sites was achieved by incorporating monoclonal antimyosin antibody. Extended circulation times were achieved by cocoating the antimyosin-liposomes with polyethylene glycol (PEG). The half-life of the immunoliposomes was 40 min, which increased to 200 min with 4% mol PEG and to approximately 1000 min with 10% mol PEG. The degree of binding of modified immunoliposomes at the target sites was also dependent on the concentration of PEG incorporated at the liposome surface. This study demonstrates the accumulation of long-circulating targeted liposomes at the area of acute rabbit experimental myocardial infarction.