Loss of myocytes due to apoptosis occurs in patients with end-stage cardiomyopathy and may contribute to progressive myocardial dysfunction.

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.

We performed radionuclide scanning after the intravenous injection of human IgG labeled with indium-111 in 128 patients with suspected focal sites of inflammation. Localization of 111In-labeled IgG correlated with clinical findings in 51 infected patients (21 with abdominal or pelvic infections, 11 with intravascular infections, 7 with pulmonary infections, and 12 with skeletal infections). Infecting organisms included gram-positive bacteria, gram-negative bacteria, Pneumocystis carinii, Mycoplasma pneumoniae, and Candida albicans. No focal localization of 111In-labeled IgG was observed in 63 patients without infection. There were five false negative results, and nine results were unusable. Serial scans were carried out in eight patients: continued localization correctly predicted relapse in six, and the absence of localization indicated resolution in two. To determine whether 111In-labeled IgG localization was specific for inflammation, we studied 16 patients with cancer. Focal localization occurred in 13 of these patients (5 with melanomas, 5 with gynecologic cancers, and 1 each with lymphoma, prostate cancer, and malignant fibrous histiocytoma). No localization was seen in patients with renal or colon cancer or metastatic medullary carcinoma of the thyroid. We conclude that 111In-labeled IgG imaging is effective for the detection of focal infection and that serial scans may be useful in assessing therapeutic efficacy. This technique may also be helpful in the evaluation of certain cancers.