Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A

Abstract

Many applications envisioned for liposomes in cell biology and chemotherapy require their direction to specific cellular targets. The ability to use antibody as a means of conferring specificity to liposomes would markedly increase their usefulness. We report here a method for covalently coupling soluble proteins, including monoclonal antibody and Staphylococcus aureus protein A (ref. 4), to small sonicated liposomes, by using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pharmacia). Liposomes bearing covalently coupled mouse monoclonal antibody against human beta 2-microglobulin [antibody B1.1G6 (IgG2a, kappa) (B. Malissen et al., in preparation)] bound specifically to human, but not to mouse cells. Liposomes bearing protein A became bound to human cells previously incubated with the B1.1G6 antibody, but not to cells incubated without antibody. The coupling method results in efficient binding of protein to the liposomes without aggregation and without denaturation of the coupled ligand; at least 60% of liposomes bound functional protein. Further, liposomes did not leak encapsulated carboxyfluorescein (CF) as a consequence of the reaction.

Differential endocytosis of T and B lymphocyte surface molecules evaluated with antibody-bearing fluorescent liposomes containing methotrexate.

“…Our method to couple monoclonal antibodies covalently to liposomes (7) has enabled us to extend this evaluation to multiple cell surface molecules and the various cell types expressing them. This study investigated separated mitogen-stimulated T and B cells from mouse spleen and evaluated the fate of class I (H-2Kk) and class II (H-2 I-Ak and I-Ek) major histocompatibility complex (MHC)-encoded determinants and of a recently described lymphoid determinant of Mr 94,000 and 180,000 implicated in the proliferation and function of T cells (p94,180) (9).…”

Detection of very low receptor numbers on cells by flow cytometry using a sensitive staining method

“…They were then chromatographed on a sepharose 4B column (1,6,9). The percentage of protein A coupled to liposomes was 8% and 2% €or SUVs and LUVs, respectively, corresponding to a n average of 5 and 31 molecules of protein A per liposome of 80 nm and 400 nm diameter.…”

“…H-2Dk, which was completely undetectable by FITC-conjugated protein A, gave a very strong signal using SW-conjugated protein A and an even stronger signal with LUV-conjugated protein A, Using this methodology as few as 500 molecules, or perhaps even fewer, may be detected without difficulty. SUVs (80 nm mean diameter) containing 20 mM recrystalized CF (13) were prepared by sonication at 50°C as described before (1,6,20). L W S were prepared by reverse-phase evaporation as described previously (9,17).…”

Immobilization of α‐chymotrypsin on sucrose stearate‐palmitate containing liposomes

“…Two different ways of protein immobilization on liposomes are known: (1) chemical modification of the protein with hydrophobic anchors with its subsequent incorporation into liposome membrane [3-51; (2) chemical coupling of protein with activated liposomes. In the second case the reactive phospholipid derivatives are used most often [6,7]. However, any other membrane-compatible reactive compound, possessing a hydrophobic moiety, can be used for protein immobilization instead of phospholipids.…”