Comparative studies of the preparation of immunoliposomes with the use of two bifunctional coupling agents and investigation of in vitro immunoliposome-target cell binding by cytofluorometry and electron microscopy.
The two coupling agents SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) and SATA (N-succinimidyl-S-acetylthioacetate) were compared in their efficiency and feasibility to couple monoclonal antibodies (Abs) via thioether linkage to liposomes functionalized by various lipophilic maleimide compounds like N-(3-maleimidopropionyl)-N2-palmitoyl-L-lysine methyl ester (MP-PL), N-(3-maleimidopropionyl)phosphatidylethanolamide (MP-PE), N6-(6-maleimidocaproyl)-N2-palmitoyl-L-lysine methyl ester (EMC-PL), and N-(6-maleimidocaproyl)phosphatidylethanolamine (EMC-PE). The composition of the liposomes was soy phosphatidylcholine (SPC), cholesterol, maleimide compounds and alpha-tocopherol (1:0.2:0.02:0.01, mol parts), plus N4-oleylcytosine arabinoside (NOAC) as cytostatic prodrug (0.2 mol parts) and a new, lipophilic and highly fluorescent dye N,N'-bis(1-hexylhfetyl)-3,4:9,10-perylenebis(dicarboximid ) (BHPD, 0.006 mol parts). From the maleimide derivatives MP-PL was the most effective in terms of preservation of the coupling activity in dependence of liposome storage. The coupling of the monoclonal A B8-24.3 (mouse IgG2b, MHC class I, anti H-2kb) and IB16-6 (rat IgG2a, anti B16 mouse melanoma) to the drug carrying liposomes was more effective and easier to accomplish with SATA as compared to SPDP. Coupling rates of 60-65% were obtained with SATA at molar ratios of 12 SATA:1 Ab:40 maleimide spacer groups on the surface of one liposome. The highest coupling rates with SPDP were obtained at the ratio of 24 SPDP:1 Ab:40 liposomal maleimide groups, with an Ab binding efficiency of only 20-25%. The optimal in vitro binding conditions to specific target cells (EL4 for B8-24.3-liposomes and B16-F10 for IB16-6-liposomes) were determined by cytofluorometric measurement of the liposomal BHPD fluorescence with SATA linked Abs. Optimal immunoliposome binding to specific epitopes on the target cells was achieved with 1-2 Ab molecules coupled to one liposome, with immunoliposome concentrations of 20-130 nM and with a small incubation volume of 0.3-0.4 ml. The specificity of the binding of B8-24.3-liposomes to EL4 target cells was visualized by scanning electron microscopy. Antibody mediated endocytic uptake of immunoliposomes could be demonstrated by transmission electron microscopy.
Schwendener
R A
RA
Department of Experimental Pathology, University Hospital, Zürich, Switzerland.
Trüb
T
T
Schott
H
H
Langhals
H
H
Barth
R F
RF
Groscurth
P
P
Hengartner
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents / Q000008:administration & dosage*; D003432:Cross-Linking Reagents; D003561:Cytarabine / Q000008:administration & dosage / Q000031:analogs & derivatives*; D004337:Drug Carriers; D005434:Flow Cytometry; D000922:Immunotoxins; D008081:Liposomes / Q000378:metabolism*; D008223:Lymphoma / Q000378:metabolism; D008301:Maleimides; D051379:Mice; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D011355:Prodrugs; D051381:Rats; D013388:Succinimides; D013440:Sulfides; D013601:T-Lymphocytes / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D004337:Drug Carriers; D000922:Immunotoxins; D008081:Liposomes; D008301:Maleimides; D011355:Prodrugs; D013388:Succinimides; D013440:Sulfides; D003561:Cytarabine; C065145:N(4)-oleylcytosine arabinoside; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; C039011:N-hydroxysuccinimide S-acetylthioacetate