Prevention of Antibody-Mediated Elimination of Ligand- Targeted Liposomes by Using Poly(Ethylene glycol)-Modified Lipids WAI MING LI, LAWRENCE D. MAYER, and MARCEL B. BALLY Department of Pathology and Laboratory Medicine (W.M.L., M.B.B.) and Faculty of Pharmaceutical Sciences (L.D.M.), University of British Columbia, Vancouver, British Columbia, Canada; and Department of Advanced Therapeutics, British Columbia Cancer Agency, Vancouver, British Columbia, Canada (W.M.L., M.B.B., L.D.M.) Received October 4, 2001; accepted November 29, 2001 This article is available online at http://jpet.aspetjournals.org ABSTRACT One of the major obstacles in the development of ligand- targeted liposomes is poor liposome circulation longevity as a result of antibody-mediated elimination of these highly immu- nogenic carriers. Because studies from our laboratory suggest that it is not possible to reduce the immunogenicity of ligand- conjugated liposomes by using surface-grafted poly(ethylene glycol) (PEG), we investigated the usefulness of PEG in protect- ing hapten-conjugated liposomes from elimination by an exist- ing immune response that was previously established against the hapten. Using biotin as a model hapten, a strong biotin- specific antibody response was generated in mice by using bovine serum albumin-biotin. When these animals were chal- lenged with liposomes containing biotin-conjugated lipid (1 or 0.1%), these liposomes were rapidly eliminated. Incorporation of PEG-lipids into these liposomes substantially reduced biotin- specific antibody binding as measured using an in vitro anti- body consumption assay. However, depending on the hapten concentration, significant reductions in antibody binding through the use of PEG-lipids may not be sufficient to protect these liposomes from rapid elimination in vivo. Complete pro- tection of liposomes was only achieved when the biotin con- centration on liposome surface was low (0.1%) and with 5 mol% of either 1,2-distearoyl-sn-glycero-3-phosphoethano- lamine-n-[methoxy(polyethylene glycol)-2000] or 1,2-dipalma- toyl-sn-glycero-3-phosphoethanolamine-n-methoxy(polyethyl- ene glycol)-2000]. The use of 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000] (up to 15 mol%) was not effective in protecting liposomes from rapid elimination in vivo, indicating the limited usefulness of this highly exchangeable PEG-lipid. In conclusion, our in vivo and in vitro data indicate that liposomes can be protected from anti- body-mediated elimination by using the right type and concen- tration of PEG-lipids. This result has important implication in the development of ligand-targeted liposomes. Active targeting can be achieved by conjugating macromol- ecules such as antibodies, peptides, and ligands of natural receptors onto liposome surfaces to improve the specificity of these drug carriers to disease sites (Vingerhoeds et al., 1994). Although active targeting of liposomes has met with some success both in vitro and in vivo (Park et al., 1995; Kirpotin et al., 1997; Gabizon et al., 1999), further development of ligand-targeted liposomes for in vivo use remains a challenge due to the immunogenicity of the drug carriers bearing sur- face ligands that function as antigenic haptens (Phillips and Emili, 1991; Phillips et al., 1994; Phillips and Dahman, 1995). Repeated administration of these liposomes becomes problematic because the pharmacokinetic and biodistribu- tion behaviors of the carrier change after subsequent injec- tions of the drug carrier (Shek and Heath, 1983; Phillips and Emili, 1991; Phillips et al., 1994; Harding et al., 1997; Tardi et al., 1997; Dams et al., 2000). Enhanced elimination of the liposomes is due to the generation of a humoral response and immunoglobulin binding to the liposomes in the plasma com- partment. We and others have shown that surface-grafted poly(ethylene glycol) cannot reduce the immunogenicity of these liposomes but can enhance the immune response to targeting molecules bound to the surface of liposomes (Phil- lips and Dahman, 1995; Li et al., 2001a) or to the terminal moiety of the grafted PEG (Harding et al., 1997). However, it This study was supported by a grant from the Canadian Institute of Health Research. ABBREVIATIONS: PEG, poly(ethylene glycol); BSA, bovine serum albumin; biotin-X-DSPE (Bx-DSPE), N-(((6-biotinoyl)amino)hexanoyl)-1,2- distearoyl-sn-glycero-3-phosphoethanolamine; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; DSPE-PEG 2000 , 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000]; DMPE-PEG 2000 , 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy- (polyethylene glycol)-2000]; DPPE-PEG 2000 , 1,2-dipalmatoyl-sn-glycero-3-phosphoethanolamine-n-methoxy(polyethylene glycol)-2000]; DSPE- PEG 5000 , 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethylene glycol) 5000]; Chol, cholesterol; ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance; 1% Bx-lipo, DSPC/Chol liposomes containing 1% biotin. 0022-3565/02/3003-976 –983$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 300, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4614/967388 JPET 300:976–983, 2002 Printed in U.S.A. 976 at ASPET Journals on April 5, 2017 jpet.aspetjournals.org Downloaded from