International Journal of Pharmaceutics 392 (2010) 218–223 Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm Pharmaceutical Nanotechnology T cell-independent B cell response is responsible for ABC phenomenon induced by repeated injection of PEGylated liposomes Hiroyuki Koide a , Tomohiro Asai a , Kentaro Hatanaka a , Shuji Akai b , Takayuki Ishii a , Eriya Kenjo a , Tatsuhiro Ishida c , Hiroshi Kiwada c , Hideo Tsukada d , Naoto Oku a,∗ a Department of Medical Biochemistry and Global COE Program, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan b Department of Synthetic Organic Chemistry, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan c Department of Pharmacokinetics and Biopharmaceutics, Institute of Health Biosciences, The University of Tokushima, 1-78-1, Sho-machi, Tokushima 770-8505, Japan d Central Research Laboratory, Hamamatsu Photonics K.K., Hamamatsu, Shizuoka, Japan article info Article history: Received 9 December 2009 Received in revised form 8 February 2010 Accepted 8 March 2010 Available online 21 March 2010 Keywords: Polyethylene glycol Liposome Accelerated blood clearance Thymus-independent type 2 antigen Nanocarriers abstract Repeated injection of polyethyleneglycol-modified (PEGylated) liposomes causes a rapid clearance of them from the bloodstream, this phenomenon is called accelerated blood clearance (ABC). In the present study, we focused on the immune system responsible for the ABC phenomenon. PEGylated liposomes were preadministered to BALB/c mice and [ 3 H]-labeled ones were then administered to them 3 days after the preadministration. Consistent with our previous results, the preadministration with PEGylated liposomes triggered the rapid clearance of [ 3 H]-labeled PEGylated liposomes from the bloodstream, but that with PEGylated liposomes encapsulating doxorubicin (Dox) did not. In addition, we found that the ABC phenomenon was observed when a mixture of free Dox and PEGylated liposomes was preadminis- tered. These data indicate that immune cells responsible for the ABC phenomenon might be selectively damaged by the Dox encapsulated in PEGylated liposomes. The ABC phenomenon was also observed in BALB/c nu/nu mice, but not in BALB/c SCID mice. The amount of anti-PEG IgM antibody induced by the stimulation with the PEGylated liposomes was significantly increased in the BALB/c nu/nu mice, but not in the BALB/c SCID ones. These data indicate that a T cell-independent B cell response would play a sig- nificant role in the ABC phenomenon. Furthermore, the present study suggests that PEGylated liposomes might be recognized by B cells as a thymus-independent type 2 (TI-2) antigen. The present study provides important information for the future development of liposomal medicines. © 2010 Elsevier B.V. All rights reserved. 1. Introduction PEGylated liposomes have been widely investigated as drug car- riers and gene delivery systems. PEG forms a water shell on the liposomal surface and provides a steric barrier to the liposomes for avoiding interactions with plasma proteins, resulting in escape from trapping by the reticuloendothelial system (Lasic et al., 1991; Torchilin et al., 1994; Van Rooijen and Van Nieuwmegen, 1980). Therefore, PEGylated liposomes have the property of long circu- lation and are useful for drug delivery to tumors and inflamed sites, resulting in improving the therapeutic indices of encapsu- lated drugs (Allen, 1994). As a representative example of liposomal Abbreviations: ABC phenomenon, accelerated blood clearance phenomenon; [ 3 H]-CHE, [ 3 H] cholesterylhexadecyl ether; MPEG-DSPE, 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000]; MPS, mononu- clear phagocyte system; PEGylated liposomes, polyethylene glycol-modified liposomes. ∗ Corresponding author. Tel.: +81 54 264 5701; fax: +81 54 264 5705. E-mail address: oku@u-shizuoka-ken.ac.jp (N. Oku). drugs, Doxil ® has been used clinically. Doxil ® is PEGylated lipo- somes encapsulating doxorubicin, which is used for reducing side effects of Dox such as cardiotoxicity and for enhancing its anti- cancer activity through enhanced permeability and retention (EPR) effect (Berry et al., 1998; Maeda et al., 2000; Muggia, 1999). On the other hand, we and others have found that a repeat injec- tion of PEGylated liposomes into certain animals such as mice, rats and rhesus monkey triggers the rapid clearance of the sec- ond dose through their accumulation in the liver (Dams et al., 2000; Ishida et al., 2003a,b). This phenomenon, called as the accel- erated blood clearance (ABC) phenomenon, is expected to have a considerable impact on the clinical use of liposomal formula- tions (Dams et al., 2000; Ishida et al., 2003a,b). A previous study of ours indicated that when rats were pretreated with a high dose (more than 5 mol phospholipids/kg) of PEGylated liposomes, the induction of the ABC phenomenon was weakened. However, when rats were pretreated with a low concentration of them (1 mol phospholipids/kg), the phenomenon was strongly induced (Ishida et al., 2006a,b,c). This phenomenon was widely observed even if the content of PEG lipid in liposomes or the length of the 0378-5173/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2010.03.022