Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced
blood clearance of a subsequent dose of PEGylated liposomes
XinYu Wang, Tatsuhiro Ishida, Hiroshi Kiwada ⁎
Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences,
The University of Tokushima, 1–78–1, Sho-machi, Tokushima 770–8505, Japan
Received 24 July 2006; accepted 20 February 2007
Available online 24 February 2007
Abstract
Earlier we reported that PEGylated liposomes lose their long-circulating characteristic when they are administrated twice in the same animal
with certain intervals (referred to as the accelerated blood clearance (ABC) phenomenon). We proposed that anti-PEG IgM, induced by the
PEGylated liposomes, is responsible for the phenomenon, based on the observation that IgM thus produced selectively binds to the surface of
PEGylated liposomes, subsequently leading to substantial complement activation. Interestingly, we found that under certain circumstances
administration of conventional liposomes without PEG-coating also caused a strong ABC response upon injection of a second dose of PEGylated
liposomes, but not of conventional liposomes. This suggests that also conventional liposomes not modified with PEG can promote an IgM
response against PEG. We report here that, irrespective of the presence or absence PEG-coating, a single first dose of liposomes is capable of
inducing a strong anti-PEG IgM response and, under certain circumstances, also weak responses against other lipid components. A good
correspondence was observed between the amount of IgM associating with both PEGylated and conventional liposomes, concomitant complement
activation triggered by those liposomes and the magnitude of the ABC phenomenon against those liposomes. Taken together, our results
demonstrate that the ABC phenomenon is fully attributable to production of anti-PEG IgM by the first dose of liposomes and the subsequent
complement activation upon a second dose of PEGylated but not conventional liposomes. Although the responsible immunogenic epitopes of the
liposomes remain to be determined, the immunogenicity of ‘empty’ liposomes presents a serious concern in the development of liposomal
formulations and their use in the clinic. Furthermore, our findings as described here raise important concerns with regard to the safety and
efficiency of liposomes currently under development for clinical use.
© 2007 Elsevier B.V. All rights reserved.
Keywords: IgM; Polyethylene glycol (PEG); Accelerated blood clearance (ABC) phenomenon; Liposomes; Repeated injection; Complement system
1. Introduction
Liposomes which are sterically stabilized with surface-
coupled polyethylene glycol (PEG) can enhance their lifetime
and that of entrapped therapeutic agents in the blood circulation
[1–3]. It is hypothesized that the presence of PEG on the
liposome attracts a water shell to the liposomal surface, pro-
viding a steric barrier against opsonins and/or recognition by
cells of mononuclear phagocyte system (MPS) [4–6]. This, in
turn, results in a decrease in the elimination rate of liposomes
from the blood stream.
We and others have found that an intravenous injection of
PEGylated liposomes causes a second dose of PEGylated lipo-
somes, injected a few days later, to lose their long-circulating
Journal of Controlled Release 119 (2007) 236–244
www.elsevier.com/locate/jconrel
Abbreviations: AUC, the area under the blood concentration–time curve;
ABC, accelerated blood clearance; CHOL, cholesterol; 3H-CHE, 3H-cholester-
ylhexadecyl ether; DGVB2+, VBS containing 0.15mMCaCl2, 1 mMMgCl2, 5%;
D-glucose and 0.1%; gelatin; DHPE, dihexadecanoylglycerophosphoethanola-
mine; HEPC, hydrogenated egg phosphatidylcholine; HRP, horseradish perox-
idase; mPEG2000-DSPE,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-
[methoxy(polyethylene glycol)-2000]; MLV, multilamellar vesicles; MPS,
mononuclear phagocyte system; PEG, polyethylene glycol; PL, phospholipid;
SRBC, sheep red blood cells sensitizedwithmonoclonal anti-sheep IgMantibody;
TI antigen, a T-cell independent antigen or thymus-independent antigen; TI-1,
type-1 TI antigen; TI-2, type-2 TI antigen; VBS, veronal-buffered saline.
⁎ Corresponding author. Tel.: +81 88 633 7259; fax: +81 88 633 9506.
E-mail address: hkiwada@ph.tokushima-u.ac.jp (H. Kiwada).
0168-3659/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2007.02.010

characteristics and accumulate extensively in liver, despite the
presence of PEG on the surface of the liposomes [7–10]. This
phenomenon is referred to as the “accelerated blood clearance
(ABC) phenomenon”. Although the details of underlying mecha-
nism are not yet elucidated, we proposed the following tentative
mechanism for the induction of theABCphenomenon on the basis
of our earlier results [11–13]: anti-PEG IgM, produced in the
spleen in response to an injected dose of PEGylated liposomes,
selectively binds to the PEG on a second dose of these liposomes,
injected several days later, and subsequently activates the com-
plement system. This, in turn, leads to opsonization of the lipo-
somes byC3 fragments and, as a consequence, to enhanced uptake
of the liposomes by the Kupffer cells in liver.
Earlier reports from our laboratory [11–14] indicate that
‘empty’ PEGylated liposomes are immunogenic and promote
antibody, especially IgM, responses against a second dose of such
liposomes. This suggests that any PEGylated liposomal formu-
lation, even if containing non-immunostimulatory payloads such
as cytotoxic agents [14], may display unexpected pharmacoki-
netic behavior upon repeated injection and, as a consequence,
may show less therapeutic efficacy or even cause undesirable side
effects. Therefore, a strategy to abrogate the immunogenicity of
PEGylated liposomeswithout significantly compromising their in
vivo performance would be highly desirable for the further
development of this otherwise promising drug delivery system.
Therefore, studies providing further insight in the mechanisms
underlying the ABC phenomenon are of great importance.
We have shown that anti-PEG IgM is responsible for the
induction of the ABC phenomenon in rats [12,13]. Similar results
had been reported. Judge et al. [15] showed that anti-PEG anti-
bodies were induced after injection of plasmid DNA-containing
PEGylated liposome in mice. Srode et al. [16] showed that anti-
PEG IgG was induced in response to the first injection of empty
PEGylated liposome in rabbits. Both reports demonstrated that
the induction of anti-PEG antibodies causes accelerated blood
clearance of subsequently injected PEGylated liposomes. How-
ever, a clear-cut relationship between the amount of anti-PEG
antibodies associated with PEGylated liposomes and the degree
of induced ABC phenomenon has not been established. In addi-
tion, our earlier studies revealed that, at higher lipid dose (5 μmol
phospholipid (PL)/kg), even conventional liposomes (without
PEG-coating) can induce enhanced clearance of a subsequently
injected dose of PEGylated, but not conventional, liposomes [17].
This raised the question if anti-PEG IgM is also produced
following injection of conventional liposomes, causing acceler-
ated clearance of a subsequent dose of PEGylated liposomes. In
the present study we therefore addressed these issues. Our find-
ings raise important concerns regarding the safety and efficiency
of PEGylated liposomes currently in use or under consideration
for clinical application.
2. Materials and methods
2.1. Materials and animals
Hydrogenated egg phosphatidylcholine (HEPC), 1,2-dis-
tearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(poly-
ethylene glycol)-2000] (mPEG2000-DSPE) and DSPE were
generously donated by Nippon Oil and Fat (Tokyo, Japan).
Cholesterol (CHOL) was of analytical grade (Wako Pure
Chemical, Osaka, Japan). All lipids were used without further
purification. Sepharose 4 Fast Flow was purchased from
Amersham-Pharmacia Biotech (Upsala, Sweden). Rhodamine-
derivatized dihexadecanoylglycerophosphoethanolamine
(DHPE) was purchased from Molecular Probes (OR, USA).
3H-Cholesterylhexadecyl ether (3H-CHE) was purchased from
NEN Research Products (MA, USA). All other reagents were of
analytical grade.
Male Wistar rats weighing 250–300 g were purchased from
Japan SLC (Shizuoka, Japan). Upon arrival, the rats were allowed
to acclimatize for at least 1 week. They had free access to water
and rat chow, and were housed under controlled environmental
conditions (constant temperature, humidity, and a 12-h dark–light
cycle). All animal experiments were evaluated and approved by
the Animal and Ethics Review Committee of The University of
Tokushima.
2.2. Preparation of liposomes
PEGylated liposomes were composed of HEPC:CHOL:
mPEG2000-DSPE (1.85:1.0:0.15 molar ratio). Conventional
liposomes were composed of HEPC:CHOL (2:1 molar ratio).
Liposomes were prepared as previously described [10]. Briefly,
the lipids were dissolved in chloroform and, after evaporation of
the organic solvent, the resulting lipid film was hydrated in
HEPES buffered saline (25 mM HEPES, 140 mM NaCl, pH 7.4)
to producemultilamellar vesicles (MLV). TheMLVwere sized by
repeated extrusion through polycarbonate membrane filters
(Nuclepore, CA., USA) with consecutive pore sizes of 400
(×3), 200 (×3), 100 (×5) and 80 (×3) nm. The mean diameters of
the prepared liposomeswere determined by using aNICOMP370
HPL submicron particle analyzer (Particle Sizing System, CA,
USA). The mean diameters for the PEGylated liposomes and
conventional liposomes were 115±11 nm (n=5) and 109±13 nm
(n=5), respectively. The concentration of phospholipid was
determined by colorimetric assay [18]. To follow the biodistribu-
tion of the second dose of liposomes, the liposomes were labeled
with a trace amount of 3H-CHE (40 μCi/μmol lipid) as a
nonexchangeable lipid phase marker. To collect liposome
fractions following gel chromatography, the liposomes were
labeled with a trace amount of rhodamine-labeled DHPE.
2.3. Biodistribution and pharmacokinetics of second (test) dose
liposomes
For the first injection, the liposomes (PEGylated or con-
ventional), at a dose of 5 or 0.001 μmol PL/kg, was injected via
the femoral vein under ether anesthesia and sterile treatment.
Control animals received HEPES buffered saline instead of
liposomes. In all experiments, to determine the biodistribution
of subsequently injected test liposomes (PEGylated or conven-
tional), pre-treated rats were cannulated via the left femoral vein
(PE-20, Natsume, Tokyo, Japan) and artery (PE-50) under ether
anesthesia and sterile treatment. Then, 3H-CHE-labeled test
237X.Y. Wang et al. / Journal of Controlled Release 119 (2007) 236–244