0022-3565/05/3123-1020 –1026$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics
JPET 312:1020–1026, 2005
Vol. 312, No. 3
78113/1191589
Printed in U.S.A.
Immunogenicity and Rapid Blood Clearance of Liposomes
Containing Polyethylene Glycol-Lipid Conjugates and
Nucleic Acid
Sean C. Semple, Troy O. Harasym,1 Kathy A. Clow,2 Steven M. Ansell, Sandra K. Klimuk,
and Michael J. Hope
Inex Pharmaceuticals Corporation, Burnaby, British Columbia, Canada
Received September 21, 2004; accepted October 29, 2004
DNA- and RNA-based therapeutics have tremendous potential for exquisite selectivity in the management of disease,
yet these agents have several properties that restrict their
application as therapeutic agents, including nuclease sensitivity, rapid plasma elimination, poor intracellular delivery,
and hemodynamic toxicities (Levin, 1999; Wang et al., 2003).
This has prompted significant research into the development
of delivery technologies for this class of drugs. Long-circulating, sterically stabilized liposomes (SSL) containing amphipathic polyethylene glycol (PEG) have been used extensively
over the past decade to improve the circulation lifetime of
lipid vesicles and entrapped therapeutic agents, provide a
1
Current address: Celator Technologies Inc., Vancouver, BC, Canada.
Current address: Memorial University of Newfoundland, St John’s, NF,
Canada.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.104.078113.
2
effect has been observed in several strains of mice and was
independent of sequence motifs, such as immunostimulatory
CpG motifs. The ODN-to-lipid ratio and ODN dose was also
determined to be important, with abrogation of the response
occurring at a ratio between 0.04 and 0.08 (w/w). Rapid elimination of liposome-encapsulated ODN from blood depends on the
presence of PEG-lipid in the membrane because the use of nonpegylated liposomes or liposomes containing rapidly exchangeable PEG-lipid also abrogated the response. These studies have
important implications for the evaluation and therapeutic use of
liposomal formulations of nucleic acid, as well as the potential
development of liposomal vaccines.
steric barrier against interactions with plasma proteins and
opsonins, improve disease-site delivery of therapeutic and
diagnostic agents, and reduce liposome uptake by mononuclear cells of the reticuloendothelial system (Allen and Hansen, 1991; Senior et al., 1991; Boerman et al., 1997; Lasic et
al., 1999). In this regard, SSL have been used in oncology
(Goren and Gabizon, 1998; Lasic et al., 1999), antimicrobial
(Bakker-Woudenberg and van Etten, 1998), and radiodiagnostic applications (Boerman et al., 1997; Goins et al., 1998).
As such, this type of delivery system is a natural candidate to
enhance the pharmacokinetic and pharmacodynamic properties of DNA- and RNA-based therapeutics (Lasic et al., 1999).
In the absence of encapsulated or surface-coupled proteins,
SSL and other nonviral lipid delivery systems are generally
considered to be nonimmunogenic (van Rooijen and van
Nieuwmegen, 1980; Alving, 1992; Harding et al., 1997). The
immunogenicity and rapid plasma elimination of protein-
ABBREVIATIONS: SSL, sterically stabilized liposomes; PEG, polyethylene glycol; ODN, oligodeoxynucleotide(s); SCID, severe-combined
immunodeficient; DSPC, distearoylphosphatidylcholine; DSPE, distearoylphosphatidylethanolamine; DODAP, 1,2-dioleoyl-3-N,N-dimethylammonium-propane; CH, cholesterol; biotin X-DSPE, N-[((6-biotinoyl)amino)hexanoyl]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine;
biotin-PEG2000-DSPE, N-[␻-biotinoylamino (polyethylene glycol)2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine; PEG-CerC14, 1-O(2⬘-(␻-methoxypolyethyleneglycol)succinoyl)-2-N-myristoylsphingosine; PEG-CerC20, 1-O-[2⬘-(␻-methoxypolyethyleneglycol)succinoyl]-2N-arachidoylsphingosine; CHE, cholesteryl hexadecyl ether; ICAM, intercellular adhesion molecule; PO, phosphodiester; PS, phosphorothioate; SALP, stabilized antisense-lipid particle(s); ELISA, enzyme-linked immunosorbent assay; PK, pharmacokinetic(s).
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ABSTRACT
Polyethylene glycol (PEG) is used widely in the pharmaceutical
industry to improve the pharmacokinetics and reduce the immunogenicity of therapeutic and diagnostic agents. The incorporation of lipid-conjugated PEG into liposomal drug delivery systems
greatly enhances the circulation times of liposomes by providing a
protective, steric barrier against interactions with plasma proteins
and cells. Here we report that liposome compositions containing
PEG-lipid derivatives and encapsulated antisense oligodeoxynucleotide (ODN) or plasmid DNA elicit a strong immune response that results in the rapid blood clearance of subsequent
doses in mice. The magnitude of this response is sufficient to
induce significant morbidity and, in some instances, mortality. This
Rapid Blood Clearance of Pegylated Liposomes Containing DNA
Materials and Methods
Mice. Female 7- to 8-week-old ICR, C57BL/6, and BALB/c mice
were obtained from Harlan (Indianapolis, IN). BALB/c nu/nu and
BALB/c SCID-Rag2 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained under pathogen-free conditions. All animals were quarantined for at least 1 week prior to use.
All procedures involving animals were performed in accordance with
the guidelines established by the Canadian Council on Animal Care.
Chemicals and Lipids. Distearoylphosphatidylcholine (DSPC),
polyethylene glycol-conjugated distearoylphosphatidylethanolamine
(PEG2000-DSPE) and 1,2-dioleoyl-3-N,N-dimethylammoniumpropane
(DODAP) were purchased from Avanti Polar Lipids (Alabaster, AL).
Cholesterol (CH) was purchased from Sigma-Aldrich (St. Louis, MO).
Biotin-X-DSPE and biotin-PEG2000-DSPE were purchased from Northern Lipids (Vancouver, BC, Canada). PEG-CerC14 and PEG-CerC20
were synthesized and purified as described previously (Wheeler et al.,
1999). [3H]Cholesteryl hexadecyl ether (CHE) was obtained from
PerkinElmer Life and Analytical Sciences (Boston, MA).
ODN and Plasmid DNA. The designations and 5⬘ to 3⬘ sequences of
the ODN used in these studies were as follows: hICAM, GCCCAAGCTGGCATCCGTCA (3⬘-untranslated region of human ICAM-1 mRNA);
mICAM, TGCATCCCCCAGGCCACCAT (3⬘-untranslated region of
murine ICAM-1 mRNA); EGFR, CCGTGGTCATGCTCC (human epidermal growth factor mRNA, receptor-translation termination codon
region); c-myc, TAACGTTGAGGGGCAT (initiation codon region of human/mouse c-myc proto-oncogene mRNA; and c-mycC, TAAGCATACGGGGTGT (c-myc scrambled control). Phosphodiester (PO) and
phosphorothioate (PS) ODN were purchased from Hybridon Specialty
Products (Milford, MA). The backbone composition was confirmed by
31
P NMR. All ODN were analyzed for endotoxin by the manufacturer
and contained less than 0.05 endotoxin units/mg. Plasmid DNA (pCM-
VLuc) was produced in Escherichia coli, isolated, and purified as described previously (Wheeler et al., 1999).
Encapsulation of ODN. Stabilized antisense-lipid particles
(SALP) composed of DSPC/CH/DODAP/PEG-CerC14 or PEG-CerC20
(molar ratio, 25:45:20:10) and encapsulated PS ODN were prepared
as described previously (Semple et al., 2000). For PS ODN, 300 mM
citrate buffer was used to dissolve the antisense, whereas 20 mM
citrate, pH 4.0 was used for PO ODN and plasmid formulations. The
lower citrate concentration was required to facilitate efficient charge
interactions between DODAP and the PO ODN or plasmid, resulting
in optimal encapsulation efficiencies (Stuart et al., 2004). At higher
citrate concentrations, these charge interactions are presumably
shielded, and significant reductions in encapsulation efficiencies are
observed. Phosphorothioate-modified ODN bind strongly to cationic
molecules, and encapsulation efficiencies are much less sensitive to
differences in buffer and salt concentrations (Stuart et al., 2004).
Plasmid formulations were not extruded, resulting in ⬃200-nm particles. DSPC/CH (molar ratio, 55:45) and DSPC/CH/PEG2000-DSPE
(molar ratio, 50:45:5) vesicles were prepared from dry lipid films by
aqueous hydration in HBS (20 mM Hepes and 145 mM NaCl, pH
7.4). Similarly, ODN encapsulation was achieved by hydration of 100
mg of lipid with 100 mg of ODN in 1.0 ml HBS, followed by five cycles
of freeze-thawing and extrusion through two stacked 100-nm filters
(Semple et al., 2000). The resulting particles were approximately 110
to 140 nm in diameter, as judged by quasi-elastic light scattering
using a model 370 NICOMP submicron particle sizer (Particle Sizing
Systems, Santa Barbara, CA). [3H]CHE, a nonexchangeable, nonmetabolizable lipid marker, was incorporated into all vesicle compositions to monitor lipid levels in the blood (Stein et al., 1980).
Liposome Elimination from the Circulation. Mice received a
single intravenous (lateral tail vein) dose of empty liposomes (50
mg/kg lipid) or liposome-encapsulated ODN (50 mg/kg lipid and 10
mg/kg ODN, unless otherwise specified) containing [3H]CHE (⬃1
␮Ci/mouse). Dosing occurred weekly unless otherwise noted. Blood
(25 ␮l) was collected 1 h postinjection by tail nicking unanesthetized
mice, using a sterile scalpel (tails were wiped with 70% ethanol prior
to nicking), and placed in 200 ␮l of 5% EDTA. The blood was then
digested (SOLVABLE, PerkinElmer Life and Analytical Sciences),
decolorized and analyzed for radioactivity according to the manufacturer’s instructions. The tail nicking procedure allowed all repeatinjection data to be collected from the same group of animals. A
comparison of this procedure with blood (⬃0.5 ml) collected weekly
by terminal cardiac puncture on anesthetized (ketamine/xylazine)
mice produced equivalent results.
ELISA. Groups of mice (n ⫽ 20 initially) were injected weekly
(i.v.) with SALP (PEG-CerC20) containing c-myc ODN or empty liposomes of the same lipid composition. One week after each injection,
a subgroup of animals (n ⫽ 5) was anesthetized (ketamine/xylazine),
blood was collected from each animal by cardiac puncture (⬃0.5 ml),
and the animals were subsequently euthanized. Plasma was collected for each animal after centrifuging the blood for 15 min at
1200g in a refrigerated centrifuge (4°C). Individual plasma samples
were pooled (n ⫽ 5), and serial dilutions were analyzed by ELISA as
described below. The results are expressed for the 1:800 plasma
dilution, which was the lowest dilution that exhibited minimal nonspecific binding to control wells.
The presence of liposome-reactive antibody was evaluated by
ELISA using biotinylated liposomes bound to streptavidin-coated
microplates. Liposome formulations bound to microtiter plates included: DSPC/CH/DODAP/PEG-CerC20/biotin-PEG2000-DSPE (molar ratio, 24.5:45:25:5:0.5), DSPC/CH/biotin-X-DSPE (molar ratio,
54.5:45:0.5), and DSPC/CH/PEG2000-DSPE/biotin-PEG2000-DSPE
(molar ratio, 49.5:45:5:0.5). Biotinylated liposomes (10 nmol of total
lipid/well) were incubated overnight at 4°C in clear SILENUS
streptavidin-coated microwell plates (Chemicon International, Temecula, CA). Plates were washed, blocked with phosphate-buffered
saline containing 3% bovine serum albumin and 0.1% Tween 20, and
subsequently incubated with serial dilutions of plasma from PEG-
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coupled liposomal systems after repeat injections have been
well documented (Aragnol and Leserman, 1986; Phillips and
Emili, 1991; Phillips and Dahman, 1995; Harding et al.,
1997; Tardi et al., 1997) and typically result from antibodymediated recognition of protein. T cell-independent antibody
responses to liposomal glycolipid antigens such as ganglioside GM1 have also been described (Freimer et al., 1993).
These responses were characterized by elevations in antibody production primarily of the IgM class, with antibody
recognition directed against the carbohydrate portion of the
ganglioside.
Nonmethylated bacterial DNA and synthetic oligodeoxynucleotides (ODN) can stimulate mononuclear cells and
lymphocytes in vitro and in vivo, resulting in the secretion of
interleukin-6, interleukin-12, interferon-␥, and IgM (Krieg,
2002). This effect has been primarily attributed to CpG and
palindromic sequence motifs, but phosphorothioate ODN all
exhibit some degree of immune stimulation in vivo (Monteith
et al., 1997). Given the relative inefficiency of nucleic acidbased therapeutics and the likely requirement for frequent
administration, we evaluated the potential immunogenicity
and circulation properties of SSL containing synthetic ODN,
plasmid DNA, or ribozyme on repeated injections. Liposome
elimination from the circulation was used as a convenient
indicator of immunogenicity in vivo. The results presented in
this study indicate that the presence of encapsulated nucleic
acid in lipid vesicles containing surface-associated PEG stimulates an immune response against the carrier, irrespective
of sequence motifs or DNA chemistry, and induces morbidity
and rapid plasma elimination of subsequent administrations.
The data further demonstrate that this response is directed
specifically against the PEG-lipid.
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Semple et al.
liposome (containing entrapped ODN)-treated animals for 1 h at
room temperature. Following multiple washes with phosphate-buffered saline/0.1% Tween 20, IgM binding was evaluated by incubation
with horseradish peroxidase-conjugated rat anti-mouse IgM monoclonal antibody (1:1000 dilution; BD Biosciences PharMingen, San
Diego, CA). The plates were washed and subsequently incubated
with 3,3⬘,5,5⬘-tetramethylbenzidine substrate (Sigma-Aldrich) and
read at 450 nm.
Results
Fig. 1. Circulation levels of PEG liposomes on repeat administrations in
immune-competent BALB/c mice (a), and immune-compromised BALB/c
nude (b) and BALB/c SCID-Rag2 mice (c). Mice were injected i.v. with
empty DSPC/CH/PEG2000-DSPE liposomes, DSPC/CH/PEG2000-DSPE liposomes containing hICAM PS ODN, empty SALP (PEG-CerC20), or
SALP (PEG-CerC20) containing hICAM ODN. Lipid doses were 50 mg/kg.
The ODN/lipid ratios for the DSPC/CH/PEG2000-DSPE and SALP (PEGCerC20) were 0.058 and 0.20, respectively. Injections were administered
weekly, and the circulation levels at 1 h postinjection were monitored by
the lipid label [3H]CHE. The bars represent the first (open bars), second
(back slash), third (forward slash), and fourth (cross-hatched) injection.
All bars represent the mean and standard deviation of eight mice.
immunostimulatory and/or nonantisense effects (Stein and
Krieg, 1997), the influence of oligonucleotide sequence was
evaluated by encapsulating a variety of PS ODN in SALP
(PEG-CerC20). Interestingly, all PS ODN encapsulated in
SALP (PEG-CerC20) induced morbidity in mice and were
rapidly removed from the circulation on repeat administrations (Fig. 2a). This was also observed for ODN that did not
contain CpG or G-quartet motifs (mICAM) or contained CpG
only (hICAM and EGFR), CpG and G-quartet (c-myc), or
G-quartet only (c-mycC).
Having demonstrated sequence independence of the rapid
elimination phenomenon, the dependence on nucleic acid
chemistry and structure was evaluated. PS or PO ODN,
ribozyme, or plasmid DNA was encapsulated in the SALP
(PEG-CerC20) lipid formulation, and the circulation levels at
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Blood Levels of PEG Liposomes Containing Encapsulated ODN. We have recently developed and characterized a novel lipid formulation that can encapsulate antisense
ODN in the aqueous space of lipid vesicles with high encapsulation efficiency and ODN-to-lipid ratios (Semple et al.,
2000). This formulation contains a PEG-ceramide lipid coating that is required for the formulation step, and we have
therefore called these vesicles SALP. Previous studies have
shown that liposomes containing PEG covalently coupled to
ceramide derivatives with 20 carbon acyl chains, a relatively
nonexchangeable lipid anchor, have circulation profiles indistinguishable from the same liposome compositions containing PEG2000-DSPE, the most commonly used PEG-lipid
anchor (Harding et al., 1997; Woodle, 1998; Semple et al.,
2000). Typically, these formulations are long-circulating,
with 75 to 95% and 30 to 40% of the administered dose
expected to remain in the circulation at 1 and 24 h, respectively. Thus, we initially compared the repeat dosing pharmacokinetics (PK) of SALP to various lipid formulations of
antisense ODN, including traditional long-circulating, SSL,
and DSPC/CH liposomes.
For liposomes and lipid-based delivery systems, monitoring the circulation properties of repeated injections is a convenient surrogate measure of carrier immunogenicity since
this parameter is invariably altered by an immune response
(Aragnol and Leserman, 1986; Phillips and Emili, 1991;
Harding et al., 1997; Tardi et al., 1997; Goins et al., 1998). To
evaluate the impact of repeated administrations on the circulation times of PEG liposomes containing hICAM ODN,
the blood levels of DSPC/CH/PEG2000-DSPE liposomes or
SALP (PEG-CerC20) were evaluated 1 h postinjection after
weekly intravenous administrations. As expected, no differences in elimination were observed for empty vesicles over
several administrations (Fig. 1a). Surprisingly, rapid elimination (⬍20% of the injected dose remained in the blood at 1 h) of
ODN-containing vesicles was observed following the second and
subsequent injections. This effect was accompanied by pronounced morbidity and, in some instances, resulted in death of
the animal within 30 min postinjection. This rapid elimination
phenomenon with PEG liposomes and ODN was observed in
several strains of mice, including outbred ICR mice and inbred
BALB/c and C57BL/6 strains (results not shown). To determine
whether an immune component was involved in this response,
these same studies were performed in T cell-deficient BALB/c
nude mice and B and T cell-deficient BALB/c SCID-Rag2 mice.
A rapid elimination response was observed in BALB/c nude
mice (Fig. 1b) but not in BALB/c SCID-Rag2 mice (Fig. 1c),
suggesting that the response depends on the presence of B cells
and immunoglobulin.
Influence of Nucleic Acid Composition on Clearance
of PEG Liposomes. Since it has been shown that PS ODN
containing CpG or G-quartet sequence motifs can exhibit
Rapid Blood Clearance of Pegylated Liposomes Containing DNA
1 h postinjection were monitored after weekly injections (Fig.
2b). Rapid elimination of the carrier was observed on the
second and subsequent injections, indicating that closed circular, double-stranded DNA was as effective at inducing the
rapid elimination as ODN with free 5⬘ and 3⬘ ends. Similarly,
the more nuclease-sensitive PO ODN also induced an immunogenic response, as did ribozyme (results not shown).
Impact of Antisense/Lipid Ratio and Dose Schedules.
In Fig. 1, the magnitude of blood clearance for DSPC/CH/
PEG2000-DSPE vesicles containing encapsulated ODN was
less pronounced than for SALP (PEG-CerC20). The major
difference between these two formulations was the encapsulation procedure and the resulting ODN-to-lipid ratio. To test
whether the amount of encapsulated ODN influences the
generation of the immune response, ODN was encapsulated
in SALP (PEG-CerC20) at different ODN/lipid ratios, and the
circulating level of vesicles was monitored after weekly injections of 50 mg/kg lipid. Interestingly, at ODN/lipid ratios
less than 0.04 (w/w), no apparent changes in elimination
were observed, whereas marked decreases in circulation levels were observed at ratios greater than 0.08 (w/w) (Fig. 3a).
Since the lipid dose was constant in these studies, the ODNto-lipid ratio and/or the total ODN dose administered significantly impact the initiation of this response.
Fig. 3. Factors influencing the onset of rapid clearance of PEG liposomes
containing nucleic acid. The influence of the DNA-to-lipid ratio (a) and
dosing schedule (b) were evaluated in ICR mice. For the DNA-to-lipid
ratio study, mice were injected weekly (i.v.) with SALP (PEG-CerC20)
containing hICAM PS ODN at various ODN/lipid ratios. The bars and
numbers of animals are indicated in the legend to Fig. 1. In the dose
schedule study, mice were injected i.v. with SALP (PEG-CerC20) containing hICAM PS ODN at various dosing schedules: daily (E), every 2 days
(F), every 3 days (䡺), and weekly (f). In both studies, the lipid dose was
adjusted to 50 mg/kg/dose, and circulation levels at 1 h postinjection were
monitored by the lipid label [3H]CHE.
Standard dosing schedules for antisense ODN therapies
typically involve repeated injections or infusions. The relevance of the dosing schedule on circulating levels of SALP
(PEG-CerC20) was examined by injecting mice daily or every
2, 3, or 7 days. Liposome levels in the blood were evaluated
1 h after each injection (Fig. 3b). For daily injections, the
plasma levels of circulating carrier increased over the first
three injections, which was not surprising given that 30 to
40% of a given dose of PEG-coated liposomes remains in the
circulation 24 h postinjection (Allen and Hansen, 1991; Harding et al., 1997; Woodle, 1998; Semple et al., 2000); however,
this increase was followed by a dramatic decline in the circulation levels of subsequent doses. In all dosing schedules,
rapid elimination of subsequent doses was observed beginning 4 to 6 days after the initial dose and was maintained for
at least 50 days.
PEG-Lipid Involvement in the Response. To evaluate
the importance of PEG-lipid on the generation of the immune
response observed in Fig. 1, PS ODN was encapsulated in
100-nm DSPC/CH vesicles or in SALP containing PEGCerC14. The CerC14 lipid anchor has shorter acyl chains than
PEG-CerC20 and exchanges more rapidly out of the lipid
bilayer (t1/2, ⬃1.1 h in vitro) (Wheeler et al., 1999). No differences in the circulation levels of these vesicles, whether
empty or containing encapsulated ODN, were observed on
repeated administrations (Fig. 4a). This was in striking contrast to SALP (PEG-CerC20), which was rapidly eliminated
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Fig. 2. Influence of nucleic acid sequence (a) and structure (b) on blood
clearance of SALP (PEG-CerC20). ICR mice were injected i.v. with SALP
(PEG-CerC20) containing PS ODN of various nucleotide sequences (a). PO
hICAM ODN and bacterial plasmid DNA were also evaluated (b). The
lipid dose was adjusted to 50 mg/kg, and the ODN/lipid ratio for each
formulation was ⬃0.20. Injections were administered weekly, and the
circulation levels at 1 h postinjection were monitored by the lipid label
[3H]CHE. The bars and numbers of animals are indicated in the legend to
Fig. 1.
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Semple et al.
after a second injection. PEG-CerC14 exchanged out of the
carrier immediately after injection, with greater than 50%
loss of PEG-lipid in approximately 3 min (Fig. 4b). This same
level was not achieved for PEG-CerC20 until 24 h. Since
neither empty DSPC/CH vesicles nor SALP containing rapidly exchangeable PEG-lipid exhibited any differences in circulation levels on repeat administrations, we conclude that
the presence of PEG-lipid in the external monolayer of the
vesicles was critical for the rapid elimination of the carrier
from the circulation.
The role of PEG-lipid was further examined in crossover
studies in which mice were sensitized with weekly injections
(total of four) of SALP containing PEG-CerC20 and encapsulated ODN. This was followed with a fifth injection of empty
vesicles of varying compositions. Crossover injections of
empty DSPC/CH/PEG-CerC20 or DSPC/CH/PEG2000-DSPE
vesicles were rapidly eliminated from the circulation,
whereas crossover injections of DSPC/CH vesicles or SALP
containing PEG-CerC14 exhibited prolonged circulation
times (Fig. 5). The rapid clearance of DSPC/CH/PEG2000DSPE vesicles from the circulation indicates that the PEG
moiety, and not the lipid anchor, was the critical component
recognized in this response. Similarly, the use of empty vesicles that had never been exposed to ODN indicates that
potential residual surface-associated ODN was not responsible for mediating elimination. Similar results were observed
in crossover studies in which, instead of SALP (PEG-CerC20),
mice were pretreated with DSPC/CH/PEG2000-DSPE liposomes containing encapsulated ODN (results not shown).
In a final set of studies designed to evaluate the presence of
Fig. 5. Crossover studies in presensitized mice. Mice were injected i.v.
with SALP (PEG-CerC20) for a total of four weekly injections. In a subsequent, final injection, mice received either SALP (PEG-CerC20), empty
SALP (PEG-CerC20), empty SALP (PEG-CerC14), empty DSPC/CH/
PEG2000-DSPE vesicles, or empty DSPC/CH liposomes. In each instance,
the lipid dose was 50 mg/kg/dose, and the circulation levels at 1 h
postinjection were monitored by the lipid label [3H]CHE. Each bar represents the mean and standard deviation of eight mice.
liposome-reactive antibody in the blood, mice were treated
four times (weekly injections) with SALP (PEG-CerC20) or
empty DSPC/CH/DODAP/PEG-CerC20 liposomes, and the
pooled plasma of these animals was examined by ELISA
using various biotinylated liposomes bound to streptavidincoated microplates. When the pooled plasma from each group
of mice was incubated with plates containing DSPC/CH/
DODAP/PEG-CerC20 or DSPC/CH/PEG2000-DSPE, increases
in liposome-reactive IgM were observed 1 week following the
first injection and generally increased over the four injections
(Fig. 6). However, when the same plasma was incubated in
plates containing DSPC/CH (i.e., no PEG-lipid), very minimal IgM binding was observed, indicating the involvement of
antibody in the clearance of PEG-liposomes containing ODN,
as well as providing additional confirmation that the response is directed against PEG and not the other lipids
comprising the formulation. The plasma from mice treated
with empty PEG liposomes showed negligible reactivity in
any of the plates.
Discussion
The majority of efficacy studies that use antisense oligonucleotides have required repeated dosing to observe biological activity in animal models. This is principally necessitated by the rapid plasma elimination of polyanionic DNA
and minimal antisense ODN delivery to the disease site. To
increase the disease site delivery of antisense ODN, we chose
to evaluate pegylated lipid-based formulations of antisense
ODN. Interestingly, we found that repeated injections of
PEG liposomes containing ODN, plasmid DNA, or ribozyme
induced a potent immunogenic response that resulted in both
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Fig. 4. Role of PEG-lipid in the rapid elimination of liposomes containing
ODN. Panel a, mice injected i.v. with empty SALP (PEG-CerC20) (a),
SALP (PEG-CerC20) (b), empty SALP (PEG-CerC14) (c), SALP (PEGCerC14) (d), empty DSPC/CH liposomes (e), or DSPC/CH containing hICAM PS ODN [ODN-to-lipid ratio, 0.081, (w/w)] (f). The lipid dose was
adjusted to 50 mg/kg/dose, and the circulation levels at 1 h postinjection
were monitored by the lipid label [3H]CHE. The bars and numbers of
animals are indicated in the legend to Fig. 1. Panel b, time course for
exchange of PEG-CerC20 (F) and PEG-CerC14 (E) evaluated by monitoring the ratio of [3H]PEG-ceramide to [14C]CHE in the plasma of mice over
24 h. The symbols represent the mean and standard deviation of six mice.
Rapid Blood Clearance of Pegylated Liposomes Containing DNA
the removal of the carrier from the circulation and pronounced morbidity in mice.
An immune component of this response was verified using
immune-compromised BALB/c nude mice, where rapid blood
clearance was observed, and in BALB/c SCID-Rag2 mice, in
which normal blood clearance was observed on repeat dosing.
The rapid elimination phenomenon and adverse clinical
signs in mice were not observed when ODN was encapsulated
in PEG-free liposomes or in liposomes containing rapidly
exchangeable PEG-CerC14. Several previous studies have
demonstrated an apparent absence of immunogenicity or
altered PK upon repeated administration of empty PEG liposomes (Harding et al., 1997; Woodle, 1998; Oussoren and
Storm, 1999), PEG liposomes containing technetium-99m
(Goins et al., 1998), or encapsulated doxorubicin (Tardi et al.,
1997). Although humoral immune responses against liposomes have been described for protein-coupled PEG liposomes, these responses are directed against the surface-
bound protein (Phillips and Emili, 1991; Phillips and
Dahman, 1995; Harding et al., 1997; Tardi et al., 1997).
Recently, a limited number of studies have demonstrated
changes in the PK of repeated injections of PEG liposomes
under certain time and dosing conditions in rats and a rhesus
monkey (Dams et al., 2000; Laverman et al., 2001). Enhanced
blood clearance of repeated doses of technetium-99m-PEG
liposomes occurred when the second injection was administered between 5 days and 4 weeks after the initial injection
(Goins et al., 1998; Oussoren and Storm, 1999; Dams et al.,
2000). Moreover, PK changes were only observed when the
doses were relatively low (⬍15 ␮mol/kg) (Laverman et al.,
2001). Ishida et al. (2003) have demonstrated the changes in
the PK behavior of SSL in mice when a second dose (25
␮mol/kg phospholipid) was given between 7 and 14 days after
the initial dose. Our results differ from that study in that our
injected lipid doses were much higher (⬃65–72 ␮mol/kg) and
the magnitude of the clearance response was much greater;
moreover, pronounced morbidity was observed on repeat injections of SSL containing high levels of DNA, which has not
been reported previously. Dams et al. (2000) have suggested
that a 150-kDa nonantibody-soluble serum factor mediates
the rapid clearance phenomenon in rats and monkeys,
whereas our data in immunocompromised mice, as well as
the IgM studies, strongly suggest an antibody response.
Whereas the mechanism of the immune response and the cell
populations that facilitate it have not yet been fully elucidated,
the studies involving nude and SCID-Rag2 mice suggest that B
cells and immunoglobulin, and not T cells, are critical. Immunecompetent and nude mice that were injected repeatedly with
PEG liposomes containing ODN showed signs of morbidity and
difficulty bleeding and, in extreme cases, exhibited rapid
breathing and seizures. These symptoms are typical of a shock
response and could be generated through a number of mechanisms. The delivery of bacterial DNA and synthetic oligonucleotides to macrophages and monocytes can result in acute cytokine release and tumor necrosis factor-␣-mediated lethal shock
in mice (Sparwasser et al., 1997). In our study, clinical signs
only developed with repeat injections and were associated with
rapid removal of the carrier from the blood, suggesting a humoral response. IgM responses typically occur 3 to 7 days after
antigen exposure, which was consistent with the onset of the
immunogenic response observed here (Fig. 3b). In this regard,
increases in PEG-reactive plasma IgM were observed in mice
treated repeatedly with PEG liposomes containing ODN. IgMantibodies against the glycolipid ganglioside GM1 are common
in patients with neuropathy and have been shown to result in
strong complement-mediated lysis of GM1-containing liposomes
(Mitzutamari et al., 1998). The generation of antibody and
putative complement activation are a likely explanation for the
rapid elimination of the vesicles from the blood. IgG, IgM, and
iC3b are prominent opsonins associated with liposome formulations that exhibit rapid plasma clearance (Chonn et al., 1992).
That the presence of encapsulated ODN in PEG liposomes
could potentially stimulate the production of antibodies is perhaps not surprising. Some phosphorothioate antisense ODN
and bacterial DNA have been shown to have strong mitogenic
properties in vitro, resulting in lymphocyte proliferation and
production of IgM (Krieg, 2002). These same ODN produced
splenomegaly and increased cellularity/activation in vivo (Monteith et al., 1997). That the presence of encapsulated DNA could
stimulate the production of antibody to the PEG-lipid is of
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 31, 2017
Fig. 6. IgM binding to pegylated liposomes. Groups of mice (n ⫽ 5) were
injected weekly (i.v.) with SALP (PEG-CerC20) containing c-myc ODN or
empty liposomes of the same lipid composition. One week after each
injection, mice were bled, plasma was collected and pooled, and serial
dilutions were subsequently analyzed on different microtiter plates as
described under Materials and Methods. Each plate contained a different
liposome formulation (indicated in each panel) bound to the wells through
biotin-streptavidin interactions. The results are expressed for the 1:800
plasma dilution. The results for mice injected with empty liposomes are
presented for the fourth and final injection.
1025
1026
Semple et al.
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Address correspondence to: Sean C. Semple, Inex Pharmaceuticals Corporation, 100-8900 Glenlyon Parkway, Burnaby, BC, Canada, V5J 5J8. E-mail:
[email protected]
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considerable interest in this study. As evidenced by a lack of
detectable elimination or IgM directed against empty PEG liposomes in mice upon repeated administration, PEG-lipid itself
is a very weak antigen (Harding et al., 1997) and is used widely
in the pharmaceutical industry to improve the PK and reduce
the immunogenicity of various therapeutic or diagnostic agents
(Harris et al., 2001). Antibody responses against PEG have
been described in mice and rabbits when PEG was coupled to
certain protein antigens; however, the responses were relatively
minor unless the PEG or PEG-protein conjugate was coadministered with a strong adjuvant such as Freund’s complete adjuvant (Richter and Akerblom, 1983; Richter and Akerblom, 1984;
Cheng et al., 1999). ODN containing CpG motifs have been
shown to act as potent adjuvants for the production of antibody
to coadministered T cell-dependent protein antigens (Weiner et
al., 1997). We did not observe any sequence or chemistry dependence when DNA or RNA was encapsulated in the carrier,
suggesting that the adjuvant effect is a general phenomenon of
nucleic acid encapsulation in lipid vesicles. Thus, the presence
of liposome encapsulated ODN or DNA may function as a novel
adjuvant system for the generation of immune responses to
weak T cell-independent antigens, such as carbohydrates and
glycolipids.
The studies reported herein have significant implications
for the use of SSL in the therapeutic evaluation of DNAbased drugs. Nonviral, lipid-based delivery systems are generally thought to be nonimmunogenic with respect to the
components of the carrier, but to our knowledge, no studies
have actually addressed this with DNA-based delivery systems. Several recent studies have demonstrated that innate
and adaptive immune responses can be enhanced through
encapsulation of immunostimulatory DNA in lipid-based delivery vehicles (Li et al., 2001; Mui et al., 2001; Whitmore et
al., 2001), increasing the importance of evaluating carrierdirected effects. SSL are known to have an enhanced ability
to accumulate at sites of infection, inflammation, and tumors
(Boerman et al., 1997; Woodle, 1998; Lasic et al., 1999; Semple
et al., 2000). However, based on the results observed in Fig. 3b,
the accumulation of later injections in sites of disease will be
significantly impaired within days. Finally, we have demonstrated that the removal of PEG-lipid from liposomes containing encapsulated DNA abrogates the immunogenic response,
thereby indicating that SSL need to be used with caution for
applications involving DNA- and RNA-based therapeutics.