Bioscience Reports, Vol. 22, No. 2, April 2002 ( 2002)
MINI REVIEW
Controlling the Physical Behavior and Biological Performance
of Liposome Formulations through Use of Surface
Grafted Poly(ethylene Glycol)
C. Allen,4,5 N. Dos Santos,2,3 R. Gallagher,1 G. N. C. Chiu,2,4 Y. Shu,1 W. M. Li,2,3
S. A. Johnstone,1 A. S. Janoff,1 L. D. Mayer,1,2,4 M. S. Webb,1 and M. B. Bally1,2,3
The presence of poly(ethylene glycol) (PEG) at the surface of a liposomal carrier has been
clearly shown to extend the circulation lifetime of the vehicle. To this point, the extended
circulation lifetime that the polymer affords has been attributed to the reduction or prevention of protein adsorption. However, there is little evidence that the presence of PEG at
the surface of a vehicle actually reduces total serum protein binding. In this review we
examine all aspects of PEG in order to gain a better understanding of how the polymer
fulfills its biological role. The physical and chemical properties of the polymer are explored
and compared to properties of other hydrophilic polymers. An evidence based assessment
of several in ûitro protein binding studies as well as in ûiûo pharmacokinetics studies involving PEG is included. The ability of PEG to prevent the self-aggregation of liposomes is
considered as a possible means by which it extends circulation longevity. Also, a ‘‘dysopsonization’’ phenomenon where PEG actually promotes binding of certain proteins that then
mask the vehicle is discussed.
KEY WORDS: Poly(ethylene glycol); liposome; drug delivery; protein adsorption;
dysopsonization.
INTRODUCTION
Due to its unique physical properties, polyethylene glycol (PEG), is commonly used
to improve the stability and biological performance of colloidal drug carriers. The
grafting of PEG to the surface of a colloidal carrier has been clearly shown to
extend the circulation lifetime of the vehicle. PEG’s ability to fulfill this role has
been attributed mostly to its physical properties such as unlimited water solubility,
large excluded volume and high degree of conformational entropy (Elbert and Hubbell, 1996; Lee et al., 1995). To this point, the link between the unique physical
1
Celator Technologies Inc., 200–604 West Broadway, Vancouver B.C., Canada V5Z 1G1.
Department of Advanced Therapeutics, British Columbia Cancer Agency, 600 West 10th Avenue, Vancouver, B.C., Canada V5Z 4E6.
3
Department of Pathology and Laboratory Medicine, Faculty of Medicine, The University of British
Columbia, Vancouver, B.C., Canada.
4
Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver,
B.C., Canada.
5
To whom correspondence should be addressed. Telephone: (604)-708-5858 ext 112; Fax: (604)-708-5883;
E-mail: [email protected]
225
2
0144-8463兾02兾0400-0000兾0  2002 Plenum Publishing Corporation
226
Allen et al.
properties of the polymer and the extended circulation lifetime that it affords has
been largely cited as the reduction or prevention of protein adsorption. PEG of a
certain molecular weight and graft density has been shown to prevent the adsorption
of selected specific proteins to a surface (Needham and Kim, 2000). However, there
is little evidence that the presence of PEG at the surface of a vehicle actually reduces
total serum protein binding. Others have shown that the steric barrier that PEG
provides prevents aggregation of colloidal carriers and thus enhances their stability
in ûiûo (Ahl et al., 1997). More recently, some groups have suggested a ‘‘dysopsonization’’ phenomenon where PEG actually promotes binding of certain proteins that
then act to mask the vehicle (Moghimi et al., 1993; Vert and Domurado, 2000). In
this same vein, attractive interactions between PEG and proteins have been reported
(Sheth and Leckband, 1997; Vert and Domurado, 2000).
The purpose of this review is to reconcile the biological phenomenon to the
chemical and physical properties of PEG in order to assist in the rational design and
application of PEG and other hydrophilic polymers for the development of effective,
systemically administered, drug carrier systems. We revisit the literature in order to
determine what has been clearly shown regarding PEG’s role in extending the circulation lifetime of carriers in ûiûo. In order to gain insight, findings from analysis
performed using theoretical models as well as work done in model in ûitro systems
and in ûiûo pharmacokinetics studies are considered. It should be noted that the role
of PEG in stabilizing lipid-based DNA-delivery vehicles have not been discussed
here. These systems are quite complex and require special consideration in the context of self-assembling systems and the orientation of PEG moieties within and on
the surface of these macromolecular structures.
UNIQUE SOLUTION PROPERTIES OF POLY(ETHYLENE GLYCOL)
Chemical and Physical Properties of PEG
In order to gain an understanding of the mechanism by which the presence of
PEG increases the circulation longevity of drug carriers we should consider the
physical and chemical properties of the polymer. As discussed below, the properties
of PEG are quite unique which explains why to date it remains unmatched as the
hydrophilic polymer of choice for extending the circulation lifetime of a carrier.
Polyethylene glycol (PEG) is a neutral, crystalline, thermoplastic polymer with
a high solubility in both water and organic solvents (Elbert and Hubbell, 1996; Lee
et al., 1995). At room temperature, its water solubility is said to be unlimited for all
degrees of polymerization (Lee et al., 1995). Under the same conditions, polymers
that are structurally related to PEG are water insoluble (e.g., poly(methylene oxide),
poly(trimethylene oxide) and polyacetaldehyde) (Lee et al., 1995). The unique high
degree of water solubility of PEG is believed to be due to its ‘‘good structural fit
with water’’ (Kjellander and Florin, 1981). The structure of water in the liquid state
is believed to approximate its solid state structure which consists of a tetrahedral
lattice of hydrogen bonded molecules. Kjellander and Florin have suggested that
PEG actually fits into the tetrahedral lattice of water facilitating hydrogen bonding
between the water molecules and the ether oxygens of PEG (Kjellander and Florin,
Physical Behavior and Biological Performance of Liposome
227
1981). Studies have also shown that water forms directional bonds with PEG such
that there is an ‘‘association of water molecules associated with a PEG chain’’
(Antonsen and Hoffman, 1992; Sofia et al., 1998). The water molecules associated
with PEG create a ‘‘hydration shell’’ where the water molecules are oriented in a
structured manner surrounding the polymer chain (Lee et al., 1995; Kjellander and
Florin, 1981). Lusse et al. used NMR to measure the water content of an ethylene
glycol chain and found that there is a maximum of one water molecule per ethylene
glycol unit (–CH2–CH2–O–) (Lusse and Arnold, 1996). On the other hand, Tirosh
et al. used a DSC technique to measure the number of water molecules bound per
chain of PEG 2000 when in the free state and grafted to the surface of micelles
(Tirosh et al., 1998). They found the number of water molecules bound per chain of
PEG 2000 to be 136J4 in the free state and 210J6 while in micelles, corresponding
to 3.1 to 4.8 waters per monomer unit, respectively.
Water is also considered to be a good solvent for PEG according to the polymer
solvent interaction parameter. The polymer solvent interaction parameter (χ ), also
known as the Flory Huggins interaction parameter, is an indication of the interaction between the polymer and the solvent and describes the state of the polymer coil
in solution (Stuart, 1998). As shown in Fig. 1, in a good solvent, χ F0.5, the polymer
coil swells due to its favorable interaction with the solvent; while, in a poor solvent,
χ H0.5, the polymer solvent interactions are weak and the polymer is collapsed. At
the theta point, where χ has a value of 0.5 the polymer is said to exist as a Gaussian
coil and behaves ideally. The value of χ for PEG in water has been found to range
between 0.4–0.5 depending on solution conditions (Lee et al., 1995, Steels et al.,
2000a). A miscible polymer solution will be found for χ values up to 0.5.
Fig. 1. A summary of the unique solution properties of poly(ethylene glycol).
A schematic diagram of the three possible states of a polymer coil in solution
(expanded, gaussion and collapsed) and a summary of the thermodynamic
parameters and solution properties of PEG in water (compiled from Elbert
et al. 1996; Brandrup et al., 1999, Lee et al., 1995). χ Gpolymer solvent interaction parameters, A2 Gsecond virial coefficient, Rf GFlory radius.
228
Allen et al.
Another parameter that is commonly used to describe a polymer in solution is
the second virial coefficient (A2) also referred to as the excluded volume parameter
(ν) and can be expressed as a function of (1–2χ ) (Lee et al., 1995, Stuart, 1998). The
value of A2 is a measure of both polymer–polymer interactions (between monomeric
units within a polymer chain) and polymer–solvent interactions. A positive value for
A2 is an indication of favorable polymer–solvent interactions or repulsive interactions between the monomeric units within the polymer chain (excluded volume or
electrostatic effects). A negative value for A2 indicates poor polymer–solvent interactions or attractive polymer–polymer interactions. At the theta point (χ G0.5) the
value of A2 is zero. For a neutral polymer such as PEG the vale of A2 is positive
owing to excluded volume effects. The values of χ and A2 may each be measured
experimentally using several different methods (Lee et al., 1995). As shown in Table I
the experimentally determined values of A2 for PEG are higher, in most cases, than
the values of A2 obtained for other hydrophilic polymers (Brandup et al., 1999).
In addition, the measured steric factor or stiffness parameter (σ ) for PEG in
water is reported to be 1.57 (at 25°C) which is close to 1; a value of 1 corresponds
to a freely rotating chain (Brandrup et al., 1999). The steric factor (σ ) is defined by
the following equation: σ G((〈r2〉o)兾(〈r2〉of))1兾2, where (〈r2〉o) is the ratio of the meansquare end-to-end distance of the polymer chain and (〈r2〉of) is the mean-square endto-end distance of a freely rotating chain (Lee et al., 1995). If a polymer chain is
sterically hindered, its rotation will be reduced resulting in a longer mean-square
end-to-end distance. Thus, more flexible chains have a steric factor that is quite close
to one. The steric factor for PEG is lower (1.57) than that reported for other hydrophilic polymers such as polyvinyl alcohol (PVA) σ G2.04, polyvinyl pyrrolidone
(PVP) σ G2.48 and polyacryl amide (PAAm) σ G2.36 (Brandrup et al., 1999; Lee et
al., 1995) (Table 1).
The experimentally determined high solubility of PEG in water along with its
high A2 value and association with water molecules suggest that in water PEG exists
as a highly hydrated polymer with a large excluded volume. The PEG chains are
also said to be in rapid motion in an aqueous medium owing to their high degree of
flexibility (Lee et al., 1995; Lang et al., 1979). In addition, the phase behavior of
PEG in water has been found to be quite complex (Sheth and Leckband, 1997). It
Table I. Parameters for Several Water Soluble Polymers in Water (adapted from Lee et al., 1995)
Water soluble parameters
χ
(Temp °C, [Ref.])
A2B104 (mol cm3兾g2)
σ
(MB10−3 g兾mole, Temp °C, [Ref.]) (Temp °C, [Ref.])
Poly(ethylene glycol) (PEG)
0.45 (27, [1]*)
0.44 (23, [1])
116–30.4
62
12
9.25
(10.9–800, 25, [2]*)
(10.1, 25, [2])
(177, 25 [2])
(300, 25, [2])
1.57 (25, [2])
Polyvinylpyrrolidone (PVP)
0.58 (25, [1])
0.49 (30, [1])
3.39
64.7–2.52
4.2
(11.6–75.4, 25, [2])
(19.5–933, 25, [2])
(310, 25, [2])
2.48 (25, [2])
Polyvinyl alcohol (PVA)
9.49 (30, [1])
3.9–5.2
(180–196, 30, [2])
2.04 (30, [2])
*Reference [1]GLee et al., 1995; [2]GBrandrup et al., 1999.
Physical Behavior and Biological Performance of Liposome
229
is not a simple structureless polymer rather it is known to take on higher order
intrachain configurations (Sheth and Leckband, 1997). Recent studies have shown
that there are different conformations that PEG may adopt in solution (Harder et
al., 1998). The helical (gauche–trans–gauche) or amorphous conformation was
found to be protein resistant whereas the ‘‘all-trans’’ conformation was not.
Now that we have outlined the chemical and physical properties of PEG, in
the section below, the physical attributes of PEG when grafted to a liposome are
considered.
Poly(ethylene glycol) Grafted to a Liposome Surface
When considering a liposome containing PEGylated lipids (in aqueous media),
it is believed that the PEG will extend away from the liposome into the solvent as
it is not attracted to the lipid bilayer (Carignano and Szleifer, 2000) and the aqueous
media is considered a ‘‘good solvent’’ for PEG. Since PEG is not attracted to the
lipid bilayer, the polymer provides kinetic rather than thermodynamic protection of
the surface (Carignano and Szleifer, 2000, Satulovsky et al., 2000). As shown in Fig.
2, deGennes has proposed that there are two regimes for polymers attached to a
surface depending on the graft density of the polymer (de Gennes, 1980). If the
density is low the polymer is said to be in the mushroom regime. The size of the
individual mushrooms are said to be Rf GaN3兾5 (where a is the size of the monomer,
which for PEG is 0.35 nm and Rf is equivalent to the radius of the polymer mushroom or half sphere). When the graft density is high the polymers are said to be in
Fig. 2. DSPC兾DSPE-PEG 2000 liposomes with <5, >5, >15 mol.% DSPEPEG-2000. A schematic of the two regimes (mushroom and brush) for polymers grafted to a surface as described by de Gennes, 1980). Rf GFlory radius,
LGextension length of polymer chain.
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Allen et al.
the brush regime. The molecular weight of the polymer as well as the graft density
will determine the degree of surface coverage and distance between graft sites.
The maximum number of PEGylated lipids (nsat) that may be incorporated into
a bilayer has been shown to depend on several parameters (Hristova and Needham,
1995). The phase behavior of several PEG-lipid兾lipid mixtures has been studied. It
has been established that at a high PEG-lipid content a micellar phase is favored
over the lamellar phase. The micelles are said to be formed as a means of decreasing
the pressure resulting from lateral interactions between PEG chains (Hristova et al.,
1995). In addition, the PEG-lipids can phase separate within a liposome such that
they are not evenly distributed over the entire surface of the carrier (Bedu-Addo et
al., 1996; Lehtonen and Kinnunen, 1995). Bedu-Addo et al. has suggested that
longer chain PEGs interact via van der Waals forces and interchain hydrogen bonding causing chain entanglement which leads to ‘‘PEG rich’’ and ‘‘PEG poor’’
domains (Bedu-Addo et al., 1996).
It should also be appreciated that the nature of the conjugation of the PEG
polymer to the lipid anchor may have an effect on the surface potential of the
resulting PEGylated liposomes. Traditional conjugation of PEG to phosphatidylethanolamine involves a carbamate linkage that results in a net negative charge
on the phosphate group of the PEG-PE at physiological pH. Comparison of the
electrophoretic mobility of liposomes containing 5 mol.% of PEG-PE, compared to
liposomes lacking a PEG-lipid but containing 5 mol.% of anionic phospholipids,
suggested that approximately 80% of the negative surface charge associated with the
PEG-PE was shielded by the PEG moiety (Webb et al., 1998).
Now that we have outlined the physical and chemical properties of PEG along
with its behavior when grafted to a liposome surface we may try to reconcile this
information to the biological phenomenon that have been attributed to incorporation of PEG lipids in a liposomal carrier.
Biological Phenomena: PEG’s Ability to Extend Circulation Lifetime
As shown in Fig. 3, the presence of PEG at the surface of liposomes has been
found to extend their circulation longevity regardless of surface charge or the
inclusion of the stabilizing component cholesterol. In cholesterol containing liposome systems the ability of PEG to increase the circulation lifetime of the vehicles
has been found to depend on both the amount of PEG incorporated and the length
Fig. 3. Elimination of liposomes from the circulation. Large unilamellar liposomes radiolabeled with [3H]
cholesterylhexadecyl ether (CHE) were administered intravenously via the dorsal tail vein of female Balb兾
c mice at an approximate dose of 150 µmoles兾kg total lipid (3A and 3B), 55 µmol兾kg total lipid (3C,
DSPC:CH:DOPS:DSPE-PEG2000 (30:45:10:15)) and 82 µmol兾kg total lipid (3C DSPC:CH:DOPS
(45:45:10)). Blood was collected at various time points by tail nick and cardiac puncture procedures. An
aliquot of plasma was used to determine liposomal lipid content. (A) Elimination of cholesterol containing liposomes (!) DSPC:CH:DSPE-PEG2000 (50:45:5), (䉫) DPSC:CH (55:45). (B) Elimination of cholesterol free liposomes (●) DSPC:DSPE-PEG2000 (95:5 molar ratio) (䊊)DSPC (100). (C) Elimination
of PS containing liposomes (■) DSPC:CH:DOPS:DSPE-PEG2000 (30:45:10:15) (䊐) DSPC:CH:DOPS
(45:45:10). Each data point represents the average lipid plasma concentrationJstandard deviation for
four mice.
Physical Behavior and Biological Performance of Liposome
231
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Allen et al.
or molecular weight of the polymer (Allen et al., 1991; Klibanov et al., 1990). In
most cases, the longer chain PEGs have allowed for the greatest improvements in
blood residence time. For example, Allen et al. reported that the blood levels were
higher for SM兾PC兾Chol兾DSPE-PEG liposomes with longer molecular weight PEG
(i.e., PEG 1900 and PEG 5000) than liposomes containing PEG-lipid with a shorter
chain PEG (i.e., PEG 750 and PEG 120) (Allen et al., 1991). The ability for PEG
to extend the circulation lifetime of liposomes has also been shown to be dependent
on the lipid anchor attached to the PEG (i.e., DSPE-PEG vs. Cholesterol-PEG)
(Yuda et al., 1996).
Recently, our group has studied the influence of PEG on the circulation longevity of cholesterol free liposomes. As shown in Fig. 4A and 4B, the percentage of
the original lipid dose remaining 24 hr post injection was 25 and 31% for the formulations containing 2 and 5 mol.% PEG 2000, respectively. For formulations containing 2 mol.% PEG 350, 550 or 750 the percentage of lipid remaining in the plasma
24 hr post injection was 10%. For formulations containing 5 mol.% PEG 350, 550
or 750 the percentage of lipid remaining in the plasma following 24 hr was 17% (of
the total lipid dose injected). The presence of PEG 2000 increased the amount of
lipid remaining in the plasma by two-fold when compared to formulations containing PEG 350, 550 or 750. It is surprising that the lipid level following 24 hr is the
same for formulations containing PEG 350 and PEG 750. This brings into question
the influence of the degree of protection provided by PEG polymers of different
molecular weights on the circulation longevity of the vehicle. At first glance one
would anticipate that a formulation containing 2 mol.% PEG 350 would have a
shorter circulation lifetime due to less surface protection than a formulation containing 2 mol.% PEG 750. As shown in Fig. 4, at 24 hr only 1% of the original dose of
the PEG free formulation (DSPC alone) remains in the circulation. How does the
surface of a liposome containing 2 mol.% PEG 350 differ from a PEG-free liposome?
In order to address this question we have calculated the area of protection of
PEG molecules of different molecular weights. From this we were able to calculate
the mole fraction of PEG-lipids in the area occupied by one polymer chain. The
calculations based on a simple approach previously established by Torchilin and
Papisov (Torchilin and Papisov, 1994) were performed as follows:
The mole fraction of PEG-lipid (χ p) in the area occupied by one polymer chain
(np G1 is)
χpG
np
npCnL
;
where np G1
where np is the moles of PEG-lipid and nL is the moles of lipid. nL may be calculated
if the surface area of the PEG chain (Ap) and the surface area of the polar lipid
headgroups (AL) are known.
nL G
Ap
AL
The surface area of the headgroup (AL ) of a DSPC lipid in the gel phase is
0.52 nm (Marsh, 1990). If we assume the polymers are in the mushroom regime, the
Physical Behavior and Biological Performance of Liposome
Fig. 4. Elimination of liposomes from the circulation. Large unilamellar liposomes
radiolabeled with [3H] cholesterylhexadecyl ether (CHE) were administered intravenously via the dorsal tail vein of female Balb兾c mice at an approximate dose of
3.3 µmoles兾mouse or 100 mg兾kg. Blood samples were taken at one and four hours post
liposome injection by tail nick, or by cardiac puncture. (A) Elimination of DSPC兾
DSPE-PEG 98兾2 liposomes (■) DPSC, (䉭) DSPE-PEG 350, (●) DSPE-PEG 550, (▼)
DSPE-PEG 750, (䊐) DSPE-PEG 2000. (B) Elimination of DSPC兾DSPE-PEG 95兾5
liposomes (■) DSPC (䉮) DSPE-PEG 350, (●) DSPE-PEG 550, (▼) DSPE-PEG 750,
(䊐) DSPE-PEG 2000.
233
234
Allen et al.
surface area protected by one PEG chain can be calculated from the Flory radius
(Rf) of the polymer. As defined by DeGennes, in the mushroom regime the polymer
‘‘occupies a half-sphere with a radius comparable to the Flory radius of a coil in a
good solvent’’ (deGennes, 1980):
Ap Gπ (Rf)2 ;
where Rf GaN 3兾5
where N is the number of ‘‘repeat’’ units in the chain (NGpolymer molecular
weight兾molecular weight of monomer (44 g兾mole)) and a is the monomer length
which is 0.35 nm for PEG. The values of Ap for PEG chains of different molecular
weights are included in Table 2A. If the total surface area of the liposome (Aliposome)
is known, the values of Ap may be used to calculate the total number of PEG molecules required to completely cover the surface of the liposome (NPEG total).
NPEG total G
Aliposome
Ap
For this calculation we assume the polymer chains are in the mushroom regime
and that the PEG lipid distributes itself evenly over the inner and outer leaflets of the
lipid membrane. The total surface area of a 100 nm, DSPC兾DSPE-PEG, liposome is
approximately 5.8B104 nm2.
The percentage of the total surface area of the liposome covered by PEG molecules in specific formulations may also be calculated knowing the total number of
lipid molecules in a liposome (Ntotal lipid).
Ntotal lipid G
4π r2û 4π (rûAT)2
C
AL
AL
where rû is the radius of the liposome vesicle and T is the thickness of the lipid
membrane (4 nm). The value for Ntotal lipid in a 100 nm, DSPC兾DSPE-PEG, gel-phase
liposome is approximately 1.1B105.
Table 2A. Calculated Mole Fraction of PEG-Lipid in Area Occupied by one PEG Chain of Different
Molecular Weights in DSPC兾DSPE-PEG Liposomes
PEG
molecular
weight
N*
Rf (nm)*
Protected area (nm)2兾
PEG molecule**
350
550
750
2000
8
13
17
45
1.22
1.63
1.92
3.44
4.68
8.35
11.58
37.18
Number PEG molecules
required to cover entire
surface
12,393
6946
5009
1560
χ p*
(×100)
10
6
4.3
1.4
*NGnumber of repeat units per polymer chain; Rf GFlory radius of PEG chain; χ p Gmole fraction of
PEG-lipid in area occupied by one PEG chain.
**Calculations of protected area are based on simple model described elsewhere (Torchilin et al., 1994).
Physical Behavior and Biological Performance of Liposome
235
Table 2B. The Percentage of Surface Area Covered in Liposome Formulations Containing 2 and 5 mol.%
PEG of Different Molecular Weights
Formulation
DSPC兾DSPE-PEG 350
DSPC兾DSPE-PEG 550
DSPC兾DSPE-PEG 750
DSPC兾DSPE-PEG 2000
Mole ratio
(%)
Total area of
liposome covered by
PEG (nm)2
Percentage of total surface
area of liposome covered
(%)
95兾5
98兾2
95兾5
98兾2
95兾5
98兾2
95兾5
98兾2
2.6B104
1.0B104
4.6B104
1.8B104
6.4B104
2.5B104
—
8.0B104
45
17
79
31
100
43
brush regime
100
Table 2B contains the calculated percentage of the total surface area of the
liposome protected by PEG in different formulations. As shown in the case of the
95兾5 formulations PEG 350 only covers 45% of the surface area of the liposome
while PEG 550 covers 79% and PEG 750 covers 100%. Yet despite the large differences in surface coverage the lipid level remaining in the plasma 24 hr post-injection
is the same for all three formulations. It is puzzling that liposomes with 55% of the
surface available (DSPC兾DSPE-PEG 350) for protein adsorption have a circulation
longevity equal to liposomes with less than 20% of the surface available (PEG 550
or PEG 750 formulations). Therefore this brings into question whether or not PEG
extends circulation lifetime by preventing protein adsorption.
To this point, all that has been shown definitively is that the inclusion of PEG
increases the circulation lifetime of both cholesterol-free and cholesterol-containing
liposomes. In most of the literature it is stated that PEG enhances the circulation
longevity of liposomes by reducing interactions with plasma proteins and cell-surface
proteins (Blume and Cevc, 1993). However, there has been no direct evidence that
PEG reduces or prevents plasma protein binding in ûiûo. In fact, in a recent study
our group found no correlation between the amount of protein bound to liposomes
in ûiûo and their circulation lifetime. In Fig. 5, there is no difference in the circulation
lifetime of three liposome formulations containing increasing amounts of PS
(DSPC兾DOPS兾DSPE-PEG; 10–50% DOPS) yet the amount of protein bound to
the liposomes ranges from 137–431 µg protein兾mg lipid. In this experiment, the
amount of protein bound to liposomes in ûiûo was measured by injecting Balb兾C
mice with liposomes at a dose of 100 mg兾kg, following one hour the mice were
sacrificed and the serum from four mice was pooled. The separation of liposome
bound protein from free protein was performed as described elsewhere (Johnstone
et al., 2001). These data, as well as other, clearly call to question the role of PEG in
mediating increases in liposome circulation longevity. In effect, graft density arguments and inhibition of serum protein binding arguments should be reconsidered in
light of these data.
There is another biological phenomenon attributed to surface grafted PEG;
observation of reduced uptake by phagocytic cell populations. There are however
conflicting reports regarding PEGs influence on the uptake of liposomes by cells of
236
Allen et al.
Fig. 5. Measure of protein binding and elimination of liposomes from the circulation.
Large unilamellar liposomes radiolabeled with [3H] cholesterylhexadecyl ether (CHE)
were administered intravenously via the dorsal tail vein of female Balb兾c mice at an
approximate dose of 3.3 µmoles兾mouse or 100 mg兾kg. Blood samples were taken at
one and four hours post liposome injection by tail nick, or by cardiac puncture. (●)
DSPC兾DOPS兾DSPE-PEG 2000 80兾10兾10, (䊊) DSPC兾DOP兾DSPE-PEG 2000 70兾20兾
10, (▼) DSPC兾DOPS兾DSPE-PEG 2000 40兾50兾10. The amount of protein bound (Pb)
to the liposomes in ûiûo was also measured.
the mononuclear phagocytic system (MPS). There have been several reports that the
inclusion of PEG reduces the MPS uptake of liposomes, resulting in a decreased
accumulation in the liver and spleen (Allen et al., 1991). However, others have
reported that although the presence of surface grafted PEG does increase the circulation longevity of the liposomes it does not alter the cumulative uptake by cells of
the MPS (Lasic et al., 1991; Patel, 1992, Mori et al., 1991). Also, it has been shown
that MPS blockade extends the circulation longevity of PEG-containing liposomes
(Parr et al., 1993).
To date a conclusive link has not been established between the chemical and
physical properties of PEG and its ability to extend circulation lifetime. Although,
the accepted opinion is that PEG increases circulation longevity of drug carriers by
reducing or preventing protein binding and兾or inhibition of cell binding兾uptake,
there is sufficient conflicting data to warrant a reassessment of the mechanism(s) by
which surface grafted PEGs provide improved liposome properties. The remaining
portions of this review will be focused on evidence based assessments of PEG
induced changes in surface properties. We believe, in brief, that grafted PEGs can
control the rate of selected protein interactions with reactive functions on the membrane surface and provide exquisite control over surface–surface interactions,
Physical Behavior and Biological Performance of Liposome
237
whether between two liposomes, a liposome and a large protein or a liposome and
a cell.
THE MECHANISMS BY WHICH PEG EXTENDS CIRCULATION
LIFETIME: INHIBITION OF SURFACE–SURFACE INTERACTIONS
The Prevention of Protein Adsorption
The adsorption or binding of protein to a surface may be via a non-specific or a
specific interaction. Non-specific adsorption includes protein binding to a neutral or
‘‘non-reactive’’ surface. Specific adsorption involves binding to a ‘‘reactive’’ surface.
We use the term ‘‘reactive’’ to refer to surfaces that contain both moieties that
promote high affinity binding such as hapten–ligand interactions (e.g., biotin-streptavidin) and charged groups that allow for electrostatic interactions. Many theoretical models have been developed to analyze the properties or behavior of non-ionic
hydrophilic polymers grafted or adsorbed to a material surface (de Gennes, 1980;
Halperin, 1999; Jeon and Andrade, 1991; Jeon et al., 1991; Szleifer, 1997; Steels et
al., 2000a, b; Satulovsky et al., 2000). Several of these models have been used to
characterize and predict polymer–protein interactions. Below, we briefly touch on
the theory in order to gain a better understanding of the in ûitro findings from
protein binding studies. Several in ûitro studies have measured the ability of PEG to
prevent non-specific and specific protein adsorption. Later, we discuss non-specific
protein binding and after we focus on specific protein adsorption to a liposome
surface.
Models for Analyzing the Protein Resistance of a Mushroom/Brush Polymer
Coated Surface
There are many models or theories that have been developed to analyze the
parameters that control the resistance of a polymer coated surface to non-specific
protein adsorption. These models aid in identifying the extent to which the properties of the polymer may be manipulated in the design of protein resistant polymer
coated surfaces. Steels et al. have analyzed the compression of polymer mushrooms
by a disc using a self-consistent field lattice model (Steels et al., 2000b). They assume,
as predicted by deGennes, that the individual polymer chains are present in a mushroom or half sphere with a radius equal to the Flory radius (Rf GaN3兾5) (de Gennes,
1980). As depicted in Fig. 6, analysis using their model predicts that upon compression a portion of the polymer chain will be ‘‘squeezed out’’ from beneath the
Fig. 6. The escape transition following protein compression of a polymer mushroom.
Based on Steels et al., 2000b.
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Allen et al.
disc. It is the increase in the conformational entropy of the escaped portion of the
polymer chain that provides the driving force for chain escape.
Several groups have also modeled the behavior of proteins at a polymer brushcoated surface. Halperin et al.,, describe the effective interaction potential between
a protein and a brush coated surface by the following equation Ueff (z)G
Ubrush(z)CUbare (z); where Ubrush(z) and Ubare(z) are defined as a purely repulsive and
a purely attractive interaction, respectively (Halperin, 1999). There are two potential
energy minima for Ueff(z); one at the external portion of the polymer brush (Uout ,
secondary minimum) and another at the surface of the material (Uin , primary minimum). For this reason, they describe two modes of adsorption; primary adsorption
at the material surface and secondary adsorption at the outer edge of the polymer
brush. The two energy minima are separated by a maximum (U*) which is the activation barrier for primary adsorption. There are various parameters that alter Uin ,
Uout , U* and thus Ueff (z) including the properties of the brush such as thickness and
graft density as well as properties of the protein such as size and shape. Their analysis finds that the mode of adsorption depends mostly on the value of the ratio of
the radius of the protein to the thickness of the polymer brush (R兾Lo). If the value
of R兾Lo is [1 then the protein will penetrate the brush causing only a minor perturbation or loss in conformational entropy to the polymer brush. Yet if R兾LoZ1
then it is more likely that the protein will engage in secondary adsorption remaining
at the outer edge of the brush. They find that it is the thickness of the
brush that controls secondary adsorption and the graft density that controls primary
adsorption.
This model and others highlight the key role that the conformational entropy
of the polymer plays in the ‘‘size dependent exclusion’’ of proteins (Halperin, 1999;
Jeon and Andrade, 1991; Szleifer, 1997; Steels et al., 2000a, b). The models are useful
in giving us an idealistic picture of the interactions between proteins and a polymer
coated surface. However, as authors outline, models are often based on various
assumptions including that the protein is a ‘‘structureless’’ sphere or cylinder and
that the polymer coat is composed of ‘‘normal’’ ‘‘simple’’ polymers (Halperin, 1999).
However, a polymer such as poly(ethylene glycol) is not a simple polymer. PEG is
complex in that it engages in strong hydrogen bonding interactions with water and
adopts higher order intrachain structure. It should also be noted that most models
developed consider the behavior of PEG attached at one end to a point that is fixed
in place. However, PEG that is linked to a lipid incorporated within a fluid-like
membrane is fixed to a mobile endpoint. This will affect its ability in controlling
protein adsorption and surface interactions.
Protein Adsorption to a Neutral Polymer Coated Surface
Many studies have shown that the presence of PEG at a material surface
reduces non-specific protein adsorption. In most studies the material surface is
exposed to protein(s) and the amount of protein adsorbed, following an incubation
period, is measured. These studies have revealed that there is an optimal molecular
weight and graft density of PEG that can reduce non-specific binding of a particular
protein to a material surface. The molecular weight and graft density of the PEG
Physical Behavior and Biological Performance of Liposome
239
polymer required to reduce non-specific adsorption has been shown to be dependent
on the physical properties of the protein such as size, shape and charge.
Recently, our group has studied the surface protective ability of various PEGs
using an established protein binding assay (Johnstone et al., 2001). In short, the
liposomes were incubated with mouse serum for one hour at 37°C (1:4 liposomes:
serum v兾v) and then separated from unbound serum protein using gel filtration
chromatography as described elsewhere (Johnstone et al., 2001). The fractions collected were analyzed for liposome as well as protein content. The peak liposome
containing fractions were pooled, the lipid was extracted and the samples were analyzed for protein content using the Micro BCA assay. For the DSPC兾DSPE-PEG
2000 formulations, 99.9兾0.1, 99兾1, 95兾5, 90兾10, the protein binding (Pb) values were
18, 18, 20, 17 µg protein兾mg lipid respectively. The data suggest that increasing the
amount of PEG incorporated into the liposome formulation does not significantly
reduce total serum protein binding. We believe that this in ûitro protein binding
assay is not measuring total protein bound but rather only the protein that is irreversibly bound.
In some cases the reduction in protein binding has been shown to be a ‘‘nonzero minimum’’ (Du et al., 1997, Efremova et al., 2000, Gref et al., 2000). For
example, Du et al. examined the adsorption of BSA, laminin and fibronectin to a
DPPC兾DSPE-PEG 5000 lipid coated glass surface (Du et al., 1997). They found
that the amount of protein adsorbed to the material surface could be reduced by
increasing the amount of PEG in the monolayer from 0 to 5 mol.%. The greatest
reduction in the total amount of protein bound was found when the mol.% PEG
was increased from 0 to 1 mol.%. A further increase in the PEG content from 1 to
5 mol.% only caused a slight decrease in the amount of protein bound. Complete
coverage of the lipid surface required the presence of only 0.7 mol.% DSPE-PEG
5000 in the mushroom regime. However, this amount of PEG was not sufficient to
completely block protein binding. At 5 mol.% DSPE-PEG 5000 the amount of protein adsorbed (expressed as number of molecules adsorbed兾cm2) was reduced to 21,
11, and 4% for BSA, laminin and fibronectin, respectively. The presence of the PEG
5000 at the surface appeared to be less effective at preventing the adsorption of BSA
compared to its effect on reducing laminin and fibronectin binding. The authors
suggested that this may be due to the fact that BSA is a smaller protein (mol. wt. G
66,000) than fibronectin (mol. wt.G220,000–250,000) and laminin (mol. wt.G
140,000). In this way, the BSA molecules, in comparison to fibronectin and laminin,
would be able to fit between the PEG molecules with less of a reduction in the
conformational entropy of the PEG polymer chains. Perhaps a higher density of a
shorter chain PEG would have been required to further reduce the amount of BSA
adsorbed to the material. Similarly, in a study by Efremova et al. the adsorption of
BPTI (globular protein, mol. wt. 6000), fibrinogen (rodlike protein, mol. wt. 340,000)
and HSA (mol. wt. 66,200) onto PEG-lipid (PEG 2000) containing lipid monolayers
were examined by surface plasmon resonance (Efremova et al., 2000). The surface
adsorption of all three proteins was found to decrease with an increase in PEG
content at the lipid surface; however, the PEG graft density required to suppress the
amount of protein bound varied for each protein. The graft density of PEG 2000
needed to reduce the binding of BPTI and HSA to a minimum was in agreement
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Allen et al.
with that predicted using a theoretical model, while the adsorption of fibrinogen to
the lipid surface was higher than expected especially at higher graft densities of PEG.
These studies highlight that grafted PEGs serve as an exclusion barrier, and, similar
to size exclusion chromatographic beads, the interactions of proteins with the surface
can be promoted or inhibited based on molecular weight (Steels et al., 2000a, b).
From these examples and many others (Steels et al., 2000a, b; Du et al., 1997;
Efremova et al., 2000; Gref et al., 2000; Harder et al., 1998; Sofia et al., 1998) we
can see that the non-specific adsorption of proteins to a PEG coated surface is
complex and often unpredictable. Also, many of these studies are performed under
ideal conditions where the PEG coated vehicle is incubated with only one type of
protein (specific molecular weight and shape). However, in ûiûo the carrier is exposed
to an entire soup of proteins at once and hence its ability to prevent binding or
adsorption of a protein with specific properties may be changed under these conditions. Perhaps more importantly, the surface properties of the PEG-lipid containing liposome may be quite different once exposed to serum protein. In fact, as will
be discussed later, the binding of proteins to liposomes with grafted PEGs may be
partly responsible for the biological properties of the carrier.
Prevention of Specific Protein Adsorption
The ability of a PEG layer to prevent specific protein–surface interactions may
be quite different from its ability to preclude non-specific protein–surface interactions. The nature of specific protein–surface interactions may supply energy required
to move proteins between the PEG chains (Chiu et al., 2001). Examples of this
concern the ability of PEG to prevent electrostatic interactions such as that found
in the phosphatidylserine-prothrombin system (Chiu et al., 2001; Chiu et al., 2002)
and the cardiolipin-Clq system (Bradley et al., 1998; Bradley et al., 1999) as well
as hapten–ligand interactions such as the biotin-avidin system (Noppl-Simson and
Needham, 1996; Needham and Kim, 2000; Li et al., 2002).
Our laboratory investigated the ability of PEG-lipids to protect liposomes containing a reactive surface lipid such as the negatively charged phospholipid, phosphatidylserine (PS) (Chiu et al., 2001). Liposomes containing PS are known to have a short
circulation lifetime in ûiûo; this is said to be due to PS enhanced plasma protein binding
and subsequent uptake by the MPS (Chiu et al., 2001). A functional in ûitro assay was
employed to measure the effect of PEG lipid on the activity of membrane bound
coagulation proteins such as the prothrombinase complex. This complex is known to
consist of Xa and Va which when assembled on a negatively charged membrane is
responsible for the activation of prothrombin to thrombin. A chromogenic substrate
was used to monitor the formation of thrombin by the prothrombinase complex in the
presence of liposomes. The rate of thrombin formation was found to be undetectable
or negligible in the presence of PS free DSPC兾Cholesterol liposomes while in PS
containing liposomes (DSPC兾Chol兾PS) the rate of formation was 1.94 mol thrombin兾min兾mol兾factor Xa. The incorporation of the 10 mol.% PEG 750 or 5 mol.%
PEG 2000 in the 10 mol.% PS liposomes did not reduce the rate of thrombin formation significantly. However, when 20 mol.% PEG-750 or 10–15 mol.% PEG 2000
Physical Behavior and Biological Performance of Liposome
241
was included, the rate of thrombin formation was dramatically reduced to
<0.465 mol thrombin兾min兾mol factor Xa.
We also studied the effect of PEG-lipids on the plasma pharmacokinetics of the
PS containing liposomes. The incorporation of DSPE-PEG 2000 and DSPE-PEG
750 was found to increase the circulation lifetime of the PS containing systems with
the PEG 2000 lipid being more effective than the PEG 750 lipid. The inclusion of
20 mol.% DSPE-PEG 750 into PS containing liposomes (DSPC兾Chol兾PS 45兾45兾
10% mol. ratio or DSPC兾Chol兾PS兾DSPE-PEG 750 25兾45兾10兾20) increased the four
hour plasma lipid concentration from 0.0019 mg兾ml for the DSPC兾Chol兾PS 45兾45兾
10 formulation to 0.035 mg兾ml for DSPC兾Chol兾PS兾PEG. However, this value is
much lower than the four hour plasma lipid concentration of 0.23 mg兾ml attained
with DSPC兾Chol (PS free). The incorporation of 10 mol.% and 15 mol.% DSPEPEG 2000 into the PS containing liposomes (10 mol.%) resulted in plasma elimination curves that were comparable to those obtained for DSPC兾Chol and DSPC兾
Chol兾DSPE-PEG (50兾45兾5), respectively. In addition the incorporation DSPE-PEG
750 or DSPE-PEG 2000 reduced the accumulation of lipid in the liver and spleen.
A five-fold reduction in lipid accumulation in these tissues was attained when 15
mol.% DSPE-PEG 2000 was incorporated into the PS containing liposomes as compared to the PEG free formulation.
Bradley et al. have also studied the ability of PEG to prevent binding to an
anionic liposome (Bradley et al., 1998). Specifically, they examined Clq binding to
liposomes (EPC兾CH兾DSPE-PEG or CH-PEG) containing the anionic lipid, cardiolipin (CL) with and without surface grafted PEG (Bradley et al., 1998; Bradley et
al. 1999). For each PEG-lipid (CH-PEG 600, CH-PEG 1000, and CH-PEG 2000) a
decrease in Clq binding to liposomes was obtained as the surface density of PEG
was increased (5–15 mol.%). Also, for a specific mol.% of PEG-lipid the amount of
Clq binding was found to be inversely related to the length of the PEG chain. The
amount of PEG required for 100% inhibition of complement activation was 15
mol.% CH-PEG 600, 10–15 mol.% CH-PEG 1000 and 5–10 mol.% DSPE-PEG
2000. Taken together these studies show that there is an optimal graft density
and molecular weight of PEG able to inhibit high affinity interactions and these
are distinct from graft densities and molecular weights relevant to non-specific
interactions.
Another approach to characterizing PEG mediated inhibition of specific protein
interactions with liposomes involves assessments of receptor–ligand interactions.
Studies in our laboratory have examined the ability of surface grafted PEG to reduce
antibody binding to biotinylated liposomes (Bx-liposomes) (Li et al., 2002). An in
ûitro assay was used to measure the binding of a monoclonal anti-biotin antibody to
Bx-liposomes. The non-haptenized DSPC兾Chol liposomes resulted in no significant
consumption of antibody while the addition of 10 µM 1% Bx-liposomes (i.e.,
1 mol.% biotin) consumed 50% of the anti-biotin antibody. By contrast, the incorporation of 5 mol.% DSPE-PEG 2000 into the 1% Bx-liposomes resulted in a 1000
fold reduction in antibody consumption when compared to the non-PEGylated formulation. Thus, although the presence of PEG reduces antibody consumption significantly it is not completely prevented. The incorporation of additional PEG-lipid
(10 mol.% DSPE-PEG 2000) did not further decrease antibody consumption. Also,
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Allen et al.
DSPE-PEG 5000 was found to be less effective than PEG 2000 at reducing antibody
binding. The circulation lifetime of the Bx-liposomes with and without PEG was
determined in mice with an established anti-biotin response. In ûiûo the incorporation of 5 mol.% PEG 2000 provided minimal protection for the Bx-liposomes.
Therefore, 5 mol.% PEG 2000 was a sufficient amount to reduce antibody consumption by 1000 fold; however, it did not completely inhibit consumption and this is
reflected in the poor circulation lifetime for this formulation in ûiûo.
The in ûitro antibody consumption assay was also used to measure the protective effect on PEG on liposomes bearing a low density of biotin (0.1 mol.% biotin).
The incorporation of 5 mol.% DSPE-PEG 2000 (or DPPE-PEG 2000) into Bx-liposomes reduced antibody consumption to the same level as that obtained for the nonhaptenized DSPC兾Chol liposomes. In the case of the low density hapten, the protective effect of PEG 2000 in ûitro was found to hold in the in ûiûo situation as well.
The addition of 5 mol.% PEG 2000 to the 0.1 mol.% Bx-liposomes prevented their
rapid elimination. Interestingly, the incorporation of 2 mol.% PEG 2000 into low
density Bx-liposomes was not as effective at reducing antibody consumption when
compared to the formulation containing 5 mol.% PEG 2000. In ûiûo the circulation
lifetime of the formulation containing 2 mol.% DSPE-PEG 2000 was significantly
lower than that containing 5 mol.% DSPE-PEG 2000. The circulation lifetime of the
5 mol.% low density Bx-liposomes was not significantly different from that of nonhaptenized DSPC兾Chol liposomes.
Needham’s group has studied the ability of PEG to reduce or prevent receptorligand interactions in giant lipid vesicles using the micropipette technique (NopplSimson and Needham, 1996; Needham and Kim, 2000). Specifically, his group has
used the binding of free avidin to biotinylated lipid incorporated in the giant vesicle
as a model system to assess the ability of different molecular weights and graft
densities of PEG to prevent specific adsorption. In order to avoid non-specific interactions, such as van der Waals attraction, the biotin molecule is connected to the
lipid via a 1–1.5 nm hydrocarbon moiety (Noppl-Simson and Needham, 1996). Their
studies revealed that ‘‘under diffusion controlled conditions’’ the incorporation of
2 mol.% PEG-750 reduces the rate of diffusion of avidin to biotin (5 mol.%) by a
factor of 5. Also, the incorporation of greater than 6 mol.% PEG-750 was sufficient
to completely block avidin binding during a two minute incubation (i.e., diffusion
conditions). At 6 mol.% PEG-750 the PEG molecules are still in the mushroom
regime. Needham explains that with the presence of PEG at the surface, the rate of
diffusion of avidin is decreased due to the energy required to push the PEG chains
aside in order to reach the biotin receptor. The surface pressure (Πp ) that PEG
provides may be defined ideally by the following equation: Πp GN兾ABkT; where
N兾A (surface density)G0.01Bmol.% PEG兾(area of lipid molecule AL). In this way,
the rate of adsorption of a molecule to a PEG coated surface may be reduced by a
factor of exp(−AAvΠp兾kT). The rate is thus dependent on both the surface pressure
that PEG provides and the area of the approaching molecule.
Prevention of Liposome–Liposome Aggregation
It is widely accepted that specific and non-specific protein binding plays a role
in liposome elimination. However, we hope that the summary provided thus far
Physical Behavior and Biological Performance of Liposome
243
clearly supports our contention that protein binding is not a defining parameter
useful in predicting the rate of liposome elimination. If this is true then we need to
consider alternative roles for PEG. The presence of PEG on the surface of liposomes
is known to prevent the aggregation of individual liposomes. Ahl et al., studied the
relationship between both the extent of aggregation and complement binding of
liposomes in ûitro and their circulation lifetime in ûiûo (Ahl et al. 1997). As shown
in Fig. 7, a strong correlation was seen between a lack of a tendency to aggregate
and circulation lifetime (i.e., a low value for turbidity corresponded to a high value
for circulation lifetime) for formulations that were found to bind low levels of complement. For instance, for DSPC, DSPC兾Chol (2:1) and DSPC兾DSPE-PEG 2000
(9:1) liposome formulations the relative turbidity was found to be 275, 185, and 15
respectively. The value of the normalized AUC for each of these formulations was
0.04J0.02, 0.34J0.04 and 1.16J0.05 (for DSPC, DSPC兾Chol (2:1) and DSPC兾
DSPE-PEG 2000 (9:1), respectively. This study is one of the few that provides direct
evidence that PEG’s role in extending circulation lifetime is due, at least in part, to
its prevention of liposome self-aggregation.
It has been found that the inclusion of PEG is necessary for the preparation of
cholesterol free liposomes (e.g., DPPC or DSPC) at temperatures below the Tc of
the acyl chain (Dos Santos et al., 2002). At least 0.5 mol.% PEG (DSPE-PEG 2000)
must be incorporated in order to characterize or study these systems. Needham
et al. have shown that poly(ethylene glycol) chains of molecular weight 1900 g兾mole
Fig. 7. Long circulating liposomes must inhibit both complement activation and selfaggregation. (Redrawn from Ahl et al., 1997 with permission.) The composition of
each formulation is as follows: DSPC兾N-glutaryl-DPPE (9:1); DSPC兾Chol兾DSPG兾
PEG 2000-DSPE (1:4:4:1); DSPC兾Chol兾DSPG (2:4:4); DSPC兾Chol兾DSPG兾Nglutaryl-DPPE (1:4:4:1); DSPC.
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Allen et al.
extend a distance of 5 nm away from the lipid bilayer. In studies where they have
employed their micropipette technique Needham’s group has demonstrated that
PEG provides an interbilayer repulsion that can overcome the van der Waals attraction between particles (Needham et al., 1992a). Specifically, they found that the
inclusion of PEGylated lipid into the lipid bilayer completely prevented any adhesive
interactions between neutral bilayers. The self-aggregation of neutral liposomes
occurs upon close approach of the vesicles resulting in adhesion due to attractive
van der Waals forces between the bilayers. The approach will only be opposed by
the hydration pressure once the bilayers are within 1–2 nm of each other; however,
at this separation the repulsive force will be overcome by attractive van der Waals
forces (Needham et al., 1992b).
The prevention of liposome aggregation is also important when considering use
and development of fusogenic liposomes. Several studies have shown that surface
grafted PEG can inhibit liposomal fusion (Holland et al., 1996; Basanez et al., 1997;
Kirpotin et al., 1996). The fusion of liposomes is said to be the result of several
events including membrane apposition, mixing of lipids from the two outer bilayers
and further lipid mixing with combination of the vesicles inner aqueous contents.
Holland et al. found that the inclusion of as little as 1 mol.% PE-PEG 5000 or
2 mol.% PE-PEG 2000 into DOPE兾POPS liposomes completely inhibits lipid mixing, a necessary step in the calcium-induced fusion of the particles (Holland et al.,
1996). Holland explains that PEG’s ability to inhibit lipid mixing is due to the steric
barrier that it provides at the liposome surface. Fusion of the lipid bilayers requires
the liposomes to approach within 1–2 nm of each other; the presence of PEG at the
surface serves to prevent this membrane apposition.
Basanez et al. studied the ability of PEG-lipid conjugates to inhibit phospholipase C-induced fusion of liposomes (Basanez et al., 1979). Specifically, they measured the extent to which the inclusion of PEG could inhibit vesicle aggregation, lipid
mixing and mixing of aqueous contents. They found that the ability of PEG to
prevent fusion was only in part due to inhibition of phospholipase C activity
resulting from a reduced access of the enzyme to the surface. More importantly, the
inclusion of PEG-PE prevents fusion by both inhibiting membrane apposition and
by stabilizing the lamellar phase which prevents the formation of a ‘‘highly curved
intermediate’’ known to form in the last stage of fusion (Basanez et al., 1997). For
liposomal delivery vehicles, the prevention of fusion is good when the vehicle is en
route to the target site but once it reaches that site it may be desirous that the vehicle
becomes ‘‘leaky’’ or fusogenic (Hu et al., 2001). Recently, programmable fusogenic
vehicles have been designed where an exchangeable PEG-lipid conjugate is used to
induce stabilization once the carrier reaches its target site (Hu et al., 2001).
A final consideration highlighting PEGs role as a molecule inhibiting surface–
surface interactions concerns its use in the development of protein conjugated liposomal carriers. PEG modified lipids have been shown to be useful in preventing vesicle–vesicle crosslinking and aggregation of ligand or protein-conjugated liposomes
(Harasym et al., 1995, Harasym et al., 1998). The aggregation of protein-conjugated
liposomes is caused by both ‘‘covalent crosslinking via a multivalent protein bridge’’
and non-covalent protein–protein interactions (Harasym et al., 1995). The incorporation of PEG-modified lipids into liposomes at concentrations below 2 mol.%
Physical Behavior and Biological Performance of Liposome
245
inhibits vesicle aggregation without reducing the protein coupling efficiency (Harasym et al., 1995, Harasym et al., 1998). It is also important to recognize PEG inhibits
cell interactions; thus the use of exchangeable PEGs need to be considered.
Taken together, the above examples outline the extent to which PEG has been
clearly shown to prevent the self-aggregation of liposomes. Although it is rarely
referred to as such, the prevention of liposome–liposome aggregation may be one of
the key means by which PEG acts to extend circulation lifetime.
THE ‘‘DYSOPSONIZATION’’ OR ‘‘CHAMELEON EFFECT’’ OF PEG
As indicated in this review recent reports have challenged the accepted opinion
that PEG’s ability to extend circulation lifetime is due to the prevention of total
plasma protein binding (Vert and Domurado, 2000; Topchieva et al., 2000; Abbott
et al., 1992; Azegami et al., 1999; Sheth and Leckband, 1997; Kokufuta and Nishimura, 1991; Xia et al., 1993; Furness et al., 1998). In fact, it has been suggested that
specific proteins may adsorb to a PEG coated surface creating a ‘‘friendly interface’’
with a ‘‘chameleon effect’’ (Vert and Domurado, 2000). Authors have reported the
existence of non-covalent PEG protein complexes, such as PEG-α chymotrypsin
(Topchieva et al., 2000) and PEG-albumin (Vert and Domurado, 2000; Azegami
et al. 1999). Other interactions between PEG and proteins have also been noted
(Sheth and Leckband, 1997; Kokufuta and Nishimura, 1991; Xia et al., 1993).
Vert and Domurado studied mixtures of PEG of different molecular weights
(600, 300, 8000, 40,000 g兾mole) with physiological concentrations of bovine fibrinogen and bovine serum albumin (Vert and Domurado, 2000). The mixture of polymer
and protein was considered to be compatible if it appeared homogeneous upon mixing and incompatible if phase separation occurred. Albumin was found to be compatible with PEG when the molecular weight of the polymer was less than 8000
g兾mole. Yet, the fibrinogen PEG mixtures phase separated regardless of the molecular weight of the polymer. The authors explain that it has been established that a
mixture of two polymers in a solvent are incompatible resulting in phase separation
unless interactions exist between the two macromolecules. The lack of phase separation in the PEG albumin mixtures suggest that there are interactions between the
two and possibly complex formation.
In addition, Azegami et al. have reported that an intrapolymer complex exists
between HSA and PEG (Azegami et al., 1999). The formation and stability of the
complex was found to be pH dependent. At pH G8 a stable HSA兾PEG complex
was formed that was measured to be 90 nm in size. The complex was determined to
consist of several HSA molecules bound to one PEG chain.
If in fact attractive interactions do exist between proteins and PEG it is puzzling
that this has not yet been acknowledged in the many in ûitro protein binding studies
that have been performed. Yet as Vert and Domurado point out most assays involve
several washing steps which may cause protein that is loosely associated with PEG
to be removed (Vert and Domurado, 2000).
In addition, others have found that surface grafted PEG can result in a ‘‘dysopsonization’’ effect whereby proteins bind to the PEG coated surface and reduce cell
uptake (Moghimi et al., 1993; Moghimi and Patel, 1989; Johnstone et al., 2001).
246
Allen et al.
Moghimi et al. coated polystyrene microspheres and colloidal gold particles with
block copolymers of poly(ethylene oxide)-b-poly(propylene oxide) (Poloxamine 908)
(Moghimi et al., 1993). The block copolymers were adsorbed to the surface of the
particles via a hydrophobic–hydrophobic interaction between the particle and the
poly(propylene oxide) block of the copolymer. The hydrophilic poly(ethylene oxide)
blocks extended outward from the particle surface providing a steric barrier which
aided in stabilizing the particles by preventing their aggregation. The blood clearance
and biodistribution of the Poloxamine-908 coated and uncoated 125-I-labelled polystyrene particles were studied. It was found that the coating of the particle with
Poloxamine-908 significantly reduced the accumulation of the particles in the liver
and extended their circulation lifetime. The uptake of the Poloxamine-908 coated
particles was examined in liver endothelial cells before and after opsonization with
plasma. The liver endothelial cells only recognized the Poloxamine-908 coated
particles prior to opsonization and did not recognize them following opsonization.
The ‘‘dysopsonic’’ activity was said to be mediated by a specific serum component
(molecular mass >100 kDa) (Moghomi et al. 1993).
In the same vein, a study in our laboratory found that the presence of serum
components reduced the uptake of PEG coated liposomes in bone marrow macrophage (BMM) cells (Johnstone et al., 2001). In these studies liposomes were incubated with mouse serum and separation of liposome bound protein from free protein
was achieved using gel filtration chromatography. The uptake of bound serum protein-liposomes and protein free liposomes were then examined by incubation with
BMM cells for four hours at 37°C. The serum treatment of PEG containing liposomes was found to reduce uptake into BMM cells for the cationic, anionic and
neutral liposomes. Specifically, the presence of serum bound protein, reduced BMM
uptake by 10 fold, 8 fold and 3 fold for the neutral (DSPC兾Chol兾PEG), anionic
(DSPC兾Chol兾PS兾PEG) and cationic (DSPC兾Chol兾DODAC兾PEG) liposomes,
respectively. Clearly, the combined effect of PEG and serum are influencing the
uptake into BMM cells.
SUMMARY
To date we only know definitively that the presence of PEG on the surface of
a carrier extends the circulation lifetime of the vehicle. As shown above, the data
suggests that the mechanism by which PEG extends the circulation lifetime may not
be prevention of protein adsorption. Research focused on the understanding of
PEGs ability to extend circulation longevity will enable the rationale design of new
hydrophilic polymers to fulfill this role. In the meantime, we do know that PEG
stabilizes drug carriers; thus we can use this to our advantage to design delivery
vehicles that are stable enough to have a circulation lifetime that allows for sufficient
target accumulation but once at the target site ‘‘leaky’’ enough to allow release of
the encapsulated contents. Several groups have carefully selected a PEG-lipid with
specific properties such that their vehicle has an extended circulation lifetime but
also releases the drug at the target site (Hu et al., 2001; Vermehren et al., 1998;
Jorgensen et al., 1999; Chiu et al., 2002). For example, Vermehren et al. have found
that the inclusion of DPPE-PEG 2000 in DPPC liposomes promotes PLA2 catalyzed
Physical Behavior and Biological Performance of Liposome
247
phospholipid hydrolysis (Vermehren et al., 1998; Jorgensen et al., 1999). Since PLA2
is at high concentrations in extravascular inflammatory tissue the incorporation of
PEG-lipid may serve as a means of promoting destabilization of the liposome at this
site. The incorporation of increasing submicellar concentrations of PEG-lipid is said
to create heterogeneous bilayers composed of lipid domains with interfacial defects.
The defect regions are more susceptible to PLA2 lipid hydrolysis owing to increased
accessibility of the enzyme to this site. Our group has reported on ‘‘transiently stabilized’’ reactive surface PS-containing liposomes (Chiu et al., 2002). The incorporation
of exchangeable PEG-lipids into the reactive surface liposome ensures their circulation longevity and then allows for ‘‘site specific’’ reaction or PS exposure at the
target site. The acyl-chain length of the PEG-lipid was found to control its rate of
exchange for the liposome. Also, as mentioned earlier, Hu et al. have developed
programmable fusogenic vesicles (PFV) for delivery of antisense oligodeoxynucleotides (Hu et al., 2001). The stability of the vesicles is controlled by inclusion of
exchangeable PEG-lipid conjugates. The loss of the PEG-lipid from the vesicle will
cause their fusogenic nature to reemerge.
In short, we believe that future formulation research will utilize PEG-modified
lipids; however, the nature of the physical attributes of the polymer-lipid conjugate
will be considered in terms of controlling surface–surface interactions.
ACKNOWLEDGMENTS
This research project is supported in part by a grant from the Canadian Institutes of Health Research. Funding was also provided through an Industrial Research
Assistance Program grant from the National Research Council to Celator Technologies Inc.
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