Progress in Lipid Research 42 (2003) 463478

www.elsevier.com/locate/plipres

Review

Stealth liposomes and long circulating nanoparticles:

critical issues in pharmacokinetics, opsonization and
protein-binding properties
S.M. Moghimia,*, J. Szebenib
a
Molecular Targeting and Polymer Toxicology Group, School of Pharmacy and Biomolecular Sciences,
University of Brighton, Brighton BN2 4GJ, UK
b
Department of Membrane Biochemistry, Walter Reed Army Institute of Research, MD 20307, USA

Abstract
This article critically examines and evaluates the likely mechanisms that contribute to prolonged circu-
lation times of sterically protected nanoparticles and liposomes. It is generally assumed that the macro-
phage-resistant property of sterically protected particles is due to suppression in surface opsonization and
protein adsorption. However, recent evidence shows that sterically stabilized particles are prone to opso-
nization particularly by the opsonic components of the complement system. We have evaluated these
phenomena and discussed theories that reconcile complement activation and opsonization with prolonged
circulation times. With respect to particle longevity, the physiological state of macrophages also plays a
critical role. For example, stimulated or newly recruited macrophages can recognize and rapidly internalize
sterically protected nanoparticles by opsonic-independent mechanisms. These concepts are also examined.
# 2003 Elsevier Ltd. All rights reserved.

Contents

1. Introduction ........................................................................................................................................................... 464

2. Circulation kinetics of stealth particles .................................................................................................................. 465

3. Protein-binding to stealth particles ........................................................................................................................ 466

3.1. A general perspective..................................................................................................................................... 466
3.2. The inuence of surface PEGylation on complement activation................................................................... 467
3.3. Can mPEG-lipids accelerate complement activation? ................................................................................... 468
3.4. Theories reconciling opsonization with stealth activity ................................................................................. 470

* Corresponding author. Tel.: +44-1273-642063; fax: +44-1273-679333.

E-mail address: s.m.moghimi@brighton.ac.uk (S.M. Moghimi).

0163-7827/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0163-7827(03)00033-X
464 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478

4. The concept of surface heterogeneity ..................................................................................................................... 471

5. Can phagocytic cells recognize and internalize stealth nanoparticles? ................................................................... 472

6. The possible fate of the internalized polymers ....................................................................................................... 475

7. Conclusions ............................................................................................................................................................ 476

References ................................................................................................................................................................... 477

Nomenclature

PEG Poly(ethylene glycol)

mPEG Methoxypoly(ethylene glycol)
RES Reticuloendothelial system
SDS Sodium dodecyl sulfate
PAGE Polyacrylamide gel electrophoresis
RBC Red blood cell
PC Phosphatidylcholine
PE Phosphatidylethanolamine
HSPC Hydrogenated soy phosphatidylcholine
DPPC Dipalmitoylphosphatidylcholine
DPPE Dipalmitoylphosphatidylethanolamine
DSPE Distearoylphosphatidylethanolamine
HIC Hydrophobic interaction chromatography
PLA2 Phospholipase A2

1. Introduction

In the past two decades we have witnessed a surge in development of long circulating vehicles
within the nanoscale size range. Numerous interesting approaches for design and engineering of
long circulating vehicles have been described [1]. Among them, surface stabilization of nano-
particles and liposomes with a range of nonionic surfactants or polymeric macromolecules has
proved to be one of the most successful approaches for keeping the particles in the blood for
prolonged periods of time [14]. Surface enrichment of nanocarriers with nonionic surfactants
can be performed by physical adsorption, incorporation during the production of the carriers, or
by covalent attachment to any reactive surface groups. The presence of such surfactants on the
particle surface strongly reduces interparticulate attractive Van der Waals forces while increasing
the repulsive barrier between two approaching particles. This steric mechanism of stabilization
involves an elastic as well as an osmotic contribution [58]. The elastic (volume restriction) con-
tribution results from loss of conformational entropy when two surfaces approach each other,
caused by a reduction in the available volume for each polymer. A positive heat of solution may
 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478 465

also occur within the region of interfacial mixing. The loss of entropy and/or increase in enthalpy
results in an increase in the free energy of mixing that causes particle separation. The osmotic
pressure contribution arises from the increase in polymer concentration on compressing two
surfaces; necessitating an inux of water into the region and hence forcing particles apart. Simi-
larly, when a protein molecule approaches the surface, the number of available conformations of
the coating polymer segments is reduced due to compression or interpenetration of polymer
chains [5,8]. Again, an osmotic repulsive force also develops, which is due to loss of conforma-
tional freedom of the polymer chains. If polymer density is high, it is probable that compression
is preferred to interpenetration, while if the surface density is low interpenetration is likely to
dominate. However, it is the mobility, uniformity and density of the molecular cloud, which
determines the extent of particleprotein interaction in biological uids. These concepts were
discussed previously [1,9,10].
It is generally thought that the macrophage-resistant property of polymer-grafted particles is
due to suppression of surface opsonization by serum or plasma proteins [1,6]. Therefore, it is not
surprising to see that polymer-grafted particles exhibit prolonged residency times in the blood. In
contrast to such views, there is growing evidence which suggests that the prolonged lifetimes of
sterically protected nanoparticles and liposomes in the circulation may not be directly related to
reduced protein adsorption (or opsonization in general) or the steric repulsion between particles
and macrophage plasma membrane receptors [6,1122]. On the contrary, opsonization of steri-
cally protected particles occurs eciently. One example is complement activation by long-circu-
lating liposomes, which results in surface opsonization with C3b and iC3b [20,21,23]. Under
certain conditions, sterically protected particles are even prone to phagocytosis in the absence of
opsonins. It is the aim of this review to critically analyse these views and examine the likely
mechanisms that contribute towards the prolonged circulation time of surface-engineered nano-
particles and vesicles. Our discussion will be limited to poloxamer-, poloxamine- and mPEG-
coated carriers; the best studied systems to date.

2. Circulation kinetics of stealth particles

Following intravenous injection, liposomes and nanoparticles are cleared rapidly from the
blood (usually within minutes) by elements of the RES, particularly the hepatic Kuper cells [1].
Conversely, stealth particles circulate for prolonged hours; reported half-lives vary from 2 to 24 h
in mice and rats and can be as high as 45 h in humans depending on the particle size and the
characteristics of the coating polymer [1,24,25]. Interestingly, a common but often ignored
observation following intravenous injection of long-circulating vesicles and polymeric nano-
particles is rapid hepatic and splenic deposition of a fraction of the administered dose. For
example, stealth liposomes (e.g. PEGylated vesicles) have been administered in doses ranging
from 0.1 to 400 mmol/kg body weight (depending on the species) with reported hepatic and sple-
nic sequestration of 1015 nmol/kg within the rst hour of injection [12,13,2629]. The concern-
ing question is why, despite the presence of the protective polymer barrier, some PEG-coated
liposomes are cleared rapidly by macrophages of the liver and the spleen. Although this obser-
vation may imply surface heterogeneity among the injected vesicles (see Section 4), with a small
population bearing insucient or no protective PEG molecules, a recent study has demonstrated
466 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478

that at very low lipid doses, 20 nmol/kg body weight, PEG2000-grafted liposomes are cleared
rapidly from the blood by macrophages of the RES [12,13,29]. The macrophage clearance of low
doses of PEG-bearing vesicles is mediated by a pool of blood opsonins [12,29]. Therefore, it
appears to be a limited pool of an opsonic factor in the blood that can interact with PEG-bearing
liposomes resulting in rapid vesicle clearance by macrophages. These studies clearly indicate that
opsonization of stealth vesicles can occur eciently in vivo. Hence, an important factor con-
tributing to the prolonged circulation times of large doses of stealth vesicles may be the limited
concentration of the unidentied opsonic protein(s).

3. Protein-binding to stealth particles

3.1. A general perspective

SDS-PAGE, two-dimensional electrophoresis and immunoblotting studies have further con-

rmed that incubation of long-circulating vesicles and polymeric nanoparticles in plasma or
serum could lead to surface enrichment with various proteins [22,3039]. The prole of adsorbed
blood proteins is distinctly dierent from the prole of plasma or serum, indicating a partitioning
eect. These dierences have been suggested to play a signicant role in particle pharmacoki-
netics. In addition, evaluation of protein binding may provide insights as to the extent of blood
compatibility and functional integrity of the administered particles. A major question with these
in vitro studies [22,3339] is to what extent the observed dierences can truly represent the in vivo
dynamic events. An important issue in this regard is the Vroman eect [4043]. The Vroman
eect is a general phenomenon reecting competitive adsorption of proteins for a nite number of
surface sites and depends on the initial concentration of plasma or serum as well as the length of
incubation period. Therefore, dierent experimental conditions could generate variable protein
adsorption prole. Another critical issue is the procedure used for protein release, particularly
from the surface of polymeric nanoparticles [3339]. The strength of protein adsorption to and
subsequent release from the nanosphere surface depends not only on the protein type but also on
the characteristics of the particle to include chemical composition of the surface, density, homo-
geneity, chain conformation and the mobility of grafted polymers. The standard practice for
protein desorption is treatment of particles with EDTA and SDS. However, to date, the majority
of studies have failed to demonstrate complete release of adsorbed proteins from polymeric
nanospheres with variable surface characteristics following such treatments [3339]. This is of
particular concern in evaluating the deposition of complement proteins on nanospheres. For
instance, following complement activation C3b and its opsonic scission products (iC3b) are
expected to be linked covalently to the particle surface (however, in some cases C3 may deposit
on the surface in a non-covalent manner; a process that may or may not lead to complement
activation). Other problems include the loss of surface-bound proteins of functional importance
during separation procedures prior to further analysis, and underestimation of some proteins by
immunoblotting. Despite these limitations, various studies have indicated association of opsonic
molecules, such as components of complement system and immunoglobulins, with stealth parti-
cles [22,3239]. These studies, however, do not indicate whether opsonic molecules are associated
with the protective polymers or other particle surface components or both. If opsonic proteins
 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478 467

can favourably interact with surface protected nanoparticles, then the question is why the
majority of such complexes are not cleared rapidly from the blood by tissue macrophages. An
interesting example is Doxil1 (also known as Caelyx1), a PEGylated liposome with encapsulated
doxorubicin [25]. This formulation, which has a biphasic circulation half-life of 84 min and 46 h,
respectively, is a strong activator of the human complement system with activation taking place
within minutes [25]. Complement activation causes opsonization of PEGylated liposomes due to
covalent deposition of the C3b factor [20]. Therefore, it appears that PEGylation does not
necessarily suppress complement osponization. We shall examine this phenomenon more closely
and discuss theories that reconcile complement activation, and opsonization in general, with
prolonged circulation times.

3.2. The inuence of surface PEGylation on complement activation

Bradley et al. [44] have examined the ability of PEG-lipids to inhibit the in vitro activation of
the complement system in diluted human serum by anionic liposomes. For example, 100 nm
liposomes composed of egg PC, cholesterol and cardiolipin (35:45:20 mole ratio) were shown to
be potent activator of the complement system resulting in 80% complement consumption at
liposome concentrations above 1 mM [44]. Conversely, these investigators [44] suggested that
complement activation can virtually be abolished by incorporating 57.5 mol% of PEmPEG2000
into the liposomal bilayer. However, a closer examination of this study reveals that the inhibitory
eect of the mPEG-lipid on complement activation is highly dependent on the liposome concen-
tration used in the complement assay. Complement consumption was signicantly above the
baseline at liposome concentrations above 4 mM [44]. For example, incubation of 4, 8 and 15
mM of liposomes (containing 5 mol% PEmPEG2000) with serum for 30 min led to approxi-
mately 20, 35 and 40% complement consumption, respectively. The assertion that the inclusion of
5% PEmPEG2000 into liposomal bilayer may not be sucient to prevent complement activation
has also been demonstrated in a series of studies by one of us (JS) where complement activation
was monitored by production of the S-protein-bound complement terminal complex, SC5b-9
[20,21]. For example, incubation of Doxil1 with 10 dierent normal human sera led to signicant
rises of SC5b-9 levels over control (phosphate buer) in 7 sera, with rises exceeding 100 to 200%
(relative to the control) in 4 subjects [20]. Doxil1 was eective at a concentration as low as 0.4 mg
lipid/ml in activating complement, and the reaction proceeded on a time scale of minutes and
reached the plateau after about 20 to 30 min [20]. Therefore, Doxil1 can cause signicant com-
plement activation in human serum in vitro, although the extent of this activation may sub-
stantially vary in dierent individuals.
Utkhede and Tilcock [27] have also claimed that the incorporation of a lipid-mPEG5000 into
liposomal bilayer prevents in vitro activation of the human complement system. Again, this
conclusion is rather questionable since no positive control for complement consumption was
performed. In our opinion, complement consumption by a positive control, such as zymosan, is
an essential criterion for validation of all CH50 assays [45]. Furthermore, the data [27] demon-
strated no complement consumption in any of the test samples, raising the concern that the
applied dilution of the test sera did not bring down the concentration of haemolytic complement
into the eective dynamic range of the CH50 assay [45]. It is known that in vivo, complement
activation can lead to cardiovascular and pulmonary adverse responses [46,47]. Therefore, to
468 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478

assess complement activation in vivo, Utkhede and Tilcock [27] also monitored haemodynamic
changes in two rabbits following i.v. injection of PEGylated liposomes. They observed no
alterations in one animal, while the second rabbit displayed a transient 23% drop in systemic
arterial pressure (from 220 to 170 mm Hg) at 10 min post liposome injection and with no further
changes at later time points. The authors concluded that liposomes caused no complement acti-
vation. However, it has been known that complement activation-related haemodynamic changes
in pigs, dogs and rats start within 12 min after injection of the activator, with most parameters
returning to normal within 1015 min [23,46,47]. Thus, while these critical changes might have
been missed by Utkhede and Tilcock [27], the observed hypotension at 10 min post liposome
injection could reasonably arise from complement activation.
Recent work by Mosqueira et al. [48] has also demonstrated that surface modication of poly-
meric nanocapsules with PEG, regardless of surface PEG chain length and density, cannot totally
prevent complement activation. However, longer PEG chains (e.g. 20 kDa PEG) were more
eective, even at lower surface density, in suppressing complement activation. This is presumably
due to a predominant brush-like PEG conguration, which may sterically suppress deposition of
large C3 convertases [49] (for instance dimensions of the convertase C3bBb is about 148 nm
[50]).

3.3. Can mPEG-lipids accelerate complement activation?

Recent studies have assessed whether the presence of 5 mol% PEmPEG2000 into liposomal
bilayer can initiate complement activation in human serum [21]. The data in Fig. 1 shows that
small unilamellar vesicle of 100 nm, prepared from HSPC and cholesterol (15:10.5 mole ratio) do
not activate the human complement system in vitro. However, incorporation of DSPEmPEG2000
into liposomes causes a signicant increase in serum SC5b-9 level over baseline. These vesicles
resemble Doxil1 [25] both in size and lipid composition, but without the presence of doxo-
rubicin. Considering that Doxil1 is negatively charged, due to the phosphodiester moiety of the

Fig. 1. Human serum SC5b-9 levels at baseline and after treatment with liposomes mimicking the composition of
Doxil1. The results are expressed as% of baseline. HSPC, hydrogenated soy phosphatidylcholine; 2K-PEG-DSPE,
PEG2000-conjugated distearoyl phosphatidylethanolamine; Chol, cholesterol; HSPG, hydrogenated soy phosphati-
dylglycerol. The numbers in brackets are the molar ratios of lipids. Reproduced with permission [21].
 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478 469

DSPEmPEG2000, these studies [21] support the notion that the negative charge on the liposome
surface may play a critical role in complement activation. Indeed, negatively charged liposomes
are potent activators of the human complement system [5153]. This is illustrated in Fig. 1 with
HSPC/cholesterol vesicles containing hydrogenated soy phosphatidylglycerol. These observations
also indicate that the surface mPEG density is not sucient to mask the negatively charged
phosphodiester moieties and protect liposomes against complement. However, incorporation of
higher concentrations of mPEG-lipid can alter the biophysical property of the lipid bilayer and
cause liposome destabilization (reviewed in Ref. 1). Therefore, future experiments with mPEG-
lipids, where the negative charge is blocked, are necessary to fully establish whether the negative
charge or the mPEG play a critical role in complement activation.
SDS-PAGE studies have also conrmed that PEG-liposomes, such as Doxil1, can generate
opsonic fragments from radiolabelled C3 in human serum [20]. As shown in Fig. 2, C3b-con-
taining complexes were generated with MW exceeding that of C3b (bands labelled as C3bnX;
iC3bnX), a phenomenon typical of complement activation by immune aggregates [54]. Fur-
thermore, the data indicate that C3b deposition and degradationto 65 and 40/43 kDa frag-
mentsreaches the plateau within 5 min, attesting to rapid complement activation and ecient
inactivation of C3b to iC3b by Factors H and I. Therefore, surface mPEG molecules do not
interfere with C3b inactivation.
mPEG may even enhance complement activation via binding of natural IgM and IgG anti-
bodies. In fact, Doxil1 can bind to immunglobulins (IgG and IgM) in human serum (Szebeni,
unpublished data). The notion that PEG grafting may augment complement activation by IgM
binding has recently received support in a related system. Bradley et al. [55] reported that
PEGylation of human RBC with cyanuric chloride activated 5 kDa mPEG reduced the immu-
nogenicity of RBCs, but it failed to protect the cells against ABO antibody-dependent, comple-
ment-mediated lysis. The haemolysis by ABO-mismatched serum was enhanced by the presence
of surface PEG molecules and this enhancement correlated well with increased IgM binding [55].

Fig. 2. Complement activation in human serum by Doxil1. Normal human serum containing 125I-labelled C3 was
incubated with Doxil1 (nal concentration: 0.2 mg/ml doxorubicin, 1.6 mg/ml lipid) for the given times, and C3
fragments were determined by SDS-PAGE. Reproduced with permission [20].
470 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478

3.4. Theories reconciling opsonization with stealth activity

In the previous section we discussed the ability of PEmPEG-containing liposomes to activate

the complement in human serum. Furthermore, the presence of surface mPEG molecules did not
interfere with the production of opsonic components from C3 (C3b and iC3b). Therefore, for-
mation of monomeric and/or polymeric iC3b on to the surface of PEG-bearing liposomes is
expected to enhance vesicle recognition by macrophages in contact with blood via complement
receptors (CR1 and CR3, CD11b/CD18) [1]. This mode of recognition should result in rapid
vesicle clearance from the blood. Remarkably, these formulations exhibit prolonged circulation
times in the blood [1,56]. How can the stealth behaviour of such complement opsonized vesicles
then be explained? One possible explanation is the steric hindrance eect, which is generated by
the surface grafted mPEG molecules [20]. Therefore, complement xation on PEG-bearing lipo-
somes appears to occur in a cryptic location inaccessible for ligation to complement receptors, a
scenario similar to complement xation by Staphylococcus aureus [57]. Another possible con-
tributor to the stealth behaviour of such vesicles is competition between the surface-bound and
free iC3b for CR3. Furthermore, degradation of surface-bound C3b to fragments inhibiting
recognition by phagocytic complement receptors might also explain the anti-phagocytic eect
[58]. Finally, at least in human blood, surface-bound C3bn may interact with CR1 expressed on
erythrocytes and this may explain their prolonged residency in the systemic circulation [59].
Similar to PEG-bearing liposomes, poloxamer 407- and poloxamine 908-coated nanoparticles
also activate rat and human complement systems via both classical and alternative pathways, and
again the majority of these particles exhibit prolonged residency in the rat circulation [1,60].
Another important factor contributing to the prolonged circulation time of nanoparticle is their
small size (<100 nm in diameter); this means geometrical factors and surface dynamics play a key
role on the initial assembly of proteins and enzymes involved in complement activation [1].
Surface enrichment of long-circulating particles with immunoglobulins (e.g. IgG) has also been
demonstrated but this deposition again fails to enhance clearance via Fc receptors [22,3335,37
39]. It is plausible that IgG binds to the surface with the Fc domain burried, rendering the
nanoparticle resistant to macrophage recognition. Fibronectin is another interesting example;
despite its large size association of bronectin with the surface of mPEG-bearing liposomes has
been well documented [22]. Detection of various apolipoproteins on the surface of polymer-fab-
ricated liposomes [22] and nanoparticles [33,35,38] is also of interest. Whether these apoproteins
can control the biological behaviour of particles remains to be evaluated. With regard to PEG-
bearing vesicles, adsorption of apolipoprotein AI is particularly intriguing since this protein is
known to play an important role in liposome destabilization [61].
The notion that surface protection with non-ionic surfactants can in general greatly suppress
protein adsorption must also be viewed cautiously [1]. For example, in a recent study, Price et al.
[22] followed brinogen adsorption from buer to four dierent liposome types (neutral, neutral
PEG2000, negatively charged, due to phosphatidic acid incorporation, and negatively charged
PEG2000) of similar size distribution. Fibrinogen adsorption from buer to negatively charged
liposomes supported the widely held view that PEG provides a steric barrier which reduces pro-
tein adsorption [13], but no such eect of PEG was seen for neutral vesicles [22]. Another
interesting observation is the serum-source dependent quantitative and qualitative dierences in
protein adsorption to long-circulating poloxamine 908-coated particles (Moghimi, unpublished
 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478 471

observations). More proteins become associated with such surface-engineered nanoparticles fol-
lowing exposure to serum derived from a poloxamine pre-dosed rat (serum obtained from an
animal 24 h after injection of 300 mg poloxamine/kg body weight) than serum from a saline-
injected animal, Fig. 3. Nevertheless, when injected in to an animal that was never exposed to
poloxamine both nanoparticle compositions exhibit prolonged and comparable circulation times.
These observations indicate that the prolonged lifetimes of stealth particles in the systemic cir-
culation may not directly be related to reduced protein adsorption or even to dierences in the
compositional prole of adsorbed proteins. Interestingly, following interstitial injection, prior
exposure of poloxamine 908-coated polystyrene particles to serum derived from a poloxamine
pre-dosed animals dramatically enhances recognition and capture by lymph node macrophages,
whereas the control serum exerts no stimulating eect [15]. On the basis of these studies in vitro
plasma or serum protein binding studies/values are unlikely to be considered as good predictors
of nanoparticle longevity in vivo.

4. The concept of surface heterogeneity

Based on recent biophysical studies, it appears that long-circulating particles are heterogeneous
with respect to their surface properties; some populations appear to have a patchy surface with

Fig. 3. Association of serum proteins with poloxamine 908-coated polystyrene nanospheres (250 nm). In (1) nano-
spheres were incubated in 50% v/v rat serum derived from an animal injected intravenously with 300 mg poloxamine
908/kg body weight. Serum was collected 24 h after the poloxamine injection and was used immediately for incu-
bation with nanospheres. In (2) serum was derived from a saline injected rat for incubation with nanospheres. Fol-
lowing incubation at 37  C for 30 min, particles were pelleted by centrifugation and washed twice in saline. Finally,
released proteins were subjected to SDS-PAGE. In both cases a protein sample of 10 mg was loaded on to an acryl-
amide gel, consisting of a 10% separating gel and a 4% stacking gel. The gels were stained with 0.1% w/v Coomassie
Blue R250.
472 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478

poor steric shielding, which allows opsonic binding to unshielded areas [49,62]. For example, a
recent study [62] demonstrated that 30% of the total population of engineered mPEG-coated
polystyrene nanoparticles are prone to phagocytosis because of inadequate surface coverage by
mPEG molecules (Fig. 4). Particle heterogeneity can be detected by HIC and dierent popula-
tions can be separated [49,62].
In a recent study, the particle population separation by HIC demonstrated a remarkable linear
relationship between the particle zeta potential and phagocytosis by J774 A1 macrophage-like
cells [49]. Microsphere populations bearing a predominant surface of mPEG molecules as high-
density mushroom-brush intermediate and/or brush conguration (Fig. 5) were most resistant to
phagocytosis and activated the human complement system poorly (see also Table 1) [49]. Con-
versely, those populations with a predominant surface mPEGs in a mushroom regime (Fig. 5)
were potent activators of the complement system and were prone to phagocytosis (Table 1) [49].
Therefore, surface heterogeneity explains why a signicant fraction of intravenously injected
long-circulating nanoparticles are cleared rapidly by macrophages of the RES. Due to surface
heterogeneity it is therefore not surprising to detect opsonic association with stealth nano-
particles. Population heterogeneity and the proportion of each population within the mixture are
likely to determine the extent of opsonization. Future eorts should identify strategies, which
yield more homogeneous nanoparticles with regard to their surface properties. For example,
surface homogeneity of PEG-bearing liposomes could perhaps be improved by adopting solvent
vaporization, rather than the thin-lm hydration method, in vesicle preparation. Nevertheless,
HIC can readily assess the extent of surface heterogeneity of PEGylated particulate drug delivery
systems and pre-select particles with optimal retention times in the blood.

5. Can phagocytic cells recognize and internalize stealth nanoparticles?

Limited in vivo studies have shown that macrophages can interact with and internalize sterically
protected polymeric nanoparticles and vesicles [1,14,16,17]. Examples include rapid recognition
of poloxamine 908-coated polystyrene particles by proliferated rat Kuper cells as well as by
newly recruited liver macrophages via an opsonic-independent mechanism [16]. Another study have
demonstrated that an interval of 3 days between two injections of poloxamine 908-coated parti-
cles can trigger rapid Kuper cell clearance of the second dose, again by an opsonic-independent
pathway [14]. Recent in vitro studies with freshly isolated Kuper cells, which are derived from rats
three days after poloxamine injection, have further conrmed their scavenging ability towards
poloxamine 908- and poloxamer 407-coated polystyrene particles in the absence of serum opso-
nins (Moghimi, unpublished observations). These stimulation protocols also enhance macrophage
recognition of PEG-bearing liposomes, Fig. 6 [18]. Therefore, it appears that macrophages express
surface receptors that can recognize and internalize sterically protected particles. It is likely that the
physiological state of macrophages plays a signicant role in blood longevity of long-circulating
carriers. Thus, with respect to particle longevity the physiological state of macrophages must be
determined or dened. Perhaps, under normal physiological conditions the putative receptors are
either down regulated or exocytosed from the cell surface and circulate in soluble form in the blood.
Studies with freshly isolated macrophages have also indicated the presence of unidentied
serum factors (dysopsonins) that act synergistically with the steric barrier of long-circulating
 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478 473

Fig. 4. Hydrophobic interaction chromatography of polystyrene nanopsheres coated with BSAmPEG5000 on a col-
umn of octyl-agarose (a) and the blood concentration of eluted nanospheres following intravenous injection to rats (b).
In (a) the elution gradient is also shown (. . .), the two numbers above each gradient step represent the molarity of
sodium chloride (left) and % v/v of Triton X-100 (right). Nanospheres were surface labelled with 125I. The biodis-
tribution of nanospheres eluted under F1 peak was dierent to those collected under F2 (b). This represent surface
heterogeneity among the two fractions. Reproduced with permission [62].
474 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478

particles, thereby further suppressing particle recognition by phagocytic cells [6]. Thus, the blood
longevity of long-circulating particles may also dependent on plasma concentration of dysopso-
nins. Limited studies have pointed towards the presence of at least two serum proteins which can
suppress the uptake of poloxamine 908-coated nanoparticles by rat liver macrophages [6]. Even
prior incubation of Kuper cells with serum or the partially puried dysopsonins could also
suppress binding and uptake of poloxamine 908-coated nanoparticles (Moghimi, unpublished
observations). Similar observations have also been reported with isolated liver endothelial cells
[6]. Presumably, under normal physiological conditions the putative cell surface receptors that
recognize long-circulating particles are blocked by dysopsonins.
Some reports have demonstrated that long-circulating particles (e.g. mPEG-liposomes) are
prone to phagocytosis by macrophages located at pathological sites with leaky vasculature (e.g.
infection sites and solid tumours) [1,63]. Although these observations may represent vesicle recogni-
tion by stimulated or activated macrophages, the elevated local concentration of phospholipases

Fig. 5. Schematic diagrams of PEG conguration on a segment of nanoparticle surface. In (a) due to low surface PEG
concentration the grafted mPEG molecules assume mushroom conguration. In (b) mPEG molecules are overlapped
and exhibit brush-like conguration and therefore extend from the surface. The mPEG surface conguration can be
determined by various techniques such as small angle neutron scattering [74] and measurements of ultrasound velocity
and absorption in colloidal suspensions [75].

Table 1
Biological performance of mPEG grafted liposomes and polystyrene particles

Particle Size (nm) mPEG displaya mPEG Complemet Phagocyte Ref.

thickness activation recognition
(nm)

Liposome 80100 Mushroom (4 mol%) 13 Unknown Unknown

Liposome 80100 Mushroom/brush 46 Moderate Poor [20,21]
(e.g. Doxil) (59 mol%)
Polystyrene 60 Mushroom/brush 46 Weak Poor [62]
Polystyrene 1000 Mushroom 1 Strong Strong [49]
Polystyrene 1000 Mushroom/brush 45 Weak Poor [49]
a
Refer to mPEG2000 or mPEG5000. For liposomes mPEG-2000 is grafted to DSPE and for polystyrene particles
mPEG-5000 is grafted to surface adsorbed serum albumin molecules.
 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478 475

Fig. 6. Scintigraphic images of rats with normal (1) and enhanced (2) macrophage activity 4 h following intravenous
injection of (99mTcO4 )-labelled long-circulating liposomes. To enhance macrophage phagocytic activity, poloxamine
908 was injected intravenously (43 mg/kg) 3 days prior to liposome injection. In (1) the images represent the circulatory
blood pool in the heart region (arrowhead) and poor localization of liposomes in both liver and spleen regions. In (2) a
large fraction of liposomes is captured by stimulated Kuper cells and splenic macrophages (arrows). Reproduced with
permission [18].

may further enhance liposome recognition and internalization by macrophages [64]. For example,
it has been shown that the mammalian PLA2 can catalyse degradation of DPPC vesicles con-
taining DPPEmPEG2000, and increasing the content of PEG-lipid further enhances phospholi-
pase-mediated lipid hydrolysis [65,66]. It seems that surface-associated PEG molecules may be
unable to provide protection to liposomes under certain pathophysiological conditions, although
this is an ideal situation for delivery of therapeutic or diagnostic agents to a selective population
of cells in the body (e.g. activated versus quiescent macrophages). Another possible application is
design and development of vesicles containing PEG-etherlipid prodrugs [67,68] as well as other
anti-cancer agents for targeting to solid tumours with elevated local concentrations of PLA2. The
PLA2-catalysed degradation will lead to drug release from the vesicles [65,66] as well as local
macrophage activation [67,68].

6. The possible fate of the internalized polymers

Surprisingly, the majority of studies to date have ignored the biological fate of non-biodegradable
polymers used in nanoparticle surface engineering. When released from the particle surface the
receivers in the blood are unknown but the major assumption is polymer excretion via the renal
system [1]. Not much is known with regard to acute and chronic pharmacological eects of such
polymers (e.g. complement activation-related pseudoallergy, modulation of gene activation, enzyme
activity, signal transduction). Factors such as polymer polydispersity, chemical contamination,
pharmacogenomics, immunogenetics and related polymorphisms could all control the extent of
these outcomes; these are discussed in detail elsewhere [1,2,19,69]. Various members of poloxamer
and poloxamine family of surfactants, including those that can prolong particle longevity in the blood,
are known to stimulate the production of various pro-inammatory cytokines by macrophages in
476 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478

a dose-dependent manner [1,2,70]. These polymers, particularly those with low polyoxyethylene
content, may even destabilize the plasma membrane or membrane of the internalized vacuoles
(pinosomes or phagolysosomes) [1]. Therefore, it is not surprising to see that such polymers have been
used as powerful adjuvants for increasing antibody formation to a variety of antigens [19,71].
Lysosomal membrane makes a major contribution to the eciency of the lysosome as an
intracellular digestive system as well as a route for cytoplasmic delivery of some nutrients. Per-
turbation of this membrane by polymers may interrupt cytoplasmic metabolic and biosynthetic
pathways. In accordance with the solutes notional hydrogen-binding capacity [72,73] it is unli-
kely for long-chain PEG or mPEG molecules to escape phagolysosomes. Gradual accumulation
of PEG molecules in lysosomes will alter organelle density and may eventually modify or mod-
ulate the activity of lysosomal enzymes, transporters and membrane glycoproteins. Such pertur-
bations could alter eux of metabolic products from the lysosomes or even lysosome fusion with
recycling vesicles. It is essential that future studies should take these possibilities into account.

7. Conclusions

The available evidence suggests that a combination of mechanisms is apparently responsible for
the long-circulating behaviour of sterically protected nanovehicles. The components of any
hydrated polymeric molecular cloud are not completely inert; they can interact with the biolo-
gical milieu via systems of collective weak associations based, for example, on Van der Waals and
hydrophobic interactions as well as formation of hydrogen bonds. Therefore, it is not surprising
to see interaction between sterically protected particles and various blood proteins as well as
macrophage cell surface receptors. These interactions can partly control the in vivo behaviour of
sterically protected particles. For example, the existing evidence at least support ecient opsoni-
zation of mPEG-grafted vesicles in the blood. It is the limited concentration of the blood opso-
nin, which partly contributes to the prolonged circulation time of mPEG-grafted vesicles in
vasculature. In addition, favourable interaction between surface-bound polymers and blood dys-
opsonins also contribute towards the prolonged blood residency of sterically protected particles.
Future studies should identify the nature of these blood factors and unravel their mode of action.
Understanding of these events is essential for design of particles with optimal circulation proles.
Another interesting observation is the complement activating nature of stealth particles.
Although complement xation may not necessarily lead to particle clearance from the blood,
complement activation is associated with the release of anaphylatoxins [23]. The latter is thought
to be responsible for the observed pseudoallergic reaction following intravenous injection of long-
circulating vesicles [23]. Complement activation is also associated with the activation of other
proteolytic plasma cascades. An example is the kallikreinkinin system, which provides a co-sti-
mulus for mast cells [23]. Therefore, it is essential to design coating systems that do not activate
complement. Understanding of the molecular mechanisms that contributes towards complement
activation by PEmPEG lipids is a logical step towards fullling this goal.
Finally, the notion that the steric barrier can prevent particle phagocytosis by macrophages
must be viewed cautiously. The principle at least holds in vitro but in vivo stimulated macrophage
can rapidly internalize sterically protected particles by opsonic-independent mechanisms.
Although the identity of these receptors are unknown, these observations imply that quiescent
 S.M. Moghimi, J. Szebeni / Progress in Lipid Research 42 (2003) 463478 477

macrophages either lack these receptors or their binding site(s) are blocked by some blood com-
ponents (e.g. dysopsonins). Therefore, the lack of receptor expression (or activity) seems to play
an important role in the pharmacokinetics of sterically stabilized particles. Understanding of
these issues is also important for the future development of this area of nanotechnology.

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