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Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonization and protein-binding properties
Progress in Lipid Research 42 (2003) 463–478 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 circulation times of sterically protected nanoparticles and liposomes. It is generally assumed that the macrophage-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 opsonization 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 influence 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: [email protected] (S.M. Moghimi). 0163-7827/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0163-7827(03)00033-X
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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 mPEG RES SDS PAGE RBC PC PE HSPC DPPC DPPE DSPE HIC PLA2
Poly(ethylene glycol) Methoxypoly(ethylene glycol) Reticuloendothelial system Sodium dodecyl sulfate Polyacrylamide gel electrophoresis Red blood cell Phosphatidylcholine Phosphatidylethanolamine Hydrogenated soy phosphatidylcholine Dipalmitoylphosphatidylcholine Dipalmitoylphosphatidylethanolamine Distearoylphosphatidylethanolamine Hydrophobic interaction chromatography 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 nanoparticles 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 [1–4]. 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 [5–8]. The elastic (volume restriction) contribution 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
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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 influx of water into the region and hence forcing particles apart. Similarly, 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 conformational 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 particle–protein interaction in biological fluids. 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 [6,11–22]. On the contrary, opsonization of sterically protected particles occurs efficiently. One example is complement activation by long-circulating 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 nanoparticles and vesicles. Our discussion will be limited to poloxamer-, poloxamine- and mPEGcoated 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 Kupffer 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 nanoparticles 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 splenic sequestration of 10–15 nmol/kg within the first hour of injection [12,13,26–29]. The concerning 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 observation may imply surface heterogeneity among the injected vesicles (see Section 4), with a small population bearing insufficient or no protective PEG molecules, a recent study has demonstrated
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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 efficiently in vivo. Hence, an important factor contributing to the prolonged circulation times of large doses of stealth vesicles may be the limited concentration of the unidentified opsonic protein(s).
3. Protein-binding to stealth particles 3.1. A general perspective SDS-PAGE, two-dimensional electrophoresis and immunoblotting studies have further confirmed that incubation of long-circulating vesicles and polymeric nanoparticles in plasma or serum could lead to surface enrichment with various proteins [22,30–39]. The profile of adsorbed blood proteins is distinctly different from the profile of plasma or serum, indicating a partitioning effect. These differences have been suggested to play a significant role in particle pharmacokinetics. 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 [22,33–39] is to what extent the observed differences can truly represent the in vivo dynamic events. An important issue in this regard is the Vroman effect [40–43]. The Vroman effect is a general phenomenon reflecting competitive adsorption of proteins for a finite number of surface sites and depends on the initial concentration of plasma or serum as well as the length of incubation period. Therefore, different experimental conditions could generate variable protein adsorption profile. Another critical issue is the procedure used for protein release, particularly from the surface of polymeric nanoparticles [33–39]. 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, homogeneity, 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 [33–39]. 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 particles [22,32–39]. 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
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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 influence 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 5–7.5 mol% of PE–mPEG2000 into the liposomal bilayer. However, a closer examination of this study reveals that the inhibitory effect of the mPEG-lipid on complement activation is highly dependent on the liposome concentration used in the complement assay. Complement consumption was significantly above the baseline at liposome concentrations above 4 mM [44]. For example, incubation of 4, 8 and 15 mM of liposomes (containing 5 mol% PE–mPEG2000) with serum for 30 min led to approximately 20, 35 and 40% complement consumption, respectively. The assertion that the inclusion of 5% PE–mPEG2000 into liposomal bilayer may not be sufficient 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 different normal human sera led to significant rises of SC5b-9 levels over control (phosphate buffer) in 7 sera, with rises exceeding 100 to 200% (relative to the control) in 4 subjects [20]. Doxil1 was effective 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 significant complement activation in human serum in vitro, although the extent of this activation may substantially vary in different 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] demonstrated 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 effective 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
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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 activation. However, it has been known that complement activation-related haemodynamic changes in pigs, dogs and rats start within 1–2 min after injection of the activator, with most parameters returning to normal within 10–15 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 modification of polymeric 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 effective, even at lower surface density, in suppressing complement activation. This is presumably due to a predominant brush-like PEG configuration, 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% PE–mPEG2000 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 DSPE–mPEG2000 into liposomes causes a significant increase in serum SC5b-9 level over baseline. These vesicles resemble Doxil1 [25] both in size and lipid composition, but without the presence of doxorubicin. 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 phosphatidylglycerol. The numbers in brackets are the molar ratios of lipids. Reproduced with permission [21].
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DSPE–mPEG2000, 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 [51–53]. 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 sufficient 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 mPEGlipids, 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 confirmed that PEG-liposomes, such as Doxil1, can generate opsonic fragments from radiolabelled C3 in human serum [20]. As shown in Fig. 2, C3b-containing complexes were generated with MW exceeding that of C3b (bands labelled as ‘C3bn–X; iC3bn–X’), a phenomenon typical of complement activation by immune aggregates [54]. Furthermore, the data indicate that C3b deposition and degradation—to 65 and 40/43 kDa fragments—reaches the plateau within 5 min, attesting to rapid complement activation and efficient 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 antibodies. 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 immunogenicity of RBCs, but it failed to protect the cells against ABO antibody-dependent, complement-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 (final 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].
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3.4. Theories reconciling opsonization with stealth activity In the previous section we discussed the ability of PE–mPEG-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, formation 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 effect, which is generated by the surface grafted mPEG molecules [20]. Therefore, complement fixation on PEG-bearing liposomes appears to occur in a cryptic location inaccessible for ligation to complement receptors, a scenario similar to complement fixation by Staphylococcus aureus [57]. Another possible contributor 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 effect [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 [22,33–35,37– 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 fibronectin with the surface of mPEG-bearing liposomes has been well documented [22]. Detection of various apolipoproteins on the surface of polymer-fabricated 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 PEGbearing 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 fibrinogen adsorption from buffer to four different liposome types (neutral, neutral– PEG2000, negatively charged, due to phosphatidic acid incorporation, and negatively charged– PEG2000) of similar size distribution. Fibrinogen adsorption from buffer to negatively charged liposomes supported the widely held view that PEG provides a steric barrier which reduces protein adsorption [1–3], but no such effect of PEG was seen for neutral vesicles [22]. Another interesting observation is the serum-source dependent quantitative and qualitative differences in protein adsorption to long-circulating poloxamine 908-coated particles (Moghimi, unpublished
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observations). More proteins become associated with such surface-engineered nanoparticles following 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 salineinjected 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 circulation may not directly be related to reduced protein adsorption or even to differences in the compositional profile 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 effect [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) nanospheres 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 incubation with nanospheres. In (2) serum was derived from a saline injected rat for incubation with nanospheres. Following 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 acrylamide gel, consisting of a 10% separating gel and a 4% stacking gel. The gels were stained with 0.1% w/v Coomassie Blue R250.
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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 different populations 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 highdensity mushroom-brush intermediate and/or brush configuration (Fig. 5) were most resistant to phagocytosis and activated the human complement system poorly (see also Table 1) [49]. Conversely, 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 significant 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 nanoparticles. Population heterogeneity and the proportion of each population within the mixture are likely to determine the extent of opsonization. Future efforts 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-film 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 Kupffer 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 particles can trigger rapid Kupffer cell clearance of the second dose, again by an opsonic-independent pathway [14]. Recent in vitro studies with freshly isolated Kupffer cells, which are derived from rats three days after poloxamine injection, have further confirmed their scavenging ability towards poloxamine 908- and poloxamer 407-coated polystyrene particles in the absence of serum opsonins (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 significant role in blood longevity of long-circulating carriers. Thus, with respect to particle longevity the physiological state of macrophages must be determined or defined. 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 unidentified serum factors (dysopsonins) that act synergistically with the steric barrier of long-circulating
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Fig. 4. Hydrophobic interaction chromatography of polystyrene nanopsheres coated with BSA–mPEG5000 on a column 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 biodistribution of nanospheres eluted under F1 peak was different to those collected under F2 (b). This represent surface heterogeneity among the two fractions. Reproduced with permission [62].
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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 dysopsonins. 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 Kupffer cells with serum or the partially purified 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 recognition by stimulated or activated macrophages, the elevated local concentration of phospholipases
Fig. 5. Schematic diagrams of PEG configuration on a segment of nanoparticle surface. In (a) due to low surface PEG concentration the grafted mPEG molecules assume mushroom configuration. In (b) mPEG molecules are overlapped and exhibit brush-like configuration and therefore extend from the surface. The mPEG surface configuration 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 thickness (nm)
Complemet activation
Phagocyte recognition
Ref.
Liposome Liposome (e.g. Doxil) Polystyrene Polystyrene Polystyrene
80–100 80–100
Mushroom (4 mol%) Mushroom/brush (5–9 mol%) Mushroom/brush Mushroom Mushroom/brush
1–3 4–6
Unknown Moderate
Unknown Poor
– [20,21]
4–6 1 4–5
Weak Strong Weak
Poor Strong Poor
60 1000 1000
[62] [49] [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.
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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 Kupffer 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 containing DPPE–mPEG2000, and increasing the content of PEG-lipid further enhances phospholipase-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 effects 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-inflammatory cytokines by macrophages in
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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 efficiency of the lysosome as an intracellular digestive system as well as a route for cytoplasmic delivery of some nutrients. Perturbation of this membrane by polymers may interrupt cytoplasmic metabolic and biosynthetic pathways. In accordance with the solute’s notional hydrogen-binding capacity [72,73] it is unlikely 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 modulate the activity of lysosomal enzymes, transporters and membrane glycoproteins. Such perturbations could alter efflux 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 biological 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 efficient opsonization of mPEG-grafted vesicles in the blood. It is the limited concentration of the blood opsonin, which partly contributes to the prolonged circulation time of mPEG-grafted vesicles in vasculature. In addition, favourable interaction between surface-bound polymers and blood dysopsonins 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 profiles. Another interesting observation is the complement activating nature of stealth particles. Although complement fixation 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 longcirculating vesicles [23]. Complement activation is also associated with the activation of other proteolytic plasma cascades. An example is the kallikrein–kinin system, which provides a co-stimulus 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 PE–mPEG lipids is a logical step towards fulfilling 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
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macrophages either lack these receptors or their binding site(s) are blocked by some blood components (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. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35]
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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 circulation times of sterically protected nanoparticles and liposomes. It is generally assumed that the macrophage-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 opsonization 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 influence 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: [email protected] (S.M. Moghimi). 0163-7827/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0163-7827(03)00033-X
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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 mPEG RES SDS PAGE RBC PC PE HSPC DPPC DPPE DSPE HIC PLA2
Poly(ethylene glycol) Methoxypoly(ethylene glycol) Reticuloendothelial system Sodium dodecyl sulfate Polyacrylamide gel electrophoresis Red blood cell Phosphatidylcholine Phosphatidylethanolamine Hydrogenated soy phosphatidylcholine Dipalmitoylphosphatidylcholine Dipalmitoylphosphatidylethanolamine Distearoylphosphatidylethanolamine Hydrophobic interaction chromatography 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 nanoparticles 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 [1–4]. 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 [5–8]. The elastic (volume restriction) contribution 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
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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 influx of water into the region and hence forcing particles apart. Similarly, 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 conformational 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 particle–protein interaction in biological fluids. 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 [6,11–22]. On the contrary, opsonization of sterically protected particles occurs efficiently. One example is complement activation by long-circulating 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 nanoparticles and vesicles. Our discussion will be limited to poloxamer-, poloxamine- and mPEGcoated 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 Kupffer 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 nanoparticles 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 splenic sequestration of 10–15 nmol/kg within the first hour of injection [12,13,26–29]. The concerning 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 observation may imply surface heterogeneity among the injected vesicles (see Section 4), with a small population bearing insufficient or no protective PEG molecules, a recent study has demonstrated
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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 efficiently in vivo. Hence, an important factor contributing to the prolonged circulation times of large doses of stealth vesicles may be the limited concentration of the unidentified opsonic protein(s).
3. Protein-binding to stealth particles 3.1. A general perspective SDS-PAGE, two-dimensional electrophoresis and immunoblotting studies have further confirmed that incubation of long-circulating vesicles and polymeric nanoparticles in plasma or serum could lead to surface enrichment with various proteins [22,30–39]. The profile of adsorbed blood proteins is distinctly different from the profile of plasma or serum, indicating a partitioning effect. These differences have been suggested to play a significant role in particle pharmacokinetics. 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 [22,33–39] is to what extent the observed differences can truly represent the in vivo dynamic events. An important issue in this regard is the Vroman effect [40–43]. The Vroman effect is a general phenomenon reflecting competitive adsorption of proteins for a finite number of surface sites and depends on the initial concentration of plasma or serum as well as the length of incubation period. Therefore, different experimental conditions could generate variable protein adsorption profile. Another critical issue is the procedure used for protein release, particularly from the surface of polymeric nanoparticles [33–39]. 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, homogeneity, 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 [33–39]. 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 particles [22,32–39]. 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
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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 influence 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 5–7.5 mol% of PE–mPEG2000 into the liposomal bilayer. However, a closer examination of this study reveals that the inhibitory effect of the mPEG-lipid on complement activation is highly dependent on the liposome concentration used in the complement assay. Complement consumption was significantly above the baseline at liposome concentrations above 4 mM [44]. For example, incubation of 4, 8 and 15 mM of liposomes (containing 5 mol% PE–mPEG2000) with serum for 30 min led to approximately 20, 35 and 40% complement consumption, respectively. The assertion that the inclusion of 5% PE–mPEG2000 into liposomal bilayer may not be sufficient 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 different normal human sera led to significant rises of SC5b-9 levels over control (phosphate buffer) in 7 sera, with rises exceeding 100 to 200% (relative to the control) in 4 subjects [20]. Doxil1 was effective 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 significant complement activation in human serum in vitro, although the extent of this activation may substantially vary in different 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] demonstrated 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 effective 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
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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 activation. However, it has been known that complement activation-related haemodynamic changes in pigs, dogs and rats start within 1–2 min after injection of the activator, with most parameters returning to normal within 10–15 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 modification of polymeric 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 effective, even at lower surface density, in suppressing complement activation. This is presumably due to a predominant brush-like PEG configuration, 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% PE–mPEG2000 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 DSPE–mPEG2000 into liposomes causes a significant increase in serum SC5b-9 level over baseline. These vesicles resemble Doxil1 [25] both in size and lipid composition, but without the presence of doxorubicin. 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 phosphatidylglycerol. The numbers in brackets are the molar ratios of lipids. Reproduced with permission [21].
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DSPE–mPEG2000, 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 [51–53]. 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 sufficient 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 mPEGlipids, 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 confirmed that PEG-liposomes, such as Doxil1, can generate opsonic fragments from radiolabelled C3 in human serum [20]. As shown in Fig. 2, C3b-containing complexes were generated with MW exceeding that of C3b (bands labelled as ‘C3bn–X; iC3bn–X’), a phenomenon typical of complement activation by immune aggregates [54]. Furthermore, the data indicate that C3b deposition and degradation—to 65 and 40/43 kDa fragments—reaches the plateau within 5 min, attesting to rapid complement activation and efficient 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 antibodies. 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 immunogenicity of RBCs, but it failed to protect the cells against ABO antibody-dependent, complement-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 (final 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].
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3.4. Theories reconciling opsonization with stealth activity In the previous section we discussed the ability of PE–mPEG-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, formation 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 effect, which is generated by the surface grafted mPEG molecules [20]. Therefore, complement fixation on PEG-bearing liposomes appears to occur in a cryptic location inaccessible for ligation to complement receptors, a scenario similar to complement fixation by Staphylococcus aureus [57]. Another possible contributor 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 effect [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 [22,33–35,37– 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 fibronectin with the surface of mPEG-bearing liposomes has been well documented [22]. Detection of various apolipoproteins on the surface of polymer-fabricated 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 PEGbearing 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 fibrinogen adsorption from buffer to four different liposome types (neutral, neutral– PEG2000, negatively charged, due to phosphatidic acid incorporation, and negatively charged– PEG2000) of similar size distribution. Fibrinogen adsorption from buffer to negatively charged liposomes supported the widely held view that PEG provides a steric barrier which reduces protein adsorption [1–3], but no such effect of PEG was seen for neutral vesicles [22]. Another interesting observation is the serum-source dependent quantitative and qualitative differences in protein adsorption to long-circulating poloxamine 908-coated particles (Moghimi, unpublished
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observations). More proteins become associated with such surface-engineered nanoparticles following 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 salineinjected 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 circulation may not directly be related to reduced protein adsorption or even to differences in the compositional profile 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 effect [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) nanospheres 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 incubation with nanospheres. In (2) serum was derived from a saline injected rat for incubation with nanospheres. Following 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 acrylamide gel, consisting of a 10% separating gel and a 4% stacking gel. The gels were stained with 0.1% w/v Coomassie Blue R250.
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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 different populations 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 highdensity mushroom-brush intermediate and/or brush configuration (Fig. 5) were most resistant to phagocytosis and activated the human complement system poorly (see also Table 1) [49]. Conversely, 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 significant 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 nanoparticles. Population heterogeneity and the proportion of each population within the mixture are likely to determine the extent of opsonization. Future efforts 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-film 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 Kupffer 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 particles can trigger rapid Kupffer cell clearance of the second dose, again by an opsonic-independent pathway [14]. Recent in vitro studies with freshly isolated Kupffer cells, which are derived from rats three days after poloxamine injection, have further confirmed their scavenging ability towards poloxamine 908- and poloxamer 407-coated polystyrene particles in the absence of serum opsonins (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 significant role in blood longevity of long-circulating carriers. Thus, with respect to particle longevity the physiological state of macrophages must be determined or defined. 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 unidentified serum factors (dysopsonins) that act synergistically with the steric barrier of long-circulating
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Fig. 4. Hydrophobic interaction chromatography of polystyrene nanopsheres coated with BSA–mPEG5000 on a column 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 biodistribution of nanospheres eluted under F1 peak was different to those collected under F2 (b). This represent surface heterogeneity among the two fractions. Reproduced with permission [62].
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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 dysopsonins. 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 Kupffer cells with serum or the partially purified 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 recognition by stimulated or activated macrophages, the elevated local concentration of phospholipases
Fig. 5. Schematic diagrams of PEG configuration on a segment of nanoparticle surface. In (a) due to low surface PEG concentration the grafted mPEG molecules assume mushroom configuration. In (b) mPEG molecules are overlapped and exhibit brush-like configuration and therefore extend from the surface. The mPEG surface configuration 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 thickness (nm)
Complemet activation
Phagocyte recognition
Ref.
Liposome Liposome (e.g. Doxil) Polystyrene Polystyrene Polystyrene
80–100 80–100
Mushroom (4 mol%) Mushroom/brush (5–9 mol%) Mushroom/brush Mushroom Mushroom/brush
1–3 4–6
Unknown Moderate
Unknown Poor
– [20,21]
4–6 1 4–5
Weak Strong Weak
Poor Strong Poor
60 1000 1000
[62] [49] [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.
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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 Kupffer 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 containing DPPE–mPEG2000, and increasing the content of PEG-lipid further enhances phospholipase-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 effects 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-inflammatory cytokines by macrophages in
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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 efficiency of the lysosome as an intracellular digestive system as well as a route for cytoplasmic delivery of some nutrients. Perturbation of this membrane by polymers may interrupt cytoplasmic metabolic and biosynthetic pathways. In accordance with the solute’s notional hydrogen-binding capacity [72,73] it is unlikely 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 modulate the activity of lysosomal enzymes, transporters and membrane glycoproteins. Such perturbations could alter efflux 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 biological 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 efficient opsonization of mPEG-grafted vesicles in the blood. It is the limited concentration of the blood opsonin, which partly contributes to the prolonged circulation time of mPEG-grafted vesicles in vasculature. In addition, favourable interaction between surface-bound polymers and blood dysopsonins 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 profiles. Another interesting observation is the complement activating nature of stealth particles. Although complement fixation 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 longcirculating vesicles [23]. Complement activation is also associated with the activation of other proteolytic plasma cascades. An example is the kallikrein–kinin system, which provides a co-stimulus 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 PE–mPEG lipids is a logical step towards fulfilling 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
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macrophages either lack these receptors or their binding site(s) are blocked by some blood components (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. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35]
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