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Published as doi: 10.1096/fj.06-6186fje.
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(The FASEB Journal. 2006;20:2591-2593.)
© 2006 FASEB

Methylation of the phosphate oxygen moiety of phospholipid-methoxy(polyethylene glycol) conjugate prevents PEGylated liposome-mediated complement activation and anaphylatoxin production

S. Moein Moghimi*,1, Islam Hamad*, Thomas L. Andresen{dagger}, Kent Jørgensen{dagger} and Janos Szebeni{ddagger}

* Molecular Targeting and Polymer Toxicology Group, School of Pharmacy, University of Brighton, Brighton, UK;

{dagger} LiPlasome Pharma A/S, Technical University of Denmark, Lyngby, Denmark; and

{ddagger} Nephrology Research Group, Hungarian Academy of Sciences and Institute of Pathophysiology, Semmelweis University, Budapest, Hungary

1Correspondence: Molecular Targeting and Polymer Toxicology Group, School of Pharmacy, University of Brighton, Cockcroft Bldg., Lewes Rd., Brighton BN2 4GJ, UK. E-mail: s.m.moghimi{at}brighton.ac.uk

ABSTRACT

Methoxy(polyethylene glycol), mPEG, -grafted liposomes are known to exhibit prolonged circulation time in the blood, but their infusion into a substantial percentage of human subjects triggers immediate non-IgE-mediated hypersensitivity reactions. These reactions are strongly believed to arise from anaphylatoxin production through complement activation. Despite the general view that vesicle surface camouflaging with mPEG should dramatically suppress complement activation, here we show that bilayer enrichment of noncomplement activating liposomes [dipalmitoylphosphatidylcholine (DPPC) vesicles] with phospholipid-mPEG conjugate induces complement activation resulting in vesicle recognition by macrophage complement receptors. The extent of vesicle uptake, however, is dependent on surface mPEG density. We have delineated the likely structural features of phospholipid-mPEG conjugate responsible for PEGylated liposome-induced complement activation in normal as well as C1q-deficient human sera, using DPPC vesicles bearing the classical as well as newly synthesized lipid-mPEG conjugates. With PEGylated DPPC vesicles, the net anionic charge on the phosphate moiety of phospholipid-mPEG conjugate played a key role in activation of both classical and alternative pathways of complement and anaphylatoxin production (reflected in significant rises in SC5b-9, C4d, and C3a-desarg levels in normal human sera as well as SC5b-9 in EGTA-chelated/Mg2+ supplemented serum), since methylation of the phosphate oxygen of phospholipid-mPEG conjugate, and hence the removal of the negative charge, totally prevented complement activation. To further corroborate on the role of the negative charge in complement activation, vesicles bearing anionic phospholipid-mPEG conjugates, but not the methylated phospholipid-mPEG, were shown to significantly decrease serum hemolytic activity and increase plasma thromboxane B2 levels in rats. In contrast to liposomes, phospholipid-mPEG micelles had no effect on complement activation, thus suggesting a possible role for vesicular zwitterionic phospholipid head-groups as an additional factor contributing to PEGylated liposome-mediated complement activation. Our findings provide a rational conceptual basis for development of safer vesicles for site-specific drug delivery and controlled release at pathological sites.—Moghimi, S. M., Hamad, I., Andresen, T. L., Jørgensen, K., Szebeni, J. Methylation of the phosphate oxygen moiety of phospholipid-methoxy(polyethylene glycol) conjugate prevents PEGylated liposome-mediated complement activation and anaphylatoxin production


Key Words: stealth liposomes • anaphylactic reactions • complement system • steric stabilization • SC5b-9 • C3a-desarg

THE RAPID SEQUESTRATION of intravenously (iv) injected liposomes by macrophages in contact with blood is problematic for efficient targeting of particulate nanocarriers to nonmacrophage cells at pathological sites (1) . Surface manipulation of liposomes with methoxy(polyethylene glycol), mPEG, affords control over vesicle interaction and fate within biological systems (1 , 2) . mPEG grafting suppresses liposome-macrophage interaction either directly or through reduced surface opsonization or both (1) . The extent of vesicle opsonization and vesicle-macrophage interactions is controlled by mPEG chain length and surface density. For example, vesicles bearing 5–7 mol% of phospholipid-mPEG2000 conjugate are usually resistant toward macrophage recognition. With shorter mPEG chain length (e.g., 350–1000 Da), bilayer enrichment with 10–25 mol% phospholipid-mPEG is necessary to avoid rapid macrophage phagocytosis (1) . As a result of such surface manipulation strategies with mPEG, the engineered vesicles exhibit prolonged residency time in the blood circulation and can escape from the vasculature. Vesicle extravasation, however, is restricted to capillaries with open fenestrations or sites where the endothelial barrier of blood capillaries is perturbed by inflammatory processes or by dysregulated angiogenesis (1) . Indeed, the first generation of long-circulating PEGylated liposomes with encapsulated doxorubicin is already on the United States and European markets for management and treatment of AIDS-related Kaposi’s sarcoma, refractory ovarian cancer, and metastatic breast cancer (3) .

One of the most frequently encountered clinical problems after infusion of PEGylated liposomes into certain subjects is initiation of non-IgE-mediated hypersensitivity reactions, which includes symptoms of cardiopulmonary distress, such as dyspnea, tachypnea, tachycardia, chest pain, hypertension, and hypotension (4 5 6) . These pseudoallergic reactions are strongly believed to arise through rapid production of anaphylatoxins C3a and C5a via complement activation leading to the release of TXA2 and other anaphylatoxin-derived mediators (6 , 7) . Despite the general view that surface camouflaging with mPEG should dramatically suppress blood opsonization processes, liposomes bearing phospholipid-mPEG conjugates in their bilayer surprisingly activate the complement system and fix complement proteins (7 , 8) . However, complement fixation seems to play a minor role in macrophage clearance of PEGylated vesicles via complement receptors since PEGylated liposomes remain intact in the blood pool for prolonged periods of time (7 , 8) . Interestingly, liposomes of the same size distribution and bilayer composition as their PEGylated counterparts but without the phospholipid-mPEG conjugates rarely activate human complement system and hence do not generate anaphylatoxin. In view of numerous medical applications for PEGylated liposomes and the clinical importance of the observed complement-mediated hypersensitivity reactions that can lead to anaphylactoid shock and cardiac anaphylaxis (9) , we sought to investigate and identify which structural features of the phospholipid-mPEG conjugate are responsible for PEGylated liposome-induced complement activation in human sera. Our efforts were particularly focused on design and synthesis of novel lipid-mPEG conjugates where the phospholipid moiety of the conjugates is made from prodrug ether lipids, as a first critical step toward development of safer long circulating vesicles for drug release at pathological sites with elevated sPLA2 activity (10 , 11) . Accordingly, we have designed PEGylated liposomes that do not activate complement both in vitro and in vivo.

MATERIALS AND METHODS

Materials
DPPE-mPEG350 and DPPE-mPEG2000 were from Avanti Polar Lipids Inc. (Alabaster, AL, USA). [14C]Inulin was from Amersham International (Amersham, UK). DPPC, DPPG, ELISA kits for plasma TXB2, and all other reagents were obtained from Sigma Chemical Company (Poole, UK). C1q-depleted human serum, purified human C1q, and ELISA kits (SC5b-9, C4d, Bb, and C3a-desarg) were from Quidel Corporation (San Diego, CA, USA). Mouse anti rat CD11b (azide free, clone MRC OX-42) and polyclonal mouse IgG were from Serotec (Oxford, UK). Mouse monoclonal antibody (mAb) against human complement factor B was obtained from Antibody (Ab) Shop A/S (Gentofte, Denmark). Near monodisperse PEGs, molecular weight = 400 and 1960 Da, were from Fluka (Gillingham, UK).

Synthesis of phospholipid-mPEG conjugates
The synthesis of 1-O-DPPE-(Me)mPEG350 (compound 5) and 1-O-DPPE-mPEG350 (compound 6) was carried out using (R)-O-benzyl glycidol as a convenient starting material as described in detail elsewhere (Fig. 1 ; ref 12 ). The synthetic approach to the two ether lipids involves the synthesis of compound 3 (Fig. 1A ), which is readily formed from compound 1 on multigram scale (12) . Preparation of the protected phosphatidylethanolamine 4 was carried out using methyl dichlorophosphate and TMP as base. Deprotection followed by coupling with mPEG350 gave the desired 1-O-DPPE-(Me)mPEG350 (5, termed Conj-B). Conj-B was converted to 1-O-DPPE-mPEG350 (6, termed Conj-A). The two conjugates were analyzed by 1H-NMR (300 MHz), 13C-NMR (75 MHz), and HPLC using an evaporative light scattering detector as described in detail earlier (12) .


Figure 1
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  		Figure 1. Synthetic pathway and chemical structures of prodrug ether lipid-mPEG conjugates (Conj-A and Conj-B). Synthetic steps are as follows: (step a): hexadecanol (C16H33OH), NaH, tetrahydrofurane (THF), dimethylformamide (DMF), 16h, 80°C, then palmitoyl chloride, pyridine, hexane, 16 h, room temperature; (step b): H2, Pd/C, ethyl acetate, 1.5 h, room temperature; (step c): methyl dichlorophosphate, TMP, toluene, 16 h, room temperature, then N-BOC-ethanolamine, TMP, 24h, room temperature; (step d): TFA, MeOH, CH2Cl2, 0.5 h, 0°C, then mPEG350 N-succinimidecarbonate, Et3N, CHCl3, 2 h, 40°C; (step e): NaI, 2-butanone, 2h, 75°C. Detailed procedures for reactions and purification steps are described by Andresen et al. (11) .

Preparation and characterization of liposomes and phospholipids-mPEG micelles
Liposomes were composed of either DPPC or 1-O-DPPC with or without various amounts of different phospholipid-mPEG conjugates. Some DPPC preparations contained 5–20 mol% DPPG. Generally, liposomes were prepared by hydrating the dried lipid film with 10 mM PBS, pH 7.2 and then extruded through polycarbonate Nuclopore filters of appropriate pore diameters using a high-pressure extruder. Some liposomal preparations contained [14C]inulin as an established aqueous space label for macrophage uptake studies (13) . To assess liposome stability and to ensure association of the radiolabel with vesicles when exposed to serum, vesicles were incubated in 25% v/v of fresh human serum for 1 h at 37°C. The mixture was then passed through the Sepharose 6B column and eluted with 0.05 mM phosphate/saline buffer, pH 7.4. The 14C radioactivity in each fraction was measured. Free inulin does not adsorb to the surface of liposomes (13) .

The critical micelle concentration of selected phospholipids-mPEG conjugates was determined by measuring pyrene solubilization. The formation of micelles is associated with pyrene solubilization, which is monitored by measuring the solution fluorescence.

Liposome and phospholipids-mPEG micelle size distribution was determined by laser light scattering using a Malvern Zetasizer 3000 (Malvern Instruments, Malvern, UK) at 25°C as described previously (13) .

Collection and treatment of serum specimens
Blood was drawn from healthy male volunteers according to approved local protocols. Blood was allowed to clot at room temperature, and serum was prepared, aliquoted, and stored at –80°C. Serum samples were thawed and kept at 4°C before incubation with test reagents.

Commercially available C1q-depleted human serum was further depleted from Factor B. This was achieved by incubating serum with mouse monoclonal anticomplement factor B antibodies coupled to activated CNBr-sepharose as described previously (14) . Depletion of factor B was monitored by measuring zymosan-induced generation of serum complement activation product SC5b-9 (see below).

Liposome-macrophage interaction
Resident peritoneal macrophages were obtained by washing the peritoneal cavity of male Wistar rats (250±30 g) with HBSS, followed by centrifugation at 150 g for 5 min. Contaminated red cells were lysed by Tris-ammonium chloride buffer (pH 7.2). Cells were washed twice in HBSS and resuspended in the same buffer. Macrophages were plated in 48-well tissue culture dishes at a density of 5 x 105 cells per well in HBSS containing 10% v/v fetal calf serum. Cells were incubated for 24 h at 37°C and afterward the medium was replaced either by a serum-free medium or a medium supplemented with 25% (v/v) human serum (37°C). Cell viability was >95%. An hour later different types of [14C]inulin-labeled liposomes were added (200 nmol phospholipid), and cells were left to incubate for an additional 3 h. At the end of the incubation, cells were washed three times with HBSS and then solubilized with 33% KOH. Samples were neutralized with HCl, and the radioactivity was measured using a suitable scintillant in a ß-counter. For each liposome type, triplicate incubations were performed. In some experiments, cells were preincubated with mouse azide-free antirat CD11b at saturation for 15 min in the absence of serum (13) . Corresponding control incubations contained a nonspecific Ab (polyclonal mouse IgG, azide-free). Unbound antibodies were removed by washing cells gently three times with the medium, which was followed by liposome addition and human serum. Liposome uptake was assessed as described above. Protein concentrations were determined by Bio-Rad assay.

Assay of in vitro complement activation
To measure complement activation in vitro, we determined the liposome-, micelle-, and PEG-induced rise of serum complement activation product SC5b-9, C4d, Bb, and C3a-desarg formation, using respective Quidel’s ELISA kits according to the manufacturer’s protocol as described elsewhere (7 , 15 , 16) . Because of substantial biological variation in serum levels of complement proteins and the large number of positive and negative feedback interactions, we monitored generation of complement activation products in sera of five healthy individuals separately. The reaction was started by adding the required quantity of liposomes, micelles, PEGs, and other required additives to undiluted serum (liposome-to-serum volume ratio of 1:4) in Eppendorf tubes (in triplicate) in a shaking water bath at 37°C for 30 min, unless stated otherwise. The final incubation volume was 150 µl. Reactions were terminated by addition of "sample diluent " provided with assay kit. SC5b-9 generation was also monitored in C1q-depleted human serum before and after addition of physiological concentration of C1q (180 µg/ml). In some experiments, factor B was immunochemically depleted from C1q-depleted serum. Control incubations contained 10 mM PBS (pH 7.2) for assessing background levels of SC5b-9, C4d, Bb, and C3a-desarg. Zymosan (5 mg/ml) was used as a positive control for complement activation.

The efficacy of liposome treatments was established by comparison with baseline levels using paired t test; correlations between two variables were analyzed by linear regression, and differences between groups (when necessary) were examined using ANOVA followed by multiple comparisons with Student-Newmann-Keuls test.

Determination of complement hemolytic activity and TXB2 level in rat blood
Before liposome injection, 1–1.5 ml blood were taken from the tail vein of male Wistar rats (310±30 g) for both plasma and serum preparation to obtain the required baseline parameters. For plasma preparation, blood was collected in EDTA/0.25 mM indomethacin-containing tubes to prevent prostaglandin metabolism. Appropriate liposome formulations were injected intravenously (80 mg/kg body wt in 0.5–1.0 ml), bolus injection within 5 s, via the opposite tail vein. Further blood samples were taken at 8 and 60 min post-liposome injection to obtain plasma and serum. Plasma TXB2 levels were determined by following the procedures supplied with the ELISA kit. Undiluted serum was used to measure CH50 using sheep red blood cell hemolysis assay as described in detail elsewhere (17) .

RESULTS AND DISCUSSION

Conjugate synthesis and rationale for liposome design
One of the key structural features in mPEG-phospholipid conjugate is the presence of a net anionic charge localized on the phosphate oxygen moiety of the mPEG-phospholipid conjugate, which could be responsible for complement activation. This hypothesis is based on previous observations that small unilamellar neutral non-PEGylated liposomes (liposomes of similar composition to clinical formulation of PEGylated vesicles but without the phospholipid-mPEG conjugate) causes no or minimal activation of the human and pig complement system (18 , 19) . On the contrary, bilayer enrichment of non-PEGylated liposomes with dicetylphosphate or a variety of anionic phospholipids, regardless of their headgroup structure, induces complement activation in both human and animal sera (7 , 8 , 20 21 22 23) . Therefore, we synthesized two types of phospholipid-mPEG conjugates bearing different charges as demonstrated in Fig. 1 . The key structural features of the first conjugate (Conj-A) mirror PE-mPEG, a conjugate with a proven capability of prolonging the circulatory half-life of liposomes in the vasculature. Thus, the phosphate moiety of Conj-A is anionic and its phosphodiester and amide linkage groups are the same as PE-mPEG. In the second conjugate (Conj-B), the phosphate oxygen is methylated to eliminate the net negative charge, thus yielding a nonionic species (Fig. 1) . The mPEG segment of both Conj-A and -B are purposely short in length (7 ethylene glycol units, molecular weight=350 kDa) for better exposure of phosphodiester linkage and liposome surface to complement proteins, thus testing the validity of the proposed complement activation hypothesis.

In contrast to classical phospholipid-mPEG conjugates, the phospholipid component of Conj-A and Conj-B bears a nonhydrolyzable ether bond in the 1-position (1-O-phospholipid). This feature is not directly applicable to complement activation hypothesis but is essential for designing long-circulating multifunctional 1-O-phospholipid-based vesicles for efficient drug delivery and trigger release at selected nonmacrophage pathological sites, in which we have an active interest. 1-O-phospholipids form highly stable liposomes with no hemotoxicity as demonstrated recently (11 , 12) . These vesicles also function as prodrugs, sPLA2 and its subtypes being the activating enzymes, and their levels are dramatically elevated in the interstitium of solid tumors as well as at various inflammatory sites (10 , 11) . sPLA2 not only acts as a trigger resulting in the release of encapsulated cytotoxic drugs from pro-drug ether lipid liposomes but also generates highly cytotoxic lysolipids that destabilizes plasma membrane of cancer cells as shown recently (11) .

We incorporated 1-O-DPPE-mPEG conjugates (Conj-A and Conj-B) into DPPC liposomes, since the resultant vesicles are highly stable in serum and in addition DPPC vesicles do not activate the human complement system (see below). Also, the bilayer of constructed vesicles is not enriched with cholesterol, since incorporation of cholesterol, particularly at >30 mol%, is known to activate the human complement system via the classical pathway after binding of anticholesterol antibodies, which are abundant in most human sera (24) . Indeed, cholesterol-rich liposomes have been shown to cause massive hemodynamic changes in pigs via complement activation (18) .

Effect of phospholipid-mPEG conjugates on liposome-macrophage interaction
Inclusion of either Conj-A or -B (5 mol%) in DPPC vesicles suppressed liposome-macrophage interaction by ~50% in the absence of serum (also comparable with vesicles containing 5 mol% DPPE-mPEG350) when compared to non-PEGylated vesicles containing 5 mol% of the negatively charged phospholipid DPPG in their bilayer (Fig. 2 A, B). Increasing the bilayer concentration of either conjugates or DPPE-mPEG350 to 15 mol% resulted in further reduction in liposome-macrophage interaction with values similar to that observed with vesicles containing 5 mol% DPPE-mPEG2000 (vesicles exhibiting maximum resistance to macrophage interaction and are known to be long circulatory in vivo). Thus, vesicles bearing 15 mol% of either conjugates in their bilayer have a sufficient density of surface mPEG350 chains to combat macrophage recognition in the absence of serum.


Figure 2
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  		Figure 2. Interaction of liposomes with rat peritoneal macrophages. Liposomes were composed of DPPC (95 mol%) and stated lipids/lipid-mPEG conjugates (5 mol%). When bilayer concentration of added lipids increased to 15 mol%, DPPC content was reduced to 85 mol% (designated values in the brackets represent mol% content of the added lipids). All liposomes were in size range of 70–110 nm in diameter. A) Comparison of effect of phospholipid-mPEG conjugate type on liposome-macrophage interaction both in the absence and presence of human serum (serum was from subject 1). Horizontal line shows extent of interaction between DPPC:DPPG (9.5:0.5) liposomes and peritoneal macrophages in the absence of serum. B) Macrophage recognition of non-PEGylated negatively charged liposomes (DPPC:DPPG, 9.5:0.5 mol ratio) both in the absence (black column) and presence of serum of subject 1 (open column). In the presence of serum, liposome recognition by macrophages decreased dramatically when cells were pretreated with complement receptor Mac-1 Ab (dotted line). C) Effect of complement inactivated sera (zymosan-treated) and complement receptor Mac-1 on PEGylated liposome-macrophage interaction. Mac-1 Ab incubations are referred to macrophages preincubated in the presence of Mac-1 Ab at saturation followed by washing and addition of liposomes and designated sera.

The binding of particulate entities opsonized with human complement proteins (e.g., iC3b) to complement receptors present on rat peritoneal macrophages (CD11b or CR3) is well established (25) . Here, we also show that human serum can stimulate macrophage recognition of DPPG containing liposomes by several folds, principally via CD11b (Mac-1) receptor (Fig. 2B ). Serum also enhanced interaction of vesicles containing 5 mol% DPPE-mPEG350 and Conj-A with macrophages when compared to their corresponding control incubation (no serum), but the extent of interaction was much lower than that of DPPG vesicles. However, the stimulatory effect of serum on liposome-macrophage interaction was dramatically reduced when the bilayer concentration of either DPPE-mPEG350 or Conj-A was increased to 15 mol%. Ab blockage of rat macrophage complement receptor (Mac-1) reduced serum-mediated interaction of both Conj-A and DPPE-mPEG350 (5 mol%)-containing liposomes by 60%, thus indicating the ability of these vesicles to activate complement and fix opsonizing complement proteins (Fig. 2C ). This suggestion is further corroborated by parallel studies with complement inactivated sera (zymosan-treated sera; Fig. 2C ). On the contrary, serum failed to exert any stimulatory effect on liposome-macrophage interaction with vesicles bearing Conj-B (at the same membrane concentration as Conj-A, 5 mol%), and further blockage of complement receptors had no effect on serum-mediated macrophage clearance of Conj-B-containing liposomes (Fig. 2A , C). Poor macrophage uptake of these vesicles in the presence of serum was not a reflection of leakage and loss of the entrapped macromolecular radiolabel from liposomes via the possible formation of the membrane attack-complex as determined by gel-permeation chromatography of the incubation medium as well as liposomes incubated with human serum; in all cases >80% of initial radioactivity was associated with liposomes during the experimental time frame.

These observations strongly suggest that the negative charge of the phosphate moiety of Conj-A and DPPE-mPEG350 plays a key role in complement activation, at least when incorporated at 5 mol% in to the liposomal bilayer. Therefore, our attention was turned toward direct complement activation measurements and anaphylatoxin production in healthy human sera with functional complement system.

Liposome-mediated complement activation in vitro
We demonstrated complement activation in sera of a number of healthy subjects with liposomes containing an anionic phospholipid (DPPC:DPPG, mole ratio 9.5:0.5) by measuring the generation of the complement activation product SC5b-9, which is a sensitive and established measure of the activation of the whole complement cascade (Fig. 3 A; ref 26 ). These observations are in line with previous reports (7 , 20 , 21) , suggesting that vesicles exhibiting a net negative charge, but not neutral DPPC vesicles, activate the human complement system. PEGylated liposomes also caused complement activation with significant rises of SC5b-9 levels over baseline, comparable to non-PEGylated DPPC:DPPG vesicles of the same size distribution in all tested sera (Fig. 3A ). With Conj-A-containing (5 mol%) liposomes SC5b-9 generation proceeded on a time scale of minutes, also identical to DPPE-mPEG350-incorporated vesicles and reached plateau at around 15–20 min (not shown). Further, Conj-A-containing liposomes were effective in causing significant rises of SC5b-9 levels in serum at a final lipid concentration of 1 mg/ml, with a trend of reaching maximal efficacy at lipid concentrations of 3–4 mg/ml (not shown). As a control experiment, we assessed SC5b-9 generation in serum after incorporation of 1-O-DPPC (5 mol%) into DPPC vesicles as well as by pure 1-O-DPPC vesicles (Fig. 3B ). Indeed, 1-O-DPPC was ineffective in raising serum SC5b-9 levels above the baseline, but bilayer enrichment of 1-O-DPPC vesicles with anionic phospholipid-mPEG conjugates raised serum SC5b-9 levels in a similar manner to PEGylated DPPC vesicles (Fig. 3B ). Thus, complement activation, as reflected by increase in serum levels of SC5b-9, proceeds with anionic lipid-mPEG conjugate-incorporated liposomes independent of mPEG molecular weight (350 and 2000 kDa) or its conjugated lipid structure (DPPC or 1-O-DPPC).


Figure 3
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  		Figure 3. Effect of liposomes on complement activation. A) SC5b-9 levels in sera of 5 healthy subjects 30 min after liposome treatment (3 mg lipid/ml) at 37°C. Zymosan (5 mg/ml) was used as a positive control. Values in brackets represent lipid mole ratios. With the exception of neutral DPPC liposomes and vesicles containing Conj-B, generated SC5b-9 levels were significantly higher than the background (control/PBS), P < 0.05. B) Effect of 1-O-DPPC bearing liposomes (70–110 nm) on SC5b-9 generation in serum of subject 5. C) Influence of lipid-mPEG conjugates (5 mol%) on DPPC liposome-mediated anaphylatoxin generation (C3a-desarg) over time in serum of subject 1. Results are expressed as percentage of control/PBS baselines. With exception of Conj-B, results were significantly different relative to control/PBS (P<0.05). All liposomes were in 70–110 nm size range.

To further confirm the role of the anionic charge of the phosphate oxygen moiety in complement activation, we demonstrated rises in C3a-desarg levels in human serum over time (Fig. 3C ). The rise of SC5b-9 levels in human sera remarkably correlates with C3a production, providing further evidence that the anionic phosphate-induced rise of SC5b-9 levels is a reflection of complement activation rather than modulation of the terminal pathway only.

The PEG moiety of liposomes, regardless of their molecular weight, plays no role in complement activation as demonstrated in experiments with endotoxin-free near monodisperse PEG1960 (polydispersity index=1.03) and PEG400 (polydispersity index=1.07). Neither PEGs (2.5 mg/ml, final concentration in serum) were able to raise serum levels of C4d, Bb, and SC5b-9 levels above the background (Fig. 4 ).


Figure 4
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  		Figure 4. Effect of near monodisperse PEG1960 and PEG400 on complement activation. PEG concentration was 2.5 mg/ml in serum, and results are expressed as percentage of respective control/PBS baselines for C4d, Bb, and SCb-9. Zymosan (5 mg/ml) was used as a positive control.

Effect of surface mPEG-density and vesicle size
Increasing the bilayer concentration of anionic Conj-A >2.5 mol% elevated serum SC5b-9 levels, and the effect was more dramatic with vesicles bearing 10–15 mol% Conj-A (Table 1 ). Similar observations were made with DPPC vesicles containing the corresponding levels of DPPE-mPEG350 (not shown). These experiments indicated the importance of surface charge density in complement activation. This is a surprising observation, since the high density of surface projected mPEG chains, which are believed to assume a mushroom-brush conformation (27 28 29 30) , should sterically shield the liposome surface against protein adsorption.


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  		Table 1. Effect of liposome size and bilayer composition on SC5b-9 generation in human serum

These observations also seem to deviate from the results of liposome-macrophage interaction in the presence of serum, since liposomes with 15 mol% DPPE-mPEG350 (or Conj-A) interacted poorly with macrophages when compared to vesicles containing 5 mol% of the corresponding lipid-mPEG conjugate (Fig. 2A ). Parallel to these experiments serum also failed to enhance the interaction of DPPE-mPEG2000 containing liposomes with macrophages (Fig. 2A ), despite earlier published observations that mPEG2000-coated vesicles are capable of fixing C3b, and the inability of surface mPEG chains to interfere with C3b inactivation (7 , 31) . Again, in all cases, poor liposome-macrophage interaction was not a reflection of leakage of the liposomal aqueous marker (80–85% of initial radioactivity was associated with the corresponding liposomal formulations in the presence of 25% v/v human serum). Since the described liposomes can activate complement, the discrepancies in cell uptake studies may be rationalized by the steric hindrance of mPEG chains to binding of opsonized vesicles to CD11b. Hence, these results are in agreement with the reported prolonged circulation times of PEGylated vesicles (1) . In contrast to anionic conjugates, increasing the bilayer concentration of nonionic mPEG-lipid (Conj-B) in DPPC vesicles to 15 mol% failed to elevate serum SC5b-9 levels above the baseline, and the results were comparable to noncomplement activating DPPC vesicles (Table 1) . This again confirms a role for the anionic phosphate oxygen in activating complement.

Vesicle size have been demonstrated to play a critical role in complement activation; smaller vesicles (sub 100 nm) are less-effective complement activators than their larger counterparts for equivalent concentrations (18 , 21 , 23) . This reflects the importance of geometric factors and surface dynamics on the initial assembly of proteins involved in complement activation. In addition, surface curvature affects projected mPEG chain conformation; this in turn may exert some control over complement consumption and activation (8 , 32) . The results in Table 1 further confirm the noncomplement activating nature of larger-sized Conj-B bearing liposomes when tested with bilayer concentrations up to 15 mol%. However, increasing size causes more complement activation with vesicles containing Conj-A.

Liposome-mediated complement activation in rats
Next, we examined complement activation in vivo. In rats, intravenous injection of liposomes containing 5 mol% of anionic phospholipid-mPEG conjugates (regardless of mPEG chain length) was associated with a significant reduction in serum total hemolytic activity on a time scale of minutes with a parallel rise of TXB2 (Fig. 5 ). TXB2 is an established and direct marker for TXA2 production (17) . Anaphylatoxins arising from complement activation (C3a and C5a) induce TXA2 release from blood cells, thus demonstration of increased serum TXB2 levels provides further evidence for in vivo PEGylated liposome-mediated complement activation (17) . On the contrary, neither in vivo TXB2 levels nor in vitro complement activity in serum (CH50 measurements) was affected with liposomes bearing nonionic lipid-mPEG conjugate (Fig. 5) , thus confirming a role for the negative charge of phosphate oxygen in complement activation.


Figure 5
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  		Figure 5. Plasma TXB2 levels in rats following bolus iv injection of liposomes (80 mg/kg in 0.5 ml). Liposomes were composed of DPPC and 5 mol% of designated conjugates (size range 80–130 nm). Similar results were obtained with liposomes bearing 10–15 mol% DPPE-mPEG350 conjugate or Conj-B (not shown). The 50% serum hemolytic complement activity (CH50) represents values at 8 min post-liposome injection and is expressed as % of respective baseline level. With the exception of Conj-B, CH50 values were significantly different from baseline (2-sided t test, P<0.05).

Involvement of both classical and alternative pathways
Having established a critical role for anionic phospholipid-mPEG conjugates in liposome-mediated complement activation, we next sought to investigate through which pathway(s) complement activation proceeds. Calcium is essential for operation of both classical and MBL pathways, whereas operation of the alternative pathway is fully maintained in EGTA-chelated (Mg2+ supplemented) serum (16 , 17) . Studies in EGTA-chelated sera of the five individuals demonstrated significant but variable reduction (15–60%, depending on serum) in SC5b-9 levels after challenge with liposomes bearing anionic phospholipid-mPEG conjugates. This confirmed a direct role for the alternative pathway, rather than amplification of C3 convertase initially triggered through calcium-sensitive pathways. To address the role of calcium-sensitive pathways, we also followed the liposome-induced changes in C4d levels in sera, and these were elevated by 1.8- to 3-fold above the background level as determined by ELISA, depending on serum source.

To further elucidate the mechanism of complement activation by anionic phospholipids-mPEG conjugates, we used C1q-depleted serum, which enables clarification of the role of C1q-dependent classical pathway activation. The results in Fig. 6 show a significant reduction in SC5b-9 generation in C1q-depleted serum with liposomes bearing anionic phospholipid-mPEG conjugates, when compared to incubations with restored physiological levels of the deficient factor (C1q), thus confirming the involvement of C1q-dependent classical pathway. As a positive control, zymosan-mediated complement activation proceeded efficiently in C1q deficient serum and the process was unaffected after the addition of C1q, as zymosan activates complement via all three pathways. Notably, liposome-mediated elevation of SC5b-9 in C1q-depleted serum was significantly above the baseline level (Fig. 6) . This was due to direct involvement of the alternative pathway, since SC5b-9 levels in EGTA-chelated (Mg2+ supplemented) C1q-depleted serum remained unchanged. Furthermore, immunochemical depletion of factor B in C1q-depleted serum totally abolished SC5b-9 generation by both liposomes and zymosan (control experiment) providing further evidence for direct involvement of the alternative pathway.


Figure 6
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  		Figure 6. Liposome-mediated SC5b-9 generation in C1q-depleted human serum. DPPC liposomes were used and contained 10 mol% of designated conjugates in their bilayer. In some incubations, C1q levels were restored to physiological concentrations (180 µg/ml), whereas in others factor B was depleted immunochemically. Zymosan (5 mg/ml) was used as positive control for activation of the alternative pathway. *P < 0.05.

We did not investigate contribution of the MBL pathway, since MBL selectively recognizes glucans, lipophosphoglycans, and glycoinositol phospholipids that contain mannose, glucose (Glc), fucose, or N-acetylglucosamine as their terminal hexose, and none of which were presented on our liposomes (23 , 33) .

Role of phospholipid head-group: A hypothesis
In contrast to PEGylated liposomes, anionic phospholipid-mPEG350 and -mPEG2000 conjugates in micellar form were incapable of rising serum SC5b-9, C4d, Bb, and C3a-desarg levels above the respective baseline (not shown). Thermodynamically, micellar solution is at equilibrium, the concentration of monomers being equal to the critical micelle concentration. Hence, neither anionic phospholipid-mPEG monomers nor micelles are capable of activating the human complement system. However, lack of complement activation by these micelles may be due to their small hydrodynamic size, which was in the region of 25–32 nm as determined by photon correlation spectroscopy. On the contrary, related nonionic micelles (2.5–10 mg/ml) of similar sizes such as those assembled from polyoxyethene/poloxypropylene block copolymers (e.g., poloxamer 407 and poloxamine 908) activate complement in human serum (2- to 4-fold rise in serum Bb and SC5b-9 levels above the respective background). On the basis of these observations, we can not exclude a possible role for liposomal zwitterionic phospholipid head-groups as an additional (or prerequisite) factor contributing to complement activation. Recent studies have demonstrated that after incubation in serum, the surface of PEGylated liposomes becomes coated with apolipoproteins, antibodies, and complement proteins (7 , 8 , 31) . We therefore propose that for binding to PEGylated liposomes, complement activating natural antiphospholipid antibodies (IgG and IgM) may require the presence of both DPPC head-groups and the anionic moiety of phospholipid-mPEG in a spatial relationship that orients the Ab into a complement activating posture. Indeed, structural modeling studies have demonstrated that the Fab/Fc orientation of IgG might be a key factor in controlling access of the C1q globular B module to the CH2 domain of human IgG1 (34 , 35) . Likewise, the globular internal A and/or C modules of C1q show preferential binding to oligomeric IgM (34 , 36) . It is also known that antibodies can activate the alternative pathway via their F(ab) portion (37 , 38) , the binding of which to C3b is also likely to depend on a two antigenic subsite-fixed orientation of IgG. Thus, on the basis of our observations, we further suggest that the steric arrangement imposed by the bulky methyl group in Conj-B either prevents simultaneous binding of antiphospholipid antibodies to the phosphate oxygen moiety of both phospholipid head-groups and phospholipid-mPEG conjugates and/or interfere with spatial organization of surface-bound antibodies for correct recognition by all three modules of globular C1q domain.

We also like to stress that the presence or absence of additional epitopes for antiphospholipid Ab binding, such as apolipoprotein-H (24 , 39 , 40) , and direct binding of C1q (41 , 42) to the anionic bilayer surface are further factors for consideration. As for the latter process, the top of the C1q head is predominantly basic and as a result C1q can function as a charge pattern recognition molecule. Indeed, the highly cationic region of C1q A chain, comprising residues 14–26, binds to cardiolipin-containing liposomes (41) . Binding of C1q to phosphatidylglycerol-containing liposomes has also been demonstrated (42) , where after binding C1q and phosphatidylglycerol colocalize into domains with characteristic radii of curvature higher than that of the surrounding bilayer, which eventually bud into small vesicles. Hydrophobic interactions and/or hydrogen bonding have also been suggested to participate in the binding of C1q to cardiolipin-containing liposomes (42) ; hydrogen bonding is presumably mediated by the central 2'-hydroxyl moiety of the glycerol backbone of cardiolipin. With respect to these observations and the results in Fig. 6 , we can not exclude a direct role for small numbers of C1q molecules in initiating complement activation by liposomes bearing anionic phospholipid-mPEG conjugates. Thus, in addition to electrostatic interactions between C1q and the anionic phosphate oxygen, the ether oxygen groups in the mobile mPEG moiety may play a role in accommodating C1q on the liposome surface via hydrogen bonding.

CONCLUSIONS

We have elaborated on the involvement of the anionic charge localized on the phosphate oxygen moiety of phospholipid-mPEG conjugates in PEGylated liposome-mediated complement activation and anaphylatoxin production. Subsequently, we designed liposomes bearing a nonionic 1-O-phospholipid-mPEG conjugate that do not activate complement in human and rat sera. This is a critical step toward development of safer zwitterionic vesicles, which are temperature sensitive as well as susceptible to degradation by sPLA2. These observations and strategies not only provide a rational conceptual basis for design of safer PEGylated liposomes for site-specific drug delivery and targeting but also highlight the importance of linkage chemistry in complement activation. The latter is of importance for surface engineering of implants and nanodevices with mPEG conjugates and related polymers for in vivo applications. We still need to fully understand how methylation of the phosphate oxygen can affect Ab and C1q accommodation on the liposome surface. Understanding of these events may eventually lead to prediction and elimination of subjects at risk and even enhance the utility of PEGylated liposomes bearing anionic phospholipid-mPEG conjugates among low risk individuals.

Others have recently synthesized a range of neutral lipopolymers, such as distearoyl glycerol (carbamate-linked)mPEG and variations thereof for liposome engineering (43) . Remarkably, preliminary investigations have also demonstrated that such lipopolymer-incorporated liposomes are, indeed, poor activators of the human and porcine complement system when compared to vesicles bearing anionic phospholipid-mPEG conjugates (44) , thus supporting the stated hypothesis. Other related uncharged lipid conjugates for construction of stealth liposomes includes mPEG-substituted synthetic ceramides (45) , but such conjugates generally exhibit poor packing into the phospholipid bilayer.

Received for publication April 28, 2006. Accepted for publication August 14, 2006.

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