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ABSTRACT In this chapter, our initial observations on activation of the complement system by poly(amino acid) (PAA)‐liposomes are reported. Complement activation by PAA‐liposomes was evaluated in vitro using an SC5b‐9 ELISA and a red blood cell lysis assay, and in a porcine model of liposome‐induced complement‐activation related cardiopulmonary distress. Like other liposome types, PAA‐liposomes also appear to activate the complement system.
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INTRODUCTION Long‐circulating, sterically stabilized liposomes (often also referred to as ʹstealthʹ liposomes) are frequently used as pharmaceutical carriers (1). Their delivery concept makes use of their passive targeting properties to tumors and sites of inflammation, where they can extravasate due to the enhanced permeability and retention (EPR) effect (2). A steric stabilization layer of a hydrophilic polymer on the liposome surface, such as the frequently used poly(ethylene glycol) (PEG), is responsible for the reduced uptake by cells of the mononuclear phagocyte system which in turn results in prolonged circulation times as compared to non‐coated liposomes. Liposomes ‐ coated but also uncoated ‐ have been reported to activate the complement system in preclinical studies (3). In the clinical setting, adverse respiratory, hemodynamic and hematological changes may occur in patients upon complement activation and potentially lead to hypersensitivity reactions (HSR) which occur immediately upon intravenous administration and are reflected in changes in systemic blood pressure and respiration rate, facial edema and chest and back pain (4). In a porcine model of liposome‐induced cardiopulmonary distress, it was demonstrated that the liposome‐induced hemodynamic reactions are very similar to the observed human hypersensitivity responses, and likely a consequence of complement activation (4). We recently designed alternative sterically stabilizing polymers based on poly(amino acid)s (PAAs), in particular poly(hydroxyethyl L‐glutamine) (PHEG) and poly(hydroxyethyl L‐asparagine) (PHEA), both coupled to a hydrophobic anchor to graft them onto the liposome bilayer (5). To evaluate whether PAA‐coated liposomes activate the complement system, initial experiments with PHEA‐ and PHEG‐liposomes in vitro were carried out, using an SC5b‐9 ELISA assay and a red blood cell lysis assay. With the SC5b‐9 ELISA activation of all three complement pathways (classical, alternative and lectin pathway) in human serum samples can be assessed (6). SC5b‐9 is the soluble, non‐lytic form of the terminal complement complex (TCC), which is generated by the assembly of the complement factors C5‐C9 and subsequent binding to the regulatory S protein (7). The amount of SC5b‐9 is proportional to the total generated TCC and therefore total complement activation. With the red blood cell lysis assay functional complement can be assessed. In addition to the in vitro experiments, the potential of PAA‐liposomes to evoke hypersensitivity reactions in the porcine model was studied.
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MATERIALS AND METHODS Materials. Poly(hydroxyethyl L‐asparagine)‐N‐succinyldioctadecylamine (PHEA‐DODASuc, average MW = 3000 Da, determined by NMR and MALDI‐ToF mass spectrometry, corresponding to an average degree of polymerization of 15) and poly(hydroxyethyl L‐glutamine)‐N‐succinyldioctadecylamine (PHEG‐DODASuc, average MW = 4000 Da, determined by NMR and MALDI‐ToF mass spectrometry, corresponding to an average degree of polymerization of 18) were synthesized as described previously (5). Dipalmitoyl phosphatidylcholine (DPPC) was donated by Lipoid GmbH, Ludwigshafen, Germany. Cholesterol was purchased from Sigma‐Aldrich Chemie BV, Zwijndrecht, The Netherlands. The SC5b‐9 ELISA kit was purchased from Quidel Co., San Diego, CA, USA. Sheep erythrocytes were obtained from the Walz farm, Hagerston, MD. Hemolysin was a product of Research Diagnostics, Inc., Concord, MA. All other reagents were of analytical grade.
Liposome preparation. PHEA‐ and PHEG‐liposomes were prepared by a film‐extrusion method (8). A lipid mixture composed of DPPC/cholesterol/PAA (molar ratio 1.85:1:0.15) was dissolved in about 10 mL of a mixture of chloroform/methanol (1:1 v/v) in a 100 mL round‐bottom flask. A lipid film was obtained by evaporation of the solvent under reduced pressure at 50 ˚C. After flushing with nitrogen, the lipid film was hydrated in 20 mL HEPES buffered saline (HBS, 10 mM HEPES, 0.9% NaCl, pH 7.4) yielding a total lipid concentration of 60 μmol/mL. Liposomes were sized by multiple extrusion through polycarbonate filters with a high‐pressure extrusion device. The mean particle size and polydispersity index of the liposome dispersions (diluted 1:10 in HBS) were determined by dynamic light scattering (DLS) using a Malvern ALV/CGS‐3 Goniometer. Phospholipids were quantified spectrophotometrically according to the method of Rouser (9).
SC5b‐9 ELISA. Serum from healthy volunteers was incubated with the PAA‐liposome formulations (4:1 v/v, 8 μmol total lipid/mL) for 30 minutes at 37 °C. After incubation the samples were diluted 20‐fold in the ‘sample diluent’ of the SC5b‐9 ELISA kit and 100 μL from this mixture were applied into the wells of the ELISA plate in triplicate. For a schematic representation of the assay principle see Figure 1 (for a detailed description of the method see (6)).
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Figure 1. Schematic representation of the SC5b‐9 ELISA assay principle. Upon possible liposome‐induced complement activation in the serum samples the SC5b‐9 complex is formed. Samples are transferred to wells of the ELISA plate, which are coated with anti SC5b‐9 antibodies. After binding of the generated SC5b‐9 to these antibodies, a horseradish peroxidase (HRP) ‐conjugated secondary antibody is added, which binds to epitopes of the SC5b‐9 complex. HRP subsequently cleaves a chromogenic substrate which can be assessed spectrophotometrically at 405 nm. SC5b‐9 concentrations in the samples were calculated using a standard curve.
Red blood cell lysis assay. Liposomes were incubated with pig serum or heparinized plasma for 30 minutes (1:4 v/v, 8 μmol total lipid /mL) and subsequently diluted five‐fold in veronal buffered saline (VBS) containing 0.2% gelatine. For sensitization, sheep erythrocytes were incubated with rabbit anti sheep erythrocyte antiserum (hemolysin) (1000:1). Sensitized erythrocytes (approximately 2×108 cells in 200 μL VBS) were added to 25 μL of the liposome‐incubated serum samples in quadruplicate in a 96‐well plate and incubated for 1 hour at 37 °C with shaking. After centrifugation at 4000 rpm for 10 min to remove intact erythrocytes, hemolysis was assessed from the optical density of the supernatant at 405 nm using a spectrophotometer. Erythrocyte lysis was calculated relative to the VBS buffer control. Figure 2 shows a schematic representation of the assay principle (for a detailed description of the method see (10)).
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Figure 2. Schematic representation of the red blood cell lysis assay principle. Liposomes are incubated with pig serum or plasma. When the complement system is activated by the liposomes, complement (C) is consumed. Samples are mixed with sensitized sheep erythrocytes. After incubation and centrifugation, activation of residual complement is assessed from hemolysis in the supernatant by spectrophotometric analysis.
In vivo assay. Experiments were performed in accordance with guidelines of the Committee on Animal Care of the Uniformed Services University of the Health Sciences. Male Yorkshire swine (weight between 10‐25 kg) were sedated with an intramuscular injection of ketamine, intubated, anesthetized with isoflurane and subsequently instrumented as described previously (4). In brief, a catheter was inserted via the right internal jugular vein into the pulmonary artery; another catheter was introduced through the right femoral artery into the proximal aortic arch to measure the central systemic arterial pressure. Furthermore, heart rate, blood oxygen and CO2 levels were monitored. PHEA‐ and PHEG‐liposomes were diluted in PBS to the desired dose (see Table 1) at an injection rate of approximately 2.5 mL/s, injected into the jugular vein in 1 mL boluses and flushed into the circulation with 10 mL PBS. Based on the changes in the systemic arterial pressure, heart rate and blood gas levels, reactions were classified as follows: none (no significant changes in any of the measured parameters), mild (transient 50% changes in at least one of the measured parameters) and lethal (circulatory collapse within 2 min requiring resuscitation with i.v. epinephrine and cardiac massage).
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RESULTS AND DISCUSSION Liposomes had a diameter of approximately 100 nm as determined by DLS, with a polydispersity index below 0.1, indicating a relatively homogenous size distribution. The SC5b‐9 ELISA assay was used to quantify the soluble form of the terminal complement complex in human serum samples after incubation with liposomes. Figure 3 shows the results of a typical experiment. PHEA‐ and PHEG‐liposomes induce formation of SC5b‐9 in a concentration‐dependent manner, indicating complement activation. In this assay, PHEG‐liposomes appear to activate the complement system to a higher extent than PHEA‐liposomes.
Figure 3. In vitro complement activation assessed by the SC5b‐9 ELISA assay. SC5b‐9 level of PHEA‐ (♦) and PHEG‐ (■) liposomes as a function of liposome concentration in a representative serum sample. Zymosan, a potent complement activator, was used as a positive control (○). Bars represent means ± SD for three wells.
Besides the SC5b‐9 ELISA, also a red blood cell lysis assay was used to assess the complement consumption by PAA‐liposomes in pig serum or plasma. This assay was adapted from the CH50 assay according to Mayer (11). It is an indirect assay that measures the ability of liposome‐incubated serum or plasma to lyse erythrocytes of a standardized suspension of sheep erythrocytes coated with anti‐erythrocyte antibodies. The stronger the complement activation induced by liposomes, the more complement factors will be consumed during incubation, leading to a reduced lysis of erythrocytes. In line with the results of the SC5b‐9 assay, PHEG‐liposomes are a stronger activator when compared to PHEA‐liposomes, whereas the latter are not significantly different from the VBS buffer control (Figure 4).
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Figure 4. Red blood cell lysis as a measure for complement consumption in pig sera/plasma relative to VBS buffer after incubation with the different liposome formulations. Bars represent means ± SD of three serum or plasma samples. Zymosan was used as a positive control (50 mg/mL).
An in vivo assay to assess complement‐related HSR was performed. The observed physiological reactions in the pigs were similar to those observed in earlier studies: temporary decreases in systemic arterial pressure, heart rate, respiration rate and changes in pCO2 (4). In Table 1 the results are shown. Both formulations induced acute HSR in the pigs, with again PHEG‐liposomes exerting a stronger effect than PHEA‐liposomes. Table 1. Hypersensitivity reactions in the porcine model.
Liposome type Lipid dose (μmol/kg)
Frequency of reactions
Severity of reactions
PHEA 0.29‐0.85 2/3 none (1), mild (2) PHEG 0.26‐1.38 3/3 mild (2), severe (1)
The blood pressure curves in Figure 5 show typical progressions as seen also for other types of liposomes and zymosan: the mean systemic artery pressure (SAP) increased slightly, and then dropped about 40 mmHg. The span between systolic and diastolic SAP was reduced at the start of reaction, which is approximately one minute after administration. The mean pulmonary artery pressure increased about 15 mmHg during the reaction. The reaction took approximately 3 minutes and was followed by a spontaneous recovery. Reactions were reproducible in the same animal (Figure 6).
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Figure 5. Pressure curves of systemic arterial pressure (SAP, light grey curve, left y‐axis) and pulmonary arterial pressure (PAP, dark grey curve, right y‐axis) after injection of PHEG‐coated liposomes (0.28 μmol total lipid/kg). Injection was given at 0 min.
Figure 6. Pressure curve of systemic arterial pressure (SAP) after repeated injection of PHEG‐coated liposomes (4.7 μmol total lipid/kg).
Thus, the in vitro and in vivo experiments reveal that PHEA‐ and PHEG‐liposomes can activate the complement system and induce HSR in the porcine model. This is in line with earlier findings on the complement activation by other types of liposomes, e.g. PEG‐liposomes (12). As yet, it is not clear how these reactions depend on factors like lipid dose/composition and animal species. As
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pointed out previously, the pigs’ uniquely sensitive reaction to liposomes represents a model of those 2‐7% hypersensitive humans who respond with severe HSR to certain liposomal drugs or diagnostic agents (4). In addition, other factors, like the role of endotoxin contamination of our preclinical formulations, need to be addressed. Regarding the latter aspect though, it has been shown previously both, in vitro and in vivo that endotoxin contamination is not responsible for liposome‐induced complement activation (13,14).
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13. J. Szebeni, L. Baranyi, S. Savay, M. Bodo, D. S. Morse, M. Basta, G. L. Stahl, R. Bunger, and C. R. Alving (2000) Liposome‐induced pulmonary hypertension: properties and mechanism of a complement‐mediated pseudoallergic reaction. Am J Physiol Heart Circ Physiol 279, 1319‐1328
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