Article pubs.acs.org/Biomac
Effects of PEGylation on Membrane and Lipopolysaccharide Interactions of Host Defense Peptides Shalini Singh,† Praveen Papareddy,‡ Matthias Mörgelin,§ Artur Schmidtchen,‡,∥ and Martin Malmsten*,† †
Department of Pharmacy, Uppsala University, SE-75123, Uppsala, Sweden Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, SE-221 84 Lund, Sweden § Division of Infection Medicine, Department of Clinical Sciences, Lund University, SE-221 84 Lund, Sweden ∥ Lee Kong Chian School of Medicine, Nanyang Technological University, 11 Mandalay Road, Singapore 308232 ‡
S Supporting Information *
ABSTRACT: Effects of poly(ethylene glycol) (PEG) conjugation on peptide interactions with lipid membranes and lipopolysaccharide (LPS) were investigated for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), an antimicrobial and anti-inflammatory peptide derived from human heparin cofactor II. In particular, effects of PEG length and localization was investigated by ellipsometry, circular dichroism, nanoparticle tracking analysis, and fluorescence/electron microscopy. PEGylation of KYE28 reduces peptide binding to lipid membranes, an effect accentuated at increasing PEG length, but less sensitive to conjugation site. The reduced binding causes suppressed liposome leakage induction, as well as bacterial lysis. As a result of this, the antimicrobial effects of KYE28 is partially lost with increasing PEG length, but hemolysis also strongly suppressed and selecticity improved. Through this, conditions can be found, at which the PEGylated peptide displays simultaneously efficient antimicrobial affects and low hemolysis in blood. Importantly, PEGylation does not markedly affect the anti-inflammatory effects of KYE28. The combination of reduced toxicity, increased selectivity, and retained anti-inflammatory effect after PEGylation, as well as reduced scavenging by serum proteins, thus shows that PEG conjugation may offer opportunities in the development of effective and selective anti-inflammatory peptides.
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activity relationship studies,1,2 identification of endogenous peptides derived from infection-derived proteolysis,4,5 and endtagging with short W/F stretches.11,12 Although these approaches can be used to some extent also for antiinflammatory functions, the mode of action of such peptide effects is more complex. For example, LPS is a major inflammatory agent, which covers >70% of the outer leaflet of Gram-negative bacteria, anchored by its lipid A moiety.13 Through its carboxyl and phosphate groups, LPS is negatively charged, and readily binds AMPs through a combination of electrostatic and hydrophobic interactions.14−17 LPS triggers inflammation through binding to lipopolysaccharide-binding protein, recognized by CD14 and Toll-like receptors, resulting in an up-regulation of NF-κB and proinflammatory cytokines.18 In addition, however, the state of LPS aggregation is important for inflammation triggering.19 Thus, anti-inflammatory effects of peptides have been correlated to their ability to disintegrate LPS aggregates, potentially due to size-dependent endocytosis
INTRODUCTION Antimicrobial peptides (AMPs) play a key role in host defense. Through direct membrane lysis, they provide fast and broadspectrum antimicrobial effects, frequently also for pathogens displaying resistance to conventional antibiotics.1 Some AMPs also display additional host defense properties, including antiinflammatory and immune modulating effects.2,3 Examples of such peptides include those derived from coagulation factors, including thrombin,4,5 heparin cofactor II (HCII),6 tissue factor pathway inhibitor,7 and other coagulation factors from the S1 peptidase family.8 For several of these peptides, the antiinflammatory effects were demonstrated not to be due to simple peptide binding to bacterial lipopolysaccharide (LPS) or its endotoxic lipid A moiety alone, but involving also other effects, including peptide-induced LPS binding to macrophage membranes and fragmentation of LPS aggregates, deflecting LPS from its inflammation-triggering binding to Toll-like receptors (TLR).9,10 A key aspect for any therapeutic compound is that of selectivity. For AMPs, this translates mainly into the issue of peptide-induced rupture of bacterial and fungal membranes, but not those of mammalian cells. Several approaches have been employed for obtaining this, including quantitative structure− © 2014 American Chemical Society
Received: December 20, 2013 Revised: February 4, 2014 Published: March 3, 2014 1337
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times with DMF, and the second Fmoc-PEG−OH was added using the same protocol. LPS from E. coli (0111:B4) and lipid A from E. coli F583 (Rd mutant) were both from Sigma (St. Louis, U.S.A.), as was PEG 2000 (Mw ≈ 1900−2200) and human serum albumin (HSA), globulin-free, lyophilized, and crystallized. Microorganisms. The bacterial isolates E. coli ATCC 25922, P. aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 29213 were obtained from the American Type Culture Collection. Scanning Electron Microscopy. Bacteria were grown in TH medium at 37 °C to midlogarithmic phase. The bacteria were washed in buffer (10 mM Tris, pH 7.4, 0.15 M NaCl, 5 mM glucose) and resuspended in the same buffer. Peptides at 30 μM were incubated with E. coli, S. aureus, or P. aeruginosa (1 × 108 bacteria) for 2 h in a total volume of 50 μL in 10 mM Tris, pH 7.4, with additional 150 mM NaCl. Bacteria were fixed and prepared for scanning electron microscopy, as described previously.5 Samples were examined with a Philips/FEI CM 100 electron microscope operated at 80 kV accelerating voltage and images recorded with a side-mounted Olympus Veleta camera at the Core Facility for integrated Microscopy (CFIM), Copenhagen University, Denmark. Fluorescence Microscopy. Membrane permeabilization was monitored by the impermeant probe FITC (Sigma-Aldrich, St. Louis, U.S.A.). E. coli ATCC 25922 bacteria were grown to midlogarithmic phase in trypticase soy broth (TSB) medium and then washed and resuspended in buffer (10 mM Tris, pH 7.4, 0.15 M NaCl) to yield a suspension of 1 × 107 colony forming units (cfu)/mL. A total of 100 μL of the bacterial suspension was incubated with 30 μM of the respective peptides at 37 °C for 60 min. Microorganisms were then immobilized on poly(L-lysine)-coated glass slides by incubation for 45 min at 30 °C, followed by the addition of 200 μL of FITC (6 μg/mL) in buffer and incubation for 30 min at 30 °C. The slides were washed and bacteria fixed by incubation, first on ice for 15 min, then in room temperature for 45 min in 4% paraformaldehyde. The glass slides were subsequently mounted on slides using Prolong Gold antifade reagent mounting medium (Invitrogen, Eugene, U.S.A.). For fluorescence analysis, bacteria were visualized using a Nikon Eclipse TE300 (Nikon, Melville, U.S.A.) inverted fluorescence microscope equipped with a Hamamatsu C4742−95 cooled CCD camera (Hamamatsu, Bridgewater, U.S.A.) and a Plan Apochromat ×100 objective (Olympus, Orangeburg, U.S.A.). Differential interference contrast (Nomarski) imaging was used for visualization of the microbes themselves. Radial Diffusion Assay (RDA). Essentially, as described earlier,26,27 bacteria were grown to midlogarithmic phase in 10 mL of fullstrength (3% w/v) trypticase soy broth (TSB; Becton-Dickinson, Cockeysville, U.S.A.). The microorganisms were then washed once with 10 mM Tris, pH 7.4. Subsequently, 4 × 106 cfu were added to 15 mL of the underlay agarose gel, consisting of 0.03% (w/v) TSB, 1% (w/v) low electroendosmosis type (EEO) agarose (Sigma, St. Louis, U.S.A.), and 0.02% (v/v) Tween 20 (Sigma-Aldrich), with or without 150 mM NaCl. The underlay was poured into a Ø 144 mm Petri dish. After agarose solidification, 4 mm diameter wells were punched and 6 μL of test sample added to each well. Plates were incubated at 37 °C for 3 h to allow diffusion of the peptides. The underlay gel was then covered with 15 mL of molten overlay (6% TSB and 1% Low-EEO agarose in distilled H2O). Antimicrobial activity of a peptide is visualized as a zone of clearing around each well after 18−24 h of incubation at 37 °C. Viable Count Analysis (VCA). As the magnitude of the clearance zone in radial diffusion assays (RDA) depends not only on antibacterial potency, but also on peptide diffusion through the polymer network, it is difficult to translate clearance zone magnitudes to expected potency in a clinical setting. In order to address this methodological shortcoming with RDA, the potency of the peptides was also assessed by viable count assay. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were grown to midexponential phase in Todd-Hewitt (TH). Bacteria were then washed and diluted in 10 mM Tris, pH 7.4, containing 0.15 M NaCl. A total of 2 ×106 cfu/mL bacteria were incubated in 50 μL, at 37 °C for 2 h, with peptides at the indicated concentrations. Serial dilutions of the incubation mixture
offering an alternative to receptor-mediated uptake and NF-κB activation.10,14,15,18 Conjugation with poly(ethylene)glycol (PEG) has been extensively investigated for a range of peptide and protein therapeutics.20 For sufficiently long and densely packed PEG chains, such “PEGylation” offers a series of advantages, including reduction of adsorption of serum proteins (notably opsonins),21 increased bloodstream circulation, and reduced uptake in tissues related to the reticuloendothelial system (RES).22 In addition, PEGylated proteins/peptides have been found to display increased resistance to proteolytic degradation, as well as reduced immune responses, aggregation, and toxicity.20 While such effects have been relatively well documented for antibodies, antibody fragments, and enzymes, much less is known about the effects of PEGylation of AMPs, apart from a couple of studies demonstrating that PEGylation offers opportunities for reducing toxicity and proteolytic susceptibility of AMPs, but also that this comes at a cost of reduced antimicrobial effect.23−25 Thus, little is still known about how PEG length and localization affect membrane binding and disruption, and how this translates into antimicrobial and cytotoxic effects. Furthermore, the effects of PEGylation on anti-inflammatory effects of AMPs remain to be elucidated, together with the underlying biophysical processes. Considering this, we here report on such investigations of the effects of PEGylation for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), a peptide derived from HCII, previously demonstrated to possess potent antimicrobial and antiinflammatory properties,10 but also issues with hemolysis. KYE28 was therefore conjugated with PEG of varying lengths and at different positions. The conjugated peptides were investigated regarding membrane, LPS, and lipid A interactions by ellipsometry, circular dichroism spectroscopy (CD), nanoparticle tracking analysis, and fluorescence/electron microscopy. The biophysical results thus obtained are correlated to results on antimicrobial and anti-inflammatory properties, as well as toxicity.
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EXPERIMENTAL SECTION
Chemicals. Peptides (Table 1) were synthesized by Biopeptide Co. (San Diego, U.S.A.) and were of >95% purity, as evidenced by mass spectral analysis (MALDI-TOF Voyager). PEGylation was achieved by using 3 equiv of Fmoc-PEG−OH/DIC/HOBt in DMF to couple to peptide resin. The completion of the reaction was checked by ninhydrin test. For two-step PEGylation, the Fmoc was removed with 20% piperidine/DMF for 10 min, whereafter the resin was washed five
Table 1. Sequence and Key Properties of the Peptides Investigated
1
IP: isoelectric point. 2Znet: net charge. 3H: mean hydrophobicity on the Kyte-Doolittle scale;41 H values for PEG conjugates not defined. 1338
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were plated on TH agar, followed by incubation at 37 °C overnight and cfu determination. In another experiment, human citrate-blood was diluted (1:1) with PBS prior to addition of 2 × 108 cfu/mL bacteria, in order to allow for the presence of highly surface active serum proteins such as fibrinogen,28 and in order to mimic the situation in a systemic infection situation. The mixture was incubated with end-overend rotation for 1 h at 37 °C in the presence of peptides (120 μM), 2% Triton X-100 (Sigma-Aldrich, St. Louis, U.S.A.) serving as positive control. The samples were then centrifuged at 800 g for 10 min and the supernatant transferred to a 96-well microtiter plate. The hemoglobin release was measured by absorbance at 540 nm and is expressed as % of Triton X-100-induced hemolysis. Hemolysis. EDTA-blood was centrifuged at 800 g for 10 min, and plasma and buffy coat removed. Erythrocytes were washed three times and resuspended in 5% PBS, pH 7.4. The cells were then incubated with end-over-end rotation for 1 h at 37 °C in the presence of peptide (60 μM). For comparison, the benchmark peptide LL-37 was included as well, while 2% Triton X-100 (Sigma-Aldrich, St. Louis, U.S.A.) served as positive control. The samples were then centrifuged at 800 g for 10 min. The hemoglobin release was measured by absorbance at 540 nm and is expressed as % of Triton X-100-induced hemolysis. LPS Effects on Macrophages In Vitro. RAW-Blue cells (1 × 106 cells/mL; InvivoGen, San Diego, U.S.A.) were seeded in phenol redfree DMEM, supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) AAS, and allowed to adhere before they were stimulated with 10−1000 ng/mL E. coli (0111:B4) LPS and with peptides at the indicated concentrations. NF-κB activation was determined after 20 h of incubation according to manufacturer’s instructions (InvivoGen, San Diego, U.S.A.). Briefly, activation of NF-κB leads to the secretion of embryonic alkaline phosphatase (SEAP) into the cell supernatant, were it was measured by mixing supernatants with a SEAP detection reagent (Quanti-Blue, InvivoGen), followed by absorbance measurement at 600 nm. Liposome Preparation and Leakage Assay. Model liposomes investigated were either anionic (DOPE/DOPG 75/25 mol/mol) or zwitterionic (DOPC/cholesterol 60/40 mol/mol). DOPG (1,2dioleoyl-sn-glycero-3-phosphoglycerol, monosodium salt), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and DOPC (1,2dioleoyl-sn-glycero-3-phosphocholine) were all from Avanti Polar Lipids (Alabaster, U.S.A.) and of >99% purity, while cholesterol (>99%) was from Sigma-Aldrich (St. Louis, U.S.A.). DOPE/DOPG is extensively used as a model system in the AMP literature29 and has been previously demonstrated to give similar results regarding peptide interactions as E. coli lipid extract membranes.12 Because of the long, symmetric, and unsaturated acyl chains of these phospholipids, membrane cohesion is good, which facilitates stable unilamellar liposomes and well-defined supported lipid bilayers, allowing detailed data on leakage and adsorption density to be obtained. Similar concerns motivated the use of (60/40) DOPC/cholesterol membranes, extensively used as eukaryotic cell mimic in literature.29,30 The lipid mixture was dissolved in chloroform, after which solvent was removed by evaporation under vacuum overnight. Subsequently, 10 mM Tris buffer, pH 7.4, was added together with 0.1 M carboxyfluorescein (CF; Sigma, St. Louis, U.S.A.). After hydration, the lipid mixture was subjected to eight freeze−thaw cycles, consisting of freezing in liquid nitrogen and heating to 60 °C. Unilamellar liposomes of about Ø 140 nm were generated by multiple extrusions (30 passages) through polycarbonate filters (pore size 100 nm) mounted in a LipoFast miniextruder (Avestin, Ottawa, Canada) at 22 °C. Untrapped CF was removed by two subsequent gel filtrations (Sephadex G-50, GE Healthcare, Uppsala, Sweden) at 22 °C, with Tris buffer as eluent. CF release from the liposomes was monitored through emitted fluorescence at 520 nm from a liposome dispersion (10 μM lipid in 10 mM Tris, pH 7.4). For the leakage experiment in the presence of LPS, 0.02 mg/mL LPS was first added to the above liposome dispersion (which did not cause liposome leakage in itself; results not shown), after which peptide was added and leakage monitored as a function of time. An absolute leakage scale was obtained by disrupting the liposomes at the end of each experiment through addition of 0.8 mM Triton X-100 (Sigma-Aldrich, St. Louis,
U.S.A.). A SPEX-fluorolog 1650 0.22-m double spectrometer (SPEX Industries, Edison, U.S.A.) was used for the liposome leakage assay. Measurements were performed in triplicate at 37 °C. CD Spectroscopy. CD spectra were measured by a Jasco J-810 Spectropolarimeter (Jasco, Easton, U.S.A.). Measurements were performed in duplicate at 37 °C in a 10 mm quartz cuvette under stirring at a peptide concentration of 10 μM. The effect on peptide secondary structure of liposomes at a lipid concentration of 100 μM was monitored in the range 200−260 nm. The α-helix content was calculated from the recorded CD signal at 225 nm using reference peptides in purely helical and random coil conformations, respectively. The 100% α-helix and 100% random coil references were obtained from 0.133 mM (monomer concentration) poly-L-lysine (Mw = 79 kDa) in 0.1 M NaOH and 0.1 M HCl, respectively. For measurements in the presence of LPS, 0.2 mg/mL was used. To account for instrumental differences between measurements, background correction was performed routinely by subtraction of spectra for buffer (with or without liposomes/LPS) from spectra of the corresponding samples in the presence of peptide. Ellipsometry. Peptide adsorption to supported lipid bilayers was studied in situ by null ellipsometry using an Optrel Multiskop (Optrel, Kleinmachnow, Germany) equipped with a 100 mW Nd:YAG laser (JDS Uniphase, Milpitas, U.S.A.). All measurements were carried out at 532 nm and an angle of incidence of 67.66° in a 5 mL cuvette under stirring (300 rpm). Both the principles of null ellipsometry and the procedures used have been described before.31 In brief, by monitoring the change in the state of polarization of light reflected at a surface in the absence and presence of an adsorbed layer, the mean refractive index (n) and layer thickness (d) of the adsorbed layer can be obtained. From the thickness and refractive index, the adsorbed amount (Γ) was calculated according to
Γ=
(n − n0) d dn/dc
(1)
where n0 is the refractive index of the bulk solution (1.3347), and dn/ dc the refractive index increment (0.154 cm3/g). Corrections were routinely done for changes in bulk refractive index caused by changes in temperature and excess electrolyte concentration. LPS-coated surfaces were prepared by adsorbing E. coli LPS to methylated silica surfaces (surface potential −40 mV, contact angle 90°32) from 0.4 mg/mL (in water) over a period of 2 h. This results in a hydrophobically driven LPS adsorption of 1.48 ± 0.38 mg/m2, corresponding to plateau in the LPS adsorption isotherm, with an approximate area per acyl group of 200 Å2, assuming an LPS molecular weight of 104, and six acyl chains per LPS. (A similar approach was previously used also for other membrane-bound lipopolysaccharides.33) Nonadsorbed LPS was removed by rinsing with Tris buffer at 5 mL/min for a period of 30 min, followed by buffer stabilization for 20 min. Peptide addition was performed at different concentrations of 0.01, 0.1, 0.5, and 1 μM, and the adsorption monitored for 1 h after each addition. All measurements were performed in at least duplicate at 25 °C. For lipid A deposition, this was solubilized with 0.25 wt % triethyl amine (TEA) under vigorous vortexing and heating to 60 °C for 10 min. Lipid A was adsorbed at methylated silica surfaces for 2 h from 5 mg/mL lipid A stock solution in 0.25% TEA at a concentration of 0.4 mg/mL in 10 mM Tris, pH 7.4, containing 150 mM NaCl. Nonadsorbed lipid A was subsequently removed by rinsing with same buffer at 5 mL/min for 15 min, followed by buffer stabilization for 20 min. This results in a lipid A adsorption of 0.8 ± 0.2 mg/m2. Peptide addition was subsequently performed to 0.01, 0.1, 0.5, and 1 μM, and adsorption monitored for one hour after each addition. (Absence of transient maxima in the time-resolved adsorption curves after peptide addition indicated competitive displacement to be unlikely for the LPS and lipid A substrates.) All measurements were performed in at least duplicate at 25 °C. The zwitterionic phospholipid bilayers were deposited on silica surfaces by coadsorption from a mixed micellar solution containing DOPC/cholesterol (60/40 mol/mol) and n-dodecyl-β-D-maltoside 1339
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(DDM; ≥98% purity, Sigma-Aldrich, St. Louis, U.S.A.), as described in detail previously.34 In brief, the mixed micellar solution was formed by addition of 19 mM DDM in water to DOPC/cholesterol (60/40 mol/ mol) dry lipid films, followed by stirring overnight, yielding a solution containing 97.3 mol % DDM. This micellar solution was added to the cuvette at 25 °C, and the following adsorption monitored as a function of time. When adsorption had stabilized, rinsing with Milli-Q water at 5 mL/min was initiated to remove mixed micelles from solution and surfactant from the substrate. By repeating this procedure and subsequently lowering the concentration of the micellar solution, stable and densely packed bilayers are formed, with structural characteristics similar to those of bulk lamellar structures of the lipids.34 As sub-bilayer and patchy adsorption resulted from the above mixed micelle approach in the case of the anionic lipid mixture, supported lipid bilayers were generated from liposome adsorption in this case. DOPE/DOPG (75/25 mol/mol) liposomes were prepared as described above, but the dried lipid films resuspended in Tris buffer only with no CF present. In order to avoid adsorption of peptide directly at the silica substrate (surface potential −40 mV, contact angle
