Article pubs.acs.org/Langmuir
Characterizing and Controlling the Loading and Release of Cationic Amphiphilic Peptides onto and from PEG-Stabilized Lipodisks Karin Reijmar,† Katarina Edwards,† Karl Andersson,‡,§ and Víctor Agmo Hernández*,† †
Department of Chemistry-BMC, Uppsala University, Box 579, Uppsala SE-75123, Sweden Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala 751 85, Sweden § Ridgeview Instruments AB, Skillsta 4, 740 20 Vänge, Sweden ‡
S Supporting Information *
ABSTRACT: Recent studies have identified PEG-stabilized lipid nanodisks (lipodisks) as promising carriers for cationic amphiphilic peptides with antimicrobial and anticancer activity. Using fluorimetric and nanogravimetric methods, we have in this work characterized the parameters describing and controlling the binding of three selected peptides (melittin, LL37, and magainin 2) onto lipodisks. It was found that the affinity of melittin for lipodisks is independent of the disk size and rim charge. On the other hand, the number of binding sites is strongly dependent on both parameters, with the highest loading being obtained for small disks with a negatively charged rim. An optimized composition of the lipodisks was utilized to study the loading of antimicrobial peptides magainin 2 and human LL37. It was observed that although magainin 2 can be loaded in large amounts, it is released very fast upon dilution, which limits future therapeutic applications. In contrast, LL37 can be loaded at relevant concentrations and the formulation is stable. This opens up for applications of LL37-loaded lipodisks as antibiotics and in anticancer treatments.
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AMPs.12−14 Several AMPs have also been shown to display selective toxicity to tumor cells. Part of the explanation for this lies in the fact that, in relation to normal cells, cancer cells tend to have increased levels of negatively charged glycoproteins, glycolipids, and phospholipids in the outer leaflet of the cell membrane.4 Similar to the case with bacterial membranes, this over-representation of anionic membrane components promotes binding of the positively charged peptides. However, in spite of these promising characteristics, there are still many challenges concerning the administration of these peptides. To obtain a significant therapeutic effect, a highenough peptide concentration near the bacteria or tumor cell membrane is required. However, the peptide in free form is subject to enzymatic degradation and often rapid renal filtration.15 This makes it difficult to maintain a therapeutically relevant concentration in the body for an extended time period. Earlier studies, by us and others, have demonstrated that the problems with rapid degradation and clearance of the peptides can be diminished if the peptides are formulated in PEGstabilized lipodisks.16,17 The lipodisks consist of a nanosized flat circular lipid bilayer surrounded by a highly curved rim.18 The disks are obtained by mixing lipids that on their own spontaneously form bilayer structures, with micelle-forming PEGylated lipids (i.e., lipids whose headgroup is attached to a poly(ethylene glycol) chain). The lipodisks are nontoxic and
INTRODUCTION Cationic amphiphilic α-helical peptides have attracted considerable interest in novel pharmaceutical applications. Among them, antimicrobial peptides (AMPs) hold potential as a new, promising class of antibiotics primarily because of the fact that they have a broad range of action and are less subject than conventional antibiotics to resistance development.1−3 In addition, several of the peptides present selective antitumor activity and have been identified as potentially effective anticancer agents.4 The mechanism of binding and the effect of these peptides on the lipid membrane have been extensively studied using liposomes and other lipid membrane-based structures and a variety of experimental techniques, as exemplified in a number of recent publications.5−8 It is generally agreed that the adsorption of cationic amphiphilic peptides on the cell membrane takes place because of both hydrophobic and electrostatic interactions. The formation of the α-helical structure is believed to constitute an important part of the driving force for the association.9 Furthermore, because of their cationic nature, these peptides bind readily onto negatively charged membranes, such as bacterial membranes, which are known to be rich in negatively charged phospholipids such as phosphatidylglycerol (PG) and cardiolipin (CL).10 In contrast, the outer leaflet of healthy human cell membranes contains mostly electrically neutral, zwitterionic phospholipids, such as phosphatidylcholine (PC) and sphingomyelin (SM).11 The negative charge, together with the lack of cholesterol, renders the bacterial membranes more susceptible to attack by the © XXXX American Chemical Society
Received: August 12, 2016 Revised: September 28, 2016 Published: October 27, 2016 A
DOI: 10.1021/acs.langmuir.6b03012 Langmuir XXXX, XXX, XXX−XXX
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However, the selected peptides differ significantly in their primary structures. Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ, cationic residues shown in bold) is a component of honey bee (Apis mellifera) venom and has a well-defined hydrophilic, positively charged region at the C-terminus and a bulky hydrophobic part composed mostly of aliphatic amino acids. Magainin 2 (GIGKFLHSAKKFGKAFVGEIMNS, cationic residues in bold, anionic residues in italics) is isolated from the skin of the African frog Xenopus laevis. In contrast to melittin, it presents an even distribution of its charges along the sequence and contains both basic (K and H) and acidic (E) residues. Finally, human LL37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) is highly charged (16 charged residues in total, 11 cationic and 5 anionic), with clusters of hydrophilic amino acids alternating with clusters of hydrophobic residues. The peptide acquires clear amphiphatic character when arranged into an α-helical structure, although the N-terminal region is believed to be found mostly in a random coil conformation.27 As mentioned, these peptides are relevant by themselves and, at the same time, illustrate the variations that can be expected with different AMPs. Therefore, the obtained results are of interest in order to allow for the design of lipodisks optimized for the transport and delivery of these peptides in particular and AMPs in general in pharmaceutical applications.
biocompatible, and the extended PEG chains surrounding the rim of the disk provide the structures with a long blood circulation time.19
Figure 1. Cross section of a lipodisk. PEGylated lipids are segregated mostly to the rim of the disk.
We have previously shown that substantial amounts of AMPs can be loaded onto lipodisks.16,18,20 Importantly, once the peptide is attached to the lipodisk, the lipid environment and the surrounding PEG chains offer protection against enzymatic degradation and provide it with a remarkable sustained release effect.16 A potential application of the peptide-loaded lipodisks in cancer therapy has recently been described by Gao et al.17 Results of this study show that melittin (a cationic amphiphilic peptide extracted from honey bee venom) loaded onto lipodisks has significant antitumor activity in vivo, whereas its usual side effect as a hemolytic peptide is strongly reduced. To be able to design lipodisks with optimal properties for the loading, protection, and controlled release of the peptide, it is necessary to fully characterize and understand the interactions between the peptides and the lipodisks. Previous work has shown that melittin, and other similar peptides, preferentially bind to the curved rim of the lipodisks.18,21 As has been shown in the cited publications, this preferential binding is caused almost exclusively by the large curvature at the rim, which promotes a stronger hydrophobic interaction between the peptides and the lipodisks. According to these studies, the charge at the rim plays only a minor role, and the interaction of the peptide with the PEG chains is negligible. The association of melittin to lipodisks with different lipid compositions and overall properties has been studied using fluorimetric determinations that take advantage of the intrinsic fluorescence of the tryptophan residue present in the peptide.22 Recently, we have developed a new nanogravimetric method by which the association isotherms of nonfluorescent peptides can be built with the help of quartz crystal microbalance with dissipation monitoring (QCM-D).20 In this report, we use a combination of the fluorimetric and nanogravimetric approaches in order to identify the most important parameters governing the interaction of three relevant cationic amphiphilic peptides (i.e, melittin, magainin 2, and human LL37) with lipodisks. These peptides were selected because of their relevance in potential therapeutic applications. Also, in spite of some common features, they have very different primary structures, thus exemplifying the diversity found between AMPs. All of the tested peptides are known to arrange into an α-helical secondary structure when bound to lipid membranes. Once in the membrane, they disrupt it via the formation of toroidal pores, although the disrupting mechanism can vary depending on the lipid bilayer composition.23−26 These features are common to several antimicrobial peptides.
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EXPERIMENTAL SECTION
Chemicals. Dry powders of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPEPEG2000), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), Npalmitoyl-sphingosine-1-(succinyl[methoxy(polyethylene glycol)2000]) (ceramidePEG2000), N-palmitoyl-sphingosine-1-(succinyl[methoxy(polyethylene glycol)5000]) (ceramidePEG5000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000amine), and 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG2000biotin) were purchased from Avanti Polar Lipids (Alabaster, AL). 11-Mercaptoundecanoic acid (MUA), N-hydroxysuccinimide (NHS), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), ethanolamine, sulfuric acid, hydrogen peroxide, sodium phosphate (mono- and dibasic), streptavidin, sodium chloride, magainin 2, and natural melittin were purchased from Sigma-Aldrich (Schnelldorf, Germany). Synthetic melittin and LL37 were purchased from Innovagen (Lund, Sweden). All aqueous solutions were prepared using deionized water (18.4 MΩ cm) obtained from a Milli-Q system (Millipore; Bedford, MA). QCM-D sensors (gold and biotynilated gold) were obtained from Q-Sense (Gothenburg, Sweden). Preparation of Lipodisks. The lipids were weighed in the desired proportions and dissolved in CHCl3. The solvent was then removed under a stream of N2 gas. The remaining solvent was evaporated in a vacuum oven overnight. The obtained lipid films were rehydrated in phosphate buffered saline (PBS, 150 mM NaCl, 10 mM phosphate, pH 7.4). The lipodisk preparation protocol for each composition was chosen in order to optimize the lipodisk formation and avoid the formation of liposomes. The preparation methods varied according to the used composition as follows. Disks composed of DPPC/DSPE-PEG2000/DSPE-PEG2000amine (75:21:4 mol/mol/mol) or DPPC/DSPE-PEG2000 (75:25 mol/mol) were prepared by subjecting the solution to five freeze−thaw cycles (liquid nitrogen−water bath at 55 °C). The suspension was then extruded 21 times through a polycarbonate 100 nm filter (Avastin; Ottawa, Canada) using a Mini-Extruder (Avanti Polar Lipids, Alabaster, AL). Disks composed of DPPC/DSPE-PEG2000 (90:10 mol/mol) were prepared by incubation with PBS buffer for 1 h at 70 °C with intermittent vortex mixing, followed by 20 min of sonication in an ice bath using a Soniprep 150 sonicator (MSE Scientific Instruments, B
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Langmuir England). The sample was then centrifuged for 2 min at 480g in order to get rid of possible metal debris from the tip. This lipodisk preparation method has recently been developed and has been shown to be able to produce stable lipodisks even with the small amounts of PEGylated lipids incorporated in the mixture.28 As reported in the cited publication, a relevant feature of lipodisks prepared in this way is that the PEG-corona surrounding the disks has a decreased density compared to that of disks formed with PEG proportions larger than 20 mol %. For the DPPC/ceramidePEG2000 and DPPC/ceramidePEG5000 (75:25 mol/mol) mixtures, the film was incubated for 30 min in PBS buffer at 70 °C, followed by sonication for 20 min in an ice bath. Finally, disks composed of DPPC/ceramidePEG2000 (90:10 mol/ mol) were prepared by incubation for 30 min in PBS buffer at 70 °C, followed by three freeze−thaw cycles and sonication for 10 min at 55 °C. The above-described methods result in samples containing almost purely lipodisks, with negligible liposome content, as confirmed by cryo-TEM. Dynamic Light Scattering (DLS). DLS was used to characterize the size distribution of the different lipodisk preparations. The setup is a Uniphase He−Ne laser operating at 25 mW (Milpitas, CA). The polarized light is emitted vertically with a wavelength of 632.8 nm. A 90° scattering angle is detected with a PerkinElmer (Quebec, Canada) diode detector and an ALV-5000 multiple digital autocorrelator (ALVlaser, Vertriebsgesellschaft, Germany). Cryogenic Transmission Electron Microscopy (Cryo-TEM). To confirm the formation of lipodisks as well as to obtain complementary information concerning their size, cryogenic transmission electron microscopy (cryo-TEM) using a Zeiss TEM Libra 120 transmission electron microscope (Carl Zeiss AG, Oberkochen, Germany) was used. Imaging was performed in zero loss bright-field mode at an acceleration voltage of 80 kV. Digital images were recorded under low-dose conditions with a BioVision Pro-SM slow scan CCD camera (Proscan, Seuring, Germany). An underfocus of 1 to 2 μm was used to enhance the contrast of the images. Details of the sample preparation are described by Almgren et al.29 For the determination of the size distribution of the lipodisk samples, at least 350 randomly selected disks in the micrographs were measured. Fluorimetric Binding Assays. Fluorimetric measurements were performed using a SPEX fluorolog 1650 0.22-m double spectrometer (SPEX Industries, Edison, NJ). A defined volume of an aqueous solution of melittin in phosphate-buffered saline (PBS) was added to a quartz cuvette. The peptide solution was then titrated with small additions (between 10 and 50 μL) from a lipodisk suspension with a known concentration. After each addition, the emission spectrum between 325 and 355 nm was acquired, with the excitation wavelength set to 280 nm. The emission fluorescence intensities at 325 and 355 nm were used to calculate the ratio α between them. This ratio α is related to the fraction of peptide associated with the lipodisks resulting from the blue shift in tryptophan emission when it is transferred from an aqueous to a lipid environment. The α value is corrected for inner filter effects. This is done by repeating the experiment in the absence of peptide and subtracting the recorded emission intensity values from those recorded when the peptide is present. The relationship between the effective associated peptide to lipid ratio after each addition, Reff, and the free peptide in the water phase can be calculated from eq 1
R αR i = eff (1 − α)[P]tot,aq [P]aq
was followed using the QCM-D-based, label-free method reported in a previous publication.20 The QCM-D technique follows changes in the oscillation frequency and dissipation factor when material is adsorbed on a sensing surface mounted on an oscillating quartz crystal. From these changes, the mass and viscoelastic properties of the adsorbed film can be determined. By determining the mass increase on a sensor surface upon immobilization of lipodisks and upon exposing the immobilized lipodisks to peptide solutions with different concentrations, the association of the peptides with the lipodisks can be characterized. Experimental details are given in the cited reference. Briefly, a QCM-D E1 (Q-Sense, Gothenburg, Sweden) instrument thermostated at 21 °C and with a controlled sample flow of 150 μL min−1 was used for all experiments. Frequency and dissipation data were collected from the fundamental sensor frequency (5 MHz) as well as the 3rd, 5th, 7th, 9th, 11th, and 13th overtones. Prior to use, QCM-D sensors with a gold working surface were cleaned according to the procedure recommended by the supplier. The sensors were treated for 10 min in a UV/ozone chamber (BioForce Nanosciences Inc., Ames, Iowa), followed by 5 min of immersion in a hot (75 °C) basic piranha solution (NH3/H2O2/water (5:1:1)). The sensors were then rinsed with Milli-Q water, dried with a stream of nitrogen, and finally treated again for 10 min in the UV/ozone chamber. After being cleaned, the sensors were dipped in a 1 mM MUA solution in absolute ethanol (Kemetyl, Uppsala) overnight. Before use, the sensor was rinsed in absolute ethanol and dried under a gentle flow of nitrogen. After the sensor was mounted, the system was equilibrated with MilliQ water until a stable baseline was obtained. The surface was then activated for 10 min with a freshly prepared 0.1 M NHS/0.4 M EDC 1:1 mixture. After activation, the system was flushed first with 80 mM acetate buffer (pH 4.5) for 10 min followed by PBS until a stable baseline was obtained. A suspension of amine-functionalized lipodisks (0.5−1 mM total lipid concentration) in PBS was then loaded and allowed to react with a flow rate of 150 μL/min until a stable value for the changes in frequency and dissipation was obtained (usually ∼1 h). The system was then rinsed with a 150 μL min−1 flow of PBS to remove any nonbound lipodisks. The final values of the frequency and dissipation changes are then used to calculate the total lipodisk mass bound to the sensor. After disk immobilization, the remaining active surface was inactivated by a 10 min flow of 1 M ethanolamine hydrochloride at pH 8.5. After the inactivation, the system was equilibrated with the working buffer (PBS). Solutions with increasing concentrations of the corresponding peptide were then sequentially loaded into the system, with rinsing steps after equilibration at each peptide concentration. Each step was coupled with changes in the measured frequency and dissipation, which are in turn associated with changes in the mass bound to the sensor and, therefore, with the binding of peptides to the immobilized lipodisks. To account for shifts arising from changes in the density and viscosity of the bulk solution (i.e., the bulk effect), frequency and dissipation displacements upon exchanging the solutions were recorded independently on an inactive sensor. For the peptides studied (magainin 2 and LL37), the bulk effect was found to be negligible even at the highest used peptide concentrations. Quantitative determinations of the immobilized amount of lipid and associated peptide were based on the approach proposed previously,20 which is in turn a linearization of the model by Voinova et al.30 for the formation of thin viscoelastic films on QCM-D sensors. The linearization is based on the following relationship
mdf0 πη (f )2 Δf =− + 1 0 (nΔD) n tqρq μ1
(1)
where [P]aq is the free peptide in the water phase, [P]tot,aq is the total peptide concentration, and Ri is the molar peptide to lipid mixing ratio after each addition. From this relationship and given that [P]tot,aq is the sum of the bound and free peptide concentrations ([P]tot,aq = Reff*[lipid] + [P]aq), Reff and [P]aq can be determined for each addition, thus allowing the construction of a binding isotherm (Reff vs [P]aq curve). Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). The association of nonfluorescent peptides with lipodisks
(2)
where md is the adsorbed mass surface density, n is the overtone number, f 0 is the fundamental oscillation frequency, tq and ρq are, respectively, the thickness and density of the quartz crystal, and η1 and μ1 are the viscosity and elastic modulus of the adsorbed layer. A plot of Δf n−1 vs nΔD at different values of n is therefore a line with a y intercept equal to −(md f 0)(tqρq)−1. All reported values were calculated from eq 2, and the Δf n−1 and nΔD values were obtained from at least four overtones. From the calculated changes in mass, the number of C
DOI: 10.1021/acs.langmuir.6b03012 Langmuir XXXX, XXX, XXX−XXX
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Langmuir immobilized lipodisks and of bound peptide were determined, allowing thus to build association isotherms (Reff vs [P]aq curve). Previous work has demonstrated that the association isotherms obtained by this method are equivalent to those obtained by wellestablished fluorimetric measurements. Besides the construction of association isotherms, the obtained time-resolved binding data were analyzed in TraceDrawer 1.6 (Ridgeview Instruments AB, Vänge, Sweden).
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RESULTS AND DISCUSSION Association of Melittin and Lipodisks. Melittin is a widely studied peptide that, in spite of its hemolytic activity, is often used as a model amphiphilic cationic peptide. The frequent use of melittin as a model peptide stems partially from its availability and the fact that it shares many properties with true AMPs. An additional important reason is that melittin contains a tryptophan residue that allows for fluorimetric studies. Melittin is protected against degradation and retains its antibacterial and antitumor effect upon formulation in lipodisks.16,17 Notably, recent studies have shown that melittin loaded onto lipodisks has reduced hemolytic activity.17 Despite these encouraging findings, it is clear that careful optimizations are needed in order to further develop melittin-loaded lipodisks as safe and effective antibiotics and anticancer agents. In the present study, we contribute to this process by investigating the effect of the lipodisk size, charge, and PEG length on the association of the peptide to the disks. Effects of Size, Charge, and Length of the PEG Chain. The binding of melittin to lipodisks with similar size but different charge at the curved rim was studied fluorometrically. Specifically, lipodisks with negatively charged edges were compared to disks with a neutral rim. For negatively charged disks, the PEGylated lipids consisted of DSPE-PEG2000 (which is anionic at the used pH), whereas uncharged lipodisks were stabilized with ceramidePEG2000 (which is noncharged). To control and vary the size of the obtained lipodisks, different amounts (either 10 or 25% mol/mol) of PEGylated lipids were included in the lipid mixtures. Finally, for one of the lipodisk compositions the PEG2000 chain was replaced with PEG5000. The obtained disks were characterized with DLS and cryoTEM. The results are summarized in Table 1. DLS curves and
Figure 2. Association isotherms of melittin with lipodisks formed by DPPC mixed with different PEGylated lipids at different molar ratios. The legend indicates the kind and molar percent of PEG lipids in the sample.
negligible. This is likely to arise from the rather large rim area per disk and the small amount of PEG lipid, providing a very low surface charge density in the case of charged disks. This, combined with the physiological salt concentration employed, reduces the extent and strength of the electrostatic interaction. On the other hand, it is observed that, for disks containing 25 mol % PEGylated lipids, the effect of the charge becomes significant, with charged disks being able to carry a much higher amount of peptide. For these disks, the PEGylated lipids are expected to sit closer together, providing, in the case of DSPEPEG lipids, a larger surface charge density. Comparing disks with 10 and 25 mol % PEGylated lipids, it is observed that for both charged and uncharged disks the effective peptide/lipid ratio at saturation increases drastically as the size of the disk decreases. As mentioned above, it has been shown that the peptide binds preferentially to the curved edge of the lipodisk. For a discoidal shape, the edge surface area/planar surface area ratio increases with decreasing disk size. This in turn means that, for a given amount of bilayer-forming lipids, the edge surface area and thus the number of potential binding sites increase as the size of the lipodisk decreases. Therefore, our observations agree with previous reports concerning the preferential binding to the disk rim. A separate QCM-D experiment was performed in which a supported planar lipid bilayer consisting only of DPPC was formed on a silica sensor. Remarkably, melittin did not bind at all to this lipid structure with zero curvature (data not shown). This further strengthens the hypothesis that melittin prefers the curved edge of the disks over the planar surface. Concerning the effect of the size of the PEG chain, the results obtained show that there are more binding sites when PEG5000 is used instead of PEG2000. This observation is most likely related to the effect of the lipodisk size discussed above. DLS data (Table 1) show that disks prepared with PEG5000 have roughly the same hydrodynamic radius as those with PEG2000. Given, however, that the contribution of PEG5000 chains to the hydrodynamic radius is larger than that of PEG2000, it is clear that the bilayer area should be smaller for the former, resulting in an increase in the edge surface area per total lipid concentration. Therefore, it is likely that the actual length of the polymer chain does not directly affect the lipodisk− peptide interaction. Further insight into the parameters governing the binding of melittin to the lipodisks was obtained from Scatchard plots generated from the isotherms shown in Figure 2. Figure 3
Table 1. PEGylated Lipid Composition and Size of the Formed Lipodisks amount and type of PEGylated lipid 25 25 25 10 10 a
mol mol mol mol mol
% % % % %
radius/nm 13.5 6.9 6.2 58.1 81.5
DSPE-PEG2000 ceramidePEG2000 ceramidePEG5000 DSPE-PEG2000 ceramidePEG2000
Hydrodynamic radius obtained from DLS. obtained from cryo-TEM.
b
± ± ± ± ±
1.6a 0.5a 1.1a 32.5b 42.7b
Planar part radius,
cryo-TEM images are shown in Figures S1 and S2 in the Supporting Information. As can be seen, increasing the amount of PEGylated lipids reduces the size of the lipodisks. The differences between DSPE-PEG- and ceramidePEG-containing lipodisks arise mainly from the fact that different preparation methods need to be used for each composition. Figure 2 shows the obtained association isotherms for the five disk compositions tested. It is observed that for disks containing 10% PEGylated lipids the effect of the charge is D
DOI: 10.1021/acs.langmuir.6b03012 Langmuir XXXX, XXX, XXX−XXX
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Table 2 shows that the value of Reff at saturation (Reff(max)) is the highest for lipodisks constituted of DPPC/DSPE-PEG2000 (75:25 mol/mol), in agreement with the trend observed in Figure 2. DPPC/ceramidePEG2000 (75:25 mol/mol) has about half the (Reff(max)) compared to DPPC/DSPE-PEG2000, thus illustrating the role of the charge on the disk rim. The affinity values (K) obtained from the Langmuir linearization are not significantly affected by charge or size. Comparisons using the Pérez-Paya model are less trustworthy, mainly because of the fact that the dependence between K and the data obtained from linear fitting is exponential (K = exp(−y intercept)). The order of magnitude of the estimated constants is, nevertheless, in all cases in agreement with the observations from the Langmuir model. The results obtained with melittin show that an optimal loading of the peptide onto lipodisks can be achieved if the size of the lipodisk is reduced and the surface charge density at the rim is increased. Therefore, lipodisks with a large DSPEPEG2000 content would be preferred over disks containing ceramidePEG and over disks with a moderate or low PEG content. In agreement with this conclusion, Figure S3 in the Supporting Information shows how the lipid concentration needed to achieve 95% loading efficiency (calculated from the K and Reff(max) values given in Table 2) is much lower for disks containing 25 mol % DSPE-PEG2000 than for the other compositions tested. In this context, it is worth noting that the results obtained with 25 mol % DSPE-PEG2000 differ strongly from those previously reported for the same lipodisk composition when using natural melittin extracted from bee venom (parameters from the Langmuir linearization: Reff(max) = 0.089 ± 0.001, K = 4.3 ± 0.4 μM−1)20 instead of the artificial peptide used in this report. A repetition of the measurements with the natural peptide confirmed this discrepancy. It is out of the scope of this report to speculate about the possible reasons for this behavior. However, it is of high relevance to point out that even though the amino acid sequence and the compound purity are similar, the binding behavior differs significantly. Unfortunately, the fluorimetric determinations used are not time-resolved. Therefore, the kinetics of the binding process cannot be studied. We have shown previously that access to kinetic data describing the association of peptides with lipodisks can be retrieved by means of QCM-D. However, these studies are, in the case of melittin, complicated by the fast binding of the peptide, its very slow release, and the rather large “bulk effect” (i.e., changes in density and viscosity) of the melittin solutions, as discussed in our previous publication.20 Therefore, no attempts were made to obtain kinetic data for the melittin association to and release from the lipodisks.
Figure 3. Scatchard plot (left axis, solid symbols) and linearization of the Pérez-Paya model (right axis, empty circles, dotted line given by the linear fitting of the data) obtained for the association of melittin with DPPC-based lipodisks containing 25% of the ceramidePEG2000 lipid.
shows an example of the obtained curves. Scatchard plots for the other lipodisk compositions and for the binding of other peptides can be found in the Supporting Information. As can be seen, the Scatchard plot deviates significantly from ideal linear Langmuir behavior. One could argue that the binding of charged peptides should affect the further binding of peptides with equal charge. This phenomenon is considered in a peptide−lipid association model developed by Pérez-Paya et al.31 According to this model, the binding can be described by an affinity constant K and a parameter w accounting for deviations from ideal behavior. As has been shown previously,20 a plot of ln([P]aq/Reff) as a function of Reff should result in a line with slope equal to the parameter w and an intercept given by −ln(K). Figure 3 shows that, indeed, a linear relationship is obtained. The deviations from ideal behavior observed in the Scatchard plots could, however, arise from artifacts when the free peptide concentration is small (among others, the inner filter effect becomes much more significant). For this reason, the binding parameters were also calculated by assuming ideal Langmuir behavior using only the data within the linear range of the Scatchard plots (observed at large Reff values). The data calculated from both the Langmuir and the Pérez-Paya association models are summarized in Table 2. It is necessary to keep in mind that both models are based on a series of assumptions that may not necessarily be fulfilled in the systems studied. However, both models are able to accurately describe the experimental data and, therefore, to predict the outcome (e.g., Reff at a certain free peptide concentration) when lipodisks and peptide are mixed in a certain ratio. The interaction parameters obtained from both approaches are therefore useful to identify relevant features of the peptide−lipodisk interaction.
Table 2. Parameters Describing the Antimicrobial Peptides Association with Lipodisks of Different Compositiona Langmuir PEGylated lipid DSPE-PEG2000 25 mol % DSPE-PEG2000 10 mol % ceramidePEG2000 25 mol % ceramidePEG2000 10 mol % ceramidePEG5000 25 mol % DSPE-PEG2000 25 mol % DSPE-PEG2000 25 mol % a
peptide melittin
magainin 2 LL37
Pérez-Paya
−1
K (μM ) 15.13 18.34 27.68 13.27 11.03 0.161 16.63
± ± ± ± ± ± ±
Reff(max)
1.36 2.7 10.76 4.6 0.84 0.01 6.7
0.039 0.009 0.020 0.009 0.029 0.40 0.026
± ± ± ± ± ± ±
0.0004 0.0001 0.0003 0.001 0.004 0.009 0.002
K (μM−1) 5.31 232.31 21.12 66.89 1.31 0.151 not determined
± ± ± ± ± ±
0.81 19.7 5.85 38.6 0.17 0.03
w 131.83 1553.7 374.75 1105.1 142.22 7.82
± ± ± ± ± ±
8.48 54.8 14.1 72.3 6.58 0.70
Data were obtained from QCM-D (for magainin 2 and LL37) and fluorimetric (for melittin) measurements. E
DOI: 10.1021/acs.langmuir.6b03012 Langmuir XXXX, XXX, XXX−XXX
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Langmuir Association of Magainin 2 with Lipodisks. Magainin 2 is an amphiphilic cationic peptide isolated from the skin of frog Xenopus laevis. This peptide is of great therapeutic interest because, in contrast to melittin, it has a very low hemolytic activity. Effective antibacterial and antitumor activities, on the other hand, have been widely reported.4,11,32,33 The binding of magainin 2 to lipid structures has not been studied to the same extent as that of melittin. A major reason for this is the lack of intrinsic fluorescence. To overcome this issue, we used the previously reported QCM-D-based method to determine the association parameters in systems containing magainin 2 and disks composed of DPPC and 25 mol % DSPE-PEG2000. This specific disk composition was chosen because it, according to the results obtained with melittin, appears to be optimal for the loading and delivery of AMPs. The obtained binding isotherm for magainin 2 is shown in Figure 4. The parameters describing the association are
binding and release data recorded with the QCM-D. This analysis was facilitated by the fact that the bulk effect of the peptide is negligible, whereas its binding and release generate time-resolved traces suitable for analysis. The binding vs time curves were analyzed using the TraceDrawer 1.6 software. Figure 5 shows the time-resolved
Figure 5. Time-resolved measurement of magainin 2 binding and release to and from lipodisks when 15 (black), 10 (red), and 3 μM (yellow) solutions of the peptide are introduced into the system followed by rinsing with PBS buffer.
traces obtained upon introducing, respectively, 3, 10, and 15 μM solutions of magainin 2 into the QCM-D flow system as well as the traces due to the release of the peptide when the system is rinsed with buffer. The solid lines represent the nonlinear fit assuming ideal Langmuir behavior. As can be seen, the fitting corresponds very well to experiment. The results from the kinetics analysis performed at different magainin 2 concentrations show that, in contrast to what would be concluded from the curved Scatchard plot, only one kind of binding site is present. This discrepancy may arise from the uncertainty in the determination of the amount of bound peptide at low peptide concentration. The rate constants of binding and release were determined to be, respectively, 1.13 × 10−3 μM−1 s−1 and 7.83 × 10−3 s−1, thus resulting in an affinity constant of K = 0.144 μM−1, corresponding well to the value of 0.161 μM −1 determined from the linear region (free concentration >3 μM) in the Scatchard plot and the Langmuir linearization. As in the case of melittin, experiments performed on a planar supported DPPC lipid bilayer showed that magainin 2 does not bind to a gel-phase membrane with zero curvature. Therefore, the only available binding site in the lipodisks is the curved rim, in agreement with what is observed with melittin. Given the large Reff values obtained at high free magainin 2 concentrations, it is clear that the rim of the disks in these cases must be highly loaded with peptide. From the size of the lipodisks and the Reff(max) value determined, it is estimated that the surface density of the peptide on the disk rim is about 1.5 molecules per nm2. Thus, a very high and compact loading of magainin 2 can be achieved. However, as can be seen from the time-resolved measurements, the peptide is rapidly released from the disks upon dilution, with the half-life time of the process being only ∼90 s. This will cause a fast increase in the concentration of free peptide, which is mostly undesirable for therapeutic applications. Mainly, it implies that a lot of material would be needed and that the peptide will likely be degraded before it reaches its target cells. Furthermore, the risk of negative side effects will increase. Thus, even though a rather
Figure 4. Adsorption isotherm of magainin 2 to DPPC lipodisks stabilized with 25 mol % DSPE-PEG2000. The inset shows the Scatchard plot (left axis, solid symbols) and the linearization of the Pérez-Paya model (right axis, open symbols), including the linear fitting to the data (dotted line).
summarized in Table 2. As can be seen from the data, the affinity of the peptide for the lipodisks is much lower than that of melittin. This finding comes as no surprise, given the higher hydrophobicity of melittin (hydrophobic amino acids: 50% of the total with only 19% charged amino acids, in contrast to only 43% hydrophobic residues and 25% charged amino acids in magainin 2). On the other hand, the amount of peptide that can be loaded onto the lipodisks is considerably higher in the case of magainin 2 (as seen in the large Reff(max) values). One can speculate that this is due to the fact that the net charge of magainin 2 at the pH used lies between +3 and +4 and is spread over a longer amino acid chain than in melittin.33 The latter has a net charge of between +5 and +6, mostly concentrated on one end of the peptide, at the used pH.24 For magainin 2, the broad distribution of the charges may lead to decreased repulsion between already-bound peptides. The high loading capacity of the lipodisks is promising concerning the use of the disks as carriers. From the association parameters shown in Table 2, it is predicted that high loading efficiencies can be achieved even at low [lipodisk]/[peptide] ratios, as illustrated in Figure S4 in the Supporting Information. To estimate the applicability of the lipodisk−magainin formulation and to characterize the peptide−lipodisk interaction more comprehensively, we analyzed the time-dependent F
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concentrations were ignored. This approximation was thus used to estimate the binding parameters, which are summarized in Table 2. It is clear from the results that the peptide has roughly the same affinity for the disks as melittin. However, the disks get saturated at a lower peptide/lipid ratio. This latter observation may be related to the size of the peptide, which is almost twice that of melittin. Given, however, that LL37 has a significant therapeutic effect at low concentrations (reported even down to the nanomolar range36), the lipodisk system is still potentially useful for the formulation and delivery of this peptide. Furthermore, high loading efficiencies can be achieved with moderate lipodisk concentrations, as illustrated in Figure S4 in the Supporting Information. Repetitions of the experiment showed reproducible results, indicating that it is possible to determine the binding isotherm of LL37 by presaturating the substrates on which the lipodisks are immobilized. This is, to our knowledge, the first time that quantitative data concerning the association of LL37 to lipid nanoparticles is provided. Unfortunately, the limited amount of available LL37 and the rather strong binding to the lipodisks did not allow an analysis of the binding and release kinetics. Given the observed similarities between the binding behavior of LL37 and melittin, it can be expected, however, that both peptides will behave similarly in the presence of bacteria and tumor cells. Further experiments to evaluate the bacterial and tumor cell killing effect of LL37-loaded lipodisks are thus clearly warranted.
high loading can be achieved, the utility of this formulation in therapeutic applications is, unfortunately, likely to be very limited. Association of LL37. LL37 is part of the immune system and the only peptide belonging to the cathelicidin family in humans.34 The net charge at physiological pH is +6. In aqueous solution, the cationic LL37 forms a random coil, but in the presence of a membrane it will form an α-helical structure and become amphipathic. This peptide presents antibacterial, antiviral, and antifungal activities. It has also been shown to have an anticancer effect.34 Because it is a very relevant peptide, several reports focusing on its effect and potential applications have been published.26,35 However, and rather surprisingly, there are not, to our knowledge, any reports characterizing its association with lipid membranes. One reason for this lack of information may arise from the fact, observed during our experiments, that LL37 binds strongly to a wide range of surfaces. Control QCM-D experiments showed that LL37 binds strongly to all of the substrates tested (silica, gold, MUAmodified gold, PEGylated silica, etc.), even at very low (nM) bulk concentrations. It is noteworthy that, regardless of the kind of substrate, the monitored changes in oscillation frequency in the QCM-D were always very similar (total frequency change = −16 Hz and negligible dissipation changes). It can be assumed that a monolayer of LL37 is formed in all cases. To circumvent this problem, we used biotinylated sensors to which we attached a layer of streptavidin. The surfaces were then saturated with LL37. It was found that the adsorption of the peptide was irreversible, also in the case of prolonged rinsing. Once the surface had been saturated this way, disks with biotinylated PEG chains were introduced into the system. These disks were cross-linked to the substrate via the already-immobilized streptavidin. Because the nonlipodisk surface is thus saturated with LL37, we can safely assume that mass changes observed when new LL37 solutions are introduced into the system would arise only because of the binding of the peptide to the disks. Under this assumption, the binding isotherm of the peptide to DPPC lipodisks containing 25 mol % DSPE-PEG2000 was determined (Figure 6). For this peptide, both the Scatchard plot and the linearization of the Pérez-Paya model showed a mostly random distribution of the experimental points. The only clear trend could be observed in the Langmuir linearization of the Langmuir isotherm when the data for the two lowest
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CONCLUSIONS Taken together, our results show that the loading of cationic amphiphilic peptides onto lipodisks is strongly dependent on both the lipodisk and the peptide properties. In agreement with previous reports, it is oberved that the peptides prefer the highly curved edge of the disks and do not bind significantly to the planar part of the structure when this consists of a gelphase, uncharged lipid bilayer. Therefore, small disks are preferred to optimize the peptide/lipid ratio. Remarkably, electrostatic interactions between the lipodisk edge and the peptides are not relevant concerning the affinity of the peptides for the lipid particles but are of relevance concerning the number of binding sites on the lipodisk edge. This observation may seem to contradict the specific affinity of these peptides for bacterial membranes, which is thought to arise from electrostatic interactions. It is, however, necessary to remark that the edge of the lipodisks is highly curved and therefore interactions between this edge and the peptides are likely to be mostly hydrophobic in nature, with the electrostatic component being negligible in comparison. This supposition is strengthened by the fact that the peptides do not bind to noncharged planar lipid membranes, although they clearly bind to the curved edge of the lipodisks even if it is not charged. The obtained data suggest that the affinity of the peptides for the lipodisks is dependent only on the nature of the peptide and not on the general properties of the lipodisk (rim charge, size, or extent of the PEG chain). Therefore, for a given peptide, the only parameter that can be controlled is the maximum number of binding sites, which is optimized by using anionic PEGylated lipids and small lipodisks. Concerning the properties of the peptides that can be loaded onto the lipodisks, our results suggest that strongly charged peptides are loaded in smaller amounts than less charged peptides. In the case of melittin, the fact that most of its charges are concentrated on one end of the peptide could be
Figure 6. Association isotherm of LL37 and DPPC lipodisks stabilized with 25 mol % DSPE-PEG2000. The inset shows the Langmuir linearization and the linear fitting of the data. G
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(5) Malmsten, M. Interactions of Antimicrobial Peptides with Bacterial Membranes and Membrane Components. Curr. Top. Med. Chem. 2016, 16, 16−24. (6) Epand, R. M.; Walker, C.; Epand, R. F.; Magarvey, N. A. Molecular mechanisms of membrane targeting antibiotics. Biochim. Biophys. Acta, Biomembr. 2016, 1858, 980−987. (7) Hädicke, A.; Blume, A. Binding of cationic peptides (KX)4K to DPPG bilayers. Increasing the hydrophobicity of the uncharged amino acid X drives formation of membrane bound β-sheets: A DSC and FTIR study. Biochim. Biophys. Acta, Biomembr. 2016, 1858, 1196−1206. (8) Sani, M. A.; Separovic, F. How Membrane-Active Peptides Get into Lipid Membranes. Acc. Chem. Res. 2016, 49, 1130−1138. (9) Wieprecht, T.; Apostolov, O.; Beyermann, M.; Seelig, J. Thermodynamics of the alpha-helix-coil transition of amphipathic peptides in a membrane environment: Implications for the peptidemembrane binding equilibrium. J. Mol. Biol. 1999, 294, 785−794. (10) Epand, R. F.; Savage, P. B.; Epand, R. M. Bacterial lipid composition and the antimicrobial efficacy of cationic steroid compounds (Ceragenins). Biochim. Biophys. Acta, Biomembr. 2007, 1768, 2500−2509. (11) Matsuzaki, K. Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim. Biophys. Acta, Biomembr. 1999, 1462, 1−10. (12) Wessman, P.; Strömstedt, A. A.; Malmsten, M.; Edwards, K. Melittin-Lipid Bilayer Interactions and the Role of Cholesterol. Biophys. J. 2008, 95, 4324−4336. (13) Strömstedt, A. A.; Wessman, P.; Ringstad, L.; Edwards, K.; Malmsten, M. Effect of lipid headgroup composition on the interaction between melittin and lipid bilayers. J. Colloid Interface Sci. 2007, 311, 59−69. (14) Matsuzaki, K. Control of cell selectivity of antimicrobial peptides. Biochim. Biophys. Acta, Biomembr. 2009, 1788, 1687−1692. (15) Diao, L.; Meibohm, B. Pharmacokinetics and pharmacokinetic− pharmacodynamic correlations of therapeutic peptides. Clin. Pharmacokinet. 2013, 52, 855−868. (16) Zetterberg, M. M.; Reijmar, K.; Pränting, M.; Engström, A.; Andersson, D. I.; Edwards, K. PEG-stabilized lipid disks as carriers for amphiphilic antimicrobial peptides. J. Controlled Release 2011, 156, 323−328. (17) Gao, J.; Xie, C.; Zhang, M.; Wei, X.; Yan, Z.; Ren, Y.; Ying, M.; Lu, W. RGD-modified lipid disks as drug carriers for tumor targeted drug delivery. Nanoscale 2016, 8, 7209−7216. (18) Lundquist, A.; Wessman, P.; Rennie, A. R.; Edwards, K. Melittin-Lipid interaction: A comparative study using liposomes, micelles and bilayer disks. Biochim. Biophys. Acta, Biomembr. 2008, 1778, 2210−2216. (19) Zhang, W.; Sun, J.; Liu, Y.; Tao, M.; Ai, X.; Su, X.; Cai, C.; Tang, Y.; Feng, Z.; Yan, X.; Chen, G.; He, Z. PEG-Stabilized Bilayer Nanodisks As Carriers for Doxorubicin Delivery. Mol. Pharmaceutics 2014, 11, 3279−3290. (20) Agmo Hernández, V.; Reijmar, K.; Edwards, K. Label-free characterization of peptide-lipid interactions using immobilized lipodisks. Anal. Chem. 2013, 85, 7377−84. (21) Wessman, P.; Morin, M.; Reijmar, K.; Edwards, K. Effect of alpha-helical peptides on liposome structure: A comparative study of melittin and alamethicin. J. Colloid Interface Sci. 2010, 346, 127−135. (22) Raghuraman, H.; Chattopadhyay, A. Interaction of melittin with membrane cholesterol: A fluorescence approach. Biophys. J. 2004, 87, 2419−2432. (23) Imura, Y.; Choda, N.; Matsuzaki, K. Magainin 2 in Action: Distinct Modes of Membrane Permeabilization in Living Bacterial and Mammalian Cells. Biophys. J. 2008, 95, 5757−5765. (24) Raghuraman, H.; Chattopadhyay, A. Melittin: A membraneactive peptide with diverse functions. Biosci. Rep. 2007, 27, 189−223. (25) Brogden, K. A. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 2005, 3, 238−250. (26) Bandurska, K.; Berdowska, A.; Barczynska-Felusiak, R.; Krupa, P. Unique features of human cathelicidin LL-37. Biofactors 2015, 41, 289−300.
responsible for the repulsion leading to low loading efficiencies On the other hand, its high affinity for all of the tested lipodisks is likely to arise from its clearly defined hydrophobic Nterminus. In contrast, magainin 2 presents a sequence in which charged (both anionic and cationic) amino acids are rather evenly distributed. The solubility of this peptide is therefore higher, and its amphiphilic character is much less marked than for melittin, leading to a lower affinity. The peptide can be loaded in large amounts, but it is released very quickly upon dilution and is therefore not suitable for therapeutic use in a lipodisk formulation. A special case is LL37, which, like melittin, has a rather large net charge but, like magainin 2, has charged amino acids (cationic and anionic) evenly spread over its sequence. It is therefore more difficult to propose an explanation concerning its high affinity but small number of binding sites. In any case, the fact that the binding parameters are similar to those calculated for melittin suggests that the loading and release behavior in vitro and in vivo may be very similar for both peptides. As a next step in our research, it will be relevant to test small charged lipodisks as carriers for melittin and LL37 and investigate their therapeutic effect, which, given the results presented in this report, appears very promising.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b03012. DLS data and cryo-TEM pictures for the studied lipodisks. Scatchard plots and linearizations of the Pérez-Paya model obtained for the binding of melittin to lipodisks of varying PEGylated-lipid composition. Curves illustrating the quantity of lipodisks needed to achieve 95% loading efficiency at different peptide concentrations. (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS K.E. acknowledges funding from the Swedish Cancer Society. We thank Dr. Jonny Eriksson (Department of Chemistry-BMC, Uppsala University) for skillful technical assistance with the cryo-TEM analyses.
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REFERENCES
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