Interaction of Peptidomimetics with Bilayer Membranes: Biophysical

Feb 16, 2012 - Xianghui Zeng , Anne Marit de Groot , Alice J. A. M. Sijts , Femke Broere , Erik Oude Blenke , Stefano Colombo , Willem van Eden , Henr...
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Interaction of Peptidomimetics with Bilayer Membranes: Biophysical Characterization and Cellular Uptake Xiaona Jing,† Marina R. Kasimova,† Anders H. Simonsen,† Lene Jorgensen,† Martin Malmsten,§ Henrik Franzyk,‡ Camilla Foged,† and Hanne M. Nielsen*,† †

Department of Pharmacy and ‡Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark § Department of Pharmacy, Uppsala University, Husargatan 3, SE-751 23, Uppsala, Sweden S Supporting Information *

ABSTRACT: Enzymatically stable cell-penetrating α-peptide/β-peptoid peptidomimetics constitute promising drug delivery vehicles for the transport of therapeutic biomacromolecules across membrane barriers. The aim of the present study was to elucidate the mechanism of peptidomimetic-lipid bilayer interactions. A series of peptidomimetics consisting of alternating cationic and hydrophobic residues displaying variation in length and N-terminal end group were applied to fluid-phase, anionic lipid bilayers, and their interaction was investigated using isothermal titration calorimetry (ITC) and ellipsometry. Titration of lipid vesicles into solutions of peptidomimetics resulted in exothermic adsorption processes, and the interaction of all studied peptidomimetics with anionic lipid membranes was found to be enthalpy-driven. The enthalpy and Gibbs free energy (ΔG) proved more favorable with increasing chain length. However, not all charges contribute equally to the interaction, as evidenced by the charge-normalized ΔG being inversely correlated to the sequence length. Ellipsometry data suggested that the hydrophobic residues also played an important role in the interaction process. Furthermore, ΔG extracted from ellipsometry data showed good agreement with that obtained with ITC. To further elucidate their interaction with biological membranes, quantitative uptake and cellular distribution were studied in proliferating HeLa cells by flow cytometry and confocal microscopy. The cellular uptake of carboxyfluorescein-labeled peptidomimetics showed a similar ranking as that obtained from the adsorbed amount, and binding energy to model membranes demonstrated that the initial interaction with the membrane is of key importance for the cellular uptake.

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experimental conditions and the cell type.6−9 Irrespective of the type of cellular uptake mechanism and further trafficking, however, it is generally believed that interaction between the CPP delivery vector, carrying a therapeutic or diagnostic cargo, and the biological membrane is a critical step preceding cellular internalization.10,11 Thus, a deeper insight into the mechanism of peptidomimetic−membrane interaction is of key interest for the design of future peptidic carriers. The inherent sensitivity of peptides to degradation by proteases has been shown to hamper the high delivery potential of some peptidic carriers, and therefore their stabilization is an important challenge to overcome in the future design of CPPs.12 Peptide backbone modifications usually confer significantly reduced sensitivity to proteolytic enzymesan approach adopted for several peptidomimetics, such as βpeptides13,14 and peptoids15−17 intended for use as carriers.18,19 Recently, enhanced cellular uptake and membrane-destabilizing properties were found for a series of α-peptide/β-peptoid peptidomimetics with superior proteolytic stability20−22 compared to a readily degradable and well-known α-peptidic

INTRODUCTION Overcoming biological membrane barriers is the main obstacle for advancing the increasing number of biopharmaceutical drug candidates into novel treatment modalities.1,2 The size and hydrophilic nature of therapeutic biomacromolecules significantly limit their permeation through biological membranes, resulting in low bioavailability when the pharmacological target site requires transmembrane transport, e.g., to the cell cytoplasm. Certain cationic peptides have been shown to possess the ability to efficiently translocate themselves as well as an associated cargo across cellular membranes of human cells. Well-known examples include polyarginines and other argininerich peptides, e.g., the Tat47−57 domain (YGRKKRRQRRR) of the HIV-1 transactivating (TAT) protein.3−5 Thus, there is currently a considerable interest in developing vectors based on such cell-penetrating peptides (CPPs) to enable a more efficient delivery of therapeutic drugs or diagnostic tools for imaging, for example. The accumulated knowledge on the internalization process points toward multiple simultaneously operating uptake mechanisms. It has become increasingly evident that uptake levels and pathways, as well as intracellular trafficking of peptidic carriers and their cargoes, depend on physicochemical properties of both the CPP and the cargo, as well as on the © 2012 American Chemical Society

Received: October 14, 2011 Revised: January 27, 2012 Published: February 16, 2012 5167

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synthesis, purified, and characterized as previously reported.21 In addition, carboxyfluorescein (CF)-labeled compounds with n = 8 (7, Figure 1) and n = 6 (6,23 Figure 1) were prepared essentially as described previously by using solid-phase synthesis with conjugation of CF to the N-terminal of the peptidomimetic.23 All compounds were purified by preparative HPLC to a purity of at least 95%, and the Mw was verified by mass spectrometry. For 5 and 7, the Mw values were confirmed by HR-MS to be 2006.24 and 3026.68 Da, respectively. Preparation and Characterization of Liposomes. Unilamellar liposomes consisting of POPC and POPG (molar ratio of 80:20) were prepared by the thin film method. The lipid stocks (5 mg/mL) were diluted with chloroform and mixed in appropriate ratios in a roundbottomed flask. The organic solvent was evaporated, and the lipid film stripped with ethanol, which was removed under vacuum overnight. For preparations used for ITC studies, the lipid film was subsequently hydrated for 1 h with vigorous agitation every tenth minute at room temperature with a buffer containing 10 mM HEPES, 150 mM KCl (pH 7.4) to give a lipid concentration of 20 mM. Upon annealing for 1 h, large multilamellar vesicles (LMVs) were extruded (Lipex Biomembranes extruder, Vancouver, BC, Canada) 10 times through two stacked polycarbonate membrane filters with 100 nm pore size (Whatman, Herlev, Denmark); the resulting LUVs were stored at 4 °C until use. For ellipsometry, the lipid film was hydrated at room temperature in a 10 mM HEPES buffer (pH 7.4). The small unilamellar vesicles (SUVs) required to create the lipid bilayer for the ellipsometry studies were prepared fresh before use, and the LMVs were freeze−thawed eight times and then extruded using the LipoFast Basic extruder (Avestin, Mannheim, Germany) 31 times through a polycarbonate membrane filter with a 30 nm pore size (Whatman, Kent, UK). For all batches used for ITC, the expected vesicle size and polydispersity index (PDI) was verified by photon correlation spectroscopy. The surface charge of the particles appropriately diluted in Milli-Q water was estimated by analysis of the ζ-potential (laserdoppler electrophoresis). The measurements were performed at 25 °C using a Zetasizer Nano ZS (Malvern, Worcestershire, UK) equipped with a 633 nm laser and 173° detection optics. Malvern DTS v. 5.10 software (Malvern, Worcestershire, UK) was used for data acquisition and analysis. For viscosity and refractive index, the values of pure water were used. Isothermal Titration Calorimetry. Thermodynamic analysis of the peptidomimetic adsorption to POPC:POPG (80:20) liposomes were performed using a Nano ITC (TA Instruments, New Castle, DE). The majority of the experiments were performed at 37 °C, although additional temperatures (27 and 47 °C) were also used to determine the heat capacity of the interaction. The reaction cell (Vcell = 1 mL), filled with the peptidomimetic dissolved in buffer (10 mM HEPES and 150 mM KCl, pH 7.4), was titrated 10 μL at a time at 5 min intervals with liposome dispersion under constant stirring at 300 rpm. This interval between injections was sufficient for complete equilibration of the system as judged by the flat appearance of the postinjection baseline (see Results section). For reference purposes, the liposome dispersion was injected into buffer showing constant nonzero heats. For the individual experiments, the exact concentrations of peptidomimetics in the samples (22.8−29.4 μM) and lipid in the titrant (17.3−18.1 mM) were used for calculations. Titrations were performed in duplicate and all solutions were degassed prior to use. Fitting of the data to the Langmuir adsorption model was done in Microsoft Excel (Microsoft, Redmond, WA). Experiments were performed in duplicate. Ellipsometry. Adsorption of the peptidomimetics to the supported bilayers was studied by null ellipsometry using an Optrel Multiskop (Optrel, Kleinmachnow, Germany) equipped with a 100 mW argon laser. The theory of null ellipsometry and the experimental procedures have been described thoroughly before.27−30 The measurements were carried out at 532 nm at an angle of incidence of 67.52° in a 5 mL cuvette under constant stirring. The adsorption process was monitored by measuring the changes in amplitude and phase of light reflected at the adsorbing surface. From these parameters, the mean refractive index, n, and the layer thickness, d, of the adsorbed layer were calculated using the optical layer models.30−32 From the thickness and

CPP.23 Noticeably, the design space for protease-stabilized peptidomimetics comprises a range of different structures, which allow for different folding patterns as a result of heterogeneous backbone design24,25 and different degree of, for example, cationic functionalization.26 The previously reported α-peptide/β-peptoid peptidomimetics contain alternating repeats of α-amino acids and N-alkylated β-alanine (β-peptoid) residues, which are designed to retain the cationic properties and hydrogen-bonding capability provided by the α-amino acid residues while the β-peptoid residues contribute to hydrophobic interactions.21−23 In the present study, a series of α-peptide/β-peptoid peptidomimetics of different length with varied N-terminal modifications was investigated with the aim of correlating membrane interaction with cellular internalization. Uptake studies using living cells provide indispensable information in themselves, whereas rigorous biophysical investigations require use of well-characterized model membranes that allow for precise control of lipid composition, thus eliminating local variation of membrane properties such as charge density and fluidity. In the present investigations, we therefore combine these approaches and extract thermodynamic parameters from biophysical investigations involving both liposomes and supported lipid bilayers using isothermal titration calorimetry (ITC) and ellipsometry, respectively. The Langmuir adsorption model for describing binding of peptidomimetics to lipid membranes was used, and as a result, thermodynamic parameters obtained from these two experimental methods could be compared for the first time. These results are related to the quantitative uptake studies and cellular distributions in order to gain an improved understanding of the molecular mechanisms responsible for the peptidomimetic−membrane interaction.

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EXPERIMENTAL SECTION

Materials. Octaarginine [R8 = H(Arg)8NH2] and N-terminally carboxyfluorescein-labeled octaarginine (CF-R8) were obtained from GenScript Corp. (Piscataway, NJ) (both of >98% purity). LysoTracker Red was from Invitrogen (Carlsbad, CA). Amino acids and coupling reagents were obtained from IrisBiotech (Marjtredwitz, Germany), while Rink amide resin for the solid-phase synthesis of peptidomimetics was from Sigma-Aldrich Chemie (Steinheim, Germany). Chloroform stocks of palmitoyloleoyl phosphatidylglycerol) (sodium salt) (POPG) and palmitoyloleoyl phosphatidylcholine (POPC) (both of >99% purity) were from Avanti Polar Lipids (Alabaster, AL). If not otherwise stated, all other compounds were of analytical grade and obtained from Sigma-Aldrich Chemie (Steinheim, Germany). Synthesis of Peptidomimetics. An N-terminal-labeled αpeptide/β-peptoid with six repeats (n = 6, i.e., 6; see Figure 1) and nonlabeled N-acetylated peptidomimetics composed of alternating achiral N-benzyl-β-alanine and L-homoarginine (1−4,21 Figure 1) with different chain lengths (n = 5−8) were prepared by solid-phase

Figure 1. Chemical structures of α-peptide/β-peptoid peptidomimetics investigated in the present study. 5168

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refractive index, the adsorbed amount (Γ) was calculated according to eq 133

Γ=

(n − n0) d dn/dc

acids (all from Sigma-Aldrich, St. Louis, MO), 1 mM sodium pyruvate (Invitrogen, Carlsbad, CA), and 10% (v/v) fetal bovine serum (FBS) (Fisher Scientific, Waltham, MA). The cells were cultured in an atmosphere of 5% CO2/95% O2 at 37 °C and the growth medium was changed three times a week. The cells were split weekly at 80% confluency. Flow Cytometric Analyses. Cell internalization of CF-labeled peptidomimetics was quantified by flow cytometry as described previously.23 HeLa cells were seeded in 24-well tissue culture plates in 0.5 mL medium at a density of 2 × 104 cells/cm2 and grown for 48 h. The medium was removed, and the cells were washed with 1 mL of phosphate-buffered saline (PBS) per well. The cells were incubated with 300 μL of premixed samples containing 10 μM peptidomimetics in fresh complete EMEM with 10% (v/v) FBS for 1 h at 37 °C. After incubation, the cells were washed and trypsinized at 37 °C for 10 min to cleave surface-adhering peptidomimetics from the cell membranes and to detach the cells from the wells. Then, 1 mL of ice-cold PBS containing 10% (v/v) FBS was added to each well, and the detached cells were centrifuged at 1076g for 10 min at 4 °C. The supernatant was removed, and the cells were resuspended and centrifuged again, after which the cells were dispersed in ice-cold PBS containing 10% (v/v) FBS and 1.5 μM propidium iodide (PI) (Invitrogen, Carlsbad, CA) for staining of dead cells. The cells were kept on ice prior to analysis. The cellular fluorescence was measured using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) at an excitation wavelength of 488 nm. Data was analyzed with the CellQuest Software (Beckton Dickinson). Dead cells were excluded on the basis of the PIstaining. The experiments were performed in triplicate, and the statistical significance of the results was validated by one-way analysis of variance (ANOVA) at a 95% significance level, followed by Tukeys post-test using SigmaPlot v. 11.0 (Systat Software, San Jose, CA). Confocal Laser Scanning Microscopy. As described above, HeLa cells grown to an 80% confluent monolayer were trypsinized and seeded at a density of 2 × 104 cells/cm2 onto 35 mm glass-bottomed Microwell dishes (MatTek, Ashland, MA). After culture for 48 h at 37 °C to 80% confluency, the cells were washed with PBS and incubated at 37 °C for 1 h with fresh complete EMEM + 10% (v/v) FBS (200 μL) containing fluorescently labeled peptidomimetics (10 μM) and LysoTracker Red (Invitrogen, Carlsbad, CA) (50 nM). The cells were then washed five times with ice-cold PBS and stored on ice in 0.5 mL of PBS. Confocal imaging of unfixed cells (to avoid artifactual localization of internalized peptidomimetics) was performed on a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany) equipped with an argon laser (458 and 488 nm) and a HeNe laser (543 nm) using the LSM 510 software and a Zeiss plan apochromat 63× oil immersion objective with a numerical aperture of 1.4.

(1) 3

29,34

and where dn/dc is the refractive index increment (0.154 cm /g) n0 is the refractive index of the bulk solution. Silica surfaces for ellipsometry were prepared from polished silicon slides (Okmetic, Vantaa, Finland). To avoid the instability caused by the spontaneous oxidation of silicon during the experiment, the silicon slides were thermally oxidized in advance to form a SiO2 layer of around 30 nm in thickness. To remove contaminations from the slides, their surface was cleaned at 80 °C for 5 min with an alkaline peroxide solution containing 3.6% NH4OH and 4.3% H2O2 and then with an acidic peroxide solution containing 4.6% HCl and 4.3% H2O2 (also at 80 °C for 5 min). These slides were stored in 99% (v/v) ethanol until use. Immediately before use, the slides were further cleaned by plasma treatment for 5 min at 18 W in low pressure residual air (0.2 mbar) in a Harrick Plasma Cleaner PDC-32G (Harrick Scientific, Ithaca, NY) Anionic bilayers were generated by adsorption of 30 nm POPC:POPG liposomes at 25 °C,35,36 while adsorption of peptidomimetics was studied at 37 °C. The characterization and verification of bilayer formation have been described previously.34,37 To avoid adsorption directly onto the silica substrate via possible defects in the supported lipid bilayer, poly-L-lysine (Mw = 167.8 kDa, Sigma-Aldrich, St.Louis, MO) was preadsorbed from water to the silica surface prior to liposome addition. Nonadsorbed poly-L-lysine was removed by rinsing with a flow of water (5 mL/min) for approximately 20 min. The water in the cuvette was then replaced with isotonic HEPES buffer containing 150 mM KCl. The liposome dispersion without KCl was added, which facilitates liposome rupture and formation of the lipid bilayer. When the liposome adsorption and fusion had stabilized, nonadsorbed and weakly adsorbed liposomes were removed by rinsing with buffer (Figure 2). The calculated average value of the surface excess of the final bilayer for all measurements was 4.2 ± 0.3 mg/m2.

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RESULTS AND DISCUSSION With the aim of performing a detailed investigation of their lipid membrane interaction, a series of enzymatically stable peptidomimetics with different chemical characteristics was applied to fluid-phase lipid bilayers with an overall negative charge, and the membrane interaction behavior and structural changes were observed and related to the actual uptake in proliferating HeLa cells. Adsorption to Vesicles Is Favored by Increasing Positive Charge and Temperature. Binding of peptidomimetics 1−5 composed of a different number of repeating units (n = 5−8) to anionic liposomes was investigated by ITC. Liposome average diameters spanned from 100 nm (PDI 0.157) to 115 nm (PDI 0.114), and their ζ-potential was around −53 mV. A representative ITC trace and the heats of interaction are shown in Figure 3. Initially, all calorimetric data were fitted to the commonly used model developed for the treatment of ligand binding to a single set of identical independent binding sites. However,

Figure 2. Deposition of a POPC:POPG bilayer on a silica surface. The graph shows the adsorbed amount of lipid, Γ, upon addition of the liposome dispersion with a lipid concentration of 20 μM (marked with an arrow). After lipid bilayer formation, the temperature was increased to 37 °C, and the external phase in the cuvette was replaced by 10 mM HEPES buffer with 150 mM KCl (pH 7.4) at a rate of 5 mL/min over a period of 30 min. When the values of the ellipsometric parameters ψ and Δ were stable,36 peptidomimetic was added first to a concentration of 0.01 μM. This initial injection was followed by three subsequent additions of peptidomimetic to give the desired final concentrations of 0.1, 0.5, and 1 μM. In all cases, the adsorption process was monitored for approximately 1 h until adsorption had stabilized. All measurements were performed in at least duplicate, and all eight data sets were used to obtain the best average fit. Cell Cultures. HeLa cells from the American Type Culture Collection (ATCC, Manassas, VA) were maintained in Eagle’s minimum essential medium (EMEM), supplemented with 100 U/ mL penicillin, 100 μg/mL streptomycin, 0.1 mM nonessential amino 5169

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study reported a good agreement between both the enthalpy and the Gibbs free energy obtained by the two approaches. Comparison of the thermodynamic parameters reported for R938 with the results obtained in this work indicates that the hydrophobic effect is an important contributor to the binding affinity, since the ΔG value of −28.4 kJ/mol found for R9 is lower in magnitude than the Gibbs free energy values for all peptidomimetics including 1, which has fewer charges than R9. The amount of POPG lipids bound per R9 reported by Conçalves et al.38 is ∼5, which agrees well to our results of 6−7 POPG per peptidomimetic (analysis results not shown). In addition to peptidomimetics, we have conducted an ITC study of interaction between POPC:POPG (80:20) liposomes and R8. However, these experiments did not provide a sufficient heat effect for a reliable analysis of the data. Indeed, the enthalpy of R9 binding to the POPC:POPG (75:25) liposomes found in the previous work was only −10.5 kJ/mol, which is 3− 4 times lower than the enthalpies obtained for the peptidomimetics. The thermodynamic parameters summarized in Table 1 indicate that at physiological temperature, the interaction between different peptidomimetics and anionic liposomes are enthalpy-driven. Both the enthalpy and the Gibbs free energy decrease with increasing peptide length (i.e., throughout the series 1−4). Since longer peptidomimetics have a higher number of α-peptide/β-peptoid repeats, they have both higher net positive charge and a higher number of hydrophobic benzyl side chains. Therefore, it is difficult to dissect the overall interaction energy into different contributions, such as those arising from electrostatic interaction or hydrophobic effect. As evident from the values of charge-normalized Gibbs free energy, ΔG/charge (Table 1), the contribution from each consecutively added charge gradually decreases upon elongation of the peptidomimetic (see Figure 1). The same observation is also valid for the Gibbs free energy normalized per benzyl side chain. This trend is apparent for peptidomimetics 1−4 and is most likely due to the entropy penalty required for immobilizing the longer peptidomimetics onto the membrane (column “−TΔS” in Table 1). For peptidomimetics of the same length, 2 and 5, the ΔG values are close; however, both the enthalpy and the entropy contributions to the overall Gibbs free energy are different. This difference can be attributed to the chemical influence of the end group of the peptidomimetics, as the nonacetylated N-terminal of 5 might well be lesssuited for penetrating into the lipophilic core of the membrane, leading to the unfavorable entropy contribution, whereas in the case of acetylated peptidomimetic 2, the entropy of binding is favorable. Finally, the total amount of adsorbed peptidomimetics, Γfit, shows a weak tendency to decrease with an increasing sequence length, which is consistent with the notion

Figure 3. A representative ITC experiment including (A) raw data showing the titration of 5 with POPC:POPG liposomes. (B) Integrated heat values as a function of injection number. The solid line corresponds to the best fit to the adsorption model.

some fundamental requirements of this model are not appropriate for a peptidomimetic−membrane interaction system (the details are discussed below). Therefore, the data treatment was repeated using the Langmuir adsorption model. In spite of the theoretical differences between the “single set” and the Langmuir models, the obtained thermodynamic parameters, such as the interaction enthalpy and the binding constant, are very similar (within 5% error), and thus, only parameters obtained in the Langmuir model are presented in Table 1. Although analysis of the data was done with the Langmuir model, it is helpful to discuss the single set model as well, as it provides a comparison to previously published works. For example, interaction of R9 to POPC:POPG (75:25) liposomes was studied by Conçalves et al.,38 where the data were analyzed with either the “single set” model or with a model that combines hydrophobic partitioning and the Gouy−Chapman electrostatic theory. Similar to our results, the authors of this

Table 1. Thermodynamic Parameters for the Adsorption of Peptidomimetics to POPC:POPG Liposomes at 37 °C Obtained by Fitting ITC Data to the Adsorption Model (see Supporting Information) peptidomitic/peptide 1 2 3 4 5d

ΔHa (kJ/mol) −29.3 −29.8 −37.5 −40.0 −38.6

± ± ± ±

1.7 0.4 3.8 6.4

K (μM−1) 0.29 0.53 0.76 1.18 0.7

± ± ± ±

0.04 0.37 0.01 0.03

Γfitb (nmol/m2) 86 90 77 75 85

± ± ± ±

2 2 2 4

ΔGc (kJ/mol) −32.4 −34.0 −34.9 −36.0 −34.7

± ± ± ±

0.4 1.2 0.1 0.1

ΔG/charge (kJ/mol)

−TΔS (kJ/mol)

−6.5 −5.7 −5.0 −4.5 −5.0

−3.1 ± 1.4 −4.3 ± 3.3 3.6 ± 2.9 3.9 ± 6.4 3.9

Adsorption enthalpy is per peptidomimetic. bThis parameter is the same as γ introduced in eq 12 of the Supporting Information. It represents the surface density of the adsorbed peptidomimetic at saturation. cΔG = −RT ln K. dn = 1. a

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that longer molecules occupy a larger surface area on the lipid bilayer. The temperature dependence of the binding parameters was investigated for peptidomimetic 5 (Figure 4). The enthalpy of

Figure 4. Enthalpy (black circles) and Gibbs free energy (gray circles) of the interaction between peptidomimetic 5 and POPC:POPG liposomes as a function of temperature.

Figure 6. Comparison of ellipsometry data for the adsorbed amount (Γ) of three selected α-peptide/β-peptoid peptoidmemetics as well as of octaarginine to supported POPC:POPG bilayers. Triangles and circles represent two independent data sets.

interaction becomes more negative as the temperature increases, resulting in a negative heat capacity value. Although the Gibbs free energy becomes more favorable with temperature as well, the absolute values for this change are smaller than for the enthalpy changes, which is due to the unfavorable entropy compensation. Adsorption to Supported Lipid Bilayer Suggests the Presence of Hydrophobic Interactions. A representative course of adsorption of peptidomimetic 4 to the bilayer is shown in Figure 5. No destabilization of the lipid bilayer was

peptides44 and guanidinium-rich peptidomimetics.45 Therefore, we compared membrane binding of a series of α-peptide/βpeptoid peptidomimetics and R8. The net charge of R8 is identical to that of 4, so this comparison addresses the importance of lipophilic side chains, which are present in 4, but not in R8. The total adsorbed amount of R8 (Γfit in Table 2) is Table 2. Thermodynamic Parameters Obtained from the Ellipsometry Data Using the Data Analysis Procedure Described in the Supporting Information peptidomitic/ peptide

K (μM−1)

Γfit (nmol/m2)a

ΔG (kJ/mol)b

ΔG/charge (J/mol)

R8 2 4 5

1.60 0.46 0.87 0.41

12 65 147 76

−36.9 −33.6 −35.3 −33.3

−4.6 −5.6 −4.4 −5.6

Γfit is the total adsorbed amount at saturation obtained from fitting. This parameter is defined as Γt in the Supporting Information and is introduced in eq 10. bΔG = −RT ln K. a

Figure 5. Representative time-resolved adsorption of 4 to a supported anionic bilayer consisting of POPG:POPC (80:20). Time points for peptide addition and the total concentration in the cuvette after each addition are indicated. Time t = 0 s designates the occurrence of the first peptidomimetic addition.

an order of magnitude lower than that of 4 (Figure 6). Thus, interaction of hydrophobic benzyl side chains with the lipid bilayer contributes significantly to the higher adsorption of 4 as compared to that of R8. Moreover, our data indicate that the presence of hydrophobic moieties is more important for increasing the absorbed amount, since the Γ value for R8 is lower than those for 2 and 5, despite the fact that these two peptidomimetics have fewer charges than R8. Modification of peptidomimetics by acetylation of the N-terminus did not significantly affect the adsorbed amount (2 vs 5 in Figure 6). This is in analogy to an ellipsometry study by Stromstedt,39 in which an antimicrobial peptide (EFK17) was investigated with respect to the influence of terminal acetylation when adsorbed to supported zwitterionic bilayers. Binding Energy is Independent of the Curvature of the Lipid Bilayer. Although ITC and ellipsometry both constitute custom methodologies for studying interactions of peptides with model lipid membranes, no previous comparative studies have, to our knowledge, been reported. In the present study, both ITC and ellipsometry were employed to study interactions of peptidomimetics with POPC:POPG lipid

observed with any of the constructs, even at the highest peptide concentration (1 μM). The threshold concentration for initial adsorption of the studied peptidomimetics was estimated to be in the range 0.01−0.1 μM. The shape of the time-resolved adsorption isotherm suggests that the adsorption is a gradual process (Figure 5), in agreement with previous findings for a range of antimicrobial peptides.26,39−43 Figure 6 summarizes the data on the interaction of peptidomimetics with supported lipid bilayers as studied by ellipsometry. The adsorbed amount of 2, 4, 5, and R8 increases as their respective concentrations increase stepwise from 0.01 to 1 μM. Previous studies have indicated that initial binding of cationic peptides to membranes rich in anionic components is important in order to achieve high uptake of arginine-rich 5171

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presence of electrostatic interactions between the bound molecules. The short Debye distance also indicates that electrostatic forces between the peptidomimetics and the lipid bilayer begin to play a role only when peptidomimetics are in close proximity or bound to the surface of the membrane. Analysis of ellipsometry data with the Langmuir adsorption model is presented in Table 2. As illustrated in Tables 1 and 2, the values of the thermodynamic parameters obtained by calorimetry and ellipsometry generally are in good agreement, suggesting that the lipid bilayer curvature does not influence the binding process considerably. This is in accordance with previous reports on lipid−peptide SUV (interaction studies on small unilamellar) vs LUV model systems,50,51 suggesting that local interactions dominate in these systems and that curvature effects in the investigated 100 nm liposomes are less important. The total amount of adsorbed peptidomimetics, Γfit, obtained by the two techniques varied to a larger extent than the Gibbs free energy. Both studies indicate that the ΔG becomes more favorable with the increase of the sequence length (the series 1−4). The effect of the end group on the binding thermodynamics can be derived from the comparison of peptidomimetics 2 and 5. As suggested by the ellipsometry results, neither ΔG nor Γfit is affected significantly by the Nterminal substitution. However, the additional parameters provided by ITC indicate that the binding thermodynamics is different; i.e., the interaction of peptidomimetic 2 is both enthalpically and entropically favorable, whereas binding of 5 is driven by enthalpy but is entropically unfavorable. Another interesting observation can be made about the nature of R8lipid interactions. Analysis of the ellipsometry data suggests that the adsorption Gibbs free energy of this peptide is similar to that of other peptidomimetics. However, this fact could not be confirmed by ITC due to the insufficient binding enthalpy, suggesting that adsorption of R8 to the lipid bilayer is entropydriven. Cellular Uptake Showed Dependence on Sequence Length. To investigate the putative correlation between the biophysical studies on binding to model lipid membrane and in vitro cell studies, the cellular uptake of fluorescently labeled R8 and peptidomimetics 6 and 7 in cell culture was quantified by flow cytometry.21,23 HeLa cells were incubated with these CF-labeled compounds. Nontreated HeLa cells were used as negative control, and the background fluorescence was subtracted, while dead cells were excluded by gating based on PI-staining and membrane-associated peptides were removed by trypsination. Since previous experiments had also shown that additional washing with heparin to remove surface-bound peptidomimetic did not influence the results,23 the obtained flow cytometry data indeed reflect the total amount of compound internalized in viable cells. The calculated mean fluorescence intensity relative to R8 thus allows for a direct comparison with this wellstudied reference CPP. As clearly seen from Figure 7, the cellular uptake of CF-labeled peptidomimetics increased significantly with chain elongation as the longer peptidomimetic 7 exhibited an uptake superior to that of its shorter counterpart 6. This also parallels previous studies on arginine oligomers,52 in which it was found that uptake efficiency increased with length up to 15 residues, whereas peptides shorter than the 6-mer lacked cell-penetrating properties. The cellular uptake of CF-labeled peptidomimetics (7 > 6 > R8) in HeLa cells indicated a ranking (8-mer peptidomimetic > 6-mer

bilayers in vesicular form or immobilized onto a silicon surface, respectively. An additional difference in the methodologies in the present setup was the final concentration of peptidomimetic employed for optimal data acquisition, which for ITC was up to 29 μM and thus much higher than for ellipsometry (1 μM). Moreover, in ellipsometry experiments the lipid bilayer was titrated by the peptidomimetic solution, whereas the experimental design was the opposite in the ITC setup. Thus, comparison of these data requires application of a common model for data fitting (Supporting Information). Typically, two major approaches are used for the data treatment of the membrane interaction with peptides/peptidomimetics: (i) the identical independent binding sites model or (ii) the surface partitioning equilibrium model. Although both models often provide good agreement with experimental data,38 theoretical foundations of their applicability are not always appropriate, particularly when the basic assumptions of one such model are not fulfilled. The uncertainties associated with applying the classic “single set” model is that the fluid lipid bilayer does not have specific peptidomimetic binding sites and thus requires a fundamentally different type of theoretical description.46−48 This feature is better accounted for by two other models, i.e., either partitioning or the Langmuir adsorption model, which do not include the concept of specificity. However, the partitioning model lacks the concept of saturation, since the partitioning process is described by the following equation: [bound peptide] = K[free peptide][lipid]. This equation implies the absence of the upper limit of the bound peptide, because the bound amount is always proportional to the peptide in solution. However, on a real membrane the binding is limited by the available surface area and thus cannot be treated as partitioning. A much better model for the description of such systems was suggested by Di Chera and Kong in ref 49. Unfortunately, due to the complexity of the mathematical theory, it could not be incorporated into an Excel-based fitting program and thus cannot be applied without the use of sophisticated programming. Thus, the Langmuir adsorption model is the best choice for treatment of the peptide−lipid interactions, because of its simplicity and because it satisfies requirements of our experimental system, such as (1) it does not include the concept of specific binding sites; (2) it includes the concept of saturation, which is defined by the total surface area of the lipid bilayer; (3) it provides a possibility of modeling the data without the need for separating the overall binding into hydrophobic partitioning and electrostatic interaction. The last point has an additional benefit for interpretation of the data. In particular, we deduce the influence of different contributions to the binding affinity by analyzing and comparing several peptidomimetics that differ by the net charge and the number of hydrophobic groups. Therefore, our approach is model-free, i.e., it does not rely on any particular model (e.g., Gouy−Chapman) for evaluation of the electrostatic effect, thus avoiding potential misinterpretation of the data. Another reason for not modeling electrostatic interactions explicitly is that we work in solutions of high ionic strength (150 mM salt). According to the calculations presented at the end of the Supporting Information, the Debye length at this salt concentration is ∼8 Å, while the distance between two bound peptidomimetics at saturation is ∼44 Å. This precludes the 5172

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ranking uptake versus membrane interaction. Since analysis of CF-labeled peptidomimetics by ellipsometry was not practically applicable, the nonlabeled compounds were used for the biophysical studies. Nevertheless, the very similar ranking of cellular uptake and adsorption to model membranes constitutes an interesting finding, since it enables several high-throughput methods to be employed for screening purposes in structure−activity studies. Although the final steps in the uptake mechanism following membrane binding are not completely elucidated, the initial interaction of peptidomimetics with the membrane constitutes a crucial step in the entire uptake process. Further evidence for cellular internalization and visual indications of the distribution patterns of CF-labeled peptidomimetics was obtained by CLSM (Figure 8), demonstrating that these constructs entered HeLa cells after incubation for 60 min at 37 °C. To exclude fixation artifacts in the internalization experiments,53,54 all confocal microscopy was performed on live cells. Both peptidomimetics were found to accumulate in the cytoplasm, but the intracellular localization patterns of peptidomimetics 6 and 7 appeared to be mostly vesicular. But under the current experimental circumstances, only a poor colocalization with LysoTracker was observed for both peptidomimetics. The nonlabeled peptidomimetics (1−4) have previously been shown to be nontoxic to proliferating HeLa cells up to concentrations at least 10 times higher than the present concentration of 10 μM.22 In addition, the cytotoxicity of the fluorophore-labeled peptidomimetics/ peptide was tested, and no toxicity was found at the tested

Figure 7. Quantitative flow cytometric analysis of the cellular uptake of a CPP and two peptidomimetics. HeLa cells were incubated for 60 min with 10 μM of CF-labeled peptidomimetics (6 and 7) and the CPP (R8) at 37 °C. Cellular mean fluorescence intensity data represent mean ± standard deviation of three samples. Data has been normalized to the mean fluorescence intensity of CF-R8 (set to 1). Significant differences from CF-R8 are indicated: ***p < 0.001.

peptidomimetic > R 8 ) similar to that deduced from ellipsometry and ITC studies using the corresponding nonlabeled peptidomimetics. The influence exerted by the Nterminal CF-labeling, necessary for the cell uptake studies, must not be completely neglected if comparing the derived values of binding constants from ellipsometry and ITC to specific values of cellular uptake, which is why the correlation is limited to

Figure 8. Confocal microscopic analysis of uptake of peptidomimetics. Intracellular localization of CF-labeled CPP (R8) and peptidomimetics (6 and 7, all at 10 μM) in living HeLa cells after incubation for 60 min at 37 °C (middle); live cells were simultaneously labeled with LysoTracker Red (left); merged images (right). Bar = 20 μm. 5173

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concentration (results not shown). Also, neither in the flow cytometry nor in the CLSM studies were observed any visual signs of cell damage.

CONCLUSIONS Adsorption of α-peptide/β-peptoid peptidomimetics to POPC:POPG (80:20) lipid bilayer is favored by an increasing number of α-peptide/β-peptoid repeats. Since each additional residue increases both the net positive charge and the number of hydrophobic side chains, this effect might be due to either electrostatic interaction or hydrophobic effect or a combination of both. The increased adsorption of peptidomimetics compared to R8 indicates that binding is promoted by the presence of the benzyl side chains, suggesting the importance of the hydrophobic effect in peptidomimetic−lipid interaction. Correlation of the thermodynamic parameters derived from ITC and ellipsometry suggests that the binding Gibbs free energy is independent of the curvature of the lipid bilayer with the lower limit set by 100 nm liposomes. The ΔG values for peptidomimetics with acetylated vs nonacetylated N-terminals are similar, and the reaction is exothermic in both cases. However, the entropic contribution into ΔG is favorable only for the acetylated peptidomimetic, suggesting that its hydrophobic N-terminus is able to access the apolar regions of the lipid bilayer. The cellular uptake of the peptidomimetics was much higher than seen for the reference peptide R8. The clear positive correlation found between cellular uptake and model membrane adsorption indicates that the initial interaction with the membrane is of key importance for the entire uptake process for these peptidomimetics. We are currently investigating the potential of the peptidomimetics for intracellular delivery of a biomacromolecular therapeutic cargo. ASSOCIATED CONTENT

S Supporting Information *

Description and assessment of the Langmuir adsorption model for combined fitting of ITC and ellipsometry data. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +45 3533 6346. Fax: +45 35 33 60 01. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Birger Brodin Larsen (Department of Pharmacy, University of Copenhagen) for scientific instruction on confocal microscopy and Dr. Lovisa Ringstad (Department of Pharmacy, Uppsala University) for scientific advice on ellipsometry. We are grateful to Lise-Britt Wahlberg (Department of Pharmacy, Uppsala University) and Maria L. Pedersen (Department of Pharmacy, University of Copenhagen) for excellent technical support. This work was funded by The Faculty of Pharmaceutical Sciences, University of Copenhagen. The Danish Agency for Science, Technology and Innovation and the Swedish Research Council are also acknowledged for funding. Equipment funding was granted by The Danish National Advanced Technology Foundation and The Danish Agency for Science, Technology and Innovation. 5174

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