Langmuir 1998, 14, 3625-3630
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Synthesis and Physicochemical Characterization of Cyclic Laminin Related Peptides A. Ferna´ndez,† Ma A. Alsina,† I. Haro,‡ R. Galantai,§ and F. Reig*,‡ Physicochemical Unit, Faculty Pharmacy, Plac¸ a Pius XII s/n, 08028 Barcelona, Spain, Department of Peptide and Protein Chemistry, CID, CSIC, Jordi Girona 18, 08034 Barcelona, Spain, and Institute of Biophysics, Semmelweis Medical University, Budapest, Hungary Received February 28, 1997. In Final Form: April 8, 1998 The synthesis of three cyclic peptides related to an active sequence of Laminin is described. These molecules have no, one, or two stearoyl residues linked to the amino terminal end of the sequence. The physicochemical properties of these peptides as well as their interactions with dipalmitoylphosphatidylcholine molecules ordered in mono- and bilayers are described. Properties based on aqueous solutions of peptides show maximum activity for the analogue with one stearoyl residue. But properties measured from organic solutions of peptides (compression isotherms), as well as those in water but at high temperatures, gave as the most surface active the distearoyl derivative. This behavior is interpreted as a strong tendency of this highly hydrophobic molecule to form aggregates (probably micelles), very stable in aqueous media. Thus, the monostearoyl-substituted peptide seems to be the most adequate for efficient insertion into mono- and bilayers.
Introduction The main cause of mortality in patients with massive malignant tumors is metastasis. One of the determinant steps in this process is the invasion of the basal membrane by the tumoral cells. Laminin (LN) is a basement membrane glycoprotein with diverse biological activities, including cell adhesion promotion, growth, neurite outgrowth, and differentiation; all of them closely related to the metastasic potential of tumor cells.1-3 From diverse biological studies it has been clearly established that Laminin and other extracellular matrix proteins are recognized by specific receptors (Integrins) on the cell surface.4 Several Integrins which share the β1 subunit are recognized to be receptors for both LN and Fibronectin (FN). A particular amino acid fragment of the Laminin molecule YIGSR recognizes a 67-KD a cell membrane receptor that influences cell attachment5 and is one of the most active in the inhibition of experimental metastasis formation.6 In addition, some cyclic derivatives have been described with enhanced activity versus lineal analogues.7 One of the problems faced when trying to use shorter active sequences to mimic the activity of a whole protein is that the peptide adopts the same conformation it has in the native state.8 The introduction of conformational restrictions is a good alternative to solve this problem, and in this sense, disulfide bridges are the method of choice.9 * To whom correspondence should be addressed. † Physicochemical Unit, Faculty Pharmacy. ‡ Department of Peptide and Protein Chemistry, CID, CSIC. § Institute of Biophysics, Semmelweis Medical University. (1) Martin, G. R., Timpl, R. and Ku¨h, K. Adv. Protein Chem. 1988, 39, 1-50. (2) Timpl, R. Eur. J. Biochem. 1989, 180, 148-502. (3) Beck, K., Hunter, I. and Engel, J. FASEB J. 1990, 4, 148-160. (4) Lowele, J. Byers Cancer Lett. 1995, 88, 67-72. (5) Rainieri, J. P. Int. J. Dev. Neurosci. 1994, 12 (8), 725-735. (6) Iwamoto, Y. Science 1987, 238, 1132-34. (7) Davies, J. S. J. Chem. Soc., Perkin Trans. 1994, 1, 201. (8) Ely, K. R.; Kunicki, T. J.; Kodandapani, R. Protein Eng. 1995, 8 (8), 823-827.
The second point to approach is how to deliver the peptide to the organism in order to ensure a good circulating time. This can be solved by the design of delivery systems and/or stabilization of the peptide. An alternative to solve both aspects is the preparation of hydrophobically derived cyclic peptides followed by their association with liposomes. According to this idea, we undertook the synthesis of cyclic Laminin YIGSR derivatives with different degrees of hydrophobicity and characterized these peptides from a physicochemical point of view. In this paper the synthesis and surface activity as well as the interaction of these peptides with mono and bilayers of lipids are described. The results obtained indicate that a medium degree of hydrophobicity is the best approach to incorporate peptides to liposomes without destroying the integrity of bilayers. Experimental Section Chemicals. Solvents and reactants used in peptide synthesis, dimethylformamide (DMF), and piperidine were from MilligenBiosearch. 9-Fluorenyl methoxycarbonyl (Fmoc) amino acids, Wang resin, diisopropylcarbodiimide (DIPCDI), and hydroxybenzotriazol (HOBT) were supplied by Novabiochem. (Dimethylamino)pyridine (DMAP), iodine (I2), methylene chloride (CH2Cl2), anisole, acetic acid, and diethyl ether were from Merck. Trifluoroacetic acid (TFA) was supplied by KaliChemie. Sodium 8-anilinonaphthalenesulfonate (ANS) and 1,6diphenyl-1,3,5-hexatriene (DPH) were from Eastman-Kodak, and dipalmitoylphosphatidylcholine (DPPC) was from Sigma. Solvents used in monolayers and fluorescence studies (chloroform, methanol, dimethyl sulfoxide, tetrahydrofuran) were Merck HPLC quality. Peptide Synthesis. Peptides were prepared following the Fmoc (OBut) methodology, working on solid phase with Wang type resins. Synthesis was carried out using the following amino acid side chain protecting groups: trityl (Trt) and acetamido methyl (Acm) for Cys; 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) for Arg; and tert-butyl for Ser and Tyr. A disulfide link (9) Gramberg, D.; Deber, C.; Beeli, R.; Inglis, J.; Bruns, C.; Robinson, J. A. Helv. Chim. Acta 1995, 78, 1588-1606.
S0743-7463(97)00222-9 CCC: $15.00 © 1998 American Chemical Society Published on Web 05/30/1998
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was formed before cleavage by oxidation with iodine followed by Fmoc deprotection and TFA/water/anisol (95/3/2) treatment for 2 h. Peptides were isolated by ether insolubilization, redissolved in 10% acetic acid, and freeze-dried. Purification was carried out by medium pressure on a Lobar system. Final characterization was carried out by amino acid analysis and mass spectrometry (electrospray).
Methods Preparation of Liposomes. Small unilamellar vesicles (SUVs) were prepared as follows: 40 mg of phospholipid (DPPC) was dissolved in 5 mL of a mixture chloroform/methanol (3:1) and dried in a rotary evaporator at 50 °C. Traces of solvents were eliminated by submitting the samples to a vacuum pump for 2 h. The dried lipid film was hydrated in 2 mL of 0.25 M sodium acetate solution (pH 7.4, isotonic), with repeated vortexmixing at 50 °C for 1 h. Multilamellar vesicles (MLVs) thus formed were sonicated in a Vibracell VC 300 ultrasounds device with a 13 mm Ti probe for 2 min eight times, leaving the samples 1 min under nitrogen atmosphere between sonication steps. To eliminate Ti particles and medium size liposomes to avoid artifacts due to turbidity, samples were centrifuged at 14000g for 1 h. In this way all samples showed homogeneous size distributions with diameters lower than 100 nm. These samples were incubated with DPH or ANS solutions (10-5 M, 50 °C) at increasing probe/PL relationship, to determine those corresponding to saturation. Phospholipid concentration was quantified following the Barlett’s method.10 Physicochemical Studies. Surface pressure (∆π) of peptide solutions (in DMSO) was measured in a circular Teflon trough, using a platinum plate connected to a Sartorius balance. The trough (6.2 cm diameter × 2 cm depth) was provided with a magnetic stir bar and has a small hole drilled at an angle through the wall to allow the addition of peptide solutions. Increasing volumes of peptide solutions were injected into the aqueous subphase (PBS, pH ) 7.4), and pressure increases were recorded for 1 h. Compression isotherms were carried out in a rectangular Teflon cuvette (28.4 cm × 17.45 cm × 0.625 cm). Peptide solutions were spread by application of small drops on the aqueous surface, using a Hamilton microsyringe. A period of 10 min was allowed for evaporation of solvent and equilibration of the monolayers before starting the compression process, with the Teflon barrier moving at a rate of 4.2 cm/min. All the surface pressure-area isotherms presented here are the average of at least three measurements; they were reproducible to within 0.05 nm2/ molecule. Polarization and fluorescence studies were carried out in a Spectrofluorometer P. E. LS50 connected to a thermostated bath (Tempette TE-8A, Techne). Liposomes (50 µL), previously saturated with DPH or ANS, were added to quartz cuvettes, containing each one 3 mL portion of peptide solution (10-5 to 10-6 M) or 0.25 M sodium acetate, located in a thermostated cuvette holder and submitted to magnetic stirring. Once the system was equilibrated, fluorescence spectrum as well as polarization were recorded in the temperature range 20-55 °C. Temperature increases were 2-3 °C, and at least 10 min was allowed for stabilization between measurements. All data were taken by triplicate in different preparations.11 Data were submitted to a statistical treatment in order to determine the significance of the differences among them.
Results Peptide Synthesis. Peptides, with sequences given in Figure 1, were prepared following solid-phase methodology, using a Wang type resin. Two different Fmoc cysteine derivatives (Trt and Acm)12 were used in order to optimize the oxidation process at the end of the (10) Barlett, G. R. J. Biol. Chem. 1959, 234, 466-68. (11) Cajal, Y.; Rabanal, F.; Alsina, M. A.; Reig, F. Biopolymers 1996, 38, 607-618. (12) Atherton, E.; Pinori, M.; Sheppard, R. C. J. Chem. Soc., Perkin Trans. 1 1985, 2057.
Figure 1. Peptide sequences. Table 1. Analytical Data of Peptides peptide
HPLCa
peptide 1
K′ ) 3.79b
peptide 2
K′ ) 10.05c
peptide 3
AAA
MW (MS-ES)
S)0.88; G)1.04 Y)0.95;C)1.23 I)1.05; R)1.39 S)0.80;G)1.14 Y)0.71; C)1.20 I)1.11; R)1.15 S)0.70; G)0.93 Y)0.67; C)1.58 I)0.95; R)0.96
[M + H]+ ) 799.4 [M + 2H]2+ ) 400.1 [M + H]+ ) 1065.8 [M + H]+ ) 1463.2
a Analytical RP-HPLC (P.E., column 4.6 mm × 250 mm, Spherisorb C-18, 10 µm). b Eluent 50:50 ACN/H2O, 0.05% TFA. c Eluent 13:85:2 ACN/DMF/H O, 0.05% TFA. 2
synthesis. This alternative of forming the disulfide link before cleavage from the resin gave better results than oxidation in solution because of the intrinsic diluting effect of the resin bulk.13 Stearoyl residues were introduced by treating stearic acid as a regular amino acid. No decrease in the reactivity of the carboxyl group, due to the hydrophobicity of the residue, was detected. No differences in reactivity were observed among the three peptides synthesized. Yields on crude peptides based on the first amino acid attached to the resin were about 85% with a purity of 80%, calculated from the area of the main peak on HPLC. Peptides 1 and 2 were purified on a medium-pressure system working with a C 18 Lobar column. Peptide 3 showed a very low solubility in any solvent, so it was not possible to control purity by HPLC. This peptide was washed extensively with ethyl ether and 10% acetic acid solution and submitted to thin-layer chromatography in several eluent systems in order to remove impurities. Finally, peptides were characterized by amino acid analysis and mass spectrometry (electrospray). These data are summarized in Table 1. The presence of stearoyl residues in peptides 2 and 3 gave bad baselines in amino acid analysis, which influenced the overall integration values. Nevertheless, mass spectra showed nice molecular peaks, thus confirming the identity and purity of peptides in addition to the results obtained by analytical HPLC. Surface Activity. As described in the experimental part, the spreading pressure was determined by injecting increasing volumes of concentrated peptide solutions into the buffer contained in the experimental cuvette. Maximum pressure increases reached after 1 h were recorded versus the concentration of the peptide in the subphase. The incorporation process of peptide molecules into the air/water interface was slow if compared with other experiences,14 and a delay in the starting of pressure increase was observed, especially at low peptide concen(13) Edwards, W. B.; Anderson, C. J.; Welch, M. J.; Fields, C. G.; Fields, G. B. J. Labelled Compd. Radiopharm. 1994, 35, 359.
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Figure 3. Effect of different concentrations of peptides 1, 2, and 3 (added to the subphase) on the surface pressure of aqueous solutions.
Figure 2. Surface pressure increase versus time recorded after injection of peptide solutions in the subphase. Peptide concentrations (µM) in the bulk of the aqueous phase are as follows: (peptide 1) 1.2, 1.5, 3, 3.7, 5, and 6; (peptide 2) 0.28, 0.71, 1.43, 2.85, 3.43, 5.12, and 6.11; (peptide 3) 0.28, 0.71, 2.8, and 4.3. Table 2. Surface Excess and Mean Interfacial Area of Peptides at the Spreading Pressure peptide conc (µM) ∆π (mN/m) 1 2 3
3.7 3.43 4.3
7.33 9.18 2.28
Γ (mol/cm2) 10-11
2.43 × 2.95 × 10-11 7.15 × 10-12
A (nm2/residue) 0.97 0.804 3.317
trations. This is illustrated in Figure 2. Surface excess (residue/cm2) was calculated by applying the Gibbs adsorption equation (eq 1), for those concentrations corresponding to the spreading pressures measured after 1 h of peptide solution injection.
Γ ) (1/RT)∆Π/∆ ln c
(1)
where R is the gas constant (8.3 × 107 erg‚K-1‚mol -1), T ) 294 (K), c is the peptide concentration in the subphase (mol or residue/L), and ∆Π is the surface pressure increase. From these values the mean interfacial area A ) 1/NΓ (nm2/residue) was also calculated. All these data are summarized in Table 2. The area/molecule values thus obtained, per amino acid residue, are higher than those calculated from compression isotherms, but as in these experiences surface pressures achieved are lower than those attained on compression (as is described in the following next paragraphs), one can assume that molecules, on the surface, are not strongly packed. As the cyclic structure of peptides involved in this study is common for all of them and the disulfide bridge is close to the hydrophobic alkyl chain, differences between them (14) Vergne, I.; Prats, M.; Tocanne, J. F.; Laneelle, G. FEBS Lett. 1995, 375, 254-58.
in area/residue seem to be due mainly to the equilibrium distribution between the surface and the bulk of the system. Figure 3 illustrates the change in the surface pressure caused by adding different amounts of peptides to the aqueous phase. Peptides 1 and 2 show similar behavior and equilibrium is reached at bulk concentrations ranging between 3 and 4 µM; on the contrary peptide 3 shows a very low surface activity and the pressure increases determined are almost constant from a 0.8 µM concentration in the subphase. Finally, according to the chemical structure of these sequences, a gradual change in hydrophobicity should be evident, and it is so when measuring properties related to isolated molecules (retention times in HPLC); but in an aqueous environment, where the tendency to form aggregates is favored for the most hydrophobic derivatives, this factor predominates over the rest, and surface properties are hidden because of the aggregation phenomena. This is the reason for the delay in surface activity experiments and for the low values of pressure increase (peptide 3), as well as the high values of area/residue determined. Binding Experiments. Phospholipid monolayers have been frequently used as membrane models to study lipid/peptide or protein interactions, since this system can simulate one side of a bilayer structure.14,15-17 Phosphatidylcholine was chosen because it is the main component of biological membranes and liposomal preparations. Besides, the selection of DPPC was a requirement of fluorescence polarization studies. The insertion of peptides into phospholipid monolayers can be measured by monitoring changes in the surface pressure (∆Π, mN/ m) of a previously spread monolayer. Though the relevant pressure in biological membranes is around 32 mN/m,18 we decided, for experimental reasons, to work at lower pressures (5, 10, and 20 mN/m), assuming that, with few exceptions,19 there is an inverse relationship between pressure increases and initial surface pressures. Concentrations in the subphase were slightly lower than those of saturation previously determined in the absence of a monolayer. After injection, pressure increases were recorded for 60 min, and values are represented in Figure 4. In all experiments there was a decrease of ∆π as πi increased. The highest initial pressure permitting peptide (15) Fonseca, M. J.; Busquets, M. A.; Alsina, M. A.; Reig, F. Langmuir 1993, 9, 3149-3153. (16) Go´mez, V.; Colome´, C.; Reig, F.; Rodriguez, L; Alsina, M. A. Anal. Chim. Acta 1994, 290, 65-74. (17) Puyal, C.; Maurin, L.; Miquel, G.; Bienvenu¨e, A.; Philippot, J. Biochim. Biophys. Acta 1994, 1195, 259-266. (18) Seelig, A. Biochim. Biophys. Acta 1987, 899, 196-204. (19) Mestres, C.; Reig, F.; Busquets, M. A.; Haro, I.; Alsina, M. A. Int. J. Pharm. 1991, 76, 145-149.
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a
Figure 4. Effect of initial surface pressure of DPPC monolayers on the penetration of peptides. The peptide concentration in the subphase (PBS, pH ) 7.4) was 1.82 µM. Each data point represents the average of three measurements.
b insertion within the phospholipid layer can be estimated by extrapolating the surface pressure increase to zero (∆π ) 0).20 An initial pressure limit of 25 and 35 mN‚m-1 for peptides 1 and 2 was obtained. Peptide 3 showed, on the contrary, a low tendency to interact with the lipid film. Comparing Figures 3 and 4, it appears that there is a relationship between the hydrophobicity order observed from surface activity experiments and the ability of these peptides to insert into a phospholipid monolayer. In both experiences DMSO was used as common solvent in order to have comparable peptide solutions. The lack of DMSO surface activity under these conditions had previously been verified. As the peptides were injected from an organic solution, molecules were not aggregated at the moment of injection, and the differences observed in the interaction with DPPC reflect their tendency to form micelles in an aqueous environment. Compression Isotherms. Peptide monolayers were formed by application of the samples dissolved in highly diluted organic solutions (10-5 to 10-7 M) of methanol/ chloroform. Results from the surface pressure-area measurements are shown in Figure 5. Peptides 2 and 3 gave isotherms mainly in the liquid condensed state that collapse at 50.4 and 58 mN‚m-1 of surface pressure, respectively, thus indicating a high stability of the monolayers. Peptide 1 forms also monolayers that collapse at 13 mN‚m-1. When applying higher amounts of peptide on the surface, initial pressures increased but no changes were observed at the collapse point. The reproducibility of these measurements was good enough to allow us to discard a partial solubilization of the monolayer. The limiting area, obtained by extrapolation of the linear part of the isotherm, for peptides 2 and 3, to π ) 0 mN‚m-1, was approximately 0.91 and 0.77 nm2/molecule, whereas the area of lift off pressure corresponded to 1.3 and 1.05 nm2/molecule. These values suggest that the presence of an extra amino acid Lys with two alkyl chains in peptide 3 modifies the orientation of the cyclic structures of peptides 2 and 3 at the air/water interface. Effect of Peptides on Model Phospholipid Membranes. The interaction of peptides with phospholipid vesicles was also determined by analyzing their effect on the thermotropic behavior of DPPC bilayers containing either DPH or ANS. The results obtained are given in Figures 6 and 7. Peptide 1 showed no interaction with bilayers (peptide/ lipid molar ratio ) 0.05), thus indicating that though it has by itself surface activity and is able to insert into monolayers, spread at medium surface pressures till 20 (20) Rafalski, M.; Ortiz, A.; Rockwell, A.; Van Hinkel, L. C.; Lear, J. D.; DeGrado, W. F.; Wilschut, J. Biochemistry 1991, 30, 10211-10220.
c
Figure 5. Isotherms of surface pressure versus area per molecule for monolayers of peptide 1 (a), 2 (b) and 3 (c), spread on PBS pH ) 7.4.
mN‚m-1 or 25 (if extrapolating), at surface pressures around 30 mN‚m-1 that are common in SUV liposomes, this interaction is not detectable. On the contrary, peptides 2 and 3 modified the phase transition profile of vesicles. Nevertheless, as these two peptides have an amphipathic chemical structure, the possibility of micelle formation should not be discarded. If so, these aggregates could also accommodate a certain number of probe molecules and as a consequence modify the overall result of polarization temperature experiments. To check for this possibility the peptides were incubated with both DPH or ANS in the absence of liposomes, and fluorescence as well as polarization was recorded.
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Figure 7. Polarization changes of DPPC/ANS vesicles after incubation with peptides: (f) plain vesicles; (9) peptide 1 (a), peptide 2 (b), and peptide 3 (c).
Figure 6. Effect of peptides 1, 2, and 3 on the transition temperature of DPPC vesicles measured through changes in the polarization (P) of DPH: ([) plain liposomes; (9) peptide 1 (a), peptide 2 (b), and peptide 3 (c); (2) peptide 2 without liposomes.
As a common trend, fluorescence intensity increased softly with temperature while polarization remained constant. Samples containing peptide 3 had to be diluted by 10 in order to have fluorescence intensity values in the range of operation. These results are in agreement with the results and interpretation of surface experiments and also indicate that the formation of these micellar structures is favored by the temperature. As both DPH and ANS do not fluoresce in water, this increase in the intensity of fluorescence must be due to the incorporation of more molecules into the hydrophobic aggregates of peptides 2 and 3. Moreover, the fact that polarization did not change with temperature can be explained by assuming that marker
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molecules are located in the central core of the micelles and are not affected by the alkyl chain motion. Concerning liposomes, the presence of peptides 2 and 3 increased the polarization values of both fluorescent probes (ANS, DPH), these data being indicative of a restriction in the motion of fluorophore molecules, due to a more viscous environment in the alkyl chain zone and at the polar heads level. For DPH experiments it was possible to adjust the values to a sigmoidal curve and, in this way, to determine, through the first derivative, the inflection point that theoretically corresponds to the transition temperature. In the case of peptide 2 this Tc increase was 2 °C, and for peptide 3 there was no apparent change although it produces a clear
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decrease in the amplitude of the observed transition, thus indicating an increase in the cooperativity of the process. The effect of peptide 3 in the interior core of liposomes began up to 30 °C; this could be explained by the stability of peptide micelles and would be in agreement with the low surface activity values detected at 20-22 °C. As a summary, one can say that hydrophobic derivatization of peptides helps their interaction with lipids. Depending on the hydrophobicity, there always exists an equilibrium between monomer and aggregates, that conditions their behavior in the different physicochemical models designed. LA970222J
