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Behavior of Lipid-Modified Peptides in Membrane-Mimetic Monolayers at the Air/ Water Interface Theodore M. Winger† and Elliot L. Chaikof*,†,‡ School of Chemical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, and Laboratory of Biomolecular Materials Research, Department of Surgery, Emory University School of Medicine, Atlanta, Georgia 30322 Received November 26, 1996. In Final Form: April 1, 1997
The biological membrane has evolved to facilitate rapid and specific physiologic responses in complex multicellular systems. As such, it has provided bioengineers with a model system for the development of biosensors, information storage and delivery devices, drug delivery systems, and surface-modified implants.1-3 As self-organizing noncovalent aggregates, membranes also offer a model for molecular engineering in which the constituent members can be controlled, modified, precisely defined, and easily assembled. In this regard, engineering a bioactive surface may be accomplished by incorporating into lipid membrane structures selected proteins, peptide sequences, and/or carbohydrate structures. This may be accomplished by passive adsorption or by a strategy of chemical conjugation to a lipid-modified building block component. Currently, both solution and solid phase strategies have been described for the synthesis of lipid-peptide conjugates.4,5 While both approaches can be utilized to lipid modify identical peptide sequences, differences in the dialkyl chain structure and the orientation of the bound peptide exist. As part of our ongoing effort to produce phospholipid-based biomimetic surfaces, two amphiphilic lipopeptides, di(C16)Glu-Succ-Gly-Arg-Gly-Asp-Tyr-OH and DSPE-COCH2SCH2CH2NH-Tyr-Asp-Gly-Arg-Gly-H, were synthesized using solid and solution phase conjugation schemes, respectively. Both conjugates contain an identical integrin receptor-activating peptide sequence (GRGDY) and were incorporated into a mixture of natural phospholipids. As many as seven different lipid types have been identified in the cell membrane. This is further complicated by incomplete data regarding the asymmetry of these components in either the outer or inner leaflet of the membrane bilayer. However, predominant lipid components include dimyristoylphosphatidylcholine (DMPC), dilaurylphosphatidylethanolamine (DLPE), and dilaurylphosphatidic acid (DLPA), which in many cell types are found in a molar ratio of 9:4:1.6-8 We have * Author to whom correspondence should be addressed. 1364 Clifton Rd. N. E., Box M-11, Laboratory for Biomolecular Materials Research and Department of Surgery, Emory University, Atlanta, GA 30322. Phone: (404) 727-8413. Fax: (404) 727-3660. E-mail:
[email protected]. † Georgia Institute of Technology. ‡ Emory University. (1) Chapman D. Biomembranes and new hemocompatible materials. Langmuir 1993, 9, 39. (2) Fuhrhop J.-H.; Ko¨ning, J. In Membranes and molecular assemblies: The synkinetic approach; Royal Society of Chemistry: Cambridge, 1994, Monographs in Supramolecular Chemistry Stoddart, J. F., Ed. Vol 5. (3) Ishihara, K.; Tsuji, T.; Kurosaki, T.; Nakabayashi, N. J. Biomed. Mater. Res. 1994, 28, 225. (4) Berndt, P.; Fields, G. B.; Tirrell, M. J. Am. Chem. Soc. 1995, 117, 9515. (5) Winger, T. M.; Ludovice, P. J.; Chaikof, E. L. Biomaterials 1996, 17, 437. (6) Zhou, Q.; Jimi, S.; Smith, T. L.; Kummerow, F. A. Biochim. Biophys. Acta 1991, 1085, 1.
S0743-7463(96)02046-X CCC: $14.00
utilized this ratio as a starting point in formulating substrate-supported membrane-mimetic structures. The purpose of the present work was to examine the mixing properties of integrin-activating lipopeptides in the presence of a membrane-mimetic mixture of natural phospholipids. This behavior will likely influence ligand accessibility and physiochemical stabilitysboth critical features in membrane-based biomaterial and biosensor design. Experimental Section Isotherms were acquired on a circular Langmuir-Blodgett trough (type 2000, Nima Technology, Coventry, England). Amphiphiles were spread using an airtight Hamilton syringe onto deionized water (18.2 MΩ‚cm) obtained from a Modulab Analytical UF/Polishing System (Continental Water Systems). Commercial organic solvents were filtered through clean glass wool prior to use. Hexanes and CHCl3 (1% ethanol stabilized) were from Fisher Scientific, and MeOH was purchased from EM Science. Di(C16)Glu-Succ-Gly-Arg-Gly-Asp-Tyr-OH (1, Figure 1) and DSPE-COCH2SCH2CH2NH-Tyr-Asp-Gly-Arg-Gly-H (2) were synthesized, purified by liquid chromatography, and chemically and physically characterized, all according to methods adapted from previously published protocols.4,5,9 A phospholipid mixture of dimyristoylphosphatidylcholine (DMPC) (3), dilaurylphosphatidylethanolamine (DLPE) (4), and dilaurylphosphatidic acid (DLPA) (5) in a 9:4:1 molar ratio was prepared in hexanes/CHCl3/ MeOH 5/4/1 v/v/v. Aliquots were taken and doped with 1 or 2 over the range 10-100% lipopeptide content. The final total lipid concentration was 2.0 mg/mL. Eight to twenty-five microliters of these solutions were spread over 200-250 cm2 at the air/liquid interface of a Langmuir-Blodgett trough. The subphase was deionized water (18.2 MΩ‚cm) at pH 5.5. Interfacial solvent evaporation was complete after 10 min. Isotherms of the amphiphilic monolayers were subsequently obtained by recording the surface pressure under a lateral compression rate of 20 cm2/min at 24 °C. All runs were performed at least in triplicate. The spreading syringe was thoroughly rinsed with CHCl3 between runs.
Results and Discussion The synthetic scheme used for compound 1, implementing a solid phase coupling methodology, followed by HPLC purification, yielded 30% of an N-terminus coupled lipopeptide. Synthesis of phospholipid conjugate 2 via a solution phase nucleophilic substitution reaction yielded 25% of conjugate exhibiting a selectively C-terminuscoupled peptide. The molecular weights, as confirmed by mass spectrometry, were 1224.6 and 1435.0 g/mol, respectively. Although the peptide sequence is identical in both conjugates, different coupling strategies yield differences in charge distribution, as well as dialkyl moiety, as illustrated in Figure 1. Surface area-pressure isotherms for both lipopeptides feature well-defined expanded and condensed phases (Figure 2). The onset of the expanded phase for 1 occurs at 165 Å2/molecule and for 2 at 93 Å2/molecule. Similarly, the onset of the condensed phase occurs at larger surface areas for 1 than for 2, 51 and 35 Å2/molecule, respectively. In both cases, the transition to a condensed phase was initiated at pressures greater than 30 mN/m. Collapse pressures for both compounds did not differ significantly (64-67 mN/m), although the area at collapse was larger for 1 than for 2 (40 vs 27 Å2/molecule). Of note, the collapse area for compound 2 is somewhat smaller than that (7) Hui, R.; Robillard, M.; Falardeau, P. J. Hypertens. 1992, 10, 1145. (8) Dominiczak, A. F.; Lazar, D. F.; Das, A. K.; Bohr, D. F. Hypertension 1991, 18, 748. (9) Winger, T. M.; Ludovice, P. J.; Chaikof, E. L. J. Liq. Chromatogr. 1995, 18, 4117.
© 1997 American Chemical Society
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Figure 1. Amphiphilic structures at pH 5.5.
Figure 2. Isotherms (24 °C) of pure 1 and pure 2 on deionized water at pH 5.5.
expected for a dialkyl amphiphile. We cannot exclude that either kinetic effects, leading to an overcompressed film, or limited solubility of the lipopeptide for the subphase contributed to this observation. Regardless, this does not alter the qualitative interpretation of the data which follows. A number of estimates of molecular structure can be derived from a consideration of simplified models of the lipid and peptide head group and correlated with the observed behavior of the amphiphilic conjugate at the airwater interface. For the purpose of this analysis, we assumed that the head group for 1 extends from the R-carbon of the glutamyl residue (which connects the two alkyl chains) to the free C-terminus of the peptide. In turn, the head group for 2 was considered to stretch from the sn-2 carbon atom of the phospholipid to the free N-terminus of the peptide. If we assume the peptide to be a fully hydrated random coil, we can estimate a molecular area of at least 211 Å2/molecule for 1 and 281 Å2/molecule for 2. In contrast, if one assumes a globular
configuration with the hydration and packing density of a normal protein, 1 would occupy an area of 156-166 Å2/molecule and 2 an area of 177-189 Å2/molecule. Finally, as a closely packed, fully stretched, and dehydrated peptide chain 1 would occupy 56-62 Å2/molecule and 2, 51-57 Å2/molecule. Similarly, the alkyl chains of compound 1 are roughly separated by 5.5 Å, while the separation distance for 2 is 4 Å. Thus, while the calculated molecular areas for the peptide head groups generally yield larger values for 2 than for 1, the dialkyl unit of compound 1 is bulkier than that for 2. Consequently, the onset of expanded and condensed phases, as well as the collapse area, appears dominated by characteristic steric features of the hydrophobic tail structure. It is of interest that the onset of the expanded phase of 1 correlates with a calculated molecular area for the peptide head group on the basis of an assumed globular protein configuration. The onset of the expanded phase for 2, however, occurs between the expected values for a globular and a closepacked peptide structure. We can speculate that the less bulky tail structure for 2 facilitates a closer packed configuration for the peptide head group. Therefore, although further experimental work is required, this observation suggests that, within a lipid assembly, the structure of the hydrophobic tail group may alter the tertiary peptide configuration. The implication for altered receptor interactions is clear. The behavior of mixed lipopeptide-lipid systems is presented in Figures 3 and 4. The average molecular area of the monolayer at low surface pressures increased with increasing content of compound 1, confirming that the addition of 1 to phospholipids 3-5 induces an expansion of the monolayer. However, at high surface pressures, the average molecular area decreased with increasing concentration of 1, indicating monolayer contraction in the crystalline state. Monolayer contraction was also noted for lipopeptide 2, with an even more dramatic effect at high lipopeptide content. An examination of the relationship between molecular area and lipopeptide content at a given surface pressure (0, 5, 25, and 50 mN/m) provides additional insight into mixing behavior. As shown in Figure 4, if the lipopeptide behaved like the other amphiphiles in the monolayer, from a purely structural point of view, the average molecular area for the monolayer would decrease linearly with a
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Figure 3. (a) Isotherms (24 °C) of DMPC:DLPE:DLPA 9:4:1 molar, spiked with 1 on deionized water at pH 5.5 (b) Isotherms (24 °C) of DMPC:DLPE:DLPA 9:4:1 molar, spiked with 2 on deionized water at pH 5.5.
Figure 4. Comparison between ideal and real molecular mixing in monolayers of DMPC:DLPE:DLPA 9:4:1, spiked with 1 or 2 on water at surface pressures of 0 (a), 5 (b), 25 (c), and 50 (d) mN/m. Negative slopes are indicative of monolayer contraction.
proportional increase in lipopeptide content. However, our analysis of the experimental data indicates that the mixing relationship is often not linear. A positive deviation from ideal mixing behavior is observed for 1 in the expanded phase (5 mN/m) at relatively high lipopeptide concentrations (40-60%), which may be secondary to repulsive peptide-peptide electrostatic interactions. In
contrast, a large negative deviation was observed in the condensed phase. This was most pronounced at relatively low lipopeptide concentrations (20%) and may be secondary to short-range intermolecular interactions between negatively charged groups in the peptide and positively charged functionalities associated with the PC and PE head group. Similarly, the mixing behavior of 2 was
Notes
characterized by a negative deviation in the gaseous and condensed phases which presumably may also be related to attractive electrostatic interactions between lipid and peptide head groups. Additional discussion on similar interactions in Langmuir-Blodgett films can be found elsewhere for lipopeptides4,10,11 and glycolipids.12 Lipid-peptide conjugates represent molecular building blocks which may facilitate the creation of functionalized surfaces for biosensor or biomedical applications. Intrinsic to the activity of the surface is the recognition of the peptide sequence by an appropriate receptor. At the level of a single lipopeptide, this recognition process is fundamentally dependent upon the correct primary amino acid sequence. The inherent flexibity of self-assembled membrane-mimetic systems offer the potential for producing functionally heterogenous structures. However, mixing and phase separation effects will likely modulate ligand recognition due to the two-dimensional organization of (10) Cha, X.; Ariga, K.; Onda, M.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 11833. (11) Cha, X.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 9545. (12) Tamada, K.; Minamikawa, H.; Hato, M. Langmuir 1996, 12, 1666.
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the surface constituents. Further, under certain packing constraints the structure of the hydrophobic tail group may alter the tertiary peptide configuration. Conclusions Our results indicate a dependence of the phase behavior of membrane-mimetic monolayers at the air-water interface on lipopeptide type and content. The steric effects of the hydrophobic moiety affects two-dimensional phase transitions, while electrostatic interactions govern short-range intermolecular mixing behavior. Both repulsive peptide-peptide and attractive peptide-lipid interactions may be responsible for observed deviations from ideal mixing behavior. The packing behavior of lipopeptides embedded in a phospholipid matrix at the air-water interface will likely influence both physiochemical stability and ligand accessibility. These observations provide relevant insights into the design of effective bioactive surfaces by membrane and surface engineering with lipopeptide constituents. LA9620465
