Article pubs.acs.org/JPCB
Interaction of Phospholipid Langmuir Monolayers with an Antibiotic Peptide Conjugate Tamás Keszthelyi,*,† Katalin Hill,‡,§ and Éva Kiss‡ †
Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1025 Budapest, Pusztaszeri út 59-67, Hungary ‡ Laboratory of Interfaces and Nanostructures, Institute of Chemistry, Eötvös Loránd University, P.O. Box 32, H-1518 Budapest 112, Hungary ABSTRACT: The interactions between phospholipid monolayers and a peptide conjugate of the antituberculotic agent isoniazide (INH) were investigated by sumfrequency vibrational spectroscopy. The primary objective of the present work was to provide a detailed picture of the molecular interactions of the INH−peptide conjugate with phospholipid monolayers by detecting the changes in the monolayer structure resulting from these interactions. In order to gain a thorough understanding, three types of experiment were performed: (i) changes induced in the structure of the precompressed phospholipid monolayer upon injection of the INH−peptide conjugate were followed; (ii) the structures of the phospholipid monolayers spread onto the solution of the INH−peptide conjugate were characterized; (iii) the structures of mixed monolayers of phospholipid and the INH−peptide conjugate were studied. Using a chain perdeuterated phospholipid, it was possible to examine the changes in alkyl chain ordering without interference from INH−peptide conjugate vibrations and investigate the effect of the INH−peptide conjugate on the ordering of the phosphocholine headgroups. We confirmed that peptide conjugation strongly influences the interactions of INH with the lipid monolayer, resulting in enhanced cell penetration ability. The interactions formed between the INH−peptide conjugate in its ordered adsorption layer and the phospholipid molecules deposited onto this solution were found to be significantly stronger than those formed by the INH−peptide conjugate with a compressed lipid monolayer. Nonetheless, both types of interaction contribute with a condensing ef fect to an increased ordering of the phospholipid alkyl chains in the monolayer.
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INTRODUCTION In traditional drug delivery, almost irrespective of the method of administration, the active pharmaceutical agent is distributed through blood circulation. This often results in nearly uniform distribution in the body, necessitating high doses in order to achieve the required drug concentration in the target organ. High drug doses put an unnecessary burden on the whole body and in some cases lead to adverse side effects such as tissue damage. The purpose of targeted drug delivery is to provide precise local concentration of the drug in the target organ or tissue, often for a prolonged period, thereby improving efficiency and reducing side effects. A promising approach within this varied and rapidly growing field is to conjugate the drug to a peptide-based carrier.1 The so-called cell-penetrating peptides are able to cross membranes and efficiently introduce drug molecules in the interior of target cells, while cell-targeting peptides possess specific binding activity for certain types of cells.2 High doses and long duration are also a problem in the treatment of tuberculosis. Tuberculosis is a bacterial infectious disease caused by Mycobacterium tuberculosis, leading to more deaths throughout the world than any other infectious disease, thus presenting a major global public health problem.3 As the pathogen can survive in the host macrophages, high drug levels in the blood are necessary in its chemotherapy to attain © XXXX American Chemical Society
therapeutically effective concentrations in infected cells. Elimination of Mycobacterium tuberculosis from the infected phagocytes could be more efficient with target cell-directed delivery of antituberculotic agents. Conjugating a drug moiety to a peptide based carrier might therefore also be a valuable method in designing an advanced treatment for tuberculosis. One of the most effective, very specific bactericidal synthetic therapeutic agents for the treatment of tuberculosis is isoniazide (isonicotinic hydrazide, INH).4 Peptide 91SEFAYGSFVRTVSLPV106, corresponding to the 91−106 region of the 16 kDa low-molecular-weight heat shock protein (Hsp16.3) expressed by M. tuberculosis, was identified as a functional T-cell epitope, and it provokes a specific immune response.5−7 This peptide is thus a promising candidate for specific delivery of INH to the infected host macrophages.8 In a previous study, using a phospholipid monolayer as a simplified membrane model, we demonstrated that conjugation to peptide 91SEFAYGSFVRTVSLPV106 significantly increases the membrane affinity of INH.9 Characterization of the interactions of therapeutic agents with lipid membranes is of particular importance as drugs have Received: February 12, 2013 Revised: May 14, 2013
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dx.doi.org/10.1021/jp401533c | J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
Article
methods. Using the chain perdeuterated phospholipid, DPPC-d62, made it possible to follow the changes in alkyl chain ordering without interference from INH−peptide conjugate vibrations and facilitated the investigation of the effect of the INH−peptide conjugate on the phosphocholine headgroups. We confirmed that peptide conjugation strongly influences the interactions of INH with the lipid monolayer, resulting in enhanced cell penetration ability. From the experimental results it was possible to estimate the course of the interactions between the phospholipid and the INH− peptide conjugate.
to be transported through several membranes to reach the infected target cell. The primary objective of the present work was to provide a more detailed picture of the molecular interactions of the INH−peptide conjugate with phospholipid monolayers. Langmuir monolayers of lipids on water or an aqueous subphase can be routinely prepared, and these monolayers are excellent simplified models of the cell membrane, as it becomes possible to separate the adsorption and penetration of biologically active molecules to the membrane surface from the actual membrane crossing and from the molecular events taking place on the far side of the bilayer.10,11 In order to gain a detailed understanding of these interactions, we performed three different kinds of experiment, each revealing a different aspect of the interactions under investigation.12 These experiments differ in the way the INH−peptide conjugate is introduced to the monolayer: (i) penetration: the phospholipid monolayer was spread onto pure water and compressed to the desired level; subsequently, the INH−peptide conjugate was injected into the subphase; (ii) spreading onto solution: the phospholipid monolayer was spread onto the solution of the INH−peptide conjugate; (iii) mixed monolayer: a solution containing the phospholipid and the INH−peptide conjugate in a given molar ratio was prepared and spread onto pure water. In the penetration method, also applied in our experiments presented in the previous article,9 the peptide conjugate is introduced to the headgroup side of a precompressed phospholipid monolayer. Thus, of the above three, this method mimics real physiological conditions best and serves as the most realistic model of the interactions between a biomembrane and a biologically active compound such as a drug or drug candidate. In the spreading onto solution method the INH− peptide conjugate is already present at the interface before the lipid film is spread and compressed. Thus, if an ordered peptide structure forms at the air/water interface, the effect of the phospholipid on this ordered peptide can be investigated. The spreading and the mixed monolayer methods are well suited to study lateral interactions within a mixed film composed of a phospholipid and a biologically active compound. We characterized the phospholipid Langmuir monolayers and their interaction with the INH peptide conjugate by tensiometry and sum-frequency spectroscopy. Infrared-visible sum-frequency vibrational spectroscopy (SFS) is a surfacesensitive second-order nonlinear optical technique which offers submonolayer sensitivity and the possibility to characterize the ordering, orientation, and conformation of molecular species in the interfacial region.13,14 Several research groups employed sum-frequency spectroscopy to study the structures and properties of lipid monolayers at the liquid/air interface15−18 and of supported lipid bilayers19−21 as well as for the molecular level characterization of the interactions of lipid monolayers22−24 and bilayers25−29 with peptides. The effect of various other substancessimple ions,30−33 surfactants,34,35 polysaccharides,36−38 even DNA39,40on the structure and organization of phospholipid monolayers at the liquid/air interface was also investigated by sum-frequency spectroscopy. In the present work we recorded surface pressure−area isotherms and measured sum-frequency vibrational spectra of saturated phospholipid (Phospholipon, DPPC-d62) and partially unsaturated (3:1 molar ratio of DPPC:POPC) phospholipid monolayers to investigate their interactions with the INH−peptide conjugate using by the above-mentioned penetration, spreading onto solution, and mixed monolayer
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EXPERIMENTAL DETAILS Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), alkyl chain deuterated 1,2-dipalmitoyl(d62)-sn-glycero-3-phosphocholine (DPPC-d62), and 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC) were obtained from Avanti Polar Lipids Inc. (Alabaster, AL) and used without further purification.41 Phospholipon 100 H, a pure solid lecithin fraction containing at least 95% of saturated phosphatidylcholine (PC), with fatty acid composition of ∼85% stearic acid and 15% palmitic acid was obtained from Phospholipid GmbH (Cologne, Germany).42 INH (purity ≥99.0%) was purchased from Sigma-Aldrich Hungary and used as received. The INH−peptide conjugate was synthesized by experts in the ELTE-MTA Peptide Chemistry Research Group. The protocol of the synthesis and the conjugation with INH was previously described in detail.8 Briefly, the epitope peptide sequence 91SEFAYGSFVRTVSLPV106 was produced manually by solid-phase synthesis on Rink-Amide MBHA resin using Fmoc/tBu strategy. The INH derivative isonicotinoylhydrazinoacetic acid was coupled directly to the N-terminal of the resin-bound 91 SEFAYGSFVRTVSLPV106 peptide. Chloroform (purity 99.8%) from Fisher Chemicals and methanol (purity ≥99.9%) from Sigma-Aldrich Hungary were used for preparing spreading solutions. Dichloromethane (purity ≥99.9%) from Spectrum-3D (Hungary) was used for cleaning the Langmuir trough. Doubly distilled water was checked by its conductivity (72.0 mN/m at 23 ± 0.5 °C). Monolayer Preparation. The lipid films were spread at the air/liquid interface in a Langmuir trough (18 cm × 6 cm × 0.6 cm) for both tensiometric and spectroscopic measurements. The trough, made of Teflon with a barrier of polyoxymethylene (POM) as suggested for lipid layers,43 was cleaned carefully with dichloromethane, methanol, and doubly distilled water. The trough was covered with a Plexiglas box in order to minimize air turbulence and possible contamination. The surface pressure was recorded with an accuracy of ±0.05 mN/ m with the aid of a Wilhelmy plate made of chromatography paper (Whatman Chr1) connected to a force transducer.44 The spreading solvent was a 3:1 v/v mixture of chloroform and methanol. Spread monolayers were formed by depositing 100 μL of the phospholipid solution (0.1 mg/mL) with a microsyringe (Hamilton Bonaduz AG, Switzerland) in the Langmuir trough containing the subphase and allowing the solvent to evaporate for 10 min. DPPC:POPC two-component lipid monolayers were spread from 3:1 chloroform:methanol solution containing 0.075 mg/mL DPPC and 0.026 mg/mL POPC, corresponding to a 3:1 molar ratio of DPPC:POPC. In the penetration experiments, following the spreading and compression of the monolayer on pure water, 2 mL aqueous B
dx.doi.org/10.1021/jp401533c | J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
Article
Figure 1. Sum-frequency vibrational spectra of (a) DPPC-d62 on pure water subphase, (b) DPPC-d62 following penetration of the INH−peptide conjugate at 15 mN/m, and (c) at 20 mN/m upon expansion at different surface concentrations (expressed as area/DPPC-d62 molecule and given in Å2 next to each spectrum). Symbols are measured data points, and solid lines are guides to the eye. The spectra have been vertically displaced for clarity.
solution (80 μM) of the INH−peptide conjugate was injected into the subphase, resulting in a final concentration of 2 μM. In another set of experiments the monolayers were formed by spreading onto a 2 μM solution of the INH−peptide conjugate. For the mixed monolayer experiments the phospholipid:INH− peptide conjugate ratio was 5:1 (mol/mol). The temperature was maintained at 23 ± 0.3 °C in all experiments. Sum-Frequency Spectroscopy. Sum-frequency spectra were collected by an EKSPLA (Vilnius, Lithuania) spectrometer,45 as described in detail in our earlier publications.9,46−48 The visible beam (532 nm) is generated by doubling the fundamental output of a Nd:YAG laser (1064 nm wavelength, 20 ps pulse with, 20 Hz repetition rate), while the tunable IR beam is obtained from an optical parametric generation/ difference frequency generation system, pumped by the third harmonic and the fundamental of the Nd:YAG laser. The IR and visible beams are temporally and spatially overlapped on the sample surface with incident angles of 55° and 60°, respectively. The beam energies at the sample were kept at 200 μJ/pulse. Sum frequency light is collected in the reflected direction through a holographic notch filter and monochromator and detected by a PMT. Spectral resolution is determined by the
