ARTICLE pubs.acs.org/JPCB
Physical Properties Affecting Cochleate Formation and Morphology Using Antimicrobial Oligo-acyl-lysyl Peptide Mimetics and Mixtures Mimicking the Composition of Bacterial Membranes in the Absence of Divalent Cations R. F. Epand,† H. Sarig,‡ D. Ohana,‡ B. Papahadjopoulos-Sternberg,§ A. Mor,‡ and R. M. Epand*,† †
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada Department of Biotechnology & Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel § NanoAnalytical Laboratory, 3951 Sacramento Street, San Francisco, California 94118, United States ‡
ABSTRACT: Several cationic antimicrobial oligo-acyl-lysyl (OAK) peptide mimetics can form cochleate structures, that is, elongated multilayered cylindrical structures, with lipid mixtures mimicking the composition of bacterial cytoplasmic membranes. These cochleate structures do not require divalent cations for their assembly. In the present work, we use light microscopy to screen for cochleate formation in several OAK-lipid systems and freezefracture electron microscopy to assess their morphological features and size. We identify several factors that facilitate a structural change in these assemblies. Dehydration of the membrane interface and a high melting temperature are features of the lipids that enhance cochleate formation in OAK-based lipid systems. In addition, we observed that there is a specific length of the hydrocarbon linker in the OAK of 8-9 carbon atoms that provides optimal formation of these structures. The biophysical properties established in this study will allow for a better understanding of their role and suitability for biological studies.
’ INTRODUCTION Oligo-acyl-lysyl (OAK) is a family of antimicrobial agents based on the covalent attachment of acyl lysines to form oligomers.1 We have recently demonstrated that the antimicrobial action of certain OAKs is highly dependent on the lipid composition of the bacterial membrane.2 The basis of this relationship was shown to be the ability of these OAKs to induce the clustering of anionic lipids. OAKs are among the best antimicrobial agents to induce the rearrangement of lipids in a bacterial membrane.3 The interaction of several antimicrobial OAKs with lipid mixtures, and in particular C12K-7R8 (structure in Figure 1), with 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE):tetraoleoyl-cardiolipin (TOCL) (75:25), has been the subject of ongoing research for their biophysical and antimicrobial properties for some time.2,4,5 A sample of the antimicrobial OAK, C12K-7R8, with the lipid mixture POPE:TOCL (75:25), sent for freeze-fracture electron microscopy, revealed the presence of cochleate structures that were further characterized by Laurdan fluorescence.6 Cochleates are lipid structures in which the planar phospholipid bilayer is induced to wrap around itself to form elongated multilayered cylindrical structures. The formation of cochleates is not a common event. They have most frequently been observed as a consequence of the bridging of an anionic lipid bilayer with divalent cations.7-9 Cochleates, formed from phosphatidylserine (PS) and Ca2þ, can entrap compounds10,11 and serve as drug delivery systems. Up to the present time, most known cochleate forms were assembled in the presence of divalent cations.12 These structures r 2011 American Chemical Society
have been shown in some cases to be more efficient in oral drug delivery than in either micellar or liposomal formulations.13 Many techniques have been employed in the past to study cochleates in addition to confocal microscopy and freeze-fracture electron microscopy, that is, FT-IR spectroscopy, Laurdan fluorescence, scanning electron microscopy, fluorescence microscopy, cryo-electron microscopy, dynamic light scattering, etc. There is particular interest in the study of cochleates made from OAKs and phospholipid mixtures because these complexes can be assembled rapidly and in the absence of divalent cations, thus also allowing replacement of PS by other lipids including mixtures containing zwitterionc-anionic or anionic-anionic lipids, by simply mixing multilamellar liposomes with OAKs at physiological pH and temperatures. In the type of cochleate we describe, addition of polyethylene glycol, dextran, or polymers to form hydrogels or emulsions, as well as reconstitution from detergents, are not required for their assembly. The general properties of several OAKs in bilayers mimicking the cytoplasmic membrane of bacteria have been previously reported.4,5 In particular, the system consisting of C12K-7R8 in POPE:TOCL 75:25 showed an increase in therapeutic efficacy when coadministered as cochleates together with erythromycin.6 Systemic treatment of neutropenic mice infected with lethal Received: November 25, 2010 Revised: January 25, 2011 Published: February 18, 2011 2287
dx.doi.org/10.1021/jp111242q | J. Phys. Chem. B 2011, 115, 2287–2293
The Journal of Physical Chemistry B
ARTICLE
Figure 1. Structures of OAKs used in the present study.
Table 1. Properties of OAKsa
as their corresponding gel to liquid crystalline transition temperatures are given in Table 2. minimal inhibitory concentration (MIC)
a
E. coli
S. aureus
charge
% hydrophobicity
(μM)
(μM)
C12K-5R8
6
49.7
3.1
50
C12K-6R8
7
50
3.1
50
C12K-7R8
8
47.5
3.1
50
C12K-8R8 C12K-9R8
9 10
48.5 48.4
3.1 6.25
>50 >50
C12K-11R8b
12
42.2
3.1
>50
C12K-7R4
8
45.2
12.5
>50
R12-7R8
9
36.9
C12K-7R12
8
55.2
12.5 >50
50 >50
Taken from ref 26. b This study.
inoculums of multidrug resistant E. coli and treated by a single intravenous dose of the OAK-phospholipid cochleate with entrapped erythromycin increased mice survival in a dose-dependent manner.6 Unlike individual treatments with free erythromycin or cochleated OAK, the coencapsulation of erythromycin in OAK-based cochleates can decrease drug toxicity and increase systemic therapeutic efficacy by orders of magnitude. In the present study, we explore the conditions required for rapid cochleate formation, with regard to the biophysical characteristics of assemblies consisting of different species of OAKs with a variety of lipid mixtures that mimic the cytoplasmic membrane of bacteria. Screening for cochleate formation was accomplished by light microscopy based on known images of cochleates obtained from PS.14 Characterization of some of these cochleates was done with freeze-fracture electron microscopy. Identification of cochleate structure formation by light microscopy can be modified for application in biotechnology for high throughput screening. The structures of the OAKs used in the present study are presented in Figure 1, and their general properties are summarized in Table 1. The lipid mixtures tested for cochleate formation as well
’ EXPERIMENTAL METHODS Materials. Phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL). The OAKs used were synthesized as previously described.15 Methods. Preparation of Samples. Lipid films containing binary mixtures of lipids were made from aliquots of stock solutions in chloroform:methanol (2:1). The solvent was evaporated with a stream of nitrogen gas, and the lipids were deposited as a film on the walls of a glass tube. The tubes were then placed under vacuum for 3 h to remove the last traces of solvent. The films were kept under argon at -20 °C. The lipid films were hydrated with buffer solutions of OAKs with extensive vortexing, so as to have a lipid to peptide molar ratio of 10, unless specified otherwise. When erythromycin was coencapsulated, a solution of this antibiotic was added to a solution of OAK at an erythromycin to OAK molar ratio of 6. Light Microscopy. Films were hydrated with 20 mM PIPES, 140 mM NaCl, pH 7.4, vortexed well, and subjected to freeze thawing three times before imaging, to ensure equilibration of OAK with the multilamellar vesicles. Freeze-thawing the samples did not affect the results obtained by light microscopy. A drop of this mixture was placed on a glass slide and covered with a glass coverslip. Light microscopy was carried out at room temperature using a Zeiss laser scanning microscopy equipped with an Axiovert 100 M microscope with a Plan-Neofluar 100/ 1.3 oil immersion objective. Differential interference contrast (DIC) images were captured with LSM 510 software and analyzed with the Zeiss LSM image browser v2.8. To optimize conditions for cochleate formation, a set of lipid mixtures were imaged by light microscopy in the presence of several different OAKs. Screening of the samples for the presence of cochleate structures was made by comparison of the type of features observed with light microscopy images reported in the literature for cochleates formed in the presence of PS, or with images taken in the presence of EDTA, which prevent their assembly.14 2288
dx.doi.org/10.1021/jp111242q |J. Phys. Chem. B 2011, 115, 2287–2293
The Journal of Physical Chemistry B
ARTICLE
Table 2. Cochleate Formation with Different Lipid Mixturesa lipid mixtures
Tm
C12K-5R8
C12K-6R8
C12K-7R8
C12K-8R8
C12K-9R8
C12K-11R8
POPE:TOCL 75:25 DOPE:TOCL 75:25 DMPE:TOCL 75:25 DPPE:TOCL 75:25 POPC:TOCL 75:25 POPE:DOPG 75:25 POPE:DOPG:TOCL 80:15:5 DMPE:DOPG 75:25 DMPC:TOCL 75:25 POPG:TOCL 75:25b DMPG:TOCL 75:25b
14-15
