Regular Article
High-resolution label-free studies of molecular distribution and orientation in ultrathin, multicomponent model membranes with infrared nano-spectroscopy AFM-IR

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Abstract

Biological membranes are undoubtedly very interesting systems. Unfortunately, their analysis is very complicated and therefore simplified artificial models, like Langmuir monolayers, are frequently applied. In this work a novel method of label-free monomolecular films analysis based on infrared nano-spectroscopy (AFM-IR) is presented. In order to verify applicability of this approach well-defined referential system of sphingomyelin (SM), cholesterol (Chol) and cyclosporin A (CsA) is applied. AFM-IR method allows to directly and chemoselectively map the distribution of components in model lipid membranes. Additionally the orientation of Chol and SM molecules in the monolayer is determined by application of two perpendicular infrared laser polarizations. This paper is the first report of using AFM-IR to analyze LB films with extremely high sensitivity.

Introduction

Biomembranes are highly organized structures displaying characteristic inhomogeneity. Mutual miscibility or immiscibility of their components determines the formation of specialized domains in the membrane, which affects the stability and dynamics of biosystems [1]; lipid rafts being as example. These systems are built from laterally arranged sphingolipids (mainly sphingomyelins) and cholesterol [2] and can bind selected peptides and proteins, which can modify their organization [3]. There are many examples of lipid phase modifications caused by peptides that result in association of lipid complexes, preservation of existing domains or their destabilization [4], [5]. Analysis of mutual miscibility of membranous components allows to conclude on interactions in the studied system, which is crucial for researching pharmaceuticals such as cyclosporin A (CsA). This hydrophobic cyclic polypeptide is known of its immunosuppressive, anti-inflammatory and antiparasitic (mainly antimalarial) properties [6]. Although its molecular targets have not been clearly elucidated, it is well recognized that CsA, due to its lipophilicity, can interact with lipid membranes.

There are various approaches to study association of biomolecules to lipid rafts. One of them is based on their isolation from natural membranes. Regrettably these domains are very dynamic; therefore their characterization is complicated. Fortunately lipid rafts can be modeled with relatively simple systems [7], [8], e.g. Langmuir monolayers [9]. In this technique, a one-molecular layer serves as a simple physical model of biological membrane [10], [11]. It is worth to mention that Langmuir monolayer technique has some restrictions. Firstly, a limited spectrum of available methods is appropriate for investigating systems in situ (directly at the liquid/gas interface) [12]. Secondly, classical analysis of the π-A isotherms does not provide an insight into the phase polymorphism and the distribution of molecules in the monolayer. Therefore, the development of new methods to get insight into these issues is of a great importance. The widely applied strategy is transferring of Langmuir monolayer onto a solid substrate using the LB technique [13].

The LB films can be topographically characterized at nanometric scale with Atomic Force Microscopy (AFM). This technique can be coupled with infrared spectroscopy (IR) to provide chemical characterization of the studied systems at the same scale. It can be achieved by the application of two different techniques, making use of: (i) infrared light scattered at a scanning probe tip (Fourier transform infrared nanospectroscopy, nano-FTIR [14]) or (ii) AFM cantilever sensitivity to sample thermal expansion caused by an absorption of IR light (atomic force microscope infrared-spectroscopy AFM-IR [15], [16]). NanoFTIR signal is strongly dependent on probe (shape and material) properties and this is the main limitation of this technique because probes at the nanoscale are not identical and – additionally - they may easily be damaged during the measurement. Nano-FTIR techniques relies on the measurement of the intensity of scattered light, which is a combination of the imaginary and real part of the incident light, while the AFM-IR technique detect only the imaginary part of the absorbed infrared light and thus the IR absorption. AFM-IR, in contrast to nano-FTIR, allows also to avoid artefacts in the spectra related with probes properties.

AFM-IR approach was applied herein. In this method local absorption spectra are generated by the amplitude variation of the cantilever oscillations as a function of the particular absorbed wavelength [17], [18]. Infrared nano-spectroscopy provides the resolution beyond the limit of conventional, diffraction-limited infrared spectroscopy. The effective spatial resolution of AFM-IR should be experimentally measured/estimated for each sample individually because several important factors determine it, including thermo-mechanical properties of the sample. Another important factor, which influences the resolution is the size of AFM tip radius (the area of interaction between the tip and the sample) typically 10–20 nm. The lateral resolution of AFM-IR mapping is additionally affected by sampling/scanning rates. The depth resolution is determined by penetration depth of the electromagnetic field, which induces thermal expansion of the sample. Infrared light penetrates the sample film to a distance in the order of the light wavelength, which is equal to micrometers. Therefore in our case the depth resolution was determined by the molecular monolayer thickness equal to 2–3 nm. The spatial resolution of AFM-IR has already been discussed in the literature [15], [19], [20], [21], [22]. An application of tunable mid-infrared quantum cascade lasers as light sources increases AFM-IR sensitivity because their pulse repetition frequency can be tuned to the resonant frequency of AFM cantilever, amplifying the AFM cantilever deflection amplitude of around two orders of magnitude (for commercially available silicon cantilevers) [16], [23], [24]. However, the mechanical resonance enhancement is not sufficient to achieve high sensitivity for LB monolayers detection [25]. Sensitivity of the AFM-IR technique may be additionally increased by an application for all spectra and maps collection of gold coated AFM tip and gold coated substrate in order to obtain an electromagnetic field enhancement in the nano-gap between the tip and the substrate [16]. Theoretical calculations proved that electromagnetic field enhancement in the 2 nm gap is as high as 2 × 105 [16], [25]. This additional enhancement allowed for successful measurements of such thin (2–3 nm) molecular monolayers. Additional advantage of AFM-IR technique is high sensitivity for molecule orientation on the surface. An excitation of molecules with light polarized in the particular direction induces special surface selection rules, enhancing spectroscopic signal from the bands oriented along the polarization direction. This can be used to determine the orientation of molecules immobilized onto the metal substrate [25]. Therefore AFM-IR enables to identify spectral markers of lipid disorder and orientation changes [17], [16], [26].

So far nanospectroscopic techniques have been applied to plethora biological systems such as malaria red blood cells [27], [28], photosystems [29], lipid droplets synthesized in glioblastoma cells upon radiation exposure [30], amyloid fibrils [31], [21], as well as to artificial models [25], [32], [33]. However, to the best of our knowledge there are no reports so far on AFM-IR application to study artificial membranes prepared with the LB technique. Up to now, LB films were analyzed only with Raman [34] and Tip Enhanced Raman (TER) [35] mapping, and by nano-FTIR [36]. The abovementioned approaches required chemically modified lipids (for signal amplification), while AFM-IR enabled us to obtain a clear signal from pure compounds organized in monomolecular layer. Moreover, AFM-IR due to its selection rules, enabled us to investigate molecular orientation, which has never been performed before.

In our recent paper we have applied systematic Langmuir approach together with AFM imaging to analyze systems containing CsA in lipid environment of SM and Chol [37]. Based on topography information and thermodynamic analysis, we have demonstrated that CsA shows affinity to lipid rafts (SM:Chol = 0.67:0.33) while causing phase separation in samples with excess of cholesterol. However, this approach is time-consuming and provides only a general picture without any details on chemical composition. Herein we have extended our investigation by presenting IR spectra acquired in nanoscale and chemical maps of films studied with AFM-IR technique. Detailed analysis using AFM-IR provides direct information on the distribution of selected components. Firstly, it enables to determine composition of coexisting phases as well as define their ordering. Secondly, this allows to define binding sites (targets) for CsA in lipid matrix. Therefore, we believe that the use of this technique can shed a new light on the mode of action of the investigated polypeptide and may be applied also to other membrane bioactive molecules. This work is just one of the first AFM-IR investigations regarding such studies.

Section snippets

Chemicals

Cyclosporin A, cholesterol (Sigma-Aldrich) and sphingomyelin (chicken egg, Avanti Polar Lipids) were of high purity (>99%) and used for experiments without any further purification.

Langmuir-Blodgett experiments

The studied compounds were dissolved in chloroform (spectroscopic grade, Sigma Aldrich) in concentration of ca. 0.3 mg mL−1. Mixtures were prepared by mixing proper volumes of respective stock solutions (Chol/SM with and without cyclosporine A, XCsA = 0.3). Langmuir monolayers were obtained by spreading the

Results and discussion

To perform pioneer chemical mapping of artificial monomolecular assembly with AFM-IR technique two representative ternary mixtures of Chol/SM/CsA in different mutual proportions (0.47:0.23:0.30 and 0.23:0.47:0.30) were selected due to their thermodynamic stability and high reproducibility of LB layers. Interestingly, in cholesterol-rich sample (transferred on mica substrate) we have observed and described a perfect visible separation of lipid phases [37], while in cholesterol-poor system CsA

Conclusions

Taking into consideration previously reported applications of AFM-IR, in this paper we have demonstrated new approach to study artificial membranes with monomolecular thickness.

AFM-IR method provided visualization of distribution of pure, non-labelled compounds on the surface of solid substrate as well as information on their relative orientation.

In this work we investigated surface pattering of Chol/SM systems influenced by CsA. AFM-IR method allowed us to directly observe homogeneous

Declaration of interest

None.

Acknowledgement

The research was performed using equipment purchased in frame of the project co-funded by the Małopolska Regional Operational Programme Measure 5.1 Krakow Metropolitan Area as an important hub of the European Research Area for 2007-2013, project No. MRPO.05.01.00-12-013/15. E. Lipiec was supported by National Science Center of the Republic of Poland (within the framework of a project No. 2014/13/D/NZ1/01014.

Authors are grateful to K. Pytlakowski for improvements in graphic design.

Author Contributions

1) these authors contributed equally to this work. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

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