Interfacial charges drive the organization of supported lipid membranes and their interaction with nanoparticles
Graphical abstract
Introduction
Gold nanoparticles (AuNPs) may adhere to cell membranes, penetrate into the cell, and lead to toxic effects, with mechanisms and damages that depend on their physical and chemical characteristics (i.e., size, shape, and ligand structure) [1,2]. If the cytotoxicity of AuNPs is measured in biomedicine by probing the macroscopic responses of cells in vitro or in vivo (e.g., in guinea pigs), there will still be a lack of knowledge of the first interactions of NPs with the cell barrier, which are at the origin of the cascade of nanotoxicity pathways. Knowledge of the fundamental interactions of NPs with the cell membrane is recognized as a prerequisite for a deep understanding of cytotoxicity responses of the whole cell [[3], [4], [5]]. In addition to fundamental knowledge, a detailed picture of the first NP/membrane contacts may also help identify the best physicochemical parameters needed by NPs to function as efficient and noninvasive components in biomedical or pharmacological applications, i.e., carriers in drug-delivery systems, contrast agents in bio-imagery techniques, or antiaging ingredients in cosmetics and skin care products [[6], [7], [8], [9], [10], [11], [12], [13], [14]].
To investigate the nanoscale system of the NP/membrane interface, one shall develop theoretical or experimental approaches, to reveal properties and processes occurring within a system a few nanometers thick. In recent years, vibrational sum frequency generation (SFG), a second-order nonlinear optical spectroscopy, has become unavoidable as a means to provide new information of membrane systems, thanks to its unique sensitivity to interfacial systems and molecular-scale responses [15]. SFG spectroscopy allows probing the phase transitions of lipid films [[16], [17], [18], [19], [20]] and enabling the study of membrane interactions with peptides and proteins [[21], [22], [23], [24]] or with salts [[25], [26], [27], [28]]; it provides new findings about the structure of water around membranes [[29], [30], [31], [32], [33], [34], [35], [36]] and demonstrates an unexpected sensitivity to bio-recognition membrane processes [37,38]. Very recently, SFG spectroscopy was introduced to the study of NP/membrane interfaces. In this domain, SFG spectroscopy succeeded in showing that AuNPs surface area and surface functionalization affect the lipid bilayer flip-flop in an SSLB of DPPC on CaF2 [39,40]. In a mixed choline/glycerol SSLB on CaF2, SFG provided evidence of how chitosan micro/NPs enter the membrane model and lead to lipid bending [41]. In SSLBs of DPPC on CaF2 and on SiO2, SFG demonstrated that cationic and anionic AuNPs damaged the conformational structure of lipid layers and modified their close water environment, with strengths depending on relative interfacial charges [42]. In Langmuir monolayers, SFG revealed how cationic superparamagnetic NPs penetrated into the anionic lipid monolayer but only remained at the surface of the neutral lipid monolayer [43].
Here, we want to take advantage of the sensitivity of SFG spectroscopy to investigate the interaction of cationic AuNPs with supported model membranes carrying negative charges (Fig. 1). In living systems, the outer and inner membrane sides of the cell are negatively charged [44]. To reproduce this membrane property, we created lipid bilayers made of anionic DPPS (1,2-dipalmitoylphosphatiserine) and neutral DPPC (1,2-dipalmitoylphosphaticholine) lipids supported on a negative substrate, namely, SiO2. With our model, i) we reproduced the bilayer structure of lipid membranes, ii) we mimicked the membrane composition using two representative components of living membranes (i.e., serine and choline phospholipids), iii) we simulated the outer membrane charges using anionic DPPS, and iv) we represented the negative charge of the inner cell environment using a negatively charged substrate (i.e., SiO2, in MilliQ-water) [[45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56]]. Due to the thin layer of water intercalated between the substrate and the inner leaflet of the bilayer, SSLBs are known to retain their lateral fluidity, which is typical of living membranes and dictates many biological functions [57]. By combining electrophoretic and dynamic light scattering in solution with SFG spectroscopy at the solid/liquid and solid/air interfaces, we investigated the structural properties of anionic SSLBs on SiO2. Indeed, cell membranes have a high conformational order of lipid aliphatic chains, which must be reproduced in model membranes. Then, using SFG spectroscopy, we probed the interaction of the organized anionic model membrane with cationic AuNPs in real time to unravel information on the kinetics and mechanism of interaction.
With this study, we have made a step forward in the comprehension of the physicochemical properties and processes of anionic lipid bilayers and their interactions with cationic NPs, thus providing new molecular understandings of how cationic AuNPs penetrate cell barriers before leading to cytotoxicity pathways. The molecular information we obtained is a fundamental piece of the complex puzzle explaining the physicochemical and biological mechanisms that lead to the toxicity of nanomaterials. The possibility to provide molecular-scale knowledge of the first interactions of nanomaterials with cell barriers is indeed a prerequisite for further understanding, predicting and controlling the macroscopic responses that nanomaterials may cause in human, animal or vegetal organisms.
Section snippets
Materials
Both 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) (Fig. 1) were supplied by Avanti®Polar Lipids, Inc. (Alabaster, AL). Dichloromethane, sulfuric acid, hydrogen peroxide and phosphate buffer solution (PBS, pH = 7.4) were purchased from Sigma-Aldrich. Gold nanoparticles were from Nanopartz, Inc. (Loveland, CO). Charges on the NPs surface were induced by a chemically bonded polymer ligand with an amine head imparting a positive zeta
Fusion of pure DPPS vesicles in water
In a first attempt, we investigated the fusion of pure DPPS vesicles on SiO2 in pure MilliQ-water. Fig. 2a shows the SFG spectra recorded at the neat SiO2/water interface (gray curve) and after adsorption of pure DPPS vesicles (black curve). The broad features near 3165 cm−1 and 3400 cm−1 are derived from the OH stretching modes of organized water molecules close to the interface. SFG spectroscopy is indeed well-known for its ability to probe those water molecules, which assemble in organized
Conclusions
Correlating zeta potentials and the structural properties of lipid films at the solid/liquid and solid/air interfaces with zeta potentials values of lipid vesicles in solution provided further insight into the mechanism of formation of the organized anionic solid-supported lipid bilayers on SiO2. Quantitative and qualitative data for the interfacial charges and structural properties of SSLBs and lipid vesicles, as obtained from SFG spectroscopy and electrophoretic light scattering, established
Funding
This work was supported by the Belgian Funds for Agricultural and Industrial Research (FRIA) and for the Scientific Research (F.R.S.-FNRS), by the University of Namur (UNamur), and by the NATO program Science for Peace and Security under the Project 985291 “Biohazards Detection”.
Author contributions
X.T.-F. carried out and analyzed the SFG experiments on the DPPS and DPPS-DPPC lipid bilayers at the solid/liquid and solid/air interface before and after interaction with 5 nm positive gold NPs. C.M. performed and analyzed the electrophoretic and dynamic light scattering experiments. F.C. designed and supervised the research, correlated the data and wrote the manuscript. All authors contributed to the final version of the manuscript.
Acknowledgments
XTF and CM thank the Belgian Fund for Agricultural and Industrial Research (FRIA) and the University of Namur (UNamur), respectively, for their fellowships. FC is Research Associate of the Belgian Fund for the Scientific Research (F.R.S.-FNRS). The authors thank the COST Action MP1302 “Nanospectroscopy” and the Lasers, Optics and Spectroscopy (LOS) technological platform of UNamur. The authors acknowledge Dr. E. Perpète (UNamur) for his help in electrophoretic and dynamic light scattering
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These authors contributed equally to this work.