Regular Article
Effects of oxidation on the physicochemical properties of polyunsaturated lipid membranes

https://doi.org/10.1016/j.jcis.2018.12.007Get rights and content

Abstract

The exposure of biological membranes to reactive oxygen species (ROS) plays an important role in many pathological conditions such as inflammation, infection, or sepsis. ROS also modulate signaling processes and produce markers for damaged tissue. Lipid peroxidation, mainly affecting polyunsaturated phospholipids, results in a complex mixture of oxidized products, which may dramatically alter membrane properties. Here, we have employed a set of biophysical and surface-chemical techniques, including neutron and X-ray scattering, to study the structural, compositional, and stability changes due to oxidative stress on phospholipid bilayers composed of lipids with different degrees of polyunsaturation. In doing so, we obtained real-time information about bilayer degradation under in situ UV exposure using neutron reflectometry. We present a set of interrelated physicochemical effects, including gradual increases in area per molecule, head group and acyl chain hydration, as well as bilayer thinning, lateral phase separation, and defect formation leading to content loss upon membrane oxidation. Such effects were observed to depend on the presence of polyunsaturated phospholipids in the lipid membrane, suggesting that these may also play a role in the complex oxidation processes occurring in cells.

Introduction

Many cellular processes and pathological conditions such as apoptosis, inflammation, infection, or sepsis, are modulated or triggered by oxidative stress through direct damage of the cell membrane [1]. Both enzymatic and non-enzymatic oxidation, the latter occurring via direct exposure to reactive oxygen species (ROS), lead to lipid peroxidation in living cells and tissues. The exposure of biological membranes to ROS results in a complex mixture of oxidized phospholipids (OxPL), including peroxyl radicals, hydroperoxides, truncated phospholipids, and acyl chain fragments such as carbonyl compounds [2]. These OxPL are not just by-products of lipid peroxidation but also active modulators of signaling processes, e.g., initiating and amplifying inflammation, and also acting as danger markers for damaged tissue [3], [4]. For instance, OxPL, and particularly oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), play an active role in the onset of atherosclerosis [5]. PAPC is one of the most abundant polyunsaturated phospholipids in mammalian tissues [6], and even low doses of its oxidized form strongly affect the permeability of endothelial cell layers, enhancing low-density lipoprotein (LDL) entry and deposition into blood vessels. PAPC also activates several pathways related to oxidation and inflammation, such as the synthesis and release of growth factors, cytokines, and chemokines [5], [7]. Many previous studies of membrane oxidation have focused on monounsaturated or saturated lipids; however, polyunsaturated fatty acids (PUFA) are much more susceptible to oxidation as they contain several methylene groups located between double bonds, known as bisallylic groups, characterized by weaker Csingle bondH bonds and so more prompt to H extraction [2]. Therefore, the inclusion of polyunsaturated phospholipids into membrane oxidation models is crucial for modelling, characterizing, and understanding the complex processes occurring in real mammalian membranes and tissues.

As shown in earlier studies, photogeneration of oxidative stress, e.g., by shortwave (λ ∼ 250 nm) ultraviolet (UV) irradiation, is a convenient way of controlling oxidation levels in different model lipid membrane systems [8]. The most common free radicals produced in aqueous media under oxidative conditions are singlet oxygen (O2-) and hydroxyl radicals (OHradical dot). After exposure of PUFA-containing membranes to ROS, in the presence of oxygen, peroxyl radicals are produced, which are subsequently transformed into hydroperoxides by reaction with other PUFA molecules. Following initiation, oxidation continues via an enzyme-independent process, resulting in a wide spectrum of OxPLs. This chain reaction, known as lipid peroxidation, continues until two radicals combine to form a stable product, or until the radicals are neutralized by a chain breaking antioxidant [2]. Final products include truncated phospholipids, which have lost some or all bisallylic methylene groups, and lipids with cyclized acyl chains. Inclusion of these lipid oxidation products has been found to change the lateral structure [9], bilayer stability and membrane permeability [10], [11], as well as to affect lipid-protein interactions [12].

Scattering techniques, particularly small-angle X-ray scattering (SAXS) and neutron reflectometry (NR), are useful tools for the characterization of lipid membrane oxidation due to their high sensitivity to the phase behaviour and structural rearrangements of lipid membranes [13], [14]. For instance, SAXS has been used to study micro- and nanoscopic 2D phase separated bilayers [15]. The effects of lipid peroxidation on the structure and organization of model PC liposomes have been previously studied by small-angle X-ray diffraction, and reductions in the bilayer thickness and potential interdigitation under oxidative stress reported [16], along with enhanced lateral phase separation in the presence of cholesterol [17]. NR has been used to study changes of supported lipid bilayers through lipid exchange, removal or degradation by exploitation of contrast variation, i.e., by adjusting the H2O/D2O ratio of the surrounding medium to highlight different parts of the bilayer [18], [19], [20]. However, despite the capacity of NR to detect compositional and structural changes at the Ångstrom level, such as phospholipid degradation or structural rearrangements, to date only a few studies have addressed oxidative degradation of lipid membranes. For instance, the oxidation of ozone-exposed lipid monolayers [21], [22] or UV-exposed supported bilayers [8], [10], [20] has been investigated using NR. These studies have focused on non- or slightly-oxidizable compositions, using lipids with saturated or monounsaturated acyl chains, such as palmitoyl or oleyl chains. In contrast, the oxidation of lipids containing PUFAs, significantly more prone to oxidation and very abundant in biological tissues, have not yet been explored using neutrons.

In this work, we therefore aim to investigate the effects of oxidative stress on the structure and stability of membranes of varying content of polyunsaturated lipids, using simple mixtures of palmitoyloleoylphosphatidylcholine (POPC) and PAPC. Lipid peroxidation was triggered by shortwave UV exposure, with or without the addition of peroxidation initiator and propagator, H2O2. These models were then investigated using a battery of biophysical and surface-chemistry techniques, including NR, SAXS, Fourier transform infrared spectroscopy with attenuated total reflection (FTIR-ATR), fluorescence spectroscopy, and light scattering, in order to obtain an overall view on structural, compositional, and stability changes of polyunsaturated phospholipid bilayers under oxidative stress.

Section snippets

Materials

The phospholipids investigated here, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0–18:1 PC, POPC) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0–22:4 PC, PAPC), both of >99% purity, were obtained from Avanti Polar Lipids (Alabaster, USA). C11-BODIPY 581/591 was purchased from Molecular Probes/Thermo Fisher Scientific (USA). Calcein (Bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein), Tris buffer (Trizma® base, ≥99.9%), D2O (>99.9%), sodium chloride (>99.5%), and all

Comparison between oxidative treatments in terms of lipid peroxidation

As a first step, a comparison between different physical treatments proposed in literature for oxidizing lipids, including shortwave UV irradiation, autooxidation by heat (60 °C), and tip probe sonication, was carried out using POPC:PAPC LUVs. These tests were performed using a standard thiobarbituric acid reactive substances (TBARS) assay, in which lipid oxidation products such as malondialdehyde (MDA) react with thiobarbituric acid to form a coloured and fluorescent product. From these

Discussion

In the present study, we have described how the degree of polyunsaturation influences bilayer oxidation, with consequences on thickness, lateral structure, and permeability. As observed by NR, there is a reduction in the stability of POPC:PAPC bilayers with increasing PAPC content. In addition, as confirmed by neutron and X-ray scattering, bilayer thicknesses are reduced, connected to corresponding increases in APM. These results are consistent with a body of literature that has characterized

Conclusions

The present investigation addresses effects of polyunsaturation on lipid oxidation, and consequences of this for membrane structure and stability. The combined use of X-ray scattering and neutron reflectometry, FTIR-ATR, DLS, and fluorescence spectroscopy demonstrates a richness in membrane oxidation effects, regardless of the simplicity of the POPC:PAPC system investigated here. Oxidation of these polyunsaturated lipid membranes results in a combination of structural, compositional, and

Acknowledgements

Financial support is acknowledged from the LEO Foundation Center for Cutaneous Drug Delivery (grant number 2016-11-01; KLM, LSED, and MM), the Novo Nordisk Foundation Interdisciplinary Synergy program SYNERGY (grant number NNF15OC0016670; SB), and travel funding for NR experiments from the Danish Natural Sciences Research Council (DanScatt). Furthermore, beamtime at ILL (DOI: 10.5291/ILL-DATA.9-13-733) is gratefully acknowledged. In addition, we would like to thank Dr. Giovanna Fragneto for

References (79)

  • A. Gomes et al.

    Fluorescence probes used for detection of reactive oxygen species

    J. Biochem. Bioph. Methods

    (2005)
  • M.L. MacDonald et al.

    Mass spectrometric analysis demonstrates that BODIPY 581/591 C11 overestimates and inhibits oxidative lipid damage

    Free Radical Biol. Med.

    (2007)
  • G.P. Drummen

    C11-BODIPY581/591, an oxidation-sensitive fluorescent lipid peroxidation probe:(micro) spectroscopic characterization and validation of methodology

    Free Radical Biol. Med.

    (2002)
  • N. Kučerka

    Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles

    Biophys. J.

    (2005)
  • W. Rawicz

    Effect of chain length and unsaturation on elasticity of lipid bilayers

    Biophys. J.

    (2000)
  • P. Heftberger

    In situ determination of structure and fluctuations of coexisting fluid membrane domains

    Biophys. J.

    (2015)
  • M. Kiselev

    A sucrose solutions application to the study of model biological membranes

    Nucl. Instrum. Methods Phys. Res., Sect. A

    (2001)
  • T. McIntosh

    Differences in hydrocarbon chain tilt between hydrated phosphatidylethanolamine and phosphatidylcholine bilayers. A molecular packing model

    Biophys. J.

    (1980)
  • B.W. Koenig et al.

    Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase

    Biochim. Biophys. Acta (BBA)-Biomembr.

    (2005)
  • N. Kučerka et al.

    Fluid phase lipid areas and bilayer thicknesses of commonly used phosphatidylcholines as a function of temperature

    Biochim. Biophys. Acta (BBA)-Biomembr.

    (2011)
  • N. Shukla

    FTIR study of surfactant bonding to FePt nanoparticles

    J. Magn. Magn. Mater.

    (2003)
  • R. Mendelsohn et al.

    Infrared reflection–absorption spectroscopy: principles and applications to lipid–protein interaction in Langmuir films

    Biochim. Biophys. Acta (BBA)-Biomembr.

    (2010)
  • M. Mosca et al.

    Effect of membrane composition on lipid oxidation in liposomes

    Chem. Phys. Lipids

    (2011)
  • J.M. Smaby

    Phosphatidylcholine acyl unsaturation modulates the decrease in interfacial elasticity induced by cholesterol

    Biophys. J.

    (1997)
  • J.F. Nagle et al.

    Structure of lipid bilayers

    Biochim. Biophys. Acta (BBA)-Rev. Biomembr.

    (2000)
  • R. Volinsky

    Oxidized phosphatidylcholines facilitate phospholipid flip-flop in liposomes

    Biophys. J.

    (2011)
  • K. Sabatini

    Characterization of two oxidatively modified phospholipids in mixed monolayers with DPPC

    Biophys. J.

    (2006)
  • H. Khandelia et al.

    Lipid gymnastics: evidence of complete acyl chain reversal in oxidized phospholipids from molecular simulations

    Biophys. J.

    (2009)
  • J. Wong-Ekkabut

    Effect of lipid peroxidation on the properties of lipid bilayers: a molecular dynamics study

    Biophys. J.

    (2007)
  • A.G. Ayuyan et al.

    Lipid peroxides promote large rafts: effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation

    Biophys. J.

    (2006)
  • L.L. Holte

    2H nuclear magnetic resonance order parameter profiles suggest a change of molecular shape for phosphatidylcholines containing a polyunsaturated acyl chain

    Biophys. J.

    (1995)
  • C. Erridge

    Oxidized phospholipid inhibition of toll-like receptor (TLR) signaling is restricted to TLR2 and TLR4 roles for cd14, lps-binding protein, and md2 as targets for specificity of inhibition

    J. Biol. Chem.

    (2008)
  • L. Cwiklik et al.

    Massive oxidation of phospholipid membranes leads to pore creation and bilayer disintegration

    Chem. Phys. Lett.

    (2010)
  • L.R. McLean et al.

    Effect of lipid physical state on the rate of peroxidation of liposomes

    Free Radical Biol. Med.

    (1992)
  • J.-P. Mattila et al.

    Oxidized phospholipids as potential novel drug targets

    Biophys. J.

    (2007)
  • M. Malmsten

    Inorganic nanomaterials as delivery systems for proteins, peptides, DNA, and siRNA

    Curr. Opin. Colloid Interf. Sci.

    (2013)
  • V.N. Bochkov

    Generation and biological activities of oxidized phospholipids

    Antioxid. Redox Signal.

    (2010)
  • S. Lee

    Role of phospholipid oxidation products in atherosclerosis

    Circ. Res.

    (2012)
  • B. Sanii et al.

    Patterning fluid and elastomeric surfaces using short-wavelength UV radiation and photogenerated reactive oxygen species

    Annu. Rev. Phys. Chem.

    (2008)
  • Cited by (26)

    • Insights into the mechanisms of interaction between inhalable lipid-polymer hybrid nanoparticles and pulmonary surfactant

      2023, Journal of Colloid and Interface Science
      Citation Excerpt :

      Curosurf® SUVs were deposited onto SiO2 crystals, and their IR absorbance was been monitored in the region between 1500 and 4000 cm−1 (Fig. S10). Due to difficulties in differentiating vibrations of PS bilayers from those of the surrounding water [45], differences in OH vibration modes (3000–3500 cm−1 and 1600–1700 cm−1, respectively) are difficult to interpret. Therefore, the discussion below focuses on: (i) the hydrocarbon vibrational peaks (between 2800 and 3010 cm−1), corresponding to the symmetric and antisymmetric stretching modes of the different C-based groups, and (ii) the stretching mode of the CO group [46,47].

    View all citing articles on Scopus
    View full text