Reversible Lifting of Surface Supported Lipid Bilayers with a

Article pubs.acs.org/Biomac

Reversible Lifting of Surface Supported Lipid Bilayers with a Membrane-Spanning Nonionic Triblock Copolymer Steven C. Hayden,†,⊥ Ann Junghans,‡,§ Jaroslaw Majewski,*,†,‡,∥ and Millicent A. Firestone*,† †

Materials Physics & Applications, Center for Integrated Nanotechnologies (CINT), Los Alamos National Laboratory, Mail Stop K771, Los Alamos, New Mexico 87545, United States ‡ Lujan Neutron Scattering Center, Los Alamos Neutron Science Center (LANSCE), Los Alamos National Laboratory, Mail Stop H805, Los Alamos, New Mexico 87545, United States § Materials Science & Engineering (MST-7), Los Alamos National Laboratory, Mail Stop H805, Los Alamos, New Mexico 87545, United States ∥ Department of Chemical Engineering, University of California Davis, Davis, California 95616, United States S Supporting Information *

ABSTRACT: Neutron reflectometry was used to monitor structural variations in surface-supported dimyristoylphosphatidycholine (DMPC) bilayers induced by the addition of Triton X-100, a surfactant commonly used to aid solubilization of membrane proteins, and the coaddition of a membrane spanning nonionic amphiphilic triblock copolymer, (PEO117−PPO47− PEO117, Pluronic F98). Surfactant addition causes slight compression of the bilayer thickness and the creation of a distinct EO layer that increases the hydrophilic layer proximal to the supporting substrate (i.e., a water and EO gap between the lipid bilayer and quartz) to 6.8 ± 0.4 Å. Addition of the triblock copolymer into the DMPC:Triton X-100 bilayer increases the complexity of (broadens) the lipid phase transition, further compresses the bilayer, and continues to expand the proximal hydrophilic layer thickness. The observed structural changes are temperature dependent with transmembrane polymer insertion achieved at 37 °C, leading to a compressed membrane thickness of 39.2 ± 0.2 Å and proximal gap of 45.0 ± 0.2 Å. Temperature-driven exclusion of the polymer at 15 °C causes partitioning of the polymer into the proximal space generating a large hydrogel cushion 162 ± 16 Å thick. An intermediate gap width (10−27 Å) is achieved at room temperature (22−25 °C). The temperature-driven changes in the proximal hydrophilic gap dimensions are shown to be reversible, but thermal history causes variation in magnitude. Temperature-driven changes in polymer association with a supported lipid bilayer offer a facile means to reversibly control both the membrane characteristics as well as the separation between membrane and solid substrate.

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INTRODUCTION

proteins) susceptible to nonspecific interactions with the solid substrate, often resulting in deleterious effects (i.e., structure and/or function) on reconstituted membrane components.6 Furthermore, the close contact between the bilayer and solid supporting substrate surface results in an insufficient ion reservoir limiting ionic transport.7 To date, various strategies for tailoring the planar lipid−solid support interface have been reported, including constructs composed of a chemisorbed monolayer and inclusion of polymer-cushions.2a,8 Polymersupported bilayers can be broadly divided into two categories: The first approach lacks direct linkages with the bilayer (e.g., independent polymer layer) and is introduced to the surface prior to lipid deposition; the second approach involves covalent coupling between the bilayer and polymer. Both of these strategies provide a suitable solvated cushion to decouple the

Supported lipid bilayers (SLBs), two-dimensional (2D) lipid bilayers physisorbed on solid supports, were introduced by McConnell and co-workers1 and have proven to be a valuable model for physiological membranes facilitating, most notably, structure−function studies of protein−lipid interactions.2 Beyond fundamental protein studies, SLBs are becoming increasingly important in biomedicine, for example, being used to guide emergent tissue architectures,3 and serving as substratum for cell analyses.4 New areas of opportunities have sought to use SLBs to enable nanoscience using them as templates for the construction of 2D lattices of DNA origami and as biocompatible coatings on nanoparticles.5 In spite of SLBs expanding areas of application, traditional SLBs suffer from significant drawbacks including mechanical instability, insufficient membrane fluidity (i.e., retarded lateral movement of individual lipids), and impeded mobility of membrane dopants.2a,6 In addition, the small water layer between the bilayer and the solid support makes embedded guests (e.g., © XXXX American Chemical Society

Received: October 3, 2016 Revised: February 15, 2017 Published: February 22, 2017 A

DOI: 10.1021/acs.biomac.6b01461 Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

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membrane from the solid support but require additional deposition/synthetic steps for the introduction of the polymeric layer. In addition, undesirable lateral phase segregation within the membrane can be facilitated by the presence of the polymer. Lastly, polymer-cushioned bilayers are not dynamic, limiting regeneration and/or modulation of the SLB architecture. Thus, significant opportunities exist for further developing novel compositions and methods for the reliable, facile preparation of polymer-supported SLBs with dynamically modulated architectures. Herein, we evaluate the adaptation of previously described thermoresponsive complex fluids (multicomponent amphiphilic mixtures) as the basis for preparing temperature-responsive surface-supported polymer−lipid bilayers.9 In the bulk phase, the complex fluids are optically transparent and self-assemble to form unoriented, freely suspended multilayers that undergo thermoreversible phase transitions.9 Moreover, the multicomponent amphiphilic mixtures allow for composition flexibility so the complex fluids can be optimized for the stable incorporation of a wide range of membrane proteins10 and/or inorganic nanoparticles.11 The specific composition studied is composed of a saturated phospholipid (dimyristoylphosphatidycholine, DMPC, Figure 1, i), a nonionic cosurfactant (Triton

Article

EXPERIMENTAL SECTION

Materials. Lyophilized dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was purchased from Avanti Polar Lipids (Alabaster, AL). Pluronic F98 was obtained from BASF Corporation (Mount Olive, NJ). Deuterated water (D2O, 99.8% D) was acquired from Acros (Pittsburgh, PA). Purified Milli-Q (18.2 MΩ·cm) water was used for sample preparation. Triton X-100 and chloroform was purchased from Sigma-Aldrich (Milwaukee, WI). All chemicals were used as received. Bilayer Deposition. A solution of DMPC (2.9 mM) in chloroform (ca. 32 μL) was spread dropwise onto the air−water interface in a Langmuir−Blodgett (LB) trough (NIMA, Coventry, UK) using a microsyringe. The chloroform was allowed to evaporate (10 min @ surface pressure of 13 mN/m), and the monolayer was compressed at a constant rate (25 cm2/min) to the target surface pressure (25 mN/ m). The monolayer was allowed to equilibrate (15 min @ 25 mN/m), and the inner and outer membrane leaflets were transferred to a polished, 3 in. in diameter single crystal quartz substrate (Institute of Electronic Materials Technology, Warsaw, Poland) using the Langmuir−Blodgett/Langmuir−Schäfer (LB/LS) technique. SLBs were deposited at intermediate surface pressure (5 mm/min dipping speed, 25 mN/m constant surface pressure) to sterically allow for subsequent polymer insertion while still achieving good bilayer structure, packing density, and surface coverage (representative surface pressure isotherms for DMPC and DMPC/Triton X-100 monolayers are given in the Supporting Information, Figure S1). All lipid membrane depositions were performed at 25 °C using ultrapure DI water (18.2 MΩ·cm) for the trough subphase. Following bilayer deposition, the quartz substrate was transferred to a flow cell underwater in order to prevent membrane collapse, and the flow cell chamber (approximate volume of 3 mL) was then slowly flushed twice with deuterated water. D2O was employed to ensure good neutron scattering contrast between the deposited membrane components and the liquid subphase. For the DMPC/Triton X-100 composed membranes, the Triton X-100 (TX100, 1:3 surfactant/lipid ratio, 0.93 mM) was dissolved along with the DMPC in chloroform, and the solution was spread on the LB trough and deposited on the quartz as described above. Polymer (F98) interactions with the deposited membrane at the quartz interface were achieved through injection of 3 mL (2.4 μM F98 in D2O) of the nonionic triblock copolymer, poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene oxide) (PEO117−PPO47−PEO117), in D2O into the subphase of the flow cell. NR Data Collection. Neutrons are highly sensitive to the scattering contrast between a hydrogenated bilayer and a deuterated subphase (D2O). During the NR experiments, neutrons penetrated through the quartz substrate, which is in contact with the liquid subphase, and impinged on the systems deposited on the quartz at a small angle, θ. NR measurements were performed at the Surface Profile Analysis Reflectometer (SPEAR) beamline at the Los Alamos Lujan Neutron Scattering Center as previously described.12 The ratio of specularly scattered neutrons to incident neutrons is measured. This ratio is defined as reflectivity, R, and it is measured as a function of the momentum transfer vector, Qz, where Qz = 4π·sin[θ]·λ−1 and λ is the neutron wavelength. Here, different values of the Qz vector were obtained via changes in both λ and θ. The reflection is recorded by a time-of-flight (ToF), position-sensitive detector. Simultaneously with the specular reflection, the off-specular signal was recorded. In our ToF NR measurements, the neutron wavelength ranged from 4.5 to 16 Å. Temperature regulation during data collection was achieved using a custom-built setup, and the sample temperature was measured with a K-type thermocouple (± 0.1 °C). Analysis of R versus Qz enables calculation of the scattering length density (SLD) distribution normal to the sample surface. SLD is a function of chemical composition and density of the material. The NR measurement and data modeling procedures have been detailed elsewhere.12,13 The presented NR data are divided by the Fresnel curve (scattering from infinitely sharp quartz/D2O interface) to preserve the normalization factor and account for the power-of-four decrease of scattering with the momentum transfer vector, Qz.

Figure 1. Molecular structure (left panel) and schematic representation (right panel) of constituents used in the preparation of thermoresponsive SLBs. (i) Dimyristoylphosphatidycholine, (DMPC); (ii) Triton X-100 (n = 9 − 10); (iii) PEO117-PPO47PEO117, referred to as Pluronic F98.

X-100, Figure 1, ii), and a nonionic triblock polymer (PEO117− PPO47−PEO117, often referred to as Pluronic F98, Figure 1, iii). The cosurfactant selected here, Triton X-100 is specifically used since it enhances the reconstitution and long-term storage of integral membrane proteins (e.g., bacteriorhodopsin, photosynthetic reaction centers) within the complex fluid.10 In this study, we systematically evaluate the use of the complex fluids for constructing thermoresponsive surface-supported bilayers. The structural changes in a DMPC SLB are monitored by neutron reflectivity (NR) as cosurfactant, and polymers are sequentially added to the lipid bilayer. The temperature modulation of the supported bilayer structure is examined, revealing that the polymer component can be reversibly intercalated into the bilayer and expelled to thereby vary the dimensions of the polymer cushion. B


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Biomacromolecules

Figure 2. (A) Fresnel divided NR scattering data (points with error bars) and the fits (solid lines) corresponding to derived SLD profiles (B) for DMPC SLBs at 15 °C (blue), 22 °C (green) and 37 °C (red). Both “smeared”, including interfacial roughness (solid red), and “un-smeared” (dashed red) SLD profiles are shown for 37 °C. Inset gives schematic of superimposed DMPC SLB and definition of fit parameters. (C) Fresnel divided NR scattering data (points with error bars) and the fits (solid lines) corresponding to derived SLD profiles (D) for DMPC/Triton X-100 SLBs at 15 °C (blue), 22 °C (green) and 37 °C (red). Both “smeared”, including interfacial roughness (solid red), and “un-smeared” (dashed red) SLD profiles are shown for 37 °C. Inset: Schematic of superimposed DMPC/Triton X-100 SLB. the best fits with the lowest χ2 values and structurally meaningful parameters for the NR data. The simplest, physically meaningful description of the polymer/lipid/cosurfactant membrane system was used to model the data. Certain models were preferentially adopted based upon prior X-ray scattering studies carried out on similar compositions, which through one-dimensional (1D) electron density mapping identified chemically distinct regions.9b In order not to overparameterize, the fitting of the NR data values of structurally similar regions (e.g., phospholipid headgroups dimensions and SLD) were linked. In the final optimization of the fits they were allowed to independently vary. The SLD value of the quartz substrate was fixed to 4.2 × 10−6 Å−2. The D2O subphase was initially set at 6.3 × 10−6 Å−2

Modeling of the SLD was performed using an open-source reflectivity package, MOTOFIT, which runs in the IGOR Pro environment.14 MOTOFIT approximates the continuous SLD function by a number of layers (also known as slabs or boxes) with constant SLDs. A Gaussian error function centered between two adjacent interfaces is used to address the interfacial roughness. Interfacial roughness values were both fitted using a single global roughness parameter and fitted separately, allowing independent variation. Typical values of independently determined interfacial roughness are provided in Table S1. A theoretical reflectometry curve can be calculated using Abeles matrix formalism. Both Genetic optimization and Levenberg− Marquardt nonlinear least-squares methods were employed to obtain C


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Biomacromolecules Table 1. Fit Parameters for DMPC SLB at 15, 22, and 37 °C Derived from the SLD Profiles Shown in Figure 2Ba interfacial layer

proximal heads

hydrocarbon tails

distal heads

temperature (°C)

dw [Å]

SLD [10−6 Å−2]

Zh,p [Å]

SLD [10−6 Å−2]

dc [Å]

SLD [10−6 Å−2]

Zh,d [Å]

SLD[10−6 Å−2]

db [Å]

χ2

15 °C 22 °C 37 °C

2.3 2.9 3.2

6.2 6.1 6.1

8.4 8.4 8.4

3.4 3.4 3.4

29.7 29.0 25.6

0.8 0.5 −0.1

8.4 8.4 8.4

3.4 3.4 3.4

46.5 45.8 42.4

2.4 2.1 1.9

a

Parameter definition is provided in Figure 2.

(1.20 × 10−6 Å−2).16 This SLD value is also considerably higher than the 2.5−2.9 × 10−6 Å−2 previously reported for fully hydrated DMPC bilayers (with 12−14 water molecules per phospholipid).15c The increased SLD found here indicates greater water infiltration (SLD of D2O is 6.35 × 10−6 Å−2) into the phospholipid region. In addition, due to established temperature-induced changes in the acyl chain packing between the gel (Lβ) to fluid (Lα) phases, variations in both the dimensions and SLD of the hydrocarbon tail layer (dc) is observed. Specifically, in the Lβ phase (15 and 22 °C), dc was found to be 29.0 ± 0.7 Å with SLD values of 0.8−0.5 × 10−6 Å−2, respectively. The theoretical SLD for the DMPC tails in the gel phase is −0.41 × 10−6 Å−2.17 Thus, here too the observed increase in SLD arises from water penetration into the bilayer, suggesting the existence of structural imperfections (i.e., voids). Given the above findings, a surface occupancy of ca. 93% can be calculated for the DMPC bilayer. A surface occupancy of 93% is consistent with the surface pressure employed during bilayer deposition (25 mN/m).15c The molecular packing of SLBs strongly depends on the lateral pressure applied during deposition. Depositions carried out at lower pressures contain a larger number of defects and induce a higher mobility in the lipid molecules.18 The bilayer thickness, dB, was determined to be 46.5 Å and 45.8 Å for DMPC at 15 and 22 °C, respectively, and is in agreement with literature values reported for incompletely packed DMPC lipid bilayers.19 The value also compares well to bilayer thicknesses determined by small-angle X-ray scattering (SAXS) of freely suspended (multilamellar) stacks of DMPC.9b Lastly, it is noted that the SLD profiles display asymmetry in the alkyl chain bilayer region, an observation attributed to differing interfacial roughness between the top (R = 6.6 Å) and bottom (R = 2.6 Å) leaflets. The difference in roughness between the two leaflets may arise from the deposition methods used to produce the bottom (i.e., deposited using a LB trough) and the top layer (Langmuir−Schaeffer). Increasing the DMPC SLB temperature to 37 °C, a value above the chain melting transition (i.e., in the Lα phase) results in an expected thinning of the bilayer to 42.4 Å. The reduction in the hydrocarbon tails dimensions (dc = 25.6 Å) is attributed to hydrocarbon chain tilting in the Lα phase. The observed thinning and increase in the area per molecule (tilting) is also accompanied by shrinkage of the D2O void zones, as evidenced by the reduction in the hydrocarbon SLD to −0.1 × 10−6 Å−2, a value that is still larger than expected for fluid-phase DMPC acyl chains (−0.3241 × 10−6 Å−2) providing additional evidence of incomplete lipid packing.17 Collectively, the NR DMPC data lays a foundation for evaluation and interpretation of SLB characteristics upon introduction of the cosurfactant and polymer. Cosurfactant Addition. Triton X-100 (TX-100) is one of the most common nonionic detergents used in biology with applications ranging from solubilization of membranes to purification and isolation of membrane proteins.20 Triton is a trade name used to represent a class of nonionic surfactants

but was allowed to vary during least-squares analysis to account for the presence of any residual H2O remaining from the subphase exchange during sample preparation. Typical values for the error in layer thickness and SLDs were ±0.4 Å and ±0.2 × 10−6 Å−2, respectively, as determined from the MOTOFIT program. The reflectivity measurements for a given Qz made during different run cycles at the neutron source, on different samples of same composition, at identical temperatures and thermal history, showed ca. ± 6.5% variation.

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RESULTS AND DISCUSSION NR is an ideal tool for the investigation of the chemical composition and relative location of molecules in SLBs. Modifications of the structural characteristics of DMPC (Figure 1, i) supported bilayers induced by the introduction of a nonionic cosurfactant (Triton X-100, Figure 1, ii) and the nonionic triblock copolymer (PEO117−PPO47−PEO117; F98, Figure 1, iii) are evaluated by NR employing the high contrast condition of deuterated water and hydrogenated amphiphiles. The systematic introduction of cosurfactant and polymer yield insights into how these molecular constituents can be used to modulate the distance between the solid support and the membrane (i.e., the interfacial layer or gap). The nature of cosurfactant and polymer association and SLB architecture is further detailed as a function of temperature, offering a strategy for addressing practical issues in the dynamic regulation of membrane permeability, stability, and water gap dimensions to reduce deleterious effects on membrane-embedded functional constituents. Surface-Supported DMPC Bilayers. The Fresnel divided NR scattering data collected on quartz-supported DMPC bilayers with a D2O subphase recorded at three temperatures around the lipid chain melting transition (Tm = 23.9 °C) are presented in Figure 2A. The reflectivity curves are best described using a five-box (slab) model that includes a D2O layer proximal to the quartz substrate (dw), the proximal bilayer leaflet phospholipid headgroups (zh,p), the bilayer hydrocarbon tails (from both leaflets) (dc), the distal phospholipid headgroups (zh,d), and the D2O subphase, as illustrated in Figure 2B. The dimensions and values for the SLDs determined by fitting the reflectivity curves employing the five-box model are given in Table 1. From these parameters the membrane thickness, dB, defined as dB = zh,p + zh,d + dc can be determined. At all temperatures examined (15 °C (blue curve), 22 °C (green curve), 37 °C (red curve)), a small water layer, dw, 2.3− 3.2 Å thick with a SLD commensurate with pure D2O (6.3 × 10−6 Å−2), was observed between the lipid bilayer and the quartz substrate (Figure 2B). The presence of a small water gap separating the solid surface and lipid bilayer has been previously determined using both X-ray and NR and reported to range from 2−8 Å.15 The dimensions of the phospholipid headgroup regions on both sides of the bilayer (i.e., zh,p, zh,d) were determined to be 8.4 Å and did not vary with temperature. Both proximal and distal phospholipid headgroup layers had an SLD of 3.4 × 10−6 Å−2, a value notably larger than expected D


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Biomacromolecules

Table 2. Fit Parameters for DMPC/TX100 SLB at 15, 22, and 37°C, Which Correspond to the SLD Profiles Shown in Figure 2D water layer temperature (°C)

dw [Å]

15 °C 22 °C 37 °C

2.6 2.8 3.0

SLD [10 Å−2] 6.1 6.2 6.3

proximal EO −6

dEO, p [Å] 4.4 4.1 3.4

SLD [10 Å−2] 4.1 4.0 3.9

proximal heads −6

Z h,p [Å] 8.4 8.4 8.4

SLD [10 Å−2]

hydrocarbon tails −6

dc [Å]

3.5 3.4 3.3

26.6 24.0 23.9

SLD [10 Å−2] 0.7 0.9 −0.1

−6

distal heads Z h,d [Å] 8.4 8.4 8.4

SLD [10 Å−2] 3.5 3.4 3.3

distal EO −6

dEO, d [Å]

SLD [10−6 Å−2]

db [Å]

χ2

4.4 4.0 3.5

4.1 4.0 3.9

43.4 40.8 40.7

1.5 1.7 2.2

Figure 3. (A) Fresnel divided NR scattering data (points with error bars) and the fits (solid lines) corresponding to derived SLD profiles for DMPC/Triton X-100 SLBs at 22 °C after 15 min (light green) and 200 min post injection of F98 (dark green) and 37 °C (red). (B) Determined SLD profiles for DMPC/Triton X-100 SLBs at 22 °C after 15 min introduction of F98 (green) compared to DMPC/Triton X-100 only constructs at 22 °C (black). (C) Comparison of SLD profiles for DMPC/Triton X-100 SLBs at 22 °C after 15 min (green) and 200 min introduction (smeared, solid lines, and unsmeared, dashed lines; dark green). Inset shows schematic of DMPC/Triton X-100 SLD after 200 min of incubation with F98. (D) Comparison of SLD determined for DMPC/Triton X-100 SLD after 200 min of incubation with F98 (dark green) to smeared and unsmeared SLD for DMPC/Triton X-100/F98 at 37 °C (red). Inset: Schematic illustration of SLB.

curves collected at three temperatures on TX-100/DMPCsupported bilayers are presented in Figure 2C. The data is best modeled using seven slabs, that is, two additional slabs accounting for the hydrophilic EO chains from the TX-100 residing on either side of the bilayer (Figure 2D; Table 2). In other words, two sublayers outside the bilayer are resolved: a pure water region and a mixed EO/water zone. The introduction of TX-100 also causes a broadening (reduction in cooperativity) in the chain melting transition of DMPC

whose basic structure is composed of a hydrophobic aromatic 4-(1,1,3,3,-tetramethylbutyl)-phenyl core and a hydrophilic variable length poly(ethylene oxide), (PEO) chain (Figure 1, ii). TX-100 possesses a PEO block of 9−10 units long. Here, the TX-100 is introduced to the supported DMPC bilayer by cospreading DMPC and TX-100 (3:1 ratio) on the Langmuir trough followed by a LB/LS transfer onto quartz. Surface pressure was maintained at 25 mN/m to ensure comparable bilayer packing density to the DMPC only construct. The NR E


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Biomacromolecules

Table 3. Fit Parameters for DMPC/TX100/F98 SLBs at 22°C (t = 15 min and t = 200 min Equilibration) and 37 °C, Which Correspond to the SLD Profiles Shown in Figure 3B,C,D water layer

proximal EO −6

proximal heads −6


−6

distal heads

dEO, d [Å]

SLD [10−6 Å−2]

db [Å]

χ2

3.4

4.1

3.9

41.1

1.2

8.4

2.3

10.2

4.4

39.5

1.5

8.4

2.6

48.6

5.5

39.2

1.7

temperature (°C)

dw [Å]

22 °C (t = 15 min) 22 °C (t = 200 min) 37 °C

2.5

6.0

4.0

3.9

8.4

3.3

24.3

0.9

8.4

--

--

10.1

4.4

8.4

2.3

22.7

0.4

--

--

45.0

4.6

8.4

2.6

22.4

0.6

SLD [10 Å−2]

dEO, p [Å]

SLD [10 Å−2]

Z h,p [Å]

SLD [10 Å−2]

(Tonset = 20.1 °C; Tm = 27.5 °C; Figure S2). Thus, the TX-100/ DMPC SLB is fully in the fluid phase only at 37 °C. The dimensions of the hydrocarbon tail region are essentially invariant, dc = 23.9 and 24.0 Å at 22 and 37 °C, respectively. The SLD does change, however, with increasing temperature, decreasing from 0.9 × 10−6 Å−2 to −0.1 × 10−6 Å−2. The decrease in SLD with increasing temperature reflects variations in the level of water penetration into the bilayer. Less water penetration into the bilayer at 37 °C arises from changes in alkyl chain packing, since the chains are only fully melted at 37 °C. As the temperature is lowered to below the Tm (15 °C) an increase in the dc to 26.6 Å is observed, and the SLD increases to 0.7 × 10−6 Å−2 consistent with changes in alkyl chain packing and improved water solubility of the EO segments. The headgroup dimensions, zh,p and zh,d, for the DMPC/TX-100 SLB are invariant with temperature and are identical to that determined for the DMPC SLB (8.4 Å). Unlike the DMPC only constructs, however, the SLD does show minor temperature variation, ranging from 3.5−3.3 × 10−6 Å−2 with increasing temperature. The symmetric EO regions dimensions were found to decrease with increasing temperature (4.4 Å to 3.4 Å). The shorter dimensions are again ascribed to the temperature-induced contraction of EO chains. The SLD of these layers varies slightly from 4.1 to 3.9 × 10−6 Å−2, values substantially larger than previously reported for TX-100 by Timmins and co-workers of 0.39 × 10−6 Å−2.21 The SLD value reported by Timmins is for all of the TX-100 molecules and thus is not appropriate to accurately describe the EO moiety segregated within this region. The value is also larger than the SLD for pure EO (0.572 × 10−6 Å−2)22 and thus must indicate well-solvated oligomeric EO units. Cosurfactant−Block Copolymer DMPC Bilayers. Triblock copolymers of PEOn−PPOm−PEOn, so-called Pluronics or Poloxamers, have been widely studied due to interest in communities ranging from materials science to medicine.23,24 Prior temperature-dependent SAXS studies conducted on complex fluids composed of DMPC, the cosurfactant lauryldimethylamino oxide (LDAO), and Pluronics of varying block architectures showed that F98 possesses sufficient molecular length in the central hydrophobic PPO block to achieve transmembrane insertion across a DMPC bilayer.9a Transmembrane insertion of the polymer acts to position the symmetric PEO blocks into the water regions on either side of the membrane.9b The insertion modality of F98 is highly temperature dependent and is determined by the water solubility of both the PPO and PEO blocks.9a Poly(propylene oxide) becomes less water-soluble with increasing temperature, transitioning from fully water-soluble at 15 °C (i.e., preferring bulk water), to intermediate solubility at 22 °C, leading to weak membrane association (at the depth of the ester linkages), and water insoluble at 37 °C, promoting full bilayer integration. Poly(ethylene oxide) undergoes temperature-dependent con-

dc [Å]

SLD [10 Å−2]

distal EO

Z h,d [Å]

SLD [10 Å−2]

−6

formational changes, transitioning from well-solvated with extended chains below 37 °C to less water-soluble, adopting a compacted coil conformational state, above 37 °C.25 The temperature-driven association of Pluronics and induced changes in membrane structure has been reported for freely suspended multilamellae; comparatively less is known about them on SLBs. F98 was introduced as a D2O mixture (2.4 μM in D2O) and injected into the subphase of the supported DMPC/TX-100 system. The NR curve obtained at 22 °C at 15 min post injection is presented in Figure 3A (light green). The ternary amphiphilic mixture (DMPC/TX-100/F98) reflectivity curve was well modeled employing seven slabs (Figure 3B, light green), a model identical to that used to describe the DMPC/ TX-100 samples (Figure 3B, black). That is, two distinct sublayers outside the bilayer composed of TX-100 EO moieties and pure D2O can be resolved. Only subtle changes are noted between the TX-100/DMPC SLBs at 22 °C and F98/TX-100/ DMPC SLBs after short time equilibration, as evidenced by the determined fit parameters presented in Table 3. The lack of an observed change in the structure of the SLB in this time frame is not unexpected and is supported by coarse-grained molecular dynamic simulations carried out by Rilano, Roccatano, and coworkers.26 Briefly, the molecular dynamics (MD) simulations examined PEO−PPO−PEO triblock copolymer association with preformed DMPC bilayers. The simulations showed polymer association is a multiphase process that can be slowed upon either increasing the polymer concentration or with large PEO block lengths.26 More specifically, the process of polymer association with the DMPC bilayer occurred in discrete stages beginning with the PEO block adsorbing to the membrane surface. In the second, rate-limiting step, association and percolation of the PPO block through the headgroup of the lipid bilayer occurs. Finally, the PPO penetrates through the bilayer upon which it remains stable. Thus, based upon these simulations, it is possible that a 15 min equilibration time is not sufficient to achieve full polymer intercalation into the SLB. Next, an increased incubation time to 200 min (at 22 °C) was examined (Figure 3A, C dark green). Additional changes are observed in the reflectivity curve and corresponding fit parameters (Table 3). Most notably, the two distinct (proximal and distal) hydrated PEO and D2O layers become indistinguishable (Figure 3C, dark green). Furthermore, the total thickness of the proximal layer (containing EO + D2O) is now larger (10.1 Å) than the summation of the two sublayers found at shorter incubation times (6.5 Å), which may indicate that the polymer has reconfigured so as to achieve vectorial projection of the PEO block normal to the bilayer, thereby enhancing the steric pressure and lift off from the solid support. Next, the phospholipid headgroup regions (both distal and proximal) show a drop in the SLD value (from 3.4 × 10−6 Å−2 to 2.3 × 10−6 Å−2). The reduction in SLD implies a reduction in water F


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Biomacromolecules

Figure 4. (A) Fresnel divided NR scattering data (points with error bars) and the fits (solid lines) corresponding to derived SLD profiles for DMPC/Triton X-100/F98 SLBs after lowering the temperature from 37 to 22 °C (orange) and 37 to 15 °C (blue). (B) Comparison of determined SLD for DMPC/Triton X-100/F98 (200 min polymer incubation; blue curve) to thermally cycled DMPC/Triton X-100/F98 from 37 to 22 °C (orange). (C) Smeared and unsmeared DMPC/Triton X-100/F98 collected at 15 °C after being heated to 37 °C. (D) Comparison of SLD of DMPC/Triton X-100/F98 37 to 22 °C (orange) and 37 to 15 °C (purple).

Table 3). A significant increase in the EO/D2O layer dimensions is observed increasing from 10.1 to 45.0 Å. Expansion of this layer is accompanied by a minor increase in SLD from 4.4 × 10−6 Å−2 to 4.6 × 10−6 Å−2. The observed increase in the extra-membrane region is consistent with prior work carried out on freely suspended multilamellar membranes containing F98 that undergo a substantial increase in the interlamellar d-spacing at 37 °C.9 The formation of an expanded lamellar structure was attributed to the transmembrane insertion of F98, which orients the constrained, symmetric compacted (mushroom conformational state) PEO blocks perpendicular to the lipid bilayer (Figure 3D, red).9b Under these conditions, sufficient steric pressure arising from the coiled, projected EO blocks drives the swelling of the interlamellar spacing. Transmembrane insertion of the F98 across the bilayer is also indicated through the minor compression in the bilayer thickness (db = 39.5 Å to 39.2 Å) and an increase in the SLD (0.4 × 10−6 Å−2 to 0.6 × 10−6 Å−2).

penetration into the region. Localization of PPO within this region would be expected to increase the SLD (PPO SLD is 0.347 × 10−6 Å−2).22 The apparent decrease of water solvation in the headgroup region is consistent with prior studies stating that the F98 PEO can act, under certain conditions, to “seal” membranes.27 A third observed change induced upon addition and extended incubation of the F98 is bilayer compression db from 41.1 to 39.5 Å (dc from 24.3 to 22.7 Å). The so-called “thinning” of the bilayer upon insertion of the polymer was observed previously by SAXS-determined electron density mapping and confirmed by coarse-grained (CG) MD simulations for triblock copolymers that span a lipid bilayer with the PEO blocks flanking the opposite sides of the bilayer and residing in the water phase. The SLD of the hydrocarbon region is decreased from 0.9 × 10−6 Å−2 to 0.4 × 10−6 Å−2 likely due to increased alkyl chain disorder. Upon raising the SLB temperature to 37 °C, the reflectivity curves and fit parameters continue to evolve (Figure 3A,D, red; G


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Table 4. Fit Parameters for DMPC/TX100/F98 SLBs Collected on a Thermal Cycle from 37 to 22 °C ; 37 to 15 °C and a Sample at 15 °C That Was Not Thermally Cycleda water layer temperature (°C)

dw [Å]

37 to 22 °C 37 to 15 °C 15 °C

----

a

SLD [10 Å−2] ----

proximal EO −6

dEO, p [Å] 26.6 177.9 145.8

SLD [10 Å−2] 4.4 4.1 4.1

proximal heads −6

Z h,p [Å] 8.4 8.4 8.4

SLD [10 Å−2]


dc [Å]

2.4 3.4 3.4

22.5 26.1 26.6

SLD [10 Å−2] 1.0 0.1 0.6

−6

distal heads Z h,d [Å] 8.4 8.4 8.4

SLD [10 Å−2] 2.5 3.4 3.4

distal EO −6

dEO, d [Å]

SLD [10−6 Å−2]

db [Å]

χ2

10.2 4.5 4.4

4.5 4.1 4.1

39.3 42.9 43.4

1.9 2.6 1.5

The parameters correspond to the SLD profiles shown in Figure 4.

Given that the SLD of PPO is reported to be 0.347 × 10−6 Å−2,22 the higher SLD value found here most likely is from intercalated PPO and increased water permeation across the bilayer. This confirms reports where transmembrane inserting nonionic triblock copolymers have been found to form hydrated pores (channels) that accelerate the passage of molecules across the membrane.27b Single channel recording studies have also indicated that F98 can function as an artificial ion channel structure.28 Reduction in the SLB temperature from 37 to 22 °C causes a noted change in the recorded reflectivity profile (Figure 4A, orange (37 °C) vs. blue (22 °C)). The NR curve is well modeled using the five-slab model (Figure 4B, orange, Table 4). Inspection of the fit parameters (Table 4) shows that the most significant change is the reduction in the bilayer to quartz gap dimension, dEO,p, (here the proximal layer contains intermixed water and EO), which decreases from 45.0 Å at 37 °C to 26.6 Å upon returning to 22 °C. The reduction in the SLB−solid substrate gap arises from the increased F98 water solubility and exclusion from the bilayer. This layer thickness at 26.6 Å is more than twice that previously determined when raising the temperature from 15 to 22 °C and incubating for 200 min (10.1 Å) yet less than that achieved at 37 °C (45.0 Å). The differences in the thickness of this region could indicate a greater concentration of polymer has partitioned into the region. The increased amount of polymer within the gap may be due to favorable electrostatic interactions between the PEO and the quartz surface.29 Recent adsorption studies carried out on PEO−PPO block copolymer with hydrophilic surfaces suggested that the process is driven by PEO block surface Si− OH interactions through possible formation of hydrogen bonds.30 The SLD for this region (4.4 × 10−6 Å−2) remains nearly the same as that determined for sample held at 22 °C for 200 min consistent with the presence of well-hydrated EO chain. The phospholipid headgroup dimensions and SLD (both proximal and distal) are also found to remain nearly the same (2.4 × 10−6 Å−2) as those determined at 22 °C, 200 min. The hydrocarbon chain, dc, and bilayer thickness, db, also remain unchanged at 22.5 and 39.3 Å, respectively. The SLD value of the hydrocarbon region does, however, increase from 0.4 × 10−6 Å−2 (determined upon raising the SLB temperature from 15 to 22 °C and holding for 200 min) to 1.0 × 10−6 Å−2 (upon lowering the SLB temperature from 37 to 22 °C). The observed increase in the SLD signals significant water penetration possibly arising from the efflux of the polymer and residual chain disordering that occurs. A reduction in temperature from 37 to 15 °C causes more significant changes in the NR curves (Figure 4C,D, orange (37 °C → 22 °C) vs. purple (37 °C → 15 °C)) and the corresponding fit parameters (Table 4). Foremost, a large increase in the thickness of the proximal EO layers is observed at 177.9 Å. This value is compared to the gap dimensions

produced upon raising the temperature to 37 °C from 22 °C, yielding a dEO,p of 45.0 Å. The increase in the gap layer occurs with only a minor decrease in the SLD of the region (4.1 × 10−6 Å−2 vs 4.6 × 10−6 Å−2). The large substrate−bilayer gap is attributed to preferential localization of the F98 within this region, as it becomes fully water-soluble and dissociates from the bilayer. The sequestration of F98 into the extra-membrane solid interface is most likely driven by its tendency to adsorb to the hydrophilic glass surface (Figure 4C, inset). This favorable interaction yields a large polymer hydrogel cushion that supports an intact bilayer, suspending it well above the quartz substrate. The intact nature of the bilayer is supported through evaluation of the dimensional and SLD fit parameters (Tables 3 and 4). The hydrocarbon layer thickness, dc, recovers to 26.1 Å, a value similar to that obtained for SLBs composed of DMPC/ TX-100 at 15 °C, confirming complete removal of the F98 from the lipid bilayer. The SLD of the bilayer hydrocarbon region concurrently decreases to 0.1 × 10−6 Å−2 as a result of the outflux of PPO. This value is lower than that determined for SLBs composed of DMPC/TX-100 at 15 °C. The reduction in the SLD could be due to increased disorder in the alkyl chains upon loss of the PPO block. The symmetric phosphate headgroup regions dimensions remain unaltered and return to the same SLD value for the DMPC/TX-100 SLB at 15 and 22 °C. It is noted that the generation of the very thick polymer layer does produce significant off-specular scattering, which arises from out-of-plane fluctuations of the lipid membrane residing on top of the large polymer cushion (data not shown). Finally, to determine whether prior F98 transmembrane insertion is required for generation of the extra-membrane polymer cushion, a measurement was made on a sample that had not undergone thermal cycling to 37 °C. Specifically, F98 was directly added to a DMPC/TX-100 SLB maintained at 15 °C. Comparison of the SLD profiles derived from fitting the NR curves obtained on a thermally cycled (37 °C → 15 °C) SLB to one isothermal held at 15 °C is presented in Figure 5 (blue (15 °C) vs. red (37 °C → 15 °C)). The large extramembrane polymer cushion was also produced without the need to attain a transmembrane insertion of the polymer. Variation in the thickness of the polymer cushion is noted between the two samples. The isothermal sample exhibits a smaller proximal hydrophilic gap containing the polymer cushion (145.8 Å) compared to one thermally cycled to 37 °C (177.9 Å). The only other observed difference is a change in the SLD of the hydrocarbon tail region. The isothermal sample construct has a larger SLD (0.6 × 10−6 Å−2) compared to the thermally cycled one (0.1 × 10−6 Å−2). The reduction in the SLD again suggests that the transmembrane insertion of the PPO into the acyl chain region causes disruption in chain packing, whereas the isothermal sample, which did not experience the incorporation of the PPO block, remains ordered. H


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Biomacromolecules

Figure 6. Comparison of SLD profiles determined for DMPC/Triton X-100/F98 heated from 15 to 37 °C (pink) to SLB that was ramped from 22 to 37 °C (red).

is close to the theoretically predicted value (−0.32 × 10−6 Å). A lower SLD value for the proximal EO region (4.7 × 10−6 Å−2) compared to that of the distal (5.6 × 10−6 Å−2) is also found, suggesting that possibly more PEO chains remain at the solid proximal membrane interface than that at the distal bulk water phase region.

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CONCLUSIONS NR was used to yield molecular details of the temperaturedependent interactions of a membrane-spanning nonionic triblock copolymer of PEO117−PPO47−PEO117 (F98) with a model SLB composed of a saturated phospholipid (DMPC) and a nonionic cosurfactant (TX-100). The nature of polymer− membrane association was found to be highly temperaturedependent and thereby provides a facile means to regulate the distance between the solid substrate (quartz) and the proximal leaflet of the bilayer (the hydration layer or gap). Specifically, at physiological temperature (37 °C) the F98 transmembrane inserts into the membrane causing the hydration gap to widen 10-fold (to 45 Å) compared to the DMPC/Triton X-100 membrane SLB gap (6.8 Å). At room temperature (22−25 °C), the hydration gap thickness is reduced to 10−27 Å, due to reorientation of the PEO blocks to residing on the same side of the bilayer (harpoon conformation), a conformational change driven by the increased water-solubility of the PPO block. Further reducing the temperature to 15 °C leads to a dramatic increase in the hydration gap to 146−178 Å. The large gap arises from expelled polymer from the membrane and preferential partitioning at the quartz-proximal bilayer leaflet interface. The large polymeric hydrogel cushion lifts the intact bilayer from the surface. While reversibility between the various gap dimensions was demonstrated, hysteresis in the actual dimensions is observed and depends on the thermal history of the sample. That is, once a sample has been held at 37 °C to achieve transmembrane insertion of the polymer, larger gap dimensions are observed, attributed to the preferential interaction of the PEO blocks to the hydroxylated substrate. Noted, however, is the near full recovery of the bilayer structure upon doping and de-doping, an observation consistent with prior X-ray scattering studies on the corresponding bulk phase

Figure 5. (A) Fresnel divided NR scattering data (points with error bars) and the fits (solid lines) corresponding to derived SLD profiles for DMPC/Triton X-100/F98 SLBs no thermal history at 15 °C (blue) and 37 to 15 °C (red). Comparison of SLD profiles determined for DMPC/TritonX-100/F98 collected at 15 °C (no thermal history) (blue) and one that has been cycled from 37 to 15 °C (red).

Reversibility of the F98 insertion into the DMPC/TX-100 bilayer was tested by cycling the sample temperature from 15 to 37 °C (Figure 6, pink) and compared to a sample held at 37 °C (Figure 6, red). Remarkably, the NR curve and experimentally determined fit parameters (Table 5) were found to fully recover, indicating that the F98 reinserted (transmembrane fashion) into the bilayer. That is, the larger polymer cushion (177.9 Å) produced upon temperature reduction to 15 °C disappears, and the gap dimension collapses to 45.3 Å, a value nearly identical to the SLB, which is cycled from 22 to 37 °C (Figure 3A, D; Table 3). The bilayer thickness, dB, is 38.9 Å, also the same as before thermal cycling at 37 °C (dB = 39.2 Å). A significantly lower SLD for the hydrocarbon region is found (−0.4 × 10−6 Å−2 compared to 0.6 × 10−6 Å−2), which suggests that an increase in alkyl chain ordering is observed by ramping the temperature from a polymer excluded state directly to membrane spanning compared to passing through the harpoon condition. In fact, the SLD determined for the acyl chain region I


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Table 5. Fit Parameters for DMPC/TX100/F98 SLBs Collected on a Thermal Cycling from 37 to 15 °C Then Back to 37 °C (System That Had Never Been Heated to 37 °C) and 37°C (System That Was Cycled from 37 to 15 °C and Back to 37 °C)a water layer temperature (°C) 37 to 15 °C to 37 °C a

EO −6

dw[Å] --

SLD [10 Å−2] --

dEO,p [Å] 45.3

heads −6

SLD [10 Å−2] 4.7

Z h,p [Å] 8.4

SLD [10 Å−2]

tails −6

2.3

dc [Å] 22.1

heads −6

SLD [10 Å−2] −0.4

Z h,d [Å] 8.4

SLD [10 Å−2] 2.4

EO −6

dEO, d [Å]

SLD [10−6 Å−2]

db [Å]

χ2

46.8

5.6

38.9

1.4

The parameters correspond to the SLD profiles shown in Figure 5B.

materials.9b Future studies taking full advantage of the noted sensitivity of neutrons to SLD differences between deuterium and hydrogen will employ contrast variation (i.e., different ratios of H2O/D2O; chain deuteration of the amphiphiles, etc.) to gain greater insights into composition and/or temperatureinduced structural changes of the multicomponent bilayers.31 Such contrast variation studies will more clearly delineate the location of the PPO block within the bilayer and more precisely “image” the conformational state of the PEO block as a function of temperature. In summary, this work establishes a facile, reversible means to control the solid−membrane interface through simple introduction of a membrane-spanning nonionic polymer coupled with modest temperature modulation.

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and tissue architecture using periosteum derived progenitor cells. Biomaterials 2013, 34 (8), 1878−1887. (4) van Weerd, J.; Karperien, M.; Jonkheijm, P. Supported lipid bilayers for the generation of dynamic cell material interfaces. Adv. Healthcare Mater. 2015, 4 (18), 2743−2779. (5) (a) Ahmed, S.; Savarala, S.; Chen, Y. J.; Bothun, G.; Wunder, S. L. Formation of Lipid Sheaths around Nanoparticle-Supported Lipid Bilayers. Small 2012, 8 (11), 1740−1751. (b) Mashaghi, S.; Jadidi, T.; Koenderink, G.; Mashaghi, A. Lipid Nanotechnology. Int. J. Mol. Sci. 2013, 14 (2), 4242−4282. (c) Suzuki, Y.; Endo, M.; Sugiyama, H. Lipid-bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures. Nat. Commun. 2015, 6, 8052. (6) Chan, Y. H. M.; Boxer, S. G. Model membrane systems and their applications. Curr. Opin. Chem. Biol. 2007, 11 (6), 581−587. (7) Raguse, B.; Braach-Maksvytis, V.; Cornell, B. A.; King, L. G.; Osman, P. D. J.; Pace, R. J.; Wieczorek, L. Tethered lipid bilayer membranes: Formation and ionic reservoir characterization. Langmuir 1998, 14 (3), 648−659. (8) (a) Sackmann, E. Supported membranes: Scientific and practical applications. Science 1996, 271 (5245), 43−48. (b) Tanaka, M.; Sackmann, E. Polymer-supported membranes as models of the cell surface. Nature 2005, 437 (7059), 656−663. (c) Richter, R. P.; Berat, R.; Brisson, A. R. Formation of solid-supported lipid bilayers: An integrated view. Langmuir 2006, 22 (8), 3497−3505. (d) McCabe, I. P.; Forstner, M. B. Polymer supported lipid bilayers. Open J. Biophys. 2013, 3, 59−69. (9) (a) Firestone, M. A.; Wolf, A. C.; Seifert, S. Small-angle x-ray scattering study of the interaction of poly(ethylene oxide)-bpoly(propylene oxide)-b-poly(ethylene oxide) triblock copolymers with lipid bilayers. Biomacromolecules 2003, 4 (6), 1539−1549. (b) Lee, B.; Firestone, M. A. Electron density mapping of triblock copolymers associated with model biomembranes: Insights into conformational states and effect on bilayer structure. Biomacromolecules 2008, 9 (6), 1541−1550. (10) Laible, P. D.; Kelley, R. F.; Wasielewski, M. R.; Firestone, M. A. Electron-transfer dynamics of photosynthetic reaction centers in thermoresponsive soft materials. J. Phys. Chem. B 2005, 109 (49), 23679−23686. (11) Firestone, M. A.; Williams, D. E.; Seifert, S.; Csencsits, R. Nanoparticle arrays formed by spatial compartmentalization in a complex fluid. Nano Lett. 2001, 1 (3), 129−135. (12) Dubey, M.; Jablin, M. S.; Wang, P.; Mocko, M.; Majewski, J. SPEAR - ToF neutron reflectometer at the Los Alamos Neutron Science Center. Eur. Phys. J. Plus 2011, 126, 110−112. (13) Penfold, J. Analysis of Neutron Reflectivity Data Using Constrained Model-Fitting. Inst. Phys. Conf. Ser. 1990, 107, 213−222. (14) Nelson, A. Co-refinement of multiple-contrast neutron/X-ray reflectivity data using MOTOFIT. J. Appl. Crystallogr. 2006, 39, 273− 276. (15) (a) Nickel, B. Nanostructure of supported lipid bilayers in water. Biointerphases 2008, 3 (3), Fc40−Fc46. (b) Koenig, B. W.; Kruger, S.; Orts, W. J.; Majkrzak, C. F.; Berk, N. F.; Silverton, J. V.; Gawrisch, K. Neutron reflectivity and atomic force microscopy studies of a lipid bilayer in water adsorbed to the surface of a silicon single crystal. Langmuir 1996, 12 (5), 1343−1350. (c) Gutberlet, T.; Steitz, R.; Fragneto, G.; Klosgen, B. Phospholipid bilayer formation at a bare Si surface: a time-resolved neutron reflectivity study. J. Phys.: Condens. Matter 2004, 16 (26), S2469−S2476.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.6b01461. Additional details regarding the analysis and reproducibility of the reflectivity data (PDF)

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AUTHOR INFORMATION

ORCID

Millicent A. Firestone: 0000-0002-8671-1502 Present Address ⊥

Dr. Steven Hayden’s current address is Aramco Research Center, Cambridge, MA 02139 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was performed, in part, at the Center for Integrated Nanotechnologies (CINT) and at the Lujan Center of Los Alamos Neutron Scattering Center (LANSCE). CINT is funded by the DOE Office of Basic Energy Sciences. LANL is operated by Los Alamos National Security, LLC, for the National Nuclear Security Administration of the U.S. Department of Energy under contract DE-AC52-06NA25396.

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REFERENCES

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Reversible Lifting of Surface Supported Lipid Bilayers with a

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