Spectroscopic and Permeation Studies of Phospholipid Bilayers

Article pubs.acs.org/Langmuir

Spectroscopic and Permeation Studies of Phospholipid Bilayers Supported by a Soft Hydrogel Scaffold Michael Grossutti, Ryan Seenath, Shannon Conlon, J. Jay Leitch, Jie Li, and Jacek Lipkowski* Department of Chemistry, University of Guelph, Guelph, Ontario N1G 2W1, Canada ABSTRACT: Polarized attenuated total reflection infrared (ATRIR) spectroscopy, fluorescence microscopy, and fluorescence spectroscopy were used to characterize a lipid coating composed of 70 mol % 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 30 mol % cholesterol, supported on a spherical hydrogel scaffold. The fluorescence microscopy images show an association between the lipid coating and the hydrogel scaffold. Fluorescence permeability measurements revealed that the phospholipid coating acts as a permeability barrier, exhibiting characteristics of a lamellar bilayer coating structure. Variable evanescent wave penetration depth ATRIR spectroscopy studies validated the determination of quantitative molecular orientation information for a lipid coating supported on a spherical scaffold. These polarized ATR-IR studies measured an average DMPC acyl chain tilt angle of ∼21−25°, with respect to the surface normal.

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INTRODUCTION Planar lipid bilayer membranes supported at a metal electrode surface have served as attractive models of biological membranes.1,2 Confining a membrane to a flat surface provides mechanical stability and allows for its investigation by an array of powerful surface analytical techniques such as infrared (IR) spectroscopy, atomic force microscopy (AFM), scanning tunneling microscopy (STM), surface plasmon resonance (SPR), and neutron reflectivity.2−15 By using a metal electrode, an electrical potential may be applied and the electric field across the membrane controlled, thereby providing a means of mimicking the electrical properties of natural biological cell membranes. Furthermore, by coupling electrochemical and surface analytical techniques, one can observe in situ, at the molecular level, electric field-induced changes in the membrane. One such example is electrochemical polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) on a gold electrode, which allows for the observation of structural changes in response to an applied electric field.4−9 A shortcoming of these models is the proximity of the electrode support to the inner leaflet of the bilayer, which can lead to several problems. Electrode-supported membranes often exhibit a lack of fluidity and inhibited lipid mobility, which can contribute to the presence of defects in the membrane.16,17 Another significant problem relates to the insertion of transmembrane proteins in the bilayer, which often denature and lose functionality when they come into contact with the metal surface.1 Considerable research has been done to mitigate the problems arising from the metal electrode support.1,18 These efforts have included the use of tethering molecules and polymer cushions to create a larger hydrophilic space between the metal and bilayer.18 © 2014 American Chemical Society

In addition to these planar biomimetic membranes, spherical supported lipid bilayers exhibiting good mechanical stability have been developed on a wide range of materials, including silica, polymer gels, and various nanoparticles.19−40 The dimensions of the spherical support have covered a range of sizes, from tens of nanometres to tens of micrometers.19 These systems often have attractive biotechnological applications not necessarily present in planar systems.41 When porous supports are used, spherical supported lipid bilayers have an enclosed internal volume, allowing for the encapsulation of hydrophilic molecules. Porous systems have attracted interest in the fields of drug delivery and controlled release.19 However, these spherical supported lipid bilayers tend to be less accessible to surface analytical and electrochemical techniques than their planar counterparts.41 The goal of this work is to develop a model membrane that overcomes the aforementioned problems associated with electrode-supported membranes while retaining their accessibility to surface analytical and electrochemical techniques. The model architecture under study is a giant scaffolded vesicle consisting of a 70 mol % 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) and 30 mol % cholesterol lipid bilayer supported by a spherical Sephadex G10 porous hydrogel with an average diameter of ∼100 μm and variable pore diameters on the order of nanometers. The chemical structures of DMPC and a deuterated analogue are shown in Figure 1. Sephadex G10 was chosen as the hydrogel scaffold for its large diameter and small pore size. The large diameter provides for the model’s accessibility to surface analytical and electroReceived: July 23, 2014 Revised: August 18, 2014 Published: August 22, 2014 10862

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Article

EXPERIMENTAL SECTION

Chemicals and Solutions. Lipid stock solutions were prepared from powdered 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (Avanti Polar Lipids), 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine (DMPC-d54) (Avanti Polar Lipids), and cholesterol (Sigma-Aldrich) dissolved in chloroform (Sigma-Aldrich). 1-Myristoyl-2-{12-[7-nitro2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-DMPC) (Avanti Polar Lipids) was purchased in chloroform. Terbium(III) chloride (Sigma-Aldrich) and dipicolinic acid (DPA) (Sigma-Aldrich) solutions were prepared in Milli-Q (Millipore) water with a resistivity of 18.2 MΩ cm. Sephadex G10 (SigmaAldrich) was hydrated in either Milli-Q water or a terbium(III) chloride solution. Sample Preparation. Dry lipid films were prepared by combining DMPC and cholesterol chloroform solutions in a test tube, at a 70:30 molar ratio. The chloroform solvent was slowly evaporated under a stream of argon while the solution was being vortexed. After evaporation, the test tubes were stored in a desiccator under vacuum for at least 24 h prior to use. These lipid films were used for fluorescence spectroscopy experiments. For ATR-IR experiments, DMPC-d54 was used in the place of DMPC. For fluorescence microscopy experiments, 1 mol % NBD-DMPC was included in the test tube at the expense of DMPC. To make vesicles, a volume of ultrapure Milli-Q water was added to the dry lipid film such that an ∼5 mg/mL solution was obtained. The mixture was sonicated for ∼5 min at ∼45 °C. The resulting vesicles were added to hydrated Sephadex G10 beads. The vesicles and Sephadex G10 were incubated at ∼45 °C for at least 1 h, while being constantly vortexed. The scaffolded vesicles were allowed to cool slowly to room temperature and incubate in the vesicle solution. The scaffolded vesicles were then thoroughly rinsed to remove excess vesicles in solution. For fluorescence spectroscopy experiments, a 1.0 mM terbium(III) chloride solution was used in the place of Milli-Q water. Fluorescence Microscopy. Fluorescence microscopy experiments were performed on an Olympus BX51 microscope equipped with a fluorescein isothiocyanate filter and QImaging Retiga 2000R camera. An Olympus U-RFL-T mercury burner served as the light source. To obtain images, fluorescent scaffolded vesicles were dropped onto a microscope slide and allowed to settle. Fluorescence Spectroscopy. Fluorescence measurements were performed with a Varian Cary Eclipse fluorescence spectrophotometer equipped with a temperature control unit (Varian Inc.). Fluorescence intensity measurements were performed immediately after approximately 50 μL of scaffolded vesicles had been deposited at the bottom of a quartz cuvette containing 2.25 mL of a 0.125 mM DPA solution at an approximately neutral pH. Fluorescence intensity measurements of the 585 nm fluorescence band where taken every 5 min for a period of roughly 4 h at a steady preset temperature of 20 ± 0.2 °C unless the effects of temperature on permeability were being investigated. Along with each sample, a control containing 50 μL of Sephadex G10 hydrated in 1.0 mM TbCl3(aq) was monitored simultaneously. Because DPA was implemented as the absorbing antenna ligand, an excitation wavelength of 270 nm was found to result in the highest Tb3+ fluorescence intensity. ATR-IR Spectroscopy. ATR-IR spectra were recorded on a Nicolet Nexus 870 spectrometer equipped with a MCT-D* detector, a VeeMax II variable-angle specular reflectance accessory (Pike Technologies), and a zinc selenide wire-grid polarizer (Pike Technologies). To collect the spectra, scaffolded vesicles (or water) are deposited on either a zinc selenide or germanium internal reflection element with a face angle of 45° (Pike Technologies). The incident angle was set to achieve the desired evanescent wave penetration depth. For each spectrum, 5000 scans were collected and averaged using an instrumental resolution of 4 cm−1. The reported spectra are averages of three independent experiments. A new batch of scaffolded vesicles was used for each experiment. The spectra are presented in absorbance units defined as A = −log(I/I0) where I and I0 are the single-beam reflected intensities for

Figure 1. Chemical structure of DMPC and DMPC-d54.

chemical techniques. The pore is smaller than the vesicles used to prepare the membrane coating, promoting vesicle fusion and bilayer formation. The mixed DMPC and cholesterol bilayer has a packing parameter close to 1, which favors the formation of a planar lipid bilayer.42 A packing parameter of ∼1 is desirable for this work, because of the large diameter of the hydrogel scaffold. The inclusion of cholesterol in membranes is known to improve the quality of lipid bilayers, producing a thicker, more uniform bilayer with fewer defects.11 The porous hydrogel scaffold is intended to provide both structural support and an internal aqueous environment. This feature should improve upon the electrode-supported models by providing for an aqueous reservoir, allowing for the incorporation of transmembrane proteins, and opening the door for membrane transport studies. Furthermore, the spherical geometry and porosity of the scaffold provide an enclosed internal aqueous volume allowing for complementary membrane permeability and encapsulation-related studies. Such studies would provide information about the completeness, permeability, and physical properties of the coating membrane. Monitoring the completeness of the lipid coating on the scaffold is necessary to proceed with the spectroscopic and electrophysiological investigations. The scaffolded vesicles have been designed to be compatible with both attenuated total reflection infrared (ATR-IR) spectroscopy (a surface analytical technique) and the electrophysiological patch-clamp technique (an electrochemical technique). A long-term goal of this work is the development of a tandem ATR-IR spectroscopy and patch-clamp analytical instrument for the study of a scaffolded vesicle. The patchclamp component is intended to control the electrical potential across the scaffolded vesicle membrane, while the ATR-IR spectroscopic component provides quantitative molecular level information about the membrane. This paper lays the groundwork for the quantitative molecular orientation ATR-IR studies of the scaffolded vesicles. This is an essential step in the development of a tandem ATRIR and patch-clamp instrument. To the best of our knowledge, the extraction of quantitative molecular orientation data from a spherical supported lipid bilayer is a novel aspect of this work. The permeability characteristics of the scaffolded vesicles were also examined. The ability to correlate the permeability characteristics of scaffolded vesicles to membrane structural information learned from ATR-IR spectroscopic studies may prove to be valuable for the study of controlled release schemes. 10863

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membrane would allow for the efflux of Tb3+, and complex formation. Figure 3 shows the fluorescence leakage profiles for scaffolded vesicles studied at room temperature and at 10 °C

hydrated scaffolded vesicles and water, respectively. Absorbance spectra for both p- and s-polarizations (Ap and As, respectively) are obtained. The absorbance spectra are baseline-corrected, typically with a quadratic function, before further analysis. For a given absorption band, the dichroic ratio is found by taking the ratio of the p- and spolarized absorbance spectra (R = Ap/As) at the band maximum. The dichroic ratio is independent of the size of the sample and of the distribution of absorbers at the surface. The size of the sample and the distribution function of absorbers on the sample affect both Ap and As to the same extent, and hence, they cancel when their ratio is calculated. For quantitative calculations of penetration depth and molecular orientation, the following refractive indices are used: 2.44 for ZnSe, 4.0 for Ge, 1.42 for DMPC acyl chains, and 1.33 for water.43,44

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RESULTS AND DISCUSSION Fluorescence Microscopy. Fluorescence microscopy was used to initially characterize the lipid coating of the hydrogel scaffold. The fluorescently tagged phospholipid NBD-DMPC was included in the lipid formulation at a concentration of 1 mol %. The fluorescent tag of NBD-DMPC is located at the end of the 2-position acyl chain. Figure 2 shows a fluorescence Figure 3. Fluorescence profile fits for uncoated Sephadex G10 beads [k = 0.15 min−1 (black)] and 70:30 DMPC/cholesterol−Sephadex G10 scaffolded vesicles at room temperature [k = 0.048 min−1 (blue)] and at 10 °C [k = 0.025 min−1 (red)]. The scaffolded vesicles were prepared at room temperature, and the rate of fluorescence intensity increase is taken as a measure of the permeability of the system. The vertical error bars represent the standard deviation of the measurement.

and a negative control that consisted of uncoated Sephadex G10 hydrogel beads. Fitting the fluorescence profiles with the following first-order rate equation allows for the determination of a rate constant (k)

Figure 2. Fluorescence microscopy image of a scaffolded vesicle containing 1 mol % NBD-DMPC.

It = Imax(1 − e−kt )

(1)

where It and Imax are the fluorescence intensities at time t and after a steady state has been achieved, respectively. The rate constant serves as a quantitative measure of membrane permeability, which in turn can be seen as a measure of coating quality. The permeability rate constants were measured at several temperatures. Figure 4 shows the dependence of the rate constant on temperature. The permeability rate constant goes through a maximum at the gel to liquid phase transition temperature (∼23 °C) for a 70:30 DMPC/cholesterol bilayer. For a lamellar coating, a maximum in permeability is expected to occur at the gel to liquid phase transition temperature because of a maximum in the number of membrane defects caused by domain boundary formation.45 To support the presence of a lipid coating on the hydrogel bead, the permeability of the scaffolded vesicle system was investigated at different temperatures. It was hypothesized that the permeability of the lipid coating should have followed the model proposed by Cuzeiro-Hansson et al. in which maximal permeability would be achieved at the transition temperature of the lipid coating.45,46 As shown in Figure 3, the rate constants tend to reach their maxima at the transition temperatures (Tm) for the DMPC/cholesterol and pure DPPC coatings (approximately 23 and 41 °C, respectively). The temperature profile of DPPC was measured to ensure the consistency of the temperature dependence in the data. It was apparent that the

image of a scaffolded vesicle, which provides direct evidence of an association between the hydrogel scaffold and lipid coating. The coronal appearance of the fluorescence suggests a vesiclelike structure, with the lipids confined to the outer surface of the hydrogel support. However, the fluorescence images do not provide information about the structure of the lipid coating itself. The images do not distinguish between a bilayer coating structure and that of adsorbed vesicles. Repeated rinsing of the scaffolded vesicles is conducted to remove extraneous unfused and/or unbound vesicles, evidenced by a dark fluorescence background. However, continued rinsing and agitation result in the progressive loss of scaffolded vesicle fluorescence intensity, suggesting damage to the lipid coating. Permeability Studies. A membrane permeability assay adapted from Rausch et. al.47 was used to probe the permeability of the scaffolded vesicles. The permeability assay exploits the fluorescence characteristics of the terbium(III) cation. An aqueous Tb3+ solution is not strongly fluorescent, but upon forming a complex with dipicolinic acid (DPA), Tb3+ displays a strong green fluorescence. Both Tb3+ and DPA are membrane impermeable. By encapsulating Tb3+ in the scaffolded vesicle interior and adding DPA to the external solution, we can study the permeability of the membrane by monitoring the fluorescence of the external solution. A highquality membrane would act as a diffusion barrier, preventing the formation of the fluorescent complex. A low-quality 10864

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bare hydrogel, the further decrease after 12 days demonstrated that a relatively high coverage (θ) was certainly possible. The coverage was estimated using the following relation θ=1−

kθ kθ= 0

(2)

where kθ, Cθ, kθ=0, and Cθ=0 are the rate constants and final concentrations of the scaffolded vesicle and uncoated hydrogel, respectively.58 Quantitative ATR-IR Spectroscopic Molecular Orientation Studies. The permeability data presented in Figure 4 show that the membrane permeability is at a low near 20 °C, which is below the main phase transition temperature. This suggests that the quality of the membrane is relatively high. Therefore, the ATR-IR experiments were performed at ∼20 °C, where the bilayer is in the gel state. The C−H stretching region is located between 3000 and 2800 cm−1 and contains information regarding the molecular conformation and orientation of the DMPC acyl chains. To avoid spectral interference from C−H stretching vibrations present in cholesterol and Sephadex G10, DMPC with deuterated acyl chains is used. DMPC-d54 displays C−D stretching vibrations between 2300 and 2000 cm−1. This region contains the molecular conformation and orientation information for the deuterated acyl chains and is free of interference from overlapping bands. Figure 6 shows a Fourier self-deconvolution and band fitting of the C−D stretching region for DMPC-d54. The symmetric

Figure 4. Permeability rate constants (k) as a function of temperature for 70:30 DMPC/cholesterol−Sephadex G10 (black) and DPPC− Sephadex G10 (blue) scaffolded vesicles. Both scaffolded vesicle systems display maxima in their leakage rate constants at their respective main phase transition temperatures (∼23 °C for DMPC and ∼41 °C for DPPC). The vertical error bars represent the standard deviation of the measurement.

scaffolded vesicle permeability characteristics mirrored those expected for lipid vesicles of the same composition. Furthermore, the data suggest that the limiting factor dictating the rate of leakage was the lipid membrane coating because at T > Tm the rate constants decreased dramatically. This trend lended support to the main hypothesis that vesicles ruptured on the hydrogel surface forming supported lamellae, covering and possibly spanning its pores. Because these temperature profiles are characteristic of a phospholipid lamella, these results suggest that the lipid coating of the scaffolded vesicles has a lamellar bilayer structure, as opposed to a film of adsorbed vesicles. An improvement in the phospholipid coating is demonstrated in Figure 5. Approximately 12 days after the initial mixing of the scaffolding hydrogel with phospholipid vesicles, a significant decrease in the rate constant (k) of Tb3+ leakage was observed. Although the rate constant was significantly decreased after the initial preparation compared to that of the

Figure 6. Fourier self-deconvolution of the C−D stretching region for 70:30 DMPC-d54/cholesterol scaffolded vesicles. The symmetric CD2 and CD3 and asymmetric CD2 and CD3 stretching bands appear in this region, as do two Fermi resonance (FR) bands.

and asymmetric methyl stretching vibrations [νs(CD3) and νas(CD3), respectively] are located at ∼2150 and ∼2215 cm−1, respectively. The symmetric and asymmetric methylene stretching vibrations [νs(CD2) and νas(CD2), respectively] are located at ∼2090 and ∼2195 cm−1, respectively. Two Fermi resonance bands located at ∼2210 and ∼2070 cm−1 are also present. The νs(CD2) and νas(CD2) bands contain the conformation and orientation information and will be the focus of this analysis. The peak maxima of the νs(CD2) and νas(CD2) bands are very well-defined and may be used to estimate the conformational order of the acyl chains. The frequencies of these methylene vibrations are sensitive to the degree of trans− gauche isomerization for the acyl chains. The νs(CD2) and ν as (CD 2 ) band positions for the all-trans acyl chain

Figure 5. Rate constant (k) of efflux of Tb3+ from the scaffolded vesicle measured over 23 days after the initial preparation. The decrease in the rate constant implied that the surface coverage (θ) improved with time in which the hydrogel beads were incubated in the vesicle solution. 10865

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conformation are 2088 and 2191 cm−1, respectively.48 In this work, the νs(CD2) and νas(CD2) band frequencies are observed at 2090 and 2195 cm−1 (±1 cm−1), respectively. The observed higher-frequency values suggest that the acyl chains are partially melted, containing both trans and gauche conformers. This is consistent with the presence of cholesterol in the lipid coating.48 The intensity of an IR band depends on the angle between the vectors associated with the transition dipole moment of a given vibration and the electric field of the incident light. This implies that the IR band intensity is inherently sensitive to molecular orientation. In an ATR-IR experiment, to extract quantitative molecular orientation information for an absorbing transition dipole moment, the direction and amplitude of the electric field at the interface must be known. The direction of the electric field is controlled by using linearly polarized light. When the penetration depth of the evanescent wave is much greater than the film thickness, the amplitude of the three spatial components of the electric field (Ex, Ey, and Ez) may be found using the thin film approximation.49 The electric amplitudes for the x, y, and z spatial directions, at the interface in the rarer medium, are found by the following expressions:49 Ex =

Ey =

chain. Therefore, each of these bands may be independently used to find the average tilt angle of the trans segments of the acyl chains, with respect to the surface normal, by the following expressions.43,49,50 Schain =

(3)

(4)

dp =

2

Ez =

2n32 sin θ1 cos θ1 (1 −

n312)1/2 [(1

+

n312)

2

sin θ1 −

n312]1/2

θdipole = cos−1

(9)

λ 2πn1 sin θ1 − n212

IRE (n1) Ge (4.0) ZnSe (2.44) ZnSe (2.44) ZnSe (2.44)

2

(10)

critical angle (deg)

incident angle (deg)

penetration depth (μm)

19 33

45 55 45 39

0.30 0.53 0.76 1.27

a

The penetration depths are calculated using an incident wavelength of 4.65 μm, which corresponds to the central frequency of the C−D stretching region.

As the penetration depth of the evanescent wave is decreased, the planar approximation should become increasingly valid. If the planar approximation is valid for the range of penetration depths studied, the apparent tilt angle of the acyl chains should remain constant. Conversely, if the planar approximation is not entirely valid, the measured tilt angle will change as the penetration depth is varied. At larger penetration depths, the apparent tilt angle would be increasingly shifted away from its true angle, toward the random angle (54.7°). As the penetration depth is decreased, the apparent acyl chain tilt angle should approach the true acyl chain tilt angle. It should be stressed that the acyl chain tilt angle is not a function of the penetration depth. Rather, varying the penetration depth merely changes how the system is observed.

(6)

2Sdipole + 1 3

2Schain + 1 3

Table 1. Experimental Parameters Used To Obtain the Four Different Penetration Depths Used in This Worka

Ex 2 − REy 2 + Ez 2 Ex 2 − REy 2 − 2Ez 2

(8)

where λ is the incident wavelength. The penetration depth was varied by changing the incident angle (θ1) and internal reflection element (n1). Four different penetration depths were used in this study. The studied penetration depths and corresponding experimental parameters are listed in Table 1.

(5)

where θ1 is the incident angle, n31 = n3/n1, n32 = n3/n2, and n1− n3 are the refractive indices of the first, second, and third media, respectively. The x−z plane is defined as the incident plane. The electric field vector for p-polarized light contains x and z contributions and lies in the incident plane. The electric field vector for s-polarized light is in the y-direction, perpendicular to the incident plane. The dichroic ratio (R) is the ratio of the absorbance of p- and s-polarized light (R = Ap/As). For planar thin films deposited directly on an internal reflection element, the dichroic ratios measured experimentally and the electric field amplitudes are calculated from the experimental parameters. For a given transition dipole moment, an order parameter (Sdipole) and orientation angle (θdipole), with respect to the surface normal, may then be determined as shown.43,49,50,51 Sdipole =

cos2 α − 1)(Ex 2 − REy 2 − 2Ez 2)

An isotropically oriented acyl chain will display Schain = 0 and θchain = 54.7°. Deviations in planarity will shift the observed orientation angle toward 54.7°. To gain quantitative information regarding the orientation of the DMPC-d54 acyl chains, the scaffolded vesicle coating is approximated as appearing planar to the ATR-IR experiment. This planar approximation is made due to the large radius of curvature of the hydrogel scaffold (∼100 μm) compared to the penetration depth of the evanescent wave (∼1 μm) and the thickness of a DMPC and cholesterol bilayer (∼5 nm). The validity of the planar approximation was tested by performing variable penetration depth experiments on the scaffolded vesicles. The penetration depth of the evanescent wave is defined as the distance from the interface at which the electric field becomes e−1, its value at the interface, and is described by the following expression.49

2 cos θ1 (1 − n312)1/2

1 (3 2

θchain = cos−1

2 cos θ1(sin 2 θ1 − n312)1/2 (1 − n312)1/2 [(1 + n312) sin 2 θ1 − n312]1/2

Ex 2 − REy 2 + Ez 2

(7)

If the direction of the transition dipole moment may be related to a molecular axis by an angle, α, the orientation of the molecular axis may be found as shown.43,49,50 This work examines the orientation of the DMPC-d54 acyl chains in the scaffolded vesicle coating. The vectors associated with the transition dipole moments of the νs(CD2) and νas(CD2) bands both lie in the plane defined by an acyl chain methylene group. Both of these vectors are oriented perpendicular (α = 90°) to the molecular axis defined by the trans segment of the acyl 10866

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Figure 7. Polarized ATR-IR spectra of 70:30 DMPC-d54/cholesterol scaffolded vesicles, at each penetration depth: (a) 1.27, (b) 0.76, (c) 0.53, and (d) 0.30 μm. The p- and s-polarized traces are indicated.

segments of the acyl chains (θchain), with respect to the surface normal. For each of the studied penetration depths, there is close agreement between the θchain values determined from the symmetric and asymmetric bands. Alternatively, θνs and θνas may be used together to find the average θchain. The transition dipole moments of the symmetric and asymmetric CD2 stretching vibrations are mutually orthogonal to the vector defined by the trans segment of the acyl chains. Therefore, the following trigonometric relationship may be used to find θchain.57

Figure 7 shows the C−D stretching region for both the pand s-polarizations, at each of the studied penetration depths. Table 2 lists the dichroic ratios and corresponding molecular Table 2. Molecular Orientation Dataa dp (μm)

vibration

R

0.30

νs(CD2) νas(CD2) νs(CD2) νas(CD2) νs(CD2) νas(CD2) νs(CD2) νas(CD2)

0.97 1.01 0.85 0.82 0.89 0.94 1.10 1.12

0.53 0.76 1.27

θdipole (deg) 77 74 72 73 69 68 64 64

± ± ± ± ± ± ± ±

4 4 3 3 2 2 1 1

θchain (deg)

θavg (deg)

± ± ± ± ± ± ± ±

21 ± 4

19 22 26 24 30 32 38 38

4 4 3 3 2 2 1 1

25 ± 3

cos2 θνs + cos2 θνas + cos2 θchain = 1

31 ± 2

(11)

Figure 8 shows the apparent θchain as a function of penetration depth. The presented angles are averages of those found from the νs(CD2) and νas(CD2) bands. The average apparent θchain values were found to be 38°, 31°, 25°, and 21° for penetration depths of 1.27, 0.76, 0.53, and 0.30 μm, respectively. These apparent θchain values decrease when the penetration depth decreases. Although the data display a quasi-linear dependence of the tilt angle on the distance, the last two lowest values are equal within the experimental uncertainty. Therefore, we consider the lowest value to represent the average tilt angle of chains in the scaffolded vesicles. The behavior of the apparent θchain with respect to penetration depth provides important information about the nature of the scaffolded vesicle coating. First, a scaffolded vesicle coating consisting of adsorbed vesicles would yield a θchain value near the random angle (54.7°). The measured θchain at each penetration depth lies well away from 54.7°, which precludes an adsorbed vesicle coating structure, and suggests an oriented bilayer. Furthermore, the θchain values progressively decrease as the penetration depth decreases and the planar

38 ± 1

R is the dichroic ratio. θdipole and θchain are the average tilt angles of the vectors associated with the transition dipole moment and hydrocarbon chain axis, respectively. θavg is the average tilt angle of the hydrocarbon chain axis as determined from the νs(CD2) and νas(CD2) absorption bands. a

orientation data for each penetration depth. The observed ordering of the νs(CD2) and νas(CD2) transition dipole moments increases as the evanescent wave penetration depth decreases. The apparent θνs increases from ∼64° to ∼77° as the penetration depth is decreased from ∼1.27 to ∼0.30 μm. Similarly, the apparent θνas increases from ∼64° to ∼74°. θνs and θνas may be used further to determine the average tilt and twist of the acyl chains. The νs(CD2) and νas(CD2) bands may each be used independently to find the average tilt angle of the trans 10867

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Figure 8. Average apparent tilt angles of the hydrocarbon chains. The vertical error bars indicate the standard deviation in the measurement. The dashed line shows the angle of random orientation, 54.7°. The gray box shows the range of acyl chain orientations found in the literature for planar DMPC and DMPC/cholesterol bilayers.54−56

Figure 9. Acyl chain twist angle with respect to evanescent wave penetration depth. The dashed line at 45° indicates the trivial twist angle, which corresponds to no preferential twisting of the acyl chains.

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SUMMARY AND CONCLUSIONS Fluorescence microscopy images revealed a clear association between the lipids and hydrogel scaffold. The permeability studies showed that the scaffolded vesicle coating acts as a permeability barrier. The observed maximum in permeability at the main phase transition temperature suggests a lamellar bilayer structure for the scaffolded vesicle coating. The ATR-IR results showed that the orientation of the DMPC-d54 acyl chains was found to be consistent with planar bilayer systems, at short penetration depths. This both suggests a lipid bilayer structure for the scaffolded vesicle coating and supports the validity of the planar approximation at the shorter penetration depths. The testing and subsequent validation of the planar approximation allows for the determination of quantitative molecular orientation data from polarized ATR-IR spectra of the spherical scaffolded vesicles. This represents an important step in the development of a tandem ATR-IR and patch-clamp analytical instrument for the study of scaffolded vesicles.

approximation becomes increasingly valid. The average θchain values observed for the two shorter penetration depths were 21° and 25°. Polarized ATR-IR studies of hydrated multibilayer DMPC films supported directly on an internal reflection element have yielded hydrocarbon chain tilt angles of ∼25°, at temperatures just below the main phase transition temperature of DMPC.54,55 DMPC-d54 films with 25 mol % cholesterol have been found to display acyl chain tilt angles of ∼20°, at temperatures just below the main transition temperature.56 These results are consistent with those found in our study. This both supports the validity of the planar approximation (at the shorter penetration depths) and suggests a bilayer structure for the scaffolded vesicle coating. For a given penetration depth, θνs and θνas are the same within experimental error. This suggests that there is no preferential twisting of the acyl chains. The twist angle (θtwist) of the trans segment of the chain is defined as the angle between the −C− C−C− plane and the plane formed by the chain axis and the surface normal.52,53 The −C−C−C− plane bisects the deuterated methylene group; the νs(CD2) and νas(CD2) transition dipole moments are perpendicular to one another, and θνs and θνas are defined with respect to the surface normal. As a result of these trigonometric relationships, the twist angle of the acyl chains may be found by the following expression:52,53 ⎛ cos θν ⎞ as ⎟⎟ θtwist = tan−1⎜⎜ cos θ ⎝ νs ⎠

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a NSERC Discovery grant. J. Lipkowski acknowledges the Canada Foundation for Innovation for Canada Research Chair Award.

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REFERENCES

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Figure 9 shows a plot of the acyl chain twist angles with respect to penetration depth. The θtwist values were found to be 51°, 43°, 46°, and 45° for penetration depths of 0.30, 0.53, 0.76, and 1.27 μm, respectively. A twist angle of 45° is termed the trivial twist angle and corresponds to no preferential acyl chain twisting.52,53 At each penetration depth, the twist angle is close to the trivial twist angle, suggesting no preferential twisting of the DMPC-d54 acyl chains. 10868

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