Article pubs.acs.org/Langmuir
Structural Features of Micelles of Zwitterionic Dodecylphosphocholine (C12PC) Surfactants Studied by Small-Angle Neutron Scattering Elias Pambou,† John Crewe,† Mohammed Yaseen,† Faheem N. Padia,† Sarah Rogers,‡ Dong Wang,§ Hai Xu,§ and Jian R. Lu*,† †
Biological Physics Group, School of Physics and Astronomy, University of Manchester, Oxford Road, Manchester M13 9PL, United Kingdom ‡ STFC ISIS Facility, Rutherford Appleton Laboratory, Didcot OX11 0QX, United Kingdom § Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266555, China S Supporting Information *
ABSTRACT: Small-angle neutron scattering (SANS) was used to investigate the size and shape of zwitterionic dodecyl phosphocholine (C12PC) micelles formed at various concentrations above its critical micelle concentration (CMC = 0.91 mM). The predominant spherical shape of micelles is revealed by SANS while the average micellar size was found to be broadly consistent with the hydrodynamic diameters determined by dynamic light scattering (DLS). Cryogenic tunneling electron microscopy (cryo-TEM) shows a uniform distribution of structures, proposing micelle monodispersity (Supporting Information). H/D substitution was utilized to selectively label the chain, head, or entire surfactant so that structural distributions within the micellar assembly could be investigated using fully protonated, head-deuterated, and tail-deuterated PC surfactants in D2O and fully deuterated surfactants in H2O. Using the analysis software we have developed, the four C12PC contrasts at a given concentration were simultaneously analyzed using various core− shell models consisting of a hydrophobic core and a shell representing hydrated polar headgroups. Results show that at 10 mM, C12PC micelles can be well represented by a spherical core−shell model with a core radius and shell thicknesses of 16.9 ± 0.5 and 10.2 ± 2.0 Å (total radius 27.1 ± 2.0 Å), respectively, with a surfactant aggregation number of 57 ± 5. As the concentration was increased, the SANS data revealed an increase in core−shell mixing, characterized by the emergence of an intermediate mixing region at the spherical core−shell interface. C12PC micelles at 100 mM were found to have a core radius and shell thicknesses of 19.6 ± 0.5 and 7.8 ± 2.0 Å, with an intermediate mixing region of 3.0 ± 0.5 Å. Further reduction in the shell thickness with concentration was also observed, coupled with an increased mixing of the core and shell regions and a reduction in miceller hydration, suggesting that concentration has a significant influence on surfactant packing and aggregation within micelles.
1. INTRODUCTION Because of their amphiphilic nature, surfactants can undergo surface and interfacial adsorption when dissolved in aqueous solution. Above their critical micellar concentrations (CMC), they can also aggregate to form micelles, with the hydrophobic tails making up the core and hydrophilic headgroups forming the shell. Micelles can adopt a variety of shapes, e.g., cylindrical, elliptical, and spherical. The exact size and shape of micelles formed depend on the molecular structure of the surfactant concerned, e.g., alkyl chain length and type of headgroup. Environmental conditions such as temperature, ion type, and concentration could also affect the micellar structure and their influencing forces, e.g., van der Waals, hydration, and electrostatic screening. As a soluble surfactant, dodecyl phosphocholine (C12PC) is able to mimic some features of natural dichain phosphocholine lipids, and it is frequently used as a membrane mimic in peptide © 2015 American Chemical Society
or drug-delivery systems. C12PC aggregation and its interaction with other molecules can be readily studied by a wide range of techniques established in colloid and interface science. The accurate determination of its structural parameters using established techniques could be useful for a number of structural studies.1,2 Different from nonionic and ionic surfactants, C12PC is zwitterionic and its surface adsorption and solution aggregation properties are generally less well understood. We have previously studied its surface adsorption using surface tension and neutron reflection, with the results indicating that C12PC and its homologues combine useful features from both nonionic and ionic surfactants. The CMCs of C12PC Received: June 6, 2015 Revised: August 24, 2015 Published: August 24, 2015 9781
DOI: 10.1021/acs.langmuir.5b02077 Langmuir 2015, 31, 9781−9789
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Langmuir
2. EXPERIMENTAL SECTION
homologues and their surface tension are little affected by the addition of salt - a feature common to nonionics. Differing from nonionics, however, its adsorption profile shows a reluctant response to temperature or pH changes, a feature common to most ionic surfactants such as SDS and C12TAB.3 Unsurprisingly, some of these features are reportedly shared by betaine surfactants,4,5 though sulfo-betaines and carboxy-betaines have some of their own characteristics that are not equally shared by the single-chain phosphocholine surfactants. The aim of this work stems from the interest in further understanding the aggregation of C12PC. In addition to being exploited in drug delivery, single-chain phosphocholine surfactants are also used in membrane protein purification and stabilization, biosensors, and other biotechnologies where biocompatible surfactants are required.6−11 Most of these applications exploit the binding and solubilization of the PC surfactants that are affected by their solution aggregation. Previous studies have examined C12PC aggregation by DSC (Differential scanning calorimetry), NMR, and computer modeling,12−17 which will be compared to our current study. However, published results using these techniques indicate large variations in micelle size and shape, illustrating the current lack of understanding of C12PC’s structural features. In this present work we have used dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryo-TEM), and small-angle neutron scattering (SANS) in combination with the isotopic (H/D) substitution of surfactant molecules to achieve a better structural determination of the C12PC micelle aggregates formed and to observe changes in their size and shape with increasing C12PC concentration. SANS has been extensively used for determining the size and shape of surfactant micelles under various conditions, particularly for ionic and nonionic surfactants and their mixtures.18−25 It has, however, never been used for C12PC or its homologues. SANS is more advantageous compared to other techniques used to elucidate or analyze complex structures on the nanometer scale as isotopic substitution can be exploited to alter a structure’s overall neutron scattering length density. As discussed by Yaseen et al.26 via isotopic substitution, surfactants can be partially or fully deuterated to exclusively investigate the distribution of C12PC alkyl chains or headgroups by contrast matching them against the rest of the micelles or bulk solution. Via the same principle, substitution can also be exploited to determine the extent of solvent penetration into a surfactant aggregate’s head and chain regions. In order to analyze the C12PC structure using SANS, a MATLAB-based software program was developed to iteratively fit the experimental data using a variety of geometrical models. The software itself is explicitly designed to model simultaneous fittings to several independent data sets measured from a particular concentration of C12PC but under different isotopic contrasts. The ability to simultaneously fit data is an important analytical tool, as treating the measured data sets individually in order to investigate the structures of surfactant micelles could reduce accuracy and reliability and hinder global assessment. Simultaneous fitting of data has been shown by numerous papers to improve the confidence of studies investigating micelles, mixed micelles, and polymer−surfactant aggregates, among other structures.27,28 The success of the mathematical models and validity of the approach were tested by comparing the obtained structural information with data obtained using alternative techniques and other common SANS software.
SANS experiments were conducted at the ISIS Neutron Facility on the time-of-flight LOQ diffractometer with a Q range of 0.009−0.250 Å−1. Measurements were performed in a 2 mm quartz cell (1 mm for H2O samples) using a 12-mm-diameter beam. The temperature for all experiments was kept constant at 295 K. On completion of each run for each set of data, the corresponding D2O or H2O (solvent) scattering profiles were subtracted from beam intensities for background correction. Dynamic light scattering (DLS) was used to provide support to the results obtained using SANS. Similar to the methodology of Padia et al,28 measurements were performed on a Malvern Instruments Zetasizer Nano using a 173° backscattering angle. Surfactant samples were loaded in standard quartz cells and were left to equilibrate for 10 min prior to the measurements at a constant temperature of 20 °C (293 K). For each sample, 5 measurements were taken consisting of 15 runs each, and the micelle radius was calculated by determining the average of each of the 5 measurements. Each run lasted 30 s. Cryo-TEM samples were prepared in a controlled-environment vitrification system (CEVS). A micropipette was used to load 5 μL of solution onto a TEM copper grid, which was blotted with two pieces of filter paper, resulting in the formation of thin films suspended on the mesh holes. After waiting for 2 s, the samples were quickly plunged into a reservoir of liquid ethane (cooled by nitrogen) at −165 °C. The vitrified samples were then stored in liquid nitrogen until they were transferred to a cryogenic sample holder (Gatan 626) and examined with a JEOL JEM-1400 PLUS TEM (120 kV) at about −174 °C. The phase contrast was enhanced by underfocus. The images were recorded on a Gatan multiscan CCD and processed with Digital Micrograph software.
Figure 1. Basic structure of dodecyl phosphocholine (C12PC).
The synthesis and purification of C12PC surfactants have been described in our previous work.29 In summary, the synthesis involved a two-step procedure. First, hydrogenated or deuterated n-dodecanol was reacted with 2-chloro-2-oxo-1,3,2-dioxaphospholane (20% molar excess) to afford the product 2-n-alkyl-2-oxo-1,3,2-dioxaphospholane. This intermediate was filtered to remove the amine salt and then purified through a silica flash column (40−60 mesh from Fluka) with petroleum ether and ethyl acetate in a 1:1 mixture. Subsequent removal of the solvent led to an intermediate product as a yellow oil. The intermediate product was then freeze-dried and reacted in a sealed thick-walled pressure tube with trimethylamine (in excess) at 75 °C for 24 h to afford the n-dodecyl phosphocholine product. The final product was purified by flash silica column chromatography initially using methanol as the mobile-phase solvent. The product was a white solid powder with a typical yield of 75%. The syntheses of the head deuterated and fully deuterated (hCndPC and dCndPC) PC surfactants have also been described previously.29 In this case, n-dodecanol was reacted with phosphoryl trichloride (in excess) and stirred for 2 to 3 h. To this mixture, an excess of 2-chloro ethanol was added to form the phosphonate ester. To the purified and dried intermediate, trimethylamine (in excess) was then added in a sealed tube and left to react for 24 h at 75 °C to form the product. The final products were purified similarly using a silica column and characterized using 1H and 31P NMR. The purity of the products and their stability against time have also been characterized previously using surface tension measurements.29,30 9782
DOI: 10.1021/acs.langmuir.5b02077 Langmuir 2015, 31, 9781−9789
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Langmuir
3. SANS DATA ANALYSIS The scattering intensity profile obtained for monodisperse samples is represented by I(Q ) = NP(Q ) S(Q ) + B
between the micelle and solvent. Similar to eq 4, the solid elliptical form factor can be extended to describe a core−shell ellipsoid. Likewise, for homogeneous triaxial shapes, the form factor is given by
(1)
P(q) = (ΔρV )2
where Q represents the momentum transfer for N scatterers, P(Q) is the particle form factor which describes the neutron scattering that occurs from any one micelle, and S(Q) is the structure factor representing intermicellar interference effects. Among the most popular and common models that can be used to approximate the intermiceller structure factor is the analytical Percus−Yevick (PY) hard-sphere model31 for weakly anisotropic interacting micelles. The Percus−Yevick function defines a micelle’s effective hard-sphere interaction radius in order to determine the interparticle interference contribution to the total measured scattering intensity. At concentrations where the micelle solution is sufficiently dilute (
