Petru
Niga
*a,
Petra M.
Hansson-Mille
a,
Agne
Swerin
ab,
Per M.
Claesson
ab,
Joachim
Schoelkopf
c,
Patrick A. C.
Gane
cd,
Jing
Dai
e,
István
Furó
e,
Richard A.
Campbell
fg and
C. Magnus
Johnson
*b
aRISE Research Institutes of Sweden – Chemistry, Materials and Surfaces, Box 5607, SE-114 86 Stockholm, Sweden. E-mail: petru.niga@ri.se
bKTH Royal Institute of Technology, Department of Chemistry, Division of Surface and Corrosion Science, SE-100 44 Stockholm, Sweden. E-mail: magnusj@kth.se
cOmya International AG, Baslerstrasse 42, CH-4665 Oftringen, Switzerland
dAalto University, School of Chemical Technology, Department of Bioproducts and Biosystems, FI-00076 Aalto, Helsinki, Finland
eKTH Royal Institute of Technology, Department of Chemistry, Division of Applied Physical Chemistry, SE-100 44 Stockholm, Sweden
fInstitut Laue-Langevin, 71 Avenue des Martyrs, CS20156, 38042 Grenoble Cedex 9, France
gDivision of Pharmacy and Optometry, University of Manchester, Manchester M13 9PT, UK
First published on 8th November 2018
Propofol is an amphiphilic small molecule that strongly influences the function of cell membranes, yet data regarding interfacial properties of propofol remain scarce. Here we consider propofol adsorption at the air/water interface as elucidated by means of vibrational sum frequency spectroscopy (VSFS), neutron reflectometry (NR), and surface tensiometry. VSFS data show that propofol adsorbed at the air/water interface interacts with water strongly in terms of hydrogen bonding and weakly in the proximity of the hydrocarbon parts of the molecule. In the concentration range studied there is almost no change in the orientation adopted at the interface. Data from NR show that propofol forms a dense monolayer with a thickness of 8.4 Å and a limiting area per molecule of 40 Å2, close to the value extracted from surface tensiometry. The possibility that islands or multilayers of propofol form at the air/water interface is therefore excluded as long as the solubility limit is not exceeded. Additionally, measurements of the 1H NMR chemical shifts demonstrate that propofol does not form dimers or multimers in bulk water up to the solubility limit.
In spite of the importance of interfacial properties of alcohol/water mixtures in a wide variety of applications9,10 the study of such interfaces remains complex, mainly due to their dynamic nature and the lack of surface-specific experimental techniques. The development of nonlinear optical methods has contributed to overcoming some of these problems and facilitated better molecular understanding of such interfaces. Specifically, vibrational sum frequency spectroscopy (VSFS) has proven to be a powerful technique for studying a wide variety of aqueous interfaces11 due to its capability of yielding surface-specific information at a molecular level.
Different types of alcohols have been studied using VSFS with respect to the stretching vibrations of the hydrocarbon chains,11–14 the fingerprint region15 and the water of hydration.16–20 It was found that for a series of increasing chain length alcohols (C1–C8), at the neat air/alcohol interface, the tails point out into the gas phase due to the amphiphilic character of alcohols, and that the presence of gauche defects is chain length dependent. At the same time a very well ordered interfacial hydrogen bonding network was detected.21 Alcohols with different chain lengths have been shown to affect the surface water structure in different ways regarding hydrogen bonding strength and the orientation of the water molecules.20 Indeed in mixtures with water, glycerol was found to partition to the interface with air, whereby disturbing the topmost water layers.18 At a hydrophobic solid surface, methanol adsorbed from its mixture with water to the interface with the C–O bond aligned to the surface normal while the water band at 3200 cm−1 weakened in strength with increasing methanol concentration.16
The knowledge of small hydrophobic but weakly amphiphilic drugs at the interface is rather limited compared with the more amphiphilic molecules. Amphiphilic molecules such as surfactants,22 proteins,23 block co-polymers24 and peptides25 have been the subject of extensive investigations. By contrast there is a lack of body of work on the surface properties of small hydrophobic drugs. For example, for ibuprofen there has been a study on its interactions with lipid bilayers26 and cholesterol27 but to our knowledge not on its adsorption properties at the air/water interface. A recent study looked at the interactions of testosterone enanthate with surfactants at the air/water interface,28 but again to our knowledge the surface properties of the drug alone have not been examined explicitly. Further, for propofol the present authors have conducted a study on its interactions with phospholipid monolayers29 but an analogous study of its adsorption properties at the air/water interface is missing. To address this shortcoming, we have decided to study its adsorption at the air/water interface.
Propofol is a small alcohol that contains both hydrophobic and hydrophilic parts and has interesting biochemical properties, e.g., it is used commonly in general anaesthesia to induce a state of reduced consciousness in patients during medical procedures.30–32 Similar in structure to propofol, phenol has been briefly characterised by VSFS at the air/water interface,15 by focusing on only the carbon–oxygen stretching region of the spectrum. It was found that phenol is present at the interface at both low and high pH, and at high pH there are both phenol and phenolate ions. Surprisingly however, according to our knowledge, there are no detailed studies of propofol at the air/water or water/hydrophobic interface despite its fundamental interest as a small amphiphilic drug with important biomedical applications.
In the present work we evaluate the adsorption isotherm from surface tension data, and the adsorbed amount is also determined using neutron reflectometry (NR). NR and VSFS data are then combined to gain structural information on the adsorbed layer and its interaction with water. We also report a bulk study of the chemical shifts of hydrogen nuclei over the entire solubility range of propofol in water in order to detect possible multimeric structures. The results represent a necessary first step in resolving the driving forces for the interactions of propofol with interfaces, and they provide complementary information to that which can be obtained from other studies involving its interactions in more complex systems (including supported lipid bilayers33,34 and membrane proteins35,36) that are closer in nature to those in practical applications of the drug.
(1) |
The solutions were prepared by dropwise adding liquid propofol to water, then gently shaking and hand warming for a few minutes until the propofol was dissolved. At least 10 min was allowed before the measurements were recorded. For higher concentrations it took longer time for propofol to dissolve completely.
(2) |
(3) |
Two different data acquisition approaches were used to determine: (1) the adsorption isotherm and (2) the interfacial structure of the adsorbed molecules at the air/water interface, following the methodology adopted in ref. 39. In the first case, measurements were made only in ACMW, and the data were reduced only over 4.5–12 Å to restrict the qz-range to 0.01–0.03 Å−1. This approach reduces the sensitivity of the analysis to details of the structure at the interface.40 Note that the surface excess of propofol could be resolved accurately even though it was used in its normal, non-deuterated form thanks to the high flux at low-qz of FIGARO. The interfacial roughness values were fixed to 3.1 Å, consistent with the outcome of the structural analysis (note that for this low-qz analysis even neglect of the roughness values resulted in a change in surface excess of <0.5%). The background was not subtracted from these data in this analysis, but its accurate determination is essential to the precise quantification of the scattering excess: its value was iterated from the lowest value possible where a layer of zero surface excess could be fitted to a measurement of pure ACMW.41
In the second case, for the structural analysis, data were recorded at a propofol concentration of 0.89 mM over a broader qz-range with measurements at θ = 0.623° and θ = 3.78° both in ACMW and D2O. The background was subtracted from these data with the use of the 2D detector on the instrument. A single-layer structural model adequately described the data. In this case the fitting parameters were the thickness of the layer and its volume fraction. The roughness values of the air/propofol and propofol/water interfaces were constrained to be equal to each other and consistent with capillary wave theory, following an approach described recently.42 The data analysis was performed exclusively using Motofit.43
The theory behind VSFS is well described in the literature.45–49 It is a nonlinear optical technique, which is able to provide surface specific information such as the nature and orientation of the species present at an interface. The intensity of the collected beam is proportional to the intensities of the incoming visible and infrared beams and the square of the effective second order nonlinear susceptibility χ(2)eff. χ(2)eff is in turn proportional to the number density multiplied by the orientationally averaged molecular hyperpolarisability 〈β(2)〉 of the probed species. We note that in order for a molecule or part of a molecule to be VSFS active it has to be both IR and Raman active, according to eqn (4):
(4) |
(5) |
Orientational information about the probed molecular species is given by elements of the second order nonlinear susceptibility tensor, specifically by using different polarisation combinations. In this study we have used SSP, PPP, and SPS, where P refers to light polarised parallel to the plane of incidence and S refers to light polarised perpendicular to the plane of incidence. The first letter corresponds to the polarisation of the sum frequency beam, the second to that of the visible beam, and the third to the infrared beam.
Fig. 2 The chemical shifts (see Fig. 1 for peak assignment) recorded in solution of propofol at different concentrations between 0.02 mM and 0.89 mM. A – aromatic H3 and H5, B – aromatic H4, C – CH(CH3)2, and D – CH3. |
The absence of such effects points to the absence of any significant aggregation/dimerisation phenomena. This is in contradiction to what has been inferred in gas-phase where “nano-micelles” containing several multimers of propofol were found.55 In addition, the NMR intensity was proportional to the concentration in the whole range which also excludes the presence of any large aggregates (for which the NMR signal could possibly be lost).
Fig. 3a shows the surface tension isotherm of aqueous propofol solutions. The decrease in the surface tension is due to adsorption of propofol at the air/water interface. The surface tension drops smoothly with concentration up to a concentration of about 1 mM, above which it remains constant. The limiting area per molecule is about 42 Å2, as seen in Fig. 3b. A discussion regarding the structure of the adsorbed propofol layer will follow in the light of the NR results presented next. Details on the area per molecule calculation are given in ESI.†
Fig. 3 (a) Surface tension isotherm of propofol and a second order polynomial fit to the data (line), and (b) area per molecule calculated using the second order polynomial fit and eqn (1). |
The surface excess was measured directly at six different bulk concentrations at low qz values in ACMW, as shown in Fig. 4a. Also shown is the measurement of pure ACMW used to determine the background level. The resulting adsorption isotherm is shown in Fig. 4b, where it is compared to the surface excess obtained from surface tension measurements. We regard the agreement as satisfactory, particularly at high propofol concentrations, considering the different evaluation methods.
Fig. 5 Neutron reflectivity, R, and model fits for propofol solutions at the air/water interface recorded in D2O (red, top) and air contrast matched water (purple, bottom). |
Propofol is considered to be of cylinder-like shape. The total volume of such cylinder, calculated from the inverse bulk density, is 285 Å3. Close packing of such cylinder-like shapes accounts for the van der Waals volume of propofol56 which is 198 Å3, 11% water (from the NR analysis) and about 22% air of the total volume. Let's consider the two extreme cases. If the molecules (thought as cylinders) are considered to stand on their ends, and if the cylinders are considered to have a cross section area of 25 Å2, slightly larger than for straight chain alkanes, then its length would be about 11 Å. The other extreme case would be a cylinder lying down giving an area per molecule of about 60 Å2 and a thickness of about 5–6 Å. Clearly, NR and tensiometry data are consistent with an intermediate situation (area per molecule 40–42 Å2, thickness 8.4 Å), suggesting a tilted orientation of propofol, and consistent at least (see below) with the OH-group towards the aqueous phase (the latter supported by the VSFS data discussed next). We are reluctant to go further and report a mean tilt angle as it would require a specific assumption about the packing of the molecules, which is not known a priori.
For alkyl chains, the aCH3 peak is normally significantly weaker than the sCH3 peak in the SSP polarisation combination due to the chain orientation.44,63,64 However, for propofol the opposite is found. The reason is that the total VSFS signal has contributions from all four closely spaced methyl groups (see Fig. 1), and due to different directions of the transition dipole moments for the symmetric and antisymmetric methyl stretches, their signal strength will be differently enhanced. The same argument rationalises that the antisymmetric methyl stretch is stronger in SSP than PPP, which normally is not observed for alkyl chains. It should be pointed out that in different environments (e.g. without water as solvent) the propofol SSP spectrum (not shown) resembles a typical spectrum from an alkyl chain, which emphasises that the unusual SSP spectrum for propofol at the air/water interface is due to the propofol orientation.
It is interesting that the IR spectrum shown in Fig. 6b shows the Fermi resonance CH3 at 2935 cm−1 which is not clearly observed in the VSFS spectra. The fitted amplitudes of the VSFS peaks are provided in the ESI.† In Fig. 6c the fitted amplitudes of the sCH3 and the aCH3 stretches are presented as a function of propofol concentration. Above 0.2 mM the fitted amplitudes of both peaks approach a plateau, a result that is consistent with the findings from surface tension isotherms, as discussed earlier (Fig. 3). The sCH3/aCH3 fitted amplitude ratio as a function of concentration is presented in Fig. 6d. The fitted amplitude depends both on the average orientation and the number of molecules probed, whereas the sCH3/aCH3 ratio is only affected by the orientation, since the number density is cancelled by taking this ratio.
We note that this ratio is almost constant over the concentration range 0.2–0.89 mM, meaning that the average orientation of the adsorbed propofol is almost constant. The SPS polarisation did not show any CH feature that could be used reliably in the fitting procedure. This, and the fact that there are four methyl groups pointing in different directions that contribute to that signal, prohibits a more detailed orientational analysis. The assignments of vibrational features of propofol in the CH stretching region are presented in Table 1.
Peak (cm−1) | Polarisation | Observed | Assignments | Ref. |
---|---|---|---|---|
2873 | SSP | VSFS/IR | sCH3 | 13, 57 and 65 |
2910 | SSP, PPP | VSFS/IR | CH isopropyl | 66 |
2965 | SSP, PPP, SPS | VSFS/IR | aCH3 | 13, 57, 61 and 62 |
3035 | SSP, PPP | VSFS/IR | arCH | 13 and 57 |
3071 | SSP | VSFS/IR | arCH | 13 and 57 |
Both the SSP and the PPP spectra of water agree with the spectra recorded before,17,44,67 where the most prominent features exhibited in the SSP polarisation are the broad band spanning over several hundred wavenumbers and centred at around 3300 cm−1 as well as the sharp peak at 3704 cm−1.
The latter peak, which is present in both the SSP and PPP polarisation, is assigned to the OH stretching mode of the topmost surface OH bonds of water molecules protruding into air and vibrating free from hydrogen bonding.68 The assignment of the broad band was for a long time a source of debate.69–71 However, it is broadly agreed now that the broad band is assigned to hydrogen bonded water molecules with varying strength and coordination meaning that the OH signal is a signature of a collective vibration of several water molecules.72–75
The SSP polarisation spectrum of a 0.89 mM propofol solution, shown in Fig. 7a, is considerably different from the pure water spectrum. At this propofol concentration, the free OH peak at 3704 cm−1 has essentially vanished, suggesting that none or very few free OH bonds remain. However, a new broad band, centred around 3670 cm−1, appears. A similar band has been observed for several amphiphilic molecules (e.g., decanol, sugar surfactants and alkyl polyethylene glycol surfactants) at the air/water interface, and has been assigned to the OH stretch of ordered water molecules weakly interacting with hydrocarbon moieties.17 This is consistent with the relatively large hydrophobic part of propofol, and suggests direct contact between water and this region of propofol. The small red shift of around 30 cm−1 compared to the free OH vibration indicates that the interaction is weak. We note that since propofol is non-ionic, any significant enhancement of the water signal as observed for ionic amphiphiles76,77 is neither expected nor seen.
In the PPP spectrum in Fig. 7b a band covering nearly the whole OH stretching region is observed, with the maximum intensity around 3600 cm−1. Thus, the maximum is red shifted with around 70 cm−1 in comparison with the SSP spectrum, which exhibits nearly zero intensity at 3600 cm−1. The OH bond populations responsible for the peaks at 3600 cm−1 and 3670 cm−1 possess weak interactions, due to proximity to the hydrophobic moieties of propofol. However, since the two peaks have their maximum at different wavenumbers, the two populations experience dissimilar environments.
An interesting observation is that the right hand side of the broad band, towards 3400 cm−1 is almost completely suppressed in the SSP polarisation combination (Fig. 7a). Instead, a new band centred at about 3175 cm−1 clearly shows up in the spectrum. The fact that this band appears at such low frequency, close to the centre frequency for ice,78,79 suggests that it is associated with interfacial water that experience strong interactions with the hydroxyl part of propofol, similar to what was found for long chain alcohols.20 A similar band, centred at 3150 cm−1 has further been observed for interfacial sugar surfactants possessing –OH groups (C10 maltoside and C10 glucoside) and assigned to OH stretching vibrations of the hydroxyl groups in the sugar rings and their hydration shells.17 In contrast, 1-decanol, which, like propofol, contains a single –OH group and a large hydrophobic moiety, exhibited a band centred at 3250 cm−1, thus indicating relatively weaker hydrogen bonds.
Moreover, the PPP spectrum of 1-decanol showed a broad band extending over the region 3000–3800 cm−1, obviously different from that obtained in presence of propofol. Accordingly, the strength of the hydrogen bonds of water that are hydrating propofol (SSP spectra) is in between those observed with the sugar surfactants and decanol, which indicates that the hydrogen bond strength depends not only on the hydrophilic group, which is the same for decanol and propofol, but also the surrounding hydrophobic environment.20
The freedom of the molecules to rotate or move around may also influence the strength of the hydrogen bond. Propofol has a larger area per molecule then decanol17 and therefore has less constraints in adopting a position that allows for stronger hydrogen bonds to be formed.
We have identified different water populations that hydrate propofol at the air/aqueous solution interface. Strong hydrogen bonds, similar to those found for the tetrahedrally-coordinated hydrogen bonding in ice, are formed between water and the OH-group of propofol. These hydrogen bonds are stronger than those found between water and decanol, but weaker than those found next to sugar surfactants. Thus, the hydrogen bond strength does not only depend on the hydrophilic group, which is the same for decanol and propofol, but also on the surrounding hydrophobic environment and the ability to adopt conformations that allow formation of strong hydrogen bonds.
Effective molecular information regarding the arrangement and hydration of propofol at the air/water interface opens the door for a more extensive examination of the interfacial properties of propofol, especially at the buried membrane/water interfaces where this may have important implications for understanding the driving forces for the underlying interactions of drugs in model systems, and they set the stage for us and others to progress with studies of the interactions of propofol and other small model drugs with more complex interfacial morphologies.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sm01677a |
This journal is © The Royal Society of Chemistry 2019 |