Determination of phase transition temperatures of lipids by light scattering

Chemistry and Physics of Lipids 139 (2006) 11–19

Determination of phase transition temperatures of lipids by light scattering Nicolas Michel a , Anne-Sylvie Fabiano a,∗ , Ange Polidori a , Robert Jack b , Bernard Pucci a,∗∗ a

b

Laboratoire de Chimie Bioorganique et des Syst`emes Mol´eculaires Vectoriels, Facult´e des Sciences, 33 Rue Louis Pasteur, 84000 Avignon, France Malvern Instruments Ltd., Enigma Business Park, Grovewood Road, Malvern, Worcestershire WR14 1XZ, UK Received 17 June 2005; received in revised form 5 September 2005; accepted 16 September 2005 Available online 12 October 2005

Abstract Various techniques have been proposed to specify the phase transition temperatures of surfactant molecules. The work reported herein deals with a new general method of Tc determination based on the optical properties’ modifications of aqueous surfactant solutions when the phase transitions occur in the phospholipid membrane. The shape alteration of supramolecular systems induced by the phase transition was correlated with the refraction and absorption coefficients of their aqueous dispersion. The mean count rate (average number of photons detected per second) measured with a Zetasizer Nano-S model ZEN1600 Dynamic Light Scattering Instrument, is representative of an emerging macroscopic phenomenon, but not directly size dependent and has been adapted to our expectations. Changes in the measured scattering intensity reflect changes in the optical properties of the material during temperature variations. Thus, this method allowed to specify the phase transition temperature of many natural or synthetic surfactants independently of their polar head or hydrophobic part. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Phase transition temperature; Surfactants; Light scattering; Non-invasive backscattering®

1. Introduction Determination of the phase transition temperatures of surfactant molecules is of crucial interest in most fields of supra-molecular studies and applications (Grigoriev et al., 2003; Toombes et al., 2002; Walde et al., 1997; Baranyai et al., 1999; Alexandridis and Holzwarth, 1997; ∗ Corresponding author. Tel.: +33 4 90 14 44 35; fax: +33 4 90 14 44 49. ∗∗ Corresponding author. Tel.: +33 4 90 14 44 42; fax: +33 4 90 14 44 49. E-mail addresses: [email protected] (A.-S. Fabiano), [email protected] (B. Pucci).

Tsuchiya et al., 2004; Jung et al., 2000; Sternin et al., 2001; Trandum et al., 1999). During the last decades pharmacological research has largely focused on the use of surfactant-derived organized systems to carry and release drugs. Various colloidal drug carriers such as micellar solutions, liquid crystal dispersions, hydrogels, vesicles or liposomes, whose properties depend entirely upon their phases, supramolecular properties and metastable behaviour, were prepared to obtain systems with optimised drug loading and release properties. In the case of liposomes or vesicles, thermotropic lipid phase transitions in biological media are correlated to discontinuities or anomalies in macroscopic physical properties such as the stability, fluidity or permeability

0009-3084/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2005.09.003

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N. Michel et al. / Chemistry and Physics of Lipids 139 (2006) 11–19

(Mouritsen and Jorgensen, 1995) of membranes which are closely dependent – among other parameters – on their phase transition temperature. A liposome becomes highly permeable near the gel to crystalline phase transition temperature of its membrane (Papahadjopoulos et al., 1973). Thus, for instance, the use of liposome carriers, or more specifically, the rational design of new liposome models acting as thermosensitive constructions (Yatvin et al., 1978; Kono, 2001), requires knowledge of their behaviour when undergoing modifications in temperature, and so their phase transition temperature (Evans and Needham, 1987). Many techniques (DSC (Nakano et al., 2003; Simon et al., 1975; Biltonen and Lichtenberg, 1993), FTIR (Khalique et al., 1992; Maeda, 2001; Maeda et al., 2001), fluorescence polarization (Hebrant et al., 1997; Van Zandvoort et al., 1997; Nassar et al., 1998; Oliger et al., 2001), NMR (MacDonald and Strashko, 1998; Dimitrova et al., 1996; Siminovitch and Jeffrey, 1981)) have been developed to obtain these values for natural phospholipids or synthetic amphiphiles. However, each of the more than 150 methods described has its own weakness. These methods are far from ideal in the routine study of phase transitions due to the complex nature of the instrumentation and the requirement to use exogenous probes or modified compounds or solvents. These drawbacks have led to the application of static light scattering methods (SLS) based on particle size analyses (Maurer et al., 1994; Nagel et al., 1992; Cao et al., 1991; Azize et al., 1994) and to the development of specific apparatus (Tsuji et al., 2001). However these measurements have generally been used in support of other more classical techniques such as DSC or 31 P NMR (Faucon et al., 1995) to show that these phase transition temperatures entail changes of macroscopic states (shape transition through the second coefficient of Viriel in SLS (Riske et al., 1997) and/or size modifications in Dynamic Light Scattering, DLS). Thus, using the latest generations of NIBS® apparatus, Majhi and Blume studied the micelles-vesicle transitions of DMPC/SDS or DMPC/DTAB mixtures versus temperature through the size modifications induced by the phase transitions of lipids. The results were correlated with DSC measurements (Majhi and Blume, 2002), which avoid the use of external probe or specific solvent. Considering all these results, our studies focused on the adjustment of DLS technique for the phase transition temperature determination without considering shape or size variations of the supramolecular systems studied. We have developed a new interpretation of the light scattering results based on the optical properties’ variations of lipids solutions. Indeed, taking into account that

a phase transition induces modifications in the optical properties of systems, one may expect a correlation with its refraction and absorption coefficients. At critical temperature Tc (pre-transition or main transition temperature), modifications of lateral diffusion, lateral expansibility, bilayer thickness, bending, permeability, etc. occur (Jutila, 2001). These alterations are produced – or at least correlated – to conformational modifications of amphiphilic compounds. Shape alterations can possibly involve size variations (specified by hydrodynamic radius measurements) but this cannot be immediately generalized if we consider fluctuations like oblate to prolate shape transition or vesicular to discotic shape transition for instance. Furthermore, surface modifications of aggregates are not always correlated with the diffusion coefficient which enters into the calculation of the hydrodynamic radius. The mean count rate (average number of photons detected per second), on the other hand, seems to be much more reliable because of its raw sight, simplicity and reproducibility. This value is representative of an emerging macroscopic phenomenon, but not directly size dependent and may be adapted to our expectations: changes in the measured scattering intensity reflect changes in the optical properties of the material. Thus, discontinuity in the mean count rate, as the temperature is altered, corresponds to a change in optical properties of the material studied (i.e. transition from initial state to another one). Thus, if each phase transition of the supramolecular system produced by a variation of the temperature can be easily characterized with this technique, the latter could be considered as an universal method of phase transition temperature determination. In order to validate this theory, we have investigated the different phase transition temperatures of a group of natural lipids. Results are presented in what follows. 2. Experimental 2.1. Materials and method All lipids were 99% purity grade (except for DMPA at 98% purity), purchased from Sigma–Aldrich and used without further purification. The solvents used (methanol and chloroform) were analytical grade. Zetasizer Nano-S model ZEN1600 (633 nm ‘red’ laser – Malvern Instruments) Dynamic Light Scattering Instrument was used to perform measurements. The position of the detector was at 173◦ relative to the laser source (backscatter detection). Closed quartz cuvettes were filled with 45 ␮l samples. Temperature in the cell was monitored by an external

N. Michel et al. / Chemistry and Physics of Lipids 139 (2006) 11–19

probe that allowed a temperature range from 2 to 90 ◦ C with temperature steps of 0.1–1 ◦ C. Experiments were driven by the 3.30 Dispersion Technology Software provided by Malvern Instruments. 2.2. Preparation of samples Lipid samples were prepared by the traditional thinfilm method for measurement reproducibility and generalization purpose (but choice of the method is of no consequence on results because non-vesicular phases – i.e. lamellar phase – are also supported without limit but the turbidity of the sample). Briefly, the lipid solutions were prepared in chloroform or chloroform/methanol mixtures in a 25 ml flask. After vortexing, the solvent was allowed to evaporate under reduced pressure. This resulted in the formation of a thin lipid film on the inside wall of the flask. The film was dried under vacuum to ensure complete evaporation of solvent. One milliliter of hydration solution (water or saline buffer) was then added into the flask in order to obtain the desired concentration (typically 10 mg ml−1 ), and was vigorously vortexed for 1 min. The flask was then placed under probe sonicator (15 min in 1:1 pulse mode). 2.3. Typical experiment A first optimisation stage was performed, where the cell positions, compensation and attenuator settings for the cell, sample and measurement type were determined

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(by default the software adjusts these values by itself – based on sample optical properties such as turbidity, etc.). This stage was repeated three times so as to achieve accurate reproducibility in the intensity of scattered light. Parameters were then introduced and locked manually (overcoming the default software settings) for the second stage of experimentation. In this step, software was used in trend mode that allows multiple measurements to be made over a range of temperatures. Parameters were selected with the first and final temperatures of the trend, temperature intervals or by choosing the number of steps, i.e. the number of measurements to be made at each step after equilibration time. The rate of experimentation – the temperature scan speed at which the determination is made – is given by these experimental conditions (depending on the desired accuracy level). Data were collected as ‘mean count rate versus temperature’ or ‘normalized mean count rate versus temperature’ for comparison purposes and treated with Boltzmann regression curves (Fig. 1). Boltzmann regression curves y=

A 1 − A2 + A2 1 + e(x−x0 )/
x

with A1 , the initial y value (initial count rate of lipids in Phase 1), A2 , the final y value (lipids in Phase 2), x0 , the centre of the distribution (y value at x0 being half way between the two limiting values A1 and A2 : y(x0 ) = (A1 + A2 )/2) and
x, the width of the slope.

Fig. 1. Schematisation of the process of phase transition as propagation of a physical macroscopic property (δ0 ) and corresponding curve as property vs. temperature.

N. Michel et al. / Chemistry and Physics of Lipids 139 (2006) 11–19

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3. Results As for bi-dimensional models of propagation obtained for emerging macroscopic physical properties (δ0 in Fig. 1), the typical profile of evolution looked like sigmoid curve (based on curve fitting with non-linear regression on raw data): several properties of membranes are known to exhibit discontinuities in the vicinity or at the temperature of their phase transition and these features have been suggested to be connected to the presence of two coexisting phases in the transition region with the presence of micro-domains (Bagatolli and Gratton, 1999). As we have said, if we consider the optical properties of each phase, transition from Phase 1 (plateau at y = A1 ) to Phase 2 (plateau at y = A2 ) involves a stage in which the two phases are present: the slope of the curve (see theoretical curve in Fig. 1 and experimental curves in Fig. 2).Let us designate the amount of lipids in the initial phase through p1 , and that of lipids in the second phase through p2 . Then the fraction of lipids in the first

Fig. 2. Determination of the main phase transition temperature of DMPG (grey triangles – Tm : 21.9 ± 0.2 ◦ C; experiment duration: 24 min), DPDPC (black circles – Tm : 33.0 ± 0.2 ◦ C; experiment duration: 28 min), DMPE (black squares – Tm : 47.9 ± 0.3 ◦ C; experiment duration: 44 min). All experiments performed in distilled water pH 5.5.

Table 1 Validation of determination of the phase transition temperatures by light scattering of a representative panel of phosphatidyl choline with different chain lengths from the lipidat database Lipids

Techniquesc DPDPC PC (15:0/15:0) Tm DSC FTIR HSDSC DSC Sub-Te

HSDSC DSC DSC

DPPC PC (16:0/16:0) Tm DSC Sub-T

Measured Tc (◦ C)b

Tc literaturea

DSC DSC, XRD DSC DSC DSC DSC DSC

Z average (nm)

Nature of transitiond

Values (◦ C)

Gel-1.c. Gel-1.c. P␤ -L␣ P␤ -L␣

31 33 34.7 36

33.0 ± 0.2

67

76

Gel-1.c.f L␤ -P␤ Lc-P␤

33.3 24.8 22.3

33.4 ± 0.4 24.1 ± 0.1 22.4 ± 0.1

130 182 81

102 86 82

Gel-1.c.

42.1

41.4 ± 0.1

95

100

Pre Pre L␤-P␤ L␤ -P␤ Pre Lc-L␤ Lc-L␤

33.8 34.5 35.3 36 28 21 21

34.9 ± 0.1

71

95

19.0 ± 0.1

127

70

Below Tc

Above Tc

All experiments performed in distilled water pH 5.5 except determination of the gel-l.c. transition of DPDPC (KCl buffer 50 mM). a From lipidat database. b Average values (three measurements). c DSC: differential scanning calorimetry, HSDSC: high sensitivity differential scanning calorimetry, FTIR: infrared Fourier transform spectroscopy, XRD: X-ray diffraction. d Attributed by authors. e Sub-transitions. f KCl buffer 50 mM.

N. Michel et al. / Chemistry and Physics of Lipids 139 (2006) 11–19

state will be: p1 α1 = p1 + p 2 and the corresponding curve (Fig. 1). Examples of experimentations are presented in Fig. 2 (see also Tables 1 and 2).

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In the case of DMPG (Fig. 2) normalization procedure we applied, enhanced the emergence of the transitions which usually revealed not very sharp with other technics. Furthermore, compounds with multiple phase transitions (i.e. sub-transitions or pre-transitions and main phase transition) displayed sums of sigmoids with each

Table 2 Validation of light scattering determination of the phase transition temperatures of a representative panel of lipids from the Lipidat database Lipids

Measured Tc (◦ C)b

Tc literaturea Nature of transitiond

Values Tc (◦ C)

Gel-l.c. Gel-l.c. Gel-l.c. Gel-fluid L␤-L␣ L␤-L␣ L␤-L␣ Lc-L␣ Lc-L␣

49 50.2 55 48 49 49.8 53 55.8 57.6

Lc-L␤

DMPG PG (14:0/14:0) Tm DSC FTIR ESR HSDSC DSC

Techniquesc

Z average (nm) Below Tc

Above Tc

47.9 ± 0.3

230

200

36

34.3 ± 1.8

95

92

Gel-l.c. Gel-l.c. L␤ -P␤ L␤ -P␤ L␤I-L␣

23 28 13 17 24

21.9 ± 0.2

66

62

DMPA PA (14:0/14:0) Tm FI (NPN) DSC DSC HSDSC Turbidity

Gel-l.c. Gel-l.c. Gel-l.c. Gel-l.c. Gel-l.c.

47 50.5 51.4 52 54

50.3 ± 0.6

150

160

DSPE PE (18:0/18:0) 31 P NMR, DSC, XRD Tm DS HSDSC HSDSC

Gel-l.c. L␤-L␣ L␤-L␣ Lc-L␣

47 73.6 70.4 75

73.7 ± 0.01

150

160

14 N NMR Raman

Gel-l.c. Main

35 37

35.5 ± 0.4

135

110

DSC

Main

45

45.5 ± 1

85

72

DMPE PE (14:0/14:0) Tm DSC HSDSC ATR-FTIR 2 H NMR 1 H NMR DS DTA HSDSC DSC Sub-Te

SM (extract) Tm DODAB Tm

DSC

All experiments performed in distilled water pH 5.5. a From lipidat database. b Averaged values (three measurements). c DSC: differential scanning calorimetry, HSDSC: high sensitivity differential scanning calorimetry, FTIR: infrared Fourier transform spectroscopy, XRD: X-ray diffraction, DTA: differential thermal analysis, ATR-FTIR: attenuated total reflectance Fourier transform infrared, FI (NPN): fluorimetry intensity of 1-N-phenylnaphtylamine, ESR: electron spin resonance, DS: densitometry. d Attributed by authors. e Sub-transitions.

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N. Michel et al. / Chemistry and Physics of Lipids 139 (2006) 11–19

Fig. 3. Evolution of the mean count rate as temperature function for the DPPC displaying three phase transitions – whole curves in inset – the main phase transition (Tm : 41.41 ± 0.01 ◦ C; experiment duration: 60 min – (a)) and two pre-transitions (Tp1 : 34.9 ± 0.1 ◦ C; experiment duration: 180 min – (b) and Tp2 : 19.0 ± 0.1 ◦ C; experiment duration: 60 min – (c)) as described in the literature. All experiments performed in distilled water pH 5.5.

transition attributed to the middle of each curve (Fig. 3). Effectively we found that this method allowed the determination not only of the main phase transition but also of sub-transitions involving under-phases (resolution of the technique allows the determination of several transitions – even low spaced if sufficiently intense). To generalize and to validate our method, we have carried out several experiments with a variety of lipids, differing in their alkyl tail length (C14 –C18 ) and polar headgroup nature (ionic, zwitterionic and neutral), in order to cover a large scale of temperatures (Tables 1 and 2). One of the most challenging problems to be dealt with while testing this method concerned the variations in referenced values and the differences in nomenclature

used to describe the lipids, the mesophases and polymorphs they form. Difficulties arose in determining how to recognize where a particular lipid or phase cited in the literature belonged. To overcome this problem we used the on-line database Lipidat, 2005 (http://www. lipidat.chemistry.ohio-state.edu/). This website is a Lipid Thermodynamic Database Project which collects in one central depository all available information on lipid mesomorphic and polymorphic transitions and miscibility. Finally, we observed a good reproducibility of the Tc determinations for any chosen lipid and whatever the sample concentration in the range from 2.5 to 30 mg ml−1 .

N. Michel et al. / Chemistry and Physics of Lipids 139 (2006) 11–19

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4. Discussions The results call for some analyses as much for their relation to phase transition temperatures already published in the literature, as for the effect trend evolutions and concentration on the values obtained: 1. In light scattering terms, the measured count rate can change for any of the following reasons: • Alterations in the structure (i.e. refractive index) of the particle membrane. • Alterations in the size of the particles. • Alterations in the concentration of the particles. As regards the effect of concentration, for all lipids the concentration was of the order of 0.1% (w/w) and did not change during measurements. Where there were step changes in the mean hydrodynamic radius, they usually coincided with a change in Z-average, but they were certainly less dramatic than for the changes in count rate. Usually the trends of these two parameters went in opposite directions – i.e. count rate decreasing while size increasing (see Fig. 4). However, the technique we propose, also allows the determination of the phase transition temperatures in cases where transition does not entail any notable variation in the size of objects. Indeed, we have been in a position to observe that mean count rate variation is not linked to the Z-average alterations when the DMPG solution temperature varies (see Fig. 5). These remarks can also be apply to the determination of pretransitions that usually do not result in any particle size alteration.

2. It is worth noting that the trend direction is of no importance since Tm is equivalent upon heating or cooling with a standard deviation of 1 ◦ C (maximum value) – Fig. 6 shows a maximum standard deviation of 0.2 ◦ C between the two measurements for DPDPC. 3. It has to be underlined that the results obtained from the SLS data are neither sensitive to the initial lipid concentration nor to the initial state from which the trend was started and so, different methods of preparation (powder or film for instance) gave the same results. 4. All results obtained by using these granulometric determinations were closely correlated to the already reported values whatever the phospholipid structure

Fig. 4. Determination of the main phase transition temperature of DODPE as normalized mean count rate vs. temperature (open circles) and as normalized Z average vs. temperature (filled squares). The experiment was performed in distilled water pH 5.5, during 120 min.

Fig. 6. Determination of the main phase transition temperature of DPDPC upon heating (black squares – Tm : 33.2 ± 0.2 ◦ C) or cooling (grey circles – Tm : 33.0 ± 0.2 ◦ C) experiment duration: 40 min each.

Fig. 5. Size (Z average – black open squares) and mean count rate (blue discs) vs. temperature evolution of DMPG solution (connection by simple B-splines for clarity). Experiment duration: 20 min.

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Fig. 7. Correlation between data from literature (lipidat database) and from experiments, visual summary of Tables 1 and 2.

(Tables 1 and 2, Fig. 7). Moreover, this method allowed the determination of pre-transition and/or sub-transition temperatures without however, providing any information on their nature. Currently, experiments are under way to specify in what conditions this method could be applied to the phase transition temperature determination of a lipids mixture. Acknowledgements The authors are grateful to Malvern Instruments for their help in the development of the method and exchanges of views, to the “Provence Alpes Cˆotes d’Azur” Regional Authorities for the financial support awarded to one of the authors (N. Michel) and to Professor Ralph Beisson (Avignon University) for his assistance. References Alexandridis, P., Holzwarth, J.F., 1997. Differential scanning calorimetry investigation of the effect of salts on aqueous solution properties of an amphiphilic block copolymer (poloxamer). Langmuir 13, 6074–6082. Azize, B., Cao, A., Perret, G., Taillandier, E., 1994. Thermal behavior and elastic properties of dimyristoyl phosphatidylcholine bilayers under the effect of pentoxifylline. Biophys. Chem. 51, 45–52. Bagatolli, L.A., Gratton, E., 1999. Two-photon fluorescence microscopy observation of shape changes at the phase transi-

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