SOLUBILISATION OF PHOSPHOLIPIDS BY NON­ IONIC SURFACTANTS AS DETERMINED BY SOLUTION

CALORIMETRY

6.1 . Introduction

The understanding o f lipid-surfactant interactions is of practical importance in drug delivery research to improve the circulation half-live of liposomes. Liposomes of conventional formulation are removed from the blood circulation directly by the macrophages of the MPS or indirectly via serum proteins (Allen, 1992). Unless the drugs encapsulated in the liposomes are directed to the MPS, the uptake of liposomes by the MPS limits their therapeutic applications. The recognition by this system can be avoided by including surfactants in the formulation which stabilize the liposomes (Allen and Choim, 1987). However, at certain concentrations, a detergent-like effect begins to appear (Allen and Choim, 1987), and the phospholipid bilayer is solubilised. The solubilising properties of surfactants are routinely used to solubilise phospholipid bilayers, integral membrane proteins and other membrane constituents (Almog et al.,

1986).

The solubilisation of the phospholipid bilayer by non-ionic and ionic surfactants has been explained using a three-stages model where the phospholipid bilayer is transferred into lipid-surfactant mixed micelles with increasing amounts of surfactant (Lichtenberg et al., 1983). In stage I of the three-stages model, at low surfactant concentrations, the surfactant distributes between the bilayer and the water phase forming mixed phospholipid-surfactant bilayers. In stage II, at higher surfactant concentrations, the bilayers saturate and start to form mixed micelles with the surfactant molecules. In stage III, a certain surfactant to phospholipid ratio is reached which converts the entire phospholipid into mixed micelles. Mixed micelles can be separated from other micelles based on their charge, size or density.

transition is connected with the resistance o f the phospholipid bilayer to encapsulated amphiphile additives or amphiphatic metabolites capable o f partitioning between water and the lipid membrane. Investigation of surfactant-induced solubilisation of phospholipids provides information in relation to the resistance of liposomes towards amphiphilic compounds and also contributes to the evaluation o f the in vitro stability o f the phospholipid bilayer against degradation products (e.g., single-tailed lysophospholipids) and the in vivo potential degradation by enzymes or bile.

In this study the interaction of three non-ionic surfactants (linear alcohol ethoxylates) with DMPC in excess water was studied by means of solution calorimetry and TEM. The interaction levels investigated comprised a range of concentrations between 0 and 100 mol % non-ionic surfactant. Measuring the heat signal at 0 and 100 mol % surfactant can provide information on the formation of pure phospholipid vesicles and pure surfactant micelles respectively. Whereas the heat signal produced at concentrations between 0 and 100 mol % surfactant would provide an insight into the interaction between surfactant and phospholipid molecules.

6. 2. Methods

6 . 2 .1 . Control experiments

The micelle formation of lysophospholipids and short chain diacylphospholipids has been associated with a heat change measurable by ITC (Heerklotz and Epand, 2001). In this study the self-aggregation of Cio(EO)3, Cio(EO)5 and Ci2(E0)y was investigated using solution calorimetry. Details of these alcohol ethoxylates can be found in Chapter 2.

Solution calorimetry experiments required firstly the preparation of the sample in a glass-crushing ampoule, and secondly dissolution (or dispersion) by mixing the surfactant with the water in the reaction vessel o f the calorimeter. An empty crushing ampoule was purged with dry nitrogen for 30 s to remove any possible moisture and the required amount of surfactant was added directly into the ampoule. Samples

contained 2 0 0 mg Cio(EO)3, 2 0 0 mg Cjo(EO)5 or 2 0 mg Ci2(EO)7.

The surfactant-containing ampoules were closed with a silicon stopper, sealed twice with beeswax and immediately analysed in the solution calorimeter at 37 °C under continuous agitation at 600 rpm. More information about the running of experiments in the solution calorimeter is outlined in section 3.6.

6. 2. 2. Interaction experiments

Interaction experiments consisted of measuring the thermal responses in the solution calorimeter of co-films containing DMPC-Cio(EO)3, DMPC-Cio(EO)5, or DMPC- Ci2(EO)7. The calorimetric data was evaluated using Microcal Origin Ver.3.5. software.

For experiments, co-films were formed in situ as explained in section 4.2.2. Briefly, the phospholipid and surfactant were added to an empty crushing ampoule, dissolved in chloroform and the organic solvent removed to deposit a co-film. Co-films were formed with 30 mg DMPC and increasing amounts o f non-ionic surfactant to give final concentrations of 15, 30, 50, 70 and 90 mol % surfactant. The ampoules containing the co-films were closed with a silicon stopper, sealed twice with beeswax and immediately mixed with deionised water in the reaction vessel of the solution calorimeter at 37 °C under continuous agitation at 600 rpm for 1 h. The temperature change associated with the process was recorded and the heat evolved by the reaction was calculated (Eqn.3.4.).

The experimental conditions for control and solubilisation experiments were exactly the same. Maintaining the experimental conditions constant allowed systematic investigation of the effect of linear alcohol ethoxylates on DMPC by comparison of the responses obtained for pure components and phospholipid-surfactant mixtures.

6. 2. 3. Transmission electron microscopy analysis

Samples collected from the reaction vessel after solution calorimetry analyses were examined immediately using TEM. The samples were negatively stained with phosphotungstic acid (1% w/v aqueous solution) as described in section 5.5.2., and viewed using a Philips CM 120 BioTWIN microscope.

6. 3. Results and discussion

6. 3.1. Preparation protocol

Mixtures of phospholipid and surfactants can be prepared according to three different protocols:

i) Dispersion of a phospholipid-surfactant co-film in water.

ii) Dispersion of a phospholipid film in a surfactant solution.

iii) Addition o f a surfactant solution to a dispersion o f phospholipid vesicles or vice versa.

Solution calorimetry is a suitable technique when one of the components to be mixed is in a solid-like state (protocols i and ii). ITC has been widely applied to study the dropwise addition of one liquid over an other as required in protocol iii. In this study the components were mixed according to protocol i because in a co-film the phospholipid and the surfactant mix at the molecular level and when dispersed in water the dispersion will reach equilibrium very quickly ensuring the reproducibility of the data (Lichtenberg et al., 1979).

6. 3. 2. Enthalpy of form ation of surfactant micelles

The solution of non-ionic surfactants in water at concentrations above the cmc results in the formation of surfactant micelles. The formation of pure surfactant micelles at 37 °C in the solution calorimeter resulted in an exothermic response for Cio(EO)3,

Cio(EO)5 and Ci2(EO)7 (Table 6.1.). For each surfactant the molar enthalpy value was

independent of the amount of sample indicating that all the material contributed to the thermal response. The enthalpies of micellisation (micelle formation) in Table 6.1. show that Ci2(EO)7 produced the most exothermic response indicating that this surfactant formed the most energetically stable micelles. On the other hand, the enthalpy values obtained for Cio(EO)3 and Cio(EO)5 showed a more exothermic response for the surfactant with the larger number o f ethoxylate groups indicating that for the same alkyl chain length, more stable micelles were formed when the polarity of the molecule increased.

Table 6.1. Enthalpies of micellisation of linear alcohol ethoxylates at 37 °C (n =3 ± s.d.) Surfactant (Commercial name) Surfactant (Chemical structure) AH (J/g) (± s.d.) Biodac® 39 Cio(E O )3 -50.5 (± 0.3) Biodac® 59 Cio(E O )5 -68.4 (± 0.7) Marlipal®MG Ci2 (E O )7 -75.3 (± 5.5)

Each plot in Fig.6.1. shows a typical power-time curve obtained in the 5 min which followed the breaking of an ampoule into the reaction vessel o f the solution calorimeter filled with 100 ml water. The ampoules contained 200 mg Ciq(E0 )3 (plot A), 200 mg Cio(EO)5 (plot B) or 20 mg Ci2(EO)7 (plot C). A single exothermic peak followed by a small endothermie peak characterized the power-time curves for the micellisation of the three surfactants. The graphs indicate that under the experimental conditions investigated the completion o f the reactions occured within less than 2

Fig.6.1. Power-time curves for the micellisation of linear alcohol ethoxylates at 37 °C. 1400 1200 ^ 1 0 0 0 I 800 r 600 200 -200 Time (min) 1400 1200 ^ 1 0 0 0 I 800 r 600 200 -200 Time (min) 140 120 100 I

I

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6. 3. 3. Enthalpy of form ation of DMPC vesicles

A discussion on the formation of DMPC vesicles can be found in section 3.10.2. Briefly, the formation of DMPC vesicles in the solution calorimeter at 37 °C from a 25 mg DMPC film resulted in an endothermie response with an enthalpy value equivalent to 48.9 (± 6.0) J/g (Table 3.3.). The power-time curve for the formation of DMPC vesicles showed a single endothermie peak. Fig.6.2. shows the response obtained in the 5 min which followed the breaking of an ampoule containing a DMPC film into the reaction vessel of the solution calorimeter.

The differences in terms of enthalpy value and features of the power-time curve between pure DMPC and pure surfactants in the solution calorimeter made them good candidates for interaction studies. The effect of increasing amounts of surfactant on the phospholipid bilayer was studied in terms o f the changes produced on the thermal response of the phospholipid.

Fig.6.2. Power-time curve for the formation of dimyristoylphosphatidylcholine vesicles at 37 °C.

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T i m e ( m i n )

6. 3. 4. Enthalpy of bilayer solubilisation

Incorporation of single chained surfactants into phospholipid vesicles in the liquid- crystalline state results in the solubilisation of the phospholipid membrane when the composition of the mixture is varied from water + 100 % lipid, to water + 100 % surfactant. Solubilisation occurs via the vesicle-to-micelle transition. This liquid- crystal to micellar phase transition was described by a three-stages model (Lichtenberg et al., 1983). A description o f this generally accepted model can be found in section 1.5.2. A schematic representation of the solubilisation process proposed by Lichtenberg et al. (1983) is shown in Fig.6.3. Very briefly, this model describes the formation o f mixed vesicles and mixed micelles in phospholipid- surfactant mixtures.

Fig.6.3. Schematic diagram of the different stages of the solubilisation of phospholipids by surfactants.

BILAYERS

_MICELLES

BILAYERS

MICELLES

S-P

s-p

_S-P

s-p

s-p

00 MOLAR RATIO (SURFACTANT/PHOSPHOLIPID)

The diagram shows the average composition o f the phases formed by a solubilising surfactant (S) and a phospholipid (P) in the presence of an excess of water. The mixed bilayer phases (B) are represented with rectangles, whereas the mixed micelle phases

The solubilisation of DMPC by alcohol ethoxylates was studied by solution calorimetry and compared to the model proposed by Lichtenberg et al. (1983). Fig.6.4. shows the enthalpic responses in the solution calorimeter for co-films formed with DMPC and non-ionic surfactant. Three different surfactants were evaluated. The red lines in Fig.6.4. show the linear behavior expected when the components do not interact with each other. On the other hand, a deviation fi*om linearity would be associated with the interaction of the components. Inspection of Fig.6.4. shows distinct behaviours for films containing DMPC and surfactant with 10 carbon atoms in the alkyl chain (Cio(EO)3, Cio(EO)5,) or DMPC and surfactant with 12 carbon atoms in the alkyl chain (Ci2(E0 )y). To simplify the discussion, firstly the results for each surfactant are discussed, and secondly a comparison between the different surfactants will be made.

For the evaluation of the behaviour o f phospholipid-surfactant mixtures, the enthalpy- concentration curves were complemented with the power-time curves. The enthalpy values and the power-time curves at various concentrations are shown in Fig.6.5. for each phospholipid-surfactant mixture. The small power-time curves in Fig.6.5. show the heat flow evolution during the first 5 min which followed the mixing of DMPC- surfactant co-films with water in the calorimeter. For any given time in the power­ time curves, the heat flow recorded corresponded to the sum of the reactions taking place. For example, a downward peak (endothermie reaction) would indicate that the endothermie reactions at that point were greater in magnitude than the exothermic ones.

ITC data has been used to determine the molecular shape o f phospholipid-surfactant aggregates (Heerklotz et al., 1997). A similar approach was followed here for the interpretation of the solution calorimetry data. The molecular shape of the aggregates is determined by the effective surfactant to phospholipid ratio in the aggregates, (Eqn.1.5.). In this study, the concentration of monomers in the water was neglected because the components were mixed at molecular level in the co-film. Therefore, according to Eqn.1.5. the surfactant to phospholipid ratio in the co-film was equal to Rg. Then, significant break points of the enthalpy-concentration curves could be directly related to the components concentration.

Fig.6.4. Enthalpy-concentration curves for co-films containing (A) DMPC C,o(EO)3, (B) DMPC-C,o(EO) 5 and (C) DMPC-C,2(EO)7.

R = 0.9452 LU -40 -60 -80 Surfactant (mol %) _ 20 O) R =0.9643 -60 -80 Surfactant (mol %) DMPC-Ci2(EO)7 40 : LU -40 R = 0.9845 -60 -80 Surfactant (mol %)

Each symbol ( • , A ,B ) represents the average enthalpy value o f three experiments. The error bars represent the standard deviation from the average value. The red lines show the linear fit expected in the absence o f interaction between the components.

Fig.6.5A. Enthalpy-concentration curve and power-time curves for co-films containing DMPC-C,o(EO)j. D I V P O C i o ( E O ) 3

A

1400 1200 1000 f 800 i 400 -200 Time (min) 1400 1200 ilOOO !800 ^600 5 400 -200 O ) 0,15,30mol%Cio(EO)3 -10 '-20 1-30 i-40 ;-50 -60 -70 -80 50, 70 mol % Cio(EO)3 -10 -20 -30 ■40 -50 -60 -70 -80 -200 Time (min) Time (min) L U

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Os

O T h e sy m b o l ( • ) re p re se n ts th e m e a n e n th a lp y v alu e o f th re e ex p e rim en ts. T he e rro r b ars re p re se n t th e sta n d a rd d e v ia tio n fro m th e m e a n valu e. T h e b lu e, g re e n an d re d line sh o w th e a p p a re n t lin ear fit for a g iv en c o n c e n tra tio n ran g e.

Fig.6.5.A shows that for DMPC-Cio(EO)3 mixtures, the enthalpy evolution followed three linear behaviours according to the components concentration. At 0-30 mol % Cio(EO)3, the enthalpy values only changed slightly and the power-time curves showed a single endothermie peak suggesting that up to 30 mol % the surfactant distributed in the DMPC bilayer and mixed vesicles were formed which had similar properties to pure phospholipid vesicles. On the other hand, higher concentrations of surfactant produced a very different response. Fig.6.5.A shows that at 50 mol % Cio(EO)3 the enthalpy value decreased significantly. For the interpretation o f the decrease in the enthalpy value it is necessary to consider that the response measured in the calorimeter corresponds to the overall sum of the processes taking place. A reduction in the enthalpy value might be related either to a decrease in the endothermie responses (e.g. formation of DMPC vesicles) or an increase in the exothermic reactions (e.g. formation of micelles), or both. At 50 mol % Ciq(E0 )3 the enthalpy value decreased and the power-time curve showed an exothermic response, together with the characteristic endothermie peak. The appearance of a small exothermic peak at 50 mol % surfactant was interpreted as the formation of mixed micelles following the saturation o f the phospholipid bilayer. Consequently, the data indicate that the onset of the lamellar to micelle transformation, or the saturation of the DMPC bilayer, occurred at a concentration between 30 and 50 mol % Ciq(E0 )3. These concentrations correspond to surfactant to phospholipid ratios equivalent to 0.4 and 1 respectively, therefore 0.4 < Rg^at < 1.0. Rg^at jg the saturating effective surfactant to phospholipid ratio and it represents the beginning of the solubilisation process. The coexistence of an endothermie and an exothermic peak at 50 and 70 mol % Cio(EO)3 was interpreted as the coexistence of mixed bilayers and mixed micelles. At 70 mol % Cio(EO)3 the endothermie response decreased, while the exothermic peak increased indicating that although mixed bilayers and mixed micelles coexisted, at this concentration the tendency was for the formation of micelles over bilayers. When 90 mol % Ciq(E0 )3 was incorporated in the film a negative enthalpy value, and a single exothermic peak were observed suggesting that at this concentration mainly micelles were formed. A significant change in the thermal response was observed between 70 and 90 mol % Cio(EO)3. 70 and 90 mol % surfactant correspond to 2.3 and 9.0 surfactant to phospholipid ratio respectively. The data indicated that the minimum surfactant to lipid ratio at which all the phospholipid converted into mixed

micelles fell between 2.3 and 9.0, being 2.3 < RgSoi « 9 0. R^soi ig the solubilising effective surfactant to phospholipid ratio; it represents the minimum surfactant to phospholipid ratio in which all the phospholipid is in mixed micelles. The exothermic response at 100 mol % surfactant corresponds to the formation of pure micelles.

For films containing DMPC and Cio(EO)5 (Fig.6.5.B) the thermal responses also depended on the ratio of components. As discussed above, the appearance of an endothermie and an exothermic peak suggested the coexistence o f mixed bilayers and mixed micelles at 30 mol % Cio(EO)5. Before mixed micelles can form it is necessary to saturate the phospholipid bilayer with surfactant (Lichtenberg et al., 1983). The data indicated that saturation occurred at a concentration below 30 mol % surfactant consequently R^sat < 0.4. Surfactant-saturated bilayers and mixed micelles coexisted over the 30-50 mol % surfactant range. At 70 and 90 mol % Cio(EO)5 the enthalpy value became negative and a single exothermic peak was observed suggesting that mainly micelles were formed. The decrease in the enthalpy value observed between 50 and 70 mol % Cio(EO)5 suggested that within this concentration range a ratio was reached in which all the phospholipid started converting into mixed micelles; conversion of these concentrations into their corresponding surfactant to phospholipid ratio indicates that 1 < RgSoi < 2 .3 for DMPC-Cio(EO)5 mixtures.

The interpretation of the results obtained for DMPC-Ci2(EO)7 mixtures is less straight forward than for DMPC-Cio(EO)n mixtures. The regression coefficient (R = 0.9845) indicated an almost linear dependence of the enthalpy of reaction on the surfactant concentration (Fig.6.4.C). Nevertheless, careful inspection o f the enthalpy- concentration graph revealed a curving tendency with small experimental error that suggested the interaction of the components. In order to evaluate the interaction between Ci2(EO)7 and DMPC, mixtures of these components were analysed using TEM. The micrographs show DMPC lamellar vesicles in the absence of surfactant (Fig.6.6.A) in contrast to disrupted lamellar structures (Fig.6.6.B) and tiny particulates (Fig.6.6.C) when 50 or 70 mol % Ci2(EO)7 were incorporated into the DMPC film. The small white or black particles observed in micrographs B and C were identified as micelles. The visualisation of distorted lamellar vesicles in the presence of Ci2(EO)7 confirms the interaction between DMPC and the surfactant.

Fig.6.5B. Enthalpy-concentration curve and power-time curves for co-films containing DMPC C,o(EO)g. 1400 1200 1000 f 800 -^600 I 400 (£ 200 70, 90 mol % Cio(EO)s

4 0

- 0, 15mol%Cio(EO)5 -10 -20 -30 -40 -50 -60 -70 -80 -200 Time (min) O ) 1400 1200 1000 t 800 -&600 i 400 (£ 200 Time (min)

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_{30,50mol%Cio(EO)5} -10 -20 -30 -50 -60 -70 -80 -200 Time (rrin)

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T h e sy m b o l ( A ) re p re se n ts th e m e a n e n th a lp y v alu e o f th re e ex p e rim en ts. T he erro r b ars re p re se n t th e sta n d a rd d ev ia tio n from th e m ean

In document A calorimetric study of the formation and stability of phospholipid vesicles (Page 151-169)