Structure-related aspects on water diffusivity in fatty acid–soap and skin lipid model systems

Journal of Controlled Release 63 (2000) 213–226 www.elsevier.com / locate / jconrel

Structure-related aspects on water diffusivity in fatty acid–soap and skin lipid model systems ´ a , *, Johan Engblom b ,1 Lars Norlen a

Department of Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden b Department of Food Technology, Chemical Center, Lund University, Lund, Sweden Accepted 26 August 1999

Abstract Simplified skin barrier models are necessary to get a first hand understanding of the very complex morphology and physical properties of the human skin barrier. In addition, it is of great importance to construct relevant models that will allow for rational testing of barrier perturbing / occlusive effects of a large variety of substances. The primary objective of this work was to study the effect of lipid morphology on water permeation through various lipid mixtures (i.e., partly neutralised free fatty acids, as well as a skin lipid model mixture). In addition, the effects of incorporating Azone  (1-dodecyl-azacycloheptan-2-one) into the skin lipid model mixture was studied. Small- and wide-angle X-ray diffraction was used for structure determinations. It is concluded that: (a) the water flux through a crystalline fatty acid–sodium soap–water mixture (s) is statistically significantly higher than the water flux through the corresponding lamellar (L a ) and reversed hexagonal (H II ) liquid crystalline phases, which do not differ between themselves; (b) the water flux through mixtures of L a / s decreases statistically significantly with increasing relative amounts of lamellar (L a ) liquid crystalline phase; (c) the addition of Azone  to a skin lipid model system induces a reduction in water flux. However, further studies are needed to more closely characterise the structural basis for the occlusive effects of Azone  on water flux.  2000 Elsevier Science B.V. All rights reserved. Keywords: Skin lipid model systems; Fatty acid–soap systems; Water diffusivity; Structure

Abbreviations: Azone  : 1-dodecyl-azacycloheptan-2-one s: crystalline phase; L a : lamellar liquid crystal; H II : reversed hexagonal liquid crystal; fSoap : volume fraction neutralised fatty acid; fAcid : volume fraction free fatty acid; fw : volume fraction water; a: lattice parameter; l: half bilayer length; d w : length of water sheet separating two bilayers; x La : weight fraction lamellar phase; x Soap : weight fraction neutralised fatty acid; xAzone  : weight fraction Azone  ; NaP: sodium palmitate; PA: palmitic acid; NaO: sodium oleate; OA: oleic acid; AS: acid–soap crystal; F: mass transfer per unit time and area (flux); D: diffusion coefficient; Deff : effective diffusion coefficient; P: permeability coefficient; K: partition coefficient; p: vapour pressure; SAXD: small-angle X-ray diffraction; WAXD: wide-angle X-ray diffraction; CI: confidence interval; b : regression coefficient *Corresponding author. ´ E-mail address: [email protected] (L. Norlen) 1 ¨ Sweden. Present affiliation: Bioglan AB, Malmo, 0168-3659 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 99 )00201-1

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1. Introduction Simplified skin barrier models are necessary to get a first hand understanding of the very complex morphology and physical properties of the human skin barrier. In addition it is of great importance to construct relevant skin barrier models that will allow for rational testing of skin toxicity and barrier perturbing / occlusive effects of a large variety of substances (e.g., for drug, barrier protecting and cosmetic formulations). It has been shown that the human skin barrier primarily is located to the intercellular lipid matrix of the stratum corneum [1–3]. The lipid composition of the intercellular spaces of human stratum corneum is unusual with respect to its high relative amount of ceramides. Other dominating lipid species are cholesterol and long chain free fatty acids [4]. The domain mosaic model for the skin barrier [5] predicts that the human skin barrier is essentially composed of a two-phase system with large crystalline regions surrounded by a liquid crystalline phase. The crystalline parts are supposed to be impermeable to water diffusion while some penetration of water is allowed through the continuous liquid crystalline part. Thus, the water permeability of human skin is predicted to be correlated to the relative amount of liquid crystalline phase present. Further, the formation of any nonlamellar morphology in this liquid crystalline lipid compartment of stratum corneum should enhance the skin permeability for polar and / or nonpolar substances. Normal liquid crystalline phases (e.g., hexagonal (H I ), discrete cubic (I 1 ) and micelles (L 1 )) are water continuous and are therefore believed to favour permeation of polar molecules. Reversed phases on the contrary (e.g., reversed hexagonal (H II ), reversed discrete cubic (I 2 ) and reversed micelles (L 2 )) have continuity in the oil domain [6, pp. 47–55]. Theoretically, substances that induce structures of bicontinuous morphology (e.g., bicontinuous cubic (V1 and V2 ), sponge phases (L 3 )) [7] should allow for rapid permeation of both polar and nonpolar substances. The mass transfer per unit time and area (F ) of a substance through the membrane is given by Fick’s first law of diffusion [8]: F 5 2 D(≠C / ≠x)

(1)

where D is the diffusion coefficient and ≠C / ≠x is the concentration gradient over the membrane. In order to study the transfer (e.g., of water) through complex media (e.g., lipid membranes) it is often more convenient to express the experimentally determined rate of transfer as permeability coefficient, P, since a single diffusion coefficient cannot be used to describe the overall process. If the effective (average) diffusion coefficient (Deff ) is constant and the mass concentrations at either side of the interface are governed by the equilibrium partition coefficient, K, [9] then P 5 Deff K

(2)

In this study, measurement of water flux were performed using an evaporimeter. Water evaporation measurement using this instrument is based on the fact that in the absence of forced convection, the process of water exchange through a stationary water permeable interface can be expressed in terms of the vapour-pressure gradient immediately adjacent to that interface [10]. The vapour-pressure gradient is approximately proportional to the difference between the vapour-pressure measured at two separate fixed points located normal to the interface and in the zone of diffusion [10]. The evaporimeter calculates the actual vapour pressure, p 5 (RH)psat

(3)

at each point of measurement from the saturated vapour pressure ( psat ), obtained with a thermistor ( psat is a function of temperature alone), and the relative humidity (RH) obtained with a capacitive sensor [10]. In order to characterise the structure and function of the complex, polymorphic lipid structures of the human skin barrier in more detail, lipid phase behaviour and morphology should be related to physical parameters of barrier function (e.g., water permeability). Therefore, we have chosen various lipid–water mixtures and measured the water flux through smears of these mixtures on nylon meshes. In particular, the effect on water flux (a) of the relative amount of lamellar liquid crystalline (L a ) to crystalline (s) phase and (b) of addition of Azone  (1-dodecyl-azacycloheptan-2-one) to a skin lipid mixture composed of human ceramide 3, cholesterol

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and free fatty acids, was studied. Small- and wideangle X-ray diffraction (SAXD, WAXD) was used for structure determinations.

2. Material and methods All experiments were performed at 218C since most of the data on lipid morphology available from literature are obtained at ambient room temperature and, in addition, only medium chain lipids (C 16 – C 18 ) were used in this study. Extracted human stratum corneum lipids were avoided due to their outspoken heterogeneity [11]. For simplicity reasons all densities have been approximated to one throughout this work.

2.1. Materials Oleic (OA; MW5282.5 g mol 21 ) and palmitic (PA; MW5257.4 g mol 21 ) acid and their corresponding sodium salts, cholesterol (MW5386.7 g mol 21 ) (all .99% grade) and NaOH (.98%), were purchased from Sigma (St. Louis, MO, USA). Human ceramide 3 (MW5597 g mol 21 ; phytosphingosine base, amide linked PA) (.95% grade) was a gift from Gist-Brocades (Cosmoferm, Netherlands) and Azone  (.96% grade) was a gift from Whitby (Richmond, VA, USA). The water used was of Millipore quality (Milli-Q-plus 185, Molsheim, France).

2.2. Methods 2.2.1. Sample preparation All samples were prepared in glass vials, sealed with rubber stoppers and metal caps. Homogenisation was achieved by centrifugation for 6 h at 2000 rpm followed by a freeze–thaw process (i.e., heating to 808C followed by freezing in liquid nitrogen, repeated three times). The samples were left to equilibrate for at least 6 months at ambient temperature. Crossed polars was used to detect any anisotropy in the samples. 2.2.2. Free fatty acid systems Ten samples of each (hydrated to 32 wt%) 59:41 (s) PA:sodium palmitate (NaP), and 59:41 (H II ) and

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35:65 (L a ) OA:sodium oleate (NaO) were prepared on molar basis [7]. In order to evaluate the swelling behaviour of the lamellar (L a ) phase, OA:NaO (35:65 mol / mol) mixtures with 25, 32, 60, 70, 80 wt% and ‘‘excess’’ water respectively, were prepared.

2.2.3. Mixed free fatty acid systems Partly neutralised OA and PA were employed to make 55 mixtures of coexisting crystalline (s) and lamellar liquid crystalline (L a ) phases (0, 5, 10, 15, . . . , 50 wt% of L a , five of each of the 11 relative concentrations555), hydrated to 32 wt% of total sample weight. 2.2.4. The skin lipid model system Ten mixtures of human ceramide 3 [4], cholesterol and free fatty acids (1:1:1 on molar basis) were prepared. The free fatty acid part was composed of equimolar amounts of PA and OA and neutralised to 53 mol%, using NaOH solution. The water content was 32 wt% of total sample weight. Zero, 5, 10, 15, 20 and 25 wt% Azone  (of total sample weight) were added to 12 mixtures of the skin lipid model system described above (two samples for each Azone  concentration). 2.2.5. Sample preparation on nylon meshes The formulations were applied on nylon meshes (nylon 66, thread diameter: 30 mm; repeat distance: 70 mm; dimension of the void spaces: 40340 mm 2 ) by use of a cover slip to produce a film of approximately uniform thickness covering the nylon mesh and filling the void spaces. The relative volume of the nylon compartment to the total volume of the unit cell is 34% (total unit cell volume: 29.4?10 4 mm 3 ; nylon compartment volume: 9.94?10 4 mm 3 ), corresponding to approximately 20 mm of the total 60 mm height of the unit cell. The equilibrium contact angles of water and n-hexane on the nylon mesh were photographically recorded. 2.2.6. Measurement of water flux through the lipid systems Our computer-based in vitro system for measurements of water evaporation has been described in detail elsewhere [12]. An evaporimeter (EP1, Ser¨ vomed, Molndal, Sweden) was used to measure

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water flux through thin smears of lipid mixtures on nylon meshes. The samples were mounted in holders, manufactured in our laboratory workshop, which were then placed on a water-filled chamber, parallel to and 1 mm above the water surface. The evaporimeter probe-head was attached to the water chamber, in direct contact with the mounted membrane. The whole unit (water chamber, sample-holder and evaporimeter probe-head) was located in a second chamber with controlled RH of 33% (achieved by a saturated solution of CaCl 2 ?6H 2 O in water) and an ambient air temperature of 218C. The temperature of the mounted membrane was controlled by a computer system with the aid of a Peltier-element, constituting the floor of the water filled chamber. The membrane temperature was set to 21.08C in all runs. Water evaporation from the mounted samples was recorded every 60 th s over a time period of 10 000 s (167 min) to ensure that the system had reached equilibrium conditions. The recorded values were sampled by the computer program and displayed graphically as a function of time. Furthermore, the mean value of the last 2000 s recording was calculated as a representative value of water flux through the membrane at steady state conditions. All pieces were weighed before and after each run to record sample weight and water loss. Twenty runs were performed on bare nylon mesh to gain reference data and to evaluate the precision of the method used.

2.2.7. X-ray diffraction A Guinier camera, modified after Luzatti et al. [13], was employed for SAXD and WAXD measure˚ ments (Cu-anode, l 51.542 A). All experiments were carried out at ambient room temperature (218C). 2.3. Statistics Means and regression coefficients, b, were given with 95% confidence intervals (CI) [14, pp. 66]. Regression analysis [14, Ch. 9] was used to evaluate effects regarding water flux, sample weight and water loss as related to (a) the relative amount of liquid crystalline to crystalline phase and (b) the relative amount of the penetration enhancer Azone  in the skin lipid model mixture, respectively. De-

parture from linearity was tested using F-test to see if the quadratic term was significant [14, pp. 398– 401]. Furthermore, linear regression was employed to detect dependence of sample weight on water flux through the samples. Analysis of variance with least significance difference test based on the Studentized range [14, pp. 234–235] was used to compare the water flux, sample weight and water loss of the different lipid formulations. All tests of hypothesis were performed on the 5% significance level (a 5 0.05). A STATISTICA 5.1 software (Statsoft, Tulsa, OK, USA) was employed for statistical calculations.

3. Results

3.1. Experimental set-up The water flux through the bare nylon mesh at 218C and 33% RH was 3961 g m 22 h 21 (95% CI of mean; n520), while the evaporation from a free water surface was 4161 g m 22 h 21 (95% CI of mean; n520). The equilibrium contact angle of water and nhexane on the nylon mesh was measured to 958 and 08, respectively. Fig. 1 shows water flux through a skin lipid model sample (human ceramide 3 [4], cholesterol and free fatty acids) as a function of time. The recording was performed at a temperature of 218C, and a relative humidity of 33%.

3.2. Free fatty acid systems Table 1 shows the obtained SAXD-patterns from aqueous swelling of the OA:NaO (35:65 mol / mol) at room temperature (218C). The visual appearance is that of a homogeneous lamellar liquid crystal throughout the whole composition region, confirmed by its birefringence using crossed polars. However, in excess water no SAXD-pattern was obtained, probably due to limitations in our X-ray facilities ˚ Furthermore, experimental data were (d ,280 A). compared to theory [15], 1 2 fwater 5 2l /a

(4)

where a is the lattice parameter and the bilayer

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Fig. 1. Water flux through a skin lipid model sample as a function of time.

˚ (from fw 50.32, half-thickness, l, is taken to be 15 A Table 1, Fig. 2). It is evident from Fig. 2 that the degree of neutralisation of the OA acid within the lamellar phase did not affect the swelling profile significantly (N.B. the included NaO / OA 8 / 2 is in fact a two-phase sample, located just below the lamellar one-phase region (cf. Ref. [7])). Water flux, sample weight, water loss and linear regression between sample weight and water flux corresponding to the lamellar (L a ) (n510), reversed hexagonal (H II ) (n510), crystalline (s) (n510) and skin lipid model (n510) samples are given in Table 2. The statistical evaluations (analysis of variance with least significance difference based on the

Studentized range) are shown in Tables 3–5. No fractures were observed by optical microscopy. The cumulated amount of water that permeates through the samples during a 3 h run was approximately 8 mg, 7 mg, 11 mg and 9 mg for the lamellar, hexagonal, crystalline and skin lipid model systems, respectively. The weight reduction of the samples (water loss) was 0.46 mg, 1.32 mg, 1.32 mg and 0.43

Table 1 Lamellar liquid crystalline (L a ) phases prepared from OA:NaO (35:65 mol / mol) mixtures with 25, 32, 60, 70, 80% (w / w) and ‘‘excess’’ water respectively

fwater

˚ Bragg spacing (A)

(wt%)

1/1

25 32 60 70 80 ‘‘Excess’’

42.3 44.8 75.4 91.6 144.9 .280 a

a

1/2

1/3

1/4

37.4 45.9 71.4

30.6 47.7

35.6

In excess water no SAXD-pattern was obtained, probably due ˚ to limitations in our X-ray facilities (d ,280 A).

Fig. 2. Swelling of lamellar liquid crystalline phases and mixtures (dotted line, data from Fig. 3) of lamellar liquid crystalline and crystalline phases in relation to water content of the samples and lamellar theory (solid line, a 5 2l /(1 2 fwater )).

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Table 2 Water flux, sample weight, water loss and linear trend between sample weight and water flux among the lamellar (L a ) (n510), reversed hexagonal (H II ) (n510), crystalline (s) (n510) and skin lipid model (n510) samples; data are tabulated as 95% CI of means and of b -values respectively Lipid phase

Lamellar (L a ) phase Reversed hexagonal (H II ) phase Crystalline (s) phase Skin lipid model a

Water flux

Sample weight

Water loss

(g 22 h 21 )

(mg)

(wt% of total)

CI for regression coefficients b between sample weight (independ.) and water flux (depend.)a

18.762.8 16.664.7 24.263.7 20.262.8

4.860.6 9.763.2 7.762.3 6.760.8

9.763.3 13.663.4 17.264.7 6.464.5

20.3060.66 20.3260.66 20.7860.43 20.4360.62

The regression coefficients are significantly different from zero when the 95% CI of b does not include zero.

Table 3 Analysis of variance with least significance difference based on the Studentized range of differences between sample means of water flux through lamellar and reversed hexagonal liquid crystals, crystalline and skin lipid model samples Water flux

Lamellar (L a )

Reversed hexagonal (H II )

Crystalline (s)

Reversed hexagonal (H II ) Crystalline (s) Skin lipid model

p.0.05 p,0.05 p.0.05

p,0.05 p.0.05

p.0.05

Table 4 Analysis of variance with least significance difference based on the Studentized range of differences between sample means of sample weight lamellar and reversed hexagonal liquid crystals, crystalline and skin lipid model samples. Sample weight

Lamellar (L a )

Reversed hexagonal (H II )

Crystalline (s)

Reversed hexagonal (H II ) Crystalline (s) Skin lipid model

p,0.05 p,0.05 p.0.05

p.0.05 p,0.05

p.0.05

mg (i.e., approximately 6%, 19%, 12% and 5% of the total amount of water evaporating from the sample surface). The total average film thickness (i.e., with correction made for the space occupied by nylon) was calculated to be 58 mm, 94 mm, 76 mm and 75 mm for the lamellar, hexagonal, crystalline and skin lipid

model samples, respectively (average sample weights after applications in the chamber divided by the density of 1.00 g cm 23 and a surface area of 113 mm 2 ). Using the octanol / water partition coefficient for water, soct 54.2?10 22 [16], the average membrane thicknesses given above, and assuming DC¯1 g

Table 5 Analysis of variance with least significance difference based on the Studentized range of differences between sample means of water loss (weight% of total weight) through lamellar and reversed hexagonal liquid crystals, crystalline and skin lipid model samples Water loss (wt% of total weight)

Lamellar (L a )

Reversed hexagonal (H II )

Crystalline (s)

Reversed hexagonal (H II ) Crystalline (s) Skin lipid model

p.0.05 p,0.05 p.0.05

p.0.05 p,0.05

p,0.05

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cm 23 , the effective diffusion coefficients (Deff 5 P/ soct ) for the various mixtures were calculated to be 72?10 29 cm 2 s 21 (L a ), 103?10 29 cm 2 s 21 (H II ), 120?10 29 cm 2 s 21 (s) and 100?10 29 cm 2 s 21 (skin lipid model mixture).

3.3. Mixed free fatty acid systems The SAXD-patterns from plain phases as well as mixtures of the lamellar and hexagonal liquid crystalline phases (OA:NaO (35:65 mol / mol and 59:41 mol / mol respectively), 32 wt% water) with the crystalline PA–soap (s) (PA / NaP (59:41 mol / mol), 32 wt% water), are shown in Table 6. ‘‘s’’ Is not a homogenous single phase, but a mixture of coexisting NaP:PA and PA crystals in water. Blending ‘‘s’’ with, e.g., lamellar (L a ) or reversed hexagonal (H II ) liquid phases will provide extra water for swelling the liquid crystals. Taking into account the total available amount of water (estimated from Fig. 3) the experimentally determined lattice parameter of the lamellar phase (L a ) in the 10 / 90, 30 / 70 and 50 / 50 (w / w) mixtures have been added in Fig. 2. Hence it is evident that a significant, however not complete, swelling does occur upon mixing (which is also evident from e.g., Ref. [7]). Water flux through 11 different combinations of the lamellar liquid crystalline (L a ) and the crystalline (s) phase are presented in Fig. 4. With increasing relative amount of lamellar (L a ) to crystalline (s) phase, the water flux did statistically

Fig. 3. Water and ion distribution of the mixed samples in relation to the relative amount (w / w) of lamellar liquid crystalline (L a ) to crystalline phases (s). fw( La) 5 0.32 /(x La 1 0.32(1 2 x La )) (solid line), x Soap 5 0.65x La 1 0.41(1 2 x La ) (dotted line).

significantly decrease (95% CI of bwater diffusion 5 20.6260.21; n555) while the sample weight did statistically significantly increase (95% CI of bsample weight 50.4160.25; n555). No statistically significant linear trends between water loss (sample weight loss) and relative composition (L a / s) (95% CI of bwater loss 5 20.1160.27; n555) and between water flux (dependent variation) and sample weight (independent variation) (95% CI of bwater diffusion vs. sample weight 5 20.436; n555) were detected. On the 5% level, there was no statistically significant departure from linearity for water flux

Table 6 The SAXD-patterns from plain phases, as well as mixtures, of the lamellar and hexagonal liquid crystals [OA:NaO (35:65 mol / mol and 59:41 mol / mol respectively), 32 wt% water] and the crystalline PA–soap mixture, referred to as ‘‘s’’ [PA / NaP (59:41 mol / mol), 32 wt% water] System

˚ Bragg spacing (A) 1/1

La H II a sa La / s 1 / 9 La / s 3 / 7

112.3 61.0

La / s 5 / 5

62.8

H II / s 5 / 5 a

54.4

a

44.8 49.2

Data from Engblom et al. [7].

1 / œ3

1/2

28.4

24.4

31.0 31.2

26.8

1/1

1/2

1/3

1/4

1/5

1/7

43.3 42.6 42.7 46.6 42.7 46.9 42.8

21.7 21.3 21.3

14.4 14.2 14.2

10.6 10.6

8.6 8.6 8.6

6.1

21.3

14.2

10.4

8.6

21.2

14.1

8.4

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Fig. 4. Water flux at steady state of mixtures of lamellar liquid crystalline (L a ) and crystalline phases (s) (n555).

with increasing relative amount of liquid crystalline phase.

weight and water flux (95% CI of bsample weight / water diffusion 5 20.5060.60; n512).

3.4. The skin lipid model system and Azone  4. Discussion The SAXD-patterns from the skin lipid mixtures with and without incorporation of Azone  are presented in Table 8 (below). WAXD shows orthorhombic chain packing (two strong lines at 3.8 and ˚ together with disordered hydrocarbon chains 4.2 A) (broad diffuse band). Furthermore, Azone  induced a significant swelling of the liquid crystalline part. Water flux, sample weight, water loss and linear trends between sample weight and water flux for the skin lipid model sample (n510) are presented in Tables 2–5. Water permeation through six different combinations of Azone  and the model skin lipid mixture are presented in Fig. 5. With increasing relative amounts of Azone  the water flux did statistically significantly decrease (95% CI of bwater diffusion 5 20.8160.36; n512) while the sample weight did increase (95% CI of bsample weight 50.6960.45; n512). The water loss did not change with increasing amount of Azone (95% CI of bwater loss 5 20.01960.62; n512). There was no statistically significant linear regression between sample

The crystalline phase (s) used in this study was most probably composed of hydrophilic lipid crystals dispersed in water, since it was melted and recrystallised in water (32 wt%) during the preparation procedure [6, p. 112; and K. Larsson, personal communication]. Consequently, the presence of an aqueous continuity in the sample may explain the higher water flux through the crystalline phase (s) compared to that of the liquid crystalline phases (L a and H II ) (Fig. 6). The equilibrium contact angle for water on the nylon mesh was 958, while instant spreading was registered for n-hexadecane. Thus, we do not expect a water film to develop between the nylon and the lipid formulations. Instead, the presence of hydrophobic surfaces like the nylon mesh can cause a splitting in the hydrophobic (hydrocarbon) tail region of the hydrophilic crystals at the interface region between nylon and lipids. This may result in a conversion of a fraction of the hydrophilic crystals present into amphiphilic crystals, directing the lipid

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Fig. 5. Water flux at steady state of mixtures of the skin lipid model sample (human ceramide 3:cholesterol:PA:OA (1:1:0.5:0.5 on a molar basis)) and Azone  (n512).

Fig. 6. Diffusional pathways for polar (e.g., water) and nonpolar molecules through (a) the crystalline phase (s), (b) the lamellar liquid crystalline (L a ) and (c) the reversed hexagonal liquid crystalline (H II ) phases.

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tails towards the nylon threads at the interface [6, p.125].

4.1. Free fatty acid systems Condensed phase diagrams of dry free fatty acid– soap mixtures show a complex phase behaviour in that molecular compounds containing both the acid and the soap with clearly unique crystal unit cells are formed below the chain melting temperature. Extensive work has been done on fatty-acid–potassium soaps over the years, and although the amount of work on the corresponding sodium soaps is minor some useful information can be found in the literature. From the PA–NaP phase diagram of McBain and Field [17, p. 332], it is evident that, except for an acid and a soap crystal, both an acid–soap crystal (AS, fSoap 50.50) and an acid–soap 2 crystal (AS 2 , fSoap 50.67) is formed in the dry state. The Bragg spacings of acid–soap crystals comply with d 5 ˚ where n is the number of carbons in 2.54n 1 4.7 A, the hydrocarbon chain [17, p. 333]. Consequently, ˚ (cf. Table 6: d(L a / s)546.6 and d(C 16:0 )545.3 A ˚ 46.9 A). According to Small [17, p. 333], the thickness of the COOH–COOK polar layer is ap˚ which should be compared to 4–5 proximately 6 A, ˚A for two COOH and 8–9 A ˚ for two COOK. Hence, if this is applicable to the protonated fatty acid crystal, assumed to be in its most stable conformation, the C-form (88 chain tilt), we expect a layer ˚ (cf. Table 6: d(s)543.3 A). ˚ spacing of d(s)543.4 A. Normally, crystalline phases composed of free fatty acids do not swell in water [6, p. 19]. Adding water to the system at room temperature should thus neither affect the Bragg spacings for the PA crystal nor the Bragg spacings for the acid–soap crystals [17, p. 337]. Potassium palmitate forms a homogeneous gel phase above approximately 20 wt% water at 258C [17, p. 329] and data from Vincent et al. [17, p. 330] indicate that below 20 wt% water layer ˚ and 34 A ˚ are to be expected spacings of around 37 A for the soap crystal and the gel phase respectively. The same behaviour should be expected with NaP. Our results (Table 6) suggest that ‘‘s’’ is a mixture of coexisting NaP:PA and PA crystals and water (0 , fSoap ,0.50, fSoap 1 fAcid 51). We do detect a lamellar crystalline phase in all the samples con-

taining ‘‘s’’ with a lattice parameter close to the ˚ for pure PA-crystals. Furthermore, estimated 43.4 A a second crystalline phase, the SAXD-pattern of which corresponds to the acid–soap, is occasionally visible on the films. The lamellar liquid crystal of OA:NaO (35:65 mol / mol) was found to possess close to infinite swelling, which in view of previously published results may seem surprising [7;17, p. 334]. However, the close resemblance with theory (Fig. 2) forms a firm base for further discussions. A conspicuously high water permeability of crystalline PA / NaP phases has earlier been recorded by Friberg and Kayali [18]. In fact, the registered water evaporation rates were of the same magnitude as those from a free water surface. This finding was attributed to dislocation lines and macroscopic fractures in the samples. In our system, the water flux through the crystalline phase (s) is statistically significantly higher than the water flux through the lamellar (L a ) and reversed hexagonal (H II ) liquid crystalline phases respectively (Table 2), which is in accordance with the results of Friberg and Kayali [18]. However, the high water flux through ‘‘s’’ may be due to the presence of hydrophilic crystals rather than macroscopic fractures. Since the reversed hexagonal (H II ) phase is oil continuous, a hydrophilic molecule like water is forced to pass through lipophilic regions during its path of diffusion. The same is true for the lamellar (L a ) phase, but here the oil phase is discontinuous. Consequently the reversed hexagonal phase should constitute a barrier for hydrophilic substances but not for lipophilic ones, while the lamellar phase does constitute a barrier both for hydrophilic and lipophilic compounds (Fig. 6). In both cases a water molecule has to pass through oil domains. For spacefilling reasons, the oil regions are thinner in the reversed hexagonal phase (Fig. 6). However, approximately the same diffusional resistance is experienced between the two phases and, apparently, the difference in lipid double layer thickness between the lamellar and the reversed hexagonal phase has only a minor influence on the barrier capacity towards water permeation.

4.2. Mixed free fatty acid systems Mixtures of partly neutralised OA and PA acids

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have previously been studied elsewhere. Engblom et al. [7] observed that when mixing the H II liquid crystal with the crystalline mixture (s), at a total water content of 32 wt%, the hexagonal phase (H II ) absorbed water to its swelling limit. In this study, various proportions of ‘‘s’’ have been mixed with the lamellar (L a ) liquid crystalline phase. The total available amount of water for swelling the liquid crystal, fw , is calculated from:

fw 5 0.32 /(x La 1 0.32(1 2 x La ))

(5)

where x La is the weight fraction lamellar phase in the mixture. The result is plotted in Fig. 3 (solid line) together with X-ray data (Table 6) to illustrate the swelling behaviour of the lamellar phase in the fatty acid mixtures (Fig. 2). Here it is evident that a significant, however not complete, swelling does occur; the swelling being more complete the larger the weight fraction of the lamellar (L a ) liquid crystalline phase. One possible explanation of the observed behaviour may be that L a and ‘‘s’’ contains fatty acids neutralised to different degrees and therefore a charge redistribution to an equilibrium mean can be expected. The rather rough-cut formula below, plotted in Fig. 3, gives an idea of what to expect:

fSoap 5 0.65x La 1 0.41(1 2 x La )

(6)

where fSoap is the average volume fraction of sodium soap in the mixture. A change in the degree of neutralisation of the fatty acid may induce a phase transition that can endanger the interpretation of our water flux results. However, except for the change in maximum water swelling of the lamellar phase (which is probably explained by the shape of the lamellar region in the phase diagram and the orientation of the related tielines) no morphological changes could be detected. For the OA-based liquid crystal, the major concern (especially with the mixture containing a small amount of the fluid phase having a low average degree of neutralised fatty acids) is a phase transition from lamellar (L a ) to reversed hexagonal (H II ) phase. From the SAXD-patterns and the lattice parameters of Table 6 it is evident that no such transition has occurred. Furthermore, varying the degree of neutralisation of the OA within the lamellar region hardly affects the lattice parameter at

223

all (Fig. 2). In the crystalline part there is a potential risk that an increase in average neutralisation could transform the system from one three-phase mixture into another, i.e., from NaP:PA and PA crystals to NaP2 :PA and NaP:PA crystals plus water (where the water is partly absorbed by the liquid crystal). However, in all diffraction patterns given in Table 6, the spectrum referred to the PA-crystal is present and we therefore conclude that no such phase transition has occurred either. In other words, mixing the lamellar (L a ) liquid crystalline and the crystalline (s) phases in the amounts we have used here does not induce phase transitions into other lipid structures. While undertaking the water flux measurements a significant water loss was observed before reaching steady state in all lipid samples (Table 2). Therefore any attempt to correlate experimental data to diffusion theory should include a correction for the enforced changes in dimensions of the respective lipid phase. The data in Table 7 are calculated from experimental data on the water loss and lattice parameter (of the liquid crystalline part) for each system. The water flux through the two-phase mixtures decreases statistically significantly with increasing relative amount of lamellar (L a ) to crystalline phase Table 7 Water loss in all lipid samples with corrections for the enforced changes in dimensions of the respective lipid phase; the data are calculated from experimental data on the weight loss (95% CI of mean) and lattice parameter of the liquid crystalline part for each system System

Weight loss (%)

fw

a ˚ (A)

l ˚ (A)

dw ˚ (A)

La

0 9.763.3 0 13.663.4 0 17.264.7 0 18.162.2 0 11.963.4 0 8.064.1

0.32 0.22 0.32 0.18 0.32 0.15 0.73 0.64 0.50 0.49 0.52 0.40

44.8 (e) 40.5 49.2 (e) 37.2 n.a.

15.2

n.a.

14.4 8.6 27.8 15.8 n.a.

112.3 (e) 83.3 61.0 (e) 58.9 62.8 (e) 50.4

15.2

81.9

15.2

30.6

15.2

32.4

H II s La / s 1 / 9 La / s 3 / 7 La / s 5 / 5

10.7 a

a ˚ l(H II ) 5 0.5a(1 2 œFw ) 5 ha 5 49.2, Fw 5 0.32j 5 10.7 A; a 5 2l /(1 2 œ(Fw )) [15]. Abbreviations: e, experimental; n.a., not applicable.

´ , J. Engblom / Journal of Controlled Release 63 (2000) 213 – 226 L. Norlen

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(s). At 50 wt% L a the water flux does not differ from that of a pure lamellar phase (mean516.4 g m 22 h 21 for L a / s (50 / 50), Fig. 4, compared to 18.762.8 g m 22 h 21 (95% CI of mean) for pure L a , Table 2). The water swelling of the lamellar phase is less complete the smaller the ratio L a / s (Table 6, Fig. 2). Consequently, for low relative amounts of lamellar phase there exist excess water that is free to evaporate from the sample. In summary, our results indicate that the morphology of the water compartment within the lipid mixtures may have strong implications for the occlusive capacity of polymorphic lipid systems.

4.3. The skin lipid model system and Azone  X-ray diffraction revealed a crystalline phase in equilibrium with a liquid crystalline phase in the skin lipid model system. The crystalline phase showed orthorhombic hydrocarbon chain packing (two strong ˚ and 4.2 A), ˚ in agreement with X-ray lines at 3.8 A ˚ data from human stratum corneum [19]. The 44 A diffraction peak (Table 8) is attributed to a crystalline packing of ceramides with fatty acids of C 16 –C 18 hydrocarbon chain length, i.e., approxi˚ mately eight carbons shorter (approximately 10 A) than endogenous human stratum corneum ceramides with amide linked fatty acids of C 24 –C 26 hydro˚ [21]). The crystalline carbon chains [20] (a564 A region is not affected by adding Azone  to the samples (Table 8). However, Azone does induce a transformation of the liquid crystalline phase, through a possible cubic phase (Pm3n or P43d), into a lamellar phase of higher water content in equilibrium with an oil phase (L 2 , i.e., reversed micelles) Table 8 SAXD-patterns of the skin lipid model mixture with 0, 15 and 25 wt% Azone  respectively xAzone 

˚ Bragg spacing (A)

(wt%)

1/1

1/2

1/3

1/1

1/2

1/3

0 15 25

48.7 58.2 a 76.0

24.4

16.3

44.5 44.3 44.6

22.2 22.2 –

14.7 14.8 14.7

a

38.4

The presence of two additional lines (40.7, weak; 37.0, medium) indicate a possible existence of a cubic phase (Pm3n or ˚ and thus a582.3 A. ˚ P43d [23]) with d 1 558.2 A

(Table 8). Consequently, the liquid crystalline phase responds to the addition of Azone  in a mode similar to the OA–sodium soap–water system [7]. The water loss (i.e., sample weight loss) is statistically significantly smaller for the skin lipid model than for the crystalline PA phase (s) despite the fact that the skin lipid mixture is mainly crystalline in nature. However, it is possible that the crystalline regions of this system is in reality a gel phase which may explain why the water loss is so conspicuously low in the skin lipid model mixture (6.464.5 wt% as compared to 17.264.7 wt% for the crystalline (s) sample (Table 2)). Our X-ray data cannot distinguish between a gel phase and a crystal, and hence further studies are required to solve this matter. As previously shown, Azone  favours reversed types of liquid crystalline phases [7]. This implies that Azone  should favour the penetration of lipophilic substances. However, Azone is also claimed to favour penetration of water soluble substances and to increase the water content of the stratum corneum when applied to skin [22]. In fact, Azone  has been shown to simultaneously induce reversed micelles and an extremely hydrated lamellar phase when added to a liquid crystal in the partly neutralised OA system [7]. If Azone  creates water rich domains it would be expected to give an increased water penetration, but this is not the case since the water flux dramatically decreased with increasing amount of Azone  (Fig. 5). The decreased water penetration cannot be explained by an increased sample thickness induced by the increasing relative amounts of Azone  since no linear trend between sample weight and water flux was registered. The water flux through the skin lipid model mixture, containing Azone (25 wt%), is surprisingly low (water evaporation of 10.2 g m 22 h 21 (mean, n52, Fig. 5)) as compared to the bare skin lipid model mixture (20.262.8 g m 22 h 21 , 95% CI of mean, Table 2). Furthermore, the water flux is far below the 95% CI of mean for both the lamellar (18.762.8 g m 22 h 21 , 95% CI of mean, Table 2) and the reversed hexagonal (16.664.7 g m 22 h 21 , 95% CI of mean, Table 2) liquid crystals. Consequently, one cannot exclude an additive occlusive effect of Azone  beyond the effect of a liquid crystalline phase. This is in agreement with the in vitro situation where it has been shown that Azone  has occlusive effects on

´ , J. Engblom / Journal of Controlled Release 63 (2000) 213 – 226 L. Norlen

water penetration through isolated stratum corneum [12]. However, further studies are needed to more closely characterise the structural basis for Azone  ’s occlusive effects on water diffusion of complex lipid systems like the human skin barrier.

5. Conclusions In our experimental model systems, the water flux through the crystalline phase is statistically significantly higher than the water flux through the liquid crystalline phases (L a and H II ), which do not differ between themselves. The lower water flux through the liquid crystalline phases can be explained by the fact that both are water discontinuous while the higher water flux through the crystalline phase can be explained by a continuous water phase, due to the presence of hydrophilic crystals. The reduction in water flux through the L a / smixtures with increasing relative amount of lamellar liquid crystalline phase (L a ) to crystalline phase (s) cannot exclusively be due to the relative reduction of the crystalline phase. Instead, our results indicate that the morphology of the water compartment of the lipid mixtures may have strong implications for the barrier function towards water diffusion of polymorphic lipid systems. The skin lipid model system consists of a crystalline phase in equilibrium with a liquid crystalline phase. The crystalline phase may be composed of ceramides with amid linked fatty acids of C 16 –C 18 hydrocarbon chain length, and is not affected by the incorporation of Azone  in the samples. On the contrary, by incorporation of Azone  into the samples, the lamellar liquid crystalline phase transforms, possibly through a cubic phase (Pm3n or P43d [23]), into a lamellar phase of higher water content in equilibrium with an oil phase (L 2 , i.e., reversed micelles). The water flux through the skin lipid model system decreased dramatically with the addition of Azone  . However, further studies are needed to more closely characterise the structural basis for the occlusive effects of Azone  on water diffusion. Our studies underline the complexity of performing and interpreting studies on skin lipid barrier models.

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Acknowledgements This investigation was made possible by a generous grant from the Swedish Council for Work life research (96-0486, 96-0110, 98-0552) and the Edvard Welander foundation. We are indebted to Margareta Andersson for outstanding technical expertise and assistance during the development of the system and during the collection of evaporimeter data throughout this work. For construction of the water evaporation chambers and for his never failing interest in the project we extend our gratitude to Lennart Wallerman. Furthermore, we thank Profes¨ and Kare ˚ Larsson sors Bo Forslind, Sven Engstrom for fruitful discussions as well as critical comments. ¨ for invaluable We also thank Professor Bo Lindstrom comments on the statistical calculations performed.

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