Influence of cholesterol on liposome fluidity by EPR

Journal of Controlled Release 68 (2000) 85–95 www.elsevier.com / locate / jconrel

Influence of cholesterol on liposome fluidity by EPR Relationship with percutaneous absorption L. Coderch a , *, J. Fonollosa a , M. De Pera a , J. Estelrich b , A. De La Maza a , J.L. Parra a a

b

IIQAB, ( CSIC), Jordi Girona 18 -26, 08034 Barcelona, Spain ` , UB, Av. Joan XXIII s /n, 08028 Barcelona, Spain Facultat de Farmacia Received 18 November 1999; accepted 1 March 2000

Abstract The influence of liposome composition on bilayer fluidity and its effect on the percutaneous absorption into the skin were investigated. Liposomes formed with saturated or unsaturated phospholipids (H-PC or PC) with varying amounts of cholesterol were prepared and their penetration behaviour into the stratum corneum was followed up by means of the stripping method. The order and dynamics of the hydrophobic domain of the vesicles were studied using electron paramagnetic resonance (EPR) methodology. Phospholipid composition and the amount of cholesterol exert a considerable influence on the penetration behaviour of the probe encapsulated in the liposomes. This behaviour is closely related to the fluidity characteristics of these liposomes studied by EPR. Therefore, a penetration mechanism of the vesicles into the skin, based on the incorporation of lipids into the skin lipids and on fluidity behaviour, is suggested.  2000 Elsevier Science B.V. All rights reserved. Keywords: Liposomes; Cholesterol; Skin; Stratum corneum; Percutaneous absorption; Stripping; EPR

1. Introduction Liposomal formulations have been widely studied in an attempt to enhance the efficiency of drug delivery via several routes of administration. The increased delivery of drugs applied to the skin entrapped in liposomes when compared with the application of the drug in a conventional delivery system is well documented [1–4]. However, there is little information on the mechanisms by which the *Corresponding author. Tel.: 134-93-400-6179; fax: 134-93204-5904. E-mail address: [email protected] (L. Coderch)

liposomes facilitate the transport of the entrapped molecules into the skin and on the manner in which the lipid composition of liposomes affects this. Some work has been done to elucidate the molecular mechanism by which the penetration of liposomal drugs is encouraged [5–7]. It seems that intact liposomes are mainly confined to the stratum corneum (SC) and do not penetrate deeply but enhance penetration of hydrophilic and especially lipophilic drugs [8]. However, some authors believe that defined liposomes can penetrate into the skin [9]. It has been suggested that one possible prerequisite for the penetration enhancement properties of phospholipid liposomes could be the fusion of the

0168-3659 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 00 )00240-6

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vesicle bilayers with the lipid layers of the stratum corneum [10]. SC lipid conformational changes and percutaneous absorption have been shown to be strongly correlated [11] and the action of several penetration enhancers has been related to their lipid fluidising effect [12]. The aim of this study was to investigate the connection between liposome bilayer fluidity and the transport of liposome-entrapped substance into the skin. Bilayer fluidity reflects the order and dynamics of phospholipid alkyl chains in the bilayer and is mainly dependent on its composition. Cholesterol has been shown to modify the order and mobility of the phospholipids in the bilayer [13,14] and other studies indicate that the cholesterol content might be of crucial importance for the effective delivery of liposome-entrapped substances into the skin [15,16]. In earlier works the ‘in vivo’ stripping technique was used to evaluate the percutaneous absorption of different liposome preparations [17]. The mechanism of liposome penetration enhancement was assessed by ATR-FTIR [11]. In the present study the influence of added cholesterol on bilayer fluidity is investigated using electron paramagnetic resonance (EPR) methods and the relationship with transport of an entrapped substance into the skin is evaluated using the stripping technique.

2. Materials and methods

2.1. Chemicals Reagent grade organic solvents, fluorescein sodium salt (NaFl), spin label 5-doxylstearic acid (5DSA) and 16-doxylstearic acid (16-DSA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Cholesterol (Chol) was obtained from Fluka Chemical Co. (Buchs, Switzerland). Lipoid S-100 (PC), whose main component is soybean phosphatidylcholine (.95%) (T m 2208C) and Lipoid S100H (H-PC), its hydrogenated form (T m 488C), were obtained from Lipoid GmbH (Ludwigshafen, Germany). All these chemicals were stored in chloroform / methanol (2:1) under freezing temperatures until use. Tris (hydroxymethyl)-aminomethane (Tris buffer) supplied by Merck (Darmstadt, Germany) was prepared as 5 mM Tris buffer adjusted to pH

7.40 with HCl, containing 100 mM of NaCl. Boric acid, potassium chloride, sodium hydroxide, sodium chloride, sodium dodecyl sulphate and hydrochloric acid were supplied by Merck.

2.2. Liposome preparation and characterisation Phospholipids (PC or H-PC) and cholesterol were dissolved in chloroform / methanol 2:1 (v / v) and appropriate volumes were combined to obtain the different lipid compositions. Lipid mixtures were evaporated to dryness in a round bottom flask in a rotatory evaporator under reduced pressure at 608C to form a thin film on the flask. For skin penetration studies, the liposomes were formed as follows. The film was hydrated with saline solution of NaFl (155 mM NaCl, 2.66 mM NaFl) to yield a final concentration of lipid of 12 mM. Multilamellar vesicle liposomes (MLV) were formed by constant vortexing for 5 min on a vortex mixer and sonication for 5 min in a bath sonicator (514 ECT Selecta; Selecta S.A., Barcelona, Spain). MLV were downsized to form oligolamellar vesicles by extrusion through polycarbonate membrane filters (Nucleopore Corp., Pleasanton, CA, USA) of variable pore size, 0.8, 0.4 and 0.2 mm under nitrogen pressures of up to 55310 5 N m 22 at 378C in an Extruder device (Lipex Biomembranes, Vancouver, Canada). Liposomes containing the encapsulated probe were purified twice by centrifugation in an ultracentrifuge TFTs 70.13 rotor of a Centrikon T-1170 (Kontron, Milan, Italy) by adding the same volume of saline solution and by centrifuging for 30 min at 40,000 rev. / min (180,0003g) at 78C and discarding the same supernatant volume. With this procedure the yield of fluorescein encapsulation was about 35% and liposome samples with a suitable lipid (|12 mM) and fluorescein (|1 mM) content were obtained to be analytically followed up during the skin penetration studies. Determination of vesicle size distribution in volume average diameters was carried out at 258C by dynamic light scattering, employing an Autosizer IIc photon correlation spectrometer (Malvern Instruments Limited, Malvern, UK). The medium in the sizing of liposomes was 155 mM NaCl. The NaFl content was determined with a spectrophotometer (Hitachi U-2000; Hitachi, Tokyo,

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Japan) at 208C by adding 1.5 ml of saline solution and 1.5 ml of SDS-borate buffer (4%, pH58.9) to 100 ml of liposomes to break them and release the NaFl ( l5493). For EPR studies, liposome samples were labelled with the doxyl nitroxides 5-doxylstearic acid (5DSA) or 16-doxylstearic acid (16-DSA), adding the spin label solution to the lipids before taken to dryness. Liposomes were formed as described above to achieve a final lipid concentration of 10 mg / ml and label (5-DSA or 16-DSA) concentration of 33 10 24 M in Tris buffer.

2.3. Penetration studies The penetration behaviour was studied ‘in vivo’ in accordance with the stripping method [18] as previously reported [11,17] applying 2.66 mM of NaFl in saline solution (155 mM NaCl) or encapsulated in different liposome formulations with 12 mM of the following lipid mixtures: PC 100%; PC / Chol 80 / 20; PC / Chol 60 / 40; H-PC 100%; H-PC / Chol 80 / 20; H-PC / Chol 60 / 40. Six Caucasian volunteers, females aged from 23 to 40 years, participated in the study. All the subjects were in good health and had no history of dermatological diseases. The experiments were carried out on the forearms of the volunteers. Topical application assays and the stratum corneum strippings were carried out in a conditioned room at 208C with a relative humidity of 60%. The subject was allowed to become acclimatised under these conditions for 15 min, prior to the test. The volume applied, using an Exmire microsyringe (ITO Corp., Fuji, Japan), was 5 ml. Preparations were applied onto areas of 4 cm 2 of the central forearm. Each formulation performed in duplicate was applied three times to each volunteer. The application area was delimited by an adhesive cell. A Dow Corning Medical Adhesive Silicone (Dow Corning, Brussels, Belgium) was placed around the area in order to avoid contamination of neighbouring tape strips and to prevent lateral diffusion of the sample, even though we have to be conscious of a possible lateral diffusion at the inner part of the skin. According to the stripping technique [18], after 30 min of contact, 15 successive tape strips of the SC

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were performed with Scotch MagicE 810 adhesive tape (3M, Cergy, France) under defined conditions (pressure: three times with a 1 kg roller; rapid stripping off; same investigator). The 15 tape strips are sufficient to remove the stratum corneum layers which in the forearm are about 10 to 15 mm thick. The extraction of the NaFl content in the different strips was done by introducing the strip or groups of strips (1st, 2nd and 3rd strip separately and 4–6, 7–9, 10–12 and 13–15 joint in these groups of three strips) into 5 ml of methanol and then shaking for 1 h before adding 5 ml of borate buffer, pH59.0 to increase the fluorescence. Fluorescence of these samples was measured by spectrofluorimetry (Shimadzu RF-540, Shimadzu, Kyoto, Japan) at ¨ Denmark) at 208C (Thermocirculator Heto Birkerod, lexc 5493 nm and lem 5515 nm. The NaFl content was determined with the help of the corresponding calibration curves. Normal distribution of each group of data was verified by the non-parametric Kolmogorov–Smirnov test. One-way analysis of variance (ANOVA) was used to determine the significant differences between the values obtained from the different treatments. (Significance level accepted P,0.05).

2.4. Electron paramagnetic resonance Electron paramagnetic resonance (EPR) techniques are used to monitor the molecular dynamics of lipids. This spectroscopic technique allows us to detect changes in the spin tropic movement of an unpaired electron. Biomolecules such as PC, H-PC or Chol, which do not contain unpaired electrons, can be studied by EPR when they are surrounded or chemically bonded to a stable free radical. This radical or spin label produces a sharp and simple EPR spectrum that yields information about the molecular environment of the label [19,20]. To this end, we used 5-doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA). These compounds consist of a radical group (doxyl) and a hydrocarbonated chain (stearic acid) which acts as a radical support (Fig. 1). Since spin probes are oriented and linked like the lipids in the bilayer [21,22], the radical in the 5 or in the 16 position of the alkyl chain will determine local motional profiles in the

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Fig. 1. Molecular formula of the spin probe 5-doxyl stearic acid (5-DSA), EPR spectrum and maximum hyperfine splitting 2A max .

two main regions of the lipidic bilayer, near the polar head group (5-DSA) or at the end of the hydrophobic chain (16-DSA). The EPR spectrum of 5-DSA incorporated into the lipidic membranes, shows an anisotropic motion, and the fluidity of the membrane can be estimated from the outermost separation between the spectral extrema, the maximum hyperfine splitting (2A max ) (Fig. 1). The value of 2A max reflects the rotational motional freedom of the phospholipids close to the polar head groups in the bilayer. This value increased with the decrease in fluidity [14]. The EPR spectrum of 16-DSA incorporated into the lipidic bilayer reflects an isotropic motion of the acyl-chain. In this case, the rotational correlation time (t ) is the parameter that can be used to measure the motion of the phospholipid acyl-chains near the hydrophobic end [23,24]. This empirical parameter is calculated with the equation expressed in Fig. 2 based on the first-derivative of the electron spin

resonance spectrum and, as in the case of 2A max , t increases with the decrease in fluidity. In the present work, EPR measurements were performed using a Varian E-109 spectrometer (Varian Associates, Palo Alto, CA, USA) working in the X-band and equipped with a Varian E-257 temperature control unit. The hyperfine splitting of the labelled liposome samples was determined with 100G scan width, 3360G of field intensity, 8 min scan time and 5 mW microwave power. The fluidity of liposome samples was determined by calculating the rotational correlation time of the different liposome formulations with 3310 24 M of 16-DSA, label concentration and 10 mg / ml of the following lipid mixtures: PC 100%; PC / Chol 80 / 20; PC / Chol 60 / 40; H-PC 100%; H-PC / Chol 80 / 20; H-PC / Chol 60 / 40. The EPR measurements were carried out with a standard quartz EPR tube 21 cm in length and with an inner diameter of 1.5 mm. The spin-labelled

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Fig. 2. Molecular formula of the spin probe 16-doxyl stearic acid (16-DSA), EPR spectrum and mathematical formula to obtain the t parameter.

liposome sample was placed in the EPR cavity for 2 min before the data acquisition to ensure thermal equilibration. After each EPR measurement, the sample was removed from the cavity and placed in the water bath at the same temperature until the cavity reached a new temperature equilibrium. Data were collected in the temperature range interval from 20 to 508C. Line widths 2A max and W0 , in Gauss (G) and heights of the mid- and high-field lines, h 0 and h 21 , respectively, were obtained from the first-de-

rivative of each absorption spectrum, and the rotational correlation time (t ) was calculated applying the equation from Fig. 2.

3. Results and discussion The liposomes were formed as described in Section 2. The values of size, polydispersity and encapsulation efficiency are shown in Table 1. In most

Table 1 Size and encapsulation characteristics of different liposomal compositions. Values are given as mean6S.D. (EE, encapsulation efficiency) Composition

Size (nm)

Polydispersity

EE (mmol / mol lipid)

PC 100% PC / Chol 80 / 20 PC / Chol 60 / 40 H-PC 100% H-PC / Chol 80 / 20 H-PC / Chol 60 / 40

19668 214611 15867 550618 224641 21262

0.1660.04 0.1760.02 0.1760.01 0.4160.05 0.2460.08 0.2160.01

50.82619.83 52.40617.55 94.2869.24 49.57622.11 61.77612.03 95.71617.31

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cases, vesicle populations were monodisperse with sizes commensurate with the last pores of the membrane used in the extrusion. Only in the case of H-PC 100% was a bimodal distribution found even though 89% of vesicles have a diameter of 210 nm and 11% 735 nm. Encapsulation efficiencies increase with the amount of cholesterol in the two cases with PC and H-PC. The stripping method was applied to six volunteers using the sodium fluoresceine encapsulated in the six different liposome formulations mentioned above and free NaFl in saline solution (155 mM NaCl) as a control. The amount of NaFl recovered from every strip or group of strips, expressed in percentage with respect to the total dose applied was determined, taking into account that no background fluorescence was obtained in the analysis of extracts from the tape strips of native skin samples (Table 2). The NaFl content in the first strip accounts for the non-penetrated content. Therefore, the high values of the NaFl content in the first strip suggest a strong SC barrier effect. The varying amounts of NaFl found in the first strip indicate the reinforcement of the SC barrier effect when the H-PC formulation is employed as well as the weakness of the SC barrier effect when the PC liposomes are applied, compared with the control, NaFl in saline solution. The presence of cholesterol in the formulations balances these two contrary behaviours. The presence of only 20% of cholesterol increases, in the case of PC, and decreases, in the case of H-PC, in about 5% the amount of NaFl found in the first strip, reaching a

similar value of 25% for all the liposomes with cholesterol. The total amount of NaFl in the SC (from the 2nd to the 15th strip) was also calculated to determine the reservoir capacity of this layer. Similar amounts of NaFl were detected in the stratum corneum when PC and PC / Chol 80 / 20 were employed. These were statistically different (P,0.001) from the values obtained when H-PC or H-PC / Chol 80 / 20 were applied. In this case, a ratio of 60 / 40 phospholipid / cholesterol is necessary to increase or decrease the amount of NaFl in the stratum corneum. If it is assumed that the amount of non-analysed NaFl penetrated into the deeper layers of skin, the results obtained of the total percentage of analysed NaFl by adding all the strips could reflect the highest penetration of PC liposomes and the influence of cholesterol. The penetration order obtained was PC100%, PC / Chol 80 / 20, control NaFl (saline), PC / Chol 60 / 40, H-PC / Chol 60 / 40, H-PC / Chol 80 / 20 and H-PC 100%. In order to follow up the penetration profile of NaFl in the different strips, the accumulative percentage of NaFl recovered from every strip or groups of strips was calculated. A regression analysis was performed and the functions y 5 a log x 1 b were obtained. Parameters a, b and the determination equation coefficient R 2 were obtained and the regression curves and the experimental points are also expressed in Fig. 3 logarithmically for the sake of simplicity. It should be noted that the accurate fit of the

Table 2 Percent recovered of applied dose in the different strips (mean values6S.D.) Strip no.

NaFl saline

PC 100%

PC / Chol 80 / 20

PC / Chol 60 / 40

H-PC 100%

HPC / Chol 80 / 20

H-PC / Chol 60 / 40

1 2 3 4–6 7–9 10–12 13–15 Total% SC%

22.564.2* 12.362.8 7.262.0 12.461.7 7.362.2 4.261.3 3.561.2 69.4610.9** 46.968.1**

19.864.6* 10.462.3 7.162.1 11.063.1 6.661.9 4.861.6 2.960.8 62.569.1** 42.7610.3**

24.765.6 11.463.0 6.061.6 11.164.2 6.261.9 4.561.3 3.260.3 67.1614.0** 42.4610.2**

25.365.5 12.061.7 7.762.2 13.662.3 8.361.5 5.661.5 4.661.7 77.068.7 51.765.9

30.465.4 20.164.2 13.261.2 14.763.7 7.162.1 4.060.9 2.361.2 91.668.6 61.266.3

26.265.7 16.962.2 10.061.3 16.961.8 9.362.0 5.861.6 4.260.9 89.367.5 63.162.3

23.868.5 15.064.4 9.862.1 15.162.0 7.461.3 4.861.1 3.460.8 79.3612.8 55.665.4

*

P,0.005 significantly lower than H-PC 100%. P,0.001 significantly lower than H-PC 100% and H-PC / Chol 80 / 20.

**

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Fig. 3. Relationship between the cumulative percentage of NaFl in the stratum corneum and the logarithm of the strip number.

experimental points has high correlation coefficients in most cases. The value of constant b, which accounts for the non-penetrated content, is low for the PC liposome, high for the H-PC liposome and has almost the same intermediate value for the control and the other formulations containing cholesterol. Slope a, which accounts for the gradient of fluorescein content inside the stratum corneum layers, is higher for the H-PC 100% and H-PC / Chol 80 / 20, suggesting its high affinity with this layer. The slight or null influence of the characteristics of the different liposomal compositions especially encapsulation efficiency from Table 1 to the results of skin penetration discussed above in Table 2 are in accordance with published data [16,25]. Furthermore, the fact that the penetration mechanism is not very related to the encapsulation capacity would support the non-penetration of liposome structures as closed vesicles suggesting their possible fusion with the stratum corneum lipids. Some authors have demonstrated that the incorpo-

ration of some enhancers or fluid lipids into the intercellular domains could interfere with the barrier function of the stratum corneum [12,26], thus lowering its phase transition temperature T m and increasing its fluidity, facilitating diffusion and transport through the skin. EPR is the most useful technique for determining fluidity and the structural changes of the lipid bilayers of liposomes. A direct relation is established between the spin movement and the viscosity in the area in which the label is surrounded, with the result that it can be related to the fluidity of the labelled liposome. The spin probes are oriented and linked like the lipids in the bilayer. Changes in membrane fluidity caused by cholesterol have been reported to be somehow different near the hydrophobic end of the acyl chains from that observed near the polar groups [14]. Therefore, although a number of studies have indicated that cholesterol greatly interferes with the most hydrophobic part of the bilayer [13,27], the two doxyl

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nitroxide 5-DSA and 16-DSA with the radical at the beginning and at the end of the hydrophobic chain were chosen to monitor changes in the fluidity in these two regions. The EPR spectra of the PC and H-PC liposome samples with varying amounts of cholesterol were obtained in duplicate at different temperatures between 20 and 508C paying special attention to the skin temperature region (358C). The maximum hyperfine splitting 2A max in G for 5-DSAlabelled samples and the correlation times t were calculated and plotted versus the temperature for each sample (Figs. 4 and 5). Since maximum hyperfine splitting and rotational correlation time are inversely related to the fluidity, a logical decrease in the 2A max and t parameters were observed for all the samples when the temperature rose. The high values obtained for the H-PC liposomes compared with the ones for the PC liposomes suggested less motion of the saturated lipids in the bilayer in the two domains of the bilayer. The effect of cholesterol in the fluidity and the thermotropic behaviour of the liposome-labelled

samples is similar in the two regions of the bilayer evaluated. However, a higher amount of cholesterol seems to be needed to affect the polar region than the hydrophobic region. A gradual decrease in fluidity (increase of 2A max and t ) can be observed as the amount of cholesterol increases in the case of the PC liposomes at all the temperatures studied. This is more marked for t in the most fluid hydrophobic region of the membrane (Fig. 5). The varying effect of the cholesterol as a function of the temperature should be pointed out for the H-PC liposomes in the two regions. At low temperatures the increase in the amount of cholesterol leads to an increase in fluidity (decrease in 2A max and t ). However, the contrary effect is observed at temperatures exceeding 458C, which is the transition temperature of H-PC (Figs. 4 and 5). This effect of cholesterol fluidising the membranes at temperatures below T m and condensing them at temperatures above T m has been reported [14,28]. This could be due to the formation of an intermediate gel state

Fig. 4. Maximum hyperfine splitting 2A max of 5-DSA of vesicles with different lipid composition, versus temperature.

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Fig. 5. Rotational correlation time t of 16-DSA of vesicles with different lipid composition, versus temperature.

caused by a hydrophobic interaction of cholesterol with the fatty acyl chains of saturated PC [29]. The fact that the skin temperature is above the T m of the PC and below the T m of the H-PC accounts for the different effect of cholesterol on the fluidity of the sample. The results obtained for the penetration study of the NaFl amount in the stratum corneum layers seems to be more related to the fluidity behaviour of the less fluid region of the liposome bilayer, since the contradictory effect of cholesterol is obtained meaningfully when it is present at 40%. Moreover, the results obtained for the total skin penetration are clearly related to the fluidity behaviour of the most fluid hydrophobic region of the bilayer. The correlation obtained between the total skin penetration results and the fluidity of the sample at skin temperature lends support to a penetration mechanism of the sample through the skin on account of the fluidity behaviour. The order and dynamics of the phospholipid alkyl chains in the bilayer depend on their composition. The bilayer

fluidity and cholesterol content may be of crucial importance for the effective delivery of liposomeentrapped substances into the skin. Most studies show that the extent of interaction between lipid vesicles and skin is highly dependent on the lipid composition of liposomes [5,12,16]. The effect of cholesterol on the fluidity of unsaturated and saturated phospholipid bilayers is consistent with our results [13,26,28]. However, the direct relationship of the lipid fluidity with skin penetration is not so clearly defined. The shape of the lipids, fusogenic cone versus rod-shaped lipids seems to be, for some authors, the main reason for inducing skin penetration. In the light of our results, considerable penetration was obtained for unsaturated lipid liposomes. However, bearing in mind that fluidity is not usually evaluated, no clear influence of cholesterol is concluded [5]. The relationship between the bilayer fluidity of liposomes and the transport of encapsulated substance into the skin has also been studied with saturated phospholipids with an ‘in vitro’ EPR

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method [16]. As in our case, the addition of at least 20% of cholesterol has been reported to significantly increase the transport of a liposome-entrapped substance. This study supports the mechanism whereby the transport of the encapsulated probe is not directly related to the solid ordered or liquid disordered state of phospholipids but to the presence of the heterogeneous structure of liposome bilayer with several coexisting domains. Thus, increasing the amount of cholesterol always favours penetration of the saturated HPC liposomes studied. The evaluation of bilayer fluidity was based on 2A max measures with a labelled spin probe near the polar group of the bilayer [16] and our results confirm that the effect of cholesterol on the bilayer fluidity is similar though not the same in the different regions. Other studies [14] support the more marked effect of cholesterol evaluated near the hydrophobic end which resulted in fluidization below T m and condensation above T m . The fluidity of stratum corneum lipids has been shown to be dramatically raised following treatment with fluid unsaturated phospholipids, whereas it is significantly decreased by solid saturated phospholipids. Their penetration behaviour through the stratum corneum or follicles has been directly related to this [26]. Since interaction of different phospholipid liposomes with lipid model mixtures for stratum corneum lipids showed different phase characteristics [10], our results suggest that the different role of vesicles in percutaneous absorption could be attributed to a preliminary incorporation of lipids into the intercellular domains and to a modification of the fluidity of skin lipids directly related to the fluidity behaviour of the vesicles.

4. Conclusion In the light of our findings it is possible to confirm the reliability of the ‘in vivo’ stripping technique, which yields the following skin penetration order decrease for the different vesicles assayed; PC100%, PC / Chol 80 / 20, PC / Chol 60 / 40, H-PC / Chol 60 / 40, H-PC / Chol 80 / 20 and H-PC 100%. The EPR technique was employed to determine fluidity and the structural changes near the polar region and in the most hydrophobic region of the lipid bilayers at varying temperatures. The high

values of the 2A max and t parameter presented for the H-PC liposomes compared with those for PC liposomes indicate less motion of the saturated lipids in the two regions of the bilayer. The cholesterol increases the fluidity of the bilayer at temperatures below T m and decreases the fluidity at temperatures above T m which is more marked in the most hydrophobic region of the bilayer. The fact that the skin temperature is above the T m of the PC and below the T m of the H-PC accounts for the different effect of cholesterol on the fluidity of the sample. The correlation obtained between the skin penetration results and the fluidity of the sample at skin temperature lends support to a penetration mechanism of the sample through the skin on account of the incorporation of lipids in the intercellular domains and the modification of the fluidity of skin lipids which can be directly related to the fluidity behaviour of the vesicles.

Acknowledgements We acknowledge the expert assessment of L. Julia´ ´ and the technical help of A. Dıez in the EPR experiments. We wish to thank the six volunteers who participated in this trial. We are indebted to N. Perez-Cullell and G. von Knorring for their expert assistance and to the DGICYT Program (PM-980119) for financial support.

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