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Preparation of liposomes containing Ceramide 3 and their membrane characteristics
Colloids and Surfaces B: Biointerfaces 20 (2001) 1 – 8 www.elsevier.nl/locate/colsurfb
Preparation of liposomes containing Ceramide 3 and their membrane characteristics Tomohiro Imura a, Hideki Sakai a,b, Hitoshi Yamauchi a, Chihiro Kaise a, Kozo Kozawa a, Shoko Yokoyama c,*, Masahiko Abe a,b b
a Faculty of Science and Technology, Science Uni6ersity of Tokyo, 2641, Yamazaki, Noda, Chiba 278 -8510, Japan Institute of Colloid and Interface Science, Science Uni6ersity of Tokyo, 1 -3, Kagurazaka, Shinjuku, Tokyo 162 -0825, Japan c Kyoritsu College of Pharmacy, 1 -5 -30, Shibakoen, Minato-ku, Tokyo 105 -8512, Japan
Received 2 February 2000; accepted 3 March 2000
Abstract Liposomes composed of Ceramide 3, [2S,3S,4R-2-stearoylamide-1,3,4-octadecanetriol], and L-a-dipalmitoylphosphatidylcholine (DPPC) were prepared by varying the amount of Ceramide 3, and the effects of Ceramide 3 on the liposome formation, particle size, dispersibility, microviscosity and phase transition temperature were examined by means of a microscopy, a dynamic light scattering method, a fluorescence polarization method, a differential scanning calorimetry (DSC) and so on. All the DPPC was able to contribute to the formation of liposomes up to 0.130 mol fraction of Ceramide 3. The particle size of liposomes was almost unaffected by the addition of Ceramide 3. The dispersibility of liposomes containing Ceramide 3 was maintained for at least 15 days. The microviscosity of liposomal bilayer membranes in the liquid crystalline state was increased with increasing the mole fraction of Ceramide 3, while that in the gel state was independent of the mole fraction of Ceramide 3. The phase transition temperature from gel to liquid crystalline states of DPPC bilayer membranes was shifted upwards with the addition of Ceramide 3, indicating a cooperative interaction between DPPC and Ceramide 3 molecules. However, a sharp DSC peak became broad and split at higher mole fractions of Ceramide 3, suggesting a phase separation in the mixed DPPC/Ceramide 3 liposomal bilayer membranes. These phenomena were suggested to be related to the previously observed fact for the mixed DPPC/Ceramide 3 monolayers that Ceramide 3 interacts with DPPC in the liquid-expanded phase with consequent phase separation accompanied with domain formation. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Ceramide 3; Phospholipid; Liposomes; Differential scanning calorimetry; Phase separation; Microviscosity
1. Introduction
* Corresponding author. Fax: +81-3-34345343. E-mail address: [email protected] (S. Yokoyama).
Human skin consists of the epidermis, dermis, subcutaneous tissue and stratum corneum [1]. Dermis is composed of more than 95% of phospholipid [1]. Epidermis consists of about 60% of
0927-7765/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 0 0 ) 0 0 1 4 9 - 1
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T. Imura et al. / Colloids and Surfaces B: Biointerfaces 20 (2001) 1–8
phospholipids and 40% of ceramides [1] and has a barrier function [2 – 5] to prevent an invasion of injurious substance to the health and the loss of the juices. The stratum corneum lipids construct the water-permeability barrier, in which the control of water permeability is essential for survival in any living system. The stratum corneum lipids are composed of: 50% ceramides, 5% cholesterol, 10% cholesterol esters and 20% free fatty acids [6]. A decreased level of ceramides in stratum corneum of atopic dermatitis has been found [5,7]. It has also been suggested that an application of small amount of ceramides may be effective to a dry skin dermatitis [6,9]. Manufacturing of ceramide product is, however, a difficult problem because ceramides can scarcely be dispersed in an aqueous medium. It is expected that the dispersibility of ceramides will be improved by the liposome formation with phospholipid. On the other hand, liposomal drug delivery systems have been researched widely for the purpose of reduction of drug toxicity and/or targeting of drugs to specific cells [8]. However, only a few studies have been done on the physicochemical properties of liposomes in contrast with many studies from the viewpoints of biochemistry and pharmacology. We think that the efficiency of liposomes as a ‘vessel’ in which drugs are included are also important, and we have studied the dispersibility, microviscosity and permeability of liposomes containing glycolipids [9], steroids [10], carboxyacyl phospholipids [11] or water-soluble polymers [12 – 15]. From these points of view, the formation of liposomes containing ceramide and their liposomal membrane characteristics were examined. In this study, Ceramide 3 was chosen as a ceramide among seven known ceramides [16,17]. We have previously studied the interaction of Ceramide 3 and phospholipid in the mixed monolayers by means of atomic force microscopy (AFM), and it has been found that Ceramide 3 interacts with phospholipid in the liquid-expanded film with consequent phase separation accompanied with domain formation [18]. So, a possible phase separation in liposomal bilayer membranes containing Ceramide 3 was also investigated by a differential scanning calorimetry and the results were dis-
cussed in connection with the previous results for the monolayers.
2. Experimental
2.1. Materials 2S,3S,4R-2-stearoylamide-1,3,4-octadecanetriol (Ceramide 3, 94% pure) as a ceramide was purchased from Cosmoferm Co. and was used without further purification. The structural formula of Ceramide 3 was shown in a previous paper [18]. L-a-Dipalmitoylphosphatidylcholine (DPPC, 99.6% pure) as a phospholipid was supplied from Nippon Oil and Fats Co., Ltd. Dicetylphosphate (DCP, 99.6% pure) as a charged material and 1,6-diphenyl-1,3,5-hexatriene (DPH, 98% pure) as a fluorescent probe were purchased from Sigma Chemical Co. Isotonic phosphate buffered saline (PBS), pH 7.4, was purchased from Nissui Pharmaceutical Co. All other chemicals were commercial products of reagent grade.
2.2. Preparation of liposomes containing Ceramide 3 The composition of liposomes containing Ceramide 3 was as follows: DPPC: Ceramide 3: DCP=10: (0 1.5):1. DCP was added to make DPPC bilayer membranes stable [19]. The liposomes were prepared according to the procedure of Bangham et al. [20]; DPPC, Ceramide 3 and DCP were dissolved in chloroform in a test tube; the solvent was then removed by blowing nitrogen gas into the test tube, and the redisual solvent was further dried overnight at room temperature in a desiccator under vacuum; PBS was added to this lipid film and warmed at 60°C above the phase transition temperature (41°C) of DPPC for 10 min; the test tube was then shaken vigorously on a vortex mixer, and the multilamellar vesicles (MLV) were obtained (liposome solution A). The multilamellar vesicles were sonicated (125 W, bath type: Branson B-220) for 1 h and subsequently extracted through a polycarbonate membrane filter (pore size 200 nm, Nuclepore Inc.) using an extruder (Lipex Biomembranes Inc.), and a homo-
T. Imura et al. / Colloids and Surfaces B: Biointerfaces 20 (2001) 1–8
geneous liposomal suspension in which almost uniform liposomal size was obtained (liposome solution B).
2.3. Determination of concentration of DPPC The concentration of DPPC in liposomes (liposome solution A) was determined by the choline oxidase-phenol method [21].
2.4. Obser6ation of liposomes Liposomes (liposome solution A) were observed using an inverted microscope with a transmitted light differential interference contrast attachment (IMT-2, Olympus Optical Co., Ltd.).
2.5. Column chromatography and thin-layer chromatography of liposome solution Column chromatography of liposome solution was performed on Sepharose CL-6b (Pharmacia Biotech Co.) with PBS as an eluent. The elution rate was 53 ml h − 1. Liposomes were detected by measuring the turbidity at 650 nm. The constituent lipids of liposomes fractionated in each fraction were qualitatively analyzed by thin-layer chromatography (TLC) with K60 thin-layer plate (Merck Co.) and chloroform/methanol (9:1) solution. The spots developed by TLC were detected with iodine reagent. The value of Rf for Ceramide 3 was 0.36.
2.6. Measurement of particle size of liposomes The particle size of liposomes (liposome solution B) was determined by a dynamic light scattering method. The distribution and time course of liposomal particle size were measured at 30°C using a NICOMP 380ZLS (Particle Sizing System Co.). The light source was a diode pump solid state laser (DPSS laser) with a wavelength of 535 nm and the scattering angle was 90°.
2.7. Measurement of micro6iscosity of liposomal bilayer membranes DPH as a fluorescent probe was dissolved in
3
tetrahydrofuran, the DPH solution was added to the dilute liposome solution B and incubated for 1 h at 37°C. The molar ratio of lipids to probe was 300:1. The microviscosity of liposomes was determined by fluorescence polarization (P), which can be calculated according to the following equation: P= (Ip − GIv)/(Ip + GIv) where Ip and Iv are the fluorescence intensities of the emitted light polarized parallel and vertical to the exciting light, respectively, and G is the grating correction factor [22]. The fluorescence intensities of Ip and Iv were measured at 30 58°C with a spectrofluorometer (RF-5000, Shimadzu Co., Ltd), and excitation and emission wavelengths were 350 and 450 nm, respectively. DPH exists in hydrophobic region and is able to evaluate the microviscosity around DPH in the liposomal bilayer membranes [23]. Fluorescence polarization is correlated to microviscosity near the fluorescent probes [24,25], which is calculated using the Perrin-Weber’s equation [26]. Microviscosity increases with increasing fluorescence polarization.
2.8. Differential scanning calorimetry (DSC) The phase transition temperature of liposomal bilayer membranes was measured with a differential scanning calorimeter (8240B, Rigaku Denki Co.). 20 mg of liposome solution (liposome solution B) was put in a sampling vessel, which is made of stainless steel (resistance to pressure at 50 atm, 3× 5 (F) mm for the vessel size), and then the vessel was sealed. The measurement conditions were 1 K min − 1 for the scanning rate, 30 60°C for the scanning range and 0.1 m cal s − 1 for the sensitivity.
3. Results and discussion
3.1. Effect of Ceramide 3 on formation of liposomes The effect of Ceramide 3 on the formation of liposomes was examined with an optical micro-
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T. Imura et al. / Colloids and Surfaces B: Biointerfaces 20 (2001) 1–8
Fig. 1. Micrographs of liposomes containing Ceramide 3. The mole fraction of Ceramide 3 is 0.09. The length of the black bar is 10 mm.
scope using the liposome solution A. Fig. 1 shows the micrograph of liposomes containing 0.09 mol fraction of Ceramide 3. The onion-like structure having an inner aqueous phase suggests the formation of MLV. Next the concentration of DPPC in the liposomes was determined, and the results are shown in Table 1. The DPPC amount at 100% means that all the DPPC is used to form liposomes. As is evident in Table 1, all the DPPC is able to contribute to the formation of liposomes containing Ceramide 3 up to 0.130 mol fraction of Ceramide 3, while not all the DPPC is utilized at higher mole fractions of Ceramide 3. Agglutination of Ceramide 3 was also visually observed for liposome solutions containing more than 0.166 mol fractions of Ceramide 3. It has been generally known [9] that more than 10 mol% of lipophilic additives bring about a destruction of liposomal structure.
3.2. Distribution of liposome size The elution curve of liposome solution B by column chromatography is shown in Fig. 2. Fig. 2
Fig. 2. Elution curve of liposomes by column chromatography. The mole fraction of Ceramide 3 is 0.09.
suggests that the liposomes are monodispersitive and that the particle size of liposomes is almost uniform. Liposome solution was fractionated in the fraction numbers 15–17. Furthermore, only in the fraction numbers 15–17, the existence of Ceramide 3 was confirmed, indicating that liposomes composed of DPPC and Ceramide 3 are indeed formed.
3.3. Effect of Ceramide 3 on particle size of liposomes The effect of Ceramide 3 on the particle size of liposomes (liposome solution B) was measured by varying the mole fraction of Ceramide 3. The distribution of liposomal particle size is shown in Fig. 3. The ordinate means the relative volume of particles when the volume of particles with 190 210 nm of diameter is defined as 1.0. No significant changes in particle size and distribution of particle size of liposomes containing Ceramide 3 (Xceramide 3 0.130) was found.
Table 1
Mole fraction of Ceramide 3 DPPC (%)
0 100
0.047
0.090
0.130
0.166
0.200
100
100
100
95
87
T. Imura et al. / Colloids and Surfaces B: Biointerfaces 20 (2001) 1–8
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Fig. 3. Distribution of particle sizes of liposomes containing various amounts of Ceramide 3. The mole fraction of Ceramide 3: (a) 0; (b) 0.047; (c) 0.090; (d) 0.130.
3.4. Effect of Ceramide 3 on dispersibility of liposomes The effect of Ceramide 3 on the dispersibility of liposomes (liposome solution B) was examined by measuring the time course of the liposomal particle size at 30°C, and the results are shown in Fig. 4. No significant changes in particle size of liposomes was observed for the system containing Ceramide 3 as well as for the DPPC alone system without Ceramide 3, indicating that the dispersibility of liposomes containing Ceramide 3 is maintained for at least 15 days.
3.5. Effect of Ceramide 3 on micro6iscosity of liposomal bilayer membranes The relationship between fluorescence polarization of DPH and temperature is shown in Fig. 5.
Fig. 4. Time courses of particle size of liposomes containing various amounts of Ceramide 3. The mole fraction of Ceramide 3: , 0; , 0.047; , 0.090; , 0.130.
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T. Imura et al. / Colloids and Surfaces B: Biointerfaces 20 (2001) 1–8
state was independent of the mole fraction of Ceramide 3. Ceramide 3 is likely to affect the microviscosity in the hydrophobic region of liposomal bilayer membranes in the liquid crystalline state. The results shown in Fig. 5 suggest that the intermolecular interactions in the liposomal bilayer membranes in the liquid crystalline state are strengthened and the packing density in the membranes becomes rigid by the addition of Ceramide 3.
3.6. Effect of Ceramide 3 on the phase transition temperature of liposomal bilayer membranes Fig. 5. Relationship between fluorescence polarization of DPH and temperature for liposomes containing various amounts of Ceramide 3. Symbols are the same as in Fig. 4.
Fig. 6. DSC curves of liposomes containing various amount of Ceramide 3. DSC curves were obtained under the conditions: scanning rate, 1 K min − 1; sensitivity, 0.1 m cal − 1 s − 1.
The fluorescence polarization of DPH in the DPPC bilayer membranes without Ceramide 3 suddenly decreased above 42°C, which agrees with the phase transition temperature of DPPC. The phase transition temperature was raised by the addition of Ceramide 3. The microviscosity of liposomal bilayer membranes in the liquid crystalline state was increased with increasing the mole fraction of Ceramide 3, while that in the gel
The DSC curves for the mixed DPPC/Ceramide 3 liposomal bilayer membranes were obtained by varying the mole fraction of Ceramide 3, and the results are shown in Fig. 6. Ceramide 3 indicated no peaks for at least 0100°C (the DSC curve was not presented in Fig. 6). This phenomenon is consistent with the fact [18] that Ceramide 3 forms only the liquid-condensed film as observed for the surface pressure measurement of Ceramide 3 monolayer. An endothermic peak at about 41°C corresponds to the gel-liquid crystalline phase transition temperature of DPPC. The phase transition temperature of DPPC bilayer membranes was shifted upwards with the addition of Ceramide 3, indicating that the liposomal bilayer membranes became thermostable by the addition of Ceramide 3. The shift of the main phase transition temperature to a higher temperature implies a cooperative interaction between DPPC and Ceramide 3 molecules, indicating also that Ceramide 3 is miscible with DPPC. The phase transition between the gel and liquid crystalline phases observed for phospholipid bilayers is related to the phase transition of phospholipid monolayers, from the liquid-expanded film to the liquid-condensed film [27]. An attractive interaction between Ceramide 3 and DPPC in the liquid-expanded films of the mixed DPPC/Ceramide 3 monolayers has been reported in our previous paper [18]. A sharp DSC peak observed for the DPPC alone system became broad in the mixed DPPC/ Ceramide 3 liposomal bilayer membranes. Furthermore, a DSC peak was noted to split at
T. Imura et al. / Colloids and Surfaces B: Biointerfaces 20 (2001) 1–8
higher mole fractions of Ceramide 3. The split peak is suggested to be caused by the phase separation in the liposomal bilayer membranes since the liposome solution used in this measurement was confirmed to be monodispersive as is shown in Fig. 2. We have shown the AFM images for the mixed DPPC/Ceramide 3 monolayers in the previous paper [18], in which elliptical domains and matrix have been found. Furthermore, new theory has been proposed in our paper [18]: the domain formation is not caused by the complete separation of one component from the other component as was observed by Dufrene et al. [28] but is caused by the phase separation between the liquid-condensed and the liquid-expanded films in their coexistence region; the domains and the matrix observed for the mixed DPPC/Ceramide 3 monolayers correspond to the liquid-condensed and the liquid-expanded films. The DSC curves shown in Fig. 6 can be considered in connection with the AFM images. As an example, in the DSC curve for the mixed DPPC/Ceramide 3 system whose mole fraction of Ceramide 3 is 0.130, the peaks at a lower temperature (about 46°C) and at a higher temperature (about 54°C) may possibly correspond to the matrix and domains in the AFM images, where the matrix and domains are poor and rich in Ceramide 3, respectively, since Ceramide 3 is miscible with DPPC. The decrease in the calorie of DSC peak at a lower temperature is also consistent with the decrease in the matrix at a higher mole fraction of Ceramide 3 in the AFM images.
4. Conclusion In the preparation of mixed DPPC/Ceramide 3 liposomes, all the DPPC was able to contribute to the formation of liposomes up to 0.130 mol fraction of Ceramide 3. No significant changes in particle size of liposomes by adding Ceramide 3 was found. The dispersibility of liposomes containing Ceramide 3 was maintained as well as DPPC liposomes without Ceramide 3. So, the liposomes composed of DPPC and Ceramide 3 may be available as a ceramide product.
7
The microviscosity of liposomal bilayer membranes in the liquid crystalline state was increased with increasing the mole fraction of Ceramide 3, while that in the gel state was independent of the mole fraction of Ceramide 3. The phase transition temperature of DPPC bilayer membranes was shifted upwards with the addition of Ceramide 3, indicating a cooperative interaction between DPPC and Ceramide 3 molecules. However, a sharp DPPC peak became broad in the mixed DPPC/Ceramide 3 systems and was split at higher mole fractions of Ceramide 3. The split peak is possibly caused by the phase separation in the liposomal bilayer membranes, since the liposome solution is monodispersive. The split DSC peak for the mixed DPPC/Ceramide 3 bilayer membranes may possibly be related to the domain formation [18] previously observed for the mixed DPPC/Ceramide 3 monolayers.
References [1] Y. Uchida, N. Kitamura, Pharmacia 34 (1998) 783. [2] L. Jiang, D. Ho¨nig, D. Mo¨bius, Chim. Chem. Lett. 3 (1992) 219. [3] M. Kerscher, H.C. Korting, K.M. Schaefer, Eur. J. Dermatol. 1 (1991) 39. [4] A. Yamamoto, Niigata Igaku-Kai Zassi (J. Med. Soc. Niigata) 110 (1996) 95. [5] J. Osawa, M. Takahashi, Z. Ikezawa, Prog. Med. 17 (1997) 31. [6] G. Imokawa, Atarashii Ganka (Recent Opthalmol.) 13 (1996) 351. [7] G. Imokawa, A. Abe, J. Kumi, Y. Higaki, M. Kawashima, A. Hidano, J. Invest. Dermatol. 96 (1991) 523. [8] G. Gregoriadis (Ed.), Liposomes as Drug Carriers. Recent Trends and Progress, Wiley, Chichester, 1988. [9] A. Sekiguchi, J. Manosroi, A. Manosroi, H. Yamauchi, M. Abe, Colloids Surf. B Biointerfaces 4 (1995) 287. [10] M. Abe, K. Hashizaki, Y. Yokouchi, Y. Takao, J. Manosroi, A. Manosroi, H. Sakai, S. Yokoyama, H. Yamauchi, J. Jpn. Oil Chem. Soc. 48 (1999) 215. [11] H. Yamauchi, I. Shimazaki, K. Hashizaki, H. Sakai, S. Yokoyama, M. Abe, J. Jpn. Oil Chem. Soc. 48 (1999) 317. [12] A. Yoshida, K. Hashizaki, M. El-Mahdy, H. Yamauchi, H. Sakai, S. Yokoyama, M. Abe, J. Jpn. Oil Chem. Soc. 47 (1998) 1323. [13] A. Yoshida, K. Hashizaki, H. Yamauchi, H. Sakai, S. Yokoyama, M. Abe, Langmuir 15 (1999) 2333.
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[14] K. Hashizaki, C. Itoh, H. Sakai, S. Yokoyama, H. Taguchi, Y. Saito, N. Ogawa, M. Abe, J. Jpn. Oil Chem. Soc. 48 (1999) 871. [15] H. Hashizaki, C. Itoh, H. Sakai, S. Yokoyama, H. Taguchi, Y. Saito, N. Ogawa, M. Abe, Colloids Surf. B Biointerfaces, 17 (2000) 295. [16] G. Imokawa, A. Abe, K. Jin, J. Invest. Dermatol. 96 (1991) 523. [17] A. Yamamoto, S. Serizawa, M. Ito, Arch. Dermatol. Res. 283 (1991) 219. [18] T. Imura, H. Sakai, H. Yamauchi, S. Yokoyama, M. Abe, Colloids Surf. B Biointerfaces, 19 (2000) 81. [19] E.M. Donald, E.R. James, J. Biol. Chem. 247 (1972) 3694. [20] A.D. Bangham, M.M. Standish, J.C. Watkins, J. Mol. Biol. 13 (1965) 238.
.
[21] M. Takayama, S. Itoh, T. Nakagaki, I. Tanimizu, Clin. Chim. Acta 79 (1977) 93. [22] T. Inoue, Y. Muraoka, K. Fukushima, R. Shimozawa, Chem. Phys. Lipids 46 (1988) 107. [23] K. Kinoshita, R. Kataoka, Y. Kimura, O. Gotoh, A. Ikegami, Biochemistry 20 (1981) 4270. [24] E.L. Pugh, R. Bittman, L. Fugler, M. Kates, Chem. Phys. Lipids 50 (1989) 43. [25] M. Abe, M. Goto, K. Ogino, J. Jpn. Oil Chem. Soc. 39 (1990) 351. [26] K. Yagi, T. Sekine, Application of Fluorescence Spectrometry to Biochemistry Research, Gakkai Shuppan Center, Tokyo, 1976, p. 34. [27] A. Georgallas, D.L. Hunter, T. Lookman, M.J. Zuckermann, D.A. Pink, Eur. Biophys. J. 11 (1984) 79. [28] Y.F. Dufrene, W.R. Barger, J.B.D. Green, G.U. Lee, Langmuir 13 (1997) 4779.
Preparation of liposomes containing Ceramide 3 and their membrane characteristics Tomohiro Imura a, Hideki Sakai a,b, Hitoshi Yamauchi a, Chihiro Kaise a, Kozo Kozawa a, Shoko Yokoyama c,*, Masahiko Abe a,b b
a Faculty of Science and Technology, Science Uni6ersity of Tokyo, 2641, Yamazaki, Noda, Chiba 278 -8510, Japan Institute of Colloid and Interface Science, Science Uni6ersity of Tokyo, 1 -3, Kagurazaka, Shinjuku, Tokyo 162 -0825, Japan c Kyoritsu College of Pharmacy, 1 -5 -30, Shibakoen, Minato-ku, Tokyo 105 -8512, Japan
Received 2 February 2000; accepted 3 March 2000
Abstract Liposomes composed of Ceramide 3, [2S,3S,4R-2-stearoylamide-1,3,4-octadecanetriol], and L-a-dipalmitoylphosphatidylcholine (DPPC) were prepared by varying the amount of Ceramide 3, and the effects of Ceramide 3 on the liposome formation, particle size, dispersibility, microviscosity and phase transition temperature were examined by means of a microscopy, a dynamic light scattering method, a fluorescence polarization method, a differential scanning calorimetry (DSC) and so on. All the DPPC was able to contribute to the formation of liposomes up to 0.130 mol fraction of Ceramide 3. The particle size of liposomes was almost unaffected by the addition of Ceramide 3. The dispersibility of liposomes containing Ceramide 3 was maintained for at least 15 days. The microviscosity of liposomal bilayer membranes in the liquid crystalline state was increased with increasing the mole fraction of Ceramide 3, while that in the gel state was independent of the mole fraction of Ceramide 3. The phase transition temperature from gel to liquid crystalline states of DPPC bilayer membranes was shifted upwards with the addition of Ceramide 3, indicating a cooperative interaction between DPPC and Ceramide 3 molecules. However, a sharp DSC peak became broad and split at higher mole fractions of Ceramide 3, suggesting a phase separation in the mixed DPPC/Ceramide 3 liposomal bilayer membranes. These phenomena were suggested to be related to the previously observed fact for the mixed DPPC/Ceramide 3 monolayers that Ceramide 3 interacts with DPPC in the liquid-expanded phase with consequent phase separation accompanied with domain formation. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Ceramide 3; Phospholipid; Liposomes; Differential scanning calorimetry; Phase separation; Microviscosity
1. Introduction
* Corresponding author. Fax: +81-3-34345343. E-mail address: [email protected] (S. Yokoyama).
Human skin consists of the epidermis, dermis, subcutaneous tissue and stratum corneum [1]. Dermis is composed of more than 95% of phospholipid [1]. Epidermis consists of about 60% of
0927-7765/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 0 0 ) 0 0 1 4 9 - 1
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T. Imura et al. / Colloids and Surfaces B: Biointerfaces 20 (2001) 1–8
phospholipids and 40% of ceramides [1] and has a barrier function [2 – 5] to prevent an invasion of injurious substance to the health and the loss of the juices. The stratum corneum lipids construct the water-permeability barrier, in which the control of water permeability is essential for survival in any living system. The stratum corneum lipids are composed of: 50% ceramides, 5% cholesterol, 10% cholesterol esters and 20% free fatty acids [6]. A decreased level of ceramides in stratum corneum of atopic dermatitis has been found [5,7]. It has also been suggested that an application of small amount of ceramides may be effective to a dry skin dermatitis [6,9]. Manufacturing of ceramide product is, however, a difficult problem because ceramides can scarcely be dispersed in an aqueous medium. It is expected that the dispersibility of ceramides will be improved by the liposome formation with phospholipid. On the other hand, liposomal drug delivery systems have been researched widely for the purpose of reduction of drug toxicity and/or targeting of drugs to specific cells [8]. However, only a few studies have been done on the physicochemical properties of liposomes in contrast with many studies from the viewpoints of biochemistry and pharmacology. We think that the efficiency of liposomes as a ‘vessel’ in which drugs are included are also important, and we have studied the dispersibility, microviscosity and permeability of liposomes containing glycolipids [9], steroids [10], carboxyacyl phospholipids [11] or water-soluble polymers [12 – 15]. From these points of view, the formation of liposomes containing ceramide and their liposomal membrane characteristics were examined. In this study, Ceramide 3 was chosen as a ceramide among seven known ceramides [16,17]. We have previously studied the interaction of Ceramide 3 and phospholipid in the mixed monolayers by means of atomic force microscopy (AFM), and it has been found that Ceramide 3 interacts with phospholipid in the liquid-expanded film with consequent phase separation accompanied with domain formation [18]. So, a possible phase separation in liposomal bilayer membranes containing Ceramide 3 was also investigated by a differential scanning calorimetry and the results were dis-
cussed in connection with the previous results for the monolayers.
2. Experimental
2.1. Materials 2S,3S,4R-2-stearoylamide-1,3,4-octadecanetriol (Ceramide 3, 94% pure) as a ceramide was purchased from Cosmoferm Co. and was used without further purification. The structural formula of Ceramide 3 was shown in a previous paper [18]. L-a-Dipalmitoylphosphatidylcholine (DPPC, 99.6% pure) as a phospholipid was supplied from Nippon Oil and Fats Co., Ltd. Dicetylphosphate (DCP, 99.6% pure) as a charged material and 1,6-diphenyl-1,3,5-hexatriene (DPH, 98% pure) as a fluorescent probe were purchased from Sigma Chemical Co. Isotonic phosphate buffered saline (PBS), pH 7.4, was purchased from Nissui Pharmaceutical Co. All other chemicals were commercial products of reagent grade.
2.2. Preparation of liposomes containing Ceramide 3 The composition of liposomes containing Ceramide 3 was as follows: DPPC: Ceramide 3: DCP=10: (0 1.5):1. DCP was added to make DPPC bilayer membranes stable [19]. The liposomes were prepared according to the procedure of Bangham et al. [20]; DPPC, Ceramide 3 and DCP were dissolved in chloroform in a test tube; the solvent was then removed by blowing nitrogen gas into the test tube, and the redisual solvent was further dried overnight at room temperature in a desiccator under vacuum; PBS was added to this lipid film and warmed at 60°C above the phase transition temperature (41°C) of DPPC for 10 min; the test tube was then shaken vigorously on a vortex mixer, and the multilamellar vesicles (MLV) were obtained (liposome solution A). The multilamellar vesicles were sonicated (125 W, bath type: Branson B-220) for 1 h and subsequently extracted through a polycarbonate membrane filter (pore size 200 nm, Nuclepore Inc.) using an extruder (Lipex Biomembranes Inc.), and a homo-
T. Imura et al. / Colloids and Surfaces B: Biointerfaces 20 (2001) 1–8
geneous liposomal suspension in which almost uniform liposomal size was obtained (liposome solution B).
2.3. Determination of concentration of DPPC The concentration of DPPC in liposomes (liposome solution A) was determined by the choline oxidase-phenol method [21].
2.4. Obser6ation of liposomes Liposomes (liposome solution A) were observed using an inverted microscope with a transmitted light differential interference contrast attachment (IMT-2, Olympus Optical Co., Ltd.).
2.5. Column chromatography and thin-layer chromatography of liposome solution Column chromatography of liposome solution was performed on Sepharose CL-6b (Pharmacia Biotech Co.) with PBS as an eluent. The elution rate was 53 ml h − 1. Liposomes were detected by measuring the turbidity at 650 nm. The constituent lipids of liposomes fractionated in each fraction were qualitatively analyzed by thin-layer chromatography (TLC) with K60 thin-layer plate (Merck Co.) and chloroform/methanol (9:1) solution. The spots developed by TLC were detected with iodine reagent. The value of Rf for Ceramide 3 was 0.36.
2.6. Measurement of particle size of liposomes The particle size of liposomes (liposome solution B) was determined by a dynamic light scattering method. The distribution and time course of liposomal particle size were measured at 30°C using a NICOMP 380ZLS (Particle Sizing System Co.). The light source was a diode pump solid state laser (DPSS laser) with a wavelength of 535 nm and the scattering angle was 90°.
2.7. Measurement of micro6iscosity of liposomal bilayer membranes DPH as a fluorescent probe was dissolved in
3
tetrahydrofuran, the DPH solution was added to the dilute liposome solution B and incubated for 1 h at 37°C. The molar ratio of lipids to probe was 300:1. The microviscosity of liposomes was determined by fluorescence polarization (P), which can be calculated according to the following equation: P= (Ip − GIv)/(Ip + GIv) where Ip and Iv are the fluorescence intensities of the emitted light polarized parallel and vertical to the exciting light, respectively, and G is the grating correction factor [22]. The fluorescence intensities of Ip and Iv were measured at 30 58°C with a spectrofluorometer (RF-5000, Shimadzu Co., Ltd), and excitation and emission wavelengths were 350 and 450 nm, respectively. DPH exists in hydrophobic region and is able to evaluate the microviscosity around DPH in the liposomal bilayer membranes [23]. Fluorescence polarization is correlated to microviscosity near the fluorescent probes [24,25], which is calculated using the Perrin-Weber’s equation [26]. Microviscosity increases with increasing fluorescence polarization.
2.8. Differential scanning calorimetry (DSC) The phase transition temperature of liposomal bilayer membranes was measured with a differential scanning calorimeter (8240B, Rigaku Denki Co.). 20 mg of liposome solution (liposome solution B) was put in a sampling vessel, which is made of stainless steel (resistance to pressure at 50 atm, 3× 5 (F) mm for the vessel size), and then the vessel was sealed. The measurement conditions were 1 K min − 1 for the scanning rate, 30 60°C for the scanning range and 0.1 m cal s − 1 for the sensitivity.
3. Results and discussion
3.1. Effect of Ceramide 3 on formation of liposomes The effect of Ceramide 3 on the formation of liposomes was examined with an optical micro-
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T. Imura et al. / Colloids and Surfaces B: Biointerfaces 20 (2001) 1–8
Fig. 1. Micrographs of liposomes containing Ceramide 3. The mole fraction of Ceramide 3 is 0.09. The length of the black bar is 10 mm.
scope using the liposome solution A. Fig. 1 shows the micrograph of liposomes containing 0.09 mol fraction of Ceramide 3. The onion-like structure having an inner aqueous phase suggests the formation of MLV. Next the concentration of DPPC in the liposomes was determined, and the results are shown in Table 1. The DPPC amount at 100% means that all the DPPC is used to form liposomes. As is evident in Table 1, all the DPPC is able to contribute to the formation of liposomes containing Ceramide 3 up to 0.130 mol fraction of Ceramide 3, while not all the DPPC is utilized at higher mole fractions of Ceramide 3. Agglutination of Ceramide 3 was also visually observed for liposome solutions containing more than 0.166 mol fractions of Ceramide 3. It has been generally known [9] that more than 10 mol% of lipophilic additives bring about a destruction of liposomal structure.
3.2. Distribution of liposome size The elution curve of liposome solution B by column chromatography is shown in Fig. 2. Fig. 2
Fig. 2. Elution curve of liposomes by column chromatography. The mole fraction of Ceramide 3 is 0.09.
suggests that the liposomes are monodispersitive and that the particle size of liposomes is almost uniform. Liposome solution was fractionated in the fraction numbers 15–17. Furthermore, only in the fraction numbers 15–17, the existence of Ceramide 3 was confirmed, indicating that liposomes composed of DPPC and Ceramide 3 are indeed formed.
3.3. Effect of Ceramide 3 on particle size of liposomes The effect of Ceramide 3 on the particle size of liposomes (liposome solution B) was measured by varying the mole fraction of Ceramide 3. The distribution of liposomal particle size is shown in Fig. 3. The ordinate means the relative volume of particles when the volume of particles with 190 210 nm of diameter is defined as 1.0. No significant changes in particle size and distribution of particle size of liposomes containing Ceramide 3 (Xceramide 3 0.130) was found.
Table 1
Mole fraction of Ceramide 3 DPPC (%)
0 100
0.047
0.090
0.130
0.166
0.200
100
100
100
95
87
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Fig. 3. Distribution of particle sizes of liposomes containing various amounts of Ceramide 3. The mole fraction of Ceramide 3: (a) 0; (b) 0.047; (c) 0.090; (d) 0.130.
3.4. Effect of Ceramide 3 on dispersibility of liposomes The effect of Ceramide 3 on the dispersibility of liposomes (liposome solution B) was examined by measuring the time course of the liposomal particle size at 30°C, and the results are shown in Fig. 4. No significant changes in particle size of liposomes was observed for the system containing Ceramide 3 as well as for the DPPC alone system without Ceramide 3, indicating that the dispersibility of liposomes containing Ceramide 3 is maintained for at least 15 days.
3.5. Effect of Ceramide 3 on micro6iscosity of liposomal bilayer membranes The relationship between fluorescence polarization of DPH and temperature is shown in Fig. 5.
Fig. 4. Time courses of particle size of liposomes containing various amounts of Ceramide 3. The mole fraction of Ceramide 3: , 0; , 0.047; , 0.090; , 0.130.
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state was independent of the mole fraction of Ceramide 3. Ceramide 3 is likely to affect the microviscosity in the hydrophobic region of liposomal bilayer membranes in the liquid crystalline state. The results shown in Fig. 5 suggest that the intermolecular interactions in the liposomal bilayer membranes in the liquid crystalline state are strengthened and the packing density in the membranes becomes rigid by the addition of Ceramide 3.
3.6. Effect of Ceramide 3 on the phase transition temperature of liposomal bilayer membranes Fig. 5. Relationship between fluorescence polarization of DPH and temperature for liposomes containing various amounts of Ceramide 3. Symbols are the same as in Fig. 4.
Fig. 6. DSC curves of liposomes containing various amount of Ceramide 3. DSC curves were obtained under the conditions: scanning rate, 1 K min − 1; sensitivity, 0.1 m cal − 1 s − 1.
The fluorescence polarization of DPH in the DPPC bilayer membranes without Ceramide 3 suddenly decreased above 42°C, which agrees with the phase transition temperature of DPPC. The phase transition temperature was raised by the addition of Ceramide 3. The microviscosity of liposomal bilayer membranes in the liquid crystalline state was increased with increasing the mole fraction of Ceramide 3, while that in the gel
The DSC curves for the mixed DPPC/Ceramide 3 liposomal bilayer membranes were obtained by varying the mole fraction of Ceramide 3, and the results are shown in Fig. 6. Ceramide 3 indicated no peaks for at least 0100°C (the DSC curve was not presented in Fig. 6). This phenomenon is consistent with the fact [18] that Ceramide 3 forms only the liquid-condensed film as observed for the surface pressure measurement of Ceramide 3 monolayer. An endothermic peak at about 41°C corresponds to the gel-liquid crystalline phase transition temperature of DPPC. The phase transition temperature of DPPC bilayer membranes was shifted upwards with the addition of Ceramide 3, indicating that the liposomal bilayer membranes became thermostable by the addition of Ceramide 3. The shift of the main phase transition temperature to a higher temperature implies a cooperative interaction between DPPC and Ceramide 3 molecules, indicating also that Ceramide 3 is miscible with DPPC. The phase transition between the gel and liquid crystalline phases observed for phospholipid bilayers is related to the phase transition of phospholipid monolayers, from the liquid-expanded film to the liquid-condensed film [27]. An attractive interaction between Ceramide 3 and DPPC in the liquid-expanded films of the mixed DPPC/Ceramide 3 monolayers has been reported in our previous paper [18]. A sharp DSC peak observed for the DPPC alone system became broad in the mixed DPPC/ Ceramide 3 liposomal bilayer membranes. Furthermore, a DSC peak was noted to split at
T. Imura et al. / Colloids and Surfaces B: Biointerfaces 20 (2001) 1–8
higher mole fractions of Ceramide 3. The split peak is suggested to be caused by the phase separation in the liposomal bilayer membranes since the liposome solution used in this measurement was confirmed to be monodispersive as is shown in Fig. 2. We have shown the AFM images for the mixed DPPC/Ceramide 3 monolayers in the previous paper [18], in which elliptical domains and matrix have been found. Furthermore, new theory has been proposed in our paper [18]: the domain formation is not caused by the complete separation of one component from the other component as was observed by Dufrene et al. [28] but is caused by the phase separation between the liquid-condensed and the liquid-expanded films in their coexistence region; the domains and the matrix observed for the mixed DPPC/Ceramide 3 monolayers correspond to the liquid-condensed and the liquid-expanded films. The DSC curves shown in Fig. 6 can be considered in connection with the AFM images. As an example, in the DSC curve for the mixed DPPC/Ceramide 3 system whose mole fraction of Ceramide 3 is 0.130, the peaks at a lower temperature (about 46°C) and at a higher temperature (about 54°C) may possibly correspond to the matrix and domains in the AFM images, where the matrix and domains are poor and rich in Ceramide 3, respectively, since Ceramide 3 is miscible with DPPC. The decrease in the calorie of DSC peak at a lower temperature is also consistent with the decrease in the matrix at a higher mole fraction of Ceramide 3 in the AFM images.
4. Conclusion In the preparation of mixed DPPC/Ceramide 3 liposomes, all the DPPC was able to contribute to the formation of liposomes up to 0.130 mol fraction of Ceramide 3. No significant changes in particle size of liposomes by adding Ceramide 3 was found. The dispersibility of liposomes containing Ceramide 3 was maintained as well as DPPC liposomes without Ceramide 3. So, the liposomes composed of DPPC and Ceramide 3 may be available as a ceramide product.
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The microviscosity of liposomal bilayer membranes in the liquid crystalline state was increased with increasing the mole fraction of Ceramide 3, while that in the gel state was independent of the mole fraction of Ceramide 3. The phase transition temperature of DPPC bilayer membranes was shifted upwards with the addition of Ceramide 3, indicating a cooperative interaction between DPPC and Ceramide 3 molecules. However, a sharp DPPC peak became broad in the mixed DPPC/Ceramide 3 systems and was split at higher mole fractions of Ceramide 3. The split peak is possibly caused by the phase separation in the liposomal bilayer membranes, since the liposome solution is monodispersive. The split DSC peak for the mixed DPPC/Ceramide 3 bilayer membranes may possibly be related to the domain formation [18] previously observed for the mixed DPPC/Ceramide 3 monolayers.
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