Molecular interactions between phospholipids and mangostin in a lipid bilayer

COLLOIDS AND U SURFACES ELSEVIER

Colloids and SurfacesB: Biointerfaces4 (1995)423 432

Molecular interactions between phospholipids and mangostin in a lipid bilayer A y u m i Y o s h i d a a, A r a n y a M a n o s r o i b, J i r a d e j M a n o s r o i b, H i t o s h i Y a m a u c h i a, M a s a h i k o A b e a,e,, a Faculty of Science and Technology, Science University of Tokyo, 2641, Yamazaki, Noda, Chiba 278, Japan b Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand Institute of Colloid and Interface Science, Science University of Tokyo, 1-3, Kagurazaka, Shinjuku, Tokyo 162, Japan Received 13 December 1994; accepted 9 February 1995

Abstract

Molecular interactions between phospholipids and mangostin in a lipid bilayer have been investigated in terms of the maximum additive concentration (MAC) of mangostin in liposomes, the surface potential, particle size, microscopicviscosity and microscopic-polarity of liposomes, and the permeability of glucose. The mangostin used is a natural product extract: 1,3,6-trihydroxy-7-methoxy-2,8-bis(3-methyl-2-butenyl)-9-xanthenenone. The MAC of mangostin was fairly dependent upon the nature of the liposomes (uncharged, negatively charged or positively charged). Solubilization of mangostin in the liposomal bilayer resulted in both an increase in the negative charge on the liposomal surface, strengthening the state of the bilayer membrane, and a depression in the release of the glucose involved. Mangostin was found to temporarily stabilize the liposomal bilayer, although the bilayer membrane is still unstable in the long run. Keywords: Liposome; Mangostin; Microscopic-viscosity; Permeability; Solubilization

1. Introduction

Liposomes are phospholipid bilayer vesicles consisting of alternating layers of aqueous medium and lipid bilayers. In 1965, Bangham et al. [1] first described liposomes as minute vesicles composed of lipid bilayers. Liposomes, which are made up of multiple concentric biomolecular lipid bilayers, were initially used in cell structure and cell function research, although the natural cell membrane is a single biomolecular layer. Liposome structure, therefore, did not completely meet the needs of the research of interest into cell structure * Corresponding author. Tel. and Fax: (+81) 471-24-8650. 0927-7765/95/$09.50© 1995ElsevierScienceB.V. All rights reserved SSDI 0927-7765(95)01198-6

and cell function at that time. Since 1971, researchers have focused on liposomes as drug delivery systems (DDSs), because liposomes have advantages over several drug carriers [ 2 - 5 ] . " F o r example, many highly toxic agents showed a reduction in normal tissue side effects when encapsulated in liposomes, An improvement in the pharmacological effects of various agents which are poorly soluble or insoluble can be achieved. So far, numerous agents, both synthetic and natural extracts, have been encapsulated in liposomes. However, mangostin, a xanthone compound, extracted from the fruit hull of Mangosteen (Garcinia mangostana, Linn. (Guttiferae)) with hexane followed by extensive rapid column chromatography [6], has never been studied in lipo-

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A. Yoshida et al./ColIoids Surfaces B: Biointerfaces, 4 (1995) 423 432

somes. The use of this fruit hull (pericarp) in many Asian (e.g. Thailand) types of folk medicine for healing skin infection and wounds, and for the relief of diarrhea, has been documented [7]. Several studies have revealed a number of pharmacological activities, i.e. cardiotonic, anti-microbial and anti-hepatotoxic effects, as well as antiinflammatory activity of mangostin and its derivatives. It was found that mangostin at a dose of 50 mg kg -1 exhibits pronounced anti-inflammatory activity in the carrageenin-induced paw edema, cotton pellet implantation and granuloma pouch models [-8]. From the molecular structure shown in Fig. 1, it can be seen that mangostin is a hydrophobic compound which is insoluble in water, but is highly soluble in many organic solvents. Hence, the entrapment of mangostin in liposomes can be of pharmacological advantage, since enhancement of the absorption through the biological membrane by entrapment or solubilization of mangostin in the bilayers of liposomes is anticipated. However, the effect of mangostin on the liposomal state has never been clarified. In this study, liposomes composed of phospholipids, cholesterol, charged materials and mangostin were prepared to study the molecular interactions between phospholipids and mangostin. The study included determination of the maximum additive concentration (MAC) of mangostin in liposomes, the particle size, surface potential, monolayer surface pressure, microscopic-viscosity, microscopicpolarity of liposomes, the permeability of glucose, and the phase transition of phospholipid. Further, the effects of mangostin on the physicochemical properties of liposomes were discussed.

0 OH ~"~ H311~O

H

Fig. 1. Structuralformulaof mangostin.

2. Experimental 2.1. Materials Phospholipid g-c~-Dipalmitoyl phosphaditylcholine (DPPC, 99.6%) Was supplied by Nippon Oil and Fats Co., Ltd., Amagasaki, Hyogo, Japan, and used without further purification. Steroid Cholesterol (Chol, 99.6%) was purchased from Sigma, St. Louis, MO, and used without further purification. Charged materials Dicetyl phosphate (DCP, 99.6%) and stearyl amine (SA, 90%) were purchased from Sigma, and used without further purification. Mangostin Mangostin (1,3,6-trihydroxy-7-methoxy-2,8-bis(3-methyl-2-butenyl)-9-xanthenenone) extracted from Garcinia mangostana, Linn. (Guttiferae), according to Ref. [6], was supplied by Professor Dr. Wiriyachitra. The extracted mangostin was identified by 1H NMR measurements with an NMR spectrometer (type JNX EX-400, JEOL Ltd., Tokyo, Japan), and the melting point with a differential scanning calorimeter. In the 1H NMR spectrum, singlet peaks at ~ = 13.8, 6.31, 6.17 p.p.m, were assigned to the OH group and d = 3.81 to the methoxy group. These peaks were in good agreement with Ref. [6]. The melting point was found to be 182.4°C (ref. 182-183°C) [9]. Fluorescent materials Fluorescent fatty acid probes, 2-(9-anthroyloxy)palmitic acid (2-AP) and 12-(9-anthroyloxy)-stearic acid (12-AS), were obtained from Sigma, and used without further purification. Pyrene (98.0%), a fluorescent probe from Sigma, was recrystallized from ethanol several times and purified by column chromatography on silica gel. In this experiment, water for injection (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) was used. Glucose was purchased from Nippon

A. Yoshidaet al./Colloids SurfacesB: Biointerfaces,4 (1995) 423-432 Rikagakuyakuhin Co., Ltd., Tokyo, Japan, and phosphate buffered saline (PBS) was purchased from Nissui Pharmaceutical Co., Ltd., Tokyo, Japan. These chemicals were commercial products of reagent grade.

2.2. Methods Composition and concentration of lipids in the liposomes In this experiment, three types of liposome were prepared: uncharged liposomes (Uric.L) which are composed of DPPC and Chol (molar ratio 1 : 1), negatively charged liposomes (Neg.L) which are composed of DPPC, Chol and DCP (7:3 : 1), and positively charged liposomes ( P o s i ) which are composed of DPPC, Chol and SA (7:3:1). The total lipid concentration of each liposome was 1 mg m1-2.

Amount of mangostin added The amount of mangostin added was 1, 2, 5, 10, 15 and 20 weight ratio to total lipid (wt.%).

Preparation of liposomes Multilamellar vesicles (MLVs) were prepared using a conventional technique similar to Bangham's method [1]. Briefly, DPPC, Chol, DCP or SA and mangostin were dissolved in chloroform in a test tube. The solvent was then removed by blowing nitrogen gas into the test tube, and the residual solvent was further dried overnight at room temperature in a desiccator under vacuum. A 5 ml portion of PBS was added to this lipid film and warmed (55-60°C) above the phase transition temperature of DPPC (T~ = 41 ° C) for 30 rain. The test tube was then shaken vigorously on a vortex mixer, and a more homogeneous liposomal suspension was obtained after 60 min of sonication (bath type: Branson B-220, Tokyo, Japan) and subsequently extracted using a polycarbonate membrane filter (pore size: 200nm, Nomura Micro Science, Tokyo, Japan).

425

Determination of DPPC and mangostin concentration in the bilayer membranes The concentration of DPPC in liposomes was determined by the choline oxidase-phenol method [10] with a spectrophotometer (type MPS-2000, Shimadzu Co., Kyoto, Japan). The concentration of mangostin in the liposomes was determined by first destroying the membrane with methanol, and then measuring the mangostin content using a spectrophotometer (2= 366nm). Mangostin in MeOH/PBS (volume ratio is 1:1) showed three absorption maxima at 366, 240 and 205 nm, respectively. Under these experimental conditions, the system was recognized to obey Beer's law [11] only at 366 nm. Measurement of the surface pressure of the monolayer Measurement of the surface pressure of the monolayer at a gas/liquid surface was performed with a surface pressure meter (type HBM-A, Kyowa Interface Science Co., Ltd., Tokyo, Japan) by the Wilhelmy method. A barrier was made of teflon, and the developing solvent used was chloroform. The total lipid concentration was 1 mmol kg-1. The sample was carefully added dropwise to the water, and the resulting mixture left to stand for 15 min. The compression rate of the adsorption equilibrium was 20 mm min -1 at the gas/liquid surface. The surface pressure (r0 was calculated according to the following equation: rt = ~ w - ~/0

(1)

where 7w and ~o are the surface tension of the water and the surface, respectively, with the monolayers [ 12,13].

Measurement of the particle size of the liposomes The particle size of the liposomes was measured using a 4700-type submicronmeter particle analyzer (Malvern Instrument Ltd., UK), with a multibit 8 Malvern correlator with delayed channels. The light source was an argon laser (Coherent Co., Innova 90), having a wavelength of 488 nm and a power of 5 W or less, and the time-dependent

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A. Yoshidaet al./ColloidsSurfacesB: Biointerfaces,4 (1995) 423-432

correlation function of the scattered light intensity was measured at a scattering angle of 90 °.

Measurement of the surface potential of the liposomes The surface potential (zeta-potential) of the liposomes was measured using a Zetasizer IIc (Malvern Instrument Ltd.) which is a laser Doppler electrophoresis apparatus. This apparatus is equipped with a multibit 8 Malvern Correlator with a delay channel and 15 mW He-Ne laser (2 = 633 nm).

Measurement of the microscopic-viscosity of the bilayer membranes Lipids, mangostin and fluorescent fatty acid probes, either 2-AP or 12-AS, were dissolved in chloroform in a test tube. Liposomes containing either 2-AP or 12-AS were prepared. The molar ratio lipid/probe was 300: 1. The microscopicviscosity of the liposomes was determined by the fluorescence polarization (P) (measured by a fluorescence spectrophotometer, type RF-5000, Shimadzu Co.; the excitation and emission wavelengths were 365 and 440 nm, respectively) which can be calculated according to the following equation: Ip - GIv P= - Ip + GIv

(2)

where Ip and Iv are the fluorescent intensities of the emitted light polarized parallel and vertical to the exciting light, respectively, and G is the grating correction factor [14].

Measurement of the microscopic-polarity of the bilayer membranes The liposomes, which solubilized 5.0 x 10 -6 mol 1-1 of pyrene in their membranes, were prepared. The microscopic-polarity of the liposomes was determined by measuring the fluorescent intensities of pyrene using the fluorescence spectrophotometer at excitation and emission wavelengths of 335 and 350-550 nm, respectively [-15].

Measurement of the entrapped efficiency and the permeability of glucose in the liposomes In order to determine the entrapped efficiency, the concentration of glucose entrapped in the liposomes was determined. The permeability of the liposomal membranes was estimated from the glucose leakage from the liposomes. Briefly, glucose, which is an aqueous model material, was entrapped in the liposomes. Any excess glucose was separated using a dialysis technique with a cellophane tube (Union Carbide Co., Chicago, IL). The liposome suspension was first put in a cellophane tube, and then dialyzed against 1 1 of saline for 3 h at 5 ° C. The saline was changed three times during dialysis. The amount of glucose entrapped was extracted into a water phase from the liposomes, according to the producer of Bligh et al. [16], and then assayed by the mutarotase glucose oxidase method [17] using the spectrophotometer.

Measurement of the phase transition temperature of DPPC The effects of mangostin on the phase transition of DPPC were investigated with a differential scanning calorimeter (type 8240B, Rigaku Co., Tokyo, Japan). Addition of cholesterol can stabilize the liposomal membrane and cause the disappearance of the T~ of phospholipids [18,19]. For this study, cholesterol was not added to the liposome constituents, so that, the effects of mangostin alone on the T~ of phospholipids could be evaluated. Lipids and mangostin were first dissolved in chloroform in a flask, followed by the removal of solvent by evaporation, to give a homogeneous mixture of lipids and mangostin. This mixture was packed in a sampling vessel made of stainless steel (resistance to pressure at 50 atm, 3 x 5~bmm for the vessel size). Water was then added and the vessel was sealed. The concentration of the mixture was 50 wt.%, and the measurement conditions were 0.5 K min -1 for the scanning rate, 20-80°C for the scanning range, and 0.1 mcal s -1 for the sensitivity. All experiments, except for the determination of the amount of DPPC and mangostin, and the DSC measurements, were carried out at 37 ° C.

A. Yoshida et aL/Colloids Surfaces B: Biointerfaces, 4 (1995) 423-432

3. Results and discussion

50xl 0-3 ]

3.1. Maximum additive concentration of mangostin in the Iiposomes

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0

0

I

I

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Weight ratio of mangostin / wt% Fig. 2. Effects of the a m o u n t s of added mangostin on D P P C formed liposomes.

o Uncharged [ ,~ Negatively

4o I I[] Positively I 30 ]

Fig. 2 shows the effects of the weight ratio of added mangostin on the DPPC-formed liposomes. The DPPC amount at 100% on the ordinate axis means that all the DPPC is used to form liposomes. In the case of Unc.L, up to the addition of 5 wt.% mangostin all the DPPC was able to contribute to the formation of liposomes. Beyond this additive amount, not all the DPPC was utilized. Similar to the case of Unc.L, the DPPC amount contributing to the formation of liposomes changed at the additive point of 15 wt.% mangostin for N e g i , and 10 wt.% mangostin for Pos.L. Fig. 3 demonstrates the amounts of mangostin solubilized in the liposomes. The broken line in this figure indicates calculated results, representing complete solubilization of all the mangostin added in the liposomes. For Unc.L, the plots fitted to the calculated line up to the addition of 5 wt.%, but beyond this additive amount they deviated from the line. It was suggested that all the mangostin added was solubilized in liposomes up to 5 wt.%, but none of the mangostins were able to solubilize when more than 5 wt.% was added. In the case of Neg.L, the additive amount of mangostin at which

b

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15

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Weight ratio of mangostin / wt% Fig. 3. Amounts of mangostin solubilizedin liposomes.

the plot deviated from the line was 15 wt.%, and for Pos.L it was 10wt.%. The limit of added mangostin for complete solubilization in the liposomes was in fair agreement with the amounts of mangostin added for which all the DPPC formed liposomes. The MAC of mangostin was found to be different in each liposome. However, no relation between the number of moles of mangostin and that of lipids was found for any of the liposomes at the MAC. Consequently, the MAC of mangostin was found: 5 wt.% mangostin was able to solubilize in Unc.L, 15 wt.% in Neg.L, and 10 wt.% in Pos.L. The DPPC and mangostin which do not contribute to the formation of liposomes were precipitated, and then removed from the system through the sizing process. Fig. 4 represents a diagram of surface pressure versus amounts of mangostin added to the lipid mixtures, with a molar ratio the same as each liposome, at 37°C. At the above-mentioned MAC (5 wt.% for Unc.L, 15 wt.% for Neg.L, and 10 wt.% for Pos.L, as shown in this figure using arrows), the surface pressure (equilibrium spreading pressure) was decreased in each monolayer. According to Tajima et al. [20], no change in surface pressure on addition of mangostin resulted from the monolayer being homogeneous. Beyond the decreasing point of the surface pressure, a cluster was consid-

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A. Yoshida et aL/Colloids Surfaces B: Biointerfaces, 4 (1995) 423-432

(a)

3.2. Effects of mangostin on the macroscopic-state of the liposomes

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Fig. 5 indicates the relation between particle size and amounts of mangostin added. In the case of Unc.L, the particle size was slightly decreased by the addition of a little mangostin, but at the MAC the particle size was increased compared to blank liposome (blank liposome refers to the liposome in which no mangostin was solubilized). For the other types of liposomes, the change in particle size resulting from the addition of mangostin was more remarkable. The particle size increment at each MAC followed the order: Unc.L (22 n m ) < Pos.L (27 nm) < Neg.L (35 rim). Fig. 6 depicts the relation between the zetapotential of liposomes and the amount of mangostin added. On the addition of mangostin, the zeta-potential of each liposome was altered as follows: Unc.L was changed from - 4 mV for blank liposome to - 1 0 mV for liposome at the MAC (Neg.L was from - 18 mV to - 3 0 mV, and Pos.L was from 18 mV to 8 mV). Hence, the change in zeta-potential followed the trend: Unc.L (6 mV) < Pos.L (10 mV) < Neg.L (12 mY). In order to evaluate the effect of mangostin on the zeta-potential of liposomes more specifically, the surface charge density of liposomes (oz) was calculated according to the following expression

Weight ratio of mangostin / wt% Fig. 4. Plot of surface pressure versus amount of mangostin added at 37°C: (a) uncharged liposomes;(b) negativelycharged liposomes; (c) positivelycharged liposomes.

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ered to be formed in the monolayer for each liposome type. Hence, that point was the maximum additive amount of mangostin for obtaining a stable monolayer. Overlapping of the monolayer resulted in the formation of a liposomal bilayer [ 12,13 ]. Accordingly, beyond the MAC the bilayer membrane was unstable, and therefore not all the mangostin was able to solubilize in each liposome (as shown in Fig. 3). The liposomes have been used for the following measurements, in which mangostin was solubilized up to the MAC.

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Fig. 5. Relationbetweenparticle size and amount of mangostin added at 37°C.

A. Yoshida et al.IColloids Surfaces B. Biointerfaces, 4 (1995) 423-432

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Fig. 6. Relation between zeta-potential and a m o u n t of mangostin added at 37°C.

Fig. 7. Relation between surface charge density and a m o u n t of mangostin added at 37°C.

[21]:

MOPAC were localized in this molecule. Atoms having high electron density were orientated at the liposomal surface. As a result, the negative charge on the liposomal surface was increased. The changes in particle size, zeta-potential, and surface charge density due to solubilization of mangostin may be a result of the different solubilization sites. For Unc.L, mangostin which is a slightly negatively charged oily material, is penetrated near the center of its lipid bilayer owing to hydrophobic-hydrophobic interactions, and the size of solubilized liposomes is slightly larger than that of blank liposomes. However, in the case of Neg.L, the mangostin is solubilized near the liposome surface, and the size is considerably larger than that of the Unc.L due to electrostatic repulsion. The solubilization site of mangostin in Pos.L is intermediate between those of Unc.L and N e g i . The above suggestion is illustrated by the solubilization model for mangostin shown in Fig. 8. The differences in solubilization site may be a result of the difference of the MAC.

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8 In [cosh(e~/4kr)] ;1/2 + (-~7 ~ j

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~c= \

~

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where e, is the relative permittivity of the medium, e 0 is vacuum permittivity, ~c is the Debye-Htickel parameter, N, is the Avogadro number, k is the Boltzmann constant, I is the ionic strength, T is the absolute temperature, e is the elementary electric charge, ~ is the zeta-potential, and a is the radius of the particle. The change in the surface charge density of the liposomes with the amount of mangostin added is shown in Fig. 7, and is similar to that of the zeta-potential shown in Fig. 6. The ionization potential of mangostin was 9.078 eV, which is calculated by MOPAC (that of xanthone is 8.42 eV [22]), so mangostin is considered to be unable to ionize by the dissociation of the hydrogen ion from the hydroxy group. The changes in the zeta-potential and the surface charge density are considered to be due to the following. There is no electric charge in mangostin. Net atomic charges determined by calculations with

3.3. Effects of mangostin on the microscopic-state of the liposomal bilayer membrane Fluorescent fatty acid probes were used in this study to interpret the microscopic-viscosity at different depths in the membranes. It is generally known that 2-AP can probe the region close to

430

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~)~.

:.

ii!,

.

Unc.L

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Neg.L

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Mangostin

Fig. 8. Schematicsolubilization model for mangostin in the lipid bilayer. the membrane surface, while 12-AS can probe the region near the bilayer center [14]. The microscopic-viscosity which is related to the fluorescence polarization can be calculated using PerrinWeber's equation [23]. It is also known that fluorescence polarization increases with increasing microscopic-viscosity. Fig. 9(a) (2-AP probe) and Fig. 9(b) (12-AS probe) denote the relation between fluorescence polarization and the amount of mangostin added. In all types of liposomes, both the microscopicviscosity at the region close to the membrane surface of the bilayer membranes and at the region near the bilayer center were increased on the addition of more mangostin. An increase in the amount of mangostin added seems to correspond to an increase in the component number of the liposomal bilayer. Mangostin solubilized in bilayers caused the fluidity of the acyl group to decrease and the microscopic-viscosity to increase, owing to the tight packing of lipids. Pyrene is a well known fluorescence probe that can exist in the region near the bilayer center. The pyrene monomer fluorescence shows five predominant peaks. Peak 3 is strong and shows minimal intensity variation with polarity. Peak 1 shows significant intensity enhancements in polar solvents. Thus, the intensity ratio (I1/I3) of peak 1 to peak 3 serves as a measure of the microscopicpolarity. The larger the I 1 / I 3 ratio, the greater the polarity [24-26].

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Fig. 9. Relation betweenfluorescencepolarization and amount of mangostin added at 37°C: (a) fluorescence polarization obtained with 2-AP; (b) fluorescence polarization obtained with 12-AS.

431

A. Yoshidaet al./Colloids Surfaces B." Biointerfaces, 4 (1995) 423-432

Fig. 10 shows the 11/13 ratio of pyrene monomer fluorescence versus the amount of mangostin added. In all types of liposomes, the microscopicpolarity of the bilayer membranes decreases with increasing amounts of added mangostin. Owing to the solubilization of mangostin, which is a lipophilic material, the hydrophobicity around the acyl group in the bilayer membrane was raised, and further, a polar material (water) was rejected. 3.4. Effects of mangostin on the permeability of the liposomes

The entrapped efficiency and the permeability of the liposomes was investigated. Here, mangostin

.1"O~x,~.

o Uncharged

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Fig. 10. Relation between I1/I 3 ratio of pyrene monomer fluorescenceand amountof mangostinadded at 37°C.

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liposome refers to the liposome solubilized MAC of mangostin in each liposome type. First, for all liposome types, the entrapped efficiency of the mangostin liposome was slightly increased compared with the blank liposome (data not shown). At the MAC, the increase in the particle size and an inner aqueous phase volume results in an increase in the entrapped efficiency of the mangostin liposome. Fig. 11 displays the glucose leakage from the liposome. In each liposome (Unc.L, Neg.L and Pos.L or blank liposome and mangostin liposome), the leakage of glucose from liposomes increased with time. However, the glucose leakage for mangostin liposomes (in all types) was depressed compared with that for blank liposomes. This was a result of the tight packing of the bilayer membrane, which was caused by mangostin solubilized between lipids. Mangostin is a large molecule (68 ~2 per molecule calculated by surface pressure measurements at 37°C). Solubilization of mangostin resulted in tight packing, but the bilayer membrane was unstable owing to the size of the molecule. The results from DSC measurements showed that in all types of liposomes, the phase transition temperature of DPPC was shifted to a lower temperature region expressed in Table 1. Solubilization of mangostin made the bilayer membrane heterogeneous and the cooperation of the gel-fluid phase transition was decreased [27], subsequently causing the phase transition to shift to a lower temperature.

40

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Incubation time / hr

Incubation time / hr

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,

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Fig. 11. Permeabilityof liposomes studied by measurementof glucose leakage from liposomes at 37°C: (a) unchargedliposomes; (b) negativelychargedliposomes;(c) positivelychargedIiposomes.

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A. Yoshida et al./Colloids Surfaces B: Biointerfaces, 4 (1995) 423-432

Table 1 Phase transition temperature (°C) of DPPC in liposomesa

Uncharged Negative Positive

Blank L.

Mangostin L.

41.5 42.1 41.2

28.3 29.6 25.6

Molar ratio of lipid in each liposome: uncharged, DPPC = 1; negative, DPPC : DCP = 7 : 1; positive, DPPC : SA = 7 : 1.

Consequently, by means of the solubilization of mangostin, bilayer membrane-packing was considered to be made rigid temporarily, although the bilayer membrane may be unstable after 7 days, when mangostin has been extracted.

4. Conclusion This work suggests that there is a MAC of mangostin for each individual liposome ( U n c i , Neg.L and Pos.L). Liposomal bilayer membranes are stabilized by the solubilization of mangostin in each liposome type. However, in the long term, mangostin is unstable in the liposomes as well as to the addition of higher amounts. From a practical viewpoint, mangostin liposomes (liposome solubilized mangostin) show possible potential as pharmaceutical agents, if they could be circulated in the living body up to the deposition of mangostin.

Acknowledgment The authors would like to thank Professor Dr. Pichaet Wiriyachitra at the Natural Product Research Center, Chiang Mai University for providing the mangostin throughout this study.

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