Gu Mingyan^a, Wang Zhengwu^a * , Chen Wenjun^b, Yuan Wenke^a, Wang Zhongni^b, Li Ganzuo^b
(^aSchool of Chemical and Material Engineering, Southern Yangtze University, Wuxi, Jiangsu 214036, ^b Key Lab of Colloid and Interface Chemistry for State Education Ministry, Shandong University, Ji'nan 250100)

Received on Mar. 16, 2005; Supported by the National Natural Science Foundation of China (No. 20473034) and the Taihu Scholar Foundation of Southern Yangtze University (2003)

1 INTRODUCTION
All-trans retinoic acid (ATRA) is one of the efficient anti-cancer drugs for the treatment on Acute Promyelocytic Leukemia£¨APL£©, as well as some skin diseases such as folliculi pili cornification, psoriasis, mite, and so on ^[1]. However, it is lipo-soluble, poisonous and unstable, especially sensitive to light, temperature as well as atmosphere and easily to be oxidized ^[2]. The desirable means to overcome these disadvantages is to use the microcapsule technology in the manufacture of ATRA medicament.
    Vesicle is a prospective drug carrier to increase the stability of drugs and the solubility of lipophilic substance in aqueous solution ^[3-4]. It also can be used to control the release ratio of drug in bodies and consequently, to enhance the effect of treatment and reduce the toxicity of drug ^[5-8].
    Since the formation of the first spontaneous vesicle from didodecyl dimethyl ammonium bromide (DDAB) by Kunitake in 1977^[9], up to date, vesicles have been obtained from diverse surfactants ^[10]: single-chain surfactants as well as dichain ones; mixtures of ionic surfactants (anionic/cationic systems) as well as of ionic with nonionic ones. As ATRA is not solvable in water but in ethanol, the spontaneous vesicle formed in high concentration of ethanol solution will be its desirable carrier. The structure of Dicetyl dimethyl ammonium chloride (DCDAC) is similar to DDAB ^[9]; therefore, it is hopeful that spontaneous vesicle can be obtained from DCDAC solution or its mixtures. However, to our knowledge, no work has been reported on the vesicles from DCDAC, nor about the formation of vesicles in high ethanol concentration. The present work is, by using atomic force microscope (AFM), negative-staining transmission electric microscope (TEM) , biologic microscope (BM) and dialysis bag-ultraviolet spectrophotonetry as the tools, firstly to explore the formation of vesicles from single DCDAC or its mixture with AOT in pure aqueous solution as well as in ethanol solution and secondly, to encapsulate ATRA with the vesicles.

2 EXPERIMENTAL              
DCDAC was donated by Witco Co. without any further purification (>98%). Sodium bis- (2-ethylhexyl) sulfosuccinate (AOT) was obtained from Fluka Co. without any further purification (>98.5%). Ethanol was A.R. grade. Water was doubly distilled.
    Vesicles were prepared with magnetic stirrer by mixing surfactants in proper proportions and concentrations. Formation of spontaneous vesicles was firstly judged by the special appearance of vesicle solution, i.e., the transparent blue color ^[10-11] and Biology Microscope(BM) (AQJ-2010, Zhengjiang Angel Fine Instrument Co.), then further confirmed by TEM (H-7000, HITACHI Japan) and AFM£¨Veeco Co. USA£©. Entrapment efficiency and release efficiency of ATRA were measured with dialysis bag (molecular weight: 14000, half girth: 33mm, Huamei Biology Medicine Co.) and Ultraviolet Spectrophotometry (TU-1901, Beijing Purkinje General Instrument Co.). Firstly ATRA was separately dissolved in the fixed volume ratio of aqua/ethanol, one example was added with surfactants but the other was not. Then the samples were separately put into dialysis bags to measure entrapment efficiency and release efficiency by using the solution with the same volume ratio of ethanol and quantity of ATRA as reference liquid. For each time 10 ml solution was used and the dialysis was immersed into 100 ml reference liquid being held in an Erlenmeyer flask and stirred at room temperature. After a certain time, 5 ml sample was taken out from the bag to measure the entrapment efficiency and release efficiency with Ultraviolet Spectrophotometry and another 5ml of aqua/ethanol solution was added into the bag to make its total volume unchanged.

3 RESULTS AND DISCUSSION
3.1 Spontaneous vesicle formation from pure DCDAC solution
The most direct method to judge whether there are vesicles formed is by the special transparent blue appearance of surfactant solution. The solution of DCDAC with a concentration 1.0¡Á10^-3 mol/L appeared blue, which indicated there were vesicles spontaneously formed. With the increase of the concentration of DCDAC the blue color and turbidity of solution both got deeper, but it could be cleared again by the addition of ethanol in the solution. Figure 1a is the TEM photograph of the DCDAC with a concentration 1.5¡Á10^-3 mol/L in 50% ethanol.
    It has been reported that many kinds of double-chain surfactant can form vesicles spontaneously, especially those with equal lengths such as C12 alkyl chains ^[9]. DCDAC has double equal length alkyl chains of C16 so that it is reasonable that it can form spontaneous vesicles in aqueous solution, the more interesting is that it even can form vesicle spontaneously in high concentration of ethanol solution.
3.2 Spontaneous vesicle formations from DCDAC/AOT solution             
Figure 1b, 1c were the TEM and AFM photographs of the DCDAC/AOT=2/8 with total concentration 1.0¡Á10^-3 mol/L in volume ratio of ethanol being 50% and aged for 2 days, in which vesicles were observed as spherical closed aggregation with uniform size and the diameter was about 200 nm measured by AFM. Table 1 was the detail appearance change of mixtures with the total surfactant concentration (1.0¡Á10^-3 mol/L) in different proportion of ethanol. For the mixtures DCDAC/AOT=1/9 and 2/8, solution appearance changed to clear when the ethanol volume proportion increases to 75%, which mean vesicles disappearing at this proportion ^[12]. For the mixtures DCDAC/AOT=3/7 and 7/3, though the double salt was dissolved by massive addition of ethanol, no any vesicles was observed. But in systems DCDAC/AOT=8/2, 9/1, transparent blue could be observed even in 75% ethanol solution.

Fig.1 Photos of vesicles (a) TEM photograph of pure DCDAC solution, C=1.5¡Á10^-3 mol/L, V_ethanol=50%;(b)TEM photograph DCDAC/AOT=2/8, C_total=1.0¡Á10^-3 mol/L,V_ethanol=50%.and (c) AFM photograph of the same sample with (b) (where the arrowhead indicates vesicles.).

    Though at the very high proportion of ethanol, vesicle formation was very difficult (clear appearance of solution), DCDAC/AOT mixture could form vesicle (bluish appearance of solution) even at much high concentration of ethanol (80%) in solution as the total surfactant concentration increase. Vesicles can be seen in the TEM photographs of vesicle with DCDAC/AOT=1/9 and 5.0¡Á10^-3 mol/L total surfactant concentration in 80% ethanol solution.
    Generally, ethanol can destroy remarkably the vesicle system/liposome when the concentration reaches around 6%-7% ^[13]. However, some vesicles systems can be stable in solution with higher concentration of ethanol ^[14] or other polar solutions such as n-propyl alcohol ^[15], isopropyl alcohol, and tetrahydropyrane ^[16]. Two effects are caused by adding polar solutions ^[12]: first decreasing the polarity of system and then inhibiting the formation of vesicle; second, decreasing the dielectric constant of solvent and increasing the electrostatic attraction between cation surfactant and anion surfactant. The electrostatic attraction is an advantage to the vesicle systems. Electrostatic attraction is stronger than the lyophobic action, which is the primary effect after addition of ethanol. So, vesicle systems can be stable in ethanol solutions.
2.3 The capability of encapsulating ARTA with DCDAC and DCDAC/AOT vesicles                 
Since the ATRA is soluble in ethanol but not in water, the simple method of encapsulating ATRA with DCDAC or DCDAC/AOT vesicles is by adding surfactants in the ethanol solution that contains ATRA. The absorption peak of ATRA in ethanol solution is 338 nm and it had been tested that the addition of DCDAC or DCDAC/AOT does not affect the absorption position. Therefore, this peak could be used to measure the entrapment efficiency of ATRA. Samples of ATRA vesicle system appeared yellow in the dialysis bag when freshly prepared, then this color got light with aging due to the escape of free ATRA from the bag, and at last, the concentration of free ATRA out/in side of the bag reached to the balance after a certain time. So the release efficiency f_r and the entrapment efficiency f_e of ATRA could be measured by the absorbency at the balance point of concentration with the following equations:
         (1)
          (2)
    Where W₀ was the quantity of ATRA outside of the dialysis bag and W_t was the total ATRA in solution.
    The release efficiency of the samples of DCDAC and DCDAC/AOT=1/9 with the total surfactant concentration 2.0¡Á10^-3 mol/L and total ATRA 0.1g/L (3.3¡Á10^-4 mol/L) in 50% volume ratio of ethanol had been measured with aging. About 4 days later, the dialysis reached the concentration balance point. The corresponding entrapment efficiency is 10% for the mixture but much higher (28%) for the single DCDAC component with the same surfactant concentration at this point. The reason may be that the bilayer structure of DCDAC/AOT mixture was much tighter and as the result, ATRA molecules were hard to get into the vesicles and leave a large quantity of ATRA as free in the solution.

4. CONCLUSION
Spontaneous vesicles could be formed either in single component of DCDAC or its mixture with AOT both in aqueous and aqueous/ethanol solvents. Deposit could be transformed to vesicles in ethanol/aqueous solution at high surfactant concentration. Entrapment efficiency of ATRA in the single DCDAC component solution was higher than that in its mixture with AOT.

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