HCO-10 vesicles

New method to prepare HCO-10 vesicles with high entrapment efficiency... 

Key words HCO-10 vesicles - heating-cooling cycle - high entrapment efHciency - narrow size distribution - calcein release... 

In the study of physicochemical properties of HCO-10 vesicles, the phase transition of the membrane caused by the dehydration of HCO-10 molecules was suggested [11-14]. In this research, we found that the fusion of the vesicles with dehydrated bilayers made HCO-10 vesicles larger in sizes, higher in entrapment efficiency and relatively uniform in size distribution. 

Freeze fracture electron micrographs were taken according to the method described previously [16], The mean diameter of the vesicles was measured by quasielastic light scattering (QLS) using Otsuka Electronics LPA 3000/3100. The calcein-entrapment efficiency of HCO-10 vesicles were determined as described [11]. 

The entrapment efficiencies and sizes of HCO-10 vesicles are shown in Table 1. When a dispersion of HCO-10 prepared either by vortex or by extrusion was heated and then cooled, both the entrapment efficiency and size of HCO-10 vesicles became increased and the size distributions were narrow (Fig. 2). We call the above procedure a heating-cooling cycle. It is apparent that the method of... 

Table 1 Calcein entrap % and sizes of HCO-10 vesicle suspensions...

Fig. 2 The sizes and autocorrelation functions in QLS measurement of HCO-10 vesicles. The vesicles were prepared by extrusion (A)-(C) and prepared by vortex (D) (E) (F) (A) (D) before the heating-cooling cycle, (B) (E) after step-by-step cooling and (C) (F) after direct cooling from 40 °C to room temperature...

In general, the entrapment efficiencies for the preparations of HCO-10 vesicles in Table 1 were lower than those described in the Hteratures for the corresponding hposome preparations. When HCO-10 vesicles prepared by vortex were sonicated, they were destructed and the suspensions separated. 

HCO-10 vesicles can be seen as particles like liposomes with an aqueous compartment. The sizes and organization of HCO-10 vesicles prepared by vortex or the heating-cooling cycle are shown in Table 1 and Fig. 3. At room temperature, the vesicles appear to consist of a few concentric bilayers with a large internal aqueous space in Figs. 3A and 3C. While, during the heating of HCO-10 suspensions small-vesicles are observed, some appear as small unilamellar vesicles (SUVs), as shown in Fig. 3B. 

The temperature dependence of the sizes of HCO-10 vesicles is shown in Fig. 4. While the suspension was being heated, the vesicle size decreased steeply from about 220 to 50 nm around 37 °C. In order to investigate further the morphology of the vesicles, a freeze-fracture electron microscope was used because ultrarapid cryofixation was able to preserve the morphology of HCO-10 vesicles at... 

Each of those preparative methods described in Table 1 produced close, discrete structures as judged from the freeze fracture electron microscopic appearances or the latency of calcein fluorescence. HCO-10 vesicles prepared by the heating-cooling cycle had a higher entrapment efficiency than those prepared by the other methods. The cUfferences in entrapment efficiency between HCO-10 vesicles presumably reflect the size or type of HCO-10 vesicles formed by each method, e.g., the vesicles prepared by the heating-cooling cycle (5% HCO-10, 440 nm, 15.4%) and the vesicles prepared by vortex (5% HCO-10, 240 nm, 6.20%). The increase in the entrapment efficiency is accompanied by an increase in the size of the HCO-10... 

Fig. 4 Variation of mean diameters and entrap % of HCO-10 vesicles with the temperatures...

In an early investigation, Horiuchi and Tajima observed with QLS that, when HCO-10 vesicle suspension was heated above 37 °C, those vesicles were transformed into small ones. They emphasized the influences of the dehydration of EOs on the vesicular membrane of HCO-10 [12]. We found that, when those small vesicles were subsequently cooled to room temperature, the larger vesicles formed, which can entrap a high amount of the solute molecule (Fig. 4). Hofland et al. prepared the large... 

Fig. 5 Temperature dependence of the calcein release from HCO-10 vesicles...

In summary, the results reported here show that the heating-cooling cycle could be a useful preparative method for HCO-10 vesicles. We took advantage of the dehydration and rehydration of HCO-10 bilayers near the phase transition temperature to improve the preparation for HCO-10 vesicles. From a methodological point of view, it is significant that the physicochemical features of NSVs are used to develop their preparations. 

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