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J. Phys. Chem. 1996, 100, 13898-13900
Mixed Vesicles of Didodecyldimethylammonium Bromide with Recognizable Moieties at the Interface C. M. Paleos,* Z. Sideratou, and D. Tsiourvas Institute of Physical Chemistry, NCSR “Demokritos”, 15310 Aghia ParaskeVi Attikis, Athens, Greece ReceiVed: May 16, 1996; In Final Form: June 17, 1996X
Mixed vesicles with complementary moieties were prepared by cosonication of didodedcyldimethylammonium bromide with 5,5-didodecylbarbituric acid and/or 9-hexadecyladenine. These vesicles, bearing recognizable moieties at the interface, were more stable than the simple vesicles prepared from didodecyldimethylammonium bromide. Phase contrast optical and AFM microscopies were used for imaging these vesicles and also for revealing that the interaction occurring between the recognizable moieties of mixed vesicles leads to the formation of larger aggregates.
Introduction Molecular recognition through hydrogen bonding is primarily effective in aprotic solvents, and it has been studied extensively in recent years.1-5 Recognition through directional hydrogen bonding is however far more important in aqueous media due to its biological relevance.6-12 For instance, such interaction has been accomplished in monolayers formed from a diaminotriazine amphiphile in which as complementary molecules were used nucleosides and nucleic bases in aqueous media.6 This unexpected interaction was attributed to the organizational characteristics occurring at air-water interface. In this connection, amphiphilic-type 5,5-didodecylbarbituric acid (DBA, Figure 1), with the well-known recognizable moiety was prepared. This molecule was prepared with the expectation of forming monolayer or bilayer structures including the formation of vesicles. Employing the conventional sonication method, vesicles could not be prepared from this compound. The formation of vesicles cannot however be completely excluded since in this preliminary study exhaustive attempts were not performed for the formation of vesicles, i.e., by employing the diversified methods that have been developed for the preparation of liposomes.13-15 The preparation of vesicles containing DBA was however achieved through the formation of mixed vesicles by cosonication of didodecyldimethylammonium bromide (DDAB) with DBA. In the same manner mixed vesicles of DDAB with 9-hexadecyladenine (HDA, Figure 1) were also prepared. A characteristic of these vesicles is the placement of the recognizable moieties of DBA and HDA at the interface of the vesicles. It could be therefore possible these vesicles to interact intervesicularly through hydrogen bonding affording new aggregates. These latter vesicles as well as the vesicles before interaction were imaged with optical and AFM microscopy. In this study vesicles prepared from DDAB are referred to as simple vesicles. The interaction of analogous barbituric acid and adenine derivatives in isotropic and aprotic media was reported16-18 several years ago, resulting in the formation of hydrogen-bonded complexes. Experimental Section Materials. 5,5-Didodecylbarbituric acid was synthesized by the reaction of bis(didodecyl) malonate with urea as described recently.19 * To whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, August 1, 1996.
S0022-3654(96)01424-4 CCC: $12.00
Figure 1. Structures of 5,5-didodecylbarbituric acid (DBA) and 9-hexadecyladenine (HDA).
9-Hexadecyladenine was prepared20 by reacting adenine with n-bromohexadecane under mild experimental conditions. Didodecyldimethylammonium bromide was purchased from Aldrich and was recrystallized from ethanol. Vesicles Formation. Mixed vesicles were formed in triply distilled water by cosonication of 0.0072 M DDAB with 0.0018 M of DBA or 0.0018 M of HDA for 20 min, in a MSE probe sonicator. Mixed vesicles in which have been incorporated both 5,5-didodecylbarbituric acid and 9-hexadecyladenine have also been prepared by the sonication method. For optical microscopy the dispersions formed were filtered through 5 µm Millipore filters while for AFM through 1.2 µm filters. Characterization. NMR spectra were obtained with a Bruker AC 250 spectrometer operating at 250 MHz for proton and 62.9 MHz for carbon-13. The NMR experiments were performed in deuterated chloroform, using the residual solvent peak as internal reference. AFM microscopic images were obtained with a Multi-Mode Nanoscope III Microscope (Digital Instruments) employing the Tapping mode. The samples were prepared by placing a few droplets of the vesicles dispersion on freshly cleaved mica, removing by evaporation the dispersing medium and finally drying the film formed on air. Results and Discussion The incorporation of DBA acid and HDA in DDAB vesicles was established by carbon-13 NMR. Thus carbon-13 NMR © 1996 American Chemical Society
Letters
J. Phys. Chem., Vol. 100, No. 33, 1996 13899
Figure 2. Typical images obtained with AFM microscopy (tapping mode) of simple vesicles originating from DDAB (A), mixed vesicles of DDAB with DAB (B) or HDA (C), respectively, and vesicles (D) obtained after the interaction of B and C aggregates.
spectra, obtained from mixed vesicular samples after their lyophilization, showed chemical shifts attributed to the presence of DBA or HDA in addition to peaks attributed to DDAB. Thus, for vesicles containing DBA the peak of C4 and C6 appeared at 173 ppm while that of C2 at 149 ppm having an intensity ratio 2:1 as expected from molecular structure (carbon atoms were numbered as shown in Figure 1). For HDA peaks of C4 at 155 ppm and C2 at 150 ppm (Figure 1) were observed. The assignment of the above peaks was made by considering analogous peaks reported in the literature.21 For the quantitative determination of the incorporated materials in DDAB vesicles the method of 13C NMR based on internal reference was employed. It was found that 68% of the initially employed concentration of DBA was incorporated, while for HDA 52% of initial concentration was solubilized in the vesicles. In Figure 2 typical images obtained with AFM microscopy (tapping mode) of simple vesicles originating from DDAB (A) and mixed vesicles of DDAB with DAB (B) or HDA (C) respectively are shown. In the same figure vesicles (D) originating from the interaction of B and C are also shown. The vesicles are spherical and their average diameters for simple and mixed vesicles are shown in Table 1. It is seen that DDAB vesicles are small enough to be seen with phase contrast microscopy equipped with image processing facility. The vesicles were imaged with the Tapping mode, which is the method of choice for systems that are susceptible to deformation
TABLE 1: Diameter of Vesicles As Determined by Phase Contrast Optical and AFM Microscopies Employing the Tapping Mode type of vesicles DDAB DDAB-DBA DDAB-HDA DDAB-DBA-HDA (simple mixing) DDAB-DBA-HDA (cosonication)
optical microscopy (diameter, µm)
AFM microscopy (diameter, nm)
1-2.5 1-2 1.5-3
20-120 25-90 20-60 50-250
0.5-1.5
30-100
by the conventional contact mode of AFM microscopy. The incorporation of DBA or HDA results in the formation of mixed vesicles which are significantly larger than DDAB. They can be observed by optical microscopy and they have almost comparable sizes (Table 1). It is interesting to note that when mixed vesicles incorporating respectively the recognizable barbituric acid and adenine derivatives were allowed to interact, larger vesicles were formed. In this case if the vesicles were not interacting, then they would have the same size as the interacting ones. The fact that larger vesicles were produced is an indication that interaction occurs between the vesicles bearing the complementary molecules. A mechanism that may be proposed is that as the particles come into contact, the recognizable hydrophilic moieties located at the external interface of the vesicles interact, leading to larger particles. One
13900 J. Phys. Chem., Vol. 100, No. 33, 1996
Letters incorporation of amphiphilic-like barbituric and adenine derivatives results in the stabilization of the simple DDAB vesicles in a way reminiscent the stabilization of the vesicles by the incorporation of cholesterol. In conclusion mixed vesicles were formed by cosonication of DDAB with DBA or HDA which were more stable than simple vesicles originating from DDAB. Phase contrast optical and AFM microscopies were employed for their imaging. By these microscopic observations it was found that due to interaction of the recognizable moieties located at the interface larger vesicles than the interacting ones were obtained. The relevance of this property with the processes occurring in biological systems is quite evident. References and Notes
Figure 3. Stability of vesicles as judged by the addition of increasing quantities of ethanol.
may envisage that the driving force for this enlargement of vesicles is the presence of the complementary molecules, otherwise the collision of the vesicles would not be effective toward the formation of larger particles. With AFM it was not possible to observe any morphological modification except the diameter increase of the vesicles. It should be noted that this enlargement is shown more explicitly with the small sized vesicles (Table 1) as observed with AFM microscopy than with the bigger aggregates observed with optical microscopy. This may be attributed to the fact that small vesicles are more labile compared to multilamellar, and therefore they have the tendency to collide effectively forming larger aggregates. For comparison purposes mixed vesicles were prepared by cosonication of the three compounds, i.e., DDAB, DBA, and HDA and found to have comparable sizes to the vesicles incorporating DDAB and either one of the recognizable molecules. The stability of the vesicles was evaluated22 by the addition of increasing quantities of ethanol to the dispersions of simple and mixed vesicles and measuring their turbidity (absorbance at 400 nm). In Figure 3 is shown the turbidity vs the quantity of added ethanol. It is seen that DDAB vesicles are the least stable, and it suffices 10% (v/v) addition of ethanol to destroy the vesicles. Mixed vesicles of DDAB with DBA or HDA or resulting from cosonication of all three components as well as those resulting by the interaction of the mixed vesicles incorporating either one of recognizable moieties exhibit excellent stability, and one has to add more than about 40% alcohol in order to destroy the vesicles. It is obvious that the
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