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Langmuir 1999, 15, 282-284
Transformation of Dihexadecyl Phosphate Vesicles to Micelles Using Guanidinium-Type Counterions Constantinos M. Paleos,* Dimitra Kardassi, and Dimitris Tsiourvas Institute of Physical Chemistry, NCSR “Demokritos”, 15310 Aghia Paraskevi, Attikis, Greece Received July 24, 1998. In Final Form: October 26, 1998
Introduction The application of liposomes and synthetic vesicles as drug delivery systems has triggered significant research in the last 20 years.1-3 The control of their size, shape, and stability has been addressed in a number of ways, including the invention of diversified methods for vesicle preparations,2 the functionalization and/or coating of their interface,3 or the formation of polymerized vesicles through addition polymerization.4 The latter method has the disadvantage of forming nonbiodegradable aggregates which, a priori, may exclude polymerized vesicles in prospected applications as drug delivery systems. A class of synthetic amphiphiles widely employed for the preparation of simple and mixed vesicles is dialkyl phosphates and specifically dihexadecyl phosphate (DHP).5,6 For vesicles derived from phosphate esters, investigations have primarily centered on the modification of their structure, size, stability, absorption, and entrapment capabilities as well as permeability as a function of the pH of the aqueous medium.7-10 Within this context, in a recent report11 we have investigated the stability and size of DHP vesicles as affected by Li+, K+, Na+, or Cs+ counterions. It was found that the bigger the counterion is, the smaller the vesicles that are formed are. Preliminary experiments with guanidinium (C+(NH2)3) counterion have shown11 that the size of DHP vesicles is also affected, i.e., becoming smaller during the preparation, in situ, of vesicles by DHP interaction with guanidinium carbonate. In the present study a series of dihexadecyl phosphateguanidinium type salts was prepared by the interaction of equimolar quantities of DHP with guanidine (1) or its derivatives, i.e., biguanide (2), 1,1-dimethylbiguanide (3), arginine (4), and dicyandiamide (5). The role of guanidinium counterion and its derivatives in affecting the size of DHP vesicles and ultimately the type of aggregates is (1) Gregoriadis, G. Trends Biotechnol. 1995, 13, 527 and references therein. (2) New, R. R. C. Liposomes, A Practical Approach, 1st ed.; IRL Press: Oxford, U.K., 1990. (3) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem. 1987; 100, 117; Angew. Chem., Int. Ed. Engl. 1988, 27, 114-158. (4) Paleos, C. M. In Polymerization in Organized Media; Paleos, C. M., Ed.; Gordon and Breach Science Publishers: Philadelphia, 1992; p 283 and references therein. (5) Carmona-Ribeiro, A. M. Chem. Soc. Rev. 1992, 209. (6) Blandamer, M. J.; Briggs, B.; Cullis, P. M.; Engberts, J. B. F. N. Chem. Soc. Rev. 1995, 251. (7) Rupert, L. A. M.; van Breemen, J. F. L.; Hoekstra, D.; Engberts, J. B. F. N. J. Phys. Chem. 1988, 92, 4416. (8) (a) Carmora-Ribeiro, A. M.; Hix, S. J. Phys. Chem. 1991, 95, 1812. (b) Carmora-Ribeiro, A. M.; Castuma, C. E.; Sesso, A.; Schreier, S. J. Phys. Chem. 1991, 95, 5361. (9) Walde, P.; Wessicken, M.; Ra¨dler, U.; Berclaz, N.; Conde-Frieboes, K.; Luigi, P. J. Phys. Chem. B 1997, 101, 7390. (10) Tricot, Y. M.; Furlong, D. N.; Sasse, W. H. F.; Davis, P.; Snook, I.; van Mengen, W. J. Colloid Interface Sci. 1984, 97, 380. (11) Kardassi, D.; Tsiourvas, D.; Paleos, C. M. J. Colloid Interface Sci. 1997, 186, 203.
investigated. In this connection it should be stressed that DHP vesicles by their neutralization with guanidine or its derivatives become carriers of these counterions including protonated arginine amino acid. Since many natural antiviral compounds, now used against HIV infection, are guanidines,12 the interest in inventing new carriers of guanidine is self-evident. It should also be mentioned that arginine residues are involved in the interaction of nucleic acid phosphodiesters with the highly charged protamines and histones.13 Experimental Section Materials. The synthesis of DHP has already been described.11 Guanidinium carbonate (Aldrich), 1,1-dimethylbiguanide hydrochloride (Sigma), L-(+)-arginine (Riedel-de Haen), and dicyandiamide (Aldrich) were commercially available whereas biguanide was prepared by a method described in the literature.14 The latter compound was used immediately since it turns yellow on exposure to the atmosphere. 1,1-Dimethylbiguanide was prepared by neutralization of 1,1- dimethylbiguanide hydrochloride in ethanol according to a literature method.15 Dihexadecyl phosphate-guanidinium type complexes were prepared by interacting in ethanol equimolar quantities of guanidine carbonate, biguanide, 1,1-dimethylbiguanide, or arginine with DHP. Crystals of the complexes precipitate slowly, the purity of which was confirmed by elemental analysis. The salt of DHP with dicyandiamide could not be obtained following the above method. Thus, when dicyandiamide was interacted with DHP, the salt was not precipitated because it was not apparently formed because the neutral character of dicyandiamide. The isolated guanidinium-based salts were used for the preparation of vesicles (see below), while in the latter case the method for the formation of vesicles was modified; i.e., DHP was dispersed in an aqueous dicyandiamide solution. Vesicles Preparation. Vesicles were prepared by a method developed by Deamer and Bangham.16 Specifically, 2 mL of DHP complexes dissolved in chloroform (3 × 10-3 M) at 40 °C is added at a rate of 0.5 mL/min to 8 mL of distilled water kept at 70 °C. Vesicles formed were filtered through a 3-µm filter. Characterization. Vesicles were imaged by video-enhanced phase-contrast optical microscopy employing an Olympus BX50 microscope. The microscope was coupled with a Kodak Megaplus model 1.4i CCD camera. Microscopic images were captured using the IC-PCI image board (Imaging Technology Inc.) and analyzed using SigmaScan Pro v4.0 image analysis software (SPSS Inc.). AFM images were obtained with a MultiMode Nanoscope III microscope (Digital Instruments) employing the tapping mode operation. Samples for AFM observation were prepared by placing droplets of vesicle dispersions on freshly cleaved mica. Dynamic light scattering measurements were conducted at 25 °C with a series 4700 Malvern system composed (12) Rama Rao, A. V.; Gurjar, M. K.; Islam, A. Tetrahedron Lett. 1993, 34, 4493. (13) Schrader, T. Chem. Eur. J. 1997, 3, 1537. (14) Karipides, D.; Fernelium,W. C. Inorg. Synth. 1963, 7, 56. (15) Slotta, K. H.; Tschesche, R. Ber. Dtsche. Chem. Ges. 1929, 62, 1390. (16) (a) Deamer, D.; Bangham, A. D. Biochim. Biophys. Acta 1976, 443, 629. (b) Carmora-Ribeiro, A. M.; Yoshida, L. S.; Sesso, A.; Chaimovich, H. J. Colloid Interface Sci. 1984, 100, 433.
10.1021/la980933x CCC: $18.00 © 1999 American Chemical Society Published on Web 12/11/1998
Notes
Langmuir, Vol. 15, No. 1, 1999 283
Table 1. Basicity of Guanidine and Its Derivatives 1,1-dimethyldicyanguanidine19 biguanide14 biguanide20 arginine21 diamide22 pk1 pk2 pk3
13.65
11.52 2.93
11.52 2.77
13.2 9.09 2.18
7
Chart 1
of a PCS5101 goniometer with a PCS7 stepper motor controller, a Cyonics variable-power Ar+ laser, operating at 488 nm and 10-mW power, a PCS8 temperature control unit, and a RR98 pump/filtering unit. A 192-channel correlator was used for accumulation of the data. The correlation function was collected at a 90° angle. Ultracentrifugation was performed by spinning the sample at 200 000g for 24 h, where even small vesicles (SUVs) sediment.2
Results and Discussion Guanidine (1) and its (2-5) derivatives are strong bases (Table 1) and interact with the phosphate groups located at the vesicles’ interface not only electrostatically but also through hydrogen bonding17 because of the complementarity of the reactants (Chart 1). Because of the organized character of the vesicular interface, hydrogen-bonding interaction between guanidinium counterions and phosphate groups is significantly more effective compared to the one achieved in isotropic media. A binding constant K of 1.4 M-1 was reported for recognition in isotropic media, while at the interface of vesicles, a K of 102-104 M-1 was determined.18 Vesicles were observed with optical as well as AFM microscopy. AFM microscopy was used to image small aggregates that could not be seen with optical microscopy. In this connection it has to mentioned that it was possible that the aggregate structure on mica surfaces could be different from that of the bulk solution due to the effect of the substrate.23 It was, however, possible to image vesicles on the mica surface, the size of which was comparable to that obtained with electronic microscopy, as was shown in previous studies.24 Vesicles originating from DHP had 0.7-1 µm diameters as observed with phase-contrast optical microscopy. We were also able to observe vesicles of 30-80 nm using AFM microscopy. Dynamic light scattering experiments have shown that hydrodynamic radii for DHP ranged from 30 to 150 nm centered at 90 nm. However, aggregates from DHP salts with guanidinium counterions originating from guanidines 2-5 were small enough that clear micellar solutions were obtained. The pH of the clear solutions (17) Hirst, S. C., Tecilla, P.; Geib, S. J.; Fan, E.; Hamilton, A. D. Isr. J. Chem. 1992, 32, 105. (18) (a) Onda, M.; Yoshihara, K.; Koyano, H.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 8524. (b) Paleos, C. M.; Tsiourvas, D. Adv. Mater. 1997, 9, 695 and references therein. (19) Williams, G.; Hardly, M. L. J. Chem. Soc. 1953, 2560. (20) Dean, J. A. Lange’s Handbook of Chemistry, 13th ed.; McGrawHill Book Company: New York, 1985; pp 5-34. (21) Budavari, S. Merck Index, 12th ed.; Merck & Co. Rahway, NJ, 1996; p 817. (22) Pinck, L. A. Inorg. Synth. 1950, 3, 43. (23) (a) Manne, S.; Gaub, H. E. Science 1995, 270, 1480. (b) Wanless, E. J.; Ducker, W. A. J. Phys. Chem. 1996, 100, 3207. (c) Jaschke, M.; Butt, H. J.; Gaub, H. E.; Manne, S. Langmuir 1997, 13, 1381. (24) (a) Azumi, R.; Matsumoto, M.; Kawabata, Y.; Ichimura, T.; Mizuno, T.; Miyamoto, H. Chem. Lett. 1995, 1925. (b) Paleos, C. M.; Sideratou, Z.; Tsiourvas, D. J. Phys. Chem. 1996, 100, 13898.
Figure 1. AFM image (obtained by the tapping mode) of DHPdicyandiamide.
was measured and found to be neutral. Therefore, the observed transformation of vesicles to micelles does not result from a pH change as reported for alkyl phosphates.8,9 In this case the size of the guanidinium ion is such that the surfactant parameter requirement,25 v/Rl < 1/3, is fulfilled and therefore spherical micelles are formed. This was confirmed by the solubilization of hydrophobic dyes such as Sudan Red 7B, which is not solubilized in vesicle dispersions. Additionally, when dynamic light scattering experiments were employed, it was not possible to detect particles in the range of the vesicles’ hydrodynamic radii. Physically, this behavior can be rationalized in light of the dependence between the size of the counterions and the size of the vesicles as applied to alkali-metal salts of dihexadecyl phosphate11 and as established26-29 for doublechained quaternary ammonium amphiphiles. Specifically, as hydrated counterions become larger, they will be located further away from the vesicle surface. This leads to increased headgroup size and intervesicle repulsion and therefore decreased size of the aggregates, leading finally to micelles. It was, however, possible with AFM to detect a small number of aggregates having diameters ranging from 7 to 25 nm. These particles represented less than 1 wt % of the clear dispersions as determined by ultracentrifugation at 200 000 g. On the other hand, the size of DHP vesicles dispersed in dicyandiamide water solutions, as determined by AFM microscopy and optical microscopy (Figure 1), was comparable to that of DHP vesicles. This behavior of dicyandiamide is attributed to its neutral, not protonated, form, which cannot be electrostatically attached at the vesicles interface. It can only be attached through hydrogen bonding. This effect on the vesicles’ size indicates that dicyandiamide is primarily dissolved in the bulk aqueous phase, with the protons acting as counterions as is the case with DHP. It is therefore evident that electrostatic binding of guanidine and its derivatives is the crucial factor affecting the transformation of vesicles to micelles. To further elucidate the effect of these counterions on the transformation of DHP vesicles to micelles, we have (25) Evans, D. F.; Ninham, B. W. J. Phys. Chem. 1986, 906. (26) Ninham, B. W.; Evans, D. F.; Wel, G. J. J. Phys. Chem. 1983, 87, 5020. (27) Brady, J. E.; Evans, D. F.; Warr, G. G.; Grieser, F.; Ninham, B. W. J. Phys. Chem. 1986, 90, 1853. (28) Brady, J. E.; Evans, D. F.; Kachar, B.; Ninham, B. W. J. Am. Chem. Soc. 1984, 106, 4279. (29) Miller, D. D.; Evans, D. F.; Warr, G. G.; Bellare, J. R. J. Colloid Interface Sci. 1987, 116, 598.
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Notes
Figure 2. AFM image (obtained by the tapping mode) of mixed vesicles resulting from DHP and the DHP-biguanide complex (1:0.25 molar ratio). Table 2. Variation of the Size of the DHP Vesicle as a Function of the Added Biguanidinium Counterion biguanide counterion (%) vesicle diameters (nm)
10 30-60
25 14-18
50 10-16
75 8-16
proceeded to the formation of dispersions on which less than equimolar quantities of guanidinium counterions were employed. Specifically, in a series of experiments 10-90% molar quantities of biguanidinium or 1,1-dimethylbiguanidinium counterions were employed relative to DHP. In all cases turbid dispersions were obtained, the microscopic observation of which indicates the formation of vesicles. As expected, it was found that the percentage of vesicles which is transformed to micelles increases as the quantity of quanidinium counterions increases. For instance, when 90 or 75% molar quantities of 1,1-dimethylbiguanidinium were employed for the neutralization of DHP, about 70 or 25 wt %, respectively, of the initial quantity was found in the clear micellar aqueous phase after ultracentrifugation. When lower quantities (50-10%) of the guanidinium counterions were utilized, micelles were not obtained. Judging from the small size (8-18 nm) of the aggregates obtained when 25-75% of guanidinium counterion was employed, it is concluded that mixed vesicles were formed. These aggregates (Figure 2) are considerably smaller compared to pure DHP vesicles. On the contrary, when only 10% of counterions is used, the size of the vesicles (30-60 nm) is comparable to that of pure DHP. The effect of the percentage of biguanidinium counterion on the vesicle size is summarized in Table 2. The vesicles obtained are stable for a period of about 1 month stored at 20 °C. Their stability was also evaluated by employing an accelerated method of the vesicles’ destruction, i.e., with the addition of increasing quantities of ethanol to their dispersions30 and measurement of their
Figure 3. Plot of absorbance at 400 nm as a function of added ethanol. Vesicles of DHP (×) and DHP with dicyandiamide (+). Mixed vesicles consisting of DHP and the DHP-biguanide complex containing various molar quantities of biguanide counterion: ([) 90%, (9) 75%, (b) 50%, (l) 25%, (1)10%.
turbidities (absorbance at 400 nm) (Figure 3). For instance, vesicle dispersions with 10 or 90% of biguanidinium counterion are not showing any change in turbidity with increasing quantities of ethanol. Their behavior is, therefore, similar to that of DHP or DHP-dicyandiamide dispersions. On the other hand, vesicles of DHP neutralized with 25-75% biguanide exhibit a different behavior, presenting a small but clearly detectable decrease in turbidity with added quantities of ethanol. It is evident that the mixed vesicles obtained in these cases have a lower stability toward ethanol. Conclusions Interaction of DHP vesicles with increasing quantities of guanidinium-type counterions decreases their size, transforming them finally to micelles. It should be noted that both liposomes and micelles are efficient carriers of the biologically significant guanidinium counterions including that of arginine amino acid. It can also be concluded that the presence of a diversity of counterions in biological systems can modify the size of these aggregates, affecting in turn their biological activity. Acknowledgment. The authors acknowledge the assistance of Dr. S. Pispas with light-scattering experiments. LA980933X (30) Regen, S. L.; Czech, B.; Singh, A. J. Am. Chem. Soc. 1980, 102, 6638.
