© Copyright 2004 American Chemical Society
AUGUST 31, 2004 VOLUME 20, NUMBER 18
Letters Aqueous Solubilization of Highly Fluorinated Molecules by Semifluorinated Surfactants Khanh C. Hoang† and Sandro Mecozzi*,†,‡ School of Pharmacy and Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53705 Received April 5, 2004. In Final Form: July 9, 2004 The physical and chemical properties of organic compounds are deeply affected by the introduction of fluorinated substituents. Perfluorinated and highly fluorinated organic molecules are both hydrophobic and lipophobic. This makes the recognition and the binding of fluorinated molecules extremely difficult to achieve through classical elements of molecular recognition. Here we show that semifluorinated watersoluble block copolymers can generate micellar structures having a fluorous phase-based inner core in aqueous solution. Furthermore, we show that these micelles can be used to encapsulate and bind highly fluorinated molecules through association in the internal fluorous phase (fluorophobic effect). We report that semifluorinated block copolymers can be used for the aqueous solubilization of the widely diffused gaseous anesthetic sevoflurane, thereby suggesting the possibility of the intravenous delivery of this commonly used anesthetic.
Introduction Semifluorinated surfactants show the typical amphiphilic behavior of the corresponding hydrogenated counterparts. However, the larger van der Waals volume of fluorinated chains, combined with the low polarizability of fluorine, makes fluorocarbons not only more highly hydrophobic than the corresponding hydrocarbons but also lipophobic.1-4 This peculiar arrangement of properties provides a driving force for perfluorinated surfactants to self-assemble in aqueous solutions into highly stable and well-organized films, bilayers, and discrete supramolecu* Corresponding author. Address: 777 Highland Avenue, Madison, WI 53705. E-mail:
[email protected]. † School of Pharmacy, University of Wisconsin. ‡ Department of Chemistry, University of Wisconsin. (1) Kissa, E. Fluorinated Surfactants and Repellents, 2nd ed.; Surfactant Science Series; Marcel Dekker: New York, 2001; Vol. 97. (2) Smart, B. E. Organofluorine Chemistry: Principles and Applications; Plenum Press: New York, 1994; pp 57-88. (3) Pavlath, A. E.; Hudlicky, M. Chemistry of Organic Fluorine Compounds II: a Critical Review; ACS Monograph No. 18; American Chemical Society: Washington, DC, 1995. (4) Marsh, E. N. G. Chem. Biol. 2000, 7, 645-652.
lar systems such as vesicles, tubules, and micelles.5-19 Finally, it is known that perfluorinated surfactants have (5) Percec, V.; Tushar, K. B. Tetrahedron 2002, 58, 4031-4040. (6) Krafft, M. P. Adv. Drug Delivery Rev. 2001, 47, 209-228. (7) Krafft, M. P.; Riess, J. G. Biochimie 1998, 80, 489-514. (8) Matsumoto, K.; Mazaki, H.; Matsuoka, H. Macromolecules 2004, 37, 2256-2267. (9) Monduzzi, M. Curr. Opin. Colloid Interface Sci. 1998, 3, 467477. (10) Schmutz, M.; Michels, B.; Marie, P.; Krafft, M. P. Langmuir 2003, 19, 4889-4894. (11) Zhang, G.; Maaloum, M.; Muller, P.; Benoit, N.; Krafft, M. P. Phys. Chem. Chem. Phys. 2004, 6, 1566-1569. (12) Krafft, M. P.; Goldmann, M. Curr. Opin. Colloid Interface Sci. 2003, 8, 243-250. (13) Oda, R.; Huc, I.; Danino, D.; Talmon, Y. Langmuir 2000, 16, 9759-9769. (14) Zarif, L.; Gulik-Krzywicki, T.; Riess, J. C.; Pucci, B.; Guedj, C. Colloids Surf., A 2003, 8, 243-250. (15) Meissner, E.; Myszkowski, J.; Szymanowski, J. Tenside, Surfactants, Deterg. 1995, 32, 261-271. (16) Tae, G.; Kornfield, J. A.; Hubbell, J. A.; Johannsmann, D.; HogenHesch, E. H. Macromolecules 2001, 34, 6409-6419. (17) Zhang, H.; Pan, J.; Hogen-Hesch, E. H. Macromolecules 1998, 31, 2815-2821. (18) Vierling, P.; Santaella, C.; Greiner, J. J. Fluorine Chem. 2001, 107, 337-354.
10.1021/la049128a CCC: $27.50 © 2004 American Chemical Society Published on Web 07/29/2004
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Figure 1. 19F NMR spectra of 6 mg/mL F8P6 in CD3OD (1) and D2O (2) obtained from a Varian 400 MHz NMR spectrometer. The polymer solutions were sonicated for 30 min for complete dissolution. Scheme 1. Synthesis of Semifluorinated Block Copolymer F8P6
lower critical micellar concentrations than their hydrogenated counterparts, implying higher stability of the corresponding aggregates.1 We are taking advantage of these properties of perfluorinated alkyl chains to design semifluorinated block copolymers that self-assemble into stable micelles and that can be used in the delivery of highly fluorinated drugs. Here we show that coupling poly(ethylene glycol) (Mn ) 6000 amu) to a perfluorinated alkyl chain generates an amphiphilic block copolymer (F8P6) that self-assembles into nanoscopic micellar structures. Poly(ethylene glycol) has been chosen because of its hydrophilic and stealth properties.20 We show that the micelles derived from F8P6 can be used to encapsulate sevoflurane, a widely used, gaseous, anesthetic drug.21 The complex micelle-sevoflurane can then be used as a tool for the intravenous delivery of sevoflurane. Experimental Section Materials. All reagents were used without further purification. 1H,1H-perfluoro-1-nonanol, benzyl bromide (98%), tosyl chloride (99.5%), poly(ethylene glycol), and palladium activated carbon were purchased from Aldrich Chemical Co. Organic solvents were purified and dried by flowing through aluminacontaining columns. FC-72 (perfluorohexanes) was purchased from SynQuest Labs., Inc. Instrumentation. Bruker REFLEX II [matrix-assisted laser desorption/ionization, time-of-flight (MALDI-TOF) analyzer] was used to determine molecular weights. 19F NMR spectra of the polymers were obtained on a Varian AC-400 spectrometer using 5 mm o.d. tubes; samples were prepared in either CD3OD or D2O containing 20 mM sodium trifluoroacetate as an internal standard. Fluorescence spectra were obtained with a F3010 Hitachi fluorometer. Micellar size was determined from dynamic light scattering on Zeta Potential/Particle Sizer Nicomp 380 ZLS. Synthesis of Benzyl-tosyl-poly(ethylene glycol). Sodium hydride (0.8 g, 10.0 mmol) was added to poly(ethylene glycol) (19) Mathis, G.; Leempoel, P.; Ravey, J.-C.; Selve, C.; Delpuech, J.-J. J. Am. Chem. Soc. 1984, 106, 6162-6171. (20) Yokoyama, M.; Miyauchi, M.; Yamada, N.; Okano, T.; Kataoka, K.; Inoue, S. J. Controlled Release 1990, 11, 269-278. (21) Halpern, D. F. In Organofluorine Chemistry: Principles and Commercial Applications; Banks, R. E., Ed.; Plenum Press: New York, 1994; pp 543-554.
Figure 2. 19F NMR spectra of F8P6 at different concentrations in D2O obtained from a Varian 400 MHz NMR spectrometer. Number of scans: 24 000 (0.5 mg/mL), 2048 (1 mg/mL), and 16 (20 mg/mL). (Mn ) 6000, 20 g, 3.3 mmol) in anhydrous tetrahydrofuran (THF). After the mixture was stirred for 10 min, benzyl bromide (0.17 g, 1.0 mmol) was added over 10 min with a syringe pump. The reaction mixture was stirred overnight and then quenched with water. THF was partially evaporated, and ethyl ether was added to precipitate monobenzyl-poly(ethylene glycol) along with dibenzylated product. Without further purification, the monoprotected product was tosylated with tosyl chloride (0.27 g, 1.4 mmol) and N,N-diisopropylethylamine (0.4 g, 3.0 mmol) in anhydrous THF. After the reaction mixture was stirred overnight, the solvent was partially evaporated and ethyl ether was added to recrystallize benzyl-tosyl-poly(ethylene glycol) with 80% yield (two steps). The purity of the product was confirmed by HPLC. Synthesis of 1H,1H-Perfluoro-1-nonanyl-poly(ethylene glycol). To 4.8 g (0.8 mmol) of benzyl-tosyl-poly(ethylene glycol) in anhydrous THF were added sodium hydride (0.5 g, 21 mmol) and 1H,1H-perfluoro-1-nonanol (0.36 g, 0.8 mmol). The reaction mixture was refluxed for 2 days and then quenched with water. The solvent was partially evaporated, and ethyl ether was added to precipitate perfluoroalkyl-benzyl-poly(ethylene glycol). The benzyl protecting group of the perfluoroalkyl-benzyl-poly(ethylene glycol) was then removed under H2 and 10% activated Pd/C in 95% ethanol overnight. The resulting mixture was filtered through a Celite545 pad to remove Pd/C powder. After the ethanol solvent was partially removed via rotary evaporation, ethyl ether was added to precipitate the polymer product. The impure polymer product was then triturated with hexane and refluxed for 2 h. The resulting precipitate was then suspended in tertbutyl methyl ether, and the resulting mixture was refluxed overnight to ensure full extraction of pure polymer from the solid. The solution phase of the tert-butyl methyl ether extraction was then collected and evaporated to obtain the pure perfluoroalkyl block poly(ethylene glycol), as was confirmed by 19F NMR, MALDI-TOF/MS, and HPLC.
Results and Discussion The synthesis of the perfluoroalkyl block poly(ethylene glycol), abbreviated as F8P6, is summarized in Scheme 1. One of the hydroxyl groups on the starting poly(ethylene
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Figure 3. Dynamic light scattering data of polymer F8P6 at different concentrations obtained from Zeta/Particle Sizer Nicomp 380 ZLS using the NICOMP analysis software and volume-distribution analysis. Each data point in this graph is obtained from statistical analysis of five data sets obtained from dynamic light scattering measurements.
glycol) was protected with a benzyl functionality. The second hydroxyl group on the poly(ethylene glycol) was tosylated to facilitate a nucleophilic substitution by the corresponding fluorinated alkoxide. Finally, the benzyl protecting group was quantitatively removed by hydrogenolysis. The final product was purified by a combination of trituration, reflux, and extraction in various organic solvents as described in the Experimental Section. The final purity was confirmed by HPLC. F8P6 has the potential to self-assemble due to the difference in hydrophobicity between the poly(ethylene glycol) chain (hydrophilic) and the perfluorocarbonic segment (super-hydrophobic) that confers an amphiphilic character to this linear copolymer. In aqueous solution, this property is manifested by the formation of self-assembling nanoscopic micellar structures. We have characterized the aggregates by studying solutions of polymer at different concentrations by 19F NMR,22 dynamic light scattering,23 and fluorescence correlation spectroscopy.24-27 The 19F NMR experiments were used to study the aggregation properties of F8P6 in water. The purified F8P6 was dissolved in either deuterated methanol or deuterium oxide, and the corresponding 19F NMR spectra were recorded. The resonance of the trifluoromethyl group of the free polymer appears as one sharp signal at δ ) -82.6 ppm in deuterated methanol (Figure 1). However, the same group appears as two different resonances in deuterium oxide, δ ) -81.2 and -83.2 ppm. Also, all the NMR resonances in deuterium oxide were much broader than those in deuterated methanol, indicating aggregation of F8P6. Assuming conditions of slow exchange, the two CF3 resonances belong to the free polymer and to the micelle. To determine which resonance belongs to the unimer or the micelle, 19F NMR experiments were performed at increasing F8P6 concentrations. The 19F NMR spectrum of the trifluoromethyl group of F8P6 in D2O shows a significant concentration dependence, as seen in Figure 2. At 20 mg/mL, only a very broad signal is visible at -83.2 ppm, whereas the signal at -81.2 ppm becomes visible at lower concentrations. Also, the signal at -81.2 ppm (22) Muller, N.; Simsohn, H. J. Phys. Chem. 1971, 75, 942-945. (23) Day, R. A.; Robinson, B. H.; Clarke, J. H. R.; Doherty, J. V. J. Chem. Soc., Faraday Trans. 1 1979, 75, 132-139. (24) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039-2044. (25) Dong, D. C.; Winnik, M. A. Can. J. Chem. 1984, 62, 2560-2565. (26) Kalyanasundaram, K. Langmuir 1988, 4, 942-945. (27) Wang, Y.; Winnik, M. A. Langmuir 1990, 6, 1437-1439.
Figure 4. Variations of the I1/I3 ratio of the pyrene vibronic fluorescence spectrum with F8P6 concentration.
appears to be sharper than the signal at -83.2 ppm. Therefore, the resonance at -81.2 ppm was assigned to the CF3 of the free polymer and the resonance at -83.2 ppm was assigned to the CF3 of the micelle, which is consistent with a larger deshielding of the CF3 in the free polymer. The size of the micelle in the F8P6 solutions and the critical micelle concentration of F8P6 were obtained by dynamic light scattering experiments. No particle was detected when the concentration of F8P6 was below 0.4 mg/mL (Figure 3). As the polymer concentration increased to 0.75 mg/mL, the measured micellar size increased to 10.7 nm with an assumption of spherical micellar formation. When the concentration of F8P6 was above 1 mg/mL, the size of the self-assembled micelles remained constant. Pyrene has been extensively used as a probe to investigate both the onset of aggregation and the extent of water penetration inside the micellar core.24-27 The ratio of the intensities (I1/I3) of the first to the third peaks of the vibronic fluorescence spectrum of the pyrene probe depends on the polarity of its immediate environment.21 Our measured I1/I3 values in water and in perfluorinated hexanes (FC-72) are equal to 1.70 and 0.85, respectively. Figure 4 shows that the I1/I3 value sharply decreases as the concentration of F8P6 increases to the critical micelle concentration value, showing the formation of a micelle characterized by an internal fluorous phase. The value of 1.35 at the concentration of surfactant corresponding to 1 mg/mL is consistent with both a previously recognized limited solubility of pyrene in a fluorophilic environment
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Figure 5. Structure of the anesthetic sevoflurane.
Figure 6. Change in chemical shift of the CF3 and CH2F resonances of sevoflurane in different F8P6 concentrations. The triangle- and square-based curves describe the changes in the chemical shifts of CF3 and CH2F of sevoflurane, respectively, in different F8P6 aqueous solutions; the circle-based curve is the change in the chemical shifts of the trifluoromethyl groups of sevoflurane in poly(ethylene glycol) aqueous solutions (control experiment).
and a hypothesized water presence close to the fluorous core due to the high solvation of the polyethyl glycol chains.26 Pyrene is presumably positioned at the interface of the poly(ethylene glycol) and fluorocarbon chains. Therefore, its fluorescence reflects an environment that is more hydrophobic than an aqueous phase but less hydrophobic than a pure fluorous phase. The corresponding I1/I3 ratio will then be bigger than the I1/I3 measured in a pure fluorous phase. Combining the dynamic light scattering data with the changes in pyrene fluorescence observed at different polymer concentrations allowed an accurate measurement of the critical micelle concentration of F8P6 as 0.75 mg/mL. The association number for the micelles (number of monomers of polymer comprising each micelle) was also measured by steady-state fluorescence quenching techniques.28 We used pyrene as the fluorophore and 3,4dimethylbenzophenone as the quencher. We found that each micelle is composed of seven polymer molecules. The discovery that F8P6 self-assembles into nanoscopic fluorous-core micelles led us to study the encapsulating (28) Winnik, F. M. Colloids Surf., A 1996, 118, 1-39.
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properties of these micelles. Highly fluorinated molecules are notoriously difficult to recognize and bind because of their fluorophilic character. Sevoflurane, 1,1,1,3,3,3hexafluoroisopropyl-fluoromethyl ether (Figure 5), the most effective and widely used anesthetic in the United States, was used in the encapsulation study. To this purpose, 2 µL of sevoflurane and sodium trifluoroacetate (internal standard) was added to 1 mL of F8P6 polymer solutions having concentrations ranging from 0.5 to 6 mg/mL. The solutions were stirred at 56 °C for 1 h in closed vials and then cooled at room temperature for 30 min to induce formation of sevoflurane-micelle complexes. NMR studies (Figure 6) of these complexes showed that the two 19F NMR resonances of sevoflurane significantly shifted upon addition to F8P6, indicating encapsulation of sevoflurane inside the fluorous core of the F8P6 micelle. The onset of formation of sevofluranemicelle complexes can be detected at the critical micelle concentration of F8P6. An increase in the concentration of the polymer and, therefore, of the number of micelles led to the full encapsulation of all the sevoflurane in solution into the micellar fluorous core. Control experiments in solutions containing poly(ethylene glycol) did not show any change in the chemical shift of sevoflurane, proving the importance of the perfluoroalkyl group of F8P6 for the recognition and encapsulation of the anesthetic (Figure 6). Figure 6 shows that 2 µL of sevoflurane can saturate the micelles in 1 mL of a 3 mg/mL polymer solution. This corresponds to 300 molecules of sevoflurane per micelle of F8P6. This large number is very promising for the proposed clinical applications of F8P6. Conclusions We have proven that a semifluorinated block copolymer consisting of a hydrophilic segment [poly(ethylene glycol), Mw ) 6000 amu] and a perfluorooctyl chain self-assembles into nanoscopic micelles in aqueous solution. The micelles have been proven to efficiently encapsulate sevoflurane, an example of a highly fluorinated drug. Studies on the morphology, stability of the nanoparticles upon dilution, and release of the encapsulated drug are currently underway. Acknowledgment. The authors thank the School of Pharmacy, the Graduate School of the University of Wisconsin-Madison, and the Wisconsin Alumni Research Foundation for generous financial support. The authors also thank Prof. Glen Kwon, Prof. Robert Pearce, and Prof. George Zografi for fruitful discussions. LA049128A
