6392
Langmuir 2003, 19, 6392-6396
Porous Vesicles Dispersible in Organic Solvents† Ji-Youn Im,‡ Duk-Bae Kim,‡ Sang Hee Lee,§ and Youn-Sik Lee*,‡ Division of Environmental and Chemical Engineering and Technology, The Research Institute of Industrial Technology, Research Institute, Chonbuk National University, Chonju 561-756, Korea, and Department of Chemistry, Kunsan National University, Kunsan 573-360, Korea Received February 4, 2003. In Final Form: May 9, 2003 The porous vesicles reported so far have hydrophilic surfaces. If the surfaces are chemically modified to be compatible with common organic solvents, their application will be extended. 1,2-Bis[9-(4vinylbenzyloxycarbonyl)nonanoyl]-sn-glycero-3-phosphocholine (BNPC) was synthesized and used as a polymerizable surfactant for the present study. Vesicles composed of BNPC and cholesterol with embedded divinylbenzene were polymerized, followed by skeletonization (removal of cholesterol) and hydrolysis (cleavage of the ester bonds in BNPC unit). The resulting vesicles (250 nm) whose surfaces contain hydroxyl groups appeared to have holes with diameters up to several tens of nanometers. They were dispersed in water, methanol, tetrahydrofuran, and chloroform. The turbidity of the dispersions decreased as follows: water > methanol > tetrahydrofuran > chloroform. The dispersion in water remained turbid with some precipitates during storage, but those in methanol and tetrahydrofuran appeared to be clear with white precipitates. On the other hand, the transparent vesicle dispersion in chloroform did not form precipitates.
Introduction Surfactant or lipid molecules can be spontaneously aggregated into spherically closed bilayers, i.e., vesicles or liposomes in water.1 The phase separation in vesicle bilayers can be induced by polymerization of bilayer membranes composed of polymerizable and nonpolymerizable surfactants.2,3 The removal of the labile (nonpolymerized) domains from the phase-separated membranes results in the formation of the skeletonized (porous) vesicles which have many holes in the polymerized membrane matrix.1,4 The surfactants in the labile domains are usually removed using organic solvents, surfactants, reducing agents, or enzymes. The porous vesicles reported so far have hydrophilic surfaces. However, it may be very interesting to prepare porous vesicles whose surfaces are compatible with organic solvents since they may be dispersible in common organic media and their application can be extended. Recently, we reported polymerization of vesicles prepared from a polymerizable surfactant, a nonpolymerizable lipid, and a cross-linking agent, which was N,N-bis[10-(4-vinylbenzyloxy)decanoylaminoethyl]-N,N-dimethylammonium chloride (BDAC) (Figure 1a), 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC), and divinylbenzene (DVB), respectively.5 When the small vesicles (mean diameter 100 nm) were polymerized, most of them retained their initial spherical shapes, but a so-called parachutelike morphology6,7 was also occasionally observed. However the parachute-like morphology was not observed when * To whom all correspondence should be addressed. E-mail:
[email protected]. Fax: +82-063-270-2306. † Part of the Langmuir special issue dedicated to David O’Brien. ‡ Chonbuk National University. § Kunsan National University. (1) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113. (2) O’Brien, D. F.; Armitage, B.; Benedicto, A.; Bennett, D. E.; Lamparski, H. G.; Lee, Y.-S.; Srisiri, W.; Sisson, T. M. Acc. Chem. Res. 1998, 31, 861. (3) Armitage, B.; Bennett, D. E.; Lamparski, H. G.; O’Brien, D. F. Adv. Polym. Sci. 1996, 126, 53. (4) Ohno, H.; Takeoka, S.; Tsuchida, E. Polym. Bull. 1985, 14, 487. (5) Yang, W. Y.; Hahn, Y. B.; Nahm, K. S.; Lee, Y.-S. Bull. Korean Chem. Soc. 2001, 22, 1291.
Figure 1. Chemical structures of (a) BDAC, (b) BNPC, and (c) cholesterol.
their large vesicles (960 nm) were polymerized.8 The skeletonization of the large polymerized vesicles resulted in the formation of very small holes or interstices in their membranes. When the skeletonized vesicles were hydrolyzed in order to remove the hydrophilic headgroup of the BDAC unit in the polymer matrix, the resulting vesicles were dispersed only in 1.0 M NaOH water/methanol (1/1). (6) Jung, M.; den Ouden, I.; Montoya-Goni, A.; Hubert, D. H. W.; Frederik, P. M.; van Herk, A. M.; German, A. L. Langmuir 2000, 16, 4185. (7) Jung, M.; Hubert, D. H. W.; van Veldhoven, E.; Frederick, P.; van Herk, A. M.; German, A. L. Langmuir 2000, 16, 3165. (8) Yang, W. Y.; Lee, Y.-S. Langmuir 2002, 18, 6071.
10.1021/la034189e CCC: $25.00 © 2003 American Chemical Society Published on Web 06/05/2003
Porous Vesicles in Organic Solvents
In a different experiment, a 4,4′-biphenol-based tetraethylammonium salt (BPAS), a bipolar surfactant, was used instead of DPPC as a nonpolymerizable component.9 The large vesicles (500 nm) were polymerized under similar conditions, and BPAS was extracted out using methanol. The headgroup of BDAC unit in the polymer matrix was cleaved off via hydrolysis in an acidic condition to yield porous vesicles whose surfaces were covered with carboxyl groups. The vesicles appeared to have many holes with diameters up to about 25 nm. However, the hydrolyzed vesicles were not dispersed in water and most organic solvents, but dispersed in methanol. It was thought that the surfaces of the hydrolyzed vesicles were still too polar for the vesicles to be dispersed in common organic solvents. 1,2-Bis[9-(4-vinylbenzyloxycarbonyl)nonanoyl]-sn-glycero-3-phosphocholine (BNPC) was chosen as a polymerizable surfactant (Figure 1b) for the present study because of the following reasons. First, many different types of polymerizable phospholipids have been reported to form vesicles in water.1,2 Second, the skeletonized vesicles were already prepared using cholesterol and a polymerizable phospholipid whose chemical structure is similar to that of BNPC.4 Third, if the vesicles composed of BNPC and cholesterol with embedded DVB are polymerized, followed by skeletonization (removal of cholesterol) and hydrolysis (cleavage of the ester bonds in BNPC unit), the resulting porous vesicles are expected to be dispersible in organic solvents because the resulting vesicle surfaces containing hydroxyl groups may be compatible with organic solvents, as shown in Scheme 1. This paper describes the preparation and dispersion behaviors of the hydrolyzed porous vesicles.
Langmuir, Vol. 19, No. 16, 2003 6393 Scheme 1. Strategy for Preparation of Porous Vesicle Surfaces Covered with Hydroxyl Groups from Skeletonized Vesicles via Hydrolysis
Experimental Section Synthesis of BNPC. A solution of 4-vinylbenzyl chloride (5.0 g, 29.5 mmol), 1,9-nonanedioic acid (5.5 g, 29.5 mmol), potassium carbonate (4.1 g, 29.5 mmol), and sodium iodide (0.50 g, 3.3 mmol) in acetone (50 mL) was refluxed for 16 h. The reaction mixture was concentrated and acidified with a 1.0 N HCl solution. After extraction with ethyl acetate, the organic phase was dried with sodium sulfate, concentrated, and separated by flash column chromatography (SiO2, hexane/ethyl acetate ) 3/1) to obtain 3.1 g of 9-(4-vinylbezyloxycarbonyl)nonanoic acid (32%). To a solution of L-R-glycerophosphorylcholine cadmium chloride complex (0.88 g, 2.0 mmol) in dimethyl sulfoxide (DMSO) (10 mL) were added 9-(4-vinylbenzyloxycarbonyl)nonanoic acid (1.84 g, 6.05 mmol), 1,3-dicyclohexylcarbodiimide (DCC) (1.65 g, 8.0 mmol), and (dimethylamino)pyridine (DMAP) (49 mg, 0.4 mmol). After being stirred for 24 h at room temperature, the reaction mixture was filtered and diluted with dichloromethane (50 mL). The resulting solution was acidified with a 1.0 N HCl solution and washed with water. After removal of dichloromethane under vacuum at room temperature, the residue was chromatographed (SiO2, from CH2Cl2 to CHCl3/MeOH/H2O ) 5/2/1). After concentration, the residue was dissolved in dichloromethane, dried with sodium sulfate, and concentrated to afford 0.75 g (45%) of BNPC as a white solid. Preparation of Bilayers for Differential Scanning Calorimetry (DSC). BNPC or a mixture of BNPC and cholesterol in a 3:1 molar ratio was dissolved in chloroform. The resulting solution was slowly evaporated to obtain a thin film on the inner wall of a vial and dried under a high vacuum for at least 2 h. The dried film was then hydrated employing the repeated freezethaw cycles (-70 to 50 °C). The viscous dispersion was transferred using a spatula with a sharp tip into an aluminum DSC pan and sealed. During the sample transfer, the less viscous part of the dispersion flowed down and off, resulting in that only the more viscous part was transferred into the DSC pan from the spatula tip. Thus the enthalpy changes of the melting transitions could (9) Im, J.-Y.; Lee, S.-H.; Ko, S. B.; Lee, K.-H.; Lee, Y.-S. Bull. Korean Chem. Soc. 2002, 20, 1616.
not be compared in this experiment since the concentrations of BNPC in the DSC pans were different from the initial ones due to the sample transfer procedure from the stocks to the DSC pans. Polymerization of Vesicles. Vesicles composed of BNPC/ cholesterol (3/1) containing 2,2′-azobisisobutyrnitrile (AIBN) were prepared as described above (BNPC/AIBN ) 5/2) (2.5 mg of BNPC/ mL of H2O). DVB was injected into the vesicle dispersion using a microliter syringe, and the resulting mixture was stirred for 2 days at room temperature to ensure a complete incorporation of the cross-linking agent into the lipid bilayers (BNPC/DVB ) 1/1). The dispersion was stirred at room temperature for 48 h and then at 50 °C for 24 h under a nitrogen atmosphere. Skeletonization and Hydrolysis of Polymerized Vesicles. The polymerized vesicle dispersions were freeze-dried and suspended in methanol. The resulting suspension was centrifuged at 13000 rpm and decanted to remove the supernatant containing cholesterol extracted out of the polymerized vesicles. The centrifugation procedure was repeated twice. A concentrated HCl solution was added to the skeletonized vesicle suspension in methanol and refluxed for 12 h, followed by centrifugation. The supernatant was decanted off, and the precipitate was resuspended in methanol and then centrifuged for 5 min. The hydrolysis-centrifugation cycle was repeated three times in order
6394
Langmuir, Vol. 19, No. 16, 2003
Figure 2.
1
H NMR spectrum of BNPC (CDCl3).
Scheme 2. Synthetic Route to BNPC
to completely cleave off the ester bond at each alkyl chain terminus of BNPC unit in the resulting polymerized vesicle matrix. Transmission Electron Microscopy (TEM). The vesicle dispersions were stained with 2 wt % phosphotungstic acid (pH 6.8 with 1.0 N KOH) for 15 min on a Parafilm (vesicle dispersion/ phosphotungstic acid)1:1, v/v) and dropped on Formvar/carbon grids. The vesicles on grids were washed with water and dried under vacuum. TEM images were obtained using a transmission electron microscope (EM10CR, Carlzeiss Co.) at 60.0 kV.
Results and Discussion BNPC was synthesized in two consecutive steps, as shown in Scheme 2. 4-Vinylbenzyl chloride was reacted with 1,9-nonanedioic acid in the presence of potassium carbonate and sodium iodide to yield 9-(4-vinylbezyloxycarbonyl)nonanoic acid which was then reacted with L-Rglycerophosphorylcholine to yield BNPC. The FT-IR spectrum of BNPC showed a strong absorption at 1735 cm-1 which corresponds to the ester bonds. The several bands at 3100-3000 cm-1 and 1650-1500 cm-1 are due to the vinyl and phenyl groups. The proton NMR spectrum of BNPC is shown in Figure 2. The resonance peaks at 7.35-7.18 ppm are due to aromatic protons, and those at 6.67-6.57, 5.70-5.65, and 5.21-5.15 ppm correspond to the olefinic protons of the 4-vinylbenzyl moieties. The peaks at 5.00 ppm and 2.30-2.14 ppm are due to the benzylic and methylene protons next to the ester groups in the alkyl chains, respectively. The trimethyl protons on the nitrogen atom appear at 3.29 ppm as a singlet. The NMR spectrum along with the FT-IR spectrum reveals
Im et al.
Figure 3. DSC thermograms of (a) BNPC and (b) BNPC/ cholesterol (3/1) bilayers. All of the thermograms were obtained from the second heating scans, and the scan rate was 10/min.
that the isolated compound has the expected chemical structure of BNPC. The BNPC molecules were self-assembled into vesicles on hydration based on the encapsulation experiment using calcein, a self-quenching fluorescent dye. Calcein was encapsulated in vesicles composed of BNPC and cholesterol (3/1) by hydrating the lipid mixture in an aqueous calcein solution (50 mM, 300 mOsm/L, pH ) 8.0), followed by ultrasonication to obtain small unilamellar vesicles. The vesicle dispersion was chromatographed on a BioGel A column to remove unentrapped calcein. The fluorescence intensity (Ex ) 490 nm, Em ) 520 nm) of the vesicle dispersion was greatly increased (several times) when a sufficient amount of Triton X-100 was added to destroy the vesicles. This result indicates that the lipid system forms vesicles spontaneously on hydration. The lipid vesicles were stable for weeks since any significant change in the transparency of the dispersion was not observed during the storage at room temperature. The DSC experiments (2910 TA Instrument) were performed in order to know the chain-melting temperature (Tm) of the lipid bilayers, and the results are shown in Figure 3. The pure BNPC bilayers exhibited a melting transition in a broad temperature range from 15 to 30 °C. The BNPC/ cholesterol (3/1) bilayers also underwent a melting transition in a broad temperature range from 5 to 20 °C, which is about 10 °C lower than that of the pure BNPC bilayers. It was reported that at concentrations of from 1 to 2025 mol % cholesterol, the DSC endotherms of linear saturated phosphatidylcholines consist of a superimposed sharp and broad component, the former corresponds to the melting of cholesterol-poor and the latter to the melting of the cholesterol-rich phosphatidylcholine domains.10 The Tm of the cholesterol-rich domains progressively increased with cholesterol content when the lipids have hydrocarbons of 16 or fewer carbons chains, but it decreased when the lipids have hydrocarbon chains of 18 or more carbon atoms. This behavior was attributed to the effects of hydrophobic mismatch between cholesterol molecule and its host phosphatidylcholine bilayer. In the present lipid system, only one broad Tm peak was observed, suggesting that BNPC and cholesterol are reasonably well mixed in the vesicle membranes. Thus the lowered Tm of BNPC/ cholesterol (3/1) bilayers compared to that of pure BNPC bilayers may be due to the hydrophobic mismatch between cholesterol and BNPC molecules. However, the mismatch in this case may result from the presence of the bulky (10) McMullen, T. P. W.; Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1993, 32, 516.
Porous Vesicles in Organic Solvents
Langmuir, Vol. 19, No. 16, 2003 6395
Figure 4. UV spectra of vesicle dispersions before and after polymerization.
Figure 5. FT-IR spectra of (a) polymerized and (b) hydrolyzed vesicles.
Figure 6. Dispersions of hydrolyzed vesicles in water, methanol, tetrahydrofuran, and chloroform (concentration ) 2.0 mg/ mL).
Figure 7. TEM images of (a) skeletonized and (b) hydrolyzed vesicles. The bars represent 250 nm. The two insets show a magnified vesicle and a vesicle with holes, respectively.
4-vinylbenzyloxycarbonyl moiety at each chain terminus because each hydrophobic chain length of BNPC (corresponding to approximately 16 carbon and 1 oxygen atoms) is shorter than that of linear 18 carbon atoms. Vesicles composed of BNPC and cholesterol in a 3:1 molar ratio with embedded DVB were polymerized in the presence of AIBN. The absorption spectrum of the resulting vesicle dispersion exhibited that the absorbance at 254 nm decreased significantly, as compared to the initial absorbance at the same lipid concentration (Figure 4). The conversion of the comonomers into cross-linked polymers was calculated to be 93% based on the UV absorbances. The polymerized vesicle dispersions were freeze-dried and washed with methanol in order to remove
cholesterol from the cross-linked membranes since cholesterol is soluble in methanol. The polymerized samples were repeatedly washed with methanol until cholesterol in the resulting suspensions was not detected with thinlayer chromatography. A concentrated HCl solution was added to the skeletonized vesicle suspension in methanol and refluxed for 12 h. The resulting suspension was centrifuged to yield a white precipitate. The precipitating procedure was repeated a few times. The FT-IR spectrum of the purified precipitate revealed that the intensity of absorption peak at 1735 cm-1 was greatly reduced but still remained noticeable after the first hydrolysis. Sells and O’Brien reported a similar result from the hydrolysis of polym-
6396
Langmuir, Vol. 19, No. 16, 2003
erized monoacryloylphosphatidylcholine vesicles using BF3 in methanol.11 Thus the hydrolysis-centrifugation cycle was repeated three times, and then the absorption peak disappeared (Figure 5), indicating that the ester bonds in the BNPC units were cleaved off and removed from the dispersion. The assemblies were large multilamellar vesicles. If they are subjected to sonication or extrusion under pressure, small unilamellar vesicles will be formed. In general, lipid molecules are less tightly packed in small unilamellar vesicles. Thus the acidic hydrolysis as well as removal of cholesterol from the smaller polymerized matrix is expected to be easier. Lipid vesicles are usually dispersed only in water. However, the hydrolyzed vesicles were more readily dispersed in methanol and tetrahydrofuran than in water but much more readily dispersed in chloroform. The hydrolyzed vesicle dispersions in various solvents are shown in Figure 6. A careful inspection reveals that the turbidity of the dispersions depends on solvents and decreases as follows: water > methanol > tetrahydrofuran > chloroform. The vesicle dispersion in chloroform looks like a solution due to its high transparency. Some hydrolyzed vesicles were slowly aggregated and precipitated from the dispersions in water, methanol, and tetrahydrofuran during storage overnight. The vesicle dispersion in water remained turbid with some precipitates, but those in methanol and tetrahydrofuran appeared clear with white precipitates. On the other hand, any precipitate was not observed from the vesicle dispersion in chloroform during the same period of time. When the dispersions containing precipitates were shaken gently, they returned to the initial dispersion states immediately. According to our previous experiment performed using BDAC, the hydrolyzed membrane surfaces were covered with carboxyl groups, and the porous vesicles were dispersed in only methanol, indicating that the vesicle surfaces were still too polar for the vesicles to be dispersible in common organic solvents.8 On the other hand, in the present study, the hydrolysis resulted in the generation of hydroxyl groups which are less polar than carboxyl (11) Sells, T. D.; O’Brien, D. F. Macromolecules 1991, 24, 336.
Im et al.
groups, leading to the good dispersion of the porous vesicles in such organic solvents. TEM experiments were performed in order to visualize the skeletonized and hydrolyzed vesicles, and the results are shown in Figure 7. The approximate spherical vesicle shapes (250 nm) were retained even after the skeletonization and hydrolysis. The vesicles appeared to have many holes with diameters up to several tens of nanometers even though it was not always possible for us to observe holes from every single vesicle on the grids (Figure 7b). The sizes of the holes are much larger than those on the previous BDAC vesicles.9 This result suggests that larger cholesterol domains were induced during the polymerization process, as compared to our previous system with BDAC. This result also indicates that BNPC vesicles were sufficiently stabilized by the cross-linking process during polymerization. Conclusions Large vesicles composed of BNPC and cholesterol with embedded DVB underwent radical polymerization in the presence of AIBN. Cholesterol was extracted out from the polymerized membranes with methanol. The alkyl chains along with the headgroups of BNPC units in the polymer membranes were cleaved off via hydrolysis. Many large holes were observed from the skeletonized and hydrolyzed vesicles. The resulting porous vesicles were readily dispersible in organic solvents such as methanol, tetrahydrofuran, and chloroform. The hydrolyzed vesicles were aggregated and precipitated slowly in methanol and tetrahydrofuran, but not in chloroform. The dispersion behaviors of the present porous vesicles are very interesting because they may be employed as a stationary phase in column chromatography, an enzyme-immobilizing material, or a catalyst-supporting material for organic reactions, mainly because of their very large surface areas and reactive functional groups. Acknowledgment. This work was supported by the Basic Research Program of the Korea Science & Engineering Foundation (Grant No. R05-2000-00343-0). LA034189E
