Langmuir 2002, 18, 6071-6074
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Surface Modification of Porous Vesicles via Hydrolysis Won Young Yang and Youn-Sik Lee* Division of Environmental and Chemical Engineering, The Research Institute of Industrial Technology, Engineering Research Institute, Chonbuk National University, Chonju 561-756, Korea Received April 1, 2002. In Final Form: May 31, 2002 The surfaces of skeletonized vesicles (or vesicles with holes) are hydrophilic. If the hydrophilic surfaces are chemically modified to have a reasonable compatibility with organic solvents, the application of vesicular colloids can be expanded. In this study, 1,2-dipalmitoyl-sn-glycro-3-phosphocholine (DPPC) and N,N-bis[10-(4-vinylbenzyloxy)decanoylaminoethyl]-N,N-dimethylammonium chloride (BDAC) were employed as a nonpolymerizable lipid and polymerizable surfactant, respectively. Divinylbenzene (DVB) was used as a cross-linking agent in the polymerization of the vesicles. Multilamellar vesicles composed of BDAC and DPPC with embedded DVB in a 3:2:3 molar ratio underwent efficient radical polymerization. DPPC was removed from the cross-linked vesicles using Triton X-100 (skeletonization). The headgroup of BDAC was removed by hydrolysis in an acidic condition. The surface of the polymerized vesicles looked very smooth, but the skeletonized vesicles looked pretty rough and showed only small interstices or holes. the DSC experiment showed that DPPC domains were widely distributed in size after polymerization. The hydrolyzed vesicle surface looked wrinkled, probably due to the enhanced fluidity of the alkyl chains. The hydrolyzed vesicles were precipitated or aggregated in many individual organic solvents but were well dispersed in a 1.0 M NaOH water/methanol (1/1) solution.
Introduction It has been known for more than 30 years that lipid molecules can be spontaneously aggregated into spherically closed bilayers, that is, vesicles or liposomes in water. These vesicular morphologies have found a multitude of applications in various scientific and applied fields in recent years. However, the stability of the vesicles is still a serious problem. Many methods have been proposed for stabilizing vesicles in the past two decades.1 One common approach is the polymerization of membrane components in the bilayers. The substantial body of literature on this subject was summarized in the outstanding reviews reported by many workers.2,3 Holes can be drilled in the membrane of a tumor cell attacked by an activated macrophage, resulting in the leakage of the cytoplasmic interior and the death of the cell. To simulate the biological process, phase-separated large vesicles were used.1,4,5 Phase separation in bilayer membranes consisting of polymerizable and nonpolymerizable lipids was induced by the direct binding of polymerizable lipids through covalent bonds on polymerization.6 As a result, the vesicle membrane was converted to a polymerized lipid matrix surrounding the labile domains of the nonpolymerizable lipids. The labile domains were removed from the polymerized membrane (skeletonization) by dissolving the nonpolymerizable lipids with surfactant or organic solvent or by cleaving them with chemical or enzymatic reactions. * To whom correspondence should be addressed. E-mail:
[email protected]. Fax: +82-063-270-2312. (1) Ringsdorf, H; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113. (2) O’Brien, D. F.; Armitage, B.; Bennet, D. E.; Lamparski, H. G.; Lee, Y.-S.; Srisri, W.; Sission, T. M. Acc. Chem. Res. 1998, 31, 861. (3) Armitage, B. A.; Bennett, D. E.; Lamparski, H. G.; O’Brien, D. F. Adv. Polym. Sci. 1996, 126, 53. (4) Chang, T. M. S. Microencapsulation (Artificial Cells). In Polymeric Materials Encyclopedia; Salemone, J. C., Ed.; CRC Press: New York, 1996; Vol. 6, p 4351. (5) Ohno, H.; Takeoka, S.; Tsuchida, E. Polym. Bull. 1985, 14, 487. (6) Takeoka, S.; Sakai, H.; Ohno, H.; Tsuchida, E. Macromolecules 1991, 24, 1279.
The surfaces of skeletonized vesicles are usually hydrophilic. If the hydrophilic surfaces are chemically modified to have a reasonable compatibility with organic solvents, the application of vesicular colloids can be expanded since the porous particles may be dispersed in organic media. The present experimental strategy for the development of novel materials is shown in Figure 1. Vesicles composed of a polymerizable lipid, a nonpolymerizable lipid, and a cross-linking agent are polymerized, followed by the skeletonization. Finally, the hydrophilic headgroups of zwitterionic or salt types in the membrane surface are cleaved off via hydrolysis. The resulting headgroups formed will be carboxyl groups, hydroxyl groups, and so forth, depending on the type of covalent linkage between the alkyl chain and original headgroup. This chemical modification of the skeletonized vesicles to change their surface from hydrophilic to organophilic is a new extension of the prior works, which has not been published yet as far as we know. To prepare multilamellar large vesicles, 1,2-dipalmitoyl-sn-glycro-3-phosphatidylcholine (DPPC) and N,Nbis[10-(4-vinylbenzyloxy)decanoylaminoethyl]-N,N-dimethylammonium chloride (BDAC) were employed as a nonpolymerizable lipid and a polymerizable surfactant, respectively. Divinylbenzene (DVB) was used as a crosslinking agent in the polymerization of the vesicles. The synthesis of BDAC will be reported elsewhere.7 This paper describes the summary of polymerization, skeletonization, and hydrolysis of the multilamellar vesicles. Experimental Section Polymerization of Vesicles. A desired volume of a stock solution of 2,2′-azobisisobutyronitrile (AIBN) in chloroform was added to a chloroform solution containing BDAC and DPPC. The resulting solution was evaporated on a rotary evaporator to form a thin film under a reduced pressure. The film was further dried under high vacuum for a few hours, followed by the subsequent (7) Im, J.-Y.; Lee, S.-H.; Ko, S.-B.; Lee, K.-H.; Lee, Y.-S. Bull. Korean Chem. Soc., submitted.
10.1021/la0203077 CCC: $22.00 © 2002 American Chemical Society Published on Web 07/16/2002
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Yang and Lee Scanning Electron Microscopy (SEM). A sample droplet was spread on an aluminum SEM stub (diameter, 10 mm) with carbon tape and dried in a vacuum oven at 50 °C, followed by platinum coating. SEM images were obtained using a fieldemission scanning electron microscope (FE-SEM, Hitachi model S-4700, Japan) at 10 kV. Differential Scanning Calorimetry (DSC). DPPC or DPPC/ BDAC (2/3) vesicle dispersions (50 wt %) were prepared by employing the freeze-thaw-vortex cycle described above. The highly viscous dispersions were transferred into aluminum DSC pans using a narrow spatula. The thermal phase transitions of the dispersions were observed with a DSC 2910 differential scanning calorimeter (TA Instrument Co.).
Results and Discussion
Figure 1. Strategy for chemical modification of vesicle membrane surface: (a) phase separation induced by polymerization, (b) formation of holes by removal of labile domains, and (c) removal of hydrophilic headgroups via hydrolysis. addition of an appropriate amount of phosphate buffered solution (pH 7.5, 2.0 mg lipid/mL). The lipid film was swollen for 1 h at room temperature and then vortexed. Finally, the mixture was subjected to a freeze-thaw-vortex cycle (-70 to 50 °C) for 10 times. DVB was injected into the vesicle dispersion using a microliter syringe, and the mixture was stirred for 2 days at room temperature to ensure a complete incorporation of the crosslinking agent into the lipid bilayers. The vesicle dispersion was polymerized under continuous stirring and nitrogen bubbling at 55 °C for at least 24 h. Skeletonization and Hydrolysis of Vesicles. The polymerized vesicle dispersion was incubated in the presence of Triton X-100 at room temperature for 1 h. The molar ratio of Triton X-100 to lipids (DMAC + DPPC) was found to be 10:1. The skeletonized vesicles were then purified by size exclusion chromatography using a column packed with Sephadex 60. Concentrated HCl solution was added to the skeletonized vesicle dispersion until the final HCl concentration attained was 12 wt %. The resulting acidic vesicle dispersion was then stirred at 50 °C for 48 h, followed by centrifugation. The supernatant was decanted, and the precipitate was dispersed with water, followed by centrifugation. The overall procedure was repeated thrice in order to remove any byproducts formed during the hydrolysis reaction.
The molar ratio of BDAC, DPPC, and DVB in the extended bilayers was 3:2:3. Polymerization was carried out using AIBN (10 mol %) as a radical initiator at 55 °C. The polymerization reaction was allowed to proceed until the absorbance at 254 nm did not decrease further. In the Fourier transform infrared (FT-IR) spectrum (JASCO FT/IR-300E spectrophotometer) of the polymerized sample (Figure 2A), the absorption peaks at 940 and 1030 cm-1 disappeared, which correspond to the C-H out-ofplane bending vibrations of the vinyl groups of BDAC and DVB. The 1H NMR spectrum of the polymerized sample also confirmed that the vinyl protons of BDAC and DVB disappeared, indicating that the conversion of the comonomers to polymers was very high. The polymerized vesicle dispersion was treated with Triton X-100 in order to remove DPPC from the vesicle membranes, followed by size exclusion chromatographic separation to eliminate mixed micelles of Triton X-100 and DPPC from the skeletonized vesicle dispersion. The unpolymerized vesicles were completely destroyed when the molar ratio of Triton X-100 to the lipids was just 2:1, but the molar ratio employed for the skeletonization was 10:1. On the other hand, the polymerized vesicles were not significantly changed in size even when the molar ratio was increased to 20:1, indicating that the polymerization greatly enhanced the vesicle stability against the surfactant. The FT-IR spectrum of the resulting vesicle dispersions revealed that DPPC molecules were removed since a characteristic peak of ester carbonyl groups in DPPC at 1734 cm-1 disappeared, as shown in Figure 2B. Calcein, a self-quenching fluorescent dye, was encapsulated in the internal aqueous phase of the vesicles. After removal of unencapsulated calcein with size exclusion chromatography, the calcein-entrapped vesicles were polymerized and treated with Triton X-100 for skeletonization. The skeletonization allowed us to observe calcein leakage from the polymerized vesicles. This result indicates that the vesicles retained the internal aqueous phase even after polymerization and the sphere was not a simple aggregate but a vesicle. The skeletonized vesicle dispersion containing 12 wt % HCl was stirred at 50 °C for 48 h and centrifuged in order to isolate hydrolyzed vesicles as a precipitate. Curve c in Figure 2B is a FT-IR spectrum of the purified precipitate. The absorption peaks of amide groups at about 3400 and 1662 cm-1 disappeared, and peaks due to carboxyl groups at about 3500-2400 and 1708 cm-1 emerged quite clearly. This result indicates that the ammonium headgroups of BDAC units in the cross-linked vesicles were removed. Our preliminary study on the polymerization of small unilamellar vesicles (100 ( 10 nm) of BDAC and DPPC with embedded DVB showed that most of the vesicles were approximately spherical, but a so-called parachute-like morphology was also occasionally observed.8 The parachute-like morphology was first reported by Jung and co-
Surface Modification of Porous Vesicles
Figure 2. FT-IR spectra of multilamellar large vesicles: (A) (a) before and (b) after polymerization; (B) (a) before skeletonization, (b) after skeletonization, and (c) after hydrolysis.
workers from the studies on the polymerization of styrene in dioctadecyldimethylammonium bromide vesicles and attributed to the phase separation of polystyrene beads from the surfactant bilayers.9 Similarly, the parachutelike structure in this system probably resulted from the (8) Yang, W. Y.; Hahn, Y. B.; Nahm, K. S.; Lee, Y.-S. Bull. Korean Chem. Soc. 2001, 22, 1291. (9) Jung, M.; Ouden, I. D.; Hubert, D. H. W.; Frederik, P. M.; van Herk, A. M.; German, A. L. Langmuir 2000, 16, 4185.
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Figure 3. SEM micrographs of the polymerized large vesicles after (a) polymerization, (b) skeletonization, and (c) hydrolysis.
polymerization of DVB proceeding in the bilayer latexlike fashion due to the phase separation between the DVB polymer and polymerized BDAC bilayer. In this study, however, polymerization was performed with multilamellar large vesicles (960 ( 70 nm) and at lower temperatures (∼55 °C). SEM micrographs revealed that the polymerized vesicles appeared to be roughly spherical, as shown in Figure 3a. The surface of the polymerized vesicles looked very smooth. The parachute-like morphology was not observed at all. At lower reaction temperatures, the diffusion rates of DVB and BDAC molecules in
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Figure 4. DSC thermograms of (a) the DPPC dispersion and the DPPC/BDAC/DVB (2/3/3) dispersion (b) before and (c) after polymerization. The surfactant concentrations of the samples were not measured. All of the thermograms were obtained from the second heating scans, and the scan rate was 5 °C/min.
the bilayer membranes should also be reduced.10 The size of vesicles may also affect the lateral diffusion rate of monomers because surfactant molecules in larger bilayer membranes are more closely packed and may diffuse more slowly. Because of the slower diffusion, the comonomers may be more randomly distributed in the resulting polymer chains, which may ultimately inhibit the phase separation between the polymerized BDAC and DVB domains.11 It was expected that large holes from the skeletonized vesicle surfaces could be observed since the content of DPPC in the vesicles was high enough (BDAC/DPPC ) 3/2). Other lipid mixtures containing a larger fraction of DPPC did not form stable vesicles. However, the skeletonized vesicle surface looked pretty rough and showed only small interstices or holes, as shown in Figure 3b. Probably, the separation of DPPC domains from the polymerized matrix on polymerization was not very efficient. Unexpectedly, the surface of the hydrolyzed vesicle looked wrinkled, as shown in Figure 3c. The interesting morphology may be due to enhanced fluidity of the alkyl chains. Initially, one end of the alkyl chain was covalently linked to the headgroup of BDAC and the other end was linked to the polymer main. In other words, BDAC polymers were cross-linked via BDAC headgroups and DVB units. When the hydrophilic headgroups are cleaved off by the hydrolysis, the cross-linking density is reduced and the alkyl chains become simple branches because the resulting polymer chains are cross-linked only via DVB units. The alkyl branches may not be packed tightly enough to retain the original vesicle membrane morphology due to the loss of rigidity. To know how efficiently DPPC domains are separated from the polymerized matrix on polymerization, DSC experiments were performed for DPPC/BDAC (2/3) vesicle systems in the presence of DVB before and after polymerization. The results are presented in Figure 4. The lipid concentrations of the DSC samples were difficult to (10) Fahey, P. F.; Webb, W. W. Biochemistry 1978, 17, 3046. (11) Jung, M.; Hubert, D. H. W.; van Veldhoven, E.; Frederik, P.; van Herk, A. M.; German, A. L. Langmuir 2000, 16, 3165.
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measure because the vesicle dispersions were not homogeneous due to high viscosity and the concentrations of the transferred dispersions in the DSC pans were different from the initial bulk concentrations (50 wt %). Thus, the enthalpy values of the melting transitions are inaccurate. The chain melting temperature (Tm) of the vesicles before polymerization was 25 °C, which is much lower than that (43.3 °C) of pure DPPC vesicles. The pure BDAC aqueous dispersions did not show any melting transition in the experimental temperature range. This result indicates that the melting transition observed in the DPPC/BDAC (2/3) vesicles corresponds to that of DPPC domains. The lowered melting temperature suggests that the DPPC domains may be quite small in size in the mixed bilayers and/or the packing of DPPC alkyl chains in the membranes may be inhibited in some degree by the presence of BDAC. On the other hand, the melting transition of the polymerized sample started from a temperature near 20 °C and ended at a temperature slightly higher than the Tm of the pure DPPC vesicle dispersion. This result indicates that the phase separation induced by the polymerization was not efficient and the DPPC domains in the polymerized matrix were widely distributed in size. This may explain why large holes were not clearly observed in this study. The skeletonized vesicles could not be dispersed in water, but they dispersed well in methanol/water (1/1). Probably, methanol molecules are adsorbed on the exposed hydrophobic surfaces of the skeletonized vesicles, whereas the hydrophilic surfaces may be wetted by water. Dispersion of the hydrolyzed vesicles was attempted using several solvents such as tetrahydrofuran, chloroform, N,N-dimethylforamide, methanol, ethanol, and water. The particles were not dispersed in any of the individual solvents. However, a very stable milky dispersion was obtained using a 0.5 M NaOH methanol/water (1/1) solution. These results indicate that the carboxyl groups at the vesicle surfaces should be ionized to be wetted efficiently by water and the exposed hydrophobic surfaces in the interstices or small holes should be shielded from water by adsorption of the methyl group in methanol. Conclusions Vesicles composed of BDAC and DPPC with embedded DVB underwent efficient radical polymerization. DPPC was removed from the cross-linked vesicles using Triton X-100. The headgroup of BDAC was removed by hydrolysis in an acidic condition. The surface of the polymerized vesicles looked very smooth, but the skeletonized vesicles looked pretty rough and showed only small interstices or holes. The DSC experiment showed that DPPC domains were widely distributed in size after polymerization. The hydrolyzed vesicle surface looked wrinkled, probably due to the enhanced fluidity of the alkyl chains. The hydrolyzed vesicles were precipitated or aggregated in many individual solvents but were well dispersed in a 1.0 M NaOH water/methanol (1/1) solution. If the holes on the vesicle surface are large enough, the surface morphology of the hydrolyzed vesicles will be more easily understood. The preparation and hydrolysis of polymerized vesicles with large holes is still in progress in this laboratory and will be reported in the near future. Acknowledgment. This work was supported by the Basic Research Program of the Korea Science & Engineering Foundation (Grant No. R02-2000-00343). LA0203077
