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Preparation of Supramolecular Graft Copolymers and the Subsequent Formation of pH-Sensitive Vesicles Jian Qian†,‡ and Feipeng Wu†,* Technical Institute of Physics and Chemistry, The Chinese Academy of Sciences, Beijing 100190, P. R. China, and Graduate UniVersity of Chinese Academy of Sciences, Beijing 100049, P. R. China ReceiVed October 12, 2008. ReVised Manuscript ReceiVed December 21, 2008
A feasible methodology for the preparation of vesicles through self-assembly of supramolecular graft copolymers (SGPs) was developed. Two types of polymeric vesicles were prepared through self-assembly of two SGPs. For the first SGP, hydrophobic poly(4-vinylpyridine) (PVPy) were used as main chains, and hydrophilic poly(N-vinylpyrrolidone) with carboxylic end groups (PNVP-COOH) were grafted onto the main chains through ionic interaction between the carboxylic groups and pyridine groups. For the second SGP, hydrophobic poly(4-acrylamidobenzoic acid) (PABA) were used as main chains, and poly(Nvinylpyrrolidone) with amino end groups were grafted onto the main chains through ionic interaction between the amino groups and carboxylic groups. Both SGPs could self-assemble to form vesicles in aqueous solution. The size of the vesicles prepared from the PVPy/PNVP-COOH (or PABA/PNVPNH2) system could be controlled through modulation of the mass ratio of PVPy and PNVP-COOH (or PABA and PNVP-NH2). Both vesicles were sensitive to pH and could be deformed by changing pH. Drug-release studies showed that release rates of the loaded drug (sunset yellow) in the two vesicles could be well-controlled by the pH of the releasing solution.
Introduction Polymeric vesicles have received a lot of attention because of their wide applicability,1-5 particularly as drug-release systems.3,6,7 To control release of guest materials in the vesicles, researchers have prepared “smart” vesicles that are sensitive to temperature,7-9 pH,6,10,11 and light6,12,13 through introducing environment-sensitive materials to construct the vesicles. In previous decades, most polymeric vesicles were successfully prepared through self-assembly of polymers with specific microstructures (e.g., block copolymers,1,14 graft copolymers15-17). To prepare these polymers, researchers have developed various polymerization strategies in the past few decades, such as * Corresponding author. E-mail:
[email protected]. † Technical Institute of Physics and Chemistry, Chinese Academy of Sciences. ‡ Graduate University of Chinese Academy of Sciences.
(1) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967–973. (2) Antonietti, M.; Fo¨rster, S. AdV. Mater. 2003, 15, 1323–1333. (3) Ro¨sler, A.; Vandermeulen, G. W. M.; Klok, H.-A. AdV. Drug DeliVery ReV. 2001, 53, 95–108. (4) Kita-Tokarczyk, K.; Grumelard, J.; Haefele, T.; Meier, W. Polymer 2005, 46, 3540–3563. (5) Graff, A.; Sauer, M.; Gelder, P. V.; Meier, W. Proc. Natl. Acad. Sci. 2002, 99, 5064–5068. (6) Gerasimov, O. V.; Boomer, J. A.; Qualls, M. M.; Thompson, D. H. AdV. Drug DeliVery ReV. 1999, 38, 317–338. (7) Qin, S.; Geng, Y.; Discher, D. E.; Yang, S. AdV. Mater. 2006, 18, 2905–2909. (8) Yin, H.; Huang, J.; Gao, Y.; Fu, H. Langmuir 2005, 21, 2656–2659. (9) Li, Y.; Lokitz, B. S.; McCormick, C. L. Angew. Chem., Int. Ed. 2006, 45, 5792–5795. (10) Borchert, U.; Lipprandt, U.; Bilang, M.; Kimpfler, A.; Rank, A.; Peschka-Su¨ss, R.; Schubert, R.; Lindner, P.; Fo¨rster, S. Langmuir 2006, 22, 5843–5847. (11) Du, J.; Tang, Y.; Lewis, A. L.; Armes, S. P. J. Am. Chem. Soc. 2005, 127, 17982–17983. (12) Liu, X.; Jiang, M. Angew. Chem., Int. Ed. 2006, 45, 3846–3850. (13) Jiang, Y.; Wang, Y.; Ma, N.; Wang, Z.; Smet, M.; Zhang, X. Langmuir 2007, 23, 4029–4034.
anionic,18 controlled radical,19,20 and group transfer polymerization.21 These techniques are based on using covalent bonds to connect the substructural polymer blocks. Developments in supramolecular chemistry regarding noncovalent interactions provide an alternative concept for preparing copolymers with specific architectures. Pioneering work by several research groups has shown that polymers functionalized with specific groups can further self-assemble and form supramolecular organization of macromolecules via noncovalent interactions of these groups, such as ionic interactions,22,23 hydrogen bonds,24-31 metal-ligand interac(14) Soo, P. L.; Eisenberg, A. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 923–938. (15) Lee, H. J.; Yang, S. R.; An, E. J.; Kim, J.-D. Macromolecules 2006, 39, 4938–4940. (16) Breitenkamp, K.; Emrick, T. J. Am. Chem. Soc. 2003, 125, 12070– 12071. (17) Dou, H.; Jiang, M.; Peng, H.; Chen, D.; Hong, Y. Angew. Chem., Int. Ed. 2003, 42, 1516–1519. (18) Hadjichristidis, N.; Pitsikalis, M.; Pispas, S.; Iatrou, H. Chem. ReV. 2001, 101, 3747–3792. (19) Matyjaszewski, K.; Xia, J. Chem. ReV. 2001, 101, 2921–2990. (20) Quinn, J. F.; Chaplin, R. P.; Davis, T. P. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 2956–2966. (21) Webster, O. W.; Hertler, W. R.; Sogah, D. Y.; Farnham, W. B.; Rajanbabu, T. V. J. Am. Chem. Soc. 1983, 105, 5706–5708. (22) Russell, T. P.; Je´roˆme, R.; Charlier, P.; Foucart, M. Macromolecules 1988, 21, 1709–1717. (23) Qian, J.; Wu, F. Macromolecules 2008, 41, 8921–8926. (24) Park, T.; Zimmerman, S. C. J. Am. Chem. Soc. 2006, 128, 13986– 13987. (25) Park, T.; Zimmerman, S. C. J. Am. Chem. Soc. 2006, 128, 11582– 11590. (26) Uzun, O.; Sanyal, A.; Nakade, H.; Thibault, R. J.; Rotello, V. M. J. Am. Chem. Soc. 2004, 126, 14773–14777. (27) Deans, R.; Ilhan, F.; Rotello, V. M. Macromolecules 1999, 32, 4956– 4960. (28) Ilhan, F.; Gray, M.; Rotello, V. M. Macromolecules 2001, 34, 2597– 2601.
10.1021/cm802777k CCC: $40.75 2009 American Chemical Society Published on Web 01/20/2009
pH-SensitiVe Vesicle Formation from Graft Copolymers
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Scheme 1. Vesicle Formation from Supramolecular Graft Copolymers
tions.32,33 It has been reported that these supramolecular macromolecules could self-assemble to form various supramolecular architectures.26,29,34,35 On the basis of this concept, two types of supramolecular graft copolymers (SGPs) were prepared through ionic interaction between the groups on the main chain and end groups of the side chains. For the first SGP, hydrophobic poly(4-vinylpyridine) (PVPy) were used as main chains, and hydrophilic poly(N-vinylpyrrolidone) with carboxylic end groups (PNVP-COOH) were grafted onto the main chain through ionic interaction between the carboxylic groups and pyridine groups. For the second SGP, hydrophobic poly(4acrylamidobenzoic acid) (PABA) were used as main chains, and poly(N-vinylpyrrolidone) with amino end groups were grafted onto the main chain through ionic interaction between the amino groups and carboxylic groups (as illustrated in Scheme 1). Both SGPs could self-assemble to form vesicles in selected solvents. Because of the pH sensitivity of PVPy and PABA, the vesicles whose shells were constructed from these two materials were sensitive to pH, and could be deformed by changing pH. These pH-sensitive vesicles are promising controlled-release drug systems. Results and Discussion Preparation of the Precursor Polymers. Hydrophobic PVPy and PABA were prepared by free-radical polymerization. Light scattering analysis showed that the weight(29) Chen, D.; Jiang, M. Acc. Chem. Res. 2005, 38, 494–502. (30) Brunsveld, L.; Folmer, B. J. B.; Meijer, E. W.; Sijbesma, R. P. Chem. ReV. 2001, 101, 4071–4097. (31) Todd, E. M.; Zimmerman, S. C. J. Am. Chem. Soc. 2007, 129, 14534– 14535. (32) Schubert, U. S.; Eschbaumer, C. Angew. Chem., Int. Ed. 2002, 41, 2892–2926. (33) Ievins, A. D.; Moughton, A. O.; O’Reilly, R. K. Macromolecules 2008, 41, 3571–3578. (34) Moughton, A. O.; O’Reilly, R. K. J. Am. Chem. Soc. 2008, 130, 8714– 8725. (35) Gohy, J.-F.; Lohmeijer, B. G. G.; Schubert, U. S. Chem.sEur. J. 2003, 9, 3472–3479.
average molecular weights of PVPy and PABA were 21670 and 15540, respectively. Hydrophilic PNVP-NH2 and PNVPCOOH were prepared by free-radical polymerization using cysteamine hydrochloride and thioglycolic acid as chain transfer reagents, respectively.36,37 Light scattering analysis showed that the weight-average molecular weights of PNVPNH2 and PNVP-COOH were 33 150 and 56 060, respectively. Vesicles Prepared from Two SGP Systems. Self-assembly behavior of the proposed SGPs was investigated in selected solvents. PVPy and PNVP-COOH with a mass ratio 4:1 were first dissolved in dimethylformamide (DMF); PABA and PNVP-NH2 with a mass ratio 3:1 were first dissolved in dimethyl sulfoxide (DMSO). PNVP-COOH could be linked with PVPy to form the SGP because of the ionic interaction between carboxylic groups and pyridine groups. Similarly, PABA and PNVP-NH2 could form the SGP through ionic interaction between carboxylic groups and amino groups (as illustrated in Scheme 1). Deionized water was added to the two polymer solutions to the desired water content. Absorbance (by UV/vis spectrophotometer at 500 nm) increases dramatically when water content is above critical water content (65.1% v/v for the PVPy/PNVP-COOH system; 33.3% v/v for the PABA/PNVP-NH2 system), which means the polymers self-assemble to form aggregates. Both of the aggregates were investigated by transmission electron microscopy (TEM). Figure 1A shows the TEM image of the vesicles obtained from the PVPy/PNVP-COOH system; the aggregates display a vesicular structure with a diameter of about 250-300 nm. Figure 1B shows the TEM image of the vesicles obtained from the PABA/PNVP-NH2 system; the aggregates also display a vesicular structure with a diameter of about 300 nm. When water was added to the two polymer solutions, the hydrophobic main chains, PVPy (PABA), aggregate to form the walls of the vesicles, whereas the hydrophilic side chains, PNVP-COOH (PNVP-NH2), (36) Chen, G.; Hoffman, A. S. Nature 1995, 373, 49–52. (37) Wei, H.; Cheng, C.; Chang, C.; Chen, W.-Q.; Cheng, S.-X.; Zhang, X.-Z.; Zhuo, R.-X. Langmuir 2008, 24, 4564–4570.
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Figure 1. TEM images of the vesicles prepared from (A) PVPy/PNVPCOOH and (B) PABA/PNVP-NH2 systems.
were anchored around the PVPy (PABA) walls through ionic bonds to form the corona and maintain the stability of the vesicles. When using a solution of 0.025 M NaCl instead of pure water as the poor solvent, precipitate was formed instead of the stable vesicular solution in the two systems. The ionic solution (Na+ and Cl-) would weaken the ionic bonds38 between the side chains and main chains, so the ordered polymer organization (SGP) could not exist in a stable manner and could not self-assemble to form regular aggregates (vesicles). These results indicated that the ionic bonds between side chains and main chains played important roles in vesicle formation, i.e., only ordered polymer organization (SGP) could self-assemble to form vesicles. The size of the vesicle could be controlled through modulation of the graft ratio of the two SGPs. In contrast with the conventional graft copolymer whose graft ratio is fixed once it is prepared, the SGP is prepared through ionic bonds, so the graft ratio can be readily modulated by changing the mass ratio of the PVPy and PNVP-COOH (or PABA and PNVP-NH2). The diameter of the vesicles obtained from the PVPy/PNVP-COOH system varied with the mass ratio of PVPy and PNVP-COOH (Figure 2A). As the mass ratio increased from 2 to 5, vesicle diameter (obtained from light scattering analysis) increased from about 150 to 370 nm. The diameter of the vesicles prepared from the PABA/PNVP-NH2 system also varied with mass ratio of the PABA and PNVP-NH2 system (Figure 2B). As the mass ratio increased from 1.5 to 4, vesicle diameter increased from about 180 to 480 nm. As mass ratio increased, the content of the hydrophilic side chains (PNVP-COOH or PNVP-NH2) in each SGP decreased. To maintain vesicle stability, more hydrophilic side chains are required when mass ratio increases, so the average SGP aggregation number of vesicles increases (i.e., vesicle size increases). pH Sensitivity. It is well-known that PVPy and PABA are sensitive to pH, so the vesicles constructed by the two polymers are also sensitive to pH. PVPy can be ionized in acidic solution, so vesicles obtained from the PVPy/PNVP(38) Ibarz, G.; Da¨hne, L.; Donath, E.; Mo¨hwald, H. AdV. Mater. 2001, 13, 1324–1327.
Figure 2. Size dependence of the two vesicles ((A) PVPy/PNVP-COOH system, (B) PABA/PNVP-NH2 system) on the mass ratio of the main chains and side chains.
COOH system could be deformed if the solution is sufficiently acid. Vesicles obtained at different graft ratios were fully deformed when the pH was 7.8 (Figure 3B). When pH was readjusted to the start point, only floccular precipitate was obtained and the stable vesicular solution could not be reformed, indicating that vesicle formation was not reversible upon pH variation. Once the vesicles were deformed, ionic bonds between the PVPy (PABA) and PNVP-COOH (PNVP-NH2) were also broken, so SGP did not exist in the solution. These scattered polymers could not self-assemble to form well-ordered vesicles because ordered polymer organizations were absent in the solution. These results indicated that ionic bonds play important roles in vesicle formation. Ionic bonds lead to the formation of ordered polymer organization (SGP) which could selfassemble to form ordered aggregates (vesicles). These results also coincided with the conclusions from the synthetic experiment in which 0.025 M NaCl solution was used as the poor solvent. Controlled Release of Drug. pH sensitivity enables the two vesicles to be used for controlled release of drugs. The water-soluble dye sunset yellow was used as the model drug.
pH-SensitiVe Vesicle Formation from Graft Copolymers
Figure 3. Transmittance of the (A) PVPy/PNVP-COOH and (B) PABA/ PNVP-NH2 vesicular solutions at various pH values.
After loading with sunset yellow, vesicles were treated in the buffer solution at different pH values. Release rates of the loaded drug in the two vesicles were very sensitive to the pH of the releasing solution. Figure 4A shows the drugrelease profile of the loaded vesicles prepared from the PVPy/ PNVP-COOH system in HAc/NaAc buffer solution at various pH values. There was virtually no release of the loaded drug from the vesicles at pH 5.00. After 50 min, only less than 1% loaded drug was released. Further results showed that less than 5% was released even after 24 h. Release rate of the drug increased enormously at pH 4.50 compared with the value at pH 5.00. It took about 30 min to totally release the loaded drug in the vesicles. At pH 4.00, the release rate of the drug was even faster than the value at pH 4.50. It took only about 8 min to accomplish total release of the drug. As the pH changed from 5.00 to 4.00, vesicle shells (which were constructed from PVPy) became gradually easier to ionize, and the drug loaded in the vesicles became increasingly free to pass through the shells. Figure 4B shows the drug-release profile of loaded vesicles prepared from the PABA/PNVP-NH2 system in NaHPO4/NaH2PO4 buffer solution at various pH values. The release rate of the drug was very low at pH 6.00, and about only 8% of the loaded drug was released after 50 min. Release rate was much faster at pH 7.00, and it took about 30 min to release all the drug in the vesicles. Release rate was even faster at pH 8.00, and it took only about 15 min to achieve total release of the drug in the vesicles. As pH changed from 6.00 to 8.00, vesicle shells (which were constructed from PABA) became gradu-
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Figure 4. Drug-release profiles of vesicles prepared from the (A) PVPy/ PNVP-COOH and (B) PABA/PNVP-NH2 systems.
ally easier to ionize, and the drug loaded in the vesicles was released through the shells with increasing ease. It is wellknown that, the pH of the stomach is below 2.0,39 whereas the pH of the small intestine is above 7.0.40 Based on the results from our study, it could be estimated that, a drug loaded in vesicles prepared from the PVPy/PNVP-COOH system could be released immediately when the vesicles enter the stomach, while a drug loaded in the vesicles prepared from the PABA/PNVP-NH2 system would barely be released in the stomach, but could be totally released when the vesicles enter the small intestine. Experimental Section Synthesis of PVPy, PABA, PNVP-COOH, and PNVP-NH2 Polymers. For the preparation of PVPy, 15.75 g of 4-vinylpyridine and 0.123 g of AIBN were dissolved in 60 mL of ethanol. After the solution was purged with nitrogen for 20 min, the vessel was sealed and the polymerization was allowed to proceed at 60 °C for 24 h. The polymers were precipitated by adding excess petrol ether. The purified polymers were dried in a vacuum at 60 °C for several hours. For the preparation of PABA, 4-acrylamidobenzoic acid, 4 g, and AIBN, 0.0172 g, were dissolved in 80 mL DMF. After the solution was purged with nitrogen for 20 min, the vessel was sealed (39) Fimmel, C. J.; Etienne, A.; Cilluffo, T.; Ritter, C. v.; Gasser, T.; Rey, ¨ ; uhler, H. W.; J.-P.; Caradonna-Moscatelli, P.; Sabbatini, F.; Pace, F. B Bauerfeind, P.; Blum, A. Gastroenterology 1985, 88, 1842–1851. (40) Bucher, G. R.; Flynn, J. C.; Robinson, C. S. J. Biol. Chem. 1944, 155, 305–313.
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and the polymerization was allowed to proceed at 50 °C for 24 h. The polymers were precipitated by adding excess water. The purified polymers were dried in vacuum at 50 °C for several hours. For the preparation of PNVP-COOH, 5 g of N-vinylpyrrolidone, 0.207 g of thioglycolic acid (Caution! It is toxic and caustic.), and 0.0738 g of AIBN were dissolved in 100 mL of dioxane. After the solution was purged with nitrogen for 20 min, the vessel was sealed and the polymerization was allowed to proceed at 60 °C for 24 h. The polymers were precipitated by adding excess petrol ether. The purified polymers were dried in vacuum at 50 °C for several hours. For the preparation of PNVP-NH2, 6.66 g of N-vinylpyrrolidone, 0.3405 gof cysteamine hydrochloride, and 0.0984 g AIBN were dissolved in 90 mL of ethanol. After the solution was purged with nitrogen for 20 min, the vessel was sealed and the polymerization was allowed to proceed at 60 °C for 24 h. After the solution was condensed to about 20 mL, 0.17 g of KOH (dissolved in 10 mL ethanol) and 3 g of anhydrous Na2SO4 were added into the solution. After 5 h, the solution was filtrated, and the polymers in the filtrate were precipitated by petroleum ether. The purified polymers were dried in a vacuum at 50 °C for several hours. Preparation of the Vesicles. Vesicles were prepared from two SGP systems, the PVPy/PNVP-COOH and PABA/PNVP-NH2 systems. For the PVPy/PNVP-COOH system, PVPy and PNVPCOOH with different mass ratios were dissolved in DMF, and the concentration of the polymers was kept at 0.01 g mL-1. The deionized water was dropwise added (about 0.3 mL per minute) into the polymer solution to 80% (v/v) water content. Then the solution was quenched into excess water (5 fold). For the PABA/ PNVP-NH2 system, PABA and PNVP-NH2 with different mass ratios were dissolved in DMSO, and the concentration of the polymers was kept at 0.01 g mL-1. The deionized water was dropwise added (about 0.05 mL per minute) into the polymer solution to 40% (v/v) water content. The solution was then quenched into excess water (10-fold). pH Sensitivity. The pH values of the vesicular solution were adjusted by 0.005 M HCl and 0.005 M NaOH solution. The transmittances of the vesicular solution at different pH values were carried out at 500 nm by the UV/vis spectrophotometer. Drug Loading Process. For PVPy/PNVP-COOH system, 1 g of sunset yellow was dissolved in 500 mL of vesicular solution prepared from the PVPy/PNVP-COOH system (mass ratio of the PVPy and PNVP-COOH was 4:1). After 72 h, the vesicles were separated by centrifugation at 14 000 rpm. After being washed with deionized water, the loaded vesicles (PVPy/PNVP-COOH system) were dried in a vacuum at 40 °C for 12 h. For the PABA and PNVP-NH2 system, 2 g of sunset yellow was first dissolved 60 mL of deionized water (as the pH of this sunset yellow solution was above 8.0, the vesicles would be deformed in the loading process, so it should be acidified). After the pH of the solution was adjusted to 4.8, the solution was added into 440 mL of vesicular solution prepared from the PABA/PNVPNH2 system (mass ratio of the PABA and PNVP-NH2 was 3:1).
Qian and Wu After 72 h, the loaded vesicles (PABA/PNVP-NH2 system) were separated by centrifugation at 14 000 rpm. After being washed with deionized water, the loaded vesicles were dried in a vacuum at 40 °C for 12 h. Drug Release Process. The releasing behavior of the two loaded vesicles was carried out at different pH values at 25 °C. For PVPy/ PNVP-COOH system, 0.1 g loaded vesicles were treated in 75 mL HAc/NaAc buffer solution with various pH values (pH 4.00, 4.50, and 5.00). To monitor the concentration of the released sunset yellow, the absorbance of the solution at different time was carried out at 482 nm by the UV/vis spectrophotometer. For PVPy/ PNVP-NH2 system, 0.1 g of loaded vesicles was treated in 50 mL of NaHPO4/NaH2PO4 buffer solutions with various pH values (pH 6.00, 7.00, and 8.00). The absorbance of the solution at different time was measured at 482 nm by the UV/vis spectrophotometer. TEM Analysis. TEM images were obtained by using a JEM200CX transmission electron microscope operating at 160 kV. The preparation of the samples for TEM measurement was as follows: one drop of vesicular solution was put onto a carbon-coated copper grid and stained with staining agent. Then the sample was dried freely at room temperature. As the vesicles obtained from the two systems were sensitive to pH, the pH values of the staining agents could influence the stability of the vesicles. Phosphotungstic acid (pH was adjusted to about 7.0 before it was used) was used as the staining agent for the vesicles obtained from PVPy/PNVPCOOH system, whereas uranyl acetate was used as the staining agent for the vesicles obtained from PABA/ PNVP-NH2 system.
Conclusion A feasible approach to prepare vesicles through selfassembly of SGPs has been demonstrated. The side chains, PNVP-COOH (PNVP-NH2), were grafted onto the main chain, PVPy (PABA), to form a SGP through ionic interaction between carboxylic groups and pyridine groups (amino groups and carboxylic groups). Both SGPs could selfassemble to form vesicles in aqueous solution. The size of the vesicles prepared from the PVPy/PNVP-COOH (PABA/ PNVP-NH2) system could be readily controlled through modulation of the mass ratio of PVPy and PNVP-COOH (PABA and PNVP-NH2). Vesicles prepared from the two systems were sensitive to pH. Drug-release studies showed that release rates of the loaded drug (sunset yellow) in the two vesicles could be well-controlled by pH of the releasing solution. These results are very promising for controlled delivery of drugs. This supramolecular strategy provides a feasible and flexible approach for the engineering of macromolecular architectures and preparation of self-assembled aggregates. CM802777K
