Preparation of Submicrometer-Sized Monodispersed

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Preparation of Submicrometer-Sized Monodispersed Thermoresponsive Core-Shell Hydrogel Microspheres Xin-Cai Xiao,† Liang-Yin Chu,* Wen-Mei Chen, Shu Wang, and Rui Xie School of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, People’s Republic of China Received November 26, 2003. In Final Form: March 17, 2004 We have successfully prepared monodispersed thermoresponsive core-shell hydrogel microspheres with a mean diameter of 200-400 nm with poly(N-isopropylacrylamide-co-styrene) [P(NIPAM-co-St)] cores and poly(N-isopropylacrylamide) (PNIPAM) shells. The submicrometer-sized monodispersed P(NIPAM-co-St) core seeds were prepared by using a surfactant-free emulsion polymerization method, and the PNIPAM shell layers were fabricated onto the core seeds by using a seed polymerization method. The particle size, morphology and monodispersity, and thermoresponsive characteristics of the prepared microspheres were experimentally studied. In the preparation of P(NIPAM-co-St) seeds, with increasing the initiator dosage, the mean diameters and the dispersal coefficients were almost at the same levels at first; however, when the initiator dosage increased further to a critical amount, the mean diameters decreased drastically and the monodispersity became worse significantly. With increasing the stirring rate, the particle diameter decreased, and when the stirring rate was larger than 600 rpm, the monodispersity became worse obviously. With increasing the phase ratio, the mean diameter became larger simply, and the monodispersity became worse first and then became better again. With increasing the reaction time, the particle sizes nearly did not change, while the monodispersity gradually became better slightly. For the core-shell microspheres, with increasing the NIPAM dosage in the preparation of the PNIPAM shell layers, the mean diameters became larger simply, the monodispersity became better, and the thermoresponsive swelling ratio of the hydrodynamic diameters increased.

Introduction Environmental stimuli-responsive polymeric hydrogels attract increasing attention because of their potential applications in numerous fields,1-5 including controlled drug delivery,6-11 chemical separations,12,13 sensors,14-16 catalysis,17 enzyme immobilization,18 and color-tunable crystals.19 For several of the potential applications of these materials, such as “smart” actuators and on-off switches, * To whom correspondence should be addressed. Tel: +86-288546-0682. Fax: +86-28-8540-4976. E-mail: [email protected]. † Current address: Department of Polymer Science, College of Chemistry and Molecule Science, Wuhan University, Wuhan, Hubei 430072, People’s Republic of China. (1) Pelton, R. Adv. Colloid Interface Sci. 2000, 85, 1. (2) Gan, D.; Lyon, L. A. J. Am. Chem. Soc. 2001, 123, 7511. (3) Gan, D.; Lyon, L. A. J. Am. Chem. Soc. 2001, 123, 8203. (4) Jones, C. D.; Lyon, L. A. Macromolecules 2000, 33, 8301. (5) Jones, C. D.; Lyon, L. A. Macromolecules 2003, 36, 1988. (6) Kwon, I. C.; Bae, Y. H.; Kim, S. W. Nature 1991, 354, 291. (7) Ichikawa, H.; Fukumori, Y. J. Controlled Release 2000, 63, 107. (8) Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Nature 1997, 388, 860. (9) Leobandung, W.; Ichikawa, H.; Fukumori, Y.; Peppas, N. A. J. Appl. Polym. Sci. 2003, 87, 1678. (10) Murthy, N.; Thng, Y. X.; Schuck, S.; Xu, M. C.; Frechet, J. M. J. J. Am. Chem. Soc. 2002, 124, 12398. (11) Vihola, H.; Laukkanen, A.; Hirvonen, J.; Tenhu, H. Eur. J. Pharm. Sci. 2002, 16, 69. (12) Kawaguchi, H.; Fujimoto, K. Bioseparation 1998, 7, 253. (13) Kondo, A.; Kaneko, T.; Higashitani, K. Biotechnol. Bioeng. 1994, 44, 1. (14) Hu, Z. B.; Chen, Y. Y.; Wang, C. J.; Zheng, Y. D.; Li, Y. Nature 1998, 393, 149. (15) Panchapakesan, B.; DeVoe, D. L.; Widmaier, M. R. Nanotechnology 2001, 12, 336. (16) van der Linden, H.; Herber, S.; Olthuis, W. Sens. Mater. 2002, 14, 129. (17) Bergbreiter, D. E.; Case, B. L.; Liu, Y.-S.; Waraway, J. W. Macromolecules 1998, 31, 6053. (18) Guiseppi-Elie, A.; Sheppard, N. F.; Brahim, S.; Narinesingh, D. Biotechnol. Bioeng. 2001, 75, 475. (19) Debord, J. D.; Eustis, S.; Debord, S. B.; Lofye, M. T.; Lyon, L. A. Adv. Mater. 2002, 14, 658.

a fast response is needed. To increase the response dynamics, several strategies have been explored. Besides improving the internal architecture or structure of the hydrogels6,20,21 and introducing linear-grafted hydrogel chain configurations with freely mobile ends,22-26 another main strategy is developing microgels or hydrogel microspheres,1-5,7,27-34 because the characteristic time of gel swelling has been reported to be proportional to the square of a linear dimension of the hydrogels.35 Furthermore, a small dimension is necessary for the stimuli-responsive hydrogels to be applied in certain applications. For example, there is a size limit for the drug delivery systems (DDS) to traverse certain organs,36 and a small particle (20) Yoshida, R.; Uchida, K.; Kaneko, Y.; Sakai, K.; Kikuchi, A.; Sakurai, Y.; Okano, T. Nature 1995, 374, 240. (21) Wu, X. S.; Hoffman, A. S.; Yager, P. J. J. Polym. Sci., Part A: Polym. Chem. 1992, 30, 2121. (22) Chu, L.-Y.; Yamaguchi, T.; Nakao, S. Adv. Mater. 2002, 14, 386. (23) Chu, L.-Y.; Park, S.-H.; Yamaguchi, T.; Nakao, S. Langmuir 2002, 18, 1856. (24) Chu, L.-Y.; Niitsuma, T.; Yamaguchi, T.; Nakao, S. AIChE J. 2003, 49, 896. (25) Chu, L.-Y.; Park, S.-H.; Yamaguchi, T.; Nakao, S. J. Membr. Sci. 2001, 192, 27. (26) Yamaguchi, T.; Ito, T.; Sato, T.; Shinbo, T.; Nakao, S. J. Am. Chem. Soc. 1999, 121, 4078. (27) Xiao, X.-C.; Chu, L.-Y.; Chen, W.-M.; Wang, S.; Li, Y. Adv. Funct. Mater. 2003, 13, 847. (28) Matsuoka, H.; Fujimoto, K.; Kawaguchi. H. Polym. Gels Networks 1998, 6, 319. (29) Matsuoka, H.; Fujimoto, K.; Kawaguchi. H. Polym. J. 1999, 31, 1139. (30) Zhu, P. W.; Napper, D. H. Langmuir 2000, 16, 8543. (31) Varga, I.; Gilanyi, T.; Meszaros, R.; Filipcsei, G.; Zrinyi, M. J. Phys. Chem. B 2001, 105, 9071. (32) Gao, J.; Hu, Z. Langmuir 2002, 18, 1360. (33) Zha, L.; Zhang, Y.; Yang, W.; Fu, S. Adv. Mater. 2002, 14, 1090. (34) Bouillot, P.; Vincent, B. Colloid Polym. Sci. 2000, 278, 74. (35) Tanaka, T.; Fillmore, D. J. J. Chem. Phys. 1979, 70, 1214. (36) Thews, G.; Mutschler, E.; Vaupel, P. Anatomie, physiologie, pathophysiologie des manschen; Wissenschaftl. Verlagsges: Stuttgart, 1980.

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size minimizes any potential irritant reaction at the injection site.37 Therefore, the fabrication of small-sized environmental stimuli-responsive microspheres is of both scientific and technological interest. Because there are many cases in which environmental temperature fluctuations occur naturally and in which the environmental temperature stimuli can be easily designed and artificially controlled, in recent years much attention has been focused on thermoresponsive hydrogel microspheres.1-5,7,27-34 Monodispersity is very important for the stimuliresponsive microspheres to improve their performance in various applications. For example, a uniform microsphere particle size is important for DDS, because the distribution of the microspheres within the body and the interaction with biological cells are greatly affected by the particle size.38 In addition, if monodispersed microspheres are available, the drug release kinetics can be manipulated, thereby making it easier to formulate more sophisticated intelligent DDS. However, few systematical investigations have been reported on the monodispersity and morphology of thermoresponsive hydrogel microspheres. In this study, submicrometer-sized monodispersed thermoresponsive core-shell hydrogel microspheres were prepared with poly(N-isopropylacrylamide-co-styrene) [P(NIPAM-co-St)] cores and poly(N-isopropylacrylamide) (PNIPAM) shells. The core seeds were prepared by a surfactant-free emulsion polymerization method, and shell layers were fabricated by a seed polymerization method. Systematical investigations were carried out on the particle size, monodispersity, and morphology of both the core seeds and the core-shell microspheres. The thermoresponsive characteristics of the core-shell microspheres were also studied. The objective of this study was to provide some valuable guidance for the preparation of small-sized monodispersed thermoresponsive core-shell hydrogel microspheres. Experimental Section Materials. The N-isopropylacrylamide (NIPAM) was kindly provided by Kohjin Co., Ltd., Japan, and was used after purifying by recrystallization in hexane and acetone and then drying in vacuo at room temperature. Styrene (St) monomer was purified by 5 wt % NaOH. Potassium persulfate (K2S2O8), which was used as an anionic initiator, was reagent grade and used as received without any further purification. Well-deionized and deoxygenated water, whose resistance was larger than 16 MΩ, was used in all the synthesis processes. Preparation of P(NIPAM-co-St) Seeds. The P(NIPAM-coSt) seeds were prepared by an emulsifier-free emulsion polymerization method. A mixture of St and NIPAM was dissolved in 185 mL of deionized water in a 250-mL four-necked round-bottom flask equipped with a condenser, a nitrogen inlet, a thermometer, and a stirrer. Nitrogen was bubbled into the solution, and the mixture was stirred for 30 min to remove oxygen from the monomeric solution. Polymerization was initiated by adding 15 mL of aqueous solution containing a certain amount of K2S2O8 at 70 °C. The reaction was allowed to proceed for 24 h at 70 °C under stirring. The resulting P(NIPAM-co-St) microsphere samples were dialyzed and purified by repetitive centrifugation, decantation and redispersion and then freeze-dried. Preparation of Core-Shell Microspheres with PNIPAM Shell Layers. The PNIPAM shell layers were fabricated on the above-prepared core seeds by a seed polymerization method. The preparation procedure of the core-shell microspheres is schematically illustrated in Figure 1. When the above-mentioned reaction for the P(NIPAM-co-St) seeds was not stopped, another 15 mL of an aqueous solution containing a certain amount of NIPAM was added, and polymerization was allowed to continue (37) Little, K.; Parkhouse, J. Lancet 1962, II (7261), 857. (38) Shiga, K.; Muramatsu, N.; Kondo, T. J. Pharm. Pharmacol. 1996, 48, 891.

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Figure 1. Schematic illustration of the preparation procedure of the core-shell microspheres. for 22 h under stirring at 200 rpm. By this method, the reaction manner was in graft polymerization because of some living radicals on the surfaces of polymerizing core particles,28,29 and the resulting shell layers of the core-shell microspheres were hairy because no cross-linker was used. The prepared coreshell microsphere samples were also dialyzed and purified by repetitive centrifugation, decantation, and redispersion and then freeze-dried. Morphological Analysis. The morphology of both the core seeds and the core-shell microspheres was observed by a scanning electron microscope (SEM S-450, Hitachi, Japan). All specimens for SEM observations were sputtered with gold at fixed conditions (time 150 s, current 20 mA, voltage 2 kV). Determination of the Mean Diameter and Size Distribution of the Microspheres. The size distribution of dry microspheres was determined using a digital image analysis system on the basis of the SEM photographs of the microspheres. In the analysis, the particle number of each sample was always more than 300. To describe the monodispersity of the microspheres quantitatively, an index named the particle size dispersal coefficient, δ, is defined as

δ)

D90 - D10 D50

(1)

where Dn (n ) 10, 50, and 90) denotes the cumulative number percentage of particles with a diameter up to Dn equal to n%. The smaller the value of δ, the narrower the size distribution, that is, the better the monodispersity. Determination of the Thermoresponsive Characteristics of the Core-Shell Microspheres. The hydrodynamic diameters of the prepared core-shell hydrogel microspheres at different temperatures were determined by temperatureprogrammed photon correlation spectroscopy (TP-PCS; Brookhaven BI-9000AT, U.S.A.). This technique has been applied extensively to the characterization of such material because it allows for in situ size characterization of soft material that cannot be reliably sized by electron microscopes as a result of deformation and dehydration under vacuum.2 The dispersed particles in water were allowed to equilibrate thermally for 10-15 min before measurements were taken at each temperature. The hydrodynamic diameters of particles were calculated from diffusion coefficients by the Stokes-Einstein equation, and all correlogram analyses were performed using the manufacturer-supplied software. In the data presented in this study, each data point at a given temperature represents the average valve of 15-20 measurements, with a 20-s integration time for each measurement.

Results and Discussion 1. Monodispersity of P(NIPAM-co-St) Microspheres. Figure 2 shows the SEM photographs of P(NIPAM-co-St) microspheres prepared with different initiator dosages, and Figure 3 shows the effect of the

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Figure 2. SEM micrographs of P(NIPAM-co-St) microspheres prepared with different initiator dosages (scale bar ) 1 µm).

Figure 3. Effect of initiator dosage on the mean diameter and monodispersity of the P(NIPAM-co-St) microspheres.

initiator dosage on the mean diameter and monodispersity of the microspheres. The SEM micrographs show that the prepared microspheres were satisfactorily spherical and with diameters in the range 200-400 nm. Different initiator dosages resulted in different sizes and distributions of particles. With increasing the initiator dosage from 0.2 to 0.5 g, the mean diameters and the dispersal coefficients of particles were almost at the same levels. However, when the initiator dosage increased further from

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Figure 4. SEM micrographs of P(NIPAM-co-St) microspheres prepared with different stirring rates (scale bar ) 1 µm).

0.5 to 0.6 g, the mean diameter of the particles decreased drastically, and the particles became more polydisperse. The initiator produced free radicals by thermal decomposition, and the free radicals initiated the polymerization reaction. The reaction was followed by chain propagation, and when the chains reached a critical length, the growing chains formed precursor particles by interwinding with each other; finally, the precursor particles grew into product particles. Termination of chain propagation occurred when two free radicals met or when a free radical contacted oxygen. When the initiator dosage was not very large (e.g., less than 0.5 g) the number of resulting free radicals was not very large, and the chance of free radicals meeting was limited. This was helpful for growth of particles. Consequently, microspheres with good monodispersity resulted. However, by increasing the initiator dosage even more (e.g., larger than 0.5 g), the number of resulting free radicals increased further, and the chance of free radicals meeting increased too much. As a result, the particles could not grow normally, the particle sizes of the resulting products decreased, and the polydispersity increased. Figures 4 and 5 show the SEM micrographs of P(NIPAM-co-St) microspheres prepared with different stirring rates and the effect of the stirring rate on the

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Figure 5. Effect of stirring rate on the mean diameter and monodispersity of the P(NIPAM-co-St) microspheres.

mean diameter and monodispersity of the microspheres, respectively. With increasing the stirring rate, the particle diameter decreased, and when the stirring rate was larger than 600 rpm, the monodispersity became worse obviously. Because a soap-free emulsion polymerization method was used in this study, the latex formation required mechanical stirring. With increasing the stirring rate, the shear forces in fluids increased and this resulted in that the latex droplets became smaller; correspondingly, the mean diameters of the produced microspheres became smaller. When the stirring rate was lower than 600 rpm, the latex droplets were uniform, and then the size distributions of the microspheres were narrow. However, when the stirring rate was higher than 600 rpm, the meeting chances between the latex droplets and between the precursor particles increased too much, which led to the product particle sizes becoming nonuniform; that is, the monodispersity of the microspheres became worse. Therefore, to fabricate small-sized monodispersed microspheres, the optimum stirring rate should be 600 rpm. Figures 6 and 7 illustrate the SEM photographs of P(NIPAM-co-St) microspheres prepared with different phase ratios and the effect of the phase ratio on the mean diameter and monodispersity of the microspheres, respectively, in which the phase ratio is defined as the mass ratio of monomers to the solvent. In this study, the monomers included both NIPAM and St, and the mass ratio of NIPAM to St was held constant at 1:9. As the phase ratio was increased, the mean diameter of the microspheres increased. An increase in the amount of monomer provided more material for particle growth. When the phase ratio was increased from 2.5 to 5.0%, the polydispersity increased, and when the phase ratio was increased to 10.0%, the polydispersity decreased. For a constant stirring rate and monomer dosage, the number of latex droplets was constant. The number of latex droplets per unit volume increased as the phase ratio increased, and the particle collisions increased leading to greater polydispersity. In the surfactant-free emulsion polymerization of this study, monomer NIPAM was hydrophilic, and its polymer played the role of providing stability and scattering action during the course of the reaction.1 When the phase ratio was not high, the precursor particles did not have enough PNIPAM chains to act as stabilizers, and then the particles aggregated and could not grow normally, which led to the monodispersity of particle size becoming worse, while with increasing the phase ratio, the opposite occured. By considering the above two factors comprehensively, with increasing the phase ratio, the general tendency of the monodispersity of microspheres became worse first and then became better again. Figures 8 and 9 show the SEM photographs of P(NIPAM-co-St) microspheres prepared with different

Figure 6. SEM micrographs of P(NIPAM-co-St) microspheres prepared with different phase ratios (scale bar ) 1 µm).

Figure 7. Effect of phase ratio on the mean diameter and monodispersity of the P(NIPAM-co-St) microspheres.

polymerization times and the effect of the polymerization time on the mean diameter and monodispersity of the microspheres, respectively. With increasing the reaction time, the particle size nearly did not change, while the monodispersity became better slightly. Figure 10a,b illustrates the thermoresponsive transmittance change of the dispersion of P(NIPAM-co-St) microspheres prepared with different polymerization times (4 and 24 h, respectively). Figure 10a shows the transmittance of the dispersion decreased with increasing the temperature,

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Figure 10. Thermoresponsive transmittance change of the dispersion of P(NIPAM-co-St) microspheres prepared with different polymerization times.

Figure 8. SEM micrographs of P(NIPAM-co-St) microspheres prepared with different polymerization times (scale bar ) 1 µm).

Figure 9. Effect of polymerization time on the mean diameter and monodispersity of the P(NIPAM-co-St) microspheres.

especially decreased obviously when the temperature was changed from 30 to 35 °C. On the other hand, Figure 10b shows that the transmittance of the dispersion hardly changed at different temperatures. It is well-known that the transmittance of PNIPAM changes rapidly at the lower critical solution temperature (LCST, around 32 °C),39 and (39) Heskins, M.; Guilleit, J. E. J. Macromol. Sci. Chem. 1968, A28, 1441.

polystyrene is always milky. Therefore, the results in Figure 10 indicates that the main materials of the products should be PNIPAM when the polymerization time was just 4 h and then P(NIPAM-co-St) when the polymerization time was increased to 24 h. That is, in the polymerization process, monomer NIPAM rapidly homopolymerized, and then St entered into the precursor particles and copolymerized gradually. At the reactive incipience, watersoluble sulfate radicals originated from K2S2O8-initiated water-soluble NIPAM monomers, which then grew in solution until they reached the critical chain length. After being longer than the critical length, the growing chains collapsed to become unstable colloidal precursor particles. They aggregated with each other until they formed particles large enough to be colloidally stable. With the reaction proceeding, St entered into PNIPAM-rich particles and started to react. With the reaction time increasing, the particle size became uniform because of the St copolymerization; as a result, the monodispersity of the microspheres became better gradually. From the above results, to prepare small-sized and monodispersed P(NIPAM-co-St) microspheres, the optimum parameters should be selected as a stirring rate of 600 rpm, an initiator dosage of 0.40 g, a phase ratio of 2.5%, and a polymerization time of 24 h, which will be used to fabricate the core seeds in the subsequent experiments. 2. Monodispersity of Core-Shell Microspheres with P(NIPAM-co-St) Cores and PNIPAM Shell Layers. Figure 11 shows the SEM photographs of the core-shell microspheres with P(NIPAM-co-St) cores and PNIPAM shell layers prepared with different NIPAM dosages in the shell fabrication, and Figure 12 illustrates the effect of the NIPAM dosage in the shell preparation on the mean diameter and monodispersity of the micro-

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Figure 11. SEM micrographs of core-shell microspheres prepared with different NIPAM dosages in the fabrication of the shell layers (scale bar ) 1 µm).

Figure 12. Effect of the NIPAM dosage in the fabrication of the shell layers on the mean diameter and monodispersity of the core-shell microspheres.

spheres. The PNIPAM shell layers were fabricated on the above-prepared core seeds by a seed polymerization method. By this method, the reaction manner was graft polymerization because of some living radicals on the surfaces of the polymerizing core particles. Because the number of core seeds per unit volume was constant, and monomer NIPAM contributed only to the PNIPAM shell formation on the seeds; therefore, the mean diameters of the core-shell microsphere products became larger simply with increasing the NIPAM dosage. On the other hand, the free radical density of smaller particles was larger relatively, which was helpful for the smaller particles to absorb more monomers or polymers with low molecular weight onto their surfaces to form larger particles. Therefore, with increasing the NIPAM dosage, the monodispersity of the core-shell microspheres became better. 3. Thermoresponsive Characteristics of the CoreShell Microspheres. Figure 13 shows the effect of the NIPAM dosage in the preparation of shell layers on the thermoresponsive swelling characteristics of the coreshell microspheres. With increasing the NIPAM dosage in the fabrication of the shell layers, the thermoresponsive

Figure 13. Effect of the NIPAM dosage in the preparation of shells on the thermoresponsive swelling characteristics of the core-shell microspheres.

swelling ratio of the hydrodynamic diameters of the coreshell microspheres at temperatures below the LCST of PNIPAM to those of the microspheres above the LCST increased. The hydrophilic groups of hairy PNIPAM chains on the core-shell microsphere surfaces formed hydrating layers by hydrogen bound with water. The longer the PNIPAM chains, which resulted from increasing the NIPAM dosage, the thicker the hydrating layer and then the larger the hydrodynamic diameter. The thickness of the hydrating layer decreased because of the breakage of hydrogen bonds with increasing temperature. When the temperature approached the LCST, hydrogen bonds were broken seriously, which led to the thickness of the hydrating layer decreasing rapidly, and then the linear PNIPAM polymer chains collapsed quickly, resulting in a rapid decrease in the hydrodynamic diameters of the core-shell microspheres. Acknowledgment. This work was supported by the National Natural Science Foundation of China (Grant 20206019), the Trans-Century Training Program Foun-

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dation for the Talents by the Ministry of Education of China (Grant 2002-48), and Sichuan Youth Science and Technology Foundation for Distinguished Young Scholars (Grant 03ZQ026-41). The authors gratefully acknowledge the help of Dr. Xu-Hong Peng at Lanzhou University for

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the hydrodynamic diameter measurements by TP-PCS and also thank the Kohjin Co., Ltd, Japan, for kindly supplying the NIPAM. LA036230J