Aqueous Microgels Modified by Wedge-Shaped Amphiphilic Molecules

Dec 4, 2009 - particles into the aqueous phase, wedge-shaped amphiphiles ionically ... wedge-shaped molecules into microgels can be controlled by ...
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Aqueous Microgels Modified by Wedge-Shaped Amphiphilic Molecules: Hydrophilic Microcontainers with Hydrophobic Nanodomains Cheng Cheng,† Xiaomin Zhu,†,‡ Andrij Pich,*,† and Martin M€oller† †

DWI an der RWTH Aachen e.V., Institut f€ ur Technische und Makromolekulare Chemie der RWTH Aachen University, Pauwelsstr. 8, D-52056 Aachen, Germany and ‡Key Laboratory of Molecular Engineering of Polymers of Ministry of Education, Department of Macromolecular Science, Fudan University, Shanghai 200433, China Received September 22, 2009. Revised Manuscript Received November 11, 2009

A simple route for the design of hydrophilic microgels comprising inner hydrophobic nanodomains has been developed based on postmodification of microgels by complexation of wedge-shaped amphiphilic molecules with complementary functional groups. Aqueous microgels functionalized with imidazole groups were transferred into an organic medium, where imidazole groups were neutralized by water-insoluble wedge-shaped molecules bearing a sulfonic acid group at the tip of the wedge and a large hydrocarbon body. After redispersion of the modified microgel particles into the aqueous phase, wedge-shaped amphiphiles ionically attached to the polymer chains self-assembled into discrete nanodomains in the interior of the polymer colloids due to the hydrophobic attraction force. The loading of the wedge-shaped molecules into microgels can be controlled by variation of the amount of imidazole groups integrated into the microgel network as well as the neutralization degree. The experimental results suggested that incorporation of hydrophobic domains into hydrophilic colloids induced dramatic changes of their properties such as swelling degree, surface charge, and responsiveness toward temperature and pH. Finally, we demonstrated that internally hydrophobized microgel particles are very effective in uptake of hydrophobic molecules in aqueous media.

Introduction Over the past decades increasing attention has been paid to the synthesis and biomedical applications of aqueous polymer-based microgels. Colloidal microgels have numerous attractive properties such as defined morphology, high porosity, and adjustable dimensions that can respond to the changes in temperature, pH, and solvent quality and ability to act as carriers for drugs, biomolecules, synthetic polymers, or inorganic nanocrystals through fluid media. These properties allow them to be increasingly important for their potential applications in drug and gene delivery, catalysis, sensing, fabrication of photonic crystals, and separation and purification technologies.1-4 Microgels are versatile because they can be synthesized to a wide range of specifications and the applications can be further widened due to the numerous postmodification possibilities on the microgels. The aqueous microgel particles have been functionalized by different *Corresponding author. E-mail: [email protected].

(1) Pelton, R. Adv. Colloid Interface Sci. 2000, 85, 1–33. (2) Nayak, S.; Lyon, L. A. Angew. Chem., Int. Ed. 2005, 44, 7686–7708. (3) Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. Adv. Mater. 2006, 18, 1345–1360. (4) Das, M.; Zhang, H.; Kumacheva, E. Annu. Rev. Mater. Res. 2006, 36, 117– 142. (5) Pich, A.; Tessier, A.; Boyko, V.; Lu, Y.; Adler, H. J. P. Macromolecules 2006, 39, 7701–7707. (6) Pich, A.; Berger, S.; Ornatsky, O.; Baranov, V.; Winnik, M. A. Colloid Polym. Sci. 2009, 287, 269–275. (7) Pich, A.; Lu, Y.; Boyko, V.; Richter, S.; Arndt, K. F.; Adler, H. J. P. Polymer 2004, 45, 1079–1087. (8) Lu, Y.; Pich, A.; Adler, H. J. P.; Wang, G.; Rais, D.; Nespurek, S. Polymer 2008, 49, 5002–5012. (9) Bysell, H.; Malmsten, M. Langmuir 2009, 25, 522–528. (10) Johansson, C.; Hansson, P.; Malmsten, M. J. Phys. Chem. B 2009, 113, 6183–6193. (11) Scott, E. A.; Nichols, M. D.; Cordova, L. H.; George, B. J.; Jun, Y. S.; Elbert, D. L. Biomaterials 2008, 29, 4481–4493. (12) Gorelikov, I.; Field, L. M.; Kumacheva, E. J. Am. Chem. Soc. 2004, 126, 15938–15939.

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reactive groups,5,6 polymer chains,7,8 and proteins.9-11 They have also been loaded by different nanoscaled materials such as noble metals,12-15 metal oxides,16 and biominerals.17,18 Controlled uptake and release is a prolific area of microgel research. The possibility of using microgels for drug delivery is of particular interest.19,20 The key factors for the successful utilization of microgel particles in this field are their colloidal stability and chemical composition, which allow controlled loading of the guest molecules into the polymeric network. The next step toward the widened applications requires that the microgel particles can not only encapsulate the hydrophilic drugs but also bind hydrophobic or water-insoluble targets. Recently, many of the reported drug carriers for hydrophobic guests are micelles self-assembled by amphiphilic molecules as well as core-shell systems, which consist either of a hydrophobic dendritic core surrounded by a hydrophilic polymeric shell21,22 or (13) Zhang, J.; Wang, H.; Yan, X. J.; Wang, J.; Shi, J. W.; Yan, D. H. Adv. Mater. 2005, 17, 1191–1193. (14) Pich, A.; Karak, A.; Lu, Y.; Ghosh, A. K.; Adler, H. J. P. Macromol. Rapid Commun. 2006, 27, 344–350. (15) Pich, A.; Karak, A.; Lu, Y.; Ghosh, A. K.; Adler, H. J. P. J. Nanosci. Nanotechnol. 2006, 6, 3763–3769. (16) Agrawal, M.; Pich, A.; Gupta, S.; Zafeiropoulos, N. E.; Rubio-Retama, J.; Simon, F.; Stamm, M. J. Mater. Chem. 2008, 18, 2581–2586. (17) Schachschal, S.; Pich, A.; Adler, H. J. Langmuir 2008, 24, 5129–5134. (18) Wehnert, F.; Pich, A. Macromol. Rapid Commun. 2006, 27, 1865–1872. (19) Nolan, C. M.; Gelbaum, L. T.; Lyon, L. A. Biomacromolecules 2006, 7, 2918–2922. (20) Wu, J. Y.; Liu, S. Q.; Heng, P. W. S.; Yang, Y. Y. J. Controlled Release 2005, 102, 361–372. (21) Liu, M. J.; Kono, K.; Frechet, J. M. J. J. Controlled Release 2000, 65, 121– 131. (22) Xu, J.; Zubarev, E. R. Angew. Chem., Int. Ed. 2004, 43, 5491–5496. (23) Hedrick, J. L.; Trollsas, M.; Hawker, C. J.; Atthoff, B.; Claesson, H.; Heise, A.; Miller, R. D.; Mecerreyes, D.; Jerome, R.; Dubois, P. Macromolecules 1998, 31, 8691–8705. (24) Radowski, M. R.; Shukla, A.; von Berlepsch, H.; Bottcher, C.; Pickaert, G.; Rehage, H.; Haag, R. Angew. Chem., Int. Ed. 2007, 46, 1265–1269.

Published on Web 12/04/2009

DOI: 10.1021/la903588p

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Cheng et al. Table 1. Amounts of Reagents Used for the Microgel Synthesis sample

PVCL/AAEM/VIm5% PVCL/AAEM/VIm4% PVCL/AAEM/VIm3% PVCL/AAEM/VIm2% PVCL/AAEM/VIm1% a SC = solid content.

VCL [g]

AAEM [g]

VIm [g]

BIS [g]

AMPA [g]

water [g]

VIm [mol %]

SCa [%]

1.783 1.801 1.820 1.839 1.858

0.321 0.325 0.328 0.331 0.335

0.071 0.057 0.043 0.028 0.014

0.06 0.06 0.06 0.06 0.06

0.05 0.05 0.05 0.05 0.05

150 150 150 150 150

4.91 3.92 2.88 1.85 0.90

1.04 1.50 1.41 0.97 1.35

hydrophobic (cross-linked) star polymers coated with hydrophilic corona.23-26 However, a common limitation of these systems is their relatively poor water solubility due to the low hydrophilic content and in particular upon guest addition they are prone to form large multimolecular aggregates, whereby the guest is not encapsulated but adsorbed at the interface of the aggregates.23-26 The application of micelles designed from amphiphilic polymers for encapsulation and transport of hydrophobic molecules was studied. Linear block copolymers (methoxypoly(ethylene glycol)-b-poly(hexyl lactides)27 and poly(acrylic acid)-b-poly(DLlactide)28 as well as brushlike polymers (poly(2-hydroxyethyl methacrylate) backbone and poly(ε-caprolactone)-b-poly(ethylene glycol) side chains)29 were prepared and in form of well-defined micellar structures (hydrophobic core/hydrophilic corona) successfully used for the uptake and release of hydrophobic substances. The delivery vehicles based on polymeric micelles exhibited better stability and controlled release profile. Basically, aqueous hydrophilic nano- or microgels containing inner hydrophobic domains could be considered as interesting candidates for loading and transport of hydrophobic molecules. However, it is difficult to incorporate hydrophobic groups during the microgel synthesis and distribute them in form of well-defined hydrophobic nanopockets in the colloidal polymer network. In this work we report a simple postmodification approach to prepare the aqueous microgel particles with integrated hydrophobic nanodomains in their interior. The poly(N-vinylcaprolactam-co-acetoacetylethyl methacrylate) (PVCL/AAEM)-based microgel system functionalized with vinylimidazole (VIm) groups (PVCL/AAEM/VIm)5 was neutralized by water-insoluble wedgeshaped amphiphilic molecules that bear a sulfonic acid group at the tip of the wedge and a large nonpolar body30 and can form stable acid-base complexes with polybases in the organic media.31 In comparison to covalent bonding, the acid-base interaction is highly specific, so almost no side reaction can be expected. It has also been shown that depending on the degree of neutralization, the complexes of wedge-shaped sulfonic acid molecules with polyvinylpyridine can self-assemble into different superstructures in bulk as well as in organic solvents. Meanwhile, the sulfonic acid group in the structure of wedgeshaped molecules guarantees a strong specific interaction with the basic groups (vinylimidazole) in the microgel particles; the large nonpolar hydrocarbon body renders the molecules insolubility in water. We apply wedge-shaped molecules in present study due to (25) Xu, S. J.; Luo, Y.; Haag, R. Macromol. Rapid Commun. 2008, 29, 171–174. (26) Yang, Z.; Liu, J. H.; Huang, Z. P.; Shi, W. F. Eur. Polym. J. 2007, 43, 2298– 2307. (27) Trimaille, T.; Mondon, K.; Gurny, R.; M€oller, M. Int. J. Pharm. 2006, 319, 147–154. (28) Xue, Y.-N.; Huang, Z.-Z.; Zhang, J.-T.; Liu, M.; Zhang, M.; Huang, S.-W.; Zhuo, R.-X. Polymer 2009, 50, 3706–3713. (29) Du, J.-Z.; Tang, L.-Y.; Song, W.-J.; Shi, Y.; Wang, J. Biomacromolecules 2009, 10, 2169–2174. (30) Zhu, X. M.; Tartsch, B.; Beginn, U.; Moller, M. Chem.;Eur. J. 2004, 10, 3871–3878. (31) Zhu, X. M.; Beginn, U.; Moller, M.; Gearba, R. I.; Anokhin, D. V.; Ivanov, D. A. J. Am. Chem. Soc. 2006, 128, 16928–16937.

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their strong hydrophobicity, accessible functionality, and ability to form supramolecular architectures. However, other functional molecules bearing sulfonic acid group and hydrophobic part without wedge-shape architecture can be applied. The most important criteria in this case are water insolubility and strong interaction energy. In this way the structure of self-assembled hydrophobic nanodomains can be varied, and finally the solubilization ability and capacity for the uptake of hydrophobic targets can be controlled. In our previous paper we showed that PVCL/AAEM/VIm microgels could be reversibly transferred from water solution to tetrahydrofuran (THF) by a simple solvent exchange procedure.32 Therefore, their modification via neutralization by wedge-shaped sulfonic acid molecules can take place in the THF solution. Now questions arise whether the resulting composite microgels can be redispersed in water after modification procedure and whether wedge-shaped sulfonic acid molecules can aggregate in microgel network forming discrete hydrophobic domains. If this is possible, then finally we end up with a porous hydrophilic colloid with numerous integrated hydrophobic “micelles” or “nanodomains”. Therefore, such hydrophobic sites can be easily accessed by hydrophobic guest molecules. This approach offers several advantages compared to micellar systems especially for the removal of harmful hydrophobic molecules from aqueous phase: (a) enhanced stability (due to the fact that active scavenging domains are locked in the polymer network of microgel); (b) simple recovery from aqueous phase (due to the large size of microgels with trapped guest molecules can be separated by centrifugation or filtration procedures); (c) the microgel concept allows incorporation of other active ingredients that will help to destroy or deactivate trapped guest molecules such as hormones or β-blocker.

Experimental Section Materials. The monomers N-vinylcaprolactam (VCL) (98%, Aldrich), acetoacetoxyethyl methacrylate (AAEM) (95%, Aldrich), and vinylimidazole (VIm) (g99%, Aldrich) were purified by distillation under vacuum. Radical initiator 2,20 -azobis(2methylpropyonamidine) dihydrochloride (AMPA) (97%, Aldrich) and cross-linker N,N0 -methylenebis(acrylamide) (BIS) (99%, Aldrich) have been used as received. The synthesis of sodium 4-N-[30 ,40 ,50 -tris(dodecyloxy)benzamido]benzene-4-sulfonic acid (TDBBSA) was described elsewhere30 tetrahydrofuran (THF) (pro analysis, Aldrich), acetone (analytical, Aldrich), and fluorescence dye Nile Red (Aldrich) were used as received. Synthesis of PVCL/AAEM/VIm Microgels. The synthesis of PVCL/AAEM/VIm microgels was carried out using a literature procedure.5 Appropriate amounts (Table 1) of VCL, AAEM, VIm, and BIS were dissolved in deionizer water. A double-wall glass reactor equipped with a stirrer and a reflux condenser was purged with nitrogen. Solution of the monomers was placed into the reactor and stirred for 1 h at 70 °C under continuous purging (32) Shen, L.; Pich, A.; Fava, D.; Wang, M. F.; Kumar, S.; Wu, C.; Scholes, G. D.; Winnik, M. A. J. Mater. Chem. 2008, 18, 763–770.

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Article Scheme 1. Incorporation of Wedge-Shaped Sulfonic Acid Molecules into Microgelsa

a (a) Preparation of complexes of wedge-shaped sulfonic acid molecules (TDBBSA) and PVCL/AAEM/VIm microgels by the reversible transfer from water to THF: (1) transfer of the aqueous microgel to THF by solvent exchange; (2) addition of TDBBSA molecules to the microgel suspension in THF and diffusion of wedge-shaped molecules into porous polymer colloids; (3) fixation of TDBBSA within microgels by complexation; (4) transfer of the modified microgel to the aqueous phase by solvent exchange. (b) Fixation of TDBBSA molecules to polymer chains of microgel network by neutralization between imidazole units and sulfonic acid units followed by self-assembly of nonpolar fragments of wedge-shaped molecules and formation of hydrophobic domains.

with nitrogen. Afterward, 5 mL of aqueous solution of initiator was added under continuous stirring. Reaction was carried out for another 8 h. The amounts of ingredients used for polymerization process are summarized in Table 1.

Incorporation of Wedge-Shaped Sulfonic Acid Molecules into Microgels. PVCL/AAEM/VIm microgel particles with different amounts of VIm (1-5 mol %) were separated from water by centrifugation (10 000 rpm, 10 min) and redispersed in THF. The microgel solutions in THF containing 0.12 mmol of imidazole units were mixed with 5 mL of THF solutions containing 20 or 2 mg/mL TDBBSA to prepare the complexes with fully neutralized (100%) or partly neutralized (10%) imidazole units, respectively. The mixtures were stirred overnight at room temperature to ensure the complete incorporation of the wedge-shape molecules into the microgels. Subsequently, the modified microgels were centrifuged to remove THF and redispersed in water. The complexes are denoted as PVCL/AAEM/VImx%/(TDBBSA)DN, where x% is the content of VIm in the microgel and DN is the degree of neutralization. Uptake of Nile Red by Microgels. Different amount of a stock solution of Nile Red (0.97 mg/mL in THF/acetone 1/2) were added to 3 mL of each microgel solutions PVCL/AAEM/ VIm5%/(TDBBSA)0, PVCL/AAEM/VIm5%/(TDBBSA)0.1, and PVCL/AAEM/VIm5%/(TDBBSA)1.0 of a given concentration of 0.2 mg/mL to evaluate the dye uptake capacity of the modified microgels. A control experiment with pure water was also carried out. The solutions were equilibrated in the darkness for 48 h; during this time THF and acetone evaporated. The Nile Red absorbance experiments in different temperature were conducted Langmuir 2010, 26(7), 4709–4716

to PVCL/AAEM/VIm5%/(TDBBSA)1.0 microgel which was fully loaded with Nile Red; the dye concentration was 3.2 μg/mL, and concentration of microgel was 0.1 mg/mL. Characterization Methods. Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet 710 FT-IR spectrometer using the technique of attenuated total reflectance (ATR) from a thin film of microgels on a germanium crystal. Hydrodynamic diameter and zeta potential of the microgel particles have been measured with a Zetasizer Nano Series, Malvern Instruments (at a scattering angle of 90°). The mean hydrodynamic diameter and its distribution were determined using Cumulant fit method. pH value of the solution was adjusted by addition of 0.01 M NaOH or 0.01 M HCl. TEM images were taken using a Hitachi S-4800 field emission scanning electron microscopy (FESEM) instrument in the TE mode operating at 30 kV and 10 μA current. UV-vis spectra of the Nile Red incubated solutions were recorded on a JASCO V-630 spectrophotometer from 400 to 800 nm and corrected with the measurements of the corresponding control solutions prepared without Nile Red (bandwidth 1.5 nm, scan speed 1000 nm/min).

Results and Discussion Incorporation of Wedge-Shaped Sulfonic Acid Molecules into Microgels. The aqueous microgel particles used in the present study are based on a copolymer of vinylcaprolactam (VCL) and acetoacetoxyethyl methacrylate (AAEM). As described in our previous studies, the microgel particles possess a heterogeneous DOI: 10.1021/la903588p

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Figure 1. Hydrodynamic diameter distribution of microgel samples at different stages of the modification process obtained from dynamic light scattering at 20 °C: (a) PVCL/AAEM/VIm5%/(TDBBSA)1.0 and (b) PVCL/AAEM/VIm5%/(TDBBSA)0.1.

structure and consist of an AAEM-rich core and a VCL-rich shell as a result of faster consumption of more reactive methacrylic monomer during precipitation copolymerization process.33 The addition of a small fraction of vinylimidazole (VIm) during PVCL/AAEM microgel synthesis allows the selective incorporation of VIm units into the swollen VCL corona. It is difficult to monitor directly the distribution of functional groups in crosslinked colloidal networks by using conventional characterization methods. At the moment we are trying to apply NMR techniques to follow the segment dynamics to evaluate the distribution of monomer units in the microgel particles. The PVCL/AAEM/VIm microgels are characterized by a narrow particle size distribution and superior colloidal stability.5 The chemical structure of the wedge-shaped sulfonic acid molecule used as the ligand to complex the microgel, namely 4-N-[30 ,40 ,50 -tris(dodecyloxy)benzamido]benzene-4-sulfonic acid (TDBBSA), is shown in Scheme 1b. It is a crystalline powder, which is not soluble in water. The general strategy for the incorporation of TDBBSA into microgel is presented in Scheme 1. In the first step, microgels synthesized in water were transferred into THF by a centrifugation/redispersion procedure. Then, a THF solution of TDBBSA was added to the microgel suspension in THF. Because of the acid-base interaction between the sulfonic acid and imidazole groups, the wedge-shaped amphiphilic molecules became entrapped in the microgel network, and the internally hydrophobized microgels PVCL/AAEM/VImx%/(TDBBSA)DN were obtained. It should be noted that even the microgels in which the imidazole units are fully neutralized with TDBBSA can still be redispersed in water. Dynamic light scattering technique was employed to monitor the effects of the solvent exchange and modification with wedgeshaped molecules on the microgel size and size distribution. Figure 1 shows hydrodynamic diameter distribution curves of the microgel samples which contain 5 mol % VIm units modified by wedge-shaped molecules at different degree of neutralization (DN = 0.1 and 1.0 for PVCL/AAEM/VIm5%/(TDBBSA)0.1 and (33) Boyko, V.; Pich, A.; Lu, Y.; Richter, S.; Arndt, K. F.; Adler, H. J. P. Polymer 2003, 44, 7821–7827.

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PVCL/AAEM/VIm5%/(TDBBSA)1.0, respectively). The four sets of measurements correspond to the four states of the modification process as shown in Scheme 1. The results showed in Figure 1 indicate that the microgel particles shrank after transfer to THF due to the worsening of the solvent quality. As far as wedgeshaped TDBBSA molecules were incorporated, the microgel size increased and the size distribution became broader in the THF solution. This might be caused by the extra charge induced by partial neutralization. As the DN increased further, the microgel size, however, decreased, but the fully neutralized microgel particles were still larger than the nonmodified ones. This should be the result of the steric repulsion of the bulky hydrocarbon part of the wedge-shaped molecules, leading to the polymer chain extension.34 After redispersion into the aqueous phase, the modified microgel particles shrank again but showed a good colloidal stability and narrow size distribution. Note that in water the sample, in which the VIm units were fully neutralized (PVCL/ AAEM/VIm5%/(TDBBSA)1.0), exhibited much smaller size compared to the original particles, while the final size of 10% neutralized sample PVCL/AAEM/VIm5%/(TDBBSA)0.1 is similar to that of the original microgel sample. The results presented in Figure 1 also confirm that the microgel particles preserve their colloidal stability during all steps of the modification process. We note here that the proposed approach allows a precise control of the microgel modification degree simply by the adjustment of the DN. Since FT-IR spectroscopy is a well-established and suitable tool to analyze the proton-transfer processes,35 this method was applied to prove the incorporation of wedge-shaped molecules in the microgels. Figure 2 presents the FT-IR spectra of the modified microgel PVCL/AAEM/VIm5%/TDBBSA1.0 and PVCL/VIm5%/ TDBBSA0.1 together with that of the pure compound TDBBSA and the original microgel PVCL/AAEM/VIm5%/TDBBSA0. The two peaks at 1008 and 1035 cm-1 appeared in the modified samples, which have been attributed to the stretching mode of (34) Frederickson, G. H. Macromolecules 1993, 26, 2825–2831. (35) Ikkala, O.; Ruokolainen, J.; Tenbrinke, G.; Torkkeli, M.; Serimaa, R. Macromolecules 1995, 28, 7088–7094. (36) Martinot, L.; Leroy, D.; Jerome, C.; Leruth, O. J. Radioanal. Nucl. Chem. 1997, 224, 71–76.

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Figure 2. FTIR spectra recorded in the (a) 850-1200 cm-1 and (b) 1700-1350 cm-1 regions for PVCL/AAEM/VIm5%/(TDBBSA)DN (DN=0, 0.1, and 1.0) microgels and TDBBSA.

Figure 3. Particle diameter of complex microgels PVCL/AAEM/ VImx%/(TDBBSA)DN (DN = 0, 0.1, and 1.0) as a function of VIm content measured by dynamic scattering at 20 °C.

sulfonate groups.31 According to the literature,36 evidence for the complexation reaction on the imidazole group is given by a shift of ring-mode vibrations for original microgel at 1480-1525 cm-1 for the complex that is clearly demonstrated in Figure 2b. On the other hand, the appearance of a new peak at 1590 cm-1 in the complexed microgel also indicates the formation of imidazolium ions.37 From FT-IR data presented in Figure 2 we conclude that TDBBSA molecules are fixed in the microgel network via acid-base interaction. Properties of the Microgels Complexed with WedgeShaped Sulfonic Acid Molecules. To explore the properties of the modified microgels, we carried out light scattering measurements for different microgel samples in water. Figure 3 compares the hydrodynamic diameter of the original microgel with that of the modified microgels as a function of VIm content. As shown in Figure 3, the size of microgels increases, if larger VIm amounts are introduced into the microgel structure. This is because the incorporation of VIm units increases the hydrophilicity of the particles leading to better solvation by water molecules and higher swelling in continuous medium.5 The microgels modified with the wedge-shaped sulfonic acid molecules, however, behave differently. The dependence of the microgel size on the VIm content at different DN exhibits a maximum at 3 mol % (37) Yamada, M.; Honma, I. Electrochim. Acta 2003, 48, 2411–2415.

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VIm. If the VIm content is lower than 3 mol % microgel size increases after modification with wedge-shaped molecules. The lower degree of neutralization results in larger swelling of microgels. Contrarily, if VIm content in microgels is above 3 mol %, the modification of the microgels leads to the particle shrinkage (at 100% neutralization) or just minimal swelling (at 10% neutralization). Moreover, the particle size of both 100% and 10% neutralized microgels decrease substantially while the VIm content increase. These results suggest that the degree of modification controlled by varying the VIm content in the microgel network and the DN value have a strong influence on the aggregation of the hydrophobic wedges of TDBBSA molecules within the hydrophilic colloids. When the concentration of TDBBSA molecules incorporated into the microgels is large enough, the hydrophobic wedges can self-assemble into nanodomains which restrict the swelling of the microgels. When the degree of modification is low, the hydrophobic interaction between the wedge-shaped molecules in this case is not strong enough to overcome the dehydration as well as the entropy loss due to polymer chain constriction in the microgels. At low DN, the microgel size is even bigger than that of the nonmodified ones. This is possibly due to the presence of noncompensated electrical charge. It has already been reported that, similar to most VCL-based microgels, the VCL/AAEM/VIm microgels are thermosensitive with a reversible transition taking place at ∼33 °C; i.e., the particles shrink drastically at this temperature.5 It is believed that the volume-phase transition in such kind of microgels occurs as a result of destruction of hydrogen bonds and hydrophobic interactions between the polymer chains. The situation, however, changed when the microgels were modified by the wedge-shaped sulfonic acid molecules. Figure 4 shows the variation of the average hydrodynamic diameter with temperature for the original and modified microgel samples. We found that after neutralizing 10% imidazole units with the wedge-shaped molecules the microgels shrank slightly with temperature, and no sharp transition was observed. By full neutralization the microgels lost completely their thermal sensitivity. This indicates that no shrinkage of the microgel took place in the whole temperature range. We assume that the complexes could have a rigid interior network between the wedge-shape molecules which restricts the movement of polymer chains and therefore hinders the response of microgel to temperature. The presence of bulky hydrophobic groups in the DOI: 10.1021/la903588p

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Figure 4. Hydrodynamic diameter of the microgel samples: (a) PVCL/AAEM/VIm5%/(TDBBSA)DN (DN=0, 0.1, and 1.0); (b) PVCL/ AAEM/VIm1%/(TDBBSA)DN (DN=0, 0.1, and 1.0) as a function of temperature.

Figure 5. Hydrodynamic diameter of microgel samples PVCL/ AAEM/VIm5%/(TDBBSA)DN (DN=0, 0.1, and 1.0) as a function of pH (T=20 °C).

Figure 6. Electrophoretic mobility of microgel samples PVCL/ AAEM/VIm5%/(TDBBSA)DN (DN=0, 0.1, and 1.0) as a function of pH.

interior of the microgel particles obviously altered the hydrophobic-hydrophilic balance of the system. Because of the presence of basic imidazole units, the pH value of the aqueous phase can also influence the dimension of the PVCL/AAEM/VIm microgels.5 The pH dependency of the hydrodynamic diameter of the original and modified microgels is presented in Figure 5. The original microgel exhibits the maximum size at pH = 4 that corresponds the maximum ionization pH of VIm units. At this pH, the microgel particles swell due to the strong electrostatic repulsion between the charged VIm groups. On the contrary, the modified microgels lost the pH sensitivity and showed almost constant particle size in the pH range of 3-9. In order to find the reason for this phenomenon, we measured the electrophoretic mobility at different pH values for the original and modified microgels (Figure 6). The experimental data for original microgel indicated that the VIm functionalized microgels were positively charged in the acidic region caused by the ionization of VIm units. The increase of pH value led to the gradual reduction of the particle charge. Interestingly, microgels modified with wedge-shaped molecules showed totally different behavior. All modified microgel samples, even those with pretty low DN (10%), exhibit strong negative surface charge in the whole pH range. It might be the result of the delocalization of the positive charge, the protons from the sulfonic acid group, within the imidazole groups in the microgel due to their high proton conductivity in the presence of water,

leading to noncompensated negative charge. The strong hydrophobic interior created by the wedge-shaped molecules may also retard the further protonation of the imidazole groups at low pH. This assumption may also explain why most modified microgels had larger particle sizes than the corresponding original microgels. The morphology of microgels has been studied by transmission electron microscopy (TEM). Figure 7 presents TEM images of microgel particles with or without modification by the wedgeshaped molecules. One can see that the original microgel particles are shrunken after water removal, while the modified microgels showed much smaller degree of shrinkage, comparing to their sizes in water. This confirms the results presented in Figures 4 and 5. In both images of the modified microgel samples showed in Figure 7, the aggregates of wedge-shaped molecules could be clearly observed as small dark dots which are located in the corona region of the microgels. That is because the VIm units are distributed mostly in the VCL-rich corona of the microgels. Uptake of Hydrophobic Dye Nile Red. Porous hydrophilic particles with hydrophobic interior can be very useful as scavengers for hydrophobic organic molecules in aqueous phase. To evaluate the scavenging properties of modified microgels, we selected a hydrophobic dye Nile Red. This dye is not soluble in water by itself but can be sequestered inside the hydrophobic pockets generated in microgels. Nile Red does not fluoresce in

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Figure 7. TEM images of (a) PVCL/AAEM/VIm5%/(TDBBSA)0, (b) PVCL/AAEM/VIm5%/(TDBBSA)0.1, and (c) PVCL/AAEM/ VIm5%/(TDBBSA)1.0 microgels.

Figure 8. (a) Absorbance spectra of the Nile Red loaded microgel solutions: (1) pure water, (2) PVCL/AAEM/VIm5%/(TDBBSA)0, (3) PVCL/AAEM/VIm5%/(TDBBSA)0.1, (4) PVCL/AAEM/VIm5%/(TDBBSA)1.0 (inset shows photograph of the solutions, Cmicrogel=0.2 mg/ mL and CNile Red=6.4 μg/mL). (b) Peak intensity of absorbance spectra at λmax=563 nm plotted vs dye concentration in the system.

Figure 9. (a) UV-vis spectra of the Nile Red loaded into PVCL/AAEM/VIm5%/TDBBSA1.0 microgel at different temperature (Cmicrogel= 0.1 mg/mL; CNile Red=3.2 μg/mL). (b) Temperature dependencies of peak position and maximum intensity of absorbance spectra.

water, but once it is encapsulated inside discrete hydrophobic domains, its aqueous solution starts to fluoresce. Nile Red has been used as fluorescence probe for investigating micelles,38,39 DNA,40 and lipid droplets.41 In our experiment Nile Red solutions in THF/acetone/water mixtures were added to the original microgel samples and microgels modified with wedge-shaped molecules, and the mixtures (38) Ghosh, S.; Irvin, K.; Thayumanavan, S. Langmuir 2007, 23, 7916–7919. (39) Che, G.; Guan, Z. J. Am. Chem. Soc. 2004, 126, 2662–2663. (40) Tainaka, K.; Fujiwara, Y.; Okamoto, A. Nucleic Acids Symp. Ser. 49, 155-156. (41) Greenspan, P.; Mayer, E. P.; Fowler, S. D. J. Cell Biol. 1985, 100, 965–973.

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were slowly agitated overnight. There is no swelling of microgels after addition of small amount of THF/acetone to microgel dispersion according to light scattering data. Also, we did not detect any particle aggregation. We performed a control experiment to clarify the solubility of wedge-shaped molecules in THF/ acetone/water mixture used for the incorporation of Nile Red. Wedge-shaped molecules are not soluble in such environment, so the possibility of their leakage from the microgel during Nile Red loading can be excluded. The modified microgels turned quickly reddish as a result of the diffusion of the Nile Red into the microgel particles, whereas nonmodified microgel and pure water remained colorless DOI: 10.1021/la903588p

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(see inset in Figure 8). This indicates clearly that wedge-shaped molecules form hydrophobic pockets in the porous hydrophilic microgels. After dye loading microgels were colloidally stable within at least 3 months. In Figure 8a the recorded UV-vis spectra for different samples are shown. The Nile Red absorbance in the visible region was much higher for 100% neutralized microgel than the 10% neutralized one, proving that higher amounts of the hydrophobic Nile Red molecules were incorporated in the 100% modified microgel. Figure 8a indicates that in the case of original microgel no adsorption of Nile Red takes place. These results lead to a conclusion that the modified microgels do have the potential to incorporate and encapsulate some hydrophobic targets. By varying dye concentration in the aqueous microgel solutions, we attempted to quantify the efficiency of the microgels in the uptake of hydrophobic dye. The results presented in Figure 8b suggest that for both microgel samples the saturation value can be reached at certain dye concentration. The increase of DN allows larger amounts of Nile Red to be incorporated into hydrophobic domains within microgels (note that microgel concentration was kept constant). From experimental results presented in Figure 8 we calculated the molar ratios of dye molecules to wedge-shaped molecules at saturation: Nile Red/TDBBSA = 0.29 at DN = 1.0 and Nile Red/TDBBSA = 0.75 at DN = 0.1. The microgel sample loaded with Nile Red was heated from 20 to 60 °C, and optical properties were detected by UV-vis spectroscopy (Figure 9a). We notice two effects, namely the blue shift of the peak maximum and increase of the peak intensity with temperature. Figure 9b presents the variation of the peak position and peak intensity with temperature derived from the spectra in Figure 9a. The position of the peak maximum undergoes blue shift as temperature increases to 40 °C and then remains constant. The intensity of the peak maximum shows opposite behavior; namely, it has constant value up to 40 °C and then rapid increases. In our opinion the effects presented in Figure 9 reflect the changes of the microgel internal structure upon heating. As shown in Figure 4a, the nonmodified microgel sample is almost in collapsed state if temperature increases above 40 °C. The modified microgel sample shows no temperature sensitivity, but the internal structure seems to undergo some changes. The shift of the peak maximum to shorter wavelength in Figure 9 indicates that interior of the microgel becomes more hydrophobic as temperature increases due to the destruction of hydrogen bonds. The increase of the peak intensity can be attributed to reorientation of dye molecules in microgel structure due to the formation of additional hydro(42) Jiang, X.; Zhao, B. Macromolecules 2008, 41, 9366–9375.

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phobic areas. Similar effects (shift of the wavelength and increase of the intensity) were reported by Zhao and co-workers,42 who studied micellization and dissociation of poly(ethylene oxide)-bpoly(methoxydi(ethylene glycol)methacrylate-co-methacrylic acid) in aqueous solution by using Nile Red. The experimental results presented in Figure 9 indicate that dye molecules loaded into microgel particles can serve as microenvironmental probes and deliver useful information about the internal structure of the polymer colloid. At the present moment the interaction between aggregates of wedge-shaped molecules and Nile Red is indirectly evidenced by the fluorescence measured after dye loading into microgel. We plan to investigate in more details the structure and morphology of hydrophobic cages from wedge-shaped molecules in microgel structure. This will help to understand how the guest molecules are oriented in hydrophobic domains and what the molecular interaction forces are between guest molecules and hydrophobic tails of amphiphatic molecules.

Conclusions The incorporation of water-insoluble wedge-shaped sulfonic acid molecules into the poly(N-vinylcaprolactam-co- acetoacetylethyl methacrylate-co-vinylimidazole) (PVCL/AAEM/VIm) microgels via acid-base interaction between imidazole units and sulfonic acid groups results the formation of porous hydrophilic colloids with hydrophobic domains localized in their interior. The formation of complexes between imidazole units and sulfonic acid groups was confirmed by FT-IR spectroscopy. Microscopy investigations confirm presence of nanodomains of wedge-shaped sulfonic acid molecules in the microgel corona. Our experimental results demonstrate that the modified microgels could retain their colloidal stability in water, and the presence of hydrophobic wedge-shaped molecules distinctly influences both the particle size and the environmental sensitivity of the microgel. The ability of the modified microgels to encapsulate hydrophobic molecules in water was confirmed by the uptake experiment of a hydrophobic dye monitored by UV spectroscopy. It indicates that the internally hydrophobized microgels can act as scavengers for hydrophobic molecules in aqueous media. The dye-loaded microgel particles showed a thermochromism that allows them to be used as biosensor. Acknowledgment. Authors thank Deutsche Forschungsgemeinschaft (DFG), VolkswagenStiftung, and SeedFund Program of RWTH Aachen University for financial support of this research. X.-M.Z. thanks the Alexander von Humboldt foundation and Senior Visiting Scholar Foundation of Key Laboratory in Fudan University for the financial support.

Langmuir 2010, 26(7), 4709–4716