Photoresponsive Nanogels Formed by the Self-Assembly of

Jun 29, 2004 - Kyoto University, Yoshida-Hommachi, Sakyo-ku, Kyoto 606-8501, Japan, ... Bioengineering, Tokyo Medical and Dental University, 2-3-10 ...
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Biomacromolecules 2004, 5, 1804-1809

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Photoresponsive Nanogels Formed by the Self-Assembly of Spiropyrane-Bearing Pullulan That Act as Artificial Molecular Chaperones Tai Hirakura,† Yuta Nomura,† Yasuhiro Aoyama,† and Kazunari Akiyoshi*,‡,§ Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Hommachi, Sakyo-ku, Kyoto 606-8501, Japan, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kannda-Surugadai, Chiyoda-ku, Tokyo, 101-0062, Japan, and Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University, Japan Received March 9, 2004; Revised Manuscript Received April 27, 2004

Novel photoresponsive nanogels were prepared by the self-assembly of spiropyrane-bearing pullulan (SpP). The solution properties of the nanogels could be controlled by photostimulation via isomerization between hydrophobic spiropyrane and hydrophilic merocyanine. The molecular chaperone-like activity of the nanogels in protein refolding was investigated. The activity of citrate synthase significantly increased when the amphiphilicity of SpP nanogels was switched by photostimulation. 1. Introduction Stimulus-sensitive hydrogels are widely used as functional smart materials in many biotechnological and biomedical applications.1,2 Due to recent advances in nanotechnology, nanosize hydrogels (nanogels), which are composed of submicrometer-scale gel particles, have attracted growing interest. Nanogels are expected to show unique properties, such as the ability to trap biomolecules inside the gel, rapidly respond to an external stimulus, and form hierarchical macrogels in a nanogel network.3 There has also been interest in applying nanogels to drug delivery systems, such as protein delivery and gene delivery.4,5 Nanogels have been prepared by the usual synthetic methods via microemulsion6 or precipitation7 polymerization and by the intramolecular cross-linking of single chains of macromolecules.8,9 We previously reported novel self-assembly methods for preparing physically cross-linked nanogels by the controlled association of hydrophobically modified polymers in water.10-12 For example, hydrophobized polysaccharides such as cholesteryl-group-bearing pullulan (CHP) form nanoparticles in dilute aqueous solution by intermolecular selfassembly. The nanoparticles were considered to be nanogels in which the associations of hydrophobic groups provided cross-linking points. At a higher concentration, they formed a macrogel.12 The amphiphilic nanogels of CHP complexed various soluble proteins or enzymes in water and behaved as a host for a guest protein.13,14 They can be used as drugcarrier systems in medicine15 and as artificial molecular * To whom correspondence should be addressed. Fax: 81-3-5280-8027, Tel: 81-3-5280-8020,E-mail: [email protected]. † Kyoto University. ‡ Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University. § Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University.

chaperones in biotechnology.16,17 We can develop tailor-made functional nanogels to create novel nanobiomaterials (nanogel engineering) by the self-assembly of functional associating polymers as building blocks. In this study, spiropyrane groups were introduced to pullulan as assembling units instead of cholesteryl groups in CHP nanogels. Spiropyrane molecules, which isomerize from hydrophobic spiropyrane (Sp) to hydrophilic merocyanine (Mer) under photo- and thermostimulation, are widely used to control the structure and function of biomaterials by light.18-20 Photocontrol of the conformation and association of various polymers using spiropyrane-bearing polymers has been reported.21-24 We report here the synthesis and characterization of spiropyrane-bearing pullulan (SpP) and its application as an artificial molecular chaperone. The molecular chaperone-like activity of the nanogels in protein refolding was investigated by using citrate synthase as a model protein. High refolding yields for guanidinium chloride (GdmCl)-denatured enzyme were achieved in the dilution method when the amphiphilicity of SpP nanogels was switched by photostimulation. To the best of our knowledge, this is the first report of the preparation of a photoresponsive nanogel by self-assembly and its ability to control the refolding of protein under photostimulation as an artificial molecular chaperone. 2. Experimental Section 2.1. Materials. Pullulan was purchased from Hayashibara Biochemical Laboratory, Inc., Okayama, Japan. The average molecular weight (Mw) of pullulan was 1.08 × 105. Citrate synthase (CS) was purchased from Roche Diagnostics Corp., Indianapolis, IN. Water was purified by a Milli-Q purification system. Commercially available organic chemicals were used without further purification. UV and visible light irradiation

10.1021/bm049860o CCC: $27.50 © 2004 American Chemical Society Published on Web 06/29/2004

Photoresponsive Nanogels

were performed using an 8 W UV lamp (254 nm) (UVP, 8 W hand-held model UVM-18) and an 8 W white light lamp (UVP, 8 W hand-held model UVM-18), respectively 2.2. Synthesis of Spiropyrane-Bearing Pullulans (SpP). The spiropyrane compound 1-(β-carboxyethyl)-3′,3′-dimethyl-6-nitrospiro (iodorine-2′,2′ [2H-1] benzopyran) (SpCOOH; 585 mg, 1.54 mmol) was reacted with dicyclohexyl carbodiimide (DCC; 318 mg, 1.54 mmol) in DMSO (10 mL) in the presence of 4-(N,N-dimethylamino) pyridine (DMAP; 30 mg, 0.25 mmol) under a N2 atmosphere at room temperature. The resulting spiropyrane anhydride was reacted with pullulan (Mw)108 000; 1.0 g, 6.2 mmol (glucose units)) in DMSO (40 mL) at 40 °C for 2 days. The product was then precipitated with acetone. The precipitate was washed with acetone and dried in a vacuum. The resulting powder was dissolved in DMSO and dialyzed with distilled water. After lyphilization, wine-red cotton-like powder was obtained (85%). The product was identified based on its IR and 1H NMR spectra. SpP IR (KBr): 1740 cm-1 (ester CdO). 1H NMR (DMSO d6/D2O ) 10/1(v/v)) δ 4.75 (s, 33H (per 100 glucose units), pullulan C1H (1-6)), 5.05 (d, 66H (per 100 glucose units), pullulan C1H (1-4)), 6.00-7.20 (m, 6H, olefinic H and aromatic H of indoline), 8.20-8.18 (m, 3H, aromatic H of nitrobenzene). Pullulan derivatives containing 1.4, 2.8, and 6.8 spiropyrane groups per 100 glucose units were obtained. SpP with 1.4 spiropyrane groups was used in this study due to its high solubility in water. 2.3. Size-Exclusion Chromatography (SEC)-Multiangle Laser Light Scattering (MALS). SEC was carried out on a chromatography system (Tosoh Co., Ltd., Tokyo, Japan) composed of a CCPD dual pump, a CO-8010 column oven, and a RI-8010 refractive index detector on a SWXL guard column (Tosoh Co., Ltd., Tokyo, Japan) connected to a MALS detector (DAWN DSP, Wyatt Technology, Santa Barbara, CA). An aqueous solution of 100 mM HEPES buffer (pH 7.5) containing 100 mM NaCl was used as the mobile phase. The molecular weight and the z-average rootmean-square radius of gyration were determined using ASTRA software on the basis of Zimm’s equation. The increase in the refractive index (dn/dc) was determined by Optilab DSP (Wyatt Technology, Santa Barbara, CA). The value of dn/dc for SpP was 0.151. The sample solutions were passed through a 0.45 µm filter (Ekicrodisk 3, Gelman Science Japan, Ltd., Tokyo, Japan) before being applied to the column. 2.4. Dynamic Light Scattering (DLS) Measurements. Light scattering measurements were performed on a DLS700 (Otsuka Electronics Co., Ltd., Osaka Japan) to determine the size of SpP nanogels. Scattering at 90° was examined to yield a profile that was related to the average intensity of light scattered as a function of time. The sample solutions were passed through a 0.45 µm filter (Ekicrodisk 3, Gelman Science Japan, Ltd., Tokyo, Japan) before the measurements. The concentration of the sample was kept constant at 1.0 mg/mL. The measured autocorrelation function was analyzed by a cumulant method. The hydrodynamic radius of nanogels was calculated by the Stokes-Einstein equation. 2.5. Surface Tension Analysis of SpP. Surface tension was measured using the Wilhelmy method with a platinum

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Figure 1. Structure of spiropyrane-bearing pullulan (SpP).

plate (DCA-100 ORIENTEC Saitama, Japan) at 25.7 °C. Mer-Sp-form SpP nanogels sample solution (1.0 mg/ml) was prepared by heating at 50 °C in 100 mM HEPES (pH 7.5) containing 100 mM NaCl and stirring for 60 min under dark conditions. After the sample was dissolved in the buffer, the SpP solution was cooled to room temperature. Each SpP nanogels sample solution was prepared by exposure to visible light for 30 min or UV light for 60 min. 2.6. Denaturation and Refolding of CS. CS was dissolved in 6.0 M GdmCl, 0.75 mM EDTA, 40 mM DTT, and 150 mM Tris-HCl buffer (pH 7.6) to give a final concentration of 1.0 mg/mL. After denaturation for 1 h at 25 °C, the mixture was diluted rapidly 50-fold with 0.75 mM EDTA, 150 mM Tris-HCl buffer (pH 7.6) containing SpP nanogels (1.0 mg/mL); the mixture was then allowed to stand for 1 h at 25 °C. The final concentration of CS was 0.02 mg/mL in 150 mM Tris-HCl buffer (pH 7.6) containing 0.12 mM GdmCl, 0.8 mM DTT, and 0.75 mM EDTA. The enzyme activity of CS was determined using an acetyl coenzyme A assay. The refolded CS solution (15 µL) was added to 0.76 mL of substrate solution containing 0.023 mM acetyl-CoA, 0.5 mM oxalacetic acid, and 0.12 mM DTNB in 0.75 mM EDTA, 150 mM Tris-HCl buffer (pH 7.6). The increase in absorbance at 412 nm was measured as a function of time. The protein concentration of CS was determined by absorbance at 280 nm with a coefficient of 1.75 (mg/mL protein)-1‚cm-1. 3. Results and Discussion 3.1. Solution Properties of Spiropyrane-Bearing Pullulans. Spiropyrane-bearing pullulan (SpP) was synthesized by reacting anhydrate spirobenzopyrane derivatives with pullulan (Figure 1). The solution properties and photochromism of SpP in water were investigated. SpP (0.1-2.5 mg/mL) readily dissolved in water at 50 °C over 10 min. After the mixture was allowed to cool to 25 °C, the absorption maximum in the UV-vis spectrum for the resulting red-colored solution was 515 nm (Figure 2). The Stokes radius of gyration (Rh) of SpP under these conditions was 120 ( 13 nm at 1.0 mg/mL and 137 ( 14 nm at 2.5 mg/mL by DLS measurement. The molecular weight of the nanoparticles in these dilute conditions was (16 ( 3) × 105,

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Figure 2. UV spectrum of the photochromism of SpP nanogels in an aqueous solution. SpP (0.25 mg/mL) was dissolved in 100 mM HEPES-KOH buffer (pH 7.5) and stirred for 30 min at 50 °C under dark conditions: (a) After cooling at room temperature under dark conditions (Mer-Sp-form, the absorption maximum was 515 nm); (b) Mer-Sp-form SpP nanogels irradiated with UV light for 10 min at room temperature (Mer-form, the absorption maximum was 535 nm); (c) Mer-Sp-form SpP nanogels irradiated with visible light for 10 min at room temperature (Sp-form).

and the radius of gyration (Rg) was 83 ( 4 nm by the SECMALS method. One nanoparticle consists of 14 ( 3 SpP macromolecules, since the molecular weight of one SpP is 1.1 × 105. The density of each nanoparticle (Øg) was estimated to be ∼0.01 mg/mL from the following equation: Øg ) Mw/NA × (4/3‚πRg3)-1, where NA is Avogadro’s number.11 These data suggest that the nanoparticles can be considered as nanogels containing ∼0.1 wt % polysaccharides in which the association of spiropyrane groups provides cross-linking points similar to those in self-assembled nanogels of cholesteryl-bearing pullulan (CHP).10-12 The solution was then irradiated with visible light. The red color disappeared, and an absorption maximum was not seen in the range of 400-600 nm within 10 min due to the conversion of the merocyanine species to the spiropyrane species (Figure 2). After irradiation, neither the size nor the molecular weight of the nanogels changed dramatically (Rg)75 ( 11 nm, Mw)(14 ( 4) × 105 g/mol). Solutions of spiropyrane-form (Sp-form) nanogels were kept in the dark, and the merocyanine species (515 nm) gradually recovered within 3 h (Figure 2). The color of Sp-form solutions changed rapidly (within a minute) upon heating at 50 °C. On the other hand, when a solution of Sp-form nanogels was irradiated with UV light for 10 min instead of being stored in the dark or heated, it became red. The absorption maximum was seen at 535 nm (Figure 2). The nanogels increased in size depending on the duration of irradiation (10 min, Rg)94 ( 5 nm, and Mw)(28 ( 4) × 105 g/mol; 30 min, Rg)104 ( 5 nm, and Mw)(41 ( 5) × 105 g/mol). The conformational change in SpP induced a reorganization of the aggregation of SpP. After the nanogel solution (535 nm species) was irradiated with visible light, the red color gradually disappeared again. This conversion took 240 min. The nanogels gradually decreased in size during irradiation and finally reached the original size of Sp-form nanogels (Rg)75 ( 5 nm, Mw)(17 ( 5) × 105 g/mol) after irradiation with visible light for 240 min.

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Figure 3. Schematic representation of the photochromism of SpP nanogels.

According to Krongauz, spiropyran molecules tend to form dimers or oligomers between Mer and Sp species that show an absorption maximum at 515 nm.25 In our experiment, the species that shows an absorption maximum of 515 nm should be a Mer-Sp dimer or oligomer. Such complex formation (Mer-Sp-form) is also suggested by the following observation. Upon irradiation of 515 nm species nanogels with UV light for 5 min, the absorption maximum shifted to 535 nm, which can be attributed to the usual value of Mer-form in polar solution. All Sp species should change to Mer-form by UV irradiation. The merocyanine species (515 nm, MerSp-form) recovered after a solution of Mer-form (535 nm) was heated at 50 °C for 5 min. In summary, spiropyrane groups in SpP nanogels are found in three states: Mer-Spform (515 nm), Mer-form (535 nm), and Sp-form (Figure 3). The photoresponsive changes in the surface properties of the nanogels were investigated in measurements of surface tension using the Wilhelmy method with a platinum plate. In the presence of Mer-Sp-form nanogels, the surface tension was 69 ( 1 dyn/cm. The surface tension of Sp-form nanogels, which were prepared by irradiation of the solution of Mer-Sp-form with visible light for 60 min, was 64 ( 1 dyn/cm. On the other hand, that of Mer-form nanogels, which were prepared by irradiation of the Mer-Sp-form with UV light for 60 min, was 58 ( 1 dyn/cm. The accessibility of hydrophobes inside nanogels to the air-water interface reflects reorganization steps of the self-assembled structure. The surface activity and reorganization of the nanogels can be controlled by photostimulation. 3.2. Interaction of a Protein with Nanogels: Molecular Chaperone-like Activity. Large amounts of biologically active recombinant proteins are produced in heterologous systems. However, in many cases, there are problems regarding the formation of insoluble inclusion bodies. The successful refolding of proteins is needed to investigate their functions. Protein aggregates are usually separated from the host cell and solubilized with a denaturant (urea or GdmCl) in vitro, and refolding is attempted by removing the denaturant. In such a process, however, exposure of the hydrophobic surface of the refolding intermediate results in irreversible aggregation. Various refolding-aid (molecular

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Photoresponsive Nanogels Table 1. Effect of Additives on Native CS or CS Refolding sample

relative enzyme activity No

Denaturationa

native CS + SpPc

1.00 (Mer-Sp-form) (Sp-form) (Mer-form)

+ Pullulan no additive Pullulan Sp-COOHd

0.98 ( 0.05 0.99 ( 0.02 1.01 ( 0.02 1.01 ( 0.02

After GuHCl Denaturetion and Dilutionb 0.32 ( 0.01 0.37 ( 0.04 (Sp-form) (Mer-form)

0.32 ( 0.06 0.34 ( 0.05

(Mer-Sp-form) (Sp-form) (Mer-form)

0.61 ( 0.01 0.52 ( 0.02 0.27 ( 0.03e 0.34 ( 0.03f 0.31 ( 0.02g

SpPc

a No denaturation; CS was diluted with 0.75 mM EDTA, 150 mM TrisHCl buffer (pH 7.6) containing pullulan (1.0 mg/mL) or some form of SpP nanogels (1.0 mg/mL). b After denaturation with GdmCl and dilution; 6.0 M GdmCl-denatured CS was diluted 50-fold with 0.75 mM EDTA, 150 mM Tris-HCl buffer (pH 7.6) containing pullulan (1.0 mg/mL) or Sp-COOH (87.7 M) or SpP nanogels (1.0 mg/mL). The refolding solution was allowed to stand for 10 h at room temperature before assay. c Mer-Sp-form SpP nanogels were prepared by dissolving in 0.75 mM EDTA, 150 mM TrisHCl buffer (pH 7.6) and stirring for 30 min at 50 °C under dark conditions. SpP-form nanogels were prepared as follows. Mer-Sp-form nanogels were irradiated with visible light for 10 min before mixing with denatured CS solutions. Mer-Sp-form nanogels were irradiated with UV light for 1 min. d Sp- or Mer-form of Sp-COOH was prepared by dissolving in 0.75 mM EDTA, 150 mM Tris-HCl buffer (pH 7.6) and irradiating with UV (10 min) or visible light (10 min), respectively. e 10 min. f 30 min. g Before mixing with denatured CS solutions.

chaperone-like) systems have been reported to overcome these problems.16,17,26-32 It is important to control the amphiphilicity of an artificial chaperone in catching and releasing the refolding intermediate-protein to prevent aggregation during refolding. Nanogels of SpP are expected to control the association with proteins under photostimulation and, as a result, to control the refolding of protein. Citrate synthase (CS), which is a 10-kDa homodimeric enzyme, was selected as a model refolding protein. CS was fully unfolded and completely inactivated in 6.0 M GdmCl, 40 mM DTT in 0.75 mM EDTA, 50 mM Tris-sulfate (pH 7.5). The refolding reaction of CS (1.0 mg/mL) was performed by rapid dilution in a renaturation buffer consisting of 0.75 mM EDTA, 50 mM Tris-sulfate (pH 7.5) in the absence and presence of SpP nanogels. The refolding activity was estimated by the rate of recovery of enzyme activity by comparing the initial rate of refolded enzyme activity to the initial rate of native enzyme activity In the absence of nanogels, CS formed an irreversible aggregate, and only 34 ( 2% of the enzyme activity was recovered upon attempted renaturation. CS refolding was not promoted by either pullulan or Sp-COOH molecules via dilution additives (Table 1). Hydrophilic Mer-form nanogels did not promote refolding (27-34%). The refolding increased in the presence of 1.0 mg/mL Sp-form nanogels (52 ( 2%) and Mer-Sp-form nanogels (61 ( 1%). Figure 4 shows a plot of refolding activity as a function of the nanogel

Figure 4. Recovered enzyme activity as a function of the concentration of SpP nanogels. Native CS was dissolved in 6.0 M GdmCl, 0.75 mM EDTA, 150 mM Tris-HCl (pH 7.6), and 40 mM DTT to give a final concentration of 1.0 mg/mL denatured CS. After denaturation for 1 h at 25 °C, the mixture was diluted rapidly 50-fold with 0.75 mM EDTA 150 mM Tris-HCl buffer (pH 7.6) containing some concentration of SpP nanogels to give a final concentration of 0-5 mg/mL of SpP nanogels. After 10 h, CS enzyme activity was determined via the acetyl coenzyme A assay.

concentration. In the presence of a higher concentration of nanogels, the refolding decreased. Refolding was promoted with an appropriate concentration of nanogels. Amphiphilic water-soluble polymers such as poly(ethylene oxide) (PEO) increase the recovery yield of native proteins during refolding with an appropriate concentration of polymer.27 The polymers appear to act as a hydrophobic buffer by blocking the exposed hydrophobic surface on the denatured protein to prevent aggregation. However, excessively strong binding to the intermediate would prevent folding to the native conformation. Thus, a delicate balance in the various folding processes is needed to achieve high efficiency. Sp-form and Mer-Sp-form nanogels show activity similar to the effect of amphiphilic polymers in protein refolding, as previously reported. Hydrophobic interaction and electrostatic interaction (anionic and cationic interactions) play important roles in the interaction of nanogels with non-native protein such as denatured or refolding intermediated protein. Non-native proteins also have both properties due to hydrophobic and hydrophilic charged amino acids residues. Therefore, not only the amphiphilic property but also the electrostatic property of Mer-Sp-form nanogels is important to show high chaperonlike activity. Mer-Sp-form nanogels potentially interact with non-native proteins by both interactions, although Sp-form nanogels mainly interact by hydrophobic interaction. As a result, Mer-Sp-form nanogels should more strongly interact with non-native proteins compared to Sp-form nanogels. The chaperone-like activity of the nanogels reflects the interaction between nanogels and non-native proteins. At lower concentration, Mer-Sp-form nanogels showed higher activity than Sp-form nanogels (Figure 4) because stronger interaction results in the effective prevention of the aggregation of proteins. However, with an increase of the concentration of nanogels, an increase of the formation of further stable complex of nanogels with non-native protein inhibits releasing and refolding of the protein. Therefore, at higher

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Table 2. Refolding Activity of SpP Nanogels after Irradiation to the SpP Nanogelsa sample

relative enzyme activity

native CS no additive SpP Mer-Sp-form Mer-Sp-form (1 min) f Sp-form Mer-Sp-form (30 min) f Sp-form Mer-Sp-form (1 min) f Mer-form Mer-Sp-form (30 min) f Mer-form Sp-form Sp-form (1 min) f Mer-Sp-form Sp-form (30 min) f Mer-Sp-form Sp-form (1 min) f Mer-form Sp-form (30 min) f Mer-form

1.00 0.32 ( 0.01 0.61 ( 0.01 0.68 ( 0.04 0.81 ( 0.01 0.38 ( 0.04 0.58 ( 0.01 0.52 ( 0.02 0.58 ( 0.01 0.50 ( 0.03 0.35 ( 0.02 0.50 ( 0.03

a 6.0 M GdmCl, 40 mM DTT-denatured CS was rapidly diluted with 1.0 mg/mL SpP nanogels solution (Mer-Sp- or Sp-form) and allowed to stand for 1 or 30 min at room temperature. This solution was then irradiated with UV (Mer-form) or visible light (Sp-form) or left under dark conditions (Mer-Sp-form). After 10 h, CS enzyme activities were determined via an acetyl coenzyme A assay.

concentration, the chaperon-like activity of Mer-Sp-form nanogels was lower than that of Sp-form nanogels. 3.3. Photoresponsive Artificial Molecular Chaperone. In living systems, molecular chaperones achieve high activity by dynamically switching the amphiphilicity of a binding site with proteins. For example, GroEL acts as a host in macromolecular self-assembly by enclosing a folding intermediate protein as a guest in its central hydrophobic cavity to prevent aggregation of the protein during the folding process.33 The host chaperone then releases the protein in a refolded form through a change in the amphiphilicity of the cavity upon stimulation by ATP and another co-chaperone. We found that the amphiphilicity of SpP nanogels can be altered by photostimulation. The effects of photostimulation on refolding activity were investigated under various conditions (Table 2). Refolding was started by dilution with MerSp-form nanogels (1.0 mg/mL) and allowed to continue for 1 or 30 min. The solution was then irradiated with visible light to change it to the Sp-form. Interestingly, under this stimulation, the refolding activity drastically increased to over 81 ( 1%. However, in the presence of a higher concentration of Mer-Sp-form nanogel, refolding activity did not change. With other combinations, i.e., from Mer-Sp to Mer, from Sp to Mer-Sp, and from Sp to Mer, a strong cooperative effect was not observed (Table 2). Figure 5 shows the time courses of refolding using various artificial chaperone systems. Compared to spontaneous refolding, nanogel-assisted refolding was kinetically slow due to the interaction of the refolding intermediate protein with the nanogels. Mer-Sp-form nanogels interact more strongly with refolding intermediates of CS than Sp-form and Merform nanogels based on the slower rate of refolding. Appropriate interactions, such as both hydrophobic interaction with the Sp-form and electrostatic interaction with the zwitterionic Mer-form, should effectively prevent the aggregation of proteins. Changing the amphiphilicity of the nanogels from Mer-Sp-form to Sp-form by photostimulation induced a change in the interaction with proteins. Electrostatic interaction between Mer-Sp-form nanogels and non-

Figure 5. Time course of the recovery of enzyme activity of GdmCldenatured CS after dilution. Native CS was dissolved in 6.0 M GdmCl, 0.75 mM EDTA, 40 mM DTT, and 150 mM Tris-HCl buffer (pH 7.6) to give a final concentration of 1.0 mg/mL denatured CS. After denaturation for 1 h at 25 °C, the mixture was diluted rapidly 50-fold with 0.75 mM EDTA, 150 mM Tris-HCl buffer (pH 7.6) (a) or 0.75 mM EDTA, 150 mM Tris-HCl buffer (pH 7.6) containing various SpP nanogels (b, c, e). The enzyme activity at each time point was estimated by an acetyl coenzyme A assay. Sample e was prepared as follows. Denatured CS was diluted 50-fold with Mer-Sp-form SpP nanogel solution and kept for 30 min at 25 °C. The conformation of SpP nanogels was then changed from Mer-Sp-form to Sp-form by irradiation with visible light.

native proteins disappear after photostimulation. This may induce less interaction with proteins and may promote their release from nanogels. As a result, total refolding activity may increase. The delicate balance in the interaction between nanogels and the refolding intermediate protein is important to achieve high refolding activity. Too much interaction with nanogels inhibits refolding of the protein. The appropriate capture-and-release of a refolding intermediate protein with SpP nanogels may be able to be controlled by photostimulation. These results suggest a new strategy for the dynamic control of protein refolding using stimuli-responsive artificial molecular chaperones. 4. Conclusions A novel photoresponsive nanogel was prepared by the selfassembly of SpP, and it showed high molecular chaperonelike activity upon photostimulation. Various hybrid nanogels can be easily obtained by simply mixing various functional associating polymers.34 Macrogels with well-defined nanostructures can also be obtained by the hierarchical selfassembly of these nanogels.12 The self-assembling method using associating polymers as building blocks should be promising as an efficient and versatile technique for preparing functional nanogels and nanobiomaterials. Acknowledgment. This work was supported by a Grantin-Aid for Scientific Research from the Japanese government (No. 15300158) and by PRESTO of JST. K.A. acknowledges financial support from Sekisui Chemical Co., Ltd.

Photoresponsive Nanogels

References and Notes (1) Kopecek, J. Nature 2002, 417, 388-391. (2) Byrne, M. E.; Park, K.; Peppas, N. A. AdV. Drug DeliVery ReV. 2002, 54, 149-161. (3) Hu, Z.; Lu, X.; Gao, J.; Wang, C. AdV. Mater. 2000, 12, 11731176. (4) Vinogradov, S. V.; Bronich, T. K.; Kabanov, A. V. AdV. Drug DeliVery ReV. 2002, 54, 135-147. (5) McAllister, K.; Sazani, P.; Adam, M.; Cho, M. J.; Rubinstein, M.; Samulski, R. J.; DeSimone, J. M. J. Am. Chem. Soc. 2002, 124, 15198-15207. (6) Antonietti, M.; Bremser, W.; Schmidt, M. Macromolecules 1995, 28, 4227-4233. (7) Gan, D.; Lyon, L. A. J. Am. Chem. Soc. 2001, 123, 7511-7517. (8) Kadlubowski, S.; Grobelny, J.; Olejniczak, W.; Cichomski, M.; Ulanski, P. Macromolecules 2003, 36, 2484-2492. (9) Kuckling, D.; Vo, C. D.; Wohlrab, S. E. Langmuir 2002, 18, 42634269. (10) Akiyoshi, K.; Deguchi, N.; Moriguchi, N.; Yamaguchi, S.; Sunamoto, J. Macromolecules 1993, 26, 3062-3068. (11) Akiyoshi, K.; Deguchi, N.; Tajima, H.; Nishikawa, T.; Sunamoto, J. Macromolecules 1997, 30, 857-861. (12) Kuroda, K.; Fujimoto, K.; Sunamoto, J.; Akiyoshi, K. Langmuir 2002, 18, 3780-3786. (13) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. Macromolecles 1994, 27, 7654-7659. (14) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. J. Am. Chem. Soc. 1996, 118, 6110-6115. (15) Ikuta, Y.; Katayama, N.; Wang, L.; Okugawa, T.; Takahashi, Y.; Schmitt, M.; Gu, X.; Watanabe, M.; Akiyoshi, K.; Nakamura, H.; Kuribayashi, K.; Sunamoto, J.; Shiku, H. Blood 2002, 99, 10, 37173724. (16) Akiyoshi, K.; Sasaki, Y.; Sunamoto, J. Bioconjugate Chem. 1999, 10, 3, 321-324.

Biomacromolecules, Vol. 5, No. 5, 2004 1809 (17) Nomura, Y.; Ikeda, M.; Yamaguchi, N.; Aoyama, Y.; Akiyoshi, K. FEBS Lett. 2003, 553, 271-276. (18) Willner, I.; Rubin, S. Angew. Chem., Int. Ed. Engl. 1996, 35, 367385. (19) Alfimov, M. V.; Fedorova, O. A.; Gromov, S. P. J. Photochem. Photobiol., A 2003, 158, 183-198. (20) Pouliquen, G.; Porcar, I.; Tribet, C.; Amiel, C. ACS Symp. Ser. 2003, 833, 262-288. (21) Menju, A.; Hayashi, K.; Irie, M. Macromolecules 1981, 14, 755758. (22) Konak, C.; Rathi, R. C.; Kopeckova, P.; Kopecek, J. Macromolecules 1997, 30, 5553-5556. (23) Ito, Y.; Sugimura, N.; Kwon, O. H.; Imanishi, Y. Nat. Biotechnol. 1999, 17, 73-75. (24) Alonso, M.; Reboto, V.; Guiscardo, L.; San Martin, A.; RodriguezCabello, J. C. Macromolecules 2000, 33, 9480-9482. (25) Krongauz, V. A.; Goldburt, E. S. Nature 1978, 271, 43-45. (26) Tandon, S.; Horowitz, P. M. J. Biol. Chem. 1986, 261, 15615-15618. (27) Cleland, J. L.; Hedgepeth, C.; Wang, D. I. C. J. Biol. Chem. 1992, 267, 13327-13334. (28) Rozema, D.; Gellman, S. H. J. Am. Chem. Soc. 1995, 117, 23732374. (29) Hashimoto, Y.; Ono, T.; Goto, M.; Hatton, A. Biotechnol. Bioeng. 1998, 57, 5, 620-623. (30) Yoshimoto, M.; Kuboi, R. Biotechnol. Prog. 1999, 15, 480-487. (31) Shimizu, H.; Fujimoto, K.; Kawaguchi, H. Biotechnol. Prog. 2000, 16, 248-253. (32) Sharma, L.; Sharma, A. Eur. J. Biochem. 2001, 268, 2456-2463. (33) Mayhew, M.; da Silva, A. C.; Martin, J.; Erdjument Bromage, H.; Tempst, P.; Hartl, F. U. Nature 1996, 379, 420-426. (34) Akiyoshi, K.; Kang, E. C.; Kurumada, S.; Sunamoto, J.; Principi, T.; Winnik, F. M. Macromolecules 2000, 33, 3244-3249.

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