Raspberry-like assembly of cross-linked nanogels for protein delivery

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Journal of Controlled Release 140 (2009) 312–317

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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

Raspberry-like assembly of cross-linked nanogels for protein delivery Urara Hasegawa a, Shin-ichi Sawada a, Takeshi Shimizu b,c, Tsunao Kishida c, Eigo Otsuji b, Osam Mazda c, Kazunari Akiyoshi a,⁎ a b c

Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan Department of Surgery, Kyoto Prefectural University of Medicine, Kamikyo, Kyoto 602-8566, Japan Department of Microbiology, Kyoto Prefectural University of Medicine, Kamikyo, Kyoto 602-8566, Japan

a r t i c l e

i n f o

Article history: Received 9 February 2009 Accepted 20 June 2009 Available online 29 June 2009 Keywords: Nanogel Protein delivery Sustained release Polysaccharide IL-12

a b s t r a c t Raspberry-like assembly of nanogels (A-CHPNG) with a high potential as a carrier for protein delivery was prepared. Cross-linking of acrylate group-modified cholesterol-bearing pullulan nanogel (CHPANG) with thiol group-modified poly (ethylene glycol) (PEGSH) by Michael addition yielded A-CHPNG with narrow size distribution. The size of A-CHPNGs was controlled in the range of 40–120 nm by changing the concentration of CHPANG and PEGSH. A-CHPNG gradually degraded by hydrolysis under physiological condition and seemed to dissociate back to original nanogel. A-CHPNG encapsulated interleukin-12 (IL-12) efficiently (96%) and stably kept it in the presence of BSA (50 mg/ml). In addition, A-CHPNG had a high potential to maintain a high IL-12 level in plasma after subcutaneous injection in mice. Therefore, A-CHPNG is a promising carrier for long-term medications. © 2009 Elsevier B.V. All rights reserved.

1. Introduction New classes of highly effective therapeutic proteins such as cytokines and antibodies have attracted a great deal of attention due to their potential applications as vaccines for cancers, allergies and infectious diseases [1,2]. Since these proteins are normally expensive to produce on a large scale and easily denatured to loose their bioactivity, it is required to develop new delivery systems to get efficient therapeutic effects at a minimum dosage. One of the promising methods is to encapsulate proteins into hydrogel nanoparticles (nanogel), which can minimize denaturation of proteins by trapping them in a hydrated polymer-network [3–7]. Among the nanogels reported so far, the self-aggregated nanogels of hydrophobic groupmodified water-soluble polymers have emerged as promising drugcarriers in protein therapies. We have reported that the nanogel of self-aggregated cholesterolbearing pullulan (CHPNG) forms a complex with various kinds of proteins spontaneously and releases them upon exposure to high concentration of other proteins such as bovine serum albumin (BSA) [8–10]. In addition, CHPNG inhibits the aggregation of the trapped proteins which often causes the irreversible denaturation and the loss of bioactivity [11–16]. These properties allow CHPNG to serve as a carrier for therapeutic proteins such as insulin [10], interleukin 12 (IL12) [17] and HER2 protein [18–21]. Though CHPNG is effective for a relatively short period of time, the use in long-term therapy is limited

⁎ Corresponding author. Tel.: +81 3 5280 8020; fax: +81 3 5280 8027. E-mail address: [email protected] (K. Akiyoshi). 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.06.025

by the destabilization of the nanogel–protein complex in vivo where high protein concentrations exist. The present paper describes a novel method for making wellordered assemblies of CHPNGs (A-CHPNG) by Michael addition of acrylate-group-modified CHPNG (CHPANG) and thiol-group-modified poly(ethylene glycol) cross-linker (PEGSH). The structure and dissociation behavior of A-CHPNG were characterized. The release profile of IL-12 from A-CHPNG was also investigated in vitro and in vivo. 2. Materials and methods 2.1. Materials Cholesterol-bearing pullulan (CHP) was synthesized as reported previously [22]. Pullulan was substituted with 1.4 cholesterol moieties per 100 anhydrous glucoside units. Pentaerythritol tetra (mercaptoethyl) polyoxyethylene (PEGSH, Mw 10,000) was purchased from NOF Corporation (Japan). Recombinant mouse IL-12 and Mouse IL-12 p70 ELISA kit were purchased from R&D systems (USA). Other reagents were obtained commercially and used without further purification. 2.2. Synthesis of acrylate group-modified CHP (CHPA) CHPAs were synthesized by condensing carboxyl acid group of acrylic acid and hydroxyl group of pullulan. CHP was reacted with acrylic acid in the presence of catalytic amount of N,N-dimethylaminopyridine (DMAP). N,N′-dicyclohexylcarbodiimide (DCC) was added to form active-ester intermediate of acrylic acid (acrylic acid: DCC = 1:1). As

2.4. Preparation of IL-12-encapsulated A-CHPNG

Table 1 Characterization of the nanogels by DLS and MALS. Sample

xa

DNG/nm (PDI)b

Mwc

Aggregation number of polymer

CHPA15NG

15.0

n.m.

n.m.

CHPA28NG

27.7

n.m.

n.m.

CHPA32NG

31.5

31.4 ± 0.3 (0.163 ± 0.004) 27.7 ± 0.2 (0.259 ± 0.006) 18.2 ± 0.1 (0.232 ± 0.004) 38.4 ± 0.2 (0.214 ± 0.010)

3.7 × 105

3.0

4.7 × 105d

4.2

CHPNG a b c d

0

313

Number of acrylate groups per 100 anhydrous glucoside units. Diameter of nanogels determined by dynamic light scattering. Molecular weight of nanogel determined by static light scattering. Data from Ref. [23].

an example, CHP (0.5 g, 3.0 mmol equivalent of anhydrous glucoside units) was dissolved in 5 ml dry dimethylsulfoxide (DMSO) containing DMAP (0.01 g, 83 μmol). Acrylic acid (613 μl, 9.0 mmol) and DCC (1.84 g, 9.0 mmol) were dissolved in 5 ml dry DMSO and stirred for 30 min at room temperature. The mixture was then added to the CHP solution and stirred for 2 days at room temperature. The reaction solution was dropped into excess ether/ethanol (97.5/2.5 by volume), and the precipitate was washed with ether/ethanol. The crude product was dissolved in DMSO, dialyzed against distilled water (MWCO 3500) and lyophilized. Degree of substitution was 31.5 per 100 glucoside units. 1H NMR (500 MHz, DMSO-d6/D2O = 10/1 (v/v), δ): 0.60–2.40 (cholesterol); 3.1–4.0 (glucose H2, H3, H4, H5 and H6); 4.70 (glucose H1 (1→ 6)); 4.90–5.10 (glucose H1 (1→ 4)); 5.9–6.3 (double bond of acrylate group).

The aqueous solutions of CHPANG with 32 acrylate groups per 100 anhydrous glucoside units (CHPA32NG) (20 mg/ml) and IL-12 (0.01 mg/ml) were mixed and kept at 25 °C for 24 h to obtain IL-12 encapsulating CHPANG. Then, aqueous solution of IL-12 encapsulated CHPANG was mixed with PEGSH and incubated at 37 °C for 24 h. The ratio of acrylate groups on CHPANG to thiol groups on PEGSH was 1:1. ELISA assay was performed to detect free IL-12 by A-CHPNG as reported previously [17].

2.5. Dynamic light scattering (DLS) The concentration of A-CHPNG was adjusted to 1 mg/ml prior to measurement. Diameter (Z-average) and polydispersity index (PDI) (=μ2 / Γ2) were determined by the cumulant method using Zetasizer Nano (Malvern, UK).

2.6. Field-flow fractionation coupled with multi-angle light scattering (FFF-MALS) Static light scattering and refractive index measurements were carried out on DAWN EOS and Optilab REX, respectively, connected in-line to the field-flow fractionation system (Wyatt Technology, USA). The channel flow rate was 1.0 ml min− 1 and the cross-flow rate decreased linearly from 1.0 ml min− 1 to 0 ml min− 1 at a 20 min interval. The eluting buffer contained 0.1 M NaNO3 and 1 mM Na2HPO4 (pH8.45). Average molecular weight (Mw) and polydispersity index (Mw / Mn) were calculated using ASTRA analysis software based on Zimm's equation for CHPANG and Berry's equation for A-CHPNG.

2.3. Preparation of the assemblies of CHPNG (A-CHPNG) CHPA was dissolved in Dulbecco's phosphate-buffered saline (PBS, pH7.4) to form self-aggregated CHPA nanogel (CHPANG). Separately, PEGSH was dissolved in PBS and added to the CHPANG solution and reacted at 37 °C for 24 h. The molar ratio of acrylate groups of CHPANG to thiol groups of PEGSH was 1:1.

2.7. Atomic force microscopy (AFM) A-CHPNG solution was dropped onto a mica surface and dried in a flow of N2. The surface was scanned with SPI300 (Seiko, Japan) using a Si probe (SI-DF20) (Seiko, Japan) with a spring constant of 15 N m− 1.

Fig. 1. Michael addition of CHPANG and PEGSH to form A-CHPNG.

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CHPA was dissolved in phosphate-buffered saline (PBS) and characterized by dynamic light scattering (DLS) and field-flow fractionation coupled with multi-angle light scattering (FFF-MALS). In the same manner as unmodified CHP, CHPA also self-aggregated to form monodisperse nanogel (CHPANG) with diameters of 20–30 nm in phosphate-buffered saline (PBS, pH7.4) (Table 1). 3.2. Formation and characterization of the assembly of nanogels (A-CHPNG)

Fig. 2. Reaction between CHPANG and PEGSH as function of time. (a) Acrylate groups per 100 glucoside units by 1H NMR. (b) Change in diameter by DLS.

2.8. Transmission electron microscopy (TEM) The cross-section image of A-CHPNG was obtained with H-600 (Hitachi, Japan) by the freeze-fracture method. The sample was prepared using the aqueous solution of A-CHPNG containing 30% glycerin and rotary-shadowed with platinum and carbon. Accelerating voltage was 100 kV. 2.9. Release profile of IL-12 from A-CHPNG in vivo IL-12, IL-12 encapsulated in CHPNG, IL-12 encapsulated in ACHPNG and BSA encapsulated in CHP were injected subcutaneously into Balb/c mice. The total amount of injected IL-12 was 0.5 μg. The serum concentration of IL-12 was assayed by ELISA as function of time. 3. Results and discussion 3.1. Characterization of acrylate group-modified CHP nanogel (CHPANG) Acrylate group-modified CHP (CHPA) was synthesized by the DCCmediated condensation of hydroxyl groups of CHP and carboxyl groups of acrylic acid. Different degree of substitution of acrylate group was obtained by changing the feed concentration of acrylic acid. The degrees of substitution (x), as determined by 1H NMR, were 15.0, 27.7 and 31.5 per 100 anhydrous glucoside units.

CHPANGs were cross-linked with PEGSH by Michael addition (Fig. 1), which offers several advantages in the fabrication of biomaterials. The cross-linking reaction proceeds rapidly under physiological conditions. Additionally, the resultant materials gradually degrade under physiological pH by hydrolysis of the β-thiopropionate linkage [24–26]. Thus, this approach has been successfully used with various biomaterials such as hydrogels to trap and release proteins [24,25] and polymeric micelles to deliver siRNA [26]. Aqueous solutions of CHPANG with 32 acrylate groups per 100 anhydrous glucoside units (CHPA32NG) and PEGSH were mixed as the molar ratio of acrylate groups to thiol groups 1:1 and incubated at 37 °C (the final concentration of CHPANG: 5.0 mg/ml). 1H NMR analysis clearly showed that acrylate groups of CHPANG disappeared within 10 h (Fig. 2a). Thus, all acrylate groups seem to react with thiols of PEGSH. The formation of disulfide bonds between PEGSHs is also expected as a side reaction in this system. However, the complete consumption of acrylate groups means that every thiols of PEGSH reacted specifically with an acrylate group and not with other thiols. We monitored the diameter change of CHPANG after addition of PEGSH by dynamic light scattering (DLS). The diameter rapidly increased within 2 h and reached ~100 nm after 24 h of incubation (Fig. 2b). The resultant particles had a narrow size distribution according to atomic force microscopy (AFM) (Fig. 3a). A more resolved picture was observed by using transmission electron microscopy (TEM). As shown in Fig. 3b, TEM images clearly showed that the small particles (10–15 nm in diameter) are gathering to form one particle with raspberry-like structure. It indicates that the resultant particle is an assembly of CHPANGs (A-CHPNG) (Fig. 3c). The number of nanogels per A-CHPNG was determined by fieldflow fractionation coupled with multi-angle light scattering (FFF-

Fig. 3. Characterization of the assembly of nanogels (A-CHPNG). (a) AFM image of A-CHPNG. (b) TEM image of A-CHPNG. (c) Schematic illustration of A-CHPNG formation.

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Table 2 Diameters of A-CHPNGs prepared at different concentrations. Nanogel

cNG/g l− 1

CHPA15NG

1.0

2.2

2.5

5.5

5.0

11.0

1.0

4.1

2.5

10.2

5.0

20.3

1.0

4.6

2.5

11.5

5.0

23.1

CHPA28NG

CHPA32NG

cPEGSH/g l− 1

DANG/nm (PDI)

DANG/DNG

61.8 ± 0.4 (0.199 ± 0.004) 81.3 ± 1.1 (0.190 ± 0.009) 112.0 ± 0.8 (0.216 ± 0.009) 50.0 ± 0.7 (0.191 ± 0.005) 70.5 ± 0.2 (0.185 ± 0.006) 111.0 ± 0.6 (0.234 ± 0.010) 41.1 ± 0.4 (0.176 ± 0.004) 58.2 ± 0.2 (0.191 ± 0.004) 103.0 ± 0.3 (0.247 ± 0.003)

1.97 ± 0.01 2.59 ± 0.04 3.57 ± 0.03 1.81 ± 0.01 2.55 ± 0.02 4.02 ± 0.02 2.26 ± 0.02 3.20 ± 0.01 5.66 ± 0.01

cNG, cPEGSH: Feed concentration of CHPANG and PEGSH. DNG, DANG: Diameter of CHPANG and A-CHPNG.

MALS). The molar mass of CHPANG and A-CHPNG were 3.7 × 105 and 2.3 × 108, respectively. Assuming that all CHPANG and PEGSH reacted completely, the number of CHPANGs per A-CHPNG is estimated to be 126. Based on the molar mass and the hydrodynamic diameter, the average polymer density of this particle is estimated to be 0.67 g/ml, which is much higher than that of CHPANG (0.20 g/ml). Therefore, A-CHPNG consists of a highly concentrated PEG network (0.53 mg/ml) as well as nanogel domains. To investigate in detail the mechanism of the nanogel association, CHPANG and PEGSH were mixed at different concentrations. Depending on the concentration, we could control the size of A-CHPNGs in the range of 40–120 nm (Table 2). Interestingly, the increase of diameter ratio (DANG/DNG) correlated with the total feed concentration of CHPANG and PEGSH (Fig. 4). Therefore, the association of CHPANG appears to be controlled in a concentration-depending manner. Based on the A-CHPNG formation, we hypothesize that two reactions occur competitively in this system, i.e., inter- and intra-nanogel crosslinking. The former reaction, by which a nanogel binds to other nanogels, increases the particle size of CHPANG. The latter reaction, which cross-links pullulan chains inside the nanogel, may limit the movement of pullulan chains and provide structural stability to CHPANG. At the initial stage of the reaction, especially at high concentration, the frequency of collision between CHPANG and PEGSH is high, and thus the inter-nanogel reaction would be enhanced. As the reaction proceeds, the number of reactive groups will decrease and the surface of nanogel will be covered with PEGSH, which may slow the inter-nanogel reaction.

Fig. 4. Association behavior of CHPANG as function of the total concentration of polymers (CHPA and PEGSH). Aqueous solution of CHPA15NG (circle), CHPA28NG (triangle) and CHPA32NG (square) were reacted with PEGSH at different concentrations for 24 h and measured by DLS.

Fig. 5. Change in the diameters of A-CHPNG by hydrolysis as function of time. Diameter of A-CHPNGs composed of (a) CHPA15NG, (b) CHPA28NG and (c) CHPA32NG were monitored by DLS. Preparation concentrations of CHPANG were 1.0 (triangle), 2.5 (circle) and 5.0 mg/ml (square).

3.3. Dissociation of A-CHPNG under physiological pH We tested whether A-CHPNG dissociates back to CHPNG under physiological pH. After preparing A-CHPNG by incubating for 24 h, we kept it in PBS at 37 °C and monitored the change in diameter as function of time. The diameters of A-CHPNG initially increased but later gradually decreased as shown in Fig. 5. This dissociation behavior suggests that as inter-nanogel cross-linking is cleaved by hydrolysis, A-CHPNG starts expanding and finally dissociates back to original nanogel. Moreover, degradation proceeded faster for A-CHPNG composed of CHPANG with less acrylate groups and prepared at a lower CHPANG concentration. The dissociation time varied from days to weeks, which is advantageous to tailor the release profile of incorporated drugs depending on a specific application. 3.4. Release of IL-12 from A-CHPNG We used IL-12 as a model protein to investigate whether A-CHPNG can serve as a carrier with prolonged release. IL-12 is an immunostimulatory cytokine that can suppress not only tumor growth and metastasis but also infection and allergy. However, it is difficult to maintain its plasma concentration at an effective level due to its short half-life in vivo. Therefore, for clinical application, multiple administrations with large dose are required, which is associated with an increased risk of serious side effects caused by highly elevated levels of serum IFN-γ [9]. First, we encapsulated IL-12 into CHPANG as reported previously and then cross-linked with PEGSH. ELISA assay revealed that 96% of IL12 was trapped into CHPANG (Fig. 6a). The main driving force for this interaction between IL-12 and CHPANG is probably hydrophobic interaction of cholesteryl group in nanogel and hydrophobic surface of proteins as we reported previously [8–10]. In addition, nanosize matrix in the nanogel should play an important role in trapping and keeping of proteins. To see the effect of a high concentration of protein on the stability of the complex of A-CHPNG and IL-12, the release profile of IL-12 was

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4. Conclusion We newly prepared the raspberry-like assemblies of nanogels (ACHPNG) with a narrow size distribution by cross-linking of acrylatemodified nanogel (CHPANG) and 4-armed PEGSH. The particle size and dissociation behavior were easily controlled by this method. To our knowledge, this is a novel and facile method without further purification to make chemically-cross-linked colloidal particles compared to the well-known methods such as precipitation, dispersion or emulsion polymerization, or intra-cross-linking of polymer assemblies [29–33]. Additionally, A-CHPNG showed high potential to encapsulate IL-12 and was able to keep it even in the presence of BSA in vitro. More importantly, the A-CHPNG gave a prolonged release profile after subcutaneous injection in mice. Therefore, A-CHPNG is a promising carrier which enables sustained release of protein drugs. The simplicity of the preparation and the high encapsulation efficiency will be very advantageous in practical applications. Acknowledgements

Fig. 6. Release of IL-12 encapsulated in CHPNG and A-CHPNG. (a) Encapsulation of IL-12 by CHPNG and A-CHPNG. Free IL-12 in the absence of CHPNG and in the presence of CHPNG and A-CHPNG. (b) in vitro release profiles of IL-12 from CHPNG (triangle) and A-CHPNG (circle) in the presence of BSA (50 mg/ml) at 37 °C. (c) in vivo release profiles of IL-12 after subcutaneous injection. Plasma concentration of IL-12 was monitored after subcutaneous injections of IL-12 alone (open square), IL-12 encapsulated in CHPNG (filled triangle), IL-12 encapsulated in A-CHPNG (open circle) and BSA encapsulated in CHPNG (filled square).

measured in the presence of BSA (50 mg/ml). As shown in Fig. 6b, even in the presence of a high concentration of BSA, A-CHPNG did not release IL-12, whereas CHPNG released all within 48 h. The slow release of IL-12 is mainly caused by PEG-cross-linking of CHPANG. As we reported previously, the uncross-linked CHPNG is stable in water. However in the presence of large amounts of proteins IL-12 was released from the nanogels mainly by exchange mechanism between IL12 and BSA. The exchange seems to be minimized by PEG chains which offer a structural stability and a protein-repellent property. Next, we compared the in vivo release profile of IL-12 from CHPNG and A-CHPNG after subcutaneous injection. As shown in Fig. 6c the plasma level of IL-12 was somewhat prolonged by CHPNG, though the initial acute elevation of the IL-12 level was observed both with and without CHPNG. By using A-CHPNG as a carrier, the IL-12 concentration increased gradually and was maintained at a high level for at least 3 days. Such release profile is preferred in order to get a prolonged therapeutic effect and avoid side effects. One of the important factors governing the prolonged release time is the mechanical stabilization of the pullulan chain inside A-CHPNG by intra-cross-linking [15]. This stabilization limits the movement of pullulan chains of nanogels and delays the release of IL-12 by inhibiting the interaction with other plasma proteins. As β-thiopropionate linkages between CHPANG and PEGSH have been shown to hydrolyze in vivo, the pullulan chains will become loose and then IL-12 can be released slowly from A-CHPNG. Also enzymes such as esterases may facilitate the hydrolysis of the ester bond as well. In addition, the protein-repellent property of PEG contributes to the lowered interaction of A-CHPNG with plasma proteins. Moreover, concerning in vivo application, the size of the particles is also important. Since A-CHPNG used in this experiment is three times bigger than CHPNG, it can affect the diffusion through subcutaneous tissue and the disposition in body [27,28]. Further in vivo study for clinical applications is under investigation.

This work was supported by the grant from the Japanese Ministry of Education, Global Center of Excellence (GCOE) Program, “International Research Center for Molecular Science in Tooth and Bone Diseases”. This work was also supported by the Japan Society for the Promotion of Science under grant-in-aid for Creative Scientific Re search (No. 18GS0421) and by the Ministry of Education, Culture, Sports, Science and Technology of Japan (20011002). We thank Mr. Masahide Nakamura (Shoko Co.) for FFF-MALS measurement. We also thank Dr. Nobuyuki Morimoto (Tokyo Medical and Dental University) for technical help and advice. References [1] H.L. Robinson, New hope for an AIDS vaccine, Nat. Rev. Immunol. 2 (4) (2002) 239–250. [2] J.A. Berzofsky, J.D. Ahlers, I.M. Belyakov, Strategies for designing and optimizing new generation vaccines, Nat. Rev. Immunol. 1 (3) (2001) 209–219. [3] N. Murthy, Y.X. Thng, S. Schuck, M.C. Xu, J.M. Frechet, A novel strategy for encapsulation and release of proteins: hydrogels and microgels with acid-labile acetal cross-linkers, J. Am. Chem. Soc. 124 (42) (2002) 12398–12399. [4] N. Murthy, M. Xu, S. Schuck, J. Kunisawa, N. Shastri, J.M. Frechet, A macromolecular delivery vehicle for protein-based vaccines: acid-degradable protein-loaded microgels, Proc. Natl. Acad. Sci. U.S.A. 100 (9) (2003) 4995–5000. [5] J.K. Li, N. Wang, X.S. Wu, Poly(vinyl alcohol) nanoparticles prepared by freezing– thawing process for protein/peptide drug delivery, J. Control. Release 56 (1–3) (1998) 117–126. [6] G. Mocanu, D. Mihai, L. Picton, D. LeCerf, G. Muller, Associative pullulan gels and their interaction with biological active substances, J. Control. Release 83 (1) (2002) 41–51. [7] M. Leonard, M.R. De Boisseson, P. Hubert, F. Dalencon, E. Dellacherie, Hydrophobically modified alginate hydrogels as protein carriers with specific controlled release properties, J. Control. Release 98 (3) (2004) 395–405. [8] T. Nishikawa, K. Akiyoshi, J. Sunamoto, Supramolecular assembly between nanoparticles of hydrophobized polysaccharide and soluble-protein complexation between the self-aggregate of cholesterol-bearing pullulan and alpha-chymotrypsin, Macromolecules 27 (26) (1994) 7654–7659. [9] T. Nishikawa, K. Akiyoshi, J. Sunamoto, Macromolecular complexation between bovine serum albumin and the self-assembled hydrogel nanoparticle of hydrophobized polysaccharides, J. Am. Chem. Soc. 118 (26) (1996) 6110–6115. [10] K. Akiyoshi, S. Kobayashi, S. Shichibe, D. Mix, M. Baudys, S.W. Kim, J. Sunamoto, Selfassembled hydrogel nanoparticle of cholesterol-bearing pullulan as a carrier of protein drugs: complexation and stabilization of insulin, J. Control. Release 54 (3) (1998) 313–320. [11] K. Akiyoshi, Y. Sasaki, J. Sunamoto, Molecular chaperone-like activity of hydrogel nanoparticles of hydrophobized pullulan: thermal stabilization with refolding of carbonic anhydrase B, Bioconjug. Chem. 10 (3) (1999) 321–324. [12] T. Hirakura, Y. Nomura, Y. Aoyama, K. Akiyoshi, Photoresponsive nanogels formed by the self-assembly of spiropyrane-bearing pullulan that act as artificial molecular chaperones, Biomacromolecules 5 (5) (2004) 1804–1809. [13] Y. Nomura, M. Ikeda, N. Yamaguchi, Y. Aoyama, K. Akiyoshi, Protein refolding assisted by self-assembled nanogels as novel artificial molecular chaperone, FEBS Lett. 553 (3) (2003) 271–276. [14] N. Morimoto, T. Endo, Y. Iwasaki, K. Akiyoshi, Design of hybrid hydrogels with selfassembled nanogels as cross-linkers: interaction with proteins and chaperone-like activity, Biomacromolecules 6 (4) (2005) 1829–1834.

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