Peptide Tag-Induced Horseradish Peroxidase-Mediated Preparation

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Peptide tag-induced horseradish peroxidase-mediated preparation of a streptavidin-immobilized redox-sensitive hydrogel Masahiro Mishina, Kosuke Minamihata, Kousuke Moriyama, and Teruyuki Nagamune Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00149 • Publication Date (Web): 16 May 2016 Downloaded from http://pubs.acs.org on May 19, 2016

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Peptide

tag-induced

horseradish

peroxidase-

mediated preparation of a streptavidin-immobilized redox-sensitive hydrogel

Masahiro Mishina,a Kosuke Minamihata,*a Kousuke Moriyama,b and Teruyuki Nagamunea,c

a

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo,

7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. E-mail: [email protected] b

Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial

Science and Technology (AIST), Tsukuba Center 5th, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan. c

Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo,

Bunkyo-ku, Tokyo 113-8656, Japan.

KEYWORDS Horseradish peroxidase, Streptavidin, Peptide-tag, Hydrogel, Redox-sensitive

ACS Paragon Plus Environment

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ABSTRACT

Several methods have recently been reported for the preparation of redox-sensitive hydrogels using enzymatic reactions, which are useful for encapsulating sensitive materials such as proteins and cells. However, most of the reported hydrogels is difficult to add further function efficiently, limiting the application of the redox-sensitive hydrogels. In this study, peptide sequences of HHHHHHC and GGGGY (Y-tag) were genetically fused to the N- and C-termini of streptavidin (C-SA-Y), respectively, and C-SA-Y was mixed with horseradish peroxidase and thiolfunctionalized 4-arm polyethylene glycol to yield a redox-sensitive C-SA-Y immobilized hydrogel (C-SA-Y gel). The C-SA-Y immobilized in the hydrogel retained its affinity for biotin, allowing for the incorporation of proteins and small molecules to hydrogel via biotin. C-SA-Y gel was further prepared within w/o emulsion system to yield a nano-sized hydrogel, to which any intracellular and cytotoxic agent can be modified, making it a potential drug delivery carrier.


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Introduction Hydrogels are three-dimensional hydrophilic networks capable of absorbing large quantities of water while maintaining their structure. Based on their unique properties, including their high water content, high permeability and biocompatibility, hydrogels have been used in numerous applications in biopharmaceutical,1-5 tissue engineering6-11 and bioengineering fields.12-18 To meet the needs of these different applications, a variety of different stimuli-sensitive hydrogels have been developed, including light-,19,20 thermal-,21,22 pH-23,24 and redox-sensitive hydrogels.2527

Among these systems, redox-sensitive hydrogels are particularly appealing because they can

be readily dissolved under mild condition in the presence of small reducing chemicals such as glutathione (GSH) and cysteine (Cys), making them desirable scaffolds for drug delivery and tissue engineering applications.28,29 Singh and co-workers recently reported a new method for the preparation of redox-sensitive hydrogels using a horseradish peroxidase (HRP) enzymatic reaction.30 In this particular study, thiol-functionalized polyethylene glycol (PEG) was cross-linked using an HRP-catalyzed thiolradical formation, which was activated by the endogenous H2O2 generated by the auto-oxidation of the thiol groups. However, given that thiols are poor substrates for HRP, the monomer used in this case required a large number of thiol groups on its side chain to form a hydrogel network and a large amount of HRP was also needed. More recently, Moriyama and co-workers reported the preparation of a redox-sensitive hydrogel using a small amount of HRP under physiological conditions by adding phenol derivatives to a mixture of HRP and thiol-functionalized 4-arm PEG, whilst avoiding the need for exogenous H2O2.31,32 In this case, the HRP was activated by the H2O2 generated by the auto-oxidation of the thiol groups and catalyzed the oxidation of the phenol molecules, which behaved as better substrates for HRP than thiols, to form phenoxy


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radicals. The phenoxy radicals subsequently underwent a radical transfer reaction with the thiol groups to give the corresponding thiol radicals, which reacted to form disulfide bonds with the concomitant formation of H2O2. Notably, all of the reactions used to form redox-sensitive hydrogels in these reports were catalyzed by HRP under mild conditions and did not require the addition of exogenous H2O2 for the gelation step. These systems are therefore suitable for the encapsulation of delicate materials, including proteins and even cells. However, in order to efficiently introduce functional molecules to the redox−sensitive hydrogel prepared by the crosslinking of thiol-groups, it requires end-thiolated or -maleimide compounds to be mixed in the monomer solution at the time of hydrogel preparation. This would inhibit the formation of the hydrogel network as the load of functional molecules increases and also modification of the hydrogels after gelation is not so efficient. For this reason, redox-sensitive hydrogel capable of being functionalized by orthogonal chemistry to the thiol groups could be an ideal platform for introducing or immobilizing delicate functional molecules. Herein, we report the development of a new method for the preparation of redox-sensitive hydrogels that can be readily functionalized with any protein or chemical compound with a high level of introduction efficiency, even after the formation of the hydrogel. To achieve this purpose, we focused on streptavidin (SA). We anticipated that the immobilization of SA on a redoxsensitive hydrogel would provide an orthogonal interface for the functionalization of hydrogel. In this way it was envisaged that it would be possible to conjugate chemicals and proteins to the hydrogel even after hydrogelation through biotin–SA interactions. Furthermore, the resulting SA-immobilized hydrogels would dissolve under reductive conditions, making them ideal candidates for the development of drug delivery systems (DDSs) capable of releasing their cargos within the cytosol.


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In this study, sequences of HHHHHHC and GGGGY (Y-tag) were genetically fused to the Nand C-termini of SA (C-SA-Y), respectively, and the C-SA-Y was mixed with HRP and thiolfunctionalized 4-arm PEG (SUNBRIGHT PTE-200SH: tT-PEG) to yield a redox-sensitive CSA-Y immobilized hydrogel (C-SA-Y gel). In this system, the phenol moiety of the Tyr residue on the Y-tag was efficiently recognized by HRP to form tyrosyl radicals, which underwent a radical transfer to the thiol group on the Cys residue at the N-terminus of C-SA-Y and the terminus of tT-PEG, resulting in the cross-linking of C-SA-Y and tT-PEG via disulfide bonds to form a hydrogel (Fig. S1). In addition, C-SA-Y gel was prepared within w/o emulsion system to yield a nano-sized hydrogel (C-SA-Y nanogel), and its properties as DDS carrier was assessed.

Materials and method Materials. SUNBRIGHT PTE-200SH was purchased form NOF Corporation (Tokyo, Japan). Horseradish peroxidase (HRP) was obtained from Sigma Aldrich (St. Louis, MO, USA). GlycylL-tyrosine Hydrate (Gly-Tyr) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Biocytin Alexa Fluor® 488 (AF488-biocytin), EZ-Link™ Sulfo-NHS-SS-Biotin (sulfoNHS-SS-biotin), Alexa Fluor® 488 Carboxylic Acid Succinimidyl Ester mixed isomers (NHSAF488) , and Opti-MEM® I Reduced Serum Media were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Cell penetrating peptide (CPP) (Biotin-G3R15GYC) was synthesized by and purchased from MBL (Aichi, Japan). Cell Counting Kit 8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan).


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Preparation of the C-SA-Y gel. Ten millimolar Tris-HCl buffer (pH 8.0) containing 100 µM of C-SA-Y, 2.5 mM of tT-PEG and 10 unit/mL of HRP was prepared in a glass tube. The same reaction was also set up using C-SA (without the Y-tag sequence at the C-terminus) instead of CSA-Y. Hydrogel formation was observed for up to 48 h at 37 °C. PEG gel was prepared by the same manner except Gly-Tyr was used instead of C-SA-Y or C-SA to initiate the hydrogelation. Conjugation of biotinylated fluorescent molecules to the C-SA-Y gel. A C-SA-Y, tT-PEG, HRP mixed solution was prepared on a glass slide printed with a highly water-repellent mark (TF2404, Matsunami Glass Ind., Ltd, Osaka, Japan) in a similar manner to that described above, but with the addition of AF488-biocytin at different concentrations. Hydrogel formation was conducted at 37 °C for 24 h. The unconjugated AF488-biocytin was removed by immersing the hydrogel in 10 mM Tris-HCl buffer (pH 8.0) and the buffer was changed 3 times at 3-h intervals. The AF488-biocytin remaining in the hydrogel was then observed using a fluorescent imaging scanner (Typhoon 9410, GE Healthcare, Little Chalfont, UK). Formation of C-SA-Y gel within w/o emulsion. Surfactants (75 mg of Span 80 and 25 mg of Tween 80) were dissolved in 3.74 ml of n-hexane, and the resulting solution was used as an organic phase. A solution of C-SA-Y (100 µM) in Milli-Q water containing 2.5 mM tT-PEG was used as an aqueous phase. The aqueous phase (30 µL) was added to 900 µL of the organic phase, and the resulting mixture was subjected to ultrasonic irradiation on a Branson Sonifier 250 (Emerson Electric Co., St. Louis, MO, USA) at a duty cycle of 30% and output control of 1 for 60 s (3 times) to form an inverse emulsion system. Cross-linking was initiated by the addition of 2 µL of HRP solution (150 unit/mL in 10 mM Tris-HCl buffer, pH 8.0), followed by 30 s of ultrasonication. The reaction was allowed to proceed for 1 hour at 37 °C with constant stirring. The remaining free thiol groups were quenched by the addition of 1 µL of 75 mM iodoacetamide.


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The separation of the resulting C-SA-Y nanogel was achieved by adding 150 µl of TBS buffer (10 mM Tris-HCl, 150 mM NaCl, pH 8.0), followed by the centrifugation of the resulting mixture at 16,000 ×g for 15 min and the decantation of the supernatant. The C-SA-Y nanogel in the aqueous layer was carefully washed four times with 500 µL of tetrahydrofuran (THF) to remove the surfactants. The remaining organic solvents were removed by dialysis. The purified C-SA-Y nanogel was stored in 10 mM Tris-HCl buffer (pH 8.0) at 4 °C. PEG nanogel was prepared in a similar manner to that described above, but 400 µM of Gly-Tyr was used instead of C-SA-Y. Transmission electron microscope (TEM) observation of C-SA-Y nanogel. TEM images were recorded on a micro grid with a specimen supporting film (COL-C15, STEM Co., Ltd, Tokyo, Japan). Two microliters of C-SA-Y nanogel were mixed with 2 µL of 5% gadolinium acetate on the micro grid. After drying the micro grid, the sample image was observed by TEM (JEM-4000FX, JEOL Ltd, Tokyo, Japan). Dynamic light scattering (DLS) analysis. The morphological characteristics of the C-SA-Y nanogel were assessed using a Zetasizer (Nano-ZS, Malvern Instrument Ltd, Worcestershire, UK). A low-volume glass cuvette (ZEN2112, Malvern Instrument Ltd) was used for the measurement of C-SA-Y nanogel, with the whole experiment conducted at 25 °C. The value of intensity was used for the observation, and the Z-average value was used as average diameter of C-SA-Y nanogel. Biotinylation and labeling with fluorescent molecules on RNase A and saporin. RNase A was modified with biotin via a disulfide linker using 0.8 equivalents of sulfo-NHS-SS-biotin in 100 mM boric buffer (pH 8.3) at RT for 3 h. An additional 2 eq. of NHS-AF488 was then added


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to the same solution, and the resulting mixture was incubated at RT for 3 h. The unreacted NHS groups in the solution were quenched by the addition of 50 µL of 1 M Tris-acetate (pH 8.0) and the buffer was replaced with 10 mM Tris-HCl buffer (pH 8.0) using an ultrafiltration membrane with a MWCO of 10 kDa. Biotinylation and labeling with AF488 on saporin was also conducted in the same manner as RNase A, but using 2 eq. of sulfo-NHS-SS-biotin in the first step instead of 0.8 eq. Fluorescence modification of biotinylated CPP. Three hundred micro molar of biotinylated CPP (Biotin-G3R15GYC-Alexa Fluor 546) was modified using a 3.3-fold molar excess of Alexa Fluor AF546-C5-maleimide in 100 mM MOPS buffer (pH 7.1). After reacting for 3 h at RT, the CPP-AF546 product was purified by HPLC using 0.1% trifluoroacetic acid (TFA) in acetonitrile (solution A) and 0.1% TFA in Milli-Q water (solution B) as the mobile phases. HPLC was performed over a YMC-Pack ODS-A column (YMC Co., Ltd, Kyoto, Japan), which was equilibrated with 5% solution A (95% solution B), followed a gradient elution with solution A from 10 to 40 % over 30 min for the purification of the products. Fractions containing CPPAF546 were lyophilized and solubilized in 10 mM Tris-HCl buffer (pH 8.0) before being stored at −30 °C. Biotinylation of anti-epiregulin IgG (9E5). 9E5 was kindly provided as a gift from Prof. Shibasaki at the Research Center for Advanced Science and Technology, the University of Tokyo, Japan. 9E5 was biotinylated with a 2.5-fold molar excess of amine-reactive sulfo-NHSLCLC-biotin in 100 mM boric buffer (pH 8.3) at RT for 3 h. The residual NHS groups were quenched by the addition of 50 µL of 1 M Tris-acetate and the buffer was subsequently changed to 10 mM Tris-HCl buffer (pH 8.0) using an ultrafiltration membrane with a MWCO of 100 kDa.


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Functionalization of the C-SA-Y nanogel. To allow for the internalization of the C-SA-Y nanogel, we added CPP-AF546 or biotinylated 9E5 to the C-SA-Y nanogel with a molar equivalency of 0.4 relative to the C-SA-Y in the nanogel, and the resulting mixture was incubated at RT for 1 h. To allow for the introduction of a cytotoxic agent to the nanogel, we added 8 molar equivalents of biotinylated saporin to the C-SA-Y nanogel suspension and allowed the resulting mixture to react for 24 h. Any unreacted saporin was removed using an ultrafiltration membrane with a MWCO of 100 kDa. Confocal laser scanning microscopy (CLSM) observation. DLD1 cells inoculated at a density of 1.0×105 cells/mL on a 3.5 cm glass-bottomed dish were precultured at 37 °C overnight in RPMI medium. The DLD1 cells were then washed with PBS three times to remove the FBS and treated with 100 µL of Opti-MEM® I Reduced Serum Media. Fifteen microliters of the C-SA-Y nanogel suspension functionalized with CPP-AF546 as a targeting molecule and biotinylated saporin as a protein drug were added to the DLD1 cells, and the resulting mixture was incubated for 1 h at 37 °C. After the incubation, the remaining nanogel was removed by washing the cells with 1 mL of PBS (3 times), followed by the addition of 1 mL of RPMI medium. The cells were then incubated for 3 h at 37 °C before being observed by CLSM (LSM510, Carl Zeiss Co., Ltd, Germany). C-SA-Y nanogel functionalized with biotinylated 9E5 was introduced to the DLD1 cells in the same manner as C-SA-Y nanogel functionalized with CPP. The only difference in this case was that RPMI medium was used instead of Opti-MEM® I Reduced Serum Media for incubating the DLD1 cells with the C-SA-Y nanogel functionalized with 9E5. Flow cytometry. DLD1 cells were plated in a 6-well flat bottom dish (Asahi Glass Co., Ltd, Tokyo, Japan) and preincubated at 37 °C overnight in RPMI medium. The cells were then


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washed and treated with 15 µL of C-SA-Y nanogel functionalized with CPP and biotinylated saporin in the same way as the material used for the CLSM analysis. After 3 h of incubation, the cells were treated with 100 µL of trypsin/EDTA for 3 min and collected in 500 µL of PBS. The cells were then sorted on a FACSCalibur flow cytometer (Becton-Dickinson, Lexington, KY, USA) and the fluorescence of the AF488 attached to the biotinylated saporin was measured. Cell viability assay. DLD1 cells prepared at the concentration of 2×104 cells/ml were plated in a 96-well flat bottom dish (Asahi Glass Co., Ltd, Tokyo, Japan) and preincubated at 37 °C overnight in RPMI medium. The medium was changed for fresh RPMI medium, and a 15-µL suspension of C-SA-Y nanogel functionalized with CPP or 9E5 and biotinylated saporin was added to each well and incubated for 1 h at 37 °C. The unreacted C-SA-Y nanogel was washed out with PBS three times and RPMI medium was added. DLD1 cells were further incubated at 37 °C for 48 h. The DLD1 cells were then treated with 10 µL of CCK-8 and incubated at 37 °C for 1 h to progress the reaction of CCK-8. The absorbance at 450 nm was measured using a multi-mode microplate reader (Molecular Devices, Sunnyvale, CA, USA). Results and discussion Preparation of C-SA-Y gel. We have successfully prepared C-SA-Y in a tetramer form, which was confirmed by SDS-PAGE analysis (Fig. S2). We initially validated the gelation of tT-PEG by adding C-SA-Y and HRP. Ten millimolar Tris-HCl buffer (pH 8.0) containing tT-PEG (5 mM), C-SA-Y (100 µM) and HRP(10 unit/mL) yielded a clear hydrogel after 9 h of incubation at 37 °C, whereas a sample containing tT-PEG, C-SA and HRP did not show any change even after 48 h of incubation (Fig. 1A, B). In addition, hydrogel was not formed in the absence of C-SA-Y or HRP (Fig. 1C, D).


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Figure 1. Preparation of redox-sensitive hydrogel composed of tT-PEG and C-SA-Y using HRP reaction. (A-D) Photographs of tT-PEG solutions (pH 8.0) after 24 hours of incubation in the presence of (A) C-SA-Y and HRP, (B) C-SA and HRP, (C) C-SA-Y, and (D) HRP.

These results suggested that the Tyr residue in the Y-tag was efficiently recognized by HRP and was therefore responsible for the gelation, working as a co-catalyst to promote the formation of disulfide bonds between the thiol groups in tT-PEG and the Cys residue of C-SA-Y. It has been reported that the Y72 residue in SA exhibits weak reactivity towards HRP treatment.33 However, given that the sample containing C-SA described above did not result in the formation of hydrogel within the time range of our experiments, the reactivity of Y72 was negligible for accelerating the formation of the hydrogel. The resulting C-SA-Y gel readily dissolved in 100 µL


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of a 10 mM dithiothreitol solution, which indicated that the hydrogel was formed by disulfide bonds (Fig.S3). Moriyama and co-workers31 previously reported that the gelation time of the redox-sensitive hydrogel using HRP and a variety of different phenols varied considerably depending on the nature of the phenol derivative being used. This result suggested that the kinetics for the conversion of phenols to the corresponding radical species by HRP recognition would have a pronounced effect on the overall rate of the hydrogel formation process. According to this report, the Tyr residue of the Y-tag on the C-terminus of SA dictated the outcome of the radical transfer reaction to generate the thiol radicals. It was envisaged that the reactivity of the Tyr residue on the Y-tag would be considerably lower than that of the small phenol molecules examined in the previous report because of steric hindrance and the low diffusion velocity caused by it being tethered to the large SA molecule. This also explains the long gelation time of this material. However, this enzymatic reaction also required oxygen for the formation of H2O2, making the gelation time dependent on the oxygen concentration in the solution. The gelation time of the C-SA-Y gel could therefore be shortened by enlarging the surface area of the solution to achieve rapid oxygen dissolution from the air to the solution through changing the shape and size of the container. We have assessed this concept by preparing PEG gel with Gly-Tyr on the two different size of a glass slide printed with a highly water-repellent mark. The first condition is the whole solution was placed on a single mark as a drop of 60 µL, and the second condition is that 4 drops of 15 µL of solution were placed in four different marks. The hydrogel prepared by second condition resulted in hydrogel formation in 41 minutes, while those prepared by first condition took 56 minutes for hydrogel formation. This strongly suggests that the large surface area will accelerate the hydrogel formation (Fig. S4).


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To further examine this hydrogelation process, we evaluated the effect of concentrations of CSA-Y and HRP on the C-SA-Y hydrogel formation rate (Fig. S5). The results showed that hydrogel formation was accelerated as the concentration of C-SA-Y increased, and it was decelerated as the concentration of HRP increased. These results were consistent with the result reported by Moriyama et al.31 Fluorescence labeling of C-SA-Y gel. C-SA-Y gel was then prepared on a glass slide, which was printed with a highly water-repellent mark (Matsunami Glass Ind., LTD., Osaka, JAPAN). AF488-biocytin was added at concentrations of 2, 4 and 6 µM, representing 0.02, 0.04 and 0.06 eq, respectively, relative to the C-SA-Y in the hydrogel. Hydrogel without C-SA-Y was also prepared for comparison by adding Gly-Tyr (500 µM) to promote the HRP-mediated hydrogelation of tT-PEG in the presence of 6 µM AF488-biocytin. Following the removal of the unconjugated AF488-biocytin by soaking the hydrogels in 10 mM Tris-HCl (pH 8.0), the samples were observed using a fluorescence imager (Fig. 2A, B). The fluorescence intensities of the C-SA-Y gels increased as the concentration of AF488-biocytin increased, whereas the hydrogel formed without any C-SA-Y showed much lower fluorescence intensity than the C-SAY gel, which suggested that the C-SA-Y in this hydrogel had retained its affinity to biotin.


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Figure 2. Evaluation of biotin binding ability of C-SA-Y gel. (A) Bright-field images and fluorescence images of C-SA-Y gels and PEG gel treated with AF488-biocytin, on a glass slide with a highly water-repellent mark. BF: bright-field images; FL: fluorescence images. (B) Result of quantitative analysis of fluorescence intensity of the C-SA-Y gels and PEG gel shown in (A) by using ImageJ. The fluorescence intensity of PEG gel was defined as 1.

Characterization of C-SA-Y gel prepared within w/o emulsion system. We investigated the preparation of a C-SA-Y gel within a w/o emulsion with the aim of controlling the size of the hydrogel particles to make them more suitable for use in DDSs. Nanocarriers with a diameter in the range of 100–200 nm are known to exhibit enhanced permeability and retention (EPR) effect-


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based accumulation properties in solid tumors.34,35 With this in mind, we selected an emulsion composed of Span 80 and Tween 80 in hexane for the reaction vessel to yield nanosized hydrogel particles with diameters in the range of 100–200 nm.30 The hydrogelation of the C-SAY gel was conducted in the w/o emulsion mentioned above for 1 h at 37 °C, followed by the quenching of the unreacted thiol groups with iodoacetamide. Particle size analysis of this C-SAY nanogel by DLS resulted in a single peak around 70 nm, with a calculated Z-average diameter of 59.93 ± 0.59 nm. (Fig. 3A) The TEM images of the C-SA-Y nanogel showed that it existed as uniformly sized spherical hydrogel particles with an average diameter in the range of 70–80 nm, which was consistent with the result of the DLS analysis. (Fig. 3B)


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Figure 3. Evaluation of morphologies of C-SA-Y nanogel. (A) Result of DLS analysis of C-SAY nanogel and the z-average diameter was 59.93±0.59 nm. (B) TEM image of C-SA-Y nanogel stained with 5 % Gadolinium acetate. The scale bar is 500 nm.

These results therefore showed that the C-SA-Y nanogel was obtained in 1 h whereas the formation of C-SA-Y gel took 9 h. The difference in these results could be attributed to the large surface area of the solution within the w/o emulsion, which would lead to an increase in the


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concentration of oxygen in the solution, thereby promoting the generation of H2O2 and the activation of HRP. We next evaluated C-SA-Y nanogel’s amenability to functionalization. We initially examined the loading capacity of the C-SA-Y nanogel by treating it with biotinylated protein and then quantifying the amount of conjugated material (Fig. S7). RNase A was modified with sulfo-NHS-SS-biotin and NHS-AF488 to yield fluorescent biotinylated RNase A; the biotin and protein of which could be separated under reductive conditions. PEG nanogel was prepared in a w/o emulsion system using Gly-Tyr instead of C-SA-Y. Biotinylated RNase A was added to the C-SA-Y and PEG nanogels, and the resulting mixture were incubated for 24 h at 4 °C. The solutions were then centrifuged through an ultrafiltration membrane with a MWCO of 100 kDa to separate the nanogel and the flowthrough containing unreacted biotinylated RNase A. The amount of unreacted RNase A was subsequently estimated by spectrofluorometry. Approximately 50% of the C-SA-Y immobilized in the nanogel was capable of interacting with the biotinylated RNase A, whereas only 20% of the RNase A was incorporated in the PEG nanogel compared with the C-SA-Y nanogel. We subsequently investigated the release of the incorporated biotinylated RNase A under reductive condition by SDS-PAGE (Fig. S8). The incubation of the C-SA-Y nanogel under non-reductive conditions did not result in the release of an RNase A, whereas the network structure of the C-SA-Y nanogel treated with 2mercaptoethanol degraded under the same conditions, resulting in the release of RNase A. These results suggested that the C-SA-Y gel did not lose its distinct properties (i.e., affinity for biotin and response to reductive agents) under these conditions, even after the being reduced in size to the nanometer scale. Cell internalization of C-SA-Y nanogel and viability assay. To verify the feasibility of using this C-SA-Y nanogel as a DDS, we selected a cell penetrating peptide (CPP) and saporin to


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provide cell internalization and cytotoxic properties to the C-SA-Y nanogel, respectively. Saporin modified with biotin, AF488 and CPP were conjugated to the C-SA-Y nanogel, and the resulting functionalized C-SA-Y nanogel was added to DLD1 cells. CLSM images of the treated DLD1 cells showed the fluorescence in the cytosol resulting from the CPP and saporin after 1 h of incubation, whereas no clear fluorescence was observed from the DLD1 cells treated with the C-SA-Y nanogel functionalized with saporin without CPP (Fig.4A). Flow cytometry analysis showed that almost all DLD1 cells uptook C-SA-Y nanogel functionalized with both saporin and CPP (94.7 %), while nanogel without attaching CPP showed small incorporation to the DLD1 cells (4.5 %), which was consistent with the CLSM results (Fig. 4B, Fig. S9).


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Figure 4. Uptake assay of the functionalized C-SA-Y nanogel on DLD1 cells. (A) CLSM images of DLD1 cells treated with C-SA-Y nanogel functionalized with CPP and saporin (left column) and C-SA-Y nanogel functionalized with saporin (right column). (B) Results of flow cytometry


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analysis of DLD1 cells treated with C-SA-Y nanogel functionalized with CPP and saporin labeled with AF488. Control: DLD1 cells without any treatment.

In addition, the C-SA-Y nanogel functionalized with biotinylated 9E5 showed good internalization towards DLD1 cells, which are known to overexpress epiregulin on their surface. In contrast the C-SA-Y nanogel functionalized with 9E5 did not show any internalization against AGS cells, which have a lower level of epiregulin expression on their surface (Fig. S10).36 These results therefore showed that C- SA-Y nanogel can be functionalized by any ligand or drug through a biotin–SA interaction. C-SA-Y nanogels without any targeting molecules did not exhibit any activity towards DLD1 cells, indicating that C-SA-Y nanogels have great potential as DDSs for the cell-specific deliver of drugs. Lastly, DLD1 cells were incubated with C-SA-Y nanogel functionalized with CPP and saporin for 48 h and the cell viability was measured using a CCK-8 assay (Fig. 5). The result of the cell viability assay revealed that the C-SA-Y nanogel prepared without CPP or saporin had no discernible impact on the viability of the DLD1 cells. In contrast, the treatment of the DLD1 cells with the C-SA-Y gel functionalized with both CPP and saporin resulted in a pronounced decrease in the proliferation of the cells, indicating that the C-SA-Y nanogel had been internalized into the cell via the CPP, allowing saporin to exhibit its cytotoxicity. The internalization ability of CPP and cytotoxicity of saporin were therefore successfully integrated on the C-SA-Y nanogel, with both properties working cooperatively to give a cytotoxic C-SA-Y nanogel.


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Figure 5. Cytotoxicity assay of DLD1 cells incubated with C-SA-Y nanogel functionalized with CPP and saporin. The viability of cells without any treatment was set as 100%. Cells without any treatment (1) and treated with C-SA-Y nanogel (2), C-SA-Y nanogel with CPP (3), C-SA-Y nanogel with saporin (4), C-SA-Y nanogel with CPP and Saporin (5), and Saporin(6).

Furthermore, the C-SA-Y nanogel functionalized with 9E5 and biotinylated saporin showed a similar trend in terms of its effect on the viability of DLD1 cells (Fig. S11), highlighting the advantage of C-SA-Y hydrogel as a DDS, which can be functionalized with a wide variety of functional molecules via biotin.

Conclusions In summary, we have demonstrated that SA-immobilized redox-sensitive hydrogels can be readily prepared using HRP-catalyzed enzymatic reaction by simply attaching a Cys residue and a Y-tag to the terminus of SA. Furthermore, this enzymatic reaction took place under mild


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conditions, allowing for the C-SA-Y immobilized in the C-SA-Y gel and C-SA-Y nanogel to retain its affinity for biotin. This meant that various other biotinylated functional molecules could be incorporated to the gels via the formation of a biotin–SA interaction, even after the gelation process. This methodology could be applied to a variety of different proteins by simply adding a Y-tag and a Cys residue to their surfaces, suggesting that hydrogels with various functionalities could be readily prepared in this way. Although the immobilization of proteins on hydrogels using an amine-coupling reaction has been reported,37,38 the amino groups in these protein are randomly modified by the molecules involved in the construction of the hydrogel networks. These processes could therefore result in a loss of protein function, as well as low immobilization efficiency for the protein of interest. In contrast, our newly developed method allowed for the efficient cross-linking of the hydrogel molecules to the target protein under mild condition through the introduction of an extra Cys residue, without inhibiting the function of the protein. Furthermore, the tethering of the Tyr residue co-catalyst in the Y-tag to the protein itself allowed for the cross-linking reaction to be initiated by HRP in close proximity to the target protein. This process therefore promoted efficient incorporation into the hydrogel network. Together with the redox-sensitive properties of the resulting hydrogel, the protein-immobilized hydrogel prepared by the method demonstrated here is promising in terms of its potential application in the field of biotechnological research.

ASSOCIATED CONTENT Supporting Information. The following information is available free of charge at ACS Publications website. Construction of expression vectors for C-SA and C-SA-Y. Expression and


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purification of C-SA and C-SA-Y. Examination of loading capacity of C-SA-Y nanogel. Protein releasing assay from C-SA-Y nanogel. Observation of tT-PEG gel solution in the presence of HRP or C-SA-Y. Reduction assay on C-SA-Y gel. CLSM observation on the C-SA-Y nanogel treated DLD1 and AGS cells. Cytotoxicity assay of DLD1 cells incubated with C-SA-Y nanogel with IgG.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected], Tel: +81-3-5841-7328, Fax: +81-3-5841-8657 Author Contributions K.M. and K. Mo. designed the research. M.M. performed all of the experiments under the supervision of K.M. and T.N. The manuscript was written by M.M. with assistance from K.M., K. Mo. and T.N. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT A part of this work was supported by “Nanotechnology Platform” (project No.12024046) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Anti-epiregulin IgG (9E5) was a kind gift from Prof. Yoshikazu Shibasaki in Research Center for Advanced Science and Technology, the University of Tokyo.


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