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Article Cite This: Biomacromolecules 2019, 20, 2350−2359

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Switching of Cell Proliferation/Differentiation in Thiol−Maleimide Clickable Microcapsules Triggered by in Situ Conjugation of Biomimetic Peptides Yuichiro Oki,† Katsuhisa Kirita,‡ Seiichi Ohta,§ Shinsuke Ohba,‡,§ Ikki Horiguchi,⊥ Yasuyuki Sakai,†,‡ and Taichi Ito*,†,‡,§ †

Department of Chemical System Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan § Center for Disease Biology and Integrative Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan ⊥ Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan

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‡

S Supporting Information *

ABSTRACT: Extracellular environments significantly affect cell proliferation, differentiation, and functions. The extracellular environment changes during many physiological and pathological processes such as embryo development, wound healing, and tumor growth. To mimic these changes, we developed novel thiol−maleimide clickable alginate microcapsules, which can introduce thiol-containing peptides by “in situ conjugation” with maleimide-modified alginate, even in serum-containing cell culture media. Additive peptides were rapidly concentrated into microcapsules by a diffusionreaction process in the capsule. The proliferation of encapsulated fibroblasts was accelerated by in situ conjugation of CRGDS, while free RGDS showed no effect. Moreover, encapsulated preosteoblastic cells started osteogenic differentiation via in situ conjugation of BMP-2 mimetic peptides such as CDWIVA and CG-BMP-2 knuckle epitope peptide, while BMP-2 did not induce differentiation of the encapsulated cells. Especially in tissue engineering, accurate and inexpensive methods for inducing cell differentiation are required. We believe that this in situ conjugation approach employing various functional peptides will be useful in biomedical, bioindustrial, and biochemical fields in the future.

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promising approach.11 Hence, many cell-adhesive12 or humoral factor-bound13,14 ECM mimetic synthetic scaffolds have been developed.15 However, the chemical structure of these previously reported scaffolds did not change during cell culture process, and it has proven difficult to mimic ECM modification mediated by reaction-diffusion. Bioorthogonal click reactions16,17 are expected to be a promising chemical tool for immobilizing new components to scaffolds. Examples include the Diels−Alder reaction,18 tetrazine ligation,19 azide−alkyne cycloaddition,20 and thiol− ene reactions.21−25 Because the click reactions proceed under physiological conditions even in the presence of cells, in situ cross-linkable hydrogels produced via click reactions were developed for tissue engineering applications.26−30 Due to their excellent biocompatibility, it is expected that bioorthogonal click reactions will become an ideal chemical tool for

INTRODUCTION

Living cells are always stimulated by a surrounding extracellular matrix (ECM)1 such as collagen, hyaluronan, laminin, or elastin. Their proliferation, differentiation, and functions are strongly affected by these cell-adhesive ECM components. Thus, the molecular composition of the ECM changes during wound healing, fibrosis in various diseases, and even embryo development.2−5 Additionally, living cells secrete various ECM-binding proteins such as osteopontin6 and thrombospondin,7 which subsequently affect their activities. This ECM modification involves chemical reaction-diffusion. ECM-binding proteins secreted from cells, such as fibronectin,8 typically penetrate and bind to the surrounding ECM, changing its properties. Furthermore, humoral factors also diffuse and adsorb to the ECM, enhancing their functions. For example, BMP-2 is reported to adsorb to heparin in the ECM, accelerating BMP-2-induced osteoblast differentiation.9 Tissue engineering and regenerative medicine are emerging research fields,10 where mimicking biological functionality using chemically synthetic materials is expected to be a © 2019 American Chemical Society

Received: March 6, 2019 Revised: May 3, 2019 Published: May 6, 2019 2350

DOI: 10.1021/acs.biomac.9b00333 Biomacromolecules 2019, 20, 2350−2359

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Biomacromolecules achieving “in situ conjugation” of functional molecules to scaffolds, which can change the extracellular environment to stimulate cultured cells/tissues. Among several click reactions, the thiol−maleimide reaction24,29 is one of the candidates for in situ conjugation due to its performance and safety in recent clinical applications of antibody−drug conjugates.25 In demonstrating the potential of in situ conjugation, rapid diffusion is also an important factor. Microcapsules are a desirable platform for tissue culture scaffolds with fast mass transfer due to their higher specific surface area and shorter diffusion distance compared with bulk hydrogels. For example, alginate microcapsules cross-linked with calcium ions enable cell encapsulation and culture via their rapid gelation and good biocompatibility.31−33 Alginate microcapsules are potentially suitable for various biomedical applications, such as islet transplantation in type I diabetes therapy.34 In the present research, we have, for the first time, stimulated cells in microcapsules via in situ conjugation of cysteine-terminated biomimetic peptides to maleimide-modified alginate (Alg-Mal), even in serum-containing cell culture media as shown in Figure 1. The cysteine-terminated peptides

from Scrum Inc. (Tokyo, Japan). The molecular weights of the above peptides were 425, 519, 533, 601, 704, 2120, and 2277, respectively. Fetal bovine serum (FBS) was purchased from Biosera, Inc., (Villebon-sur-Yvette, France). MEM Alpha (αMEM) with nucleosides, G418, and 5-((2-(and-3)-S-(acetyl mercapto)succinoyl)amino) fluorescein (SAMSA-FL) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The molecular weight of SAMSA-FL was 521. EDTA·2Na (2NA) was purchased from Dojindo Laboratories (Tokyo, Japan). NIH-3T3 was purchased from RIKEN Cell Bank (Tsukuba, Japan). Col1a1GFP-MC3T3E1 cells were developed from MC3T3E1 cells (RIKEN Cell Bank, Tsukuba, Japan) in our previous work.35 Synthesis of Alg-Mal. Alg-Mal was synthesized via a carbodiimide reaction according to a previous study.23 Briefly, 500 mg of Alg was dissolved in 100 mL of 0.1 M MES buffer. WSCD/HCl (2.35 g, 12.3 mmol) and NHS (1.43 g, 12.4 mmol) were added to the solution, and the resulting mixture was stirred for 30 min. N-(2aminoethyl) maleimide hydrochloride (88.3 mg, 0.50 mmol) dissolved in 5 mL of 0.1 M MES buffer was then added to the mixture, followed by pH adjustment to 6.0 with 0.1 M NaOH. After reacting for 20 h at room temperature, the reaction was quenched by increasing the pH to 7.5 with sodium hydrogen carbonate. The reaction mixture was subsequently dialyzed, first against 50 mM NaCl for 3 days and then against deionized water for 3 days using the dialysis membrane. The polymers were obtained as white foams after lyophilization for 3 days. The synthesis of Alg-Mal was confirmed by 1 H nuclear magnetic resonance (NMR; α500, JEOL, Tokyo, Japan) and Fourier-transform infrared spectroscopy (FT-IR; FT/IR 4000, JASCO, Tokyo, Japan). D2O was used as a solvent for 1H NMR analysis. FT-IR spectra were obtained using a KBr tablet of the polymer. The degree of modification (DM), defined as the number of maleimide groups per disaccharide ring of Alg, was determined by 1H NMR. In the 1H NMR spectrum, the signals at δ = 4.6 and 5.0 ppm were assigned to the proton on carbon-1 of mannuronic acid and guluronic acid of Alg, respectively.36 On the other hand, the signal at δ = 6.2 ppm was assigned to the vinyl protons of the maleimide moieties.37 Thus, the DM was calculated using the ratio of these two integral values. Preparation of Alg-Mal Hydrogels and Young’s Modulus Measurement. Aqueous Alg-Mal (2 wt %) was injected into rubber molds sandwiched between two Nuclepore membranes with pore sizes of 1 μm. The molds were then immersed in 50 mM aqueous CaCl2 overnight to obtain Alg-Mal hydrogel disks with diameter of 1.2 cm and thickness of 3.5 mm. To investigate the effect of peptide conjugation on the properties of Alg-Mal hydrogels, Alg-Mal was also conjugated with CRGES or CRGDS38,39 prior to hydrogel fabrication; the same number of moles of CRGES or CRGDS as maleimide groups of Alg-Mal was added to the 2 wt % Alg-Mal aqueous solution and incubated for 6 h at room temperature, followed by hydrogel preparation via the same procedure without further purification. For Young’s modulus measurements, the obtained hydrogels were cut to 8 mm diameter with a disposable biopsy punch (Kai. Industry Co., Ltd., Gifu, Japan). The Young’s moduli of these hydrogels were determined by indentation testing using a rheometer (CR-3000EXs, Sun Scientific Co., Ltd., Tokyo, Japan). Preparation of Alg-Mal Microcapsules and Cell Encapsulation. Alg-Mal microcapsules were prepared using a coaxial two-fluid nozzle, based on previously reported procedures.33 Aqueous Alg-Mal (2 wt %) was discharged from the inner nozzle (internal diameter 0.27 mm, 26G) into 200 mL of 50/100 mM aqueous CaCl2 stirred at 300 rpm under flowing N2 gas (2.0 L min−1) from the outer nozzle (internal diameter 1.3 mm, 16G). The total ionic strength of the aqueous CaCl2 was adjusted with NaCl to maintain the physiological osmotic pressure. The obtained microcapsules were washed gently with saline containing 1.0 mM CaCl2. The obtained microcapsules were observed by optical microscopy (IX73, OLYMPUS, Tokyo, Japan) immediately after the fabrication. The diameter of the microcapsules was measured by using ImageJ. The number of measured microcapsules was 100 and the data is presented as the mean ± standard deviation.

Figure 1. Concept of in situ conjugation of biomimetic peptides using clickable microcapsules.

diffuse into Alg-Mal microcapsules and bind to immobilized maleimide groups via a thiol−maleimide reaction, stimulating the encapsulated cells. Switching of cell proliferation/differentiation by in situ conjugation of functional peptides will be demonstrated using this system.

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EXPERIMENTAL SECTION

Materials. Alg (IL-6G, Mw = 750−850 kDa) was kindly gifted by Kimica Co. (Tokyo, Japan). Alginate lyase was purchased from SigmaAldrich (St. Louis, MO, USA). Water-soluble carbodiimide hydrochloride (WSCD)/HCl, Dulbecco’s modified Eagle’s medium (DMEM), penicillin−streptomycin−amphotericin B (PSA), recombinant human BMP-2, and 0.4 w/v% trypan blue solution were purchased from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan). N-Hydroxysuccinimide (NHS) and N-(2-aminoethyl) maleimide hydrochloride were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Dialysis membrane (Spectra/Por, MWCO = 6000−8000 Da) was purchased from Spectrum Laboratories Inc. (Rancho Dominguez, CA, USA). Peptides RGDS, CRGDS, CRGES, DWIVA, CDWIVA, KIPKASSVPTELSAISTLYL, and CGKIPKASSVPTELSAISTLYL were purchased as dry powders of the trifluoroacetic acid salt with purities above 95% 2351

DOI: 10.1021/acs.biomac.9b00333 Biomacromolecules 2019, 20, 2350−2359

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Figure 2. In situ conjugation of thiolated fluorescent molecules to the microcapsules examined experimentally and via simulations. (A) Confocal microscopy images of Alg and Alg-Mal microcapsules during in situ conjugation of thiolated fluorescent molecules. The microcapsules were incubated with excess thiolated fluorescent molecules. (B) Simulated distribution of conjugated maleimides in Alg-Mal capsules during in situ conjugation. Experimental conditions for (A) were used for the calculation. (C) Time change in the degree of conjugation of thiolated fluorescent molecules to maleimide moiety in Alg-Mal capsules in various media (N = 3). 1 equivalent of the thiolated fluorescent molecule to maleimide was added to the microcapsule. (D) Simulated time change in the degree of maleimide conjugation and the free concentration of thiolated molecules in media. The thiol concentration was normalized by the initial concentration. Experimental conditions for (C) were used for the calculation. Distribution of conjugated maleimides inside the microcapsule is also shown in Figure S6. Swelling of the microcapsules in DMEM was evaluated at 37 °C according to changes in their diameter observed by microscopy after 7 days of incubation. To investigate the effect of in situ conjugation of peptides on the swelling behavior, Alg-Mal capsules were also in situ conjugated with CRGES or CRGDS 1 day after capsule fabrication, followed by observation of the swelling as described above. For culture experiments, NIH-3T3 cells were suspended in 2 wt % aqueous Alg-Mal and encapsulated in the microcapsules at a cell density of 2 × 106 cells/mL. The microcapsules were immersed in DMEM with 10% FBS at 37 °C. Col1a1GFP-MC3T3E1 cells were similarly encapsulated in the microcapsules at a cell density of 1 × 107 cells/mL, using αMEM with 10% FBS, 1% PSA, and 400 μg/mL G418 as a culture medium. In Situ Conjugation of Thiolated Fluorescent Dye to Microcapsules. As a maleimide-reactive fluorescent dye, SAMSAFL was used to evaluate the in situ conjugation. SAMSA-FL was deprotected with 0.1 M NaOH and neutralized with 6 M HCl before use. Alg-Mal microcapsules (20 mg/mL, about 100 microcapsules/ mL) were suspended in 1 mM SAMSA-FL solution, which was in approximately 30 times molar excess relative to the maleimide group. The microcapsules were sampled at each time point and washed repeatedly with saline containing 1 mM CaCl2. The sampled microcapsules were observed by confocal laser scanning microscopy (LSM 510, Carl Zeiss, Oberkochen, Germany). The in situ conjugation was further quantified by fluorescence measurements. Alg-Mal microcapsules (20 mg/mL, about 100 microcapsules/mL) were suspended in saline, DMEM, or DMEM with 10% FBS in a 6-well cell culture plate. Then 35 μM SAMSA-FL, which was one equivalent relative to the maleimide group, was added to the suspension. To evaluate the time course of the degree of conjugation, the microcapsules were sampled at different time points

and washed as described above. The sampled microcapsules were observed via confocal laser scanning microscopy, followed by the image analysis of fluorescent intensity spatial distribution in the microcapsules by ImageJ. The concentration of conjugated SAMSAFL inside the microcapsules was measured with a fluorophotometer (ARVO X3, PerkinElmer, Waltham, MA, USA) after dissolving the microcapsules by chelation of Ca2+ with 10 mM aqueous EDTA. The effect of the added SAMSA-FL on the degree of conjugation was evaluated by adding various amounts of SAMSA-FL followed by fluorescence measurement after incubating for 2 h. Furthermore, the kinetics of the in situ conjugation in the microcapsule were modeled based on a reaction-diffusion mass transport model (Figure S1). The model was solved using a commercial finite element package, COMSOL Multiphysics version 5.4 (COMSOL Multiphysics Burlington, MA). The calculation details are described in the Supporting Information. In Situ Conjugation of RGD Motif Peptides to the Microcapsules and Evaluation of Cell Functions. One day after encapsulating the NIH-3T3 cells, RGDS as a nonreactive peptide, CRGES as an Alg-Mal reactive but noncell-adhesive peptide, or CRGDS as an Alg-Mal reactive and cell-adhesive peptide was in situ conjugated as with SAMSA-FL. The capsules were incubated in DMEM with 10% FBS at 37 °C in 5% CO2. The encapsulated cells were stained using MEBCYTO Apoptosis Kit (MBL, Nagoya, Japan) and analyzed via flow cytometry (BD LSR II, Nippon Becton Dickinson, Tokyo, Japan) to examine the cell apoptosis 1 day after in situ conjugation. Cells treated with 500 μM H2O2 were used as a positive control. The encapsulated cells were collected with alginate lyase. The amount of DNA was determined by DAPI assay (Dojindo Laboratories, Tokyo, Japan) to count the number of cells in microcapsules 4 and 7 days after in situ conjugation. On day 7, the 2352

DOI: 10.1021/acs.biomac.9b00333 Biomacromolecules 2019, 20, 2350−2359

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Biomacromolecules cells were stained using a LIVE/DEAD viability/cytotoxicity kit (Lonza, Basel, Switzerland) to evaluate the cell viability. The cells were also stained with sirius red solution (MUTO PURE CHEMICALS CO., Ltd., Tokyo, Japan) to determine the collagen deposition on day 7. Gene expression of bFGF, TGF-β, and Col1a1 in the cells was measured after capsule degradation with alginate lyase on day 7. First, total RNA extraction and reverse-transcription were performed using RNeasy mini kit (Qiagen, Courtaboeuf, France) and Takara Prime Script RT reagent Kit (TaKaRa Biomedicals, Japan), respectively. Quantitative polymerase chain reaction (RT-qPCR) was subsequently performed with the SYBRFast qPCR Mix Kit (TaKaRa Biomedicals, Japan) and ABI 7500 Fast Real-Time PCR System (Applied Biosystems, CA). The relative expression levels were calculated using the comparative CT method. Glyceraldehyde 3phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. Primer sequences are shown in Table S1. The encapsulated cells were also collected with alginate lyase and reseeded on a cell culture dish to evaluate the proliferation rate after recovery from the 3D cell culture in Alg-Mal microcapsules. Trypan blue staining was used to count live cells for determining a reseeding cell density. The proliferation rate was measured by counting cells via trypan blue staining after a certain time. In Situ Conjugation of BMP-2 Mimetic Peptides to Microcapsules and Evaluation of Osteogenic Differentiation. Additionally, we evaluated osteogenic differentiation by in situ conjugation of BMP-2 mimetic peptides using previously developed preosteoblastic cells, Col1a1GFP-MC3T3E1, which express GFP only on differentiation into osteoblastic cells.35 One day after encapsulating the Col1a1GFP-MC3T3E1 cells, DWIVA,40 KIPKASSVPTELSAISTLYL (BMP-2 KE Pep)41 as a nonreactive peptide, CDWIVA, or CGKIPKASSVPTELSAISTLYL (CG-BMP-2 KE Pep) as an Alg-Mal reactive and osteo-inducive peptide were in situ conjugated as described above. On day 7, confocal microscopy images of the AlgMal microcapsules were obtained by confocal laser scanning microscopy (TCS SP2, Leica Microsystems, Wetzlar, Germany). The GFP fluorescence intensity of encapsulated cells was also measured by flow cytometry (BD LSR II, Nippon Becton Dickinson, Tokyo, Japan). For comparison, Col1a1GFP-MC3T3E1 cells were also seeded on a cell culture dish at 1000 cells/cm2 in the culture media with or without 100 ng/mL recombinant human BMP-2.23 The GFP fluorescence of the cells in the 2D culture was evaluated by confocal laser scanning microscopy and flow cytometry. Statistical Analysis. All data are presented as mean ± standard deviation (N = 3 or 4). Statistical analysis was performed using Student’s t test for two groups. ANOVA followed by Tukey posthoc test were used for the analysis of three or more groups.

Fluorescent Molecules. To visualize and quantify the in situ conjugation, a thiolated fluorescent molecule, SAMSA-FL, was added to Alg-Mal microcapsules rather than the peptides. Figure 2A shows confocal microscopy images of Alg-Mal microcapsules incubated with excess SAMSA-FL at various time points. The Alg-Mal microcapsules rapidly showed fluorescence due to conjugation with SAMSA-FL, while no fluorescence was observed in Alg microcapsules as shown in Figure 2A. At 15 min after starting in situ conjugation, the central region of the microcapsules was fully stained, which was consistent with the estimation of the characteristic time for diffusion (Supporting Information). Time change of the in situ conjugation was further examined using lower concentration of SAMSA-FL (an equal amount to the maleimide group) as shown in Figure S4. The conjugation proceeded from the edge of Alg-Mal microcapsules in a diffusion-limited manner, consistent with its Thiele modulus of 5.1 (details are described in the Supporting Information). The in situ conjugation was also simulated based on a diffusion-reaction model, and the simulation showed similar trend with the experimental results (Figure 2B and Figure S4). On the other hand, the reaction front in the simulation was clearer than that in the experiment. This would be due to the decrease in the rate constant of thiol−maleimide reaction by fixing maleimides to the Alg microcapsules. Clearer spatial control would be expected by utilizing faster bioorthogonal reactions, such as Tetrazine-TCO ligation.42 The conversion of the in situ conjugation in the microcapsules was further quantified via fluorescence measurement by adding an equal amount of SAMSA-FL to the maleimide group. The degree of conjugation rapidly increased to 70 mol % of the initial maleimide groups of Alg-Mal at 30 min and reached a plateau conversion of 85 mol % at 60 min in saline as shown in Figure 2C. The plateau conversion had an almost linear relationship with the amount of added SAMSA-FL, and then approached to ca. 90% of the degree of conjugation calculated from 1H NMR (Figure S5). It is noteworthy that the stability of thiol−maleimide adducts are not so high especially in the presence of thiol-containing compounds, due to a retroMichael reaction or thiol exchange.43 Therefore, since the cell culture media and serum, including DMEM and FBS, contained thiol compounds including cysteines and albumins, the degree of conjugation in DMEM or DMEM+FBS decreased by 10−15 mol % compared to that in saline. Additionally, the time change in the degree of conjugation was simulated using the diffusion-reaction model and showed good agreement (Figure 2D). The simulation results further demonstrated that the free thiol concentration decreased with the reaction progress, approaching 0%. This result, together with the degree of conjugation shown in Figure 2C, suggested that almost all the SAMSA-FL was finally transferred and conjugated inside the Alg-Mal microcapsules. This was because the penetrated SAMSA-FL was rapidly consumed by the conjugation, thereby maintaining the concentration gradient of SAMSA-FL from outside to inside the microcapsules until the free SAMSA-FL was depleted. This led to condensation of SAMSA-FL inside the microcapsules, minimizing the loss of bioactive peptides, which could reduce the costs of differentiation-inducing processes in the future. Avoidance of Anoikis Induced by in Situ Conjugation. To demonstrate in situ conjugation in the presence of cells, SAMSA-FL was added to NIH-3T3-encapsulated microcapsules. Cells were stained red with a cell tracker before

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RESULTS AND DISCUSSION Fabrication of Alg-Mal Microcapsules. Alg-Mal was synthesized according to previous reports.23 Figure S2 shows 1 H NMR and FT-IR spectra of Alg-Mal. The presence of δ = 6.2 (COCHCHCO) and δ = 2.3, 3.3 (CH2CH2 NH) were confirmed in the 1H NMR spectrum. It is noteworthy that the peaks at δ = 2.3 and 3.3 would also be derived from methylene protons of unremoved WSCD. Peaks derived from CN stretching at 1250 cm−1, CC stretching at 1550 cm−1, and CO stretching at 1720 cm−1 were confirmed in the FT-IR spectrum. These results indicated that Alg-Mal synthesis was successful. The DM was calculated as 5.3% in Alg from the 1H NMR spectrum. By using the obtained Alg-Mal, Alg-Mal microcapsules were fabricated for the first time via ejection through the coaxial two-fluid nozzle and subsequent cross-linking with Ca2+. The Alg-Mal microcapsules were spherical with a diameter of 490 ± 221 μm as shown in Figure S3. Rapid Penetration and Click Reaction Visualized, Quantified and Simulated by in Situ Conjugation of 2353

DOI: 10.1021/acs.biomac.9b00333 Biomacromolecules 2019, 20, 2350−2359

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Figure 3. (A) Confocal microscopy images of NIH-3T3-encapsulating Alg and Alg-Mal microcapsules 1 h after in situ conjugation of SAMSA-FL (green). Cells were stained using CellTracker (red). (B) Live cell staining of NIH-3T3 cells encapsulated in Alg-Mal microcapsules 7 days after adding RGDS, CRGES, or CRGDS using calcein (green). Results for the sample without peptide addition are also shown as no peptide.

hydrogels did not spread.45 It was hypothesized that the void space was not sufficient to spread for the cells. This explanation is applicable to our study. Note that cell morphology might be different in higher spatial resolution; and thus, detailed investigation, such as immunostaining of cytoskeleton, will be meaningful in further research. A potential concern regarding the addition of adhesion factors is competition with pre-existing cell-matrix adhesion, leading to apoptosis caused by the loss of cell-matrix interactions, known as anoikis. It was reported that nonimmobilized RGD peptides induce fibroblast apoptosis by disrupting cell adhesion on the underlying scaffold.46 However, Figure 3B shows that most of encapsulated cells were alive in Alg-Mal microcapsules. In addition, few apoptotic cells were observed under all the conditions regardless of the peptide addition by flow cytometric analysis using Annexin V-FITC/PI staining (Figure S8B). These results suggested the avoidance of anoikis in Alg-Mal microcapsules. Note that necrotic cells were found in all the conditions with similar probability as shown in Figure S8C in the Annexin V-FITC/PI staining. Since most cells were alive in Alg-Mal microcapsules as shown in Figure 3B, necrosis would be induced by the cell collection from Alg-Mal microcapsules using alginate lyase. In Situ Conjugation Did Not Affect Swelling Behavior or Young’s Modulus. To investigate the effect of in situ conjugation of cell-adhesive peptides on the mechanical properties of the microcapsules, we measured the swelling degree of the microcapsules and the Young’s modulus of the hydrogels after in situ conjugation. The diameter of Alg-Mal microcapsules in situ conjugated with CRGES, CRGDS, or without conjugation increased to about 140% after 7 days of

encapsulation in microcapsules. Confocal microscopy images of Alg and Alg-Mal microcapsules are shown in Figure 3A. Red fluorescence derived from cells occurred with both Alg and Alg-Mal microcapsules, indicating successful cell encapsulation. Green fluorescence derived from SAMSA-FL was homogeneously observed in Alg-Mal microcapsules, while Alg microcapsules showed minimal green fluorescence. These results suggested that in situ conjugation progressed in Alg-Mal microcapsules even in the presence of cells, and part of the conjugated peptides were certainly overlapped with the places encapsulated-cells occupied. Since the density of in situ conjugated peptides in Alg-Mal capsules (5 mM) was higher than that of conjugated RGD peptides in Alg hydrogels (2 mM) in a previous report,44 we concluded that the in situ conjugated CRGDS peptides sufficiently associated with the encapsulated cells. It is noteworthy that green fluorescence was observed from some cells with Alg microcapsules. This could be because SAMSA-FL was nonspecifically adsorbed on cell membranes. To verify cell viability after in situ conjugation of CRGDS peptides in Alg-Mal microcapsules, these were observed via live cell staining. One day after cell encapsulation, CRGDS as a cell-adhesive peptide, CRGES as a noncell-adhesive peptide, or RGDS as a nonreactive peptide was added to Alg-Mal microcapsules. Live cells were stained green with calcein 7 days after cell encapsulation. Confocal microscopy images of Alg-Mal microcapsules are shown in Figure 3B. Green fluorescence was observed under all the conditions, indicating survival of encapsulated cells. As shown in Figure S7, the cell morphology was rounded under all the conditions. It was previously reported that MSCs cultured inside 3D alginate 2354

DOI: 10.1021/acs.biomac.9b00333 Biomacromolecules 2019, 20, 2350−2359

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To determine whether we can collect and further culture the cells proliferated in the microcapsules, we degraded the microcapsules with alginate lyase. The proliferation rate of the collected cells after reseeding on a cell culture dish is shown in Figure S10. There was no difference in the proliferation rates of cells precultured in the microcapsules and on TCPS. These results suggested that the cell encapsulation-release procedure with the Alg-Mal microcapsule system enabled collection and reculture of the encapsulated cells. Enhancement of Gene Expression by in Situ Conjugation of Cell-Adhesive Peptide in Fibroblasts. The mRNA expression of Col1a1 (Col I), bFGF, and TGF-β in the encapsulated cells was measured at day 7 by RTq-PCR. Relative mRNA expression in the control without added peptide is shown in Figure 5. Compared with the control, the

incubation in DMEM (Table 1). The Young’s modulus of AlgMal microcapsules in situ conjugated with CRGES, CRGDS, or Table 1. Effect of in situ Conjugation on Diameter Change of the Microcapsules Caused by Their Swelling in DMEM and Young’s Modulus of the Hydrogels (N = 3) Microcapsules Alg-Mal without in situ conjugation Alg-Mal in situ conjugated with CRGES peptides Alg-Mal in situ conjugated with CRGDS peptides

Diameter of the microcapsules normalized by the initial diameter 7 days after in situ conjugation (%)

Young’s moduli (kPa)

145 ± 8

8.65 ± 1.62

138 ± 6

9.01 ± 1.75

142 ± 7

8.44 ± 1.18

without conjugation was about 8−9 kPa (Table 1). Under all the conditions, there was no change in the swelling behavior or Young’s modulus of the hydrogels. This suggested that the mechanical properties of the hydrogels were not altered by in situ conjugation due to the high water solubility and relatively low molecular weights of CRGES and CRGDS. Therefore, in this case, in situ conjugation enables investigation of the effect of these biofunctional peptides. Proliferation Switch by in Situ Conjugation of CellAdhesive Peptide in Fibroblasts. The effect of in situ conjugation of cell-adhesive peptides on the proliferation of encapsulated NIH-3T3 cells was investigated by measuring the amount of DNA. The change in the DNA amount during microcapsule culture is shown in Figure 4. Compared with the

Figure 5. Effect of in situ conjugation of various peptides on mRNA expression of Col1a1, bFGF, and TGF-β in NIH-3T3 cells encapsulated in Alg-Mal capsules. The mRNA expression level under each condition was normalized to that in control cells, which received no added peptide. (N = 3, *: p < 0.05, compared with the control without added peptides).

gene expression of Col I, a cell adhesion protein, slightly decreased in the presence of CRGDS. However, the gene expression of both growth factors, bFGF and TGF-β, significantly increased in the presence of CRGDS. It is noteworthy that an increase in the gene expression of TGF-β was also observed with CRGES, presumably due to nonspecific interactions, but this was much lower than that with CRGDS. Collagen is an ECM component that induces proliferation and differentiation of adhered cells. It was reported that adding RGD-polyamidoamine (PAMAM) conjugates downregulated the mRNA expression of Col I in NIH-3T3 cells under 3D spheroid culture.47 This report is consistent with our result showing Col I expression in NIH-3T3 cells. However, our sirius red staining also showed that production of collagen was increased by adding CRGDS (Figure S11). Although posttranscriptional processes of Col I mRNA are not yet completely clarified,48 we speculated that mRNA expression of Col I was suppressed after sufficient collagen production. It is known that bFGF has potent mitogenic activity against fibroblasts and endothelial cells and stimulates fibroblast proliferation and production of angiogenic factors.49 TGF-β is a cytokine that controls the proliferation and differentiation of cells and maintains homeostasis in living organisms. It plays important roles in ontogeny, tissue remodeling, wound healing, inflammation/immunity, and cancer invasion/metastasis. It was reported that TGF-β induces proliferation in human renal

Figure 4. Effect of in situ conjugation of various peptides on proliferation rate of encapsulated NIH-3T3 cells, determined by the amount of DNA. The amount of DNA at each time point was measured by DAPI assay, and then normalized by the initial DNA amount (N = 3, *: p < 0.05).

control without an added peptide, the cell proliferation rate in microcapsules with added RGDS and CRGES peptides did not differ significantly. However, adding CRGDS peptides increased the cell proliferation rate compared with other conditions, indicating that in situ conjugation of cell-adhesive peptides promoted proliferation of encapsulated cells. The dependency of the cell proliferation rate on the amount of added CRGDS is shown in Figure S9. Compared with the control, the cell proliferation rate increased in a dosedependent manner and significantly improved when the ratio of CRGDS peptide exceeded 2. 2355

DOI: 10.1021/acs.biomac.9b00333 Biomacromolecules 2019, 20, 2350−2359

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Biomacromolecules

Figure 6. (A) Confocal microscopy observation of GFP fluorescence in Col1a1GFP-MC3T3E1 cells cultured in microcapsules after in situ conjugation of BMP-2 mimetic peptides. (B) Effect of in situ conjugation of BMP-2 mimetic peptides on encapsulated-cell differentiation evaluated via fluorescence intensity measured by flow cytometry. GFP fluorescence intensity under each condition was normalized to the control without peptide addition (N = 4, *: p < 0.05). Scale bar: 100 μm.

fibroblasts by bFGF induction.50 Additionally, proliferative synergism by sequential bFGF and TGF-β costimulation was reported in vitro with human bronchial smooth muscle cells.51 Based on the above, it was suggested that up-regulating the gene expression of both bFGF and TGF-β by in situ conjugation of cell-adhesive peptides induced the promotion of encapsulated-cell proliferation in microcapsules. Differentiation Induction by in Situ Conjugation of BMP-2 Mimetic Peptides in Osteoblast Precursor Cells. Induction of cell differentiation via in situ conjugation of soluble factor mimetic peptides to Alg-Mal microcapsules was further demonstrated. Preosteoblastic cells, Col1a1GFPMC3T3E1 cells were previously established as a convenient cell line to increase the expression levels of osteogenic differentiation markers such as ALP and OC mRNAs, concurred with the GFP fluorescent intensity, suggesting that expression of GFP reliably reflected only on differentiation into osteoblastic cells.35 Therefore Col1a1GFP-MC3T3E1 cells were employed to evaluate osteogenic differentiation via fluorescence. As a mimetic of osteo-inducive growth factor BMP-2, DWIVA, BMP-2 knuckle epitope peptide (BMP-2 KE Pep), with and without terminal cysteine, was used. Figure 6 shows the GFP fluorescence of encapsulated cells evaluated by confocal laser scanning microscopy and flow cytometry. Compared with the control without added peptide, GFP fluorescence was significantly increased by adding CDWIVA (DWIVA with cysteine) or CG-BMP-2 KE Pep (BMP-2 KE Pep with cysteine). However, no significant difference in GFP fluorescence was observed after adding DWIVA or BMP-2 KE Pep, due to the lack of reactive thiol groups. Additionally, the GFP fluorescence of cells treated with BMP-2 was not significantly different from that of the control. BMP-2 was reported to induce osteoblast differentiation in various cells. In this study, we used BMP-2 mimetic peptides, DWIVA and BMP-2 KE Pep. It was reported that DWIVA40 and BMP-2 KE Pep41 prefixed to a scaffold induce osteogenic differentiation. Our results demonstrated that the osteoinductive function of these peptides can be introduced via in situ conjugation to Alg-Mal microcapsules, based on the reaction-diffusion process. Since cysteine-containing BMP-2 mimetic peptides were concentrated into Alg-Mal microcapsules via in situ conjugation, osteogenic differentiation was expected to be induced more efficiently by conjugated BMP-2

mimetic peptides than soluble BMP-2 mimetic peptides without cysteine. This condensation process of conjugated BMP-2 mimetic peptides was suggested to be a similar manner with the adsorption of BMP-2 to heparin in ECM. Our results also indicated that adding BMP-2 did not significantly induce osteogenic differentiation of Col1a1GFPMC3T3E1 cells encapsulated in Alg-Mal microcapsules. We believe that this is due to inactivation of BMP-2 before its diffusion into Alg-Mal microcapsules. The diffusion coefficient of BMP-2 is reported as 2.3 × 10−12 m2/s in alginate hydrogels,52 leading to a characteristic diffusion time of 30 h in Alg-Mal capsules with diameter 500 μm, according to eq 1 from the Supporting Information. However, regarding BMP-2 inactivation, it was reported that the concentration of BMP-2 in the culture medium rapidly decreased to below 50% of the original concentration within 1 h and was almost undetectable after 10 h.53 The above estimations suggest that the in vitro half-life of BMP-2 is much shorter than the characteristic time for BMP-2 diffusion, and thus BMP-2 would be inactivated before diffusing into the Alg-Mal microcapsules. Under 2D culture where BMP-2 can freely access cells, the GFP fluorescence of Col1a1GFP-MC3T3E1 cells cultured with BMP-2 significantly increased to about twice that of the control (Figure S12). To mimic the dynamism of the ECM, a few in situ reactive scaffold hydrogels were recently developed for tissue engineering. DeForest et al.27 introduced cell-adhesive RGD motif peptides into encapsulated cells by photoinduced thiol−ene reaction. Gandavarapu et al.28 achieved reversible modification of RGD motif peptides by chain transfer between an allyl sulfide functional group and a thiyl radical. Gramlich et al.54 and Wade et al.55 reported that the hydrogel stiffness was increased by in situ cross-linking using a photoinduced thiol− norbornene reaction. Wylie et al. 56 reported in situ immobilization of sonic hedgehog and ciliary neurotrophic factors into encapsulated cells in an agarose hydrogel via barnase−barstar and streptavidin−biotin using coumarin-caged thiols. In these studies, the in situ reactivity depended on the photochemical reactions, and the form of the hydrogels was a thin film. In another case, Hao et al.57 reported in situ matrix stiffening or RGD tagging in bulk hydrogel via tetrazine ligation without external triggers. However, the present simple in situ conjugation via combination of the click reaction and 2356

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Biomacromolecules Notes

microcapsule shown in Figure 2 will enable easy scale-up in shaking or gyratory culture without a specialized apparatus. As presented, we demonstrated that in situ conjugation of ECM mimetic or soluble factor mimetic peptides can induce a cell proliferation/differentiation switch of fibroblasts or preosteoblastic cells. In our in situ conjugation system, transplantation of collected cells can be achieved for biomedical applications. The use of relatively cheap peptides compared to original growth factors will also reduce the cost of a stem cell culture. This advantage is further reinforced by the efficient condensation of peptides in microcapsules (Figure 2), owing to the high conversion of in situ conjugation. Additionally, transplantation of the conjugated microcapsules is possible due to the biocompatibility of alginate. Due to the simple reaction-diffusion phenomena used in this research, various click reactions, many peptides and functional molecules, and matrix materials can be applied to this culture system. Therefore, our in situ conjugation system is predicted to become a versatile platform for introducing a library of biomimetic peptides to trigger multiple cell functions.

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CONCLUSIONS

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ASSOCIATED CONTENT

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We deeply thank Kimica Co. for supplying Alginate. This work utilized the core research facility of Center for Disease Biology and Integrative Medicine, The University of Tokyo, which was organized by The University of Tokyo Center for NanoBio Integration entrusted by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan.

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To mimic in vivo extracellular changes, we developed novel “clickable” alginate microcapsules, which can introduce thiolcontaining peptides by “in situ conjugation” between maleimide and thiol groups. Proliferation and gene expression of encapsulated fibroblast cells were accelerated by in situ conjugation of CRGDS, while free RGDS showed no effect. Additionally, differentiation of encapsulated preosteoblastic cells was triggered by in situ conjugation of CDWIVA and CGBMP-2 knuckle epitope peptide, which are BMP-2 mimetics. We expect this in situ conjugation approach using various functional peptides and click reactions to be promising for future biomedical, bioindustrial, and biochemical fields. S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.9b00333. Details for (i) the estimation and simulation of the kinetics of in situ conjugation in Alg-Mal microcapsules, (ii) primer sequence used in RT-qPCR, additional data for the characterizations of Alg-Mal, (iii) additional images of Alg-Mal microcapsules, (iv) experimental/ simulated time change of in situ conjugation using 1 equivalent of SAMSA-FL, (v) enlarged view of cellencapsulated Alg-Mal microcapsules, (vi) Annexin VFITC/PI staining, dependency of cell proliferation on the amount of CRGDS, (vii) cell proliferation after reseeding encapsulated-cells, (viii) sirius red staining, and (ix) the effect of BMP-2 on GFP fluorescence (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*T. Ito. E-mail address: [email protected]. Tel: +81-35841-1425. Fax: +81-3-5841-1697. ORCID

Taichi Ito: 0000-0002-1589-8242 2357

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