Self-Assembly of Thermoreversible Hydrogels via Molecular

Subscriber access provided by Warwick University Library

Article

Self-assembly of thermo-reversible hydrogels via molecular recognition toward a spatially organized co-culture system Ryota Tamate, Kotomi Takahashi, Takeshi Ueki, Aya Mizutani Akimoto, and Ryo Yoshida Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01672 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 10, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Self-assembly of thermo-reversible hydrogels via molecular recognition toward a spatially organized co-culture system Ryota Tamate,†,a Kotomi Takahashi,†,a Takeshi Ueki,b Aya Mizutani Akimoto,*,a and Ryo Yoshida*,a a

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

Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan. b

National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan.

KEYWORDS: block copolymers, hydrogels, self-assembly, molecular recognition, co-culture

ABSTRACT

In this study, we present the spontaneous adhesion of two thermo-reversible physically crosslinked hydrogels via molecular recognition under a physiological condition. We successfully prepared two types of hydrogels generated using two kinds of well-defined ABA type triblock copolymers: CAT-ABA and PBA-ABA, which contain catechol and phenylboronic acid groups as

1 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 25

functional side chains, respectively. Both types of ABA triblock copolymers were able to undergo sol-to-gel transition with the increase in temperature resulting from the formation of physical cross-links at a physiological temperature, which enables easy cell encapsulation in the hydrogel. It was determined that the cell encapsulating hydrogels exhibited spontaneous macroscopic adhesion through the formation of boronic esters between phenylboronic acid and catechol at the hydrogel interface. This novel system likely represents a promising method to construct a precisely organized, three dimensional co-culture system to enable the reconstruction of complicated tissues such as the liver in vitro.

INTRODUCTION In the tissue engineering field, naturally derived hydrogels such as fibrin, collagen, and Matrigel are broadly used for in vitro cell culture.1,2 Although natural hydrogels are advantageous from the view point of their inherent biocompatibility, there are certain risks to their use in vivo such as immunological reactions and pathogen transmission. In addition, it is difficult to control the molecular structure of natural hydrogels, which consequently leads to batch-to-batch variability. To overcome these issues, there has been a growing demand in tissue engineering to utilize synthetic hydrogels as extracellular matrix mimics, the architectures and functions of which can be fully controlled.3–5 In particular, to fabricate complicated tissue structures in vitro, the precise spatial control of different types of cells in an organized manner is essential. Toward this aim, the co-culture of different types of cells has received much attention; however, traditional co-culture systems commonly involved the simple random seeding of two or more types of cells.6–8 Although several


Page 3 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

studies have been published regarding the spatial control of two dimensional co-culture systems,9– 12

few reports have achieved three dimensional spatial control of cells.13–15 Considering the three

dimensionally-organized structures of living tissues such as the liver, there is a large requirement to establish new co-culture systems by using synthetic hydrogels that can construct spatially organized three-dimensional structures. Alternatively, recent developments of functional synthetic hydrogels have allowed the construction of highly organized hydrogel structures by means of the spontaneous adhesion of multiple different hydrogels. For example, Harada et al. have developed pioneering works for the directed self-assembly of hydrogels with the aid of the concept of molecular recognition.16–22 Specifically, they fabricated chemically cross-linked hydrogels that contained complementary functional groups for molecular recognition-based interaction such as host-guest interactions of cyclodextrin,16,18–20 metal-ligand interactions,17 and diol-boronate complexions.21 In these systems, complementary hydrogels with a size order of mm through cm can selectively adhere to each other via directed molecular recognition between the functional groups incorporated into hydrogel building blocks, resulting in the establishment of the macroscopic self-assembly of hydrogels. Additionally, Asoh et al. also demonstrated a novel strategy of electrophoretic hydrogel adhesion through polyion complex formation at the hydrogel interfaces.23–25 In the current study, we combine these modalities to describe a novel self-assembled hydrogel system toward the establishment of a 3D co-culture system, based on the recent developments for the self-assembly of synthetic hydrogels as a building block. Two types of well-defined, thermoresponsive ABA triblock copolymers, CAT-ABA, and PBA-ABA, were successfully synthesized via reversible addition fragmentation chain transfer (RAFT) polymerization and the subsequent post-modification of a functional group as a molecular recognition unit. The terminal A blocks of


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 25

both ABA triblock copolymers contain poly(N-isopropylacrylamide) (PNIPAAm), which undergoes a lower critical solution temperature (LCST) transition thus providing thermoreversible sol-gel transition in an aqueous solution. By incorporating tert-butylacrylamide (tBAAm) as a hydrophobic comonomer into the PNIPAAm main chain, the sol-gel transition temperature could be precisely modulated to form physically cross-linked hydrogels at physiological temperatures. In turn, the hydrophilic B middle blocks for CAT-ABA and PBAABA contain a small amount (1–3 mol%) of catechol and phenylboronic acid, respectively, as molecular recognition units along with a hydrophilic N, N-dimethylacrylamide (DMAAm) main monomer. Both the ABA triblock copolymers undergo sol-to-gel transition upon heating and form physically cross-linked hydrogels at physiological temperatures, which enables the capability of facile cell encapsulation. We demonstrated that these two types of physically cross-linked hydrogels from CAT-ABA and PBA-ABA could exhibit spontaneous adhesion by simple contact between the surfaces of the two gels, owing to the molecular complex formation of boronic ester with phenylboronic acid and the catechol units incorporated into the block copolymers. Therefore, by encapsulating two different cell types into CAT-ABA and PBA-ABA gels, respectively, the coculture of cells in a three dimensionally ordered manner could be realized. This may allow the possibility of reconstructing organized tissues such as the liver, wherein the endothermal foregut and mesenchymal vascular structures are mediated by heterotypic interaction. In addition, the nature of the physically cross-linked gels would allow removal of the matrix polymers via a structural transformation from gel to sol by simply reducing the temperature subsequent to tissue formation in the hydrogels. The co-culture system proposed here thereby represents a promising methodology for the future fabrication of cell-laden self-assembled hydrogels for tissue reconstruction in vitro.


Page 5 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

EXPERIMENTAL SECTION Materials N-Isopropylacrylamide (NIPAAm) was kindly provided by KJ Chemicals (Japan) and purified by recrystallization from toluene and n-hexane. N-Succinimidyl acrylate (NAS) was purchased from Tokyo Chemical Industries (Japan) and used as received. Acrylic acid (AA) and N, Ndimethylacrylamide (DMAAm) were purchased from Wako Pure Chemical Industries (Japan) and purified by passing through an activated basic alumina column prior to use. 2-1-(Carboxy-1methylethylsulfanylthiocarbonylsulfanyl)-2-methylpropionic acid (CMP) was synthesized according to the literature.26 Dulbecco’s modified Eagle’s medium with low glucose (DMEM-LG), Dulbecco’s

phosphate

buffered

saline

(PBS),

penicillin-streptomycin,

and

trypsin-

ethylenediaminetetraacetic acid were purchased from Sigma-Aldrich (MO, USA). CellTracker™ Red CMTPX or CellTracker™ Green CMFDA were from Life Technologies (CA, USA). CalceinAM was purchased from Dojin Chemicals (Japan) and ethidium bromide was from Nippon Gene (Japan). All other materials were purchased from Wako Pure Chemical Industries and used as received. Synthesis of P(NIPAAM-r-tBAAm) macro-CTA CMP (50.0 mg, 0.177 mmol), NIPAAm (7.87 g, 69.5 mmol), and tert-butylacrylamide (tBAAm, 0.983 mg, 7.73 mmol) were placed in a round bottom 2-way 100 mL flask and were dissolved in 1,4-dioxane (38.5 mL). A 100-L aliquot of 2,2’-azobis(2,4-dimethylvaleronitrile) (ADVN, 0.586 mg, 2.36 mol) in dioxane was added to the solution and bubbled with argon (Ar) for 30 min at room temperature. Then, RAFT polymerization was carried out at 70 °C for 2 h. The reaction mixture was diluted with acetone and precipitated into n-hexane. The resulting P(NIPAAm-r-


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 25

tBAAm) macro-CTA was further purified by reprecipitation from acetone/n-hexane three times (5.27 g, 59% yield). Synthesis of P(NIPAAm-r-tBAAm)-b-P(DMAAm-r-NAS)-b-P(NIPAAm-r-tBAAm) (NASABA) and P(NIPAAm-r-tBAAm)-b-P(DMAAm-r-AA)-b-P(NIPAAm-r-tBAAm) (AA-ABA) P(NIPAAm-r-tBAAm) macro-CTA (2.00 g, 0.0765 mmol), DMAAm (3.13 g, 31.6 mmol), and NAS (165 mg, 0.977 mmol) were dissolved in 32.1 mL 1,4-dioxane. A solution of ADVN (0.203 mg, 0.817 mol) in 0.5 mL dioxane was added to the solution, which was then bubbled with Ar for 30 min at room temperature. RAFT polymerization was allowed to proceed at 70 °C for 4 h. The reaction mixture was next diluted with acetone and precipitated into n-hexane. After drying under vacuum, the ABA triblock copolymer P(NIPAAm-r-tBAAm)-b-P(DMAAm-r-NAS)-bP(NIPAAm-r-tBAAm) (NAS-ABA) was purified by reprecipitation from acetone as a good solvent and diethyl ether as a poor solvent (3.73 g, 70% yield). P(NIPAAm-r-tBAAm)-b-P(DMAAm-r-AA)-b-P(NIPAAm-r-tBAAm)

(AA-ABA)

was

synthesized in a manner similar to NAS-ABA, except that DMAAm was copolymerized with AA instead of NAS (3.72 g, 71% yield) Conjugation of dopamine hydrochloride (DOPA) with NAS-ABA (CAT-ABA synthesis) NAS-ABA (2.00 g, 0.0288 mmol), DOPA (0.247 g, 1.30 mmol), and triethylamine (1.32 g, 13.0 mmol) were dissolved in ethanol (20.0 mL). The conjugation reaction was conducted at room temperature for 16.5 h. The reaction mixture was then poured into n-hexane to precipitate crude polymer and dried under vacuum. The crude polymer was purified by dialysis against water and finally CAT-ABA was obtained by lyophilization (1.52 g, 68% yield). Conjugation of aminophenylboronic acid (APBA) with AA-ABA (PBA-ABA synthesis)


Page 7 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

AA-ABA (2.00 g, 0.0276

mmol),

APBA

(0.130

g, 0.946 mmol),

and

N-(3-

dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (0.362 g, 1.89 mmol) were dissolved in MES buffer (2-(N-morpholino)ethanesulfonic acid, 100 mM, pH = 5.4). The reaction solution was stirred at room temperature overnight and the resultant aqueous polymer solution was then dialyzed against water for 6 days. PBA-ABA was finally obtained by lyophilization (1.59 g, 75% yield). Polymer characterization The polydispersity indices (PDI) of the obtained polymers were calculated by using a gel permeation chromatography (GPC) system (Tosoh, Japan). The GPC columns were calibrated using polyethylene oxide standards. Dimethylformamide with 3 mM LiBr and 0.5 % triethylamine was used as a carrier solvent. The GPC traces of P(NIPAAm-r-tBAAm) macro-CTA, CAT-ABA and PBA-ABA are shown in Figure S1. 1H nucleic magnetic resonance (NMR) measurements were conducted using a JEOL ECS-400 spectrometer (JEOL, Japan) at room temperature. Mn of P(NIPAAm-r-tBAAm) macro-CTA was calculated based on monomer conversion determined by 1

H NMR. Monomer conversion of NIPAAm was calculated by comparing the integral intensity of

the vinyl proton of NIPAAm (1H,  = 5.55–5.60 ppm) to the total integral intensity of the isopropyl group of NIPAAm (1H,  = 3.90–4.20 ppm), and that of tBAAm was by comparing the integral intensity of the vinyl proton of tBAAm (1H,  = 5.51–5.55 ppm) to the total integral intensity of the tert-butyl group of tBAAm (9H,  = 1.25–1.40 ppm). The ratios of [CAT]/[DMAAm] in CATABA and [PBA]/[DMAAm] in PBA-ABA were calculated from integrated signal intensities for the aromatic group of catechol (3H,  = 20–7.00 ppm), one of the peaks of the aromatic group of phenylboronic acid (1H,  = 7.77–7.90 ppm) and the dimethyl group of DMAAm (6H,  = 2.75– 3.25 ppm). The 1H NMR spectra for CAT-ABA and PBA-ABA are shown in Figure S2 and S3.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 25

Dynamic light scattering (DLS) measurement For DLS measurements, 0.1 wt% CAT-ABA and PBA-ABA triblock copolymers were dissolved in PBS. Prior to use, the solutions were filtered through 0.25-m filters to remove dust. The measurements were conducted using a DLS/SLS-5000 compact goniometer (ALV-GmbH, Germany). A He-Ne laser (JDS Uniphase Co., CO, USA, λ0 = 632.8 nm) was used as a light source. At each temperature, polymer solutions were equilibrated for 15 min. The collected data were analyzed by the cumulants method27 as well as by CONTIN analysis. Rheological measurement For rheological measurements, 20 wt% CAT-ABA and PBA-ABA were dissolved in the culture medium (DMEM-LG containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin). Temperature sweep tests were performed using an Anton Paar Physica MCR 301 rheometer (Anton Paar, Austria). A 50-mm parallel plate geometry was used with a gap spacing of ca. 0.1 mm. The polymer solution was heated from 10 °C to 40 °C at a heating rate of 2 °C/min with a strain amplitude of 2% and a frequency of 1 Hz. Adhesion test of CAT-ABA and PBA-ABA hydrogels encapsulating stained cells HeLa cells were routinely cultured using DMEM-LG containing 10% FBS and 1% penicillinstreptomycin as the culture medium on tissue culture polystyrene (TCPS) dishes in an incubator (37 °C, 5% CO2). Prior to encapsulation, cells were stained with CellTracker™ Red CMTPX or CellTracker™ Green CMFDA. ABA triblock copolymers were sterilized by heating at 100 °C for 2 h before being dissolved in the culture medium. To encapsulate cells in the ABA triblock copolymer hydrogels, the triblock copolymer solution and cell suspensions were prepared separately. Then, the polymer solutions and cell suspensions were mixed in an ice bath.


Page 9 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Subsequently, cell-laden hydrogels were formed by increasing the temperature of the mixture to 37 °C in the incubator. The final polymer concentration and cell density were 20 wt% and 6 × 105 cells/mL, respectively. Cell-laden hydrogels were molded in a cuboid shape and the molded CATABA and PBA-ABA hydrogels were then resuspended in a petri dish filled with the culture medium at 37 °C. Next, hydrogel adhesion was tested by gently placing the hydrogels in contact. After confirming hydrogel adhesion, the hydrogels as well as the encapsulated cells were observed using optical microscopy (VHX-900, Keyence, Japan) and fluorescent microscopy (DM-IL, Leica, Germany). Cell viability test (Live/Dead assay) Cell viability in the hydrogels was evaluated by using a standard Live/Dead assay. Cell-laden hydrogels were prepared similar to the method described above, except that the cell density was 1 × 105 cells/mL and the culture medium for the CAT-ABA contained 20 mM glutathione to prevent oxidation of the catechol units. The hydrogels were washed with warm PBS and incubated with Live/Dead dyes (20 M calcein-AM and 40 M ethidium bromide) in PBS for 30 min. Cells were observed by fluorescence microscopy and cell viability was calculated by image analysis using Image J software (National Institutes of Health, MD, USA).

RESULTS AND DISCUSSION Preparation and characterization of ABA triblock copolymers ABA triblock copolymers were successfully synthesized by sequential RAFT polymerization followed by post-modifications of the molecular recognition functional groups (Figure 1, Scheme S1 and S2). P(NIPAAm-r-tBAAm) was synthesized by RAFT copolymerization from the bifunctional chain transfer agent (CTA) as the first step, leading to the generation of a P(NIPAAmr-tBAAm) macro chain transfer agent (macro-CTA). Here, in order to manipulate the sol-gel


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 25

transition temperature of the resultant ABA triblock copolymer based on the LCST nature of NIPAAm, we selected tBAAm as a hydrophobic comonomer for NIPAAm. As the random copolymerization of hydrophobic monomers such as tBAAm with NIPAAm is known to decrease its LCST-type phase transition temperature,28 the monomer ratio in the reaction solution in the feed was fixed as NIPAAm:tBAAm = 90:10. Subsequently, the RAFT copolymerization of DMAAm with NAS and with AA from P(NIPAAm-r-tBAAm) macro-CTA was conducted to introduce P(DMAAm-r-NAS) (NAS-ABA) and P(DMAAm-r-AA) (AA-ABA) as middle blocks, respectively. The two types of target ABA triblock copolymers, CAT-ABA (Figure 1(a)) and PBA-ABA (Figure 1(b)), were successfully prepared by post modification of NAS-ABA with DOPA and of AA-ABA with APBA, respectively. The results of polymer characterization are summarized in Table 1. The narrow PDI calculated from the GPC measurements suggests that the RAFT polymerization process was well controlled. It was also important that the prepared block copolymer be well-defined, as a block copolymer with a low PDI is expected to provide the sharpness of the sol-gel transition.29

Figure 1. Chemical structures of (a) CAT-ABA and (b) PBA-ABA.


Page 11 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Table 1. Characterization of macro-CTA, CAT-ABA, and PBA-ABA.

P(NIPAAm-r -t BAAm) macro-CTA CAT-ABA PBA-ABA

M na [CAT]/[DMAAm]a [PBA]/[DMAAm]a PDI (M w/M n)b (per mol%) (per mol%) [kDa] 33 1.22 17-40-17 1.41 3.6 17-40-17 1.40 1.3

a

calculated from 1H NMR spectra. bcalculated by GPC.

DLS assessment of thermo-responsive self-assembly of the ABA triblock copolymers in dilute concentration The thermo-responsive behaviors of the ABA triblock copolymers were investigated by DLS measurement. Figure 2a shows the temperature dependence of the hydrodynamic radius (Rh) of the particles in the 0.1 wt% CAT-ABA and PBA-ABA triblock copolymers in PBS solution. At low temperature, below the lower critical micellization temperature (LCMT)-type micellization temperature, the Rh of the diffusion component in the PBS solution was below 10 nm in both CATABA and PBA-ABA triblock copolymers, consistent with the block copolymer single polymer chains having a molecular weight of 74 kDa. The reduced second cumulant values (2/2), indicative of the polydispersity of the particle size, were relatively broad (2/2 = 0.925 for CATABA and 2/2 = 0.660 for PBA-ABA, respectively, at 20 °C). The broad 2/2 for thermoresponsive ABA triblock copolymers at low temperature were previously reported.30, 31 It appeared that almost all of the particles in the PBS solution below the LCMT-type micellization temperature were individually dissolved as polymer chains with only a very small amount of aggregates, which might result from solubility differences owing to chain-to-chain variations in comonomer distribution and composition in the random copolymer block. When the temperature was increased


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 25

above the LCMT-type micellization temperature, the Rh of the particles increased considerably to several tens of nanometers. This suggested that the triblock copolymers self-assembled into flower-like micelles with P(NIPAAm-r-tBAAm) cores surrounded by well-solvated hydrophilic middle block shells at higher temperatures, consistent with expectations;32 this structure model was also supported by the narrow distribution of particle size (2/2 = 0.077 for CAT-ABA, and

2/2 = 0.168 for PBA-ABA, respectively at 37 C). As the LCMT-type micellization temperatures for CAT-ABA and PBA-ABA were almost comparable, the chemical structure difference of the B block did not affect the thermo-sensitivity of the A block. Furthermore, when solutions of CAT-ABA and PBA-ABA were mixed at physiological temperature (i.e., 37 °C), the Rh of the particles in the mixed solution increased up to 80 nm. This implied that larger micelle aggregation comprised of CAT-ABA and PBA-ABA micelles were formed, driven by the boronic ester formation between the phenylboronic acid and catechol groups introduced into the outer B blocks of the micelles. The aggregation number of the micellar aggregate was roughly estimated to be 4.4 micelles per aggregate on average, based on the knowledge of the Rh of the aggregate and its constituent micelles. Figure 2b shows the corresponding Rh distribution function obtained by the CONTIN analysis of the particles from CAT-ABA and PBA-ABA solutions prior to mixing, together with that from the mixture of CATABA with PBA-ABA solutions at 37 °C, indicating large micellar aggregate formation.


Page 13 of 25

Hydrodynamic radius (nm)

(a) 80

○: CAT-ABA ●: PBA-ABA ■: Mixed

70 60 50 40 30 20 10 0 10

20

30 40 Temperature (℃)

50

(b) ○: CAT-ABA ●: PBA-ABA ■: Mixed

1.0 0.8 G(-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

0.6 0.4 0.2 0.0 1

10

100

1000

Rh

Figure 2. (a) Temperature dependence of Rh values for 0.1 wt% CAT-ABA (open circles) and PBA-ABA (closed circles) in PBS. The square indicates the Rh value after mixing the solutions of CAT-ABA and PBA-ABA at 37 °C. (b) Distribution of Rh values at 37 °C for CAT-ABA, PBAABA, and mixed solutions obtained by CONTIN analysis.

Rheological properties of the ABA triblock copolymers in concentrated solutions In order to explore the gelation capability of the triblock copolymers, we characterized the rheological properties of CAT-ABA and PBA-ABA triblock copolymers in concentrated solutions. Figure 3 shows the temperature dependence of storage (G’) and loss (G”) moduli for the CATABA and PBA-ABA triblock copolymers. The polymer concentration was set at 20 wt%, which


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 25

was higher than the chain-overlap concentration necessary to bridge the copolymers between micelles. The sample solutions for the rheological measurements were prepared using the cell culture medium to determine the rheological responses toward temperature changes with a condition similar to that of the actual cell culture system. In both polymer solutions, the value of G” exceeded that of G’ at lower temperatures, indicating that the solutions were viscous liquids (i.e., the sol state). Conversely, although both G’ and G” started to increase at approximately 25 C with increased temperature, G’ finally became greater than G” in the high temperature region, indicating solid (gel) properties. The results confirmed the formation of a three-dimensional polymer network at higher temperature through physical cross-linking points that consisted of the thermo-sensitive A segments.33 As a result, both ABA triblock copolymers underwent sol-to-gel transition concomitant with the temperature increase, resulted in physically cross-linked hydrogels at a physiological temperature (37 °C). The gelation temperature (Tgel) was defined as the temperature where the value of G’ became greater than G”. The Tgel values for CAT-ABA and PBA-ABA were determined to be 27.2 C, and 25.9 C, respectively. The excellent reversibility of the sharp sol-gel transition mediated by changes in temperature was also confirmed. In contrast to conventional chemically cross-linked hydrogels, the sharp sol-gel transition within a narrow range of temperature at approximately physiological conditions could permit the encapsulation of cells within the hydrogels as a 3D matrix as well as the recovery of cells grown in the hydrogel matrix through straightforward temperature changes.


Page 15 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 3. Temperature variation of storage (G’) and loss (G”) moduli for (a) CAT-ABA and (b) PBA-ABA 20wt% solutions, respectively.

Adhesion tests of the CAT-ABA and PBA-ABA hydrogels encapsulating stained cells Macroscopic adhesion tests between the CAT-ABA and the PBA-ABA hydrogels were conducted under physiological conditions. As a model cell, HeLa cells previously stained with CellTracker™ Red CMTPX or CellTracker™ Green CMFDA were encapsulated in the CATABA and the PBA-ABA hydrogels, respectively. In order to fabricate cell-laden hydrogels, we utilized the thermo-processable nature of physically cross-linked hydrogels. First, cell suspensions and block copolymer solutions were separately prepared and then the two solutions were mixed


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 25

together in an ice bath. Subsequently, the mixture solutions were warmed to 37 °C in an incubator to form cell-laden hydrogels. Next, the gels were molded and resuspended in a petri dish filled with the culture medium at 37 C. When the cell-laden hydrogels from CAT-ABA and PBA-ABA were gently placed in contact, the two gels spontaneously adhered to each other to form one larger gel (Figure 4a). After the gel adhesion had completed, the gel could not be separated into two pieces of gel or further recombined, even if moderate shaking was applied to the gel suspension. As shown in Figure 4b, fluorescence microscopy further confirmed that the two versions of the stained cells were separately encapsulated in each type of hydrogel. Notably, in this system the possibility exists of the formation of boronic ester from interaction of the phenylboronic acid with glucose contained within the culture medium.34 However, the previously reported association constant between catechol and phenylboronic acid was much larger than that between phenylboronic acid and glucose over a wide range of pH values (For example, Keq = 830 M−1 for catechol and 4.6 M−1 for glucose at pH = 7.4).35 Therefore, it is clear that the boronic ester formation between CAT-ABA and PBA-ABA preferentially occurs, even in the presence of a certain amount of glucose. As controls, we further conducted adhesion tests between two CAT-ABA or two PBA-ABA hydrogels, from which no spontaneous adhesions were observed even when the two gel surfaces were strongly contacted with each other. These results indicated that the different types of cells loaded in the hydrogels could be macroscopically assembled with the aid of a microscopic trigger of molecular recognition leading to hydrogel adhesion. Furthermore, the hydrogels immediately melted (i.e., gel-to-sol transition) within the culture medium by simply reducing the temperature, allowing the recovery of the encapsulated cells. Figure 4c demonstrates that the recovered cells could adhere on TCPS in the same manner as those prior to encapsulation. Therefore, if cells were able to successfully grow and form tissue-


Page 17 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

like structures in hydrogels, it might be possible to remove only the polymers and exclusively obtain the remaining self-assembled tissue-like structures.

Figure 4. (a, b) Optical (b) and fluorescence (b) microscope images of the adhered hydrogels encapsulating stained HeLa cells. Green cells were encapsulated in the PBA-ABA hydrogel, whereas red cells were within the CAT-ABA hydrogel. (c) Phase contrast image of HeLa cells seeded onto the TCPS dish that had been recovered following the gel-to-sol transition of adhered CAT-ABA and PBA-ABA hydrogels.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 25

Cytotoxicity of the ABA triblock copolymer hydrogels Finally, hydrogel cytotoxicity toward HeLa cells was evaluated using a Live/Dead assay. Figure 5 shows the cell viability shortly after cell encapsulation and following 24 h incubation in the hydrogels. As a control, we conducted cell viability tests using an ABA triblock copolymer hydrogel in which no functional groups for molecular recognition (catechol or phenylboronic acid) had been introduced (i.e., P(NIPAAm-r-tBAAm)-b-DMAAm-b-P(NIPAAm-r-tBAAm) triblock copolymer). The data indicated that although all cells remained alive shortly after cell encapsulation, the cell viability decreased in the CAT-ABA and PBA-ABA hydrogels after 24 h of incubation. The control experiment clearly confirmed that the cytotoxicity of the catechol and phenylboronic acid groups utilized in the block copolymers were associated with a reduced survival rate of cells.36,37 Therefore, in order to decrease hydrogel cytotoxicity, it is necessary to reduce the total amount of phenylboronic acid and catechol groups loaded therein. For this purpose, improvement of the mechanical stability of hydrogels sufficient to provide a stable cellencapsulating environment with low polymer concentration is required. One possibility might be the preparation of ABC-type triblock copolymers that carry thermo-sensitive A blocks together with hydrophobic C terminal blocks known to form efficient micellar bridging inside the solution, yielding hydrogels with low polymer concentration.38 Another possible strategy might be to increase the length of the middle B block along with a reduction of the composition of phenylboronic acid and catechol. It is well-known that the critical gelation concentration of loaded ABA triblock copolymers decreases when the molecular weight of the micelle bridging B block increases.33,39 Alternatively, it would be possible to use hydrogels formed by ABA triblock copolymers without catechol and phenylboronic acid groups to encapsulate cells and coat them with a thin layer of CAT-ABA or PBA-ABA hydrogels for adhesion.


Page 19 of 25

120 100 Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

N.S.

*

CAT‐ABA PBA‐ABA Control

80 60 40 20 0 0 24 Incubation time (h)

Figure 5. Cell viability shortly following encapsulation (0 h) and after 24 h of incubation in hydrogels (N = 3). Control shows cell viability upon encapsulation in P(NIPAAm-r-tBAAm)-bPDMAAm-b-P(NIPAAm-r-tBAAm) triblock copolymer hydrogels. N.S., not significant; *, P < 0.01.

CONCLUSIONS In this study, we successfully realized spontaneous self-assembly of cell-laden hydrogels through molecular recognition-based adhesion. Two types of physically cross-linked hydrogels were fabricated using hydrogels generated from thermo-responsive ABA-type triblock copolymers. From these, two kinds of totally synthetic ABA triblock copolymers, CAT-ABA and PBA-ABA, were prepared, which possessed the same NIPAAm-base thermo-responsive A block but different


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 25

hydrophilic B blocks including catechol and phenylboronic acid groups, respectively. Upon heating the block copolymer aqueous solution, the ABA triblock copolymers underwent LCMTtype micellization. The block copolymer “flower-like” micelles consisted of hydrophobic terminal A blocks surrounded by the hydrophilic B blocks, exposing the molecular recognition unit outer surfaces of the micelles. At high polymer concentration, the triblock copolymers exhibited sharp sol-to-gel transition concomitant with increases in temperature. We demonstrated that cell-laden hydrogels constructed from CAT-ABA and PBA-ABA triblock copolymers could exhibit macroscopic self-assembly through microscopic molecular recognition of the boronic ester formation between the catechol and phenylboronic acid groups. Our strategy might therefore represent a promising approach to construct tissue-like assembled structures in vitro. In particular, independent encapsulation of different types of cells in the hydrogels and assembling these various hydrogels through molecular recognition may make it possible to construct assembled cellular structures in hydrogels. Additionally, the sharp sol-gel transition of physically cross-linked hydrogels that occurs around physiological conditions would allow the specific removal of only the polymers after the formation of tissues in hydrogels by a straightforward temperature decrease. One of the remaining issues was related to the potential cytotoxicity of the hydrogels. The results of a Live/Dead assay indicated that both CAT-ABA and PBA-ABA exhibited cytotoxicity that could be attributed to the molecular recognition units, catechol and phenylboronic acid. For further improvement of the concept presented in this study, our ongoing studies involve the analysis of more sophisticated architectures of polymer design as well as the identification of molecular recognition processes with low cytotoxicity. Accordingly, the present study overall offers a new strategy to construct complex cellular structures based on the molecular recognition of hydrogels.


Page 21 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

ASSOCIATED CONTENT The following files are available free of charge. Synthesis schemes, GPC traces and 1H NMR spectra of polymers. AUTHOR INFORMATION Corresponding Author E-mail: [email protected]. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. †These authors contributed equally. ACKNOWLEDGMENT This work was supported in part by Grants-in-Aid for Scientific Research (No. 26620164 and No. 15H05495 to T. U., and No. 15H02198 to R. Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (No. 14J02019 for R. T.). We appreciate Prof. Toshiya Sakata for providing HeLa cells. We also thank Prof. Ung-il Chung and Prof. Takamasa Sakai for access to the DLS and rheological instrumentations. REFERENCES


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(1)

Page 22 of 25

Rice, J. J.; Martino, M. M.; De Laporte, L.; Tortelli, F.; Briquez, P. S.; Hubbell, J. A. Adv.

Healthc. Mater. 2013, 2, 57–71. (2)

Thiele, J.; Ma, Y.; Bruekers, S. M. C.; Ma, S.; Huck, W. T. S. Adv. Mater. 2014, 26, 125–

148. (3)

Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol. 2005, 23, 47–55.

(4)

Tibbitt, M. W.; Anseth, K. S. Biotechnol. Bioeng. 2009, 103, 655–663.

(5)

Baker, B. M.; Chen, C. S. J. Cell Sci. 2012, 125, 3015–3024.

(6)

Guguen-Guillouzo, C.; Clément, B.; Baffet, G.; Beaumont, C.; Morel-Chany, E.; Glaise,

D.; Guillouzo A. Exp. Cell Res. 1983, 143, 47–54. (7)

Shimaoka, S.; Nakamura, T.; Ichihara, A. Exp. Cell Res. 1987, 172, 228–242.

(8)

Goulet, F.; Normand, C.; Morin, O. Hepatology 1988, 8, 1010–1018.

(9)

Bhatia, S. N.; Balis, U. J.; Yarmush, M. L.; Toner, M. FASEB J 1999, 13, 1883–1900.

(10) Tsuda, Y.; Kikuchi, A.; Yamato, M.; Chen, G.; Okano, T. Biochem. Biophys. Res. Commun. 2006, 348, 937–944. (11) Hui, E. E.; Bhatia, S. N. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 5722–5726. (12) Kaji, H.; Camci-Unal, G.; Langer, R.; Khademhosseini, A. Biochim. Biophys. Acta 2011, 1810, 239–250. (13) Du, Y.; Lo, E.; Ali, S.; Khademhosseini, A. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 9522–9527.


Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(14) Du, Y.; Ghodousi, M.; Qi, H.; Haas, N.; Xiao, W.; Khademhosseini, A. Biotechnol. Bioeng. 2011, 108, 1693–1703. (15) Matsunaga, Y. T.; Morimoto, Y.; Takeuchi, S. Adv. Mater. 2011, 23, H90–H94. (16) Harada, A.; Kobayashi, R.; Takashima, Y.; Hashidzume, A.; Yamaguchi, H. Nat. Chem. 2011, 3, 34–37. (17) Kobayashi, Y.; Takashima, Y.; Hashidzume, A.; Yamaguchi, H.; Harada, A. Sci. Rep. 2013, 3, 1243. (18) Nakahata, M.; Takashima, Y.; Harada, A. Angew. Chem. Int. Ed. 2014, 53, 3617–3621. (19) Nakamura, T.; Takashima, Y.; Hashidzume, A.; Yamaguchi, H.; Harada, A. Nat. Commun. 2014, 5, 4622. (20) Harada, A.; Takashima, Y.; Nakahata, M. Acc. Chem. Res. 2014, 47, 2128–2140. (21) Nakahata, M.; Mori, S.; Takashima, Y.; Hashidzume, A.; Yamaguchi, H.; Harada, A. ACS Macro Lett. 2014, 3, 337–340. (22) Nakahata, M.; Takashima, Y.; Hashidzume, A.; Harada, A. Chem. Eur. J. 2015, 21, 2770– 2774. (23) Asoh, T. A.; Kikuchi, A. Chem. Commun. 2010, 46, 7793–7795. (24) Asoh, T. A.; Kawai, W.; Kikuchi, A. Soft Matter 2012, 8, 1923–1927. (25) Asoh, T. A. Polym. J. 2016, DOI: 10.1038/pj.2016.85 (26) Lai, J. T.; Filla, D.; Shea, R. Macromolecules 2002, 35, 6754–6756.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 25

(27) Koppel, D. E. J. Chem. Phys. 1972, 57, 4814–4820. (28) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163–249. (29) Sugihara, S.; Hashimoto, K.; Okabe, S.; Shibayama, M.; Kanaoka, S.; Aoshima, S Macromolecules 2004, 37, 336−343. (30) Kitazawa, Y.; Ueki, T.; Niitsuma, K.; Imaizumi, S.; Lodge, T. P.; Watanabe, M. Soft Matter 2012, 8, 8067−8074. (31) Tamate, R.; Ueki, T.; Kitazawa, Y.; Kuzunuki, M.; Watanabe, M.; Akimoto, A. M.; Yoshida, R. Chem. Mater. 2016, 28, 6401–6408. (32) Balsara, N. P.; Tirrell, M.; Lodge, T. P. Macromolecules 1991, 24, 1975–1986. (33) Kirkland, S. E.; Hensarling, R. M.; McConaughy, S. D.; Guo, Y.; Jarrett, W. L.; McCormick, C. L. Biomacromolecules 2008, 9, 481–486. (34) Matsumoto, A.; Yoshida, R.; Kataoka, K. Biomacromolecules 2004, 5, 1038–1045. (35) Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291–5300. (36) O’Brien, P. J. Chem. Biol. Interact. 1991, 80, 1–41. (37) Jin, X.; Zhang, X.; Wu, Z.; Teng, D.; Zhang, X.; Wang, Y.; Wang, Z.; Li, C. Biomacromolecules 2009, 10, 1337–1345. (38) Zhou, C.; Hillmyer, M. A.; Lodge, T. P. J. Am. Chem. Soc. 2012, 134, 10365–10368. (39) Semenov, A. N.; Joanny, J. F.; Khokhlov, A. R. Macromolecules 1995, 28, 1066–1075.


Page 25 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Table of Contents


Self-Assembly of Thermoreversible Hydrogels via Molecular

Recommend Documents