Dynamic Bonds between Boronic Acid and Alginate - ACS Publications

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Dynamic Bonds between Boronic Acid and Alginate: Hydrogels with Stretchable, Self-healing, Stimuliresponsive, Re-moldable, and Adhesive Properties Sang Hyeon Hong, Sunjin Kim, Joseph P. Park, Mikyung Shin, Keumyeon Kim, Ji Hyun Ryu, and Haeshin Lee Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00144 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on March 31, 2018

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Dynamic Bonds between Boronic Acid and

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Alginate: Hydrogels with Stretchable, Self-healing,

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Stimuli-responsive, Re-moldable, and Adhesive

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Properties

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Sang Hyeon Hong†, Sunjin Kim†, Joseph P. Park†, Mikyung Shin†, Keumyeon Kim‡, Ji Hyun Ryu

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7 8 9 10

⊥

, and Haeshin Lee*,†

†

Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305–701, South Korea ‡R&D

Center InnoTherapy Inc. 25, Sonyou 13th Rd, High-tech CityII Suite #2019, Youngdeungpo-Gu 07282, South Korea ⊥

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Department of Carbon Fusion Engineering, Wonkwang University, Iksan, Jeonbuk, 54538, South Korea

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KEYWORDS: Hydrogel, self-healing, stretchable, boronic acid.

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ABSTRACT: For the increasing demand of soft materials with wide ranges of applications,

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hydrogels have been developed exhibiting variety of functions (e.g. stretchable, self-healing,

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stimuli-responsive, and etc.). So far, add-in components such as inorganic nanoparticles, carbon

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materials, clays, and many others to main polymers have been used to achieve various unique

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functions of hydrogels. The multi-component hydrogel systems often exhibit batch-dependent

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inconsistent results and problems in multi-component mixings, require labors during

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preparations, and accompany unpredictable cross-talk between the added components. Here, we

ACS Paragon Plus Environment

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developed ‘single polymeric component’, alginate-boronic acid (alginate-BA) hydrogel to

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overcome the aforementioned problems. It exhibits unprecedented multi-functionalities

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simultaneously such as high stretchable, self-healing, shear-thinning, pH- and glucose-

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sensitivities, adhesive, and re-shaping properties. Multi-functionalities of alginate-BA hydrogel

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is resulted from the reversible inter-, intra-molecular interactions by dynamic equilibrium of

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boronic acid-diol complexation and dissociation, which was proved by single molecule level

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Atomic Force Microscopy (AFM) pulling experiments. We also found that the alginate-BA gel

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showed enhanced in vivo retentions along gastrointestinal (GI) tract. Our findings suggest that

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rational polymer designs can result in minimizing the number of a participating component for

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multi-functional hydrogels, instead of increasing complexity by adding various additional

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components.

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INTRODUTION

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Since the development of the first hydrogel by Wichterle and Lim in 1960,1 hydrogels have been

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used in a variety of biomedical applications, such as scaffolds for tissue engineering2,3, matrixes

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for cell culture systems,4–6 and vehicles for drug delivery.7 However, recent progress in hydrogel

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development has focused on extra functions, such as the ability to sensitively respond to external

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stimuli,8 self-heal,9 stretch,10–12 and remold shape 13,14 for a wide range of applications.

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In general, the physicochemical properties of hydrogels depend greatly on gelation chemistry,

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which is classified into two types15,16: covalent and physical crosslinking. The primary purpose

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of using chemical crosslinking in hydrogels is to achieve strong mechanical properties and


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stability. However, hydrogels with unique, dynamic characteristics, such as self-healing or

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injectable properties, utilize physical crosslinking (e.g., hydrogen/ionic bonds, hydrophobic

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interactions, and mechanical entanglements). Reversibility in bond association and dissociation

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helps to reconstruct polymeric networks within hydrogels that exhibit self-healing, stimuli-

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responsive, or injectable properties.

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To achieve these dynamic properties in hydrogels, two or three components are typically used. A

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mixture of clay, sodium polyacrylate, and dendrimers (3 components) led to hydrogels with

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exceptional mechanical strength accompanied by self-healing ability 17. Additionally, tri-n-

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butylphosphine, glycidyl pluronic, and polysulfide (3 components) generated self-healing and

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remoldable gels18. Unprecedented stretchability was achieved by a mixture of calcium, alginate,

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and poly(acrylamide), in which the reversible ionic bonds reconstructed internal polymeric

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networks 12. Poly(propylene glycol), a furan-functionalized component, and bismaleimide

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exhibited self-healing properties 19. Several other studies have utilized three components to

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construct hydrogels with dynamic properties. Functional, dynamic hydrogels with two

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components have also been reported. For example, host-guest recognitions, such as cyclodextrin-

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cholic acid 20 or dibenzo[24]crown-8-bisammonium21, resulted in self-healing hydrogels. In

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addition, dynamic self-healing properties were achieved by dynamic bond exchange of the

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Schiff-base linkage formation between hydroxyl-terminated PEG with 4-formylbenzoic acid and

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the glycol chitosan,22 catechol-Fe3+ complexation23 .

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Although preparation methods aimed at creating functional, dynamic hydrogels have been

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successfully developed, the use of multiple components in hydrogels may require significant

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degrees of considerations such as solubility, multi-step synthesis, mixing, and so on compared to

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a single polymeric component system. The advantages of using a single polymeric component


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are numerous. Uncertainties, including inhomogeneous mixing of viscous polymeric solutions,

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interference between functional groups due to unexpected interactions, or leaching of ionic

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components, such as calcium, into the environment, can be significantly reduced in a single

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polymeric component system. Furthermore, multiple components often decrease injectable

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properties and require a tedious amount of labor. In fact, attempts to reduce the number of

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polymeric components for multi-functional hydrogel exist. However, tethering chemistry to a

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polymer backbone has been rather complex. For example, self-healable and injectable hydrogel

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using a copolymer of 2-(dimethylamino)-ethyl methacrylate (DMAEMA) and 2-(3-(6-methyl-4-

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oxo-1,4-dihydropyrimidin-2-yl)ureido)ethyl methacrylate (SCMHBMA)24. Also, a hydrogel with

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glucose responsive and self-healing ability was achieved by acrylate copolymerization of

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phenylboronic acid and amine group followed by glucose tethering for boronic aicd-glucose

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complexation25.

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In this study, we developed a highly simplified single polymeric component hydrogel that

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consisted of an alginate-boronic acid conjugate (alginate-BA) utilizing intrinsic cis-diol existing

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along alginate backbones. The prepared alginate-BA hydrogel simultaneously exhibited multiple

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dynamic functions of self-healing, stretchability, re-shaping, stimuli-sensitivity, and adhesive

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properties. The boronate and cis-diol interaction is well known26 and has been applied to various

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applications, such as sugar sensors27, chromatography28, drug delivery systems29, and cell surface

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labeling30. Interestingly, when preparing alginate-BA, moieties of cis-diol and boronic hydroxyl

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groups co-exist in the same polymer, which can cause spontaneous self intra- and/or inter-

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polymeric interactions. However, we hypothesized that the backbone stiffness of alginate31 and a

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high alginate-BA concentration might minimize intra-cis-diol-boronate interactions. In

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particular, the dynamic equilibrium of the bond rearrangement/transfer between the cis-diol and


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boronic acid hydroxyls resulted in a distinctive natural rubber-like viscoelastic alginate-BA

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hydrogel. The alginate-BA hydrogel demonstrated unprecedented multi-functionality by

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simultaneously exhibiting, high stretchability, self-healing, shear-thinning, pH- and glucose-

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sensitivity, adhesiveness, and re-shaping properties that were achieved by the single polymeric

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component, alginate-BA. Utilizing these features, oral administration of the hydrogel resulted in

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long-lasting in vivo mucosal adhesions compared to the unmodified alginate. We expect that this

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novel material will satisfy the demanding biological and handling requirements for specific

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hydrogel uses.

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EXPRIMENTAL SECTION

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Materials. Alginate sodium salt from brown algae (Sigma, USA), 3-Aminophenylboronic acid

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hydrochloride (Sigma), N-Hydroxysuccinimide (NHS, Sigma), and 1-(3-Dimethylaminopropyl)-

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3-ethylcarbodiimide hydrochloride (EDC, TCI, Japan) were used as received.

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Preparation of alginate-boronic acid. 3-Aminophenyl boronic acid (BA) was conjugated onto

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the alginate backbone by EDC/NHS reactions. To conjugate the boronic acid, 1 g of alginate was

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dissolved in 0.1 M MES buffer solution, and the pH of the solution was adjusted to 5.5 using 0.1

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M HCl. And then, 700 mg of EDC and 100 mg of NHS were added to the alginate solution in a

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drop wise manner. Boronic acid (300 mg) was added to the mixture solution and reacted for 12

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hours at room temperature. The resulting reaction solution was dialyzed (MWCO: 3,500 Da,

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Spectra/Por), and the pH of the solution was maintained at 5.5 during purification. The degree of

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substitution (DOS) of boronic acid moiety was determined using UV absorbance at 295 nm using

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an UV-Vis spectrophotometer (Hewlett-Packard 8453, Groton, CT, USA). Boronic acid was

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used as a standard molecule from 0.1 mM to 0.5 mM (Figure S1). The Fourier transfer infrared


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(FT-IR) analysis of the polymers were conducted on a Nicolet is50 FT-IR spectrometer (Thermo

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Fisher Scientific Co., Waltham, MA, USA) in the range 4,000-400 cm-1 and 1H nuclear magnetic

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resonance (NMR) data were collected in D2O using Agilent-400 MHz NMR instrument

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(Agilent Technologies Inc., USA). In addition, the polymers were characterized with Agilent

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1200 series using gel permeation chromatography (GPC) column (SB-804 HQ, Shodex,

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Germany). The flow of eluent was 0.5 mg / min, and the injection volume was 50 µl. The eluent

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was monitored by refractive index (RI) detector.

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Alginate-BA hydrogel formation. For the preparation of alginate-BA hydrogels, the polymers

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were dissolved in DDW at 3 wt %, and then 100 mM of PBS buffer (pH 7.4, except for

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hydrogels used in the rheometer measurements) was added to the alginate-containing solutions to

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yield a final concentration of 2.3 wt%. The hydrogels were formed within 1 min and stabilized

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for subsequent 30 min. After that, the manipulation of the hydrogel such as elongation by

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tweezers, shape molding or injection through 18G needle was performed.

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Rheological studies of alginate-BA hydrogel. The rheological properties (storage modulus, G’

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and loss modulus, G’’) of the alginate-BA hydrogels were monitored using a rotating rheometer

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(Bohlin Advanced Rheometer, Malvern Instruments, Worcestershire, U.K.). For the frequency

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sweep measurements, the sweep frequency varied from 0.1 to 10 Hz. The moduli were measured

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at pH 7.4, 10, and 12 to investigate the pH-dependent modulus changes of the hydrogel. The pH

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reversibility was tested by addition of small amount of acid (1 M HCl) to dissolve hydrogel and

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subsequent addition of base (1 M NaOH) to re-from hydrogel. The modulus was also measured

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with the amount of glucose (200, 400, 600, 800, and 1000 mM) to observe subsequent decrease

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of storage modulus of fully cured hydrogel at pH 12. And also, viscosity was measured witin the

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range of shear rate (0.001-10 s-1)


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Test for self-healing property. To evaluate self-healing ability, the alginate-BA hydrogel was

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shaped into heart. The sample was partially cut by a razor and then manually re-attached.

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Additional 5 min was used for stabilization of the hydrogels at room temperature before picking

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up by a tweezers. To quantitatively investigate the self-healing property, responses to applied

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shear strain was observed by step-strain experiments with a rheometer. The step-strain

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experiments were performed at 1 % strain 5 % 1 % 10 % 1 % 20 % 1%.

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UTM study for the adhesive force. To examine the adhesion of alginate-BA hydrogels, a UTM

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test was conducted (Instron 5583, USA) with a 150 N load cell. The 20 µl of hydrogel for the test

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were prepared between glass capped cylindric probes (diameter: 8 mm) and was compressed

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with 0.1 mN of normal force. The probes were detached at a rate of 10 mm min−1. The adhesion

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force (kPa) was determined as the maximum load (kN) divided by the area (m2).

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AFM experiment for molecular mechanism study. AFM pulling experiments were performed

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using Asylum MFP-3D-BIO AFM instrument (Asylum Research, Santa Barbara, CA) equipped

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with silicon nitride tips with a spring constant of 250 pN/nm (Bruker AFM Probes, Madison,

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WI). For surface preparations, alginate-BA was spin-coated (750 rpm) on Si substrates to for

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thickness of ~ 10 nm. Subsequently, force-distance curves were measured by bringing the tip

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down to the surfaces with a speed of 0.5 µm/sec and then stayed on surface (surface dwell time =

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60 sec) to allow the polymer adsorption onto the tip surfaces in 10 mM Tris (pH 9). The pulling

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speed was the same as 0.5 µm/sec.

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In vivo oral administrations and tissue histology. All animal care and experimetnal

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procedures were followed the ethical protocol given by the Korean Ministry of Health and

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Welfare. Nude mice (BALB/c nude mouse, male, 9-10weeks Central Lab. Animal Inc., Korea)


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were starved 12 hr before experiments. Alginate and alginate-BA were dissolved in DDW at 3

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wt% rhodamine-dextran solution (1 mg / ml) was mixed with alginate or alginate-BA solution at

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the ratio of 10 v/v% for visualization. Doses of 10 µl of the samples were orally administered to

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mice. Afterward, 50 µl of PBS buffer was orally injected into the mice. The mice were then

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sacrifieced after 30 min or 24 h. Fluorescent images were obtained with an IVIS 200 imaging

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system (Xenogen, CA, USA). Three mice for each group were used in vivo test.

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Alginate solution and alginate-BA hydrogel were also subcutaneously implanted on the backs of

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nude mice. At day 3 and 7 post implantations, the mice were euthanized, and then the tissues

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surrounded by the hydrogel-implanted areas were collected. The tissue sections were fixed with

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10% formalin, and dehydrated in a graded sucrose solution (10, 20, and 30 w/v %). For the

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cryosection, the dehydrated tissues were embedded in optimum cutting temperature (OCT)

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compound (Sakura Finetek, USA) followed by freezing process using liquid nitrogen. Serial

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cryosections of 10 µm were obtained using a Leica CM3050s Cryostat (Leica Biosystems,

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Germany). For histological analysis, the sectioned tissues were stained with hematoxylin and

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eosin. The stained sections were observed using a Nikon Eclipse TS100 inverted routine

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microscope (Nikon, Japan).

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RESULTS AND DISCUSSION

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To

introduce

the

boronic

acid

moiety to

the

alginate

backbone,

1-ethyl-3-(3-

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dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used as

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carboxyl activating agents. Alginate was employed as a backbone material due to its

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biocompatibility and structural suitability from a cis-diol group in a repeating unit. The degree of

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substitution was quantified by UV–vis absorbance at 295 nm using 3-aminophenylboronic acid


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as a standard molecule. The result showed that the degree of boronic acid conjugation was

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approximately 10 ± 1.5 % (Figure S1). To confirm the presence of boronic acid moiety, the

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alginate-BA was further characterized by FT-IR and NMR spectroscopy. As shown in Figure.

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S2a, the newly appeared peak at 1,670 cm-1 was observed in the spectrum (alginate: black,

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alginate-BA: red line), which were attributed to the amide-linked C=O stretching vibrations.

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Also, the C=C bending of phenyl group and the B-O stretching vibrations in boronic acid moiety

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appeared at 1,500-1,600 cm-1 and 1,340 cm-1 respectively. The peak at 700 cm-1 was assigned to

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the C-H bending vibration of the phenyl group of BA. The 1H NMR (D2O) spectra confirmed the

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conjugation by showing the phenyl group protons at 7.3 to 7.7 ppm of the alginate-BA (blue)

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(Figure S2b), which corresponded to control boronic acid protons shown in the red spectra. In

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addition, GPC measurements of the synthesized alginate-BA (0.5 mg/ ml) were conducted, and

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the eluent was monitored by a refractive index (RI) detector. As shown in Figure S2c, the

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elution time (10.8 min) of alginate-BA was earlier than that of unconjugated alginate (11.8 min),

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indicating that the interactions between boronic acid and cis-diol in alginate-BA resulted in an

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increase in the hydrodynamic volume. Then, Alginate-BA was dissolved in 25 mM phosphate

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buffered saline (PBS, pH 7.4) at a concentration of 2.3 wt %, which formed viscoelastic

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hydrogels.

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While most common hydrogels suffer from mechanical failures under large strain due to their

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rigid polymer network, the alginate-BA hydrogel exhibited superior stretchable property (Figure

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1a). The hydrogel (200 µl) was stretched by two tweezers and could be stretched by 23 times (1

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23 cm) in the longitudinal direction without any fracture. Chemically unmodified alginate or

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the well-known conventional alginate- Ca2+ hydrogel did not show the similar stretching

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behavior (Figure S2). Another unique characteristic of the hydrogel is that the alginate-BA gel is


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injectable by a ‘shear-thinning’ mechanism. As shown in the rheometer study, the viscosity of

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the alginate gel was rapidly decreased (i.e. thinned) at a strain rate started from 0.1 sec-1 (1450 to

3

45 Pas) (Figure S3). Due to the shear-thinning properties, the alginate gel was injectable via 18-

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gauge needle, showing a fiber-like, one-dimensional long hydrogel (Figure 1b). The gel was

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visualized by red dye, monoazo-chromophores. This macroscopically observed result could be

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attributed to the reversible inter-alginate chain interactions between the cis-diol in the alginate

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backbone and the diols of boronic acid. To explain the observed stretchability at a molecular

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level, we performed pulling experiments using atomic force microscopy (AFM). Previously, the

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cis-diol and boronate interaction has been reported[22-26], but a single-molecule level

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demonstration of the alginate inter-chain interaction has not been conducted. Alginate-BA was

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deposited onto a silicon substrate by spin-coating (1 mg/mL, 750 rpm for 30 sec). This process

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provided a thin film of alginate-BA with a dry thickness of ~ 10 nm. Subsequently, an alkaline

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buffer (pH = 9.0, Tris) was added to the alginate-BA adsorbed Si surfaces to trigger cis-diol-

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boronic acid binding as well as swelling. After that, a bare Si3N4 AFM tip was placed on the

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surface (0.5 µm/sec) and then stayed on the surface for 60 sec to allow alginate-BA adsorption

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onto the tip surface before it was pulled (0.5 µm/sec). Subsequently, the tip surface adsorbed

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alginate-BA was stretched by AFM pulling (Figure 1c). In particular, in an external-force

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applied environment by AFM pullings, the cis-diol/boronic acid bond dissociation is inevitably

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observed. Importantly however, the dissociated bond moved along the direction of applied force

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indeed reforms the same type of a bond (i.e. cis-diol/boronic acid) due to its reversible character.

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The aforementioned bond dissociation and reformation cycles were repeated along the applied

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force direction by AFM, accomplishing the outstanding stretchability of the alginate-BA gel

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(Figure 1d). The dissociation at a molecular level exhibited a saw-tooth pattern as shown in


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Figure 1e. The force required for dissociation between cis-diol and boronic acid was in the range

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of 130~360 pN. The saw-tooth pattern in the AFM pulling experiments was similar to the pattern

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for multi-domain proteins, such as titin32, or multimers of small proteins with disulfide bonds33,

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but the pattern was not a typical result for a polysaccharide pulling experiment34. In general, a

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polysaccharide with a 1,4-glycosidic bond exhibited a pure elastic chain extension corresponding

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to a worm-like model (Figure 1e, 2nd curves, ref: 30). It is worthwhile to mention that those

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curves did not show any saw-tooth pattern, whereas a clear saw-tooth pattern was

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unprecedentedly observed in the alginate-BA pulling experiments (Figure 1e, 1st curve). The

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pattern indicates the energy required for breaking an individual (or multiple) boronic acid-diol

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complex (boronate complex) during pulling of the alginate-BA chain. Note that the high

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frequency of the peak appearance might represent repetitive cycles of reversible formation-

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dissociation of boronic acid-diol complexes.

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In contrast, without external force, the thermodynamic mobility of polymer chains allowed for

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the new formation of boronic acid-cis-diol bonds. Unlike common hydrogels composed of a rigid

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covalent network, the dynamic bond rearrangements within the alginate-BA hydrogels

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continuously occur at a molecular level (Figure 2a). Thus, the dynamic bond formation-

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dissociation cycles can result in various unique hydrogel properties, such as self-healing and re-

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shaping. First, alginate-BA hydrogel was capable of self-healing, as shown in Figure 2b. The

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hydrogel was initially molded into a heart shape and then partially cleaved (1st and 2nd photos).

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Subsequently, the cleaved area was re-attached for 5 minutes, and the healed part was monitored.

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The healed hydrogel was mechanically strong enough to be picked up by a tweezers without

22

fracturing (Figure 2b, 3rd photo). The self-healing property was also quantitatively measured by

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continuous step-strain measurement at a frequency of 1 Hz using a rheometer (Figure 2c). We


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performed strain-sweeping experiments in an up-and-down manner by 1 (317.8 Pa) to 5 % (17.5

2

Pa), down back to 1 (213.6 Pa) to 10 % (4.8 Pa), and down back to 1 (194.4 Pa) to 20 % (1.2 Pa)

3

until the gel exhibited mechanical failure. For the last strain relaxation step (20 1 %), the gel

4

recovered the storage modulus (G’, 141.9 Pa) despite the sudden large changes in strain value. In

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order to analyze the self-healing efficiency of the hydrogel with a view point to its mechanical

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strength recovery, we synthesized the alginate-BA having different degree of substitution (DS =

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5 and 18 %). The healing efficiency was evaluated by storage modulus (G’) determined by step-

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strain rheometeric experiments for the gels after re-attachment. As shown in Figure S5, the

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healing efficiency increased with increasing DS of polymer. For the 10 % DS hydrogel, only 44

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% of the storage modulus was recovered, whereas 98.3 % of modulus were recovered with 18 %

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DS hydrogels. This rapid and complete self-healing in bulk hydrogel may also be attributed by

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the aforementioned dynamic network reorganization processes. On the other hand, in the case of

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hydrogel having a 5 % of DS, the mechanical property was too weak for proper measurement (~

14

10 Pa). Second, a re-shaping characteristic was also observed in the alginate-BA hydrogel, as

15

described in Figure 2d. The prepared hydrogel was molded into various shapes, and the

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morphology changed from the sphere to a dolphin in a one-step molding process (top left and top

17

right). Subsequently, due to the reversible bond reformation, the hydrogel shape could be

18

continuously changed from a dolphin to a heart to a duck shape (bottom right and left).

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Bonds between cis-diol and boronic acid exhibit pH-dependent properties35. In general, the

20

phenylboronic acids show pKa values approximately 7.0 to 8.5 depending on the substituted

21

moieties. Above a corresponding pKa value of a given boronic acid derivative, boronate is

22

formed, which binds strongly to cis-diol, and below the pKa value, boronic acid is formed, which

23

weakly binds to cis-diol. First, we hypothesized that the alginate-BA hydrogel might show pH-


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sensitive properties that were particularly sensitive for mechanical strength. We performed

2

frequency sweep experiments from 0.1 to 10 Hz as a function of pH (7.4, 10, and 12; 2.3 wt%).

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At pH 7.4, the hydrogel exhibited viscoelastic properties. In detail, a G’ (292 Pa at 1 Hz) < G”

4

(421 Pa at 1 Hz) trend was observed at a rotating frequency region lower than 3 Hz, but G’ (821

5

Pa at 5 Hz) > G’’ (629 Pa at 5 Hz) was detected at a frequency higher than 3 Hz (Figure 3a).

6

This result indicates that the hydrogel network was not fully formed. On the other hand, the

7

value of both the storage modulus and loss modulus (G’ = 1996 Pa, G’’ = 1265 Pa at 1 Hz) were

8

increased by approximately 5-fold, and the gel-like region also expanded (~ 0.2 Hz) as pH

9

increased to 10 (Figure 3b). At pH 12, no such crossover point was found. For the overall

10

rotational frequency region, G’ (representative value of 80,260 Pa at 1 Hz) was always higher

11

than G’’ (7,099 Pa at 1 Hz) and demonstrated almost complete boronate bond formation (Figure

12

3c). The observed pH-dependent mechanical property changes of the hydrogel can be reversible,

13

and a sol-gel transition was also observed in sudden pH shift experiments, such as shifting pH 12

14

down to pH 2 and back up to pH 12. By measuring G’ values at a frequency of 1 Hz, the G’

15

values were reversible in response to the pH values with G’ decreasing from 34,560 Pa to 3.5 Pa

16

and then increasing to 42,600 Pa after the addition of small amount of 1 M HCl or NaOH

17

solution (Figure 3d). In addition, we lowered (2.3 wt% -> 1.0 wt%) and increased (2.3 wt% ->

18

4.0 wt%) the polymer concentrations at pH 7.4 (Figure S6). When decreasing the concentration,

19

G’ value was lowered from 292 Pa for 2.3 wt% down to 22.5 Pa for 1 wt% at 1 Hz oscillation

20

condition. Increasing the concentration resulted in expanding gel-like region from 3 Hz for 2.3

21

wt% 0.13 Hz for 4 wt%, and G’ value was increased as expected (433 Pa for 4 wt% at 1 Hz).

22

Second, we also hypothesized that the alginate-BA hydrogel might show glucose-sensitive

23

properties in which the 1,2-cis-diol of glucose specifically bonded to boronic acids. As such, the


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Page 14 of 30

1

glucose bonds would act as crosslinking inhibitors, which would result in decreases in the

2

mechanical properties of alginate-BA hydrogels (Figure 3e). We prepared solutions with various

3

concentrations of glucose: 200, 400, 600, 800, and 1,000 mM in PBS buffer (pH = 12). As

4

expected, the added glucose inhibited existing crosslinking, which resulted in decreases in the

5

storage moduli from 80,260 Pa (for 0 mM glucose) to 15,620 Pa (for 200 mM), 7,363 Pa (for 400

6

mM), 5,579 Pa (for 600 mM), 2,558 Pa (for 800 mM), and 1,290 Pa (for 1,000 mM) (Figure 3f).

7

These results showed that an alginate-BA hydrogel can be utilized as a glucose-sensitive

8

material.

9

Furthermore, with the application of slight pressure, the alginate-BA hydrogel was capable of

10

serving as a pressure-sensitive adhesive (PSA)36, which is generally attributed to a viscoelastic

11

material, such as acrylic resins. Viscoelastic materials make intimate contact followed by

12

adherence onto surfaces via mechanical interlocking or van der Waals forces. The boronic acid-

13

mediated binding force demonstrated in AFM experiments might be beneficial for enhancing

14

bulk properties (i.e., increases in pulling resistance between substrates) as a PSA material in

15

addition to the intrinsic viscoelastic nature of alginate-BA hydrogels37. Therefore, we evaluated

16

the adhesiveness of the hydrogel in a universal testing machine (UTM). The hydrogel was

17

prepared between two identical glass-capped probes with a probe traveling speed of 1 cm/min, as

18

shown in Figure 3g. The detachment force value was ~ 32 kPa (Figure 3h, black), whereas the

19

use of alginate itself without boronic acid conjugations showed a significantly low adhesion

20

force of ~ 3 kPa (red). This adhesiveness was comparable to previously reported polymer-based

21

hydrogel adhesives

22

Also, the elongation behavior was observed in the long-strain region of the graph (black)

23

showing the significant stretchability as the probe moved upward from the bottom probe.

38,39

. Yet, the magnitude of adhesion might be suitable as a biological glue.


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Utilizing the aforementioned adhesive properties, we tested mucoadhesion properties of the

2

alginate-BA hydrogels. We orally administered alginate-BA to mice (Figure 4a). According to

3

the previous studies, alginate itself has been used in oral drug delivery systems due to its

4

resistance to strong acidic environments in stomach40. Thus, it is fairly reasonable model system

5

for alginate-BA hydrogel as a new oral administrative polymer. Also we expect that the adhesive

6

nature demonstrated by alginate-BA hydrogels would increase residual time in the body.

7

We prepared 3 wt% of alginate-BA and alginate solution as a negative control respectively,

8

which were visualized by mixing rhodamine B isothiocyanate-dextran (Rho-dex) (1 mg/mL, 1

9

µl). The alginate-BA (10 µl) was fed to mice and then additional PBS buffer (pH 7.4, 50 µl) was

10

administered. The same volume of chemically unmodified alginate was used as a control. The in

11

vivo retention along a gastrointestinal (GI) tract of the polymer was monitored by IVIS Lumina

12

at 30 minutes and 24 hours after the administrations (n=3). At an early timeframe (< 30 minutes),

13

a clear alginate-BA fluorescent signal was detected at the esophagus region (Figure 4b, right

14

panel, yellow circle). Whereas no fluorescence was observed for alginate oral administration

15

(Figure 4b, left panel). It is because alginate-BA gelation was expected to occur in the

16

esophagus region due to the pH upshift to 7.4, resulting in an increase of in vivo retention, but

17

alginate was simply washed off (top, left panel). In addition, overall remained amount of

18

alginate-BA was high compared with that of alginate. The increased in vivo retention of alginate-

19

BA was continuously observed for subsequent 24-hr observation after the feeding. Strong

20

fluorescent signals (e.g. red dots) of alginate-BA hydrogel were observed in intestine region.

21

However, weak dispersive residual fluorescent signals were remained for the case of alginate

22

(Figure 4b, bottom left panel). We also performed additional a negative control experiment.

23

Rho-dex (1 mg/ml, 1 µl) itself did not exhibit mucoadhesive property. One µl of Rho-dex


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1

solution was orally administered with 9 µl of PBS buffer instead of alginate or alginate-BA, and

2

then a 50 µl of PBS was injected consistently with the previous experiment. After 30 minutes,

3

fluorescent image was obtained, and signals were not observed in both untreated mouse (Figure

4

S7, left) and Rho-dex fed mouse (right). Thus, the fluorescent signals shown in Figure 4b were

5

indeed the alginate-BA’s mucoadhesion with which the labelling Rho-dex was complexed in

6

vivo. These results indicate that the pH-responsible gelation and the sticky nature of alginate-BA

7

hydrogel allow one to utilize alginate-BA as a potential useful material for mucoadhesive oral-

8

delivery applications. Finally, alginate solution and alginate-BA hydrogels were implanted

9

subcutaneously under mice skins for evaluation of inflammatory responses by histological study.

10

The sectioned tissues were stained by hematoxylin-eosin (H&E), where inflammatory cells show

11

purple and normal tissues show pink. Severe inflammatory responses at day 3 were observed

12

both alginate and alginate-BA (Figure 4c, 1st and 2nd pictures, black triangle), however, immune

13

responses were mostly disappeared in day 7 (Figure 4c, 3rd picture). The data indicates that the

14

hydrogel has low toxicity.

15

CONCLUSONS

16

In conclusion, we developed a single polymeric component, alginate-BA hydrogel that

17

exhibited unprecedented multifunctional properties, including stretchability (Figure 1a), self-

18

healing (Figure 2b), stimuli-responsiveness (pH: Figure 3a-c, glucose: Figure 3f), re-shaping

19

(Figure 2d), shear-thinning (Figure 1b), and adhesive properties (Figure 3h). The carboxyl

20

group of the alginates was chemically conjugated with the amine group of boronic acid to take

21

advantage of the reversible boronate-cis-diol complexation, which resulted in reversible self-

22

crosslinking both with and without external force. Based on our results, the alginate-BA

23

hydrogels have excellent potential for a wide range of applications, including pressure-sensitive


16

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1

biological glues to biomedical platforms that require stretchability, self-healing, and multi-

2

responsive nature.

3


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Figure 1. Macroscopic and molecular-level studies of alginate-BA hydrogels: shear-thinning and stretchable properties in the presence of an external force. (a) A series of photos showing the high stretchability and (b) injectability demonstration by a shear-thinning mechanism applied via an 18-gauge needle of alginate-BA hydrogels (macroscopic phenomenon). (c) Schematic illustrations of molecular behavior at a microscopic level in the AFM pull-off experiment, and


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1 2 3 4 5

Biomacromolecules

schematics of (d) bonding transfer along the direction of an external force. (e) force-distance curves of molecular interactions between alginate-BA chains (top) and unmodified polysaccharides (bottom, the spectrum was adapted from ref: 34) obtained in the AFM pull-off experiment. Extensions were normalized by the length determined at a force of 3.0 nN (top) and1.5 nN (bottom) and the tick interval on the y-axis is 1.5 nN.

6


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1 2 3 4 5 6 7 8

Figure 2. (a) Schematic illustrations of the boronic acid-cis-diol bond dissociation followed by association via dynamic chain movement without external force. (b) Demonstration of selfhealing properties. (c) Quantitative analysis of self-healing properties. The gel was fractured under strain (top: sequential strain steps, 1 % 5 % 1 % 10 % 1 20 % 1 %) and the recovered storage moduli were determined (bottom). (d) Photos demonstrating the re-shaping capability of the hydrogel.

9


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Biomacromolecules

1 2 3 4 5 6 7 8

Figure 3. Experiments examining the tunable rheological properties of alginate-BA hydrogels by pH-dependent changes in modulus (G’: closed, G’’: open) at (a) pH 7.4, (b) pH 10, and (c) pH 12 by a frequency sweep from 0.1 to 10 Hz and (d) a reversible sol-gel transition from a pH shift. (e-f) Addition of glucose resulted in a decrease of storage modulus (0 mM: closed circle, 200 mM: open circle, 400 mM: closed triangle, 600 mM: open triangle, 800 mM: closed square, 1000 mM: open square). (g-h) Pressure sensitive adhesive force measured by UTM (black: alginateBA, red: alginate).

9 10


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Figure 4. (a) Schematic illustrations of oral administration of alginate-BA and alginate. (b) BALB/c mice were orally administered with Rho-dex containing alginate (left) and alginate-BA (right) solutions and euthanized after 30 min and 24 hrs. The fluorescent images were obtained by IVIS. (c) Photos of H&E stained subcutaneous tissue sections after 3 and 7 days of implantations of alginate-BA hydrogel and alginate (3 days). The inflammatory response was indicated black triangles.

8


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Biomacromolecules

1 2

ASSOCIATED CONTENT

3

Supporting Information.

4

Supporting Information is available from the ACS Publications website.

5

UV-Vis, FT-IR, NMR spectrometry data of alginate-BA; GPC; Fracturing of alginate-Ca2+

6

hydrogel; Rheology data; Fluorescent images of in vivo test

7 8

AUTHOR INFORMATION

9

Corresponding Author

10

*E-mail: [email protected]

11

Notes

12

The authors declare no competing financial interest

13 14

ACKNOWLEDGMENT

15

This work was supported by a grant from the National R&D Program for Cancer Control, The

16

Ministry for Health and Welfare, Republic of Korea (1631060) and Disaster and Safety

17

Management Institute (MPSS-CG-2016-02).

18

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For Table of Contents Use Only

3

Dynamic Bonds between Boronic Acid and

4

Alginate: Hydrogels with Stretchable, Self-healing,

5

Stimuli-responsive, Re-moldable, and Adhesive

6

Properties

7

Sang Hyeon Hong†, Sunjin Kim†, Joseph P. Park†, Mikyung Shin†, Keumyeon Kim‡, Ji Hyun Ryu

8

⊥

, and Haeshin Lee*,†

9

10 11


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Dynamic Bonds between Boronic Acid and Alginate - ACS Publications

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