VanadylâCatecholamine Hydrogels Inspired by Ascidians and Mussels

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Vanadyl-Catecholamine Hydrogels Inspired by Ascidians and Mussels Joseph Paul Park, In Taek Song, Juwon Lee, Ji Hyun Ryu, Yunho Lee, and Haeshin Lee Chem. Mater., Just Accepted Manuscript • Publication Date (Web): 05 Dec 2014 Downloaded from http://pubs.acs.org on December 5, 2014

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Chemistry of Materials

Vanadyl-Catecholamine Hydrogels Inspired by Ascidians and Mussels Joseph P. Park,†, §,|| In Taek Song,‡,§,|| Juwon Lee,‡ Ji Hyun Ryu,† Yunho Lee,‡ Haeshin Lee*,†,‡,§ †

‡

Graduate School of Nanoscience and Technology and Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, South Korea §

Center for Nature-inspired Technology (CNiT) in KAIST Institute NanoCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, South Korea ABSTRACT: In general, mechanical properties and gelation kinetics exhibit a positive correlation with the amount of gelation reagents used. Similarly, for catechol-containing hydrogels, which have attracted significant attention due to their unique dual properties of cohesion and adhesion, increased amounts of crosslinking agents, such as organic oxidants and/or transition metals (Fe3+), result in enhanced mechanical strength and more rapid gelation kinetics. Here, we report a new metal-ligand crosslinking chemistry inspired by mussels and ascidians that defies the aforementioned conventional stoichiometric concept. When a small amount of vanadium is present in the catechol-functionalized polymer solution (i.e., [V] > [catechol]), the catechol remains chemically intact by coordination that inhibits gelation. Thus, a large amount of crosslinking agent is not necessary to prepare mechanically strong, biocompatible hydrogels using this system. This new chemistry may provide insight into the biological roles of vanadium and its interaction with catechol-containing molecules (i.e., determination of the liquid vs. solid state). Excess amounts of vanadium ([V] >> [catechol]) coordinate with catechol, which may result in a liquid state for ascidian blood, whereas excess amounts of catechol ([V] 8), the catechol is autooxidized to catecholquinone, which can subsequently undergo formation of catechol-catechol adducts, catechol-amines, or catechol-thiol adducts.5,6 Other methods for preparing catechol-containing hydrogels include addition of oxidizing agents such as periodate ions (IO4-)6,7 and addition of metal ions (e.g., Fe(III) ions).8-12 The reason for using an organic oxidizing agent such as IO4- is to form covalently crosslinked (i.e. irreversible) hydrogels, which often causes toxicity due to the use of excess amount of IO4-. In addition, the reason for using a transition metal ion such as Fe(III) is to induce coordinationbased (i.e. reversible) hydrogels. A typically stoichiometric ratio has been one (Fe): three (catechol).8-10 When the amount of iron is decreased, the number of coordina-

tion bond is also decreased, resulting in no gelation. In contrast, when the amount of iron is increased, the coordination might be retained (or decreased to a mono- or bi-dentate configuration), but the metal-induced toxicity is inevitably increased. Therefore, the development of improved crosslinking chemistry is in-dispensable for improving the practical utility of catechol-containing hydrogels. Vanadium is present in marine environments at concentrations ranging from approximately 1 to 3 μg/L.13,14 Despite the low ambient vanadium concentration in sea water, tunicates such as ascidians or sea squirts are known to accumulate vanadium at high levels in their blood cells. In particular, Ascidia nigra can accumulate an extremely high concentration of vanadium of up to 106-107 times the concentration in sea water.15,16 It has been suggested that vanadium might play an active role in oxygen transport because it is known to form complexes with the catechol-containing ligand tunichrome. By contrast, the byssus of marine mussels contains a variety of metals including vanadium in trace amounts.17,18

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when lower concentration of vanadium was added. Lowering the amount of vanadium lowered the cytotoxicity. Thus, in this study, we developed a new hydrogel system using catechol-functionalized polymers and a new transitional metal, vanadium, with increased biocompatibility based on the stoichiometry of vanadium and catechol. EXPERIMENTAL SECTION

Figure 1. a) Stoichiometric difference in vanadium and catechol present in ascidians and mussel and b) colorimetric differences in the reaction solutions with varying [catechol]/[V] from 0.1 to 10 immediately after mixing and after 6 hr.

Similar to ascidians, mussels possess catecholic ligands in the form of the amino acid 3,4-dihydroxy-L-phenylalanine (DOPA). Though vanadium have been reported to be accumulated in mussels and its’ byssal threads, the exact biological role of vanadium in the mussels have not been reported due to the small amount of vanadium present.19,20 Considering the common presence of catecholic compounds and vanadium at different ratios in ascidians and mussels, as shown in Figure 1a, we hypothesized that the biological role of vanadium might be dependent upon the stoichiometry between the catecholic ligand and the vanadium. Under this hypothesis, the biological roles of vanadium in both marine organisms may be revealed, providing important new insights for bio-inspired materials. Interestingly, we found that reactive organic radical species were effectively generated when the vanadium ion is present in stoichiometrically smaller amounts than catechol (i.e. [cat]/[V] > 1).In contrast, the reactive radical formation was inhibited by coordination between catechol and vanadium when the amount of vanadium was in excess. These findings may be applicable to hydrogels in that the use of a small amount of crosslinking agents, such as vanadium, can significantly reduce cytotoxicity. In fact, when the above observation was applied to catecholcontaining hydrogel systems (chitosan-catechol, abbreviated by CHI-C), hydrogels with better mechanical properties were produced using smaller amounts of vanadium. This stands in opposition to the general trends reported in previous studies using oxidizing agents as a crosslinker, because the mechanical properties was improved

Materials. Dopamine hydrochloride, vanadium (III) chloride, iron (III) chloride, sodium phosphate monobasic, sodium phosphate dibasic and sodium chloride were purchased from Sigma-Aldrich. 1-Ethyl-3-(3dimethylamino propyl) carbodiimide (EDC) was purchased from Tokyo Chemical Industry (TCI), and chitosan was purchased from Heppe Medical Chitosan (HMC). Preparation of Vanadyl Solution. Vanadium stock solution (110 mM VCl3) was prepared by adding 863 mg of VCl3 powder to DDW 50ml. 55, 22, 16.5, 13.2, 11, 8.8, 5.5, 2.75, 1.1, and 0.11 mM VCl3 solutions were serially diluted. The solutions were stored at room temperature for one week. Color of the solutions was changed to light-blue confirming the conversion of the vanadium ion state to vandyl state. Colorimetric Analysis and Surface Coating. To observe the colorimetric difference at varying [catechol]/[V] ratios, the concentration of vanadium was fixed to 11mM and varying concentrations of dopamine were added. . For the surface coating experiments, 20 mg/ml (110 mM) of the dopamine solution was prepared as a 10X stock in PBS buffer (10X, pH 7.4). One ml of the vanadyl solution and 0.1 ml of the dopamine solution were then mixed with varying [catechol]/[V] ratios. For the surface coating experiment, titanium (Ti) surfaces were incubated with the different reaction solutions. As a control, conventional dopamine coating was performed (dopamine 2 mg/ml in 10 mM Tris buffer pH8.5). The thicknesses of the coated Ti surfaces were measured by ellipsometry. Electron Paramagnetic Resonance (EPR) Analysis. The EPR (X-band CW-EPR, Bruker) parameters were as follows: temperature of 20 K; microwave frequency of 9.64 GHz; microwave power of 0.94 mW; modulation amplitude of 10 G; modulation frequency of 100 kHz; and five scans. The solution was mixed with glycerol until being 20% glycerol solution. X-band CW-EPR machine was used for EPR analysis. Synthesis of Catechol-Conjugated Chitosan. Catechol was conjugated to a chitosan backbone using a previously reported method.21,22 The catechol moieties were conjugated onto the chitosan backbone using standard EDC chemistry. Chitosan (0.5 g) was dissolved in pH 5.5 deionized and distilled water (DDW, 50 ml). Then, 3,4dihydroxyhydrocinnamic acid (591.0 mg) in DDW and EDC (1244.9 mg) in ethanol were consecutively added to the chitosan solution and reacted for 12 h. The reaction solution pH was maintained at 5.5 by adding HCl. The product was then purified via dialysis (MWCO: 12 00, SpectraPor) against a pH 5.0 HCl solution in DDW for 2 days and in DDW for 4 h. The final product was freezedried and stored in a moisture-free desiccator until used.


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The 1H-NMR was used to calculate the degree of catechol conjugation. Gelation Test and Rheometer Analysis. The synthesized CHI-C was dissolved in DDW. For the rheometer analysis, CHI-C with degree of catechol conjugation of 12% was dissolved in DDW to 4 w/v % solution. 100 μl of CHI-C solution was mixed with 10 μl of the vanadyl solutions (110, 11, and 5.5 mM) resulting in various [catechol]/[V] ratios were incubated for 20 min. As a comparison, 100 μl of CHI-C solution was mixed with 10 μl of the iron solutions (110, 11, and 5.5 mM). Storage modulus and loss modulus were measured by rheometer (Bohlin Advanced Rheometer, Malvern Instrument). In Vitro Cytotoxicity Test for CHI-C Hydrogels. The in vitro cytotoxicity test of various CHI-C hydrogels were evaluated using the standard CCK assay (Cell counting kit-8, Dojindo Laboratories, Japan). The CHI-C hydrogel extracts of CHI-C hydrogels were obtained by incubating the hydrogel fragments to DMEM media at a concentration of 0.2g/ml at 37°C for 24 hrs. CHI-C (4% wt/v%) solution, V110-CHI-C, V5.5-CHI-C, Fe110-CHI-C, Fe5.5-CHI-C and CHI-C hydrogel with NaOH were made and used to prepare the extracts. The hydrogel extracts were then sterilized by filtration (0.22 μm, Millipore, USA). NIH3T3 fibroblasts used in the experiments were cultured at 37°C with 5% CO2 in culture plate containing DMEM culture media with 10% fetal bovine serum (FBS) prior to use. NIH3T3 fibroblast cells were seeded onto flat-bottomed 96-well microculture plates (5000cells/well) and incubated for 24 hr at 37°C in 5% CO2 humidified incubator. After 24 hr incubation, 100 μl of prepared various CHI-C hydrogel extracts were added to the 96-well plate and further incubated for 12 hrs at 37°C. Samples and controls were tested in sextuplets. These plates were then incubated for 24hr and 48hr before the in vitro cytotoxicity of various CHI-C hydrogel were evaluated using CCK assay. The DMEM media without any exposure to the hydrogel extract were used as a negative control. After the respective incubation times, the optical density of each well was read using a microplate reader (Varioskan Flash, ThermoScientific, USA) at absorbance wavelength of 450 nm to quantify the viability of cells. Also the morphology of the cells was observed under the microscope and were compared with negative and positive control. RESULTS AND DISCUSSION In this study, we investigated the stoichiometry between catechol and vanadium. To demonstrate the effects of vanadium:catechol stoichiometry, we designed a simple experiment where we mixed vanadium with the catecholic ligand dopamine as a function of the [catechol]/[V] ratio from 0.1 to 10. The vanadium stock solution (VCl3, 110 mM) was prepared ahead of time and stored under aerobic conditions at room temperature for one week before the experiments. This changed the vanadium state from vanadium (III) to vanadyl (VO2+), producing a change in the color of the solution from light brown to blue, which was characterized by UV-Vis spectroscopy

(Figure S1). In aqueous solutions, the thermodynamically stable species of vanadium is vanadyl. Thus, vanadium in the vanadyl state

Figure 2. a) UV/Vis absorbance spectra of vandyl/dopamine solutions with different [cat]/[V] ratios of 0.5, 1, 2, 4 and 10. b) The elipsometric analysis of Ti substrates incubated with dopamine and vanadyl/dopamine solutions with [cat]/[V] ratios of 0.2, 1, 2, and 4.

was used in all experiments to closely reflect the marine biochemistry. The vanadium stock solution was serially diluted and subsequently added to the dopamine solution to prepare reaction solutions with [cat]/[V] ratios of 0.1, 0.2, 0.5, 1, 2, 3, 5, and 10. Spontaneous and rapid color changes were observed after mixing the vanadium and catechol solutions (Figure 1b). Interestingly, the color change occurred only with [catechol]/[V] ratios greater than 1 (Figure 1b). As the [cat] became excessive (i.e., [cat]/[V] ratios of 5 and 10), the solution became dark blue-violet. When the [cat]/[V] ratio approached one, the solution became a paler blue. However, after 6 hr incubation, all of the solutions that initially exhibited bluish coloration deepened to a dark blue-violet color (Figure 1b). The obvious distinction in the color change at [cat]/[V] ratios greater than 1 indicates that the catecholvanadyl interaction is dependent on the stoichiometry. Ultraviolet/visible (UV/Vis) adsorption spectroscopy was employed to determine the chemical configuration of the



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vanadium-catechol complex (Figure 2a). The UV/Vis spectra of vanadyl/dopamine solutions with different [cat]/[V] ratios of 10, 4, 2, 1, and 0.5 showed that a new peak appeared at approximately 560 nm for the solutions with [cat]/[V] ratios greater than 1 (Figure 2a, grey for [cat]/[V] = 2, orange for 4, and light blue for 10). Previous studies showed that coordination of the vanadyl with three catecholic ligands (denoted as [V(cat)3]), produced an absorbance peak at approximately 550 – 560 nm.23,24 Thus, we can tentatively conclude that the new chemical species with an absorbance of approximately 560 nm can be assigned as [V(cat)3]. Additionally, the absorption intensity at 560 nm was enhanced as the catechol concentration increased. Another unexpected observation was the surface modification caused by the vanadyl/dopamine solution (Figure S2). Surface modification of the glass was observed for the vanadyl/dopamine solution with [cat]/[V] ratios greater than 1. The degree of surface modification was greater than the control poly(dopamine) coating (2 mg/ml dopamine hydrochloride in 10 mM Tris, pH 8.5), as shown by the darker color of the glass substrate. By contrast, no surface modification was observed when the [cat]/[V] ratio was below 1. The phenomenon of surface modification seems to be similar to poly(dopamine) (pDA) coating, which is due to the similar coating color.3,25 Based on this observation, the surface coating ability of the vanadyl/dopamine solutions with various [cat]/[V] ratios of 0.2, 1, 2, and 4 was explored by immersing titanium (Ti) substrate in the solutions for up to 24 hrs. As a control, pH-triggered pure pDA coating was also performed. The thickness of the Ti substrates was measured by ellipsometry (Figure 2b). Surface coating was observed only in the solutions ([cat]/[V] ratios of 2 and 4) that developed color. Furthermore, the thickness of the coating rapidly increased with time compared to the typical pDA coating. When the Ti substrate was incubated with a solution with a [cat]/[V] ratio of 4, the coating of the Ti substrate was approximately 7 times thicker than the pDA coating (58.1 nm vs. 8.6 nm). Coating did not occur for solutions that did not develop color changes ([cat]/[V] ratios of 0.2 and 1). X-ray photoelectron spectroscopy (XPS) of the Ti substrate showed that the chemical composition of the coating was similar to the pHtriggered pDA coating (Figure S3). Significant adsorption/incorporation of vanadium on the surface was originally expected, but only trace amounts (< 1%) of V2p3/2 (517 eV) were measured, suggesting that the coating process is vanadyl-triggered. Thus, it can be concluded that the [V(cat)3] complex triggers the oxidation and polymerization of dopamine. The rapid kinetics of dopamine polymerization and the increased coating thickness (approximately 7 times) also suggest that the trisvanadyl/dopamine complex generates organic radicals during the process of dopamine oxidation. To test this, electron paramagnetic resonance (EPR) spectroscopy was performed. All of the test solutions for EPR analysis were frozen in 20% glycerol. EPR analysis of the vanadyl solution without dopamine showed an EPR spectrum typical of vanadyl, with eight distinct hyperfine splitting sites arising from the interaction of one unpaired

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electron spin (S=1/2) and the 7/2 spin of the vanadium nucleus (Figure 3a, black). Importantly, the same vanadyl species was detected for the solution with a [cat]/[V] ratio of 0.5 (Figure S4). However, the EPR spectrum of the solution with a [cat]/[V] ratio of 10 exhibited a significantly different EPR spectrum compared with the vanadium solution (Figure 3a, red). This suggests the generation of organic radicals from the [V(cat)3] complex. As a positive control, we also performed the EPR experiment using an [Fe(cat)3] complex solution with a [cat]/[Fe] ratio of 10. The iron and catechol moieties are also known to form tris-complexes, in which reactive radical species are generated.11, 26-28 Thus, iron is considered an important chemical species for adhesion and cohesion of the byssal threads and adhesion pads. Surprisingly, the intensity of the radical peak of the [V(cat)3] complex was significantly stronger than that of the [Fe(cat)3] complex (Figure 3a, blue, inset). Therefore, we can conclude that vanadium generates more organic radicals than iron when the coordination complexes are formed, indicating that low amounts of vanadium might also contribute to the mechanical properties of the byssal threads and might prevent ascidian blood from clotting (Figure 3b). Furthermore, the organic radicals generated by [V(cat)3] complexes can also be useful for producing new biomaterials.

Figure 3. a) EPR spectra of the vanadium solution (black line), [V(cat)3] complex solution (red line), and [Fe(cat)3] complex solution (blue line). An enlargement of the EPR spectrum is provided for better comparison. b) Proposed changes in the complex state of vanadium/catechol as a function of the vanadium:catechol stoichiometry.

So far, iron has been the most widely used metal to prepare catechol-containing bio-inspired materials.8-12 The use of vanadium in biomaterial preparation has not been well studied. We hypothesized that only a small amount of vanadium might be required for hydrogel preparation compared to the amount of iron needed due to the enhanced organic radical formation ability of vanadium, as shown in Figure 3a. A chitosan-catechol (CHI-C) conjugate was chosen as a model polymer system because


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it exhibits good responsiveness in terms of its mechanical properties upon catechol-mediated crosslinking.21,22

CHI-C was synthesized using 1-ethy-3-(3-dimethyl aminopropyl)carbodiimide (EDC) chemistry by coupling the carboxylic acid group of 3,4-dihydroxyhydrocinnamic acid

Figure 4. a) Schematic representation of the gelation mechanism of Chitosan-catechol (CHI-C) with vanadium. Rheological analysis was performed 20 minutes after mixing 4 wt% CHI-C with various concentrations (110, 11, and 5.5 mM) of b) vanadium and c) iron. The concentration-dependent gelation showed opposite trends for vanadium and iron.

to the primary amine groups in the chitosan (Figure S5a). The degree of catechol substitution in the CHI-C conjugate was 12%, as determined by 1H-NMR (Figure S5b). The CHI-C was dissolved in distilled water to 4 wt% for all of the hydrogel experiments. We then prepared two types of metal ion solutions (vanadium and iron) at concentrations of 5.5, 11, and 110 mM. The volume ratio of CHI-C/metal was fixed at 10 in the final hydrogel-forming solutions. The rheological properties were measured 20 min after mixing the metal ions with the CHI-C solution using a rotating rheometer (Bohlin, Malvern Instruments, UK). In agreement with the previous stoichiometric experiments (Figure 1 to 3), reducing the amount of vanadi-

um ion increased the storage moduli (G’) of the vanadium-containing hydrogels (V-CHI-C). In contrast, increasing the amount of the conventional metal crosslinker (i.e., iron) increased the storage modulus (G’) (Fe-CHI-C). For example, the CHI-C solution mixed with 110 mM vanadium (V110-CHI-C) exhibited a sol state (Figure 4b, left), whereas the 5.5 mM V5.5-CHI-C solution rapidly formed a hydrogel (Figure 4b, right). The G’ values were increased from 6.5 Pa for V110-CHI-C to 1,454 Pa for V5.5-CHI-C at 1 Hz rotational frequency. In comparison, when mixed with 5.5 mM Fe, the Fe5.5-CHI-C solution remained in the sol state (right). Increasing the iron concentration to 110 mM resulted in the formation of a hydrogel (Fe110-CHI-C)



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(left). The G’ values decreased from 13,530 Pa for Fe110CHI-C to 536 Pa for Fe5.5-CHI-C at 1 Hz. The rheological data for the V5.5-CHI-C hydrogel showed that the crosslinking reactions were not completed for the first 20 min, as shown by the further increase in the storage modulus (G’) after 20 min. When the V5.5-CHI-C gel was left in ambient conditions for 48 hrs, the G’ value reached 10,450 Pa independent of the sweeping frequency, demonstrating complete crosslinking (Figure S6). From a rheological analysis of the various mixtures of CHI-C and metal ions, we can conclude that lower amounts of vanadium ions are needed for potent crosslinking compared with iron. This might be advantageous for forming biocompatible hydrogels. In contrast to the conventional iron crosslinker used in catechol-containing hydrogels (i.e., a large amount of iron), the new vanadium crosslinking approach requires only 10% vanadium to generate organic radicals for hydrogel preparation. We expected the in vitro cytotoxicity of the V-CHI-C hydrogels to be lower than the ironinduced hydrogels because of the aforementioned stoichiometry. To evaluate the cytotoxicity, we performed a CCK assay along with microscopic observations. The CHIC extracts were prepared by mixing CHI-C (4 wt%) with either 110 mM vanadium or iron. Extracts were also prepared with either 5.5 mM vanadium or iron. The NIH3T3 fibroblast cytotoxicity of each mixture was evaluated, as shown in Figure 5. The cells incubated with the V5.5-CHIC extracts exhibited similar adhesion and spreading compared with the negative control cells. By contrast, cells incubated with extracts of V110-CHI-C and Fe110-CHI-C were round in shape, with less spreading, indicating significant cytotoxic effects due to the high concentration of metal ions (110 mM). A CCK assay was employed to quantify the cell viability of NIH3T3 cells exposed to the various extracts. The CCK assay utilizes tetrazolium salt, 2-(2methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium (WST-8) to produce water-soluble formazan by way of the mitochondrial enzymes present in viable cells. More than 90% of cells were viable for the V5.5-CHI-C hydro-gel extract. However, for the Fe110-CHI-C hydrogel, only approximately 50% of cells were alive. As shown by the CCK assay and morphological observations, the V5.5-CHI-C hydrogel displayed virtually no cytotoxicity relative to the negative control. It is interesting to note that overall cytotoxicity of vanadium is lower than iron when the concentration is same from the cell viability test results. The findings in this study provide an important insight into the stoichiometry of vanadium in marine organisms, such as ascidians and mussels. In ascidian blood, vanadium is present in excess compared to the catecholcontaining tunichrome ligand (i.e., [cat]/[V] > 1). In this condition, the catechol is readily oxidized by vanadium-

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induced radical-generation of [V(cat)3], resulting in chemical crosslinking. Thus, we anticipate that low concentrations of vanadium might play an important role in the liquid-to-solid phase transition. Furthermore, high concentrations of iron might play a role in the rapid solidification of the byssal liquid precursor, similar to the results shown in Figure 4.

Figure 5. a) Morphology of NIH3T3 fibroblasts cultured with Fe/V-CHI-C solutions. b) Cytotoxic effect of the CHI-C hydrogel extracts on NIH3T3 fibroblast cells, as assessed by quantification of the CCK assay after 24 hrs of incubation with the extracts.

Although vanadium and iron are similar in terms of ionic radius, aqueous redox activity, and their tendency to form tris-complexes with catechol, their stoichiometric gelation behaviors exhibit completely opposite trends. Application of this new insight can be useful in biomaterials science. Most studies have used iron to induce metal ion-catechol crosslinking to investigate the configuration of polymers, the effect of environmental pH, and the surface adhesion.8-12 In the previous studies, the stoichiometric effects were largely overlooked, with large amounts of iron used to induce catechol-mediated crosslinking. In contrast, the use of a large amount vanadium resulted in an adverse effect on catechol-mediated crosslinking. This stoichiometric interaction of vanadium and catechol might lead to new approaches for controlling the mechanical properties of hydrogels, overcoming the previous toxicity issue induced by metal ion usage. Thus, this new stoichiometrically controlled vanadium hydrogel offers new possibilities for creating advanced biomaterials with increased biocompatibility.


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CONCLUSION In conclusion, we revealed interesting stoichiometric dependence between catechol and vanadium that may be useful for preparing non-toxic hydrogels and explaining sol-gel transitions in ascidian blood and byssal threads. When a small amount of vanadium is present in the catechol-functionalized polymer solution (i.e., [V] > [catechol]), the catechol remains chemically intact by a process that inhibits gelation. Based on this stoichiometric result, a novel vanadyl-catecholamine hydrogel system with reduced cytotoxicity was developed. Considering the long history of iron research in the formation of byssal threads and adhesive pads in ascidians, our results might provide new insights into the molecular mechanisms of the rapid solidification of the byssal threads in mussels and the maintenance of the liquid state of ascidian blood.

ASSOCIATED CONTENT Supporting Information. Glass surface coating experiment images, XPS analysis of coated Ti substrates, EPR analysis of vanadyl/catechol reaction solutions, NMR analysis of catechol functionalized chitosan. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Author Contributions ||

These authors contributed equally.

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ACKNOWLEDGMENT This work is supported from National Research Foundation of South Korea: Mid-career Scientist Grant (2014002855). This work is in part supported by the Ministry of Health and Welfare of Korea (A120170).

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