Gauging and Tuning Cross-Linking Kinetics of Catechol-PEG

Article pubs.acs.org/Biomac

Gauging and Tuning Cross-Linking Kinetics of Catechol-PEG Adhesives via Catecholamine Functionalization Julieta I. Paez,† Oya Ustahüseyin,† Cristina Serrano,† Xuan-Anh Ton,† Zahid Shafiq,†,⊥ Günter K. Auernhammer,† Marco d’Ischia,∥ and Aránzazu del Campo*,†,‡,§ †

Max- Planck Institut für Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany INM − Leibniz Institute for New Materials, Campus D2 2, 66123 Saarbrücken, Germany § Saarland University, 66123 Saarbrücken, Germany ∥ Department of Chemical Sciences, University of Naples Federico II, Via Cintia, I-80126 Naples, Italy ‡

S Supporting Information *

ABSTRACT: The curing time of an adhesive material is determined by the polymerization and cross-linking kinetics of the adhesive formulation and needs to be optimized for the particular application. Here, we explore the possibility of tuning the polymerization kinetics and final mechanical properties of tissue-adhesive PEG gels formed by polymerization of endfunctionalized star-PEGs with catecholamines with varying substituents. We show strong differences in cross-linking time and cohesiveness of the final gels among the catecholamine-PEG variants. Installation of an electronwithdrawing but π-electron donating chloro substituent on the catechol ring resulted in faster and more efficient cross-linking, while opposite effects were observed with the strongly electron-withdrawing nitro group. Chain substitution slowed down the kinetics and hindered cross-linking due either to chain breakdown (β-OH group, in norepinephrine) or intramolecular cyclization (α-carboxyl group, in DOPA). Interesting perspectives derive from use of mixtures of catecholamine-PEG precursors offering further opportunities for fine-tuning of the curing parameters. These are interesting properties for the application of catecholamine-PEG gels as tissue glues or biomaterials for cell encapsulation.

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INTRODUCTION The curing time of an adhesive material is the time required for fully cross-linking and complete hardening. Depending on the particular application, the curing time can be adjusted to seconds or hours by changing the chemical formulation. Slow curing allows reallocation of the fixing parts to correct misalignment and stress relaxation during hardening. Fast curing favors immediate mechanical stabilization of the bonded parts and shortens working times. In medical applications using mussel-inspired catecholamine modified star-poly(ethylene glycol) (PEG),1,2 polymerization kinetics is adjusted by changing the oxidant concentration, or by changing the pH of the precursor mixture or the application environment.3,4 However, changes in pH or oxidant concentration also affect the polymerization degree and cohesiveness of the final adhesive material, compromising its mechanical performance in the application. There are different medical scenarios where slow crosslinking but strong mechanical response of the final adhesive are required.5 For example, in the context of peripheral nerve surgery reconstruction, where a slow cross-linking adhesive mixture could act as a sealant in neighboring injuries during operation, and fix the tissue only at the end of the surgery process. Also in cell encapsulation and cell therapies, where a © 2015 American Chemical Society

slow curing would provide sufficient handling time for preparation and injection, and allow gentle and tunable hardening at the application site. Looking for cross-linking strategies of catecholamine modified PEGs with independent tuning of polymerization kinetics and final polymerization degree, we decided to test mixtures of PEGs functionalized with different catecholamine variants having different oxidation properties, intermediate chemistry and reactivity. Dopamine (Dop) oxidation proceeds at fast rate at pH 8.5 and has been shown to involve both dimerization and cyclization to 5,6-dihydroxyindole intermediate.6,7 Similarly, polymerization of DOPA (3,4-dihydroxyphenylalanine) involves 2-carboxylic acid-5,6-dihydroxyindole formation in addition to 5,6-dihydroxyindole in variable proportions depending on reaction conditions.8 Inclusion of a β-OH group on the dopamine system, as in norepinephrine (NE), causes differences in its oxidative pathway. Oxidative breakdown of the catecholamine chain occurs at the beta reactive site9 to form small benzaldehyde fragments able to copolymerize via Schiff base formation.10 We have recently Received: August 20, 2015 Revised: November 17, 2015 Published: November 19, 2015 3811

DOI: 10.1021/acs.biomac.5b01126 Biomacromolecules 2015, 16, 3811−3818

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Figure 1. Chemical structure and nomenclature of PEG-catechol derivatives studied. NMR Studies. 1H NMR measurements were performed with a 5 mm PAQXI 1H/19F/D-13C/15N with z-gradient on a 700 MHz Bruker Advance III spectrometer. 1H spectra were recorded with 128 transients. The 90° pulse was 13.8 μs long, with a spectral width of 12600 Hz and a recycling delay of 5 s. The spectra were measured in D2O and were referenced by the residual HDO signal at δ(1H) = 4.79 ppm. The temperature was hold at 333 K and regulated by a standard 1 H methanol NMR sample using the Topspin 3.1 software (Bruker). 1 H NMR spectra of PEG-catechol derivatives were acquired in D2O before and after addition of oxidant at increasing reaction times. 600 μL of a 25 mg mL−1 polymer solution in D2O were mixed with 7.5 μL of a 200 mM NaIO4 solution in D2O in the NMR tube and the spectra were measured at 333 K. The molar concentration of catechol and NaIO4 in the mixture was 2.5 μM, and the catechol/NaIO4 molar ratio was (4:1). UV−Vis Studies. UV−vis spectra were recorded at room temperature on a Varian Cary 4000 UV−vis spectrometer (Varian Inc. Palo Alto, CA) in water before and after addition of oxidant at increasing reaction times. PEG derivatives dissolved at 200 mg mL−1 concentration in water were mixed with 18 mM NaIO4 in water at 1:1 volume ratio. The catechol/NaIO4 molar ratio was (8:1). Rheological Measurements. The shear moduli of PEG-catechol and oxidant mixtures were measured with a homemade piezorheometer.17 The sample was placed between two glass slides connected to two piezoactuators: one piezoactuator applied the shear deformation to the sample and the other one measured the stress transmitted through sample. The gap between the glass slides was 100 μm, and it was filled with 15 μL of a solution of the polymer at 100 mg mL−1 concentration. The measurements were performed at room temperature and 0.32% strain. In the time sweep measurements, moduli at 10 Hz were used. In order to prepare the polymer solution, the PEG-catechol derivatives were dissolved at 200 mg mL−1 concentration in water and mixed with 18 mM NaIO4 in water at 1:1 ratio, unless otherwise stated. The mixtures were fluid when they were placed to the sample chamber. To avoid drying of hydrogels during measurements, hydrogels were sealed with low viscous polydimethylsiloxane oil (PDMS). The catechol/NaIO4 molar ratio was (4:1). Preparation of PEG-ClDop:PEG-Dop Mixtures. In preparation of PEG-catechol mixtures, total solid content in solution was kept constant. Solutions of PEG-catechol derivatives were prepared in volume of 10 μL and mixed at 1:3, 1:1 or 3:1 ratio. This mixture of PEG-catechol derivatives was oxidized with 18 mM NaIO4 in water at 1:1 volume ratio. Adhesion Measurements. The adhesion strength of PEG-Dop and PEG-ClDop hydrogels on pig skin were measured with a Zwick Roell Z005 Universal Testing Machine (UTM) equipped with a 1 kN load cell. The protocol of adhesion test was adapted from the American Society for Testing and Materials (ASTM) standard F225805. Samples of porcine skin were cut into disks of 13 mm diameter, and had their fat removed. PEG-Dop or PEG-ClDop was dissolved in 0.01 M phosphate-buffered saline (pH 7.4) at 300 mg/mL concentration. 80 μL of the solution was spread on the skin sample. 80 μL of 47 mM NaIO4 in water containing 10% NaOH (0.4 M) was added to induce gelation. Both solutions mixed immediately, and the hydrogel was immediately recovered by a disk of porcine skin, to allow curing between two pieces of skin. The sample was loaded with a force of 0.2 N. and allowed to cure for 30 min. The two disks of skin were then pulled away at a rate of 1 mm/min. A number of samples between 3 and 10 were measured for obtaining the adhesion values. The adhesion strength was determined by the maximal stress and

demonstrated that the inclusion of substituents in the catechol ring of Dop also leads to significant changes in the reactivity and reaction products of catechol derived materials.11−13 Substitution at the 6-position of the catechol ring decreases the formation of high molecular weight aggregates and πelectron delocalization, leading to thinner polycatechol films. The effect is more pronounced with the strong electronwithdrawing nitro group (NDop, 6-nitrodopamine) and moderate with chloro (ClDop, 6-chorodopamine). In this Article, we study and compare the polymerization kinetics and final mechanical properties of PEG gels obtained by polymerization of different catecholamine end-functionalized star-PEGs (Figure 1). Four-arm functionalized PEGs have previously proved ideal as models for gelation studies due to their inert and controlled molecular structure,14 biocompatibility, and noncytotoxicity.15 We show strong differences in cross-linking time and cohesiveness of the final gels among the catecholamine-PEG variants. By mixing different catecholamine-PEGs, the polymerization kinetics and the final crosslinking degree can be tuned. These are interesting properties for the application of catecholamine-PEG gels as tissue glues or biomaterials for cell encapsulation.

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EXPERIMENTAL SECTION

Synthesis of PEG-Catechol Derivatives. Four-arm PEG succinimidyl carboxymethyl ester (PEG-NHS, Mw 10 kDa) was purchased from Jenkem Technology. All other chemicals were purchased from Sigma-Aldrich (Germany) and used as received. The synthesis of PEG-catechol derivatives was performed at 250 mg scale following reported procedures16 with minor modifications. The corresponding catechol (6 equiv) was dissolved in dry DMF (5 mL) and reacted with N-methyl morpholine (NMM, 10 equiv) for 15 min at rt under Ar atmosphere, followed by the addition of PEG-NHS (1 equiv) in dry DMF (5 mL). The mixture was stirred at rt under Ar overnight. The mixture was concentrated under reduced pressure, dissolved in distilled water, purified by dialysis (MWCO 3.5 kDa) against distilled water pH ca. 4 (adjusted with HCl), and lyophilized. The degree of catechol substitution was obtained by 1H NMR (Bruker Advance III, 700 MHz) end-group determination. The integral of the signal corresponding to the PEG backbone (4.2−3.9 ppm) was set to 220H and compared with the integral of the catechol protons (H2, H5). Functionalization degrees of 80−90% and yields of 70−85% were obtained in all cases. PEG-Dop: 1H NMR (700 MHz, D2O) δ (ppm): 7.25 (d, H5), 7.19 (s, H2), 7.10 (d, H6), 4.39 (s, Hf), 4.42−3.9 (PEG), 3.86 (t, He), 3.14 (t, Hd). PEG-ClDop: 1H NMR (700 MHz, D2O) δ (ppm): 7.29 (s, H5), 7.23 (s, H2), 4.40 (s, Hf), 4.20−3.9 (PEG), 3.88 (m, He), 3.26 (ps-t, Hd). PEG-NDop: 1H NMR (700 MHz, D2O) δ (ppm): 8.03 (s, H5), 7.22 (s, H2), 4.38 (s, Hf), 4.25−3.9 (PEG), 3.88 (m, He), 3.49 (ps-t, Hd). PEG-DOPA: In this case, dry DMSO was used as solvent instead of DMF. 1H NMR (700 MHz, D2O) δ (ppm): 7.13 (d, H5), 7.07 (s, H2), 6.99 (d, H6), 4.90 (ps-t, He), 4.36−4.28 (m, Hf), 4.15−3.9 (PEG), 3.45−3.40 (m, Hd1), 3.25−3.20 (m, Hd2). PEG-NE: 1H NMR (700 MHz, D2O) δ (ppm): 7.28 (m, H2+H5), 7.19 (m, H6), 5.11 (m, Hd), 4.38 (s, Hf), 4.40−3.9 (PEG), 3.85 (m, He). 3812


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Figure 2. Shear moduli (G′, G″) of catechol functionalized PEGs in water as a function of cross-linking time. The noise level of the rheological measurements is ∼10 Pa (a) at 100 mg mL−1 polymer concentration with different oxidant concentrations and (b) at 9 mM oxidant concentration with decreasing polymer concentrations. (c) Comparison of curing process of the different PEG-catecholamine derivatives at 100 mg mL−1 polymer concentration and 9 mM oxidant concentration, (4:1) catechol/NaIO4 molar ratio. integral area of the stress vs strain curve divided by the initial contact area (132.7 mm2) between the hydrogel and the skin.

catechol/NaIO4 ratio window, such as (4:1) ratio (corresponding to 9 mM oxidant concentration) and (7:1) ratio (5 mM). Figure 2b shows the curing curves for PEG-Dop gels at different polymer concentration. Curing time increased and mechanical stability of the final gel decreased at lower polymer concentrations. These results show that a reduction in the oxidant or polymer concentration for improving handling negatively impact the cohesive properties of the adhesive, and viceversa. Similar experiments were performed with the different PEGcatechol derivatives. We used mixtures of 100 mg mL−1 polymer concentration and 9 mM NaIO4 concentration for the measurements. These conditions allowed a comfortable time window for experimentation and a comparison of the cross-linking kinetics between all systems. The cross-linking kinetics and the mechanical properties of the cross-linked gel significantly varied with the type of catecholamine end-group functionalization (Figure 2c). PEG-ClDop and PEG-Dop achieved the highest G′ after cross-linking (104 Pa). However, curing was complete within a few minutes in the case of PEGClDop, but it took almost 10 h for PEG-Dop. PEG-NDop reached a lower G′ after cross-linking (∼3 × 103 Pa) than PEG-Dop and PEG-ClDop. The rheological curve

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RESULTS AND DISCUSSION The cross-linking kinetics of the PEG-catechol homopolymers was first tested by measuring their mechanical properties during curing by piezorheology. This technique allows determination of the shear moduli (G′, G′′) of soft gels (101−105Pa) with small sample volumes (15 μL). The mixture of PEG-catechol (100 mg mL−1) and different concentrations of NaIO4 oxidant (5−9 mM) was placed between the two plates and the shear moduli were measured until the cross-linking was completed, i.e. a stable plateau was reached in the rheological curves. Figure 2a shows the data obtained for PEG-Dop at decreasing oxidant concentrations. The slope of the rheology curve reflects the polymerization kinetics, which decreased with decreasing oxidant concentration. The final value of the shear modulus reflects the mechanical stability of the gel after curing and is indicative of the final cross-linking degree, the latter depending on the catechol/oxidant ratio used. Decreasing oxidant concentration leads to a lower number of oxidized catechol groups and this translates into lower cross-linking degree. This observation was possible because we worked on a relevant 3813


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Biomacromolecules showed a particular shape. G′ reached a maximum value of 5 × 103 after 4 h cross-linking, but it decreased for longer crosslinking times until it reached the plateau value after 10 h. This behavior was reproducible. Such curve suggests a gelation process where two cross-linking mechanisms with different kinetics compete. A fast cross-linking reaction of PEG-NDop (time scale 1−2 h) is reflected in the steep initial slope in the rheological curve. A second, slower cross-linking mechanism contributed to the stiffening of the hydrogel in a time scale of 5−10 h under our experimental conditions. The reason for the partial drop in the shear modulus observed after 4 h before the plateau value of G′ is reached remains unclear at this point. It is important to note that the fast cross-linking mechanism of PEG-NDop is already visible during sample preparation. While all other solutions of PEG-catechol derivatives maintain their fluid properties right after mixing with the oxidant for at least a few seconds (PEG-ClDop) to hours (all the others), the PEGNDop does not longer flow right upon mixing with the oxidant. This observation is in agreement with recently published work on PEG-NDop gels by Lee et al.14 who observed faster gelation on PEG-NDop than on PEG-Dop (tested by inverting the vial), but lower shear modulus in the final PEG-NDop gel. These authors suggested that NDop mainly forms dimers through oxidative cross-linking while unsubstituted Dop can form higher cross-linked products. Our data complement this picture by providing information about the kinetics of the cross-linking mechanism. However, the nature of the cluster forming intermediates and that of the slower cross-links remains to be elucidated. A possible explanation for the anomalous behavior of PEGNDop can be found in the mechanism of polymerization of nitrodopamine and nitrocatechols reported in previous studies.18 According to those reports, the electron-withdrawing nitro group decreases the oxidation potential of the catechol ring, which accounts for a greater stability to autoxidation. However, once semiquinone or quinone intermediates are generated under forcing conditions, oxidative coupling may occur at rather fast rates leading to dimeric species. These latter may then evolve via slower processes involving nucleophilic attack of the phenolic −OH groups onto the quaternary nitrosubstituted 6-position of the aromatic ring, possibly in a quinonoid form, with sp2 to sp3 conversion (see Supporting Information Figure S1). The overall outcome of this process is the weakening of the C−N bond causing the possible release of nitrite ions with rearrangement of the dimer structure and an alteration of the overall properties of the cross-linked product.18 The rheological curves of PEG-NE and PEG-DOPA showed a pronounced sigmoidal shape, typical of processes with slow cross-linking kinetics. Full cross-link was achieved after 20 and 80 h, respectively. A G′ of ∼3 × 103 Pa was observed after curing, significantly lower than the final G′ of PEG-Dop under comparable experimental conditions. From these data it could be concluded that the introduction of substituents (−Cl, −NO2) in the catechol ring, or in the alkyl chain of the catecholamines (−COOH, −OH) strongly affects the cross-linking kinetics and cross-linking degree of the PEG-catecholamine derivatives. The cross-linking kinetics followed the sequence: PEG-ClDop ≫ -NDop, -Dop > -NE > -DOPA. The cross-linking degree (i.e., the final G′ and the cohesiveness of the material) followed the sequence: PEGClDop ≈ -Dop > -NDop, -DOPA, -NE. In order to get additional information about the cross-linking mechanism, solution 1H NMR spectra of the cross-linking

mixture were taken before and after addition of the oxidant and at different time points during gelation (0 to 24 h). Note that gelation experiments in the NMR tube had to be performed at lower solution concentration (25 mg mL −1) than the rheological measurements (100 mg mL−1) in order to obtain well-resolved spectra. However, the oxidant/catechol ratio was the same as in the rheological experiments and, therefore, results can be compared. Representative 1H NMR spectra of PEG-Dop at increasing reaction times are shown in Supporting Information, Figure S2. Upon addition of the oxidant to the PEG-catechol derivatives, we observed a clear decrease in the integral of the catechol protons H2, (H6) and H5 over time with respect to the integral corresponding to the protons of the PEG chain (Figure 3). This indicates consumption of the

Figure 3. Decay of the normalized NMR integral corresponding to H2 proton of PEG-catecholamine derivatives. Conditions: 25 mg mL−1 polymer solution in D2O, (4:1) catechol/NaIO4 molar ratio, 333 K.

catechol groups, presumably by oxidation to quinone followed by further reactions. The half-life times (τ1/2) of the different catechol groups were extracted from the integral decay of H2 protons (Table 1). The following sequence of τ1/2 was obtained Table 1. Half-Life Time Values (τ1/2) for Catechol Consumption in PEG-Catecholamine Derivativesa PEG-catecholamine derivative

τ1/2 [min]

Dop ClDop NDop DOPA NE

20.5 1.6 13.8 28.8 73.8

a

Estimated from the decay of the H-2 NMR integral. Conditions: 25 mg mL−1 polymer solution in D2O, (4:1) catechol/NaIO4 molar ratio, 333 K.

for the different derivatives: PEG-ClDop ≪ -NDop < -Dop < -DOPA ≪ -NE. This sequence reflects significant differences in the kinetics of oxidation and reaction among the different PEGcatechol derivatives, which is expected to impact the observed cross-linking kinetics in the rheological experiments (Figure 2c). In fact, this sequence correlates with that obtained for cross-linking kinetics, except for the DOPA derivative. This indicates that catechol oxidation effectively translates into cross3814


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Figure 4. UV−vis spectra of PEG-catecholamine derivatives (200 mg mL−1 polymer solution in water and NaIO4 9 mM, (8:1) catechol/NaIO4 molar ratio) as a function of oxidation time. (a) PEG-Dop, (b) PEG-ClDop, (c) PEG-NDop,(d) PEG-DOPA, and (e) PEG-NE. Q, quinone; QM, pquinomethane; DH, α,β-dehydrodopamine amide; DD, dopamine amide dimers. (f) Time evolution of the absorbance at 320 nm.

linking and mechanical strength for most catechol derivatives. In DOPA, the oxidation products seem to undergo other types of reactions that do not lead to chain extension and crosslinking. Since PEG-DOPA differs from the other catecholamine derivatives in that it possesses a carboxyl group on the side chain, a realistic option would be that just formed, the oquinone is partly diverted from the intermolecular coupling route toward an entropically favored intramolecular cyclization reaction brought about by the carboxylate anion to generate a lactone derivative amenable to further reactions different from coupling. In particular, intramolecular cyclization might account for a decrease in reactive sites available for cross-linking. The residual value of the H2 integral after 24 h reflects the number of unreacted end-groups in the final gel. The obtained values for the different polymers followed the sequence: PEGClDop < -Dop < -NDop < -NE ≈ -DOPA. These values inversely correlated with the mechanical stability of the gel (G′

values), corroborating the parallelism between the rheological and spectroscopic measurements. In parallel to the decay in the integral of the protons of the reactant during oxidation and cross-linking, new peaks appeared in the aromatic region (see Supporting Information Figure S2). The intensity of these signals increased first and decreased at longer cross-linking times, reflecting the presence of oxidation products and reaction intermediates that accumulated and disappeared during reaction. The appearance of high-field doublets in the region between 6.6 and 6.9 ppm supports this view. Due to the complexity of catechol reactivity we could not identify the reaction intermediates by unambiguous peak assignment. Most of these signals were no longer visible at the end of the cross-linking process. The overall integral of all aromatic protons decreased over time (Figure S3). This result indicates a loss of aromatic protons during reaction, as expected in self-condensation reactions of the catechol groups. The remaining integral value 3815


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(λ > 400 nm), N-QM (λ > 500 nm), and N-DH (λ ca. 340 nm); which appear red-shifted compared to the unsubstituted ones. Although the presence of nitrocatechol dimers at 405 and 423 nm has been informed14,24 these bands were not clearly present in our studies. In summary, the kinetics and extent of cross-linking of PEGcatecholamines strongly varies with the chemical nature of the catechol group, as reflected in our rheological and spectroscopic data. This is a consequence of the differences in their oxidation, acidity and reactivity. Given the complexity of catechol chemistry and the combined action of these parameters, the observed differences cannot be explained in terms of single molecular mechanisms but are likely to reflect concurrent competing pathways. However, some general conclusions can be drawn. The cross-linking kinetics measured for the different PEG-catechol derivatives via rheology or changes in the spectroscopic signals were parallel, indicating that the three techniques can be used to follow curing kinetics of catecholamine functionalized materials. From the chemical viewpoint, different effects were observed depending on the site and type of functionalization. Ring functionalization allowed for effective tuning of the cross-linking process both with regard to kinetics and product features. In particular, the chloro substituent in PEG-ClDop induced a faster cross-linking relative to PEG-Dop, while the nitro group in PEG-NDop exerted an opposite effect. In the latter, the formation of dimers instead of high order cross-links14 and their subsequent evolution by slow processes may account for the observed rheology. Conversely, chain functionalization resulted invariably in a decrease in the reaction kinetics and degree of cross-linking, as evidenced by the slow reactions observed with PEG-NE and PEG-DOPA. In these latter cases, side reactions such as chain breakdown in PEG-NE and cyclization for PEG-DOPA may be exploited to achieve more complete control over cross-linking degree and kinetics. Finally, it is worth noting that all these catechol derivatives have been extensively proved valuable for biocompatible and noncytotoxic biomaterials preparation.11,12,25−30 We tested the possibility of tuning the kinetics of PEG gelation using mixtures of catecholamine-PEGs with different curing kinetics. We selected the fast cross-linking derivative PEG-ClDop and mixed it with the slower cross-linking derivative PEG-Dop in different ratios (ClDop/Dop = 100:0, 75:25, 50:50, 25:75, 0:100) keeping a constant solid content of the gel (100 mg mL−1). As controls, we also measured the curing kinetics of the monocomponent gels at different solid contents, that is, ClDop/Dop = 75:0, 50:0, 25:0 and 0:75, 0:50, 0:25. Figure 5 presents the curing curves of the mixtures as measured by piezorheology. The fast polymerization kinetics of PEG-ClDop was successfully slowed down in a 25:75 mixture with PEG-Dop, while the final mechanical properties of the gel (104 Pa) were retained. This demonstrates that the rheological behavior of the 25:75 mixture reflects the synergy effect of the two comonomers during cross-linking that can be exploited for the control of polymerization kinetics without compromising the mechanical properties of the final gel. We propose the following mechanism behind the observed rheological behavior: the fast cross-linking ClDop groups form gel clusters within the sample at low cross-linking times and cluster aggregation to form the stable gel is done by the Dop groups at a slower rate. Interestingly, polymer mixtures with higher concentrations of ClDop (50:50, 25:75) did not show a significantly slower kinetic than the homopolymer PEG-ClDop gel, only a lower G′

when cross-linking was completed should be an indirect measure of the catechol groups that did not participate in cross-linking reactions, and should inversely correlate with the observed G′. Out of Figure S3, the concentration of not-selfcondensed catechols decreased as PEG-ClDop < -Dop < -NDop < -DOPA < -NE, which roughly correlates with the rheological data. Note that as cross-linking proceeds, the mobility of the protons located in close proximity to new crosslinking points may also be restrained. This could lead to broadening of the proton signals (which is indeed observed in same cases) and an additional decay in the overall integral value. Interestingly, in PEG-NE the appearance of a transient singlet at 9.93 ppm was observed. This signal may correspond to benzaldehyde intermediate, which has been described during NE oxidation due to nonmelanogenic chain breakdown.19 The oxidation and cross-linking of PEG-catecholamine derivatives was also followed by UV−vis spectroscopy of the solutions (Figure 4). Reported studies revealed that selfpolymerization of N-acetyl dopamine does not involve cyclization to 5,6-dihydroxyindole intermediate, instead it undergoes oxidative and cross-linking pathways through other intermediates in a process that resembles insect cuticle sclerotization20,21 (Figure S4, Supporting Information). Therefore, we expect these reaction pathways are taking place in the oxidation of our PEG-catecholamine series, since here the catecholamine unit is connected to the PEG core by an amide bond (i.e., an N-acylated dopamine). In PEG-Dop (Figure 4a), the absorption maxima at ca. 400, 480−500, and 320 nm would correspond to quinones (Q), pquinomethane (QM), and α,β-dehydrodopamine amide (DH) transient oxidation intermediates, respectively. Additionally, products of aryl−aryl coupling leading to the formation of dopamine amide dimers (DD) were seen at 280 and 480−520 nm. These features were also found in the UV spectra of PEGClDop, PEG-DOPA, and PEG-NE, except for a few details. In PEG-ClDop, the band located at 287 nm in the starting material (DD species) shifts to 269 nm, possibly due to the formation of chlorocatechol and catechol species, the latter due to chlorine expulsion. In PEG-DOPA, an additional band at 370 nm steadily increases, supporting the formation of 3-aminoesculetin (6,7-dihydroxycoumarin) derivatives resulting from the cyclization of α,β-dehydroDOPAacetyl moieties22 (Figure S5). The formation of a well-detectable red-shifted band above 500 nm in the oxidation mixture from PEG-DOPA is peculiar and suggests a specific pathway for quinone evolution. Assuming that intramolecular cyclization of the carboxyl group onto the o-quinone does occur, as suggested previously, it is possible that the resulting lactone undergoes slow hydrolysis to give a hydroxyquinone derivative23 contributing to the observed chromophore (Figure S6). The time evolution of the absorbance at 320 nm (assigned as DH intermediate) for the four derivatives was compared (Figure 4f) and the observed kinetics followed the sequence -ClDop > -Dop > -NE > -DOPA. This sequence correlates with the cross-linking kinetics of the corresponding gels (Figure 2c), indicating that DH intermediate is crucial in the cross-linking reaction of the aminocatechols tested. The oxidation of PEG-NDop, however, looks different (Figure 4c). The initial nitrocatechol bands at 310 and 354 nm evolve into new bands located at 283, 337, 370 and λ > 450 nm. These new bands are believed to correspond to the nitroanalogues of the described transient intermediates, such as N-Q 3816


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CONCLUSIONS Ring and/or chain functionalization of catecholamine components has been explored as a convenient strategy to gauge and tune cross-linking kinetics in catechol-PEG derivatives for new design rules. Comparing the polymerization kinetics and final mechanical properties of PEG gels obtained by polymerization of catecholamine end-functionalized star-PEGs with different substituents at specific ring and chain positions, it was possible to draw some general rules for controlling and tuning crosslinking processes and product properties. In particular, installation of an electron-withdrawing but pi-electron donating chloro substituent on the catechol ring resulted in faster and more efficient cross-linking, while opposite effects were observed with the strongly electron-withdrawing nitro group. Chain substitution, on the other hand, interfered with the process by slowing down the kinetics and hindering crosslinking, due to either chain breakdown (β-OH group) or intramolecular cyclization (α-carboxyl group). Interesting perspectives derive from use of mixtures of catecholaminePEG precursors offering further opportunities for fine-tuning. Mixing fast gelling PEG-ClDop with slow gelling PEG-Dop, a 10 kPa cured tissue glue was obtained that cured in ca. 2 h. This system can be particularly interesting in surgery applications where first a sealant and then a gluing function are desired at the operation point with a single material. In addition, this material has inherent antibacterial properties,12 representing a multifunctional gluing system with simple composition but relevant added value for medical applications.

Figure 5. Shear moduli (G′, G″) of PEG-ClDop/PEG-Dop mixtures at different ClDop/Dop ratios as a function of cross-linking time (100 mg mL−1 polymer concentration and 9 mM oxidant concentration, (4:1) catechol/NaIO4 molar ratio).

after curing. At these higher ClDop concentrations, the clusters quickly formed by the ClDop reactive sites seem to have hindered the motion of the Dop groups in such a way that they could not find reaction partners on different chains. Whether this led to dangling ends or to intrachain reactions is unclear. Nevertheless, these bonds did not contribute to the shear modulus, they were not force-carrying bonds. From the rheological data we however conclude that too dense clusters formed by the ClDop groups hinder the efficiency of the Dop groups in forming force-carrying bonds. As control, Figure S7 shows the polymerization kinetics for 75:0 mixture (i.e., without PEG-Dop), which occurs as rapidly as in 100:0 but achieves lower mechanical stability. Finally, we tested the adhesive properties of PEG-Dop and PEG-ClDop on porcine skin, in order to demonstrate that the presence of substituents in the ring does not disturb the tissueadhesion properties of these materials. We found no significant differences in the adhesive performance of PEG-Dop and PEGClDop, as confirmed by statistical analysis using t test and oneway ANOVA (Figure 6). Both materials failed cohesively, indicating that the strong interfacial properties of dopamine for tissue gluing are maintained in the ClDop derivative, and that the adhesive performance is limited by the mechanical strength, which is similar in both cases (according to the rheological data in Figure 2c).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01126. Additional figures as described in the text (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 681 9300 510. Fax: +49 681 9300 223. E-mail: [email protected]. Present Address ⊥

Z.S.: Bahauddin Zakariya University, Multan 60800, Pakistan.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Oya Ustahüseyin thanks the Max-Planck-Graduate Center for financial support. REFERENCES

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Figure 6. Adhesion strength of PEG-Dop and PEG-ClDop hydrogels on porcine skin. The square represents the average value, and the line represents the median value. 3817


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Gauging and Tuning Cross-Linking Kinetics of Catechol-PEG

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