A comparative study of enzyme initiators for crosslinking phenol-functionalized hydrogels for cell encapsulation
© The Author(s). 2016
Received: 26 July 2016
Accepted: 1 September 2016
Published: 5 October 2016
Dityrosine crosslinking in proteins is a bioinspired method of forming hydrogels. This study compares oxidative enzyme initiators for their relative crosslinking efficiency and cytocompatibility using the same phenol group and the same material platform. Four common enzyme and enzyme-like oxidative initiators were probed for resulting material properties and cell viability post-encapsulation.
All four initiators can be used to form phenol-crosslinked hydrogels, however gelation rates are dependent on enzyme type, concentration, and the oxidant. Horseradish peroxidase (HRP) or hematin with hydrogen peroxide led to a more rapid poly (vinyl alcohol)-tyramine (PVA-Tyr) polymerization (10–60 min) because a high oxidant concentration was dissolved within the macromer solution at the onset of crosslinking, whereas laccase and tyrosinase require oxygen diffusion to crosslink phenol residues and therefore took longer to gel (2.5+ hours). The use of hydrogen peroxide as an oxidant reduced cell viability immediately post-encapsulation. Laccase- and tyrosinase-mediated encapsulation of cells resulted in higher cell viability immediately post-encapsulation and significantly higher cell proliferation after one week of culture.
Overall this study demonstrates that HRP/H2O2, hematin/H2O2, laccase, and tyrosinase can create injectable, in situ phenol-crosslinked hydrogels, however oxidant type and concentration are critical parameters to assess when phenol crosslinking hydrogels for cell-based applications.
KeywordsOxidative enzyme Hydrogel Phenol-crosslinking Cell encapsulation
Hydrogels have great potential for biomedical applications and have been extensively explored for drug delivery, cell encapsulation and tissue adhesion for wound closure. Crosslinking is a necessary step in hydrogel formation, converting soluble polymers into more stable polymer networks with high water content. This stabilisation can be achieved through a variety of approaches including physical crosslinking such as in freeze-thaw processes , formation of ionic complexes such as in calcium crosslinked alginate , and self-assembly of peptides. However, covalent crosslinking in physiological conditions is the preferred approach for fabrication of biomedical hydrogels with stable crosslinks and robust mechanical properties [3–8]. In particular, enzymatic crosslinking [9–11] is favoured due to the mild physiological conditions under which the reactions occur.
HRP is one of the most commonly used enzymes to crosslink phenol residues. HRP is normally combined with the oxidant hydrogen peroxide (H2O2), where HRP extracts oxygen from the peroxide leading to a change in the oxidation state of its heme group. The HRP reaction can be simply summarized in the following equation: 2RH + H2O2 → 2R• + 2H2O where RH represents the phenol and R• represents the free radical formed (Fig. 1c). The combination of HRP and H2O2 in the presence of phenolic hydroxyl groups enables crosslinking of the aromatic ring by C-C and C-O coupling, and ultimately leads to hydrogel formation [9–11]. A suggested alternative to using HRP is hematin, which is an enzyme-like molecule that is clinically approved for the management of porphyria attacks [22, 23]. Hematin is an iron-containing porphyrin, with a Fe (III) compound structure similar to the prosthetic iron protoporphyrin IX found in HRP. Similarly to HRP, hematin can be used for the oxidative crosslinking of phenol compounds in the presence of H2O2 [22–24]. Some oxidoreductase enzymes, such as laccase and tyrosinases do not require H2O2, but instead use molecular oxygen to crosslink phenol residues. Laccase interacts with molecular oxygen and forms a tightly bound H2O2, which is then used to form di-phenol crosslinks . Tyrosinase is an enzyme widely found in animals and plants and is responsible for the production of various types of melanin in animal skin and blackening of fruits [26, 27]. Tyrosinase crosslinks phenol residues in a two-step process that is different than the radically-mediated HRP, hematin, and laccase enzymes (Fig. 1d). The key mechanism of the oxidative tyrosinase reaction is the conversion of phenol into catechol by adding an additional hydroxyl group on the aromatic ring using molecular oxygen. Further oxidation of catechol generates a reactive quinone, which readily forms a covalent bond with other quinones .
Many of the studies using oxidative enzyme crosslinkers to crosslink phenols use protein or polysaccharide-based hydrogels or biological-synthetic hybrid hydrogels. However proteins and polysaccharides can act as antioxidants, obscuring the effects of the initiating system on cells during encapsulation [13, 21]. Therefore, when evaluating initiators for oxidative, phenol crosslinking the selection of a “blank slate” hydrogel that does not interfere with the initiating system is advantageous. PVA modified with tyramine (PVA-Tyr) groups is an ideal candidate because PVA is a hydrophilic, cytocompatible synthetic polymer for medical applications that is relatively bioinert. As a synthetic polymer, PVA does not natively interact with cells and therefore provides a direct platform for observing the effects of oxidative crosslinking systems on hydrogel formation and cell encapsulation.
The aim of this work was a comparative study that illuminates both the advantages and challenges of various enzyme initiators used to crosslink phenol containing hydrogels using one hydrogel platform, based on a purely synthetic, “blank slate” material. Specifically we wanted to 1) characterize variations in material properties as a function of enzyme type and concentration and 2) evaluate the impact of oxidative, enzyme encapsulation on cells after hydrogel crosslinking. Some of the most commonly used enzyme initiating systems, namely HRP/H2O2, hematin/H2O2, laccase, and tyrosinase, were selected to crosslink PVA-Tyr hydrogels. Kinetics were spectroscopically monitored to demonstrate how different concentrations of enzymes and oxidants determine the reaction rate and the final hydrogel quality (i.e., mass loss, swelling, and modulus). Fibroblasts were encapsulated within PVA-Tyr hydrogels using each enzyme initiating system to show the impact of oxidative crosslinking on long-term cell viability and proliferation. Overall, this study demonstrates that a thorough evaluation of enzyme initiators should be performed, especially when using hydrogels crosslinked with oxidants, as the initiator will impact the quality of the hydrogel formed and cellular proliferation, which is critical for biomedical applications.
Poly (vinyl alcohol) (PVA) (13–23 kDa, 98 % hydrolysed), succinic anhydride, triethylamine, 1,3-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), tyramine, molecular sieves (4 Å), dialysis tubing (10 kDa molecular weight cut-off), Dulbecco’s Phosphate Buffered Saline (PBS), horseradish peroxidase type VI, hematin porcine, hydrogen peroxide, laccase from trametes versicolor, tyrosinase from mushroom, Dulbecco’s Modified Eagle’s Medium (DMEM), Penicillin-Streptomycin, Trypsin-EDTA Solution 1X, Calcein-AM, Propidium Iodide, Dithiothreitol (DTT), 2-[N-morpholino] ethanesulfonic acid (MES), Tris base, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), phosphoric acid and ammonium sulfate were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased from Moregate Biotech. Dimethyl sulfoxide (DMSO) dried over 4 Å molecular sieves, ethanol, methanol, and acetone were bought from Ajax Chemicals. Hydrogel disc molds were made from silicone sheets (Silastic®Sheeting, reinforced medical grade silicone rubber, Dow Corning). MTS reagent (CellTiter 96® AQueous One Solution Cell Proliferation Assay) was purchased from Promega. Protein molecular weight markers (Precision Plus Protein™ All Blue Prestained Protein Standards) were purchased from BioRad. Carboxy-H2DFFDA, Bis-Tris NuPAGE® SDS-PAGE gels, and Coomassie Blue G250 were purchased from ThermoFisher Scientific.
PVA-tyramine was synthesized in a two-step reaction as described previously . Briefly, PVA was dissolved in dry DMSO at 60 °C under nitrogen purging and succinic anhydride and triethylamine were added and stirred for 24 h. The carboxylated PVA was precipitated in a 10-fold excess of ethanol and then dialyzed against water prior to lyophilization. Dried carboxylated PVA was dissolved in dry DMSO at 60 °C, and nitrogen purged. The solution was cooled to room temperature and DCC and NHS were added and allowed to react for 24 h, followed by the addition of tyramine for another 24 h. PVA-tyramine was precipitated in 10-fold excess of acetone, re-dissolved in water, and vacuum filtered to remove the dicyclohexylurea byproduct followed by dialysis against water and lyophilization. The PVA-tyramine used in this article was characterized to be 2 % tyraminated (7 tyramine per PVA chain) using 1H NMR (300 MHz Bruker Advance DPX-300 spectrometer in D2O).
PVA-tyramine hydrogel formation
PVA-tyramine (5 % w/w) was dissolved in PBS at 80 °C. Upon complete dissolution, the polymer solution was cooled to room temperature and the initiators were added to the solution under gentle vortexing to ensure homogenous gelation. Initiators used were HRP (0–0.5 U/mL) with H2O2 (0–12 mM), hematin (0–0.08 % w/w) with H2O2 (0–12 mM), laccase (0–25 U/mL) and tyrosinase (0–2000 U/mL). The macromer solution was then placed into silicone molds (1 mm height x 6 mm diameter) between silicone sheeting (0.1 mm thick). The samples were then placed at 37 °C in a humidified incubator (37 °C and 5 % CO2) to gel for 4 h.
Kinetics of oxidative crosslinking were measured by UV-vis spectroscopy. PVA-tyramine hydrogel precursor solutions were formulated as described above and 50 μL of solution was added to the bottom of 96-well plates. UV-vis spectra were recorded on a BMG LABTCH SPECTROstar Nano spectrophotometer for up to 6 h during gelation at 37 °C. Plates were covered with an adhesive plate cover to minimize evaporation.
Hydrogel swelling and mass loss
Fully formed hydrogel samples (4 h gelation) were swollen in a sink of PBS and incubated at 37 °C for two days prior to forming rheological measurements. A Kinexus Pro rheometer from Malvern was used to obtain the magnitude of the (complex) dynamic shear modulus (|G*|) at an oscillation frequency of 1 Hz and a strain of 0.5 % at room temperature (21 °C). Parallel plate geometry with a diameter of 20 mm and a sample gap of 1.0 mm was used. All experiments were performed within the linear viscoelastic region.
Cell viability and proliferation following encapsulation
L929 murine dermal fibroblasts were trypsinized and resuspended in PBS using aseptic technique. The enzymes were added with gentle mixing to separate sterile macromer solutions at final concentrations of 0.2 U/mL HRP with 6 mM H2O2, 15 U/mL laccase, or 750 U/mL tyrosinase. The cell suspension was then added to the macromer solution to give a final density of 1x106 cells/ml, of which 50 μL was added to the bottom of wells in a 96-well plate (6 mm diameter). The samples were immediately then placed in a humidified incubator (37 °C and 5 % CO2) and rotated for 4 h at 4 rpm (modified MACSmix Rotator) to ensure homogenous cell suspension during polymerization. The samples were then immersed in media (DMEM, 10 % v/v FBS, 1 % v/v Penicillin-Streptomycin) and cultured for up to 7 days.
Cell viability within gels was determined over one week of culture qualitatively via a Live-dead assay and quantitatively via the MTS assay. At 0 and 7 days post-encapsulation, the samples were stained with 1 μg/ml of Calcein-AM and 1 μg/ml Propidium Iodide in PBS. After 10 min incubation with the stains, the gels were imaged using a confocal microscope (Leica, DM LFSA) while hydrated. After 0, 1, 3, and 7 days cell proliferation was assessed by adding MTS reagent (20 μL) to the cell cultures for 4 h prior to measuring the absorbance at 490 nm. The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture.
Intracellular ROS production
The cell-permeant dye, carboxy-H2DFFDA was used to quantify and visualize intracellular reactive oxygen species (ROS) due to H2O2 and encapsulation with enzyme initiators, respectively. To measure cell generated ROS by 0–12 mM H2O2, fibroblasts in suspension culture were incubated with 10 μM carboxy-H2DFFDA for 20 min to allow for its transport into cells and subsequent cleavage of the diacetate followed by several rinses in PBS via centrifugation. Cells (1x106 cells/mL) were resuspended in PBS with 0–12 mM H2O2 and transferred to 96-well plates (80 μl/well). The plate was placed in a humidified incubator (37 °C and 5 % CO2) and assayed on a Tecan Infinite F200 plate reader at 485 nm excitation 535 nm emission.
Cell proliferation in the presence of H2O2
L929 murine dermal fibroblasts were trypsinized, resuspended in PBS containing 0–12 mM H2O2 and placed in a humidified incubator (37 °C and 5 % CO2). After 4 h cell culture media was added to the wells and cells were then cultured for up to a week. Cell proliferation was assessed by adding MTS reagent to the cell cultures for 4 h prior to measuring the absorbance at 490 nm.
A one-way or two-way analysis of variance (ANOVA) was performed to compare multiple conditions with Tukey’s post-hoc analysis. Significance was tested using GraphPad Prism 6 (GraphPad Software) and results of p < 0.05 were considered significant. Experiments were performed in triplicate and experiments were repeated three times. Quantitative data are expressed as mean with error bars representing standard deviation (mean ± SD).
Results and discussion
Gelation, swelling, mass loss, and mechanical properties of phenol-modified hydrogels crosslinked with various enzyme initiators
Synthetic hydrogels based on PVA modified with the phenol molecule tyramine (Fig. 1a) were successfully crosslinked using the initiators HRP/H2O2, hematin/H2O2, laccase and tyrosinase under physiological conditions (37 °C, pH 7.4) (Fig. 1b). For all of the different initiating systems examined, there is a significant increase in absorbance around 325 nm during the crosslinking process, indicating the formation of dityrosine crosslinks . Therefore, this increase in absorbance at 325 nm is monitored over the crosslinking period as a measure of the polymerisation kinetics, where complete gelation is defined as the time when no further changes in absorbance is observed.
Physical properties of PVA-Tyr hudrogels crosslinked via varying enzyme types and concentrations
Time to gel (min)a
Final absorbance at gelation (325 nm)b
Sol fraction (% mass loss at 48 h)
Swelling (q, at 48 h)
Crosslinking density (ρx, mmol/L)c
Dynamic shear modulus (G*, Pa)
1.4 ± 0.2
32 ± 7 %
29 ± 3
138 ± 43
2.0 ± 0.2
31 ± 5 %
23 ± 1
219 ± 30
1.2 ± 0.1
36 ± 7 %
33 ± 5
89 ± 24
0.3 ± 0.0
76 ± 8 %
168 ± 36
5 ± 1
0.7 ± 0.2
63 ± 10 %
121 ± 25
11 ± 3
1.1 ± 0.1
49 ± 11 %
84 ± 16
38 ± 5
2.4 ± 0.2
33 ± 6 %
26 ± 4
97 ± 34
2.9 ± 0.1
24 ± 10 %
21 ± 3
145 ± 22
1.7 ± 0.1
26 ± 5 %
44 ± 7
145 ± 49
2.0 ± 0.2
20 ± 8 %
35 ± 10
169 ± 56
PVA-Tyr hydrogels fabricated using 0.1 and 0.2 U/mL HRP (6 mM H2O2) had significantly different final absorbance values and were further compared in terms of the physico-mechanical properties. Soluble (sol) fraction or mass loss after initial swelling, swelling and modulus of fully gelled hydrogels are shown in Fig. 2c, d, e, respectively. The mass loss within PVA-Tyr hydrogels crosslinked with HRP/H2O2 as an initiation system have ~30 % mass loss (Fig. 2c), similar to the initial mass loss within other hydrogel studies . As the HRP concentration was increased (0.1 to 0.2 U HRP/mL, 6 mM H2O2) swelling significantly decreased, which corresponds with the varying degrees of gelation demonstrated using spectrophotometry (Fig. 2d). The final dynamic shear modulus (G*, Fig. 2e) follows the opposite trend, where a decrease in swelling of HRP/H2O2 crosslinked PVA-Tyr gels results in a significant increase in mechanical properties and vice versa. This result agrees with the literature where an increase in crosslinking density is reflected by a decrease in equilibrium water content (swelling). This decrease in the equilibrium water content (swelling) results in an increase in the crosslinking density (ρx) and an increase in the mechanical properties, such as shear modulus . Overall, it can be seen that the HRP/H2O2 initiating system can be used to effectively crosslink phenol-modified macromers and that the concentration of HRP is critical to determining the rate and degree of polymerization.
Overall, it can be seen that all enzymes and enzyme-like initiators evaluated were able to crosslink phenol-modified macromers into hydrogels. Gelation monitored using spectroscopy showed that varying the initiator/oxidant type and concentration led to final polymerization times ranging from 10 min to over 6 h under physiological conditions (37 °C, pH 7.4). A higher enzyme and oxidant concentration generally led to a higher degree of polymerization, with the exception of HRP where too much oxidant is known to reduce reaction efficiency [20, 25]. The sol fraction was evaluated versus absorbance, as a relative measure of crosslinking efficiency of all four enzyme and enzyme-like initiators. A higher PVA-Tyr hydrogel absorbance corresponded to lower hydrogel mass loss which suggests a larger fraction of PVA chains being crosslinked into the gel up until an absorbance of ~1.2 (Additional file 1: Figure S1). It is hypothesized all PVA chains functionalized with tyramine continued to further crosslink as the absorbance increased from 1.2 to 2.9 as suggested by a decrease in swelling (Table 1), however all of these samples had ~25 % mass loss because of unfunctionalized PVA that came out as the sol fraction. HRP and hematin with H2O2 as an oxidant both led to rapid gelation of PVA-Tyr hydrogels within 30 min, whereas laccase and tyrosinase which use dissolved O2 as an oxidant gelled significantly slower (2.5+ hours). Although the use of H2O2 as an oxidant leads to rapid polymerization which is advantageous for in situ clinical gelation, H2O2 is also known to reduce cell proliferation and be cytotoxic at high concentrations which leads to questions about its use for cell encapsulation and/or for wound closure [21, 35].
Cell viability and proliferation after encapsulation in phenol-modified hydrogels crosslinked with various enzyme initiators
Although HRP/H2O2, hematin/H2O2, laccase and tyrosinase can all form robust hydrogels from phenol-modified polymers, it is also critical for biomedical applications to evaluate the impact of the enzyme-mediated crosslinking process on cells. Based on the hydrogel material properties, enzyme initiators that resulted in the highest degree of polymerization were selected for cell encapsulation (i.e. sol fraction ~25 %, swelling q ~ 25, G* ~ 180 Pa). Hematin/H2O2 crosslinked gels did not reach the same material properties as gels formed from the other initiators, and was not evaluated for cell encapsulation because of the high H2O2 concentrations required. PVA-Tyr hydrogels crosslinked for 4 h with HRP/H2O2 (0.2 U/mL/6 mM), laccase (15 U/mL), and tyrosinase (750 U/mL) had similar swelling and mechanical properties, and thus a similar crosslinking density . Having a similar crosslinking density will allow for direct comparison between the fully formed hydrogels because it implies that oxygen and nutrient diffusion will be similar within all hydrogels.
This study provided a single bioinert, hydrogel platform to systematically evaluate four different enzyme, and enzyme-like, initiators for their ability to crosslink phenol-based macromers and the influence of this oxidative crosslinking on cells during encapsulation. These in situ forming, enzymatically crosslinked phenol-containing hydrogels can be used for a large variety of biomedical applications including as adhesives for wound closure , for drug delivery , and for cell encapsulation for engineering tissues such as cartilage  and neural  tissue. All enzyme initiators evaluated (HRP/H2O2, hematin/H2O2, laccase, tyrosinase) can be used to form phenol-crosslinked hydrogels, however gelation rates are dependent on enzyme type, concentration, and the oxidant. HRP or hematin with hydrogen peroxide led to a more rapid PVA-Tyr polymerization because a high oxidant concentration was dissolved within the macromer solution at the onset of crosslinking, but the HRP/H2O2 initiating system also led to decreased cell survival. Whereas laccase and tyrosinase require oxygen diffusion to crosslink phenol residues and took longer to gel, cells encapsulated in these gels proliferated over time. Overall this study demonstrates that there are many available enzyme and enzyme-like initiators to create injectable, in situ phenol-crosslinked hydrogels. However care must be taken when selecting the appropriate enzyme as the oxidoreductase and hematin initiators evaluated in this study require oxidants to polymerize phenol residues, which can affect cells during encapsulation and may alter the long-term therapeutic potential of the cells for tissue engineering applications.
(Complex) dynamic shear modulus
Analysis of variance
Dulbecco’s modified eagle’s medium
Fetal bovine serum
- H2O2 :
- Md :
Dried sample mass
- Mi :
Initial wet mass
- Mi,d :
Initial macromer fraction
- Ms :
Swollen sample mass
Dulbecco’s phosphate buffered saline
Mass swelling ratio
Reactive oxygen species
Sodium dodecyl sulfate
The authors would like to thank Professor Lynn Bilston and Dr. Lauriane Jugé at Neuroscience Research Australia for assistance with rheometry.
Research reported in this publication was supported by a Whitaker International Scholarship Grant and University of New South Wales Early Career Researcher Grant awarded to J.J.R. K.L is supported by the Health Research Council of New Zealand under grant agreement HRC 15/483.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files, or is available from the corresponding author on reasonable request.
JJR executed and analyzed the materials preparation, materials analysis and and a portion of the cell studies. PN executed and analyzed a portion of the cell studies. KSL contributed to study design and the intellectual content of the manuscript. LPW contributed to the intellectual content of the manuscript. JJR and PJM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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