Article pubs.acs.org/Biomac
Enzymatically Cross-Linked Hyperbranched Polyglycerol Hydrogels as Scaffolds for Living Cells Changzhu Wu,† Christine Strehmel,‡ Katharina Achazi,† Leonardo Chiappisi,‡ Jens Dernedde,⊥ Marga C. Lensen,‡ Michael Gradzielski,‡ Marion B. Ansorge-Schumacher,§ and Rainer Haag*,† †
Institut für Chemie und Biochemie, Freie Universität Berlin, Takustraße 3, 14195 Berlin, Germany Institut für Chemie, Technische Universität Berlin, Straße des 17. Juni 124, 10623 Berlin, Germany ⊥ Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany § Institut für Mikrobiologie, Technische Universität Dresden, 01062 Dresden, Germany ‡
S Supporting Information *
ABSTRACT: Although several strategies are now available to enzymatically cross-link linear polymers to hydrogels for biomedical use, little progress has been reported on the use of dendritic polymers for the same purpose. Herein, we demonstrate that horseradish peroxidase (HRP) successfully catalyzes the oxidative cross-linking of a hyperbranched polyglycerol (hPG) functionalized with phenol groups to hydrogels. The tunable cross-linking results in adjustable hydrogel properties. Because the obtained materials are cytocompatible, they have great potential for encapsulating living cells for regenerative therapy. The gel formation can be triggered by glucose and controlled well under various environmental conditions.
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INTRODUCTION Hydrogels that have been built into a hydrophilic polymeric network can store a great amount of water in their porous scaffolds and still perfectly maintain their three-dimensional (3D) shape.1,2 The hydrogels’ biophysical similarity to living tissues and extracellular matrix has made them particularly valuable for tissue engineering,3,4 drug/gene delivery,5−7 biocatalysis,8−11 and regenerative medicine.12,13 Traditionally, hydrogels are either physically gelled via hydrophobic14 and ionic15 interactions, or chemically cross-linked by photo-crosslinking,16 Michael-type addition reactions,17 Schiff-base formation,18 and click reactions.19 Physical gelation generates hydrogels with weak interactions under mild conditions, thus avoiding damaging the encapsulated guest molecules during cross-linking. However, these hydrogels cannot preserve the guests in the scaffolds strongly enough during biological applications, which has largely prohibited their use as drug/ cell/protein carriers.20 In contrast to physical interactions, chemical cross-linking can maintain the interior structure of hydrogels for long periods, and the encapsulated molecules can © XXXX American Chemical Society
be transported or released in a controllable fashion. However, the chemical cross-linking approach is also limited in many biorelated applications because it often generates side products, utilizes toxic reagents, and requires harsh reaction conditions.12 Therefore, there has been a quest to establish a reliable and efficient methodology to construct stable hydrogels under physiological conditions. Unlike traditional techniques, biological enzymes can catalyze polymer cross-linking to hydrogels via strong covalent bonds under mild conditions with an overall high chemo-, regio-, and stereoselectivity.21 This mild cross-linking neither triggers any toxic side reactions nor releases substantial heat, thus minimizing damage to the surrounding tissues. Additionally, the enzymatic reaction rate can be easily controlled to give an adjustable gel formation time. Because of these advantages, enzymatic cross-linking has been studied to produce injectable Received: May 15, 2014 Revised: August 16, 2014
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Scheme 1. Formation of hPG-HPA Hydrogels by HRP Cross-Linking
Table 1. HRP-Catalyzed Cross-Linking of Functionalized hPG
hydrogels for regenerative medicine,13 biodegradable gels for drug delivery,22 biocompatible materials for bone repair,23 and so forth.24−27 So far, a variety of enzymes such as transglutaminases and peroxidases have been successfully applied to mediate hydrogel formation.21 Among them, horseradish peroxidase (HRP) has been mostly used because it is highly stable, active, and commercially available.21,28 Park and Kobayashi’s groups have shown in their profound study of HRP-triggered oxidative polymerization that phenol-/anilinederivative conjugates can be coupled by HRP with hydrogen peroxide (H2O2).29,30 However, most hydrogel formation by HRP has only been reported for the polymer conjugates of either tyramine (TA) or 3-(4-hydroxyphenyl) propionic acid (HPA). Nevertheless, tremendous achievements have been made to advance HRP-cross-linked hydrogels for biomedical applications.21 Despite numerous studies focused on HRP-cross-linked hydrogels, little is known to date about HRP’s substrate specificity with regard to polymer structure and functionality. It has been reported that linear and star-shaped polymers can be cross-linked to hydrogels by HRP,8,13,25 but it is not known whether HRP can accept sterically demanding dendritic polymer substrates. Enzyme kinetics depicts the dependency of reaction rate on enzyme concentration, temperature, and inhibitors. So far, only reactants and enzymes have been
controlled to follow gel formation for injectable purposes,13,22−24,26,27 but the influence of temperature and the presence of inhibitors, which highly relates to enzyme activity, has not been disclosed until now. Furthermore, H2O2, a toxic agent, is used to react with HRP to form highly reactive intermediates for cross-linking. However, there are no clear data that report on the residual content of H2O2 in hydrogels. In this work, we are the first to study the enzymatic crosslinking of dendritic polymers to hydrogels with HRP. Dendritic polymers, which are highly branched and symmetrical macromolecules, are very interesting for biomedical applications because of their unique and significantly different properties from their linear counterparts.31,32 Herein, we used hyperbranched polyglycerol (hPG) that had been functionalized with 3-(4-hydroxyphenyl) propionic acid (HPA) for cross-linking (Scheme 1). hPG is a highly water-soluble and biocompatible polymer with multiple hydroxyl functionality, which therefore facilitates encapsulation of cells.33 Enzyme-mediated hPG crosslinking contributed to the time-tunable gel formation in response to the change of cross-linking conditions. The obtained hPG gels were evaluated with regard to their rheological behavior and cellular toxicity. Glucose-triggered gel formation was also investigated under various environmental conditions to control the gelation time. B
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another 50 h using a RTCA device. Each sample was analyzed in 4 replicates, and the results were reported as the mean value. Sample preparation of MTT assay was similar to RTCA. Briefly, 100 μL medium suspension containing 7500 L929 cells was added into each well of a 96-well plate, followed by 24 h incubation (37 °C and 5% CO2). The medium was then replaced by a fresh medium containing different substances, and incubated for another 48 h. Subsequently, 25 μL MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, 5 mg/mL in PBS) was directly added into each well. After 2 h incubation at 37 °C, the medium was removed and 100 μL DMSO was added to dissolve the blue formazan crystals. Optical density was detected by a microplate reader (Spectra Max Gemini, Molecular Devices) at 490 nm. The wells without adding any substance were considered as control. H2O2 Detection. The residual H2O2 content in gels was detected by Pierce quantitative peroxide assay kits according to the enclosed protocol with minor modifications. Hydrogels (50 μL) were formed with several repetitions in a typical manner (100 mg/mL hPG-HPA@ 5%, 5 U/mL HRP, and 12.5 mM H2O2). Immediately after the gel formation was completed, 500 μL distilled water was added to extract the residual H2O2 under slow shaking. After 5, 10, 20, and 30 min as well as 2, 4, 6, 12, and 24 h, a 200 μL fraction of the solution was transferred into 1 mL working reagent (WR) solution provided by the supplier. The mixture was incubated at room temperature for 20 min, and subsequently absorbance at 560 nm was recorded. The residual H2O2 concentration was calculated according to two calibration curves (0.1−1 μM and 0.5−10 μM), shown in the Supporting Information. In addition, H2O2 stability in water at room temperature was evaluated as well (Supporting Information). Cytocompatibility of hPG-HPA Gels. The viability of L929 cells cultured on top of the hPG-HPA gel surface and the viability of encapsulated cells were investigated by a live/dead assay (see below). For these studies, gels were prepared with 100 mg/mL hPG-HPA, 5 U/mL HRP, and 12.5 mM H2O2. Smooth gels were directly polymerized onto the observation area of a μ-dish (35 mm in diameter). Subsequently, 500 μL of a cell suspension (containing 30 000 L929 cells) were seeded on the gel surface and incubated for 24 and 48 h at 37 °C under a 5% CO2 atmosphere at 95% humidity. Experiments were performed in triplicate, and the cell viability was determined by cell counting. Cell entrapment in hPG-HPA gels was obtained by quickly mixing 10 000 cells into a 50 μL solution containing 100 mg/mL hPG-HPA, 5 U/mL HRP, and 12.5 mM H2O2 and subsequently pouring onto the observation area of a μ-dish. After gelation, 500 μL cell culture medium was added into the μ-dish and incubated at 37 °C under a 5% CO2 atmosphere for 24, 48, and 72 h. Experiments were carried out in triplicate and live/dead assay was performed to determine cell viability. The live/dead assay was performed using a standard protocol with slight modification.25 The cells were stained with 300 μL of a staining solution containing fluorescein diacetate (0.5 mg/mL stock solution in acetone) and propidium iodide (0.5 mg/mL stock solution in DPBS). Viable and dead cells were detected by confocal laser scanning microscopy (CLSM, Leica TCS SP5 II, Leica Microsystems) using an excitation wavelength of 495 nm (fluorescein) and 536 nm (propidium iodide), and emission was recorded at 519 nm (fluorescein) and 617 nm (propidium iodide). Viable cells expressed green fluorescence, which indicates transformation of fluorescein diacetate into fluorescein by the action of esterases inside the living cells. Propidium iodide passed through damaged plasma membranes and bound to nucleic acids resulting in red fluorescence. Cell viability was statistically calculated from a number of CLSM images (at least five images for one sample) and expressed as the mean value. 2D images were taken from the single level of gel surfaces, and 3D images were created from a stack of 2D images with a scanning distance of approximately 0.3 cm inside the gels. Cell Attachment on/in hPG-HPA Gels in the Presence of Fibronectin. Hydrogels were prepared with 100 mg/mL hPG-HPA@ 5% or 10%, 5 U/mL HRP, and 12.5 mM H2O2. For both 2D and 3D studies, a 50 μL mixture of polymer, HRP, H2O2, and fibronectin (Fn, 4000 μg/mL as stock for use) solution was directly polymerized onto a
MATERIALS AND METHODS
Materials. General chemicals were purchased from Sigma-Aldrich and used as received. Anhydrous pyridine and dimethylformamide (DMF) were purchased from Acros Organics. RPMI 1640 cell culture medium, fetal bovine serum (FBS), penicillin/streptomycin (PS), Dulbecco’s phosphate buffered saline solution (DPBS), and trypsinEDTA were from PAA Laboratories GmbH. Cell culture flasks and μdishes were obtained from PAA Laboratories GmbH and Ibidi GmbH, respectively. hPG (Mw = 6 kDa) was synthesized by anionic, ringopening multibranching polymerization of glycidol under slow monomer addition.34,35 Polyethylene glycol (PEG, Mw = 6 kDa) was obtained from Acros Organics. Horseradish peroxidase (HRP, type VI-A, approximately 250 U/mg lyophilized powder (using pyrogallol)) was purchased from Sigma-Aldrich. Glucose oxidase (GOD, Amano 5, 146 U/mg) was generously donated by Amano Enzyme (Nagoya, Japan). Mouse fibroblasts, cell line L929, were kindly provided by Dr. J. Lehmann (Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig). Synthesis. hPG-NH2 was synthesized according to the literature with 2.5%, 5%, and 10% amine functionality, respectively.36,37 hPGHPA (compound 1 in Table 1) was synthesized through amide coupling. Typically, HPA (1.31 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 1.31 mmol), triethylamine (TEA, 1.31 mmol), and N-hydroxysuccinimide (NHS, 0.87 mmol) were mixed in 50 mL anhydrous DMF under argon protection, and the mixture was stirred for 5 h at room temperature. One gram hPG-NH2@5%, approximately 0.68 mmol amine groups on the hPG surface, was subsequently introduced into the mixture. After 20 h, the product was purified by dialysis (membrane Mw cut off 2 kDa) against methanol for 3 days (3 times solvent exchange per day), and finally dried for use. The synthesis of other conjugates (compounds 2, 3, and 4 in Table 1) is described in the Supporting Information (SI). 1 H NMR (CD3OD, 500 MHz) of hPG-HPA@5%: δ 7.04 (d, Ar), 6.72 (d, Ar), 3.40−4.00 (m, −CH2 and −CH, PG scaffold), 2.82 (t, −CH2−Ar), 2.46 (t, −CH2CONH−), 1.39 (s, −CH2 core), 0.87 (s, −CH3 core), (shown in SI Figure S1). Rheological Measurements. Rheology data were measured with a Malvern Instruments Gemini 200 HR with a plate−plate geometry of an 8 mm plate and a 0.35 mm gap. All measurements were carried out at 25 °C unless otherwise stated. A delay time of 2 s was chosen and the integration was performed over three periods. Measurements were performed at least twice in order to obtain the average value of data. Frequency sweeps were performed with a strain of 0.01, which always stayed in the linear viscoelastic range. The kinetics study of enzymatic gelation was performed under different cross-linking conditions by the variation of HRP, polymer, and inhibitor concentration as well as temperature. All kinetic experiments were performed with an oscillation frequency of 1 Hz and a strain of 0.01. Data recording was started immediately after mixing all compounds and stopped when G′ reached a decent plateau. The mechanical strength of formed gels was measured under different concentration of HRP, H2O2, and hPGHPA. Cell Culture. L929 cells were cultured in RPMI 1640 cell culture medium containing 10% FBS and 1% PS at 37 °C and 5% CO2 in a humidified incubator. The cells were grown in 75 cm2 cell culture flasks, the medium was refreshed every second day, and the culture was subcultured when the cells were confluent. Real-Time Cell Analysis (RTCA) and MTT Assay. The RTCA was conducted by an RTCA SP instrument (Roche Diagnostics GmbH, Mannheim, Germany) which was placed in a humidified incubator at 37 °C and 5% CO2.38 Cell proliferation and viability were dynamically monitored in a 96-well E-plate (Roche Diagnostics GmbH, Mannheim, Germany) in real time. Initially, 50 μL of cell-free RPMI 1640 medium containing 10% FBS and 1% PS was added into each well in order to measure background impedance. Subsequently, 5000 L929 cells in 50 μL medium suspension were seeded into each well, and incubated for 24 h. Then, the cell culture medium was aspirated and the different test substances diluted in the cell culture medium were added. The toxicity of these substances was observed for C
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Figure 1. Rheological study of hPG-HPA@5% gels (measured at f = 1 Hz and a strain of 0.01): (a) G′ of hPG-HPA@5% hydrogels recorded over time at the different HRP concentration while the other conditions (100 mg/mL hPG-HPA@5%, 12.5 mM H2O2, and 25 °C) were fixed; (b) G′ of hPG-HPA@5% hydrogels recorded over time at different hPG-HPA@5% concentration while the other conditions (1.2 U/mL HRP, 12.5 mM H2O2, and 25 °C) were fixed; (c) G′ of hPG-HPA@5% hydrogels recorded over time at different temperature while the other conditions (100 mg/ mL hPG-HPA@5%, 1.2 U/mL HRP, and 12.5 mM H2O2) were fixed; (d) G′ of hPG-HPA@5% hydrogels recorded over time at different NaN3 concentration while the other conditions (100 mg/mL hPG-HPA@5%, 1.2 U/mL HRP, 12.5 mM H2O2, and 25 °C) were fixed; (e) the influence of HRP concentration on G′ and G″ under the cross-linking conditions with 100 mg/mL hPG-HPA@5%, 12.5 mM H2O2, and 25 °C; and (f) the influence of H2O2 concentration on G′ and G″ under the cross-linking conditions with 100 mg/mL hPG-HPA@5%, and 1.2 U/mL HRP.
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μ-dish. The other preparations, such as cell cultivation and live/dead assay, were the same as the procedure mentioned above for the cytocompatibility studies of gels without the use of Fn. Glucose-Triggered Gel Formation. The experiments were performed with a total volume of 50 μL gel mixture containing 100 mg/mL hPG-HPA@5% with the combined use of HRP and GOD in the presence of glucose. The gelation time was recorded using the vial tilting method.24,27 In general, the dependence of gel formation on glucose concentration was carried out by the addition of glucose (15− 40 mM) to the mixture of GOD (25 or 250 U/mL), HRP (30, 40, or 50 U/mL) and hPG-HPA@5%. The influence of enzyme inhibitors on gelation time was analyzed upon the addition of 160 mM respective inhibitors (NaN3, CuSO4, and AgNO3) to the mixture of 50 U/mL HRP, 250 U/mL GOD, 40 mM glucose, and 100 mg/mL hPG-HPA.
RESULTS AND DISCUSSION
Synthesis of hPG Phenolic Derivatives for HRP CrossLinking. Four different synthetic strategies have been applied for the preparation of phenol functionalized hPG for HRP cross-linking (Table 1). The linear polymer conjugates of 3-(4-hydroxyphenyl) propionic acid (HPA) or tyramine (TA) are quite often reported for HRP-catalyzed gel formation, whereby HPA and TA are the accepted substrates of HRP.22,24,25 Therefore, we also initially applied their grafting to hPG for HRP crosslinking. hPG-HPA (compound 1 in Table 1) was synthesized with an amide coupling reaction between hPG-amine and HPA (Materials and Methods, SI Figures S1 and S2), while hPG-TA D
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enzyme-catalyzed product formation.40,41 This illustrates that network formation is directly connected to the applied amount of enzyme activity, which is therefore a perfect parameter for controlling gelation time. Similar to the effect of the HRP concentration, increasing the polymer concentration from 50 to 150 mg/mL could obviously shorten the gelation time by about a factor of 5 (Figure 1b), which is in a good accord with the description of the enzymatic reaction rate theory.39,40 It is unexpected that one would observe a linear dependence of the plateau value of G′ on the polymer concentration (SI Figure S15). Since an almost linear dependence occurs in the case of the initial rate of an enzymatic reaction versus substrate concentration at low value,10,42 the plateau value of G′ may somehow correlate with the reaction rate. These findings, on the other hand, may indicate the possibility of using rheological measurements to assess the enzyme kinetics with a gel formation reaction. Nevertheless, the control of the hPG-HPA concentration is a good method to tune gelation time and the mechanical strength of the finally formed gels, both of which are important parameters for applications in regenerative medicine. In addition to enzyme and substrate concentration, the enzymatic cross-linking was also strongly affected by the temperature as well as by the addition of NaN3 that is a wellknown HRP inhibitor (Figure 1c and d). For example, by just decreasing the cross-linking temperature from 25 to 10 °C, the gelation time increased by a factor of 2, while it was further sped up by a factor of 2 when the temperature was elevated from 25 to 37 °C (Figure 1c). These findings illustrate that the temperature control is an efficient and convenient way to allow the mixture of hPG-HPA polymer and enzyme solution injectable at the low temperature and quickly cross-linkable at the physiological temperature. On the other hand, the HRP activity was largely suppressed by the addition of 50−100 μM inhibitors, and completely deactivated if 500 μM NaN3 was added (Figure 1d). Although NaN3 is toxic, its inactivation of HRP cross-linking proves the concept of the multiple control options of enzymatically cross-linked hPG-HPA hydrogels. In a further experiment on hPG-HPA@5% hydrogel formation, G′ and G″ remained constant when the HRP concentration was increased from 0.01 to 0.16 mg/mL (Figure 1e). This is in agreement with the literature that G′ is not affected above a certain HRP activity level.25,43,44 The mechanical strength of hydrogels, on the other hand, strongly depends on the H2O2 concentration (from 2.5 to 12.5 mM) with G′ and G″ changing over 3 orders of magnitude (Figure 1f). However, for the concentration higher than 12.5 mM H2O2, G′ and G″ reached a plateau, thus apparently achieving the maximum gel strength. The dependence of gel mechanical properties on the H2O2 concentration is expected because H2O2 is another substrate of the enzymatic reaction and thereby controls the cross-linking density. These findings indicate that fine control of the mechanical strength of these materials is accessible, which is a fundamental requirement for different biorelated applications. From the high frequency limit G0 of the storage modulus G′, the number density 1N of the effective network connections can be calculated by eq 1:45,46
(compound 2 in Table 1) was obtained by coupling TA with hPG-succinic acid in two steps (Supporting Information). The HRP-catalyzed oxidative cross-linking successfully took place in the presence of H2O2 for both hPG-HPA and hPG-TA (SI Figure S8). These results, along with others on HRP-crosslinked linear polymers from the literature,13,22,24,25 clearly show that HRP-catalyzed oxidative cross-linking is suitable for dendritic polymers regardless of the polymer’s architecture. In order to see if other phenolic derivatized hPGs are suitable for HRP-catalyzed gel formation, hPG-HBA (compound 3 in Table 1) was synthesized by coupling 4-hydroxy-benzaldehyde (HBA) with hPG-amine and subsequently reducing the imine using NaBH4 (Supporting Information). Surprisingly, in contrast to hPG-HPA, HRP-catalyzed oxidative cross-linking did not occur in the case of hPG-HBA (SI Figure S11). This can be explained by the fact that the HRP-catalyzed polymerization rate of HBA is much lower than that of HPA (SI Figures S9 and S10). Consequently, the success of HRP oxidative cross-linking of dendritic polymers would rely on the local functionality of polymers, but is independent of the polymer macroscopic structure. Apart from the possibility of partially loading phenolic moieties, the multiple hydroxyl bearing hPG scaffolds also allow further modification of the polymer surface with other biomedically interesting molecules. For example, naproxen (NAP), a hydrophobic nonsteroidal anti-inflammatory drug (NSAID), could be conjugated to hPG-HPA@5% scaffolds (compound 4 in Table 1; see the synthesis in the Supporting Information). The water solubility of the conjugates could be tuned according to NAP loading. hPG-HPA@5% loading 1% NAP was water-soluble as well as enzymatically cross-linkable (SI Figure S12), while higher than 5% NAP loading resulted in water-insoluble polymers. Nevertheless, the enzymatically cross-linkable hPG-HPA@5%−NAP@1% demonstrates multiple functionality and good water solubility of hPG-HPA scaffolds, which is, on the other hand, hardly achieved by most linear polymers with few functional end groups for enzymatic cross-linking. Rheological Study of hPG-HPA Hydrogels. hPG-HPA was used for all the following HRP cross-linking experiments because of its superior water solubility (Table 1). The rheological properties of the hPG-HPA@5% (HPA/hydroxyl = 5%) gels were investigated in terms of the storage modulus (G′) and loss modulus (G″). Classic enzyme kinetics depicts that the reaction rate is highly dependent on enzyme, substrate, and inhibitor concentration as well as reaction temperature.39,40 Therefore, the HRP-catalyzed gelation kinetics, i.g., G′ versus time, was studied by the variation of the above-mentioned conditions (Figure 1a,b,c,d). The profile of G′, which accounts for the elastic properties of the gels, reflects the gradual formation of the enzymatically cross-linked hydrogel network in three stages: (i) a pregelation period for which G′ = 0, (ii) a gelation period in which a rapid increase of G′ was measured, which indicates a percolation transition and network formation, and (iii) a postgelation period in which G′ reaches a plateau. Data from Figure 1a shows that gelation time decreased upon increasing HRP concentration and took approximately 10 min to complete gelation at a concentration of 0.8 U/mL HRP. This slow gelation makes these materials injectable (Figure 1a and SI Figure S13). However, gel formation occurred within seconds when more than 4 U/mL HRP was used. The dependency of network formation on HRP-concentration as illustrated in SI Figures S13 and S14 strongly resembles typical kinetics of
G0 = 1N ·k·T
(1)
where k and T are the Boltzmann constant and temperature, respectively. E
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Figure 2. Proliferation of L929 cell: (a) in the presence of PEG (6 kDa), hPG (6 kDa), and hPG-HPA derivatives (6 kDa) analyzed by RTCA assay, and (b) in the presence of 0.1−2 μM H2O2.
Figure 3. Viability (live/dead assay) and the morphology of L929 cells (48 h of cultivation) on the gel surfaces of hPG-HPA@5% and hPG-HPA@ 10% with and without the addition of 360 μg/mL Fn.
mechanical properties of hydrogels are tunable and highly depend on cross-linking conditions. An optimal gel formation during the enzymatic cross-linking can be easily controlled to meet the specific needs of different cells during encapsulation, which would eventually improve therapeutic functions. Toxicity Study. Fibroblast cells were chosen to evaluate the cytotoxicity because they constitute part of the connective tissues and play a crucial role in wound healing,49 which is a prospective application of functionalized hydrogels. The relative cytotoxicity was evaluated by real-time cell analysis (RTCA) and conventional MTT assay using the fibroblast murine cell line L929. RTCA data (Figure 2a) proved that cells incubated with hPG (500 μg/mL, 6 kDa), PEG (500 μg/mL, 6 kDa), hPG-HPA@ 2.5% (500 μg/mL, 6 kDa), and hPG-HPA@5% (500 μg/mL, 6
An effective structural size can be determined from this which corresponds for the highest modulus of hPG-HPA@5% gels (cross-linked with 100 mg/mL polymers) of approximately 35.4 kPa to 4.9 nm. This means that the elastic network present in these gels can be described as a 3D network, where an elastic network point, i.e., a cross-linking point, is formed every 4.9 nm. Here it should be noted that eq 1 is an idealized equation and the real network density might deviate for several reasons somewhat from 1N of eq 1, but the deduced characteristic length will be of the right magnitude, especially as in calculating one takes the cubic root of 1N. Mooney et al. have demonstrated the high importance of mechanical properties of hydrogels as a matrix to encapsulate cells, e.g., stem or immune cells, for tissue engineering.47,48 Our findings from Figure 1 reveal that both gelation time and F
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Figure 4. Viability (live/dead assay) and the morphology of gel-encapsulated L929 cells (48 h of cultivation) in hPG-HPA@5% and hPG-HPA@ 10% with and without the addition of 360 μg/mL Fn.
irritative observation even when 70 mM H2O2 was initially applied for enzymatic cross-linking.22 Moreover, Park et al. recently disclosed the cytocompatible properties of enzymatically cross-linked gels to the MC3T3-E1 cell line with the initial use of 18.5 mM.27 Therefore, the initially applied H2O2 concentration (12.5 mM) used for hPG-HPA cross-linking should be a safe level.22,27 With the entire cytotoxicity study on polymer, HRP, and residual H2O2 taken together, it can be concluded that the applied process to prepare the hydrogels is biocompatible. Cytocompatibility of hPG-HPA Gels. Encouraged by the results obtained from the cytotoxicity analyses, the cytocompatibility of hPG-HPA gels was further investigated. A live/dead assay was used to evaluate the viability of fibroblast cells that were applied to gel surfaces as well as embedded into gels. Figure 3a shows that L929 cells remained viable (98%) on the hPG-HPA@5% gel surfaces for a cultivation period of 2 days. Although the round-shaped cell morphology (Figure 3b) indicates that L929 cells could not adhere to the surface due to the nonfouling characteristics of the gels,54−61 cellular viability was not affected during the time of observation, which is in good accord with the published data of other polyglycerol gels.8,57 The mechanism of protein/cell nonfouling effect has not been fully understood until now, but some empiric data indicate that the surface hydrophilicity plays an important role.58−61 Interestingly, the Cooper-White group recently invented a simple method to enhance cell adhesion on the cell nonfouling PEG gels by incorporating a peptide fragment of fibronectin (Fn), a protein known to enhance cell adhesion.25 Inspired by this finding, a cell adhesion study was also performed on hPG-HPA@5% gels with the addition of 360 μg/mL Fn; Figure 3c showed that cell attachment was then observed on the surface, which is in line with the finding by
kDa) showed no reduction in cell proliferation and viability compared to the untreated control. This indicates that hPG modified with less than 5% HPA is cytocompatible. Data from the MTT assay (SI Figure S19) confirmed the finding that hPG-HPA@5% and hPG were as cytocompatible as PEG, the current gold standard. However, toxicity slightly increased when the same amount of hPG-HPA@10% (6 kDa) was applied to the cells. These results are in agreement with data from the literature reporting increased material toxicity for hPG excessively modified with functional groups such as amine.50,51 The cytocompatibility of HRP was also tested by RTCA. No toxicity to L929 cells was observed (SI Figure S20) when incubated with 5 U/mL HRP, which was the highest concentration used in the experiments so far. Unlike hPG-HPA and HRP, H2O2 is known to be toxic to cells at least at high concentration by inducing apoptosis.52 For this reason, the toxicity was analyzed in more details (SI Figures S21, S22, and S23), and furthermore, the residual H2O2 level in the gels was determined by a quantitative peroxide assay (Materials and Methods) with a detection limit of 0.1 μM H2O2 (SI Figures S24 and S25). Interestingly, 5 min after the crosslinking, the residual H2O2 level in gels was undetectable (
