Nonswelling Click-Cross-Linked Gelatin and PEG Hydrogels with

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Non-Swelling Click-Crosslinked Gelatin and PEG Hydrogels with Tunable Properties Using Pluronic Linkers Vinh X. Truong, Kelly M. Tsang, and John S Forsythe Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01601 • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

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Non-Swelling Click-Crosslinked Gelatin and PEG Hydrogels with Tunable Properties Using Pluronic Linkers Vinh X. Truong1*, Kelly M. Tsang1, John S. Forsythe1* 1

Department of Materials Science and Engineering, Monash Institute of Medical Engineering,

Monash University, Clayton 3800 VIC, Australia KEYWORDS Non-swelling hydrogel, gelatin, bio-orthogonal click, nucleophilic thiol-yne addition, fibroblasts

ABSTRACT

Swelling of hydrogels leads to a decrease in mechanical performance coupled with complications in solute diffusion. In addition, hydrogel swelling affects patient safety in biomedical applications such as compression of tissue and fluid blockage. A conventional strategy for suppressing swelling is to introduce a thermoresponsive polymer with a lower critical solution temperature (LCST) within the network structure to counter the water uptake at elevated temperature. However, altering the gels mechanical strength via modification of the network structure often affects the water uptake behavior and thus a non-swelling platform with

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tunable mechanical properties suitable for various biomedical applications is desirable. In this study we applied the commercially available triblock PEG-PPG-PEG (Pluronic®) as a crosslinker for the preparation of nucleophilic thiol-yne click crosslinked hydrogels with suppressed swelling at physiologically relevant temperature. The mechanical properties and degradation rate of these non-swelling hydrogels can be tuned by judicious combinations of the available linkers. The Pluronic® linkers can be applied to prepare biologically relevant gelatin based hydrogels with suppressed swelling under physiological conditions that support attachment of fibroblast cells in 2D culture and controlled release of albumin, paving the way for the development of reliable and better performing soft biomaterials.

INTRODUCTION Hydrogels have emerged as an important biomaterial platform in bioengineering strategies for various applications including delivery of therapeutic compounds,1-3 cell culture platforms,4 and tissue regeneration.5, 6 Most hydrogels, including the high-strength double network hydrogels,7-12 swell when they come into contact with water, significantly decreasing the mechanical strength in the swollen state. This swelling leads to a dynamic increase in network mesh size, yielding complex and unpredictable release profiles of macromolecules in delivery applications.8, 13, 14 In tissue engineering applications involving cell-matrix interactions, dynamic changes in gel stiffness when the materials absorb water may impart unwanted mechanical signaling cues to cells.15,

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Importantly, uncontrolled swelling of the hydrogel may affect patient safety in

biomedical applications,17-19 such as blockage of fluids or compression and damage to nearby tissues.


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Hydrogel swelling can be suppressed by incorporating a hydrophobic segment within the network structure such as using an amphiphilic polymer to reduce polymer-solvent (water) compatibility at elevated temperature, or by increasing the crosslinking density.7,

8, 20-23

For

example, Kamata et al. reported the synthesis of a tetra-arm thermoresponsive amphiphilic copolymer, via ring opening polymerization, crosslinked with a 4-arm hydrophilic poly(ethylene glycol) polymer by succinimide ester-amine coupling.7 The swelling of the resultant gels in water at 37 °C could be tuned by varying the ratio of the amphiphilic copolymer and hydrophilic polymer precursors. The well-defined [4+4] network structure results in a hydrogel with high water content (90 wt%) and exceptional mechanical strength (ability to withstand a compressive stress of 60 MPa) which could be retained after submerging the gel in water at 37 °C. More recently, Langer and coworkers synthesized [3+2] hydrogels crosslinked by thiol-acrylate addition using a short chain 3-arm PEG linker with a molar mass of ca. 1000 g mol-1 and PEGbisacrylate with molar mass in the range of 575 to 1000 g mol-1.8 The resultant gels have very dense network structures and high concentrations of hydrophobic crosslinking points to prevent water sorption at physiological temperature. These materials are promising candidates for tunable release of macromolecules, however a stiff network structure may not be ideal for cell culture applications. Designing a non-swelling hydrogel platform with tunable strength and degradation will significantly expand the usefulness of the materials in bioengineering, for example hydrogels used for studies of cell-based interactions or as implanted soft materials. In this work, we present a facile preparation of click-crosslinked, PEG-based or gelatin-based hydrogels with suppressed swelling under physiological conditions by using linkers derived from commercial poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol)s (PEG-PPGPEG or tradename Pluronic®). The nucleophilic thiol-yne addition was selected as the


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crosslinking chemistry, as it was previously utilized in the presence of living cells in addition to being fast and efficient.10, 24 Varying the hydrogel properties including modulus and degradation profile without being compromised by swelling is demonstrated and the interaction of fibroblasts with the gelatin-based hydrogels investigated in 2D and 3D cultures.

EXPERIMENTAL General considerations: 4-arm PEG-OH (molar mass = 5000 g mol-1) was purchased from Jenkem Technology and 5,5'-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, Sigma Aldrich) was recrystallized from ethanol solution (35 %) as yellow needles. Other chemicals were purchased from Sigma-Aldrich and used as received. 1H NMR spectra were recorded on a Bruker Advance III 400 with a 5 mm broadband auto-tuneable probe with Z-gradients at 293 K. Chemical shifts are reported as δ in parts per million (ppm) and referenced to the chemical shift of the residual solvent resonances (CDCl3 1H NMR: δ = 7.26 ppm). Synthesis of pluronic-bispropiolate (Plu19-Alk):

Pluronic® L-35 (PEG-PPG-PEG, 50 wt%

PEG, molar mass = 1900 g mol-1, 19 g, 0.01 mol), propiolic acid (2.8 g, 0.04 mol) and p-toluene sulfonic acid (p-TsOH, 0.1 g, catalytic amount) were mixed with cyclohexane (100 mL) in a round bottom flask fitted with Dean-Stark condenser. The mixture was heated to refluxing (at 90 °C) for 20 h. Cyclohexane was decanted and the mixture was taken up in CH2Cl2 (50 mL), washed with saturated NaHCO3 solution (100 mL), brine (100 mL), dried (MgSO4), and concentrated in vacuo to give product as a slight yellow oil (yield 17.4 g, 87%). 1H NMR 4.324.35 (t,

3

JHH = 6.7 Hz, O=COCH2-), 3.65-3.67 (m, -OCH2CH2O-), 3.40-3.50 (m,

OCH3CHCH2O) 2.99 (s, CH≡CC(O)O-), 1.10-1.15 (m, CHCH3).


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Plu29-Alk and PEG20-Alk were synthesized using a similar procedure as Pluronic® L-64 (PEGPPG-PEG, 40 wt% PEG, molar mass = 2900 g mol-1) and linear PEG-OH (molar mass = 2000 g mol-1) to give product as slight yellow oil (yield 82%) and slight yellow solid (yield 89%) respectively. PEG20-bisacrylate was synthesized from linear PEG-OH (molar mass = 2000 g mol-1) according to previously reported procedure.25 Synthesis of 4-arm PEG50-SH: 4-arm PEG (molar mass = 5000 g mol-1, 10 g, 2 mmol), 3mercaptopropionic acid (1.7 g, 16 mmol), p-TsOH (0.1 g, catalytic amount) were mixed with cyclohexane (100 mL) in a round bottom flask fitted with Dean-Stark condenser. The mixture was heated to refluxing (at 90 °C) for 20 h. Cyclohexane was decanted and the mixture was taken up in CH2Cl2 (50 mL), washed with saturated NaHCO3 solution (100 mL), brine (100 mL), and dried (MgSO4). The solution was concentrated to ca. 10 mL and precipitated by drop-wise addition in diethyl ether (200 mL) to give product as white powder (yield 9.67 g, 95%). 1H NMR (400 MHz, CDCl3) δ, ppm: 1.64 (t, 3JHH = 7.42 Hz, -SH), 2.65 (t, 3JHH = 5.92 Hz, O=CCH2), 2.68-2.88 (m, CH2S), 2.97 (t, 3JHH = 4.78 Hz, CCH2O) 3.57-3.70 (m, CH2CH2O), 4.22 (t, 3JHH = 4.76, O=COCH2). Synthesis of gelatin-SH: Gelatin (1 g, Type B from bovine skin, gel strength ~225 g Bloom, Sigma) was dissolved in water (100 mL) and cysteamine hydrochloride (0.1 g, 0.73 mmol) was added, followed by EDC.HCl (1 g, 5.2 mmol) and N-hydroxysuccinimide (NHS, 0.6 g, 5.2 mmol). The pH was adjusted to 4.5 by HCl solution (1 M) and the solution was stirred at ambient temperature for 16 h. Dithiothreitol (DTT, 1.5 g, 9.7 mmol) was added and the pH of the solution was adjusted to 9 using NaOH solution (1 M).

The solution was then stirred at ambient

temperature for 16 h and dialyzed against HCl solution (5 mM) using dialysis tubing with a


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molecular weight cut-off of 3500 Da. Subsequently, the solution was freeze-dried to give product as off-white fluffy solid (yield: 0.74 g, ca. 74%). The thiol content was determined by treatment with Ellman’s reagent in tris(hydroxymethyl) aminomethane-HCl buffered solution (pH = 8) and the absorbance was measured at 410 nm. A calibration curve was obtained from the cysteine solution at known concentrations. Cloud point titration For determination of the cloud points of the polymer solutions, the corresponding polymer was dissolved in deionized water at a concentration of 10 mg mL-1 and filtered via syringe through a membrane with a pore size of 0.45 µm into a cuvette. The cuvette was sealed and placed in a circulated water bath with a digitally controlled thermostat at 5 °C. The temperature was subsequently increased at 0.5 °C intervals at which the sample was equilibrated for 5 min before the transmittance at 580 nm was measured using a UV-VIS spectrophotometer. The cloud point was determined by the temperature at which the transmittance was below 80%, this is also the point where the solution becomes turbid. Hydrogel preparation and swelling study Hydrogels were prepared by mixing two polymer solutions in PBS pH 7.8 solution. The molar stoichiometry of thiol to alkyne was kept at 1 unless stated otherwise. The resultant solution was vortex mixed for 5 s before transferring to a mould (typically a plastic syringe capped at one end using paraffin) and allowed to cure at ambient temperature. For the swelling study, the as-prepared hydrogels (circular shape with a volume of 100 µL and diameter of ca. 15 mm) were placed in excess deionized water or PBS pH 7.8 solution and the


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weight was recorded after a predetermined time. Samples used for rheology measurements were allowed to swell for 12-16 h after which the weight of the hydrogels remained constant. Hydrogel degradation was monitored by measuring the mass of the examined gels in PBS pH 7.8 solution. Rheological analysis Rheology studies were carried out using an Anton Paar Physica rheometer with a plate-plate configuration with the lower plate made of quartz and the upper plate made of stainless steel having a diameter of 15 mm. Frequency and strain sweeps were carried out on hydrogels to ensure rheological tests were performed in the linear viscoelastic regime. For analysis of gelation kinetics, a solution (50 µL) containing a mixture of polymer precursors was quickly placed on the lower plate and the upper plate was lowered to a measurement gap of 0.2 mm. A layer of paraffin oil was placed outside the upper plate to prevent drying of the samples and the test was started by applying a 1% strain with a frequency of 1 Hz on the sample. Protein Release FITC-conjugated albumin (Sigma Aldrich, molecular weight albumin 66 kDa) was dissolved in PBS pH 7.8 solution at a concentration of 1 mg mL-1 and the resultant solution was used to prepare gelatin containing hydrogels (volume of 100 µL) as described above. Hydrogels containing FITC-albumin were subsequently placed in 1 mL of PBS pH 7.8 and incubated at 37 °C. The media was replaced daily to maintain a sink condition and the fluorescent intensity of the solutions was analyzed (excitation/emission of 490/525 nm) using a plate reader (Clariostar) to determine the amount of FITC-albumin being released.


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Cell culture studies Cell culture was carried out using commercial human foreskin fibroblasts HFF-1 (ATCC® SCRC-1041TM). Cells were cultured on tissue culture flasks as per manufacturers’ instructions, then trypsinised with Tryple Express to detach from the culture surfaces. The cells were centrifuged for 3 mins at 0.3 g and the supernatant discarded. For cell seeding studies, 10 µL each of gelatin and Pluronic components were aliquoted to a 24 well plate and mixed in situ. The gels were rested for 30 minutes to allow for crosslinking, then rinsed 3x with cell culture media and incubated overnight 37 ⁰C and 5% CO2. The next day, the wells were rinsed twice again with cell culture media, and cells were seeded at 20,000 cells per well. Media was changed at day one and then once every 3-4 days. Procedures for the 3D encapsulation experiments and MTS assay are provided in the SI. Live dead staining and imaging At 1 and 3 days after seeding, samples were stained with live/dead solution consisting of 4 µM ethidium homodimer-1 and 2 µM calcein AM (Life Technologies) which were made up in DPBS. The media was removed from the wells, and the gels were incubated in live/dead solution (100 µL per well) for 30 mins 37 ⁰C and 5% CO2. The cells were rinsed once with PBS and imaged under either a Nikon Eclipse Ti-Li with Spot Xplorer camera or Nikon Eclipse LV 100ND with Nikon DS-Ri2 camera. Statistical analysis All measurements were undertaken in triplicate unless stated otherwise and data are reported as mean ± standard deviation. Statistical analysis was determined using a paired t-test or one-way


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ANOVA followed by Tukey’s post-hoc test using GraphPad InStat. Statistical significance was determined as p < 0.05.

RESULTS AND DISCUSSION

Preparation and Characterization of PEG-Containing Hydrogels Polymer precursors with necessary thiol and activated ester alkyne functionality for clickcrosslinking were first synthesized by simple Fischer esterification (Figure 1A). The alkynecontaining polymers were synthesized from commercially available linear Pluronic® L-35, Pluronic® L-64, and PEG-OH with a molar mass of 1900 g mol-1, 2900 g mol-1, and 2000 g mol1

respectively (henceforth the polymer products are called Plu19-Alk, Plu29-Alk and PEG20-

Alk) whilst the thiol-containing crosslinker was prepared from 4-arm PEG-OH having a molar mass of 5000 g mol-1, thus this product is called 4-arm PEG50-SH). Pure polymer product can be isolated by simply washing with NaHCO3 solution and dried in vacuo. The purification of the Pluronic products gave lower yield than the PEG products due to a smaller percentage of amphiphilic copolymers that remained as an emulsion in the aqueous phase during washing. Nevertheless, we have developed a highly efficient procedure (yield in the range of 82-95%) that can produce polymer precursors containing either thiol or propiolate end-groups in multi-gram scale using cyclohexane as the solvent and pTsOH as the catalyst. After the reaction, cyclohexane can simply be decanted, dried with MgSO4, and reused. The polymer end group modification is highly efficient with complete conversion of the hydroxyl group to the corresponding propiolate or mercaptopropionate as evident in the NMR analysis (Figure 1B, Figure S1 and S2).


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Figure 1. (A) Scheme of the synthesis of Plu19 (or 29)-Alk and 4-arm PEG50-SH; and (B) 1H NMR of the polymer precursors (400 MHz, CDCl3). We first prepared hydrogels by crosslinking 4-arm PEG50-SH with PEG20-Alk, Plu19-Alk and Plu29-Alk having equivalent moles of the reacting groups in the polymer mixture in PBS solution pH 7.8 (Gel-A, Gel-B and Gel-C in Table 1). Modification of the hydroxyl end-group on the triblock copolymers led to a reduction in lower critical solution temperature (or cloud point) from 73 °C and 58 °C (provided by supplier) to 17 °C and 15 °C for Plu19-Alk and Plu29-


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Alk respectively (Figure S1 and S2). Thus the mixtures containing amphiphilic copolymers were turbid at ambient temperature (22-24 °C), however physical gelation of the Plu19-Alk and Plu29-Alk in PBS solution was not observed even at very high concentration (25 wt%) and up to 50 °C. Crosslinking via nucleophilic thiol-yne occurred effectively at room temperature without cooling the solution containing triblock copolymers. After crosslinking at room temperature, all formed gels were transparent indicating a homogeneous network structure (Figure 2A).

Figure 2. (A) Solution containing 4-arm PEG50-SH and Plu29-Alk before and after crosslinking; and (B) Schematic presentation of the thiol-yne crosslinking reaction and network formation. The nucleophilic thiol-yne reaction only proceeds to mono-addition, with high efficiency, in a weakly basic environment such as PBS pH 7.8 (Figure 2B),24 thus equivalent molar ratios of the thiol and alkyne theoretically resulted in complete conversion of the reacting groups. Gel-A and Gel-B formed within 1 min while Gel-C formed within 5 min of mixing the solutions containing


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polymer precursors. PEG-bisacrylate was also prepared from linear PEG-OH with molar mass of 2000 g mol-1 to crosslink with 4-arm PEG50-SH, however a hydrogel did not form at a polymer concentration of 10 wt% after 24 h at ambient temperature, most likely due to the slow reaction rate of the Michael thiol-ene addition in PBS solution pH 7.8. Indeed, a previous study utilized a very high concentration of the polymer precursors (25 wt%) and short chain PEG linker (molar mass of 1100 g mol-1) to facilitate efficient crosslinking using Michael thiol-ene reaction.8

Table 1. Hydrogel terminology and corresponding polymer precursor compositionsa Hydrogel 4-arm PEG50SH [SH] (mM)

a b c

PEG20Alk [Alk] (mM)

Plu19-Alk [Alk] (mM)

Plu29-Alk [Alk] (mM)

[Polymer] (mM)b

Full curing time (s)c

Gel-A

0.041

0.041

-

-

0.041

30 ± 2

Gel-B

0.043

-

0.043

-

0.043

35 ± 3

Gel-C

0.035

-

-

0.035

0.035

246 ± 5

Gel-D

0.036

-

0.029

0.007

0.036

84 ± 3

Gel-E

0.043

-

0.004

0.039

0.043

224 ± 4

Gel-F

0.042

-

-

0.028

0.035

280 ± 8

Referred to molar ratio of the thiol to alkyne Total molar concentration of the polymer precursors, the final weight concentration was fixed at 10 wt% Determined by vial inversion method at which is the time point the polymer solution stops moving


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Figure 3. Gelation kinetics followed by evolution of storage moduli (G’, empty circle symbol) and loss moduli (G” filled circle symbol) of Gel-A (A), Gel-B (B), Gel-C (C), Gel-D (D), Gel-E (E), and Gel-F (F) as a function of time. The gelation behaviour was next examined by rheological analysis and the kinetics of gelation was followed by evolution of the storage modulus (G’) and loss modulus (G”) as a function of time (Figure 3). Gel-A and Gel-B displayed an almost identical gelation profile with a G’ value of ca. 12.5 kPa at full curing time, indicating the triblock Plu19-Alk and linear PEG20-Alk with similar molar mass (ca. 2000 g mol-1) react similarly with 4-arm PEG50-SH. The gelation point, defined as the cross-over between G’ and G”, was not observed for Gel-A and Gel-B due to the very fast crosslinking of polymers which had already occurred during the process of transferring the mixed solution to the rheometer (approximately 30 s). The gel point for Gel-C was ca. 46 s at ambient temperature and a storage modulus of ca. 2 kPa was determined at full gelation. The longer gelling time and lower storage modulus at complete gelation of Gel-C is due to Plu29-Alk


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linker having a longer chain length (theoretical molar mass of ca. 3000 g mol-1) and thus lower crosslinking density.

Figure 4. (A) Images of as-prepared and fully swollen Gel-A, Gel-B and Gel-C at 37 °C. The gels were dipped in a solution of red dye for visualization. Swelling ratios of the prepared gels at different temperatures for (B) Gel-A (black circle), Gel-B (blue triangle down), and Gel-C (green triangle up); and (C) Gel-D (cyan star), Gel-E (yellow diamond), and Gel-F (red square), the


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faint blue lines are drawn to show swelling at 37 °C. Note that at 37 °C the swelling of Gel-D, Gel-E, and Gel-F are identical and have a Q value of 1. The swelling ratios (Q) of the prepared gels in deionized water are shown in Figure 4. The PEG only hydrogel (Gel-A) absorbed water across the temperature range from 5 °C to 37 °C and the swelling ratio decreased with increasing temperature. At 50 °C, Gel-A showed expulsion of water and the swelling ratio dropped to 0.85. This decrease in water sorption of PEG hydrogels at elevated temperature is due to the dissociation of water molecules bound to the ethylene glycol monomer unit leading to the dehydration of the PEG chains (note that PEG has a LCST in the range of 100-120 °C and the hydrophobic interaction of the PEG chains does not change considerably at the investigated temperature range).26 This syneresis property of PEG hydrogels was reported to be dependent on the crosslinking density,8,

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i.e. stronger syneresis at higher

concentration of the crosslinked groups due to stronger hydrophobic interactions of the free polymer chains and crosslinking points. As expected, Pluronic® containing hydrogels, Gel-B and Gel-C, exhibited a more profound thermoresponsive syneresis with a swelling ratio of 1.1 and 0.4 respectively at 37 °C (Figure 4B). Gel-C displayed a stronger syneresis compared to Gel-B at 37 °C despite having a lower crosslinking density due to the amphiphilic copolymer having a higher percentage of hydrophobic component (Plu19-Alk contains 40% PPG by weight while Plu29-Alk contains 50% PPG by weight). All hydrogels had maintained their transparency after water expulsion at 50 °C, which indicates that the network homogeneity was retained. This is different from physically-crosslinked thermoresponsive hydrogels such as poly(Nisopropylacrylamide) containing hydrogels which become opaque at temperatures above the LCST of the polymer due to phase separation.27, 28


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From the observation that PEG20-Alk and Plu19-Alk have similar reactivity, at similar gel content toward 4-arm PEG50-SH, and PEG-containing gel swells while Plu29-containing gel contracts at 37 °C, we speculated that the hydrogel physical properties could be tuned while retaining the non-swelling behavior of the hydrogels at physiological temperature at which the materials are expected to be utilized. Thus we prepared three types of gels containing various concentrations of the Plu29-Alk (Gel-D, Gel-E and Gel-F in Table 1) and adjusted the concentration of PEG20-Alk or Plu19-Alk to counter the triblock copolymer contraction of Plu29-Alk in water due to the hydrophobic effect so that the swelling of the materials would reach unity at 37 °C. The polymer mixtures showed different gelation profiles and G’ values at full gelation, indicating different crosslinking densities of the resultant materials (Figure 3D-F). All three mixed hydrogels showed near zero water absorption at 37 °C and different swelling ratios at 5 °C and 50 °C due to the gels having different concentrations of the Plu29-Alk (Figure 4C). Specifically, Gel-E and Gel-F which have a higher concentration of Plu29-Alk, showed stronger water expulsion at 50 °C with Q values of 0.45 and 0.43 respectively, whilst Gel-D had a swelling ratio of 0.48. This observed non-swelling property at physiological temperature could not be achieved even in the hydrogel platform having a dense network structure, for example the lowest Q for thiol-acrylate [2+3] crosslinked hydrogel was reported to be c.a. 2.1.8 Further, we repeated the swelling measurements of all samples in PBS solution pH 7.8 and obtained similar swelling ratios as measured in deionized water at different temperatures, which suggests that the ionic and slight basic conditions do not have any significant effect on the water uptake of PEG and PEG copolymerized with PPG hydrogels.


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Figure 5. Storage moduli of the as-prepared gels (black bars) at ambient temperature and gels in the fully swollen state at 37 °C (red bars) (**p < 0.01, ***p < 0.001 relative to the modulus before swelling). The mechanical properties of the gels in the original state and fully swollen state at 37 °C were evaluated by measuring the storage modulus (Figure 5). We found the G’ values of the hydrogels were highly dependent on both the crosslinking density and swelling ratio. In particular, a reduction in storage modulus, from 12.8 ± 0.5 kPa to 4.1 ± 0.6 kPa, for PEG-only hydrogel (Gel-A) was recorded when the gels were fully swollen in water at 37 °C. For Gel-B, the storage modulus dropped from 12.4 ± 0.4 kPa to 8.2 ± 0.5 kPa. This reduction is a result of a slight increase in swelling ratio of the material (Q = 1.1). In contrast, the storage modulus of GelC increased from 2.0 ± 0.3 kPa in the original state to 4.5 ± 0.6 kPa in the fully swollen state which is likely a result of contraction of the hydrophobic segment when the hydrogel de-swelled. As expected, the storage moduli of Gel-D, Gel-E, and Gel-F varied significantly from 7.8 kPa to 1.2 kPa but these values remained constant for gels in the fully swollen state at 37 °C. A possible contribution to the changes in moduli may originate from hydrolysis of the ester groups during the swelling measurement which occurred over a 12-16 hr period. Regardless of this, these


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results demonstrate that non-swelling hydrogels at physiological temperature with tunable crosslinking concentration and mechanical strength could be fabricated by judicious combination of the readily available PEG and triblock PEG-PPG-PEG precursors.

Figure 6. Degradation profiles followed by measuring hydrogel swelling ratios as a function of time for Gel-A (black circle), Gel-B (blue triangle down), Gel-C (green triangle up), Gel-D (cyan star), Gel-E (yellow diamond), and Gel-F (red square). Hydrogel degradation can affect network mesh size and water uptake capacity, consequently affecting materials performance over time. Degradation of synthetic hydrogels is mostly facilitated by hydrolysis of the ester functionality.7, 8, 10, 20, 21, 27, 29, 30 Thus the stability of our hydrogels in PBS solution pH 7.8 at 37 °C was investigated by monitoring the swelling ratio over time until complete disintegration (Figure 6). The degradation of the gels are highly influenced by the crosslinking density and hydrophobicity content of the network structure. Specifically, Gel-A and Gel-B completely degraded in less than 1 week whilst Gel-C, with lower crosslinking density but higher content of the hydrophobic PPG, was stable in PBS solution for more than 2 weeks. The non-swelling gels in PBS at 37 °C also displayed different degradation


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profiles due to the difference in crosslinking density and hydrophobic content. It is worth noting that Gel-C and Gel-F both showed non-swelling behaviour before degradation onset and lower water uptake than other gels before complete degradation, likely due to the hydrophobic moiety being preserved within the structure after partial hydrolysis of the ester linkages. The observed degradation behavior for our hydrogels is similar to the ester-containing hydrogels previously reported.7, 8, 20, 21, 29, 31

Preparation and Characterization of Low Swelling Gelatin-Containing Hydrogels


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Figure 7. (A) Synthesis of gelatin-SH and subsequent crosslinking; Gelation kinetics followed by evolution of storage moduli (G’, empty circle symbol) and loss moduli (G” filled circle symbol) of Gel-G1 (B), Gel-G2 (C), Gel-G3 (D), and Gel-G4 (E). Table 2. Composition of gelatin-based hydrogels Hydrogel

Gelatin-SH

PEG20-Alk

Plu19-Alk

Plu29-Alk

Polymer

[SH]a (mM)

[Alkyne]b (mM)

[Alkyne]b (mM)

[Alkyne]b (mM)

Concentration (wt%)

Gel-G1

0.017

0.017

-

-

6.3

Gel-G2

0.017

-

0.017

-

6.3

Gel-G3

0.017

-

-

0.017

7.4

Gel-G4

0.017

0.01

-

0.07

6.8

Because PEG-based hydrogels do not support cell adhesion and growth in cell culture experiments, we sought to apply the Pluronic® crosslinker in the preparation of gelatin-based hydrogels due to gelatin having inherent binding sequences within the polymer structure for cell attachment.29,

31-36

Thus gelatin was first modified by EDC/NHS coupling with cysteamine

hydrochloride followed by DTT reduction to confer the thiol group to the gelatin structure (Figure 7A). The thiol content was determined, via Ellman’s titration, to be 0.57 mmol g-1, and this was lower than the thiol content of 4-arm PEG50-SH (0.8 mmol g-1). Although gelatin-SH in PBS solution at basic pH was reported to crosslink by disulfide formation upon exposure to air,37 gel formation was not observed for this material at a concentration of 4 wt% in PBS pH 7.8 solution at ambient temperature for more than 2 h. At concentrations higher than 4 wt%, the gelatin-SH solution solidified at room temperature due to physical crosslinking which could be reversed by warming the mixture to 37 °C. Thus for fabrication of hydrogel via thiol-yne crosslinking, we selected the gelatin-SH concentration to be 4 wt% and equivalent molar ratio of


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thiol to alkyne (Table 2). Crosslinking of gelatin-SH with PEG20-Alk or Plu19(29)-Alk resulted in gelation in 3-10 min with a gelation time 60-80 s (Figure 1B-E). A slower gelation time and lower storage moduli at complete gelation were observed for gelatin based hydrogels compared to PEG based hydrogel. This is due to the lower crosslinking density of the gelatin based hydrogel and the less well-defined nature of the gelatin polymer chain.

Figure 8. Swelling ratios of the gelatin based hydrogels at different temperatures for Gel-G1 (black circle), Gel-G2 (blue triangle down), Gel-G3 (green triangle up) and Gel-G4 (cyan square) in deionized water (A) and in PBS solution pH 7.8 (B).


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The swelling of Gel-G1, Gel-G2, and Gel-G3 followed a similar trend to Gel-A, Gel-B and Gel-C in which the swelling ratio decreases with increasing temperature. It is interesting to note that the thermoresponsive swelling of gelatin hydrogels is not profound as observed in the PEG based hydrogels. In particular, both Plu-Alk containing hydrogels displayed swelling ratios well above 1 at 37 °C (Q = 1.8 for Gel-G2 and 1.6 for Gel-G3) and 50 °C (Q = 1.5 for Gel-G2 and 1.4 for Gel-G3). This is likely because gelatin has an upper critical solution temperature (UCST) of 14 to 22 °C depending on the concentration of the gelatin solution38-40 and the gelatin chain becomes more hydrophilic at higher temperature thus countering the LCST effect of PEG and PEG-PPG-PEG polymers. A higher swelling ratio of the gelatin-based hydrogels compared to PEG-based hydrogels is due to the lower crosslinking density of gelatin-based gels. In contrast to PEG based hydrogels, the swelling of gelatin hydrogels were strongly affected by the ionic condition. In particular, we observed a decrease in the Q values of Gel-G1, Gel-G2, and Gel-G3 in PBS solution (Figure 8B), however a pH range from 7 to 8 has minimal effect on the swelling ratio. We believe the decrease in swelling is due to the salting out effect in which the peptide chains of the gelatin become more hydrophobic due to the increase in the ionic strength of the solution. This observation is consistent with previous reports in which increasing ionic concentration of the solution resulted in a switch of the gelatin chains from electrostatic domain to hydrophobic domain.38, 41 Thus the swelling ratio of Gel-G2 and Gel-G3 in Dulbecco PBS solution used for cell culture was more strongly affected by the thermoresponsive properties of the triblock copolymers and a larger decrease in swelling ratio at 37 °C and 50 °C was observed. We thus prepared a hydrogel using PEG20-Alk and Plu29-Alk linkers to produce a non-swelling gel in PBS solution at physiological temperature (Gel-G4, Table 2). As expected,


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Gel-G4 showed an increase in swelling ratio in the temperature range of 4-50 °C in deionized water, however displayed a Q value of 1 at 37 °C in PBS solution (Figure 8B).

Figure 9. Storage moduli of the as-prepared gelatin based gels (black bar) at ambient temperature using PBS pH 7.8 as the solvent and gels in the fully swollen state in water (red bar) and PBS solution pH 7.8 (green bar) at 37 °C (**p < 0.01, ***p < 0.001 relative to the modulus before swelling). Similar to PEG-based hydrogels, gelatin-based hydrogels displayed a change in storage moduli in response to the change in swelling ratio at different temperatures. All gelatin based gels displayed a decrease in the storage modulus in their fully swollen stage in water at 37 °C (Figure 9). In PBS solution Gel-G1 and Gel-G2 showed a decrease in the modulus whilst the modulus of Gel-G3 increased due to the shrinking of the materials. As expected, Gel-G4 showed a negligible change in storage modulus after being treated with PBS solution at 37 °C. Also similar to PEGonly hydrogels, the stability of gelatin-PEG hydrogels is highly enhanced by the incorporation of the hydrophobic Pluronic linker as compared to PEG linker (Figure 10). The gelatin hydrogels are slightly more stable than the PEG-based hydrogel despite having a lower concentration of the


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crosslinking groups. This is because the thiol group was attached to gelatin chain via amide conjugation (Figure 7) instead of ester conjugation, thus the gelatin based hydrogels have lower percentage of the hydrolysable ester groups compared to the PEG-only hydrogels.

3

Q

2

1

0 0

5

10

15

20

Time (day)

Figure 10. Degradation profiles in PBS pH 7.8 at 37 °C followed by measuring hydrogels swelling ratio as a function of time for Gel-G1 (black circle), Gel-G2 (blue triangle down), GelG3 (green triangle up) and Gel-G4 (cyan square).

120

Percentage Release

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100 80 60 40 20 0

0

2

4

6

8

10

12

14

16

18

Time (day)


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Figure 11. Cumulative release profiles of FITC-conjugated albumin from Gel-G1 (black circle), Gel-G2 (blue triangle down), Gel-G3 (green triangle up) and Gel-G4 (cyan square) in PBS pH 7.8 and at 37 °C. To evaluate the utility of the gelatin-PEG hydrogels in a controlled delivery application, we investigated the release of FITC-conjugated albumin (molar mass of 66 kDa) encapsulated in the materials during the gel formation process. As seen in Figure 11, the release profiles of FITCalbumin are highly dependent on the swelling and the degradation properties of the hydrogels. Rapid release of FITC-albumin was observed from Gel-G1 and Gel-G2 which have high swelling properties in the investigated media (Q value of 1.5 and 1.2 respectively). In contrast the release of FITC-albumin in non-swelling gel, Gel-G3 and Gel-G4, is negligible until the degradation onset (day 10 and day 11) at which a burst release of the protein was observed. The complete release of the FITC-albumin from hydrogels in PBS media was seen to vary from 3 to 16 days, demonstrating that controlled delivery profiles can be achieved by tailoring the ratio of hydrophilic PEG to amphiphilic PEG-PPG-PEG linkers. The low- to non- swelling properties of the Pluronic containing gelatin hydrogels make them valuable for applications where biomaterials are inserted in a confined space and surrounded by delicate tissue.


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Figure 12. Fluorescence images of human foreskin fibroblasts with live-dead staining on gelatin based hydrogels after 3 days, scale bar = 50 µm. The gelatin-based hydrogels were further evaluated for suitability in biological systems. Fibroblasts seeded on top of the hydrogels, which had previously been washed, showed high cell viability after 3 days culture. We observed fibroblast seeding and spreading on all gelatin-based hydrogels (Figure 12) in addition to displaying a high metabolic activity (Figure S6) confirming that after crosslinking and washing, the hydrogel materials are non-toxic, capable for supporting cell growth can be used for further cells studies. 3D encapsulated experiments were also performed. Gel-G1 which consists of PEG and gelatin showed high cell viability with over 95% live cells, indicating that the click crosslinking reaction is highly bio-orthogonal (Figure S8). However, a high percentage of cell death was observed in


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Gel-G2, Gel-G3 and Gel-G4, which suggest that unreacted Pluronic linkers are toxic to cells. Indeed, the MTS assay which measures metabolic activity, showed high toxicity to fibroblasts when exposed to the Pluronic precursors, before and after esterification with propiolic acid, even at very low concentrations (Figure S6 and S7). The reason for this toxicity is unknown and we are screening different Pluronic polymers having different molar mass and composition for polymer with lower toxicity to cells.

CONCLUSION In conclusion, we applied commercial triblock amphiphilic PEG-PPG-PEG polymers as the polymer precursor for the preparation of nucleophilic thiol-yne click-crosslinked hydrogels with suppressed swelling in water and at elevated temperature. We demonstrated that the mechanical properties of the hydrogels could be tuned while maintaining non-swelling properties under physiological conditions. Additionally, the non-swelling behaviour can be maintained during much of the degradation of the hydrogel. Further, the Pluronic® linkers can be used to prepare low- to non-swelling gelatin hydrogels under physiological conditions to be used in biomedical applications such as controlled delivery of therapeutic proteins. Although the toxicity of the Pluronic@ polymers used in this study rendered the system not suitable for 3D cell encapsulation, the hydrogels themselves are non-toxic and can be used controlled release of therapeutics such as protein for 2D cell culture platforms to study cell-materials interactions at different mechanical strength or implanted soft biomaterials. Improvement of the hydrogel systems for 3D cell study can be used by selecting amphiphilic copolymers, such as poly(lactic acid)-PEG-poly(lactic acid) or other Pluronic linkers with different molar mass and weight percentage of PPG, which may have lower toxicity to cells. Screening of such linkers and their use in 3D cell culture are currently under investigation.


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ASSOCIATED CONTENT Supporting Information. NMR data of Plu29-Alk, PEG20-Alk, and gelatin-SH, cloud point titration data, MTS assay data and cell encapsulation results. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] Author Contributions VXT, JSF developed the concept, designed the experiment and wrote the manuscript. VXT, KMT carried out the experiments and analyzed the data. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENTS The authors wish to thank Prof George Simon and Prof Richard Boyd for useful discussions and advice. This research was supported under the Australian Research Council's Linkage Projects funding scheme (LP130101142), Cell Medicine Australia and Shunxi Investment Group Company International Pty. Ltd.

ABBREVIATIONS REFERENCES


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Nonswelling Click-Cross-Linked Gelatin and PEG Hydrogels with

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