Letter pubs.acs.org/macroletters
Efficient In Situ Nucleophilic Thiol-yne Click Chemistry for the Synthesis of Strong Hydrogel Materials with Tunable Properties Laura J. Macdougall, Vinh X. Truong,† and Andrew P. Dove* Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, United Kingdom S Supporting Information *
ABSTRACT: Synthetic hydrogel materials offer the ability to tune the mechanical properties of the resultant networks by controlling the molecular structure of the polymer precursors. Herein, we demonstrate that the nucleophilic thiol-yne click reaction presents a highly efficient chemistry for forming robust high water content (ca. 90%) hydrogel materials with tunable stiffness and mechanical properties. Remarkably, optimization of the molecular weight and geometry of the poly(ethylene glycol) (PEG) precursors allows access to materials with compressive strength up to 2.4 MPa, which can be repeatedly compressed to >90% stress. Beyond this, we demonstrate the ability to access hydrogels with storage moduli ranging from 0.2 to 7 kPa. Moreover, we also demonstrate that by a simple precursor blending process, we can access intermediate stiffness across this range with minimal changes to the hydrogel structure. These characteristics present the nucleophilic thiol-yne addition as an excellent method for the preparation of hydrogels for use as versatile synthetic biomaterials.
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network in a double network hydrogel formed by two orthogonal click reactions.48 We postulated that the high efficiency and rapid nature of the nucleophilic thiol-yne addition reaction was largely responsible for the excellent mechanical properties of the double network, including the repeatable compression and hence further investigation of this approach would enable the synthesis of robust hydrogel materials with excellent mechanical properties in a simple, bioorthogonal manner. Furthermore, we demonstrate that the mechanical properties of the hydrogels can be readily tuned across a wide range of stiffness and pore size/structure by a simple blending procedure that could potentially lead to a new method for the preparation of versatile, injectable synthetic biomaterials. Alkyne and thiol-terminated poly(ethylene glycol), PEG, precursors with 2, 3, and 4 arms were prepared by simple Fischer esterification methodologies (Scheme 1). In situ forming PEG hydrogels were subsequently prepared by mixing solutions containing a 1:1 molar ratio of alkyne to thiol polymer precursors in phosphate buffered saline (PBS) solution at pH 7.4. The polymer content was kept at 10 wt % and injected into suitable molds to create materials for testing. The nomenclature for each hydrogel was determined by the structure and molecular weight of the PEG alkyne and thiol precursor. Each hydrogel is principally named relating to the number of arms on the alkyne (A) and thiol (S) precursor. The subscript denotes the molecular weight of the polymer and functional group, for example, PEG 42A24S is a hydrogel formed
ailoring the characteristics of a material to meet the needs of a biological environment is a key goal in biomaterials science.1−3 Hydrogels, with their high water content and synthetic flexibility are promising candidates to fulfill this goal.4−7 Over the past decade, research into hydrogels has expanded and their true potential as synthetic biological materials has been revealed,8−10 with applications ranging from use as scaffolds for tissue engineering11−13 to carriers for drug delivery.14,15 Targeting predefined and specific hydrogel properties is an essential goal to respond to the demands of a particular biological environment or to elicit a desired cellular response,16−21 and materials in which these properties can be readily tuned, are essential to the advancement of this area. With the introduction of metal free bioorthogonal click reactions to hydrogel synthesis,10,22−24 a range of reactions including the strain- or electron-withdrawing, group-promoted azide−alkyne [3 + 2] cycloaddition (SPAAC),25−29 inverse electron-demand Diels−Alder,30−32 oxime32−36 and thiol−ene coupling reactions23,37−39 have been implemented to form hydrogels under physiological conditions. The biorthogonality of these click cross-linking methods, in addition to the easily accessible functional groups, lend themselves to be very suitable for biomaterial applications.40 Recently, the nucleophilic thiol-yne click reaction has been demonstrated as an attractive reaction for the synthesis of biomaterials.41,42 Like the thiol-ene reaction, the thiol-yne mechanism can be carried out via both radical and nucelophilic pathways. The more popular radical pathway has been adopted to synthesize a range of materials from linear43 and multifunctional brush polymers 44 to block copolymers. 45 As a consequence of its high speed and efficiency, the nucleophilic pathway has begun to attract attention for the synthesis of elastomer46 and hydrogel materials,47 including as the dense © XXXX American Chemical Society
Received: November 11, 2016 Accepted: January 11, 2017
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Scheme 1. (a) Nucleophilic Base-Catalyzed Reaction between an Alkyne and Thiol; (b) Schematic of the PEG Precursors Synthesized for Crosslinking; and (c) Schematic of Exemplar Hydrogel Networks
Table 1. Gelation Times and Swelling Kinetics of PEG Hydrogels Made at 10 wt % in PBS hydrogela PEG PEG PEG PEG PEG PEG
31A21S 31A22S 31A24S 42A21S 42A22S 42A24S
molar concentration of end group (mM)
gelation timeb (s)
2.69 1.38 0.51 1.75 0.95 0.40
210 ± 7 290 ± 10 600 ± 12 87 ± 5 125 ± 6 238 ± 10
gel fraction (GF; %) 73.9 80.2 71.8 94.8 98.8 99.3
± ± ± ± ± ±
2.6 1.9 0.2 0.6 0.7 0.7
equilibrium water content (EWC; %) 95.2 94.2 95.1 91.9 92.4 94.2
± ± ± ± ± ±
0.9 0.8 2.4 0.5 0.2 0.1
mesh sizec (§; nm) 5.80 6.36 9.74 5.55 6.38 8.53
± ± ± ± ± ±
0.49 0.16 0.93 0.17 0.07 0.06
Young’s modulus (kPa) 27 35 37 66 72 46
± ± ± ± ± ±
5 8 9 25 20 6
a
The hydrogel naming convention used (PEG XzAYzS) denotes the hydrogel structures, where X = number of arms on the alkyne precursor, Y = number of arms on the thiol precursor, and Z = the molecular weight of the PEG precursor, e.g., PEG 42A24S is a hydrogel formed from a 4-arm alkyne at 2 kg mol−1 cross-linked with a 2-arm thiol at 4 kg mol−1. Hydrogels prepared in a 1:1 molar ratio of alkyne to thiol end groups. bMeasured via the vial tilt method. cCalculated from the Flory−Rehner equation.49,50
from a 4-arm alkyne at 2 kg mol−1 cross-linked with a 2-arm thiol at 4 kg mol−1 (Scheme 1). Several combinations of alkyne and thiol-terminated PEG were investigated for their hydrogel formation and resultant properties. The single addition to form the thioalkene product, in line with that expected from previous literature,41,46 was demonstrated by the addition of excess 2-mercaptoethanol to PEG21A in deuterated PBS (Figure S7). As determined by the vial tilt method, all of the hydrogels formed within 10 min after mixing (Table 1). The gelation time was observed to increase with higher molecular weight precursors which is most likely a result of the decreased end-group concentration in the final prenetwork (Table 1). The kinetics of the gelation process were further examined by monitoring the evolution of storage (G′) and loss (G″) moduli of the mixture with time (Figure 1). The gelation times were defined by the crossover point between G′ and G″ and examination of the plateau of the G′ and G″ with respect to time reveals that the hydrogels become viscoelastically stable within 30 min (Figures 1 and S8 in Supporting Information). To confirm that the thiol-yne hydrogels remain viscoelastically stable and are fully cross-linked when formed, rheological frequency sweep tests were conducted. The results showed no change in G′ and G″ over 0 to 100 rad/s, confirming the hydrogels were fully cross-linked and stable (see Figures S9 and S10 in Supporting Information). The gel fraction (GF) for each hydrogel was measured to evaluate the efficiency of the cross-linking process. The GF was found to be high for all the synthesized hydrogels (>72%) which suggests that the nucleophilic thiol-yne chemistry is a highly efficient polymer cross-linking reaction in aqueous conditions, highlighting its suitability for hydrogel synthesis. The GF was higher for the hydrogels comprised of a 4-arm alkyne precursor compared to those that were primarily
Figure 1. Evolution of storage (G′) and loss (G″) moduli as a function of time for the nucleophilic thiol-yne PEG hydrogels.
composed from a 3-arm alkyne precursor (99% and 80%, respectively, Table 1). This reveals that a more efficient thiolyne click reaction takes place with the 4-arm alkyne precursor. This could be a consequence of the greater number of functional groups per molecule which leads to an increased probability of cross-linking, thus lowering the amount of unreacted precursors in the hydrogel and increasing the GF. However, direct comparisons cannot be made because of the difference in molecular weight between the two alkyne precursors, 3-arm alkyne at 1 kg mol−1 compared to 4-arm alkyne at 2 kg mol−1. 94
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Figure 2. Cryo-SEM images of the PEG thiol-yne click hydrogels (scale bar = 2 μm).
In order to identify the water permeability and surface properties of the materials, the hydrogels were further characterized by measurement of the equilibrium water content (EWC). High EWC percentages (>92%, Table 1) confirmed that all of the hydrogels contained porous structures with the ability to hold large amounts of water. The average mesh size of the thiol-yne click hydrogels was calculated using the Flory− Rehner equation49,50 and ranged from 5.6 to 9.7 nm. The results followed the same trend demonstrated by the gelation time and EWC data. The longer, more flexible 2-arm PEG thiol precursors were capable of creating a large pore structure in the hydrogels owing to the increased chain length between crosslinks. Cryogenic scanning electron microscopy (cryo-SEM) was undertaken to further study the structure of the prepared hydrogels. (Figure 2). As a consequence of the limitations of cryo-SEM it cannot be conclusively ascertained that there is a trend between the geometry and molecular weight of the functionalized PEG precursors and the resultant hydrogel however the images suggest that the hydrogels have a heterogeneous and porous structure. Many studies have focused on the design of hydrogels for biomedical applications, such as tissue engineering scaffolds or to influence cell behavior in which the ability to control the mechanical properties (e.g., Young’s modulus, ultimate compressive strength, and G′) to suit an application or differentiate a cell culture are essential. G′ and G″ were measured at different amounts of strain using a rheological amplitude sweep. As the strain on the hydrogel is increased, the hydrogel’s internal structure may break. This is observed as a decrease in G′ as the stored energy from the material is released, which indicates that the cross-linking in the network has broken. Amplitude sweep tests were carried out on the thiol-yne click hydrogels at a constant frequency of 10 rad/s with G′ and G″ (Figures 3 and S11) measured as the strain was ramped logarithmically from 0.1 to 100%. It was observed that none of the hydrogels displayed a significant change in storage modulus even after application of 100% strain. These results demonstrate that these materials can withstand maximum strain without releasing any stored energy thus resulting in a flexible hydrogel that is capable of withstanding large amounts of strain. Most notably, the moderation in length of the 2-arm PEG thiol precursors leads to a remarkable range of accessible G′
Figure 3. Amplitude sweep charts the PEG 31A2S hydrogels synthesized from different molar ratios of blended of 1 and 4 kg mol−1 PEG dithiol (ratio stated as 1:4 kg mol−1 precursor).
values, almost 3 orders of magnitude from about 180 to 7000 Pa. While access to a wider range of precursors would no doubt enable selection of specific gel stiffness, we postulated that a simple blending approach may yield hydrogels with intermediate stiffness thus allowing access to hydrogel materials with highly tunable stiffness from the same chemistry. To investigate this concept, we blended a 25:75, 50:50, and 75:25 molar ratio of the 2-armed PEG thiol precursors with Mn = 1 and 4 kg mol−1 (21S and 24S, respectively) and formed hydrogels with the 3-armed PEG alkyne precursor, 31A. Measurement of G′ revealed intermediate values of G′ (G′ = 250, 1000, and 2000 Pa respectively) between the observed G′ for PEG 31A21S and PEG 31A24S materials (Figure 3). This concept demonstrated the highly tunable nature of the materials’ modulus over this very large range in a predictable manner (Figure S12 in Supporting Information) thus providing materials with a range of potential applications. To further evaluate the mechanical strength of the thiol-yne click hydrogels, uniaxial compressive tests were undertaken to determine the ultimate compressive stress and Young’s modulus of each hydrogel. The hydrogels were synthesized in 95
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Cyclic compression video of PEG 31A215 at 298 K with a preload force of 0.1 N. Each cycle compressed the hydrogel to 5% of the original height (AVI). Cyclic compression video of PEG 31A225 at 298 K with a preload force of 0.1 N. Each cycle compressed the hydrogel to 5% of the original height (AVI). Cyclic compression video of PEG 31A245 at 298 K with a preload force of 0.1 N. Each cycle compressed the hydrogel to 5% of the original height (AVI).
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Andrew P. Dove: 0000-0001-8208-9309 Present Address
Figure 4. Stress/strain data for the nucleophilic thiol-yne PEG hydrogels.
†
Department of Materials Science and Engineering, Monash University, Clayton, Victoria 3800, Australia.
molds then left to cure for 1 h before a known force was applied to a hydrogel of a known area and the resultant stress and strain were measured (Figure 4 and Table 1). Similar to the storage and loss modulus, the thiol-yne click hydrogels were found to have a wide range of strengths (0.6−2.4 MPa). Interestingly, with lower molecular weight PEG precursors the PEG 42A21S and PEG 41A22S hydrogels ruptured at about 80% strain, whereas the PEG 31A21S, PEG 31A22S did not. With the 4 kg mol−1 PEG dithiol precursor, neither the PEG 31A24S and PEG 42A24S hydrogels ruptured (up to 95% strain). Among the gels that did not rupture, a clear trend could be observed with increasing maximum compressive strength as the molecular weight of the precursors increased such that the PEG 42A24S displayed the highest compressive strain (2.4 MPa). Further mechanical testing revealed that, similarly to the double network hydrogels, in which these systems are the dense single network,48 the 3A2S and 42A24S hydrogels could be compressed repeatedly to 90% strain, with the 3A2S systems able to withstand 95% strain, up to 10 times without breaking (see Movies S1, S2, and S3 in Supporting Information). The ability for this 3A2S system to tolerate higher strain could be a result of the geometry and molecular weight of the 3-arm PEG alkyne. With fewer, shorter chain ends, the 3-arm PEG precursor is less likely than the 4A precursor to form loops or leave unreacted chain ends, which are not taken into account in the GF measurement. As such, we postulate that the 3A2S system forms a more perfect network,51−53 in which less elastic defects (e.g., loops or catenanes) are present. In summary, we have demonstrated that the nucleophilic thiol-yne click reaction can be used to prepare robust hydrogels. Most importantly, with suitable molecular geometry, these hydrogels display high mechanical strength with repeatable compression without rupture. Furthermore, by judicious choice of the PEG precursor the modulus of the materials can be tuned over almost 3 orders of magnitude. Consequently, these versatile hydrogels can be tailored to the needs of specific biological applications.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS EPSRC and BBSRC are thanked for a DTP studentship to L.J.M. and funding to support V.X.T., respectively. The Royal Society is acknowledged for an Industry Fellowship. L.J.M. thanks Dr. Maria C. Arno and Rebecca J. Williams (University of Warwick) for their assistance and scientific discussion. Mr. Steve York (University of Warwick) is thanked for assistance with cryo SEM measurements.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00857. Experimental and characterization details (PDF). 96
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