We present a novel series of hydrolytically-degradable PEG hydrogels. We have characterized the hydrogels’ degradability, mechanical properties and potential utility as 3D cell scaffolds. Because the ultimate application of these hydrogels relies on predictable rates of cross-linking that occurs under conditions compatible for cell and protein encapsulation, we explored these concepts in depth to yield a scaffolding system with tunable physical properties and known rates of degradation. Below, we first discuss aspects critical to cross-linker and hydrogel synthesis. Then, characterization of the PEG hydrogel series is discussed in relation to variables of the hydrogel cross-linking reaction.
Though the cross-linker and hydrogel synthesis procedures are relatively straightforward, care must be taken to ensure hydrogels with reproducible properties. All synthesized materials were sensitive to humidity and oxygen and thus were stored under inert gas at −20°C at all times. The aliquot of PEG-VS in pH 8 TEA was found to be stable for over a year without reducing its reactivity. However, aliquots of PEG-diester-dithiol or non-degradable PEG-dithiol (also in TEA, pH 8) were found to have very limited stability (~min) and thus were used immediately upon preparation. This instability is due to the chemical structure of the PEG-diester-dithiols and the non-degradable PEG-dithiol. In basic solution, the ester bond rapidly hydrolyzes and the free thiols form disulfide bonds due to thiol deprotonation and conversion to a more reactive thiolate group.
In order to achieve tunable hydrogel properties, we designed cross-linkers to impart three mechanisms for manipulating hydrogel degradation time. First, to test the hypothesis that degradation time will be inversely proportional to cross-linker molecular weight, we designed cross-linkers based on PEG of three different molecular weights, 3.4, 6 and 8 kDa. Second, our cross-linkers were designed to contain hydrolytically degradable ester bonds and it is well-known that the environment local to the ester impacts the rate of hydrolysis. Thus, our second strategy was to vary the number of methylene moieties between the ester and the thiol group of the cross-linker, hypothesizing that this approach will provide greater control over hydrogel degradation rate. And lastly, we speculated that hydrogel degradation time will be proportional to polymer density and therefore tested total polymer densities of 5%, 10%, and 15% w/v. With these strategies, we have achieved hydrogel degradation times that span from several hours to several days. As shown by our experiments, the presented materials demonstrated degradation times comparable to other available PEG hydrogels. For instance, PEG-diacrylate hydrogels also formed by Michael-type addition via cross-linking with DTT had shown degradation times of up to 21 d39
and PEG-PLA of increasing number of lactoyl repeat units had shown degradation times of 4 to 17 d respectively.12
We were aware that these cross-linker and hydrogel parameters will also affect certain physical properties of the hydrogel including but not limited to elasticity (G
′), swelling ratio and mesh size. Hence, we monitored hydrogel degradation by measuring these parameters at predetermined time intervals, which allowed further insight into the interplay between initial hydrogel structure, degradation kinetics, and hydrogel physical properties known to impact cell function and protein diffusivity. For example, hydrogel stiffness influences cell proliferation, motility and morphology.40
Thus, hydrogel G
′ was an important parameter for characterization and also was used to indirectly measure the extent of hydrogel degradation over time. The values of initial G
′ for all cases described below was in the range of 1000 – 3000 Pa, which correlated with values reported in the literature for similar systems. For example, 10% w/v hydrogels made with 20 kDa 4-arm PEG-VS (as opposed to 10 kDa for our system) and MMP-2 sensitive peptides exhibited G
′ of 290 Pa30, 36
while 40% w/v hydrogels made of 14.8 kDa 4-arm PEG-acrylate and 3.4 kDa PEG-dithiol (same molecular weight as some of our cross-linkers) exhibited a G
′ of 10 000 Pa.3
As shown by our experiments, the G′ decreased for all hydrogel types as the degradation proceeded. This was an expected behavior since G′ is directly related to cross-link density. (Degradation of the hydrogel leads to a lower cross-link density, resulting in a lower G′). Based on the non-linear dependence of G′ on degradation time, we could speculate that the hydrogels undergo bulk degradation due to their high water content. From our swelling data, we calculated that all tested PEG hydrogels were highly hydrophilic and contained ~96% of water upon complete swelling. When exposed to water the PEG chains are subjected to random scission at the ester bonds and each ester bond has the same probability of being broken via hydrolysis. As the hydrogel degrades, the water content increases, which further promotes the rate of hydrolysis. Thus for equivalent chemical structures, the rate of hydrolysis should depend on the water content of the swollen network and number of hydrolyzable groups.
We found that cross-linker molecular weight was directly correlated with hydrogel degradation rate and hence inversely proportional to the rate of change in G
′ as well as initial G
′ (). These trends, also observed by others,41
could be explained by the lower cross-linking density resulting from the higher molecular weight cross-linkers since all of the tested hydrogels were 10% w/v in total polymer density and a stoichiometric ratio of VS to SH groups. In addition, each cross-linker would have one ester at each end separated by a long PEG chain. Therefore, a lower cross-link density would correspond to a lower total concentration of ester bonds. The calculated theoretical concentration of ester groups for PEG-SH 2 3.4, PEG-SH 2 6 and PEG-SH 2 8 was 23.5 mM, 18.3 mM, and 15.5 mM respectively. Because fewer ester bonds are present in hydrogels synthesized with higher molecular weight cross-linkers, these hydrogels degrade faster and have greater rates of change in G
′. Additionally, this trend could also be explained by the fact that in order for a cross-linker to be completely released from the hydrogel structure, two ester bonds need to be hydrolyzed. For the 4-arm PEG polymer to be released, four ester bonds need to be hydrolyzed. Therefore, a change in G
′ may only be measurable when sufficient ester bond hydrolysis had occurred and the rate of change in G
′ would increase further as sufficient PEG diffusion had occurred.
We also found that degradation and hence G
′ was strongly affected by the number of methylene units between the ester and the thiol moieties of the cross-linker (). The interplay of several factors can explain this phenomenon. It is well known that the hydrophobicity of the ester environment affects the rate of hydrolysis. By increasing the number of methylene groups, we effectively increased the hydrophobicity of the group adjacent to the ester making it less accessible to water. In addition, due to inductive effects, the carbonyl group of the glycolate (one methylene group) is more acidic (pKa, 7.68)42
than that of the propionate (two methylene groups; pKa, 10.48)43
, rendering the glycolate more susceptible to nucleophilic attack and resulting in more rapid hydrolysis as compared to the propionate. In conformity with our findings, it has been noted previously that increase in the number of carbons from 1 to 2 between a thiol and an ester in a thiol-acrylate polymers decreases the rate of hydrolysis 3-fold.28
Schoenmakers et. al.27
has also reported hydrolysis rate occurring as a function of the number of methylene groups present between a thiol and an ester. Comparing between three and four methylene groups, the probability of hydroxyl attack on the carbonyl group was attributed to the atomic charge on the carbon atom of the carbonyl group: as the number of methylene groups increased, the atomic charge and therefore the hydrolysis of the ester decreased. Through molecular modeling it was shown that the atomic charge on the carbonyl carbon decreased as the number of methylene groups increased rendering the ester less hydrolyzable. Therefore, we speculate that additional increase in the number of methylene groups between the thiol and ester groups in our in-house synthesized PEG-diester-dithiol cross-linkers would lead to further decrease in the degradation rate and thus further impacting the tunability of our materials.
We also showed that both G′ (the elastic component) and G″ (the viscous component) were independent of frequency in the low frequency range studied (). This finding confirmed that the hydrogels were fully cross-linked and swollen at the time of the measurements. Further, the value of G′ was 2 – 2.3 orders of magnitude larger than the value of G″, indicating that the PEG hydrogels remained intact and elastic during the experiment.
In addition, hydrogels synthesized with cross-linkers containing esters were correlated with decreased values of G
′ as compared to hydrogels made with the non-degradable PEG-dithiol (PEG-SH 3.4). This effect may be explained by the fact that the carbonyl oxygen of the ester is a strong hydrogen bond acceptor, which may increase the water content in the hydrogel network and result in increased initial swelling ratios. Further, G
′ is inversely proportional to the polymer volume fraction, ν2
thus supporting the fact that hydrogels containing ester groups had lower initial values of G
We also monitored degradation indirectly by investigating the change in swelling ratio of the hydrogels. The initial values of QM
were in the range of 18 – 30 for the various hydrogel systems studied, which correlated well with values reported in the literature for similar systems. For example, ~ 100% w/v hydrogels made of 10 kDa 8-arm PEG-VS and DTT (0.15 kDa) exhibited QM
while 10% w/v hydrogels made of 4-arm PEG-acrylate and 3.1 kDa PEG-dithiol exhibited a QM
Based on their inverse proportionality, the same factors discussed above that contributed to the decrease in G
′ would also contribute to the increase in QM
. Moreover, swelling ratio is a measure of the hydrophilicity of the polymer and was also used to calculate the mesh size of the hydrogels. We found that hydrogels synthesized with cross-linkers of greater molecular weight (e.g., 6 and 8 kDa) were associated with a slight increase in initial swelling ratio, but this difference became more pronounced as the degradation proceeded (); this relationship is in agreement with the G
′ findings discussed above. It is interesting to note that varying cross-linker molecular weight from 3.4 to 8 kDa resulted in ~2× change both in the hydrogels’ degradation times as well as in ΔQM
(), but the effect of the number of methylene units had even more pronounced effect (): at 16 h, hydrogels made with PEG-SH 1 3.4 had a 2× greater swelling ratio and had degraded completely whereas the PEG-SH 2 3.4 hydrogels required 6 d to achieve the same change in properties. We also confirmed that polymer density could be exploited to control hydrogel degradation rate and QM
. As expected,44
both swelling ratio and degradation rate were greater in gels synthesized with greater polymer density ().
Cross-linker type not only altered the PEG hydrogel degradation profiles and mechanical properties but also affected gelation times. Acknowledging that Michael-type addition cross-linking reaction is very susceptible to pH of the environment we determined gelation time at various pH for hydrogels made with the different cross-linkers (). Our findings were in agreement with work carried out by Lutolf et al. where rheological measurements were used to establish that gelation time was inversely correlated with pH for gels made with PEG-VS and cysteine functionalized peptide cross-linkers.24
For our PEG-VS hydrogels cross-linked with PEG-diester-dithiol, gelation time was also inversely correlated with pH (). Under acidic conditions (pH 6.9) the gelation time was on the order of minutes, and decreased to seconds under basic conditions (pH 10). All hydrogels behaved similarly except the hydrogels made with PEG-SH 1 3.4 cross-linker, which gelled in the matter of seconds at all pH conditions possibly due to inductive effects associated with the closer proximity of the ester to the thiol moiety within this cross-linker. We also observed that gelation time correlated with cross-linker molecular weight (); this trend could be explained by the increased entropy of activation due to the increased polymer chain flexibility and rotation. For our work, we found pH 7.4-8 to be optimal as these conditions are cytocompatible and result in gelation times of 10 min or less for all cross-linker types.
To evaluate the feasibility of future use of these hydrogels as tissue engineering scaffolds, we performed several cytotoxicity experiments. First, we cultured Balb/3T3 fibroblasts in indirect contact with the degradable hydrogels and found that there was not a significant difference in cell viability for any of the hydrogel types (). It should be noted that during the 24-h culture period, the hydrogels underwent different rates of degradation and the hydrogels made with PEG-SH 1 3.4 cross-linker had completely degraded. The cell viability at the time of the measurement was 90% or above indicating that neither the products of degradation nor any byproducts of the synthesis were toxic to the cells. In a separate experiment we tested the toxicity of the unreacted cross-linkers in cell culture medium and found relevant concentrations of the cross-linkers to be non-toxic to cells as well.
Most of the hydrogel characterizations for this project were carried out for the simplest case when no biological factors were added to the gels. We chose this strategy to first unveil major structure-function trends and begin to identify favorable conditions for cell and protein encapsulation. However, we recognize that PEG is inert and therefore not fit to sustain the survival and proliferation of anchorage-dependent cells. Thus, to demonstrate that basic biological functionality can be incorporated into the hydrogels, we chose to implement one of the most commonly used polypeptide sequences RGD45
to promote cell adhesion in the hydrogels. When encapsulated within the RGD-modified hydrogels synthesized with all types of degradable PEG-diester-dithiol cross-linkers, fibroblasts demonstrated >90% viability after 10 h or 24 h of culture () suggesting that all of the reported hydrogels are equally suitable as materials for tissue engineering scaffolds. We note that when adhesive ligand was omitted from the hydrogels, the cell viability dropped to 1.6% (data not shown) further supporting the assumption that PEG provided the structural backbone of the hydrogel scaffold but is completely devoid of biological activity, hence offering a potential for independent control of physical and biological properties of the system. Additionally, note that hydrogels made with PEG-SH 1 3.4 cross-linker were still present at 10 h but had completely degraded in 24 h and the cells had retained high viability. These results indicate that fibroblasts survived encapsulation and provide further evidence that the components released from degrading hydrogels are not toxic.
Lastly, we tested if the incorporated RGD ligand influences the hydrogel mechanical properties. We found that the effect of the ligand covalently bound to the hydrogel structure was closely related to the molecular weight of the hydrogel cross-linker. For the lower molecular weight cross-linker (3.4 kDa) the covalent addition of ligand affected hydrogel storage modulus () and swelling ratio (); however, this effect was not noticeable for hydrogels made with higher molecular weight cross-linkers (6 and 8 kDa). We hypothesize that the added electrostatic charge associated with the RGD ligand may be related to the altered hydrogel properties and further investigation has been undertaken46
in order to confirm this hypothesis or determine other possible mechanisms that underlie this behavior.