3.1. Incorporation of ligand into the PEG hydrogel
The fluorescent ligand 5FAM-GRCD-RGDS-PD was incorporated into the PEG hydrogel and its fluorescence was measured over time to estimate whether any unbound peptide was released from the hydrogel. shows that the fluorescence of the ligand in the hydrogel or in the supernatant did not change appreciably over the course of the experiment (4 and 24 h). The average fluorescence of the PBS solution was lower than the average fluorescence of the hydrogel indicating that some ligand was not bound covalently to the hydrogel and therefore was free to diffuse out of the hydrogel during the soaking in PBS. shows that the fluorescence of the hydrogel did not change over 72 h which also indicated that all of the ligand was released initially (in < 4 h) and the ligand remaining in the hydrogel was stably incorporated; therefore, characterization of hydrogel properties (e.g., G′) was not affected by unbound ligand. Additionally, fluorescent ligand was reacted with PEG-VS and after the reaction was complete, the solution was filtered to recover the unbound ligand. The unbound ligand from this experiment was estimated to be 36.1 ± 0.3%.
Figure 2 PEG hydrogel with covalently incorporated fluorescent ligand was used to determine that unbound ligand: a) was released in the supernatant PBS initially prior to 4 h, but b) was not released from the hydrogel over time (i.e., differences between sample (more ...)
There are several possible explanations for the apparent incomplete incorporation of the ligand. First, it is possible that a small amount of free dye is present in solution. The excess dye was cleaned with a one-step high-performance liquid chromatography (HPLC) purification (CPC Scientific) and the purification efficiency was not tested subsequently. Second, it is possible that despite the preventative measures, a fraction of the ligands had reacted with each other forming dimers by disulfide bonding thus taking up thiol groups that otherwise would be covalently bound to the VS group of the PEG polymer. Disulfide formation could have occurred in the aliquot or after the ligand was added to the PEG-VS solution. The competitive disulfide formation after the peptide was added to the PEG-VS solution would be closely related to the initial concentration of ligand: more ligand would lead to the formation of more disulfides [34
]. Lastly, the reaction efficiency of the cysteine thiol binding to the VS group of the PEG by Michael-type addition strongly depends on the surrounding amino acids and their charges: Lutolf et al. [34
] demonstrated that positively charged amino acids positioned near the thiol resulted in a faster addition reaction rate. Specifically, the binding efficiency constant for the sequence GRCD (used in our project) to PEG-diacrylate (PEGDA) was 58.3 L/mol.min compared to 124.0 L/mol.min for GRCR. Therefore, it is possible that choosing a peptide with a sequence such as GRCR-RGDS
-PD, would improve the binding efficiency.
3.2. Effect of ligand type on material properties: general findings
QM is defined as the mass of the swollen hydrogel divided by the mass of the dry hydrogel and is a measure of the hydrophilicity of the polymer. When the polymer is cross-linked and forms a 3D structure, QM is also a measure of how much water is incorporated into the structure (i.e., the water to polymer ratio). Given a mesh network (a good approximation of the PEG hydrogel) as shown in : more cross-link points will result in tighter mesh; consequently, less water will be incorporated in the network. On the other hand, if some of the cross-links are disrupted, local “pockets” with larger mesh would be created that are able to incorporate more water. Hence, by adding ligand and consequently inhibiting the complete cross-linking of the hydrogel, we should see increase in QM (up to a certain maximum peptide concentration that would occupy sufficient cross-linking sites to prevent polymer-polymer bonds and result in a soluble macropolymer). If this hypothesis holds true, it would indicate that the properties of the ligand and the PEG polymer are comparable and there is no ligand-polymer interaction.
Figure 3 a) Schematic representation of hydrogel mesh disruption upon covalent binding of ligand or PEG-SH to the 4-arm PEG-VS; b) Influence of ligand type on PEG hydrogel swelling ratio (QM). All hydrogels were prepared as 10% w/v polymer with 100 μM (more ...)
It is also likely that QM would not be affected by the incorporation of ligand. Note that at 100 μM ligand or PEG-SH, only up to 0.7% of the VS groups would be occupied. The rest of the VS groups would be free for gelation with the PEG-dithiol cross-linker. This decrease in available cross-linkable VS sites may have a negligible effect; thus the net homogeneity of the resultant hydrogel network would not be affected and QM would not be altered.
shows the influence of ligand type on QM of the PEG hydrogel. Compared to hydrogels made without ligand or PEG-SH, QM was ~14% higher when PEG-SH was incorporated into the hydrogel, which was expected because the PEG-SH occupied cross-link sites but was not expected to interact with the surrounding PEG network. For the peptide ligands, the addition of ligand either had no effect (as for RGDS and IKVAV), or decreased QM (as for YIGSR and YIGSRPD).
For all general purposes, especially cell attachment and protein adhesion, PEG is considered an inert polymer [47
], an observation which would not explain the above findings. However, Kokufuta et. al. [48
] and Xia et. al. [49
] have shown that PEG was able to form a complex with pepsin under the appropriate conditions. The complex formation had been attributed to hydrogen bonding of the carboxyl and phenolic OH groups in pepsin. However, the hydrogen bonding would only be possible when these acidic side chains are protonated, i.e., this effect would depend on the pH of the surrounding solution. The pH of the PEG solution was 8.2 during gelation and 7.4 after gelation when the fully cross-linked 3D hydrogel was soaked in PBS. Considering the full amino acid sequence of all ligands, one may note that the only amino acid present that contains a carboxylic side chain is D (aspartic acid). It has a pKa of 3.9 [50
] and thus would be protonated at pH ~3 or below. Therefore, under the selected experimental conditions, the carboxylic acid of D would be unprotonated and could not participate in hydrogen bonding with the ether oxygen of the PEG polymer. Furthermore, D is present in all ligands (in the flanking sequences GRCD and PD) and thus could not explain the differences in their influence over the material properties.
All ligands as well as the control PEG-SH were of similar molecular weight (~1000 Da), thus it is not likely that ligand size alone could explain the observed results. Therefore, the effect of ligand incorporation could not be generalized and specific ligand properties must be considered to explain their various effects. We first examined the individual properties of the ligand peptide sequences used in this work (). Because we observed a completely different influence associated with addition of PEG-SH to the hydrogel as compared to addition of peptide ligands, we examined the major properties that clearly distinguish the two. First, we considered the net charge of the ligands (). PEG itself (including PEG-SH) is uncharged, while each of the ligands contained charged polar amino acids. However, considering net charge alone, we did not observe obvious trends in the data.
Influence of ligand type on PEG hydrogel swelling ratio (QM). Each plot highlights a different ligand property: a) net charge; b) hydrophobicity index; c) pI. All hydrogels were prepared as 10% w/v polymer with 100 μM ligand.
Next, we considered the possibility that negatively charged or polar uncharged amino acids were forming ionic or weaker Van der Waals bonds with the partially positively charged ether oxygen of the PEG. An interaction of this type could potentially cause the ligand to align parallel to the PEG polymer chain or even create a weak bond (temporary or permanent) with an opposing PEG chain and thus influence the mesh structure. Such bond is more likely to be created at solution pH close to that of the ligand pI. Therefore, we examined whether any trends existed between QM and ligand pI (). Again, we did not observe a clear trend in the data but in general the ligands with the highest pI, YIGSR and YIGSRPD, were the ones that had the greatest effect on QM versus gels without ligand (see also ). The YIGSR ligand with the largest influence (20% lower QM than hydrogels with no ligand) had the highest pI of 8.9, which is the closest to the pH of the hydrogel solution during cross-linking (pH 8.2). Therefore, it is possible that some ligand types (in our case YIGSR and YIGSRPD) bonded via weaker ionic or Van der Waals interactions with the 4-arm PEG-VS after they had been covalently incorporated into the PEG network (via the unpaired cysteine residue). It is also possible that these interactions resembled the manner of a cross-linker, connecting (permanently or temporarily) opposing PEG chains or creating folds within a single PEG chain. Such an interaction would not only result in a disrupted non-homogeneous mesh but would also lead to a smaller mesh since the molecular weight of the ligand (1000 Da) is 3-fold smaller than that of the cross-linker used (3400 Da).
Because QM is a measure of the hydrophilicity of the network environment, it is possible that the addition of ligand introduced local areas of hydrophobicity to the otherwise fully hydrophilic polymer. To explore this hypothesis, we specifically examined the hydrophobicity index of each ligand type (). We did not observe a clear trend in the data. In fact, it appeared that the ligand with highest hydrophobicity index, RGDS, did not exhibit a significant influence on QM versus hydrogels without ligand. Therefore, change in the hydrophobicity of the hydrogel environment alone could not explain the observed results.
To explore other possible effects of ligand type on hydrogel properties, we also determined hydrogel ξ and G′. The concept of QM is closely related to ξ and these two hydrogel properties are proportional. shows the effect of ligand type on ξ of the hydrogel, and as expected, the data followed the same trends observed in . As discussed earlier, addition of PEG-SH to the hydrogel occupied sites that otherwise would be available for cross-linking thus leading to an increase in ξ. On the other hand, ligands also occupied sites available for cross-linking but interactions with the PEG chains either counter-balanced that effect or lead to a decrease in the overall ξ of the hydrogel.
a) Influence of ligand type on PEG hydrogel mesh size; b) Influence of ligand type on PEG hydrogel storage modulus (G′). All hydrogels were prepared as 10% w/v polymer with 100 μM ligand. Asterisks designate significant differences.
Another important scaffold property that could be influenced by the addition of ligand is G′. shows the influence of the ligand type on PEG hydrogel G′. Based on the data presented in and we expected that the addition of the control PEG-SH would lead to an increase in G′ but we did not see a significant difference in G′ between the hydrogels made without ligand and hydrogels made with PEG-SH. This result could be explained with the fact that even though PEG-SH occupied cross-linking sites, the overall polymer density was not altered. As with QM and ξ, addition of RGDS and IKVAV also did not have a significant effect on G′ but the influence of YIGSRPD and YIGSR ligands in this case was most pronounced and resulted in 10% and 19% increases in G′, respectively. This outcome confirmed our initial observation that some inherent property of the YIGSR and YIGSRPD ligands may be responsible for altering PEG hydrogel properties.
3.3. Effect of ligand type and pH on material properties: focus on YIGSR
To further explore the concept that the difference in material properties observed upon addition of ligand is inherently linked to ligand type, we examined the two ligands that had the most effect on QM
, namely YIGSR and YIGSRPD. The unique amino acids that they have in common but do not share with the other ligands were Y (tyrosine; polar, uncharged and contains a benzene ring) and I (isoleucine; non-polar and hydrophobic). Y is also unique in that it contains a phenolic OH group [50
]. The pKa of the Y side chain is 10.1 [50
]. Under the current experimental conditions, the side chain would be protonated and therefore able to form a hydrogen bond with the ether oxygen of the PEG polymer ().
Figure 6 a) Schematic representation of hydrogen bonding between the phenolic OH group of the tyrosine (Y) amino acid of the YIGSR and YIGSRPD ligands and the ether oxygen of the PEG polymer; b) Influence of buffer pH on the swelling ratios of PEG hydrogels containing (more ...)
The impact of a bond between Y and the PEG network could have several outcomes. For example, this hydrogen bond could reduce polymer hydrophilicity by altering the nature of the ether oxygen of the backbone chain or more importantly reducing the mesh size of the network by connecting opposing PEG chains or creating entanglements in a single PEG chain. A hydrogen bond between the YIGSR or YIGSRPD and the PEG polymer chain could therefore explain the decrease in swelling ratio and mesh size and increase in G′ upon addition of these ligands to a PEG-based hydrogel.
A pH experiment was specifically designed to test whether the interaction between the YIGSR ligand and the PEG polymer was due to hydrogen bonding between the phenolic OH group of the Y amino acid and the ether oxygen of the PEG repeat unit (). shows the effect of pH on PEG hydrogels made with or without YIGSR ligand. In the pH range of 5.6–10.5 we noted three distinct effects of YIGSR incorporation. First, in the acidic pH range (5.6 and 6.6) there was no difference in the QM of the hydrogels made with and without YIGSR. This finding supported our hypothesis that an acidic environment provides an abundance of protons that would compete with the phenolic OH group of the Y amino acid, occupy hydrogen bonding sites and thus decrease or completely eliminate the influence of the ligand on the bulk PEG properties. As noted previously (), at pH 7.4 the presence of ligand lead to a decrease in hydrogel QM. However, in a basic environment (pH 8.6 and 10.5), we observed a shift in the hydrogel QM: hydrogels made with YIGSR had higher QM than hydrogels without ligand. The difference between the two types of hydrogels was significant at pH 10.5, which is above the pKa of the Y side chain. This shift could be explained by the fact that the phenolic OH groups became deprotonated at basic pH and were no longer available for hydrogen bonding with the PEG polymer. With deprotonated phenolic OH groups, the YIGSR ligand behaved more like the control PEG-SH (i.e., did not interact with the PEG polymer but simply occupied cross-linking sites).
3.4. Effect of ligand concentration on material properties
All of the above data was collected at a single ligand concentration. However, in building tissue engineering scaffolds, ligands are used at a range of concentrations. For example, comparing between RGDS, YIGSR and IKVAV ligands, Gunn et. al. [37
] has shown that the concentration of ligand required for optimal neurite extension on 2D PEGDA hydrogels depended on which specific ligand was incorporated. In this work, the authors had also linked the extent of neurite outgrowth to the change in mechanical properties of the hydrogels without attempting to establish a connection between the ligand concentration and the resulting hydrogel mechanical properties. In general, adhesive ligand concentration which influences neurite outgrowth independent of scaffold mechanical properties, has not been consistently controlled, nor quantitatively measured [51
]. Thus, we have chosen an array of RGDS, IKVAV and YIGSR concentrations widely used in building tissue engineering scaffolds (10, 100, 300 μM) [35
] and tested the influence of ligand concentration on hydrogel materials properties. (Between the two similar sequences, YIGSR and YIGSRPD, we chose YIGSR for further testing as it showed the greatest impact on material properties.)
shows that the only RGDS concentration to have a significant influence on QM was 300 μM, which led to a 12% increase in QM compared to hydrogels synthesized without ligand. On the other hand, indicate that addition of IKVAV and YIGSR ligand either had no effect, or in fact resulted in reduced values of QM. The greatest influence of IKVAV was noted at 10 μM ligand (20% decrease in QM versus hydrogels without ligand), whereas the greatest impact of YIGSR was observed at 100 μM (21% decrease). Hence, in contrast to the results presented in (which showed that the effect of YIGSR on QM was larger than that of IKVAV), we saw that both IKVAV and YIGSR had the same overall influence on the hydrogel QM but at different concentrations. shows that addition of 10 μM of the control PEG-SH did not affect the hydrogel QM while addition of 100 and 300 μM both lead to a 20% increase in QM, which is consistent with .
Influence of ligand concentration on swelling ratio (QM) of PEG hydrogels: a) RGDS ligand; b) IKVAV ligand; c) YIGSR ligand; d) PEG-SH control. All hydrogels were prepared as 10% w/v polymer. Asterisks designate significant differences.
Thus, we observed that both IKVAV and YIGSR exhibited an “optimal” concentration at which their influence on the material QM was at its maximum, but RGDS ligand did not exhibit the same behavior within the range of ligand concentrations explored in this study. In fact, at the highest concentration (300 μM) the RGDS influence on the material properties resembled that of the PEG-SH. Based on this observation, it is possible that RGDS did not react in any significant way with the PEG hydrogel and did not change the hydrogel network environment.
Note that RGDS has the highest hydrophobicity index. Since the addition of a high quantity of the ligand resulted in an increase in QM rather than a decrease, we can conclude that the decrease in the overall hydrophilicity of the PEG network upon addition of ligand under these conditions was negligible and not responsible for the overall change in hydrogel properties. Additionally, the pI of the RGDS ligand (4.2, the lowest for all studied ligand types), however, was far below the pH of the hydrogel solution upon cross-linking (8.2) and after cross-linking (7.4). Therefore, it is unlikely that the RGDS ligand would interact with the PEG polymer (apart from the covalent addition of the cysteine thiol to the PEG’s VS reactive group).
shows the influence of ligand concentration on hydrogel ξ. These data follow the same trend as measurements of QM (). The addition of RGDS did not significantly influence ξ at concentrations below 300 μM (). Both IKVAV (at 10 μM) and YIGSR (at 100 μM) lead to ~3 nm decrease in ξ (, respectively). Addition of the control PEG-SH did not significantly alter in the studied concentration range ().
Influence of ligand concentration on mesh size of PEG hydrogels: a) RGDS ligand; b) IKVAV ligand; c) YIGSR ligand; d) PEG-SH control. All hydrogels were prepared as 10% w/v polymer. Asterisks designate significant differences.
shows the influence of ligand concentration on G′. The addition of RGDS did not significantly alter G′ at concentrations below 300 μM (). Both IKVAV (at 300 μM) and YIGSR (at 100 μM) lead to ~15% increase in G′ (, respectively). Addition of the control PEG-SH did not change G′ significantly in the studied concentration range ().
Influence of ligand concentration on storage modulus (G′) of PEG hydrogels: a) RGDS ligand; b) IKVAV ligand; c) YIGSR ligand; d) PEG-SH control. All hydrogels were prepared as 10% w/v polymer. Asterisks designate significant differences.
– indicate that IKVAV influenced the hydrogel properties in a similar fashion as YIGSR, albeit at different concentrations. Upon considering the amino acid sequence for IKVAV, we note that the amino acids that are not shared with the other ligands are lysine (K), valine (V) and aspartic acid (A). A and V are non-polar hydrophobic amino acids, but as mentioned above, we hypothesize that the hydrophobicity of the tested ligands does not significantly alter hydrogel material properties. K is a basic amino acid with a pKa of the side chain of 10.79. Therefore, it was possible that at the pH of the hydrogel microenvironment, these fully protonated basic groups form hydrogen bonds with PEG, a phenomenon that had been observed by Azegami et. al. [52
] when mixing human serum albumin with PEG at pH 8.
The other basic amino acid found in all tested ligands was R (arginine) where it was present in the flanking sequence GRCD and was also part of the active sequence for YIGSR, YIGSRPD and RGDS. However, based on its proximity to cysteine (C), the site of covalent bonding to 4-arm PEG-VS, we would expect that only the R of YIGSR and YIGSRPD would be available for hydrogen bonding. (In the case of GRCD sequence, R was an α substituent and in the case of RGDS (GRCDRGDSPD) it was a β substituent and potentially physically restricted, i.e., not available for hydrogen bonding).
In summary, the results of the ligand types and concentrations presented in – lead to the conclusion that ligand concentration must be considered when discussing the influence of ligand type on the material properties of hydrogels because a single concentration may not result in similar effects or trends in all cases.
3.5. Effect of polymer density on material properties
depicts the influence of YIGSR ligand on material properties examined in hydrogels of 5–20% w/v polymer density. At the lowest polymer density, the addition of YIGSR did not have an effect on QM (), ξ (), or G′ (). For higher polymer concentrations (10, 15, 20% w/v), addition of ligand resulted in decreased QM and ξ and increased G′ (though the difference in G′ at 20% was not significant). Since ligand concentration was kept constant for all polymer densities, and in the range of 10–20% w/v polymer, no other trends were noted, these results ruled out the possibility that the ratio of ligand to polymer concentration had a significant influence. Moreover, if the influence of the ligand on hydrogel properties was due to consumption of cross-linking sites, the extent of this effect would have been inversely proportional to polymer density; this result was generally not observed. The lack of influence of ligand on the properties of the 5% w/v hydrogels could be explained by the very large QM of the hydrogel at this polymer density. With high swelling, it is possible that weak bonds (such as hydrogen bonds) were insufficiently strong and temporary in effect; thus, the overall contribution of entanglements resulting from such bonds could be negligible.
Comparison between PEG hydrogels with varying polymer density prepared with 100 μM YIGSR ligand or no ligand and the effects on: a) swelling ratio, b) mesh size, c) and storage modulus. Asterisks designate significant differences.
3.6. Effect of ligand on bulk diffusion in the PEG hydrogels
shows the effect of ligand on BSA diffusivity in the PEG hydrogels. All ligand types resulted in decreased diffusivity by ~30%, whereas addition of the control PEG-SH decreased diffusivity of BSA by ~60%. The charge and hydrophobicity of the ligand amino acids likely interact with BSA and obstruct its diffusivity in the otherwise inert hydrogel. However, the discussion above may suggest another ligand-specific mechanism that could restrict the diffusion of soluble molecules in the PEG hydrogels. The ~2-fold decrease in BSA diffusivity in hydrogels made with PEG-SH as compared to hydrogels made with peptide ligands could be explained by the fact that covalently-bound PEG-SH did not interact with the PEG polymer chain. Instead, it may act as an additional free chain rotating in the mesh of the hydrogel, thus obstructing the diffusion of BSA inside the hydrogel. The peptide ligands on the other hand, may interact with PEG polymer chains as described above. In this way, the net effect of ligands in the hydrogel was a reduced obstacle to the diffusion of BSA inside the mesh.
Figure 11 Effect of ligand type on BSA diffusivity (De) in PEG hydrogels. All hydrogels were prepared as 10% w/v polymer with 100 μM ligand. Asterisks designate significant differences. The BSA diffusivity in the hydrogel is normalized by that of BSA diffusivity (more ...)