Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

The methods previously illustrated are invaluable for the synthesis of PEGDM and methacrylamide functionalization of peptides or other amine-containing compounds. These materials can then be used for regenerative medicine and drug delivery applications. Due to the hydrophilic nature of PEG, hydrogels formed from PEG macromers have a high water content similar to many tissues in the body². This quality makes PEG very resistant to protein adsorption and therefore inert in the body³. However, the hygroscopic nature of PEG can prove troublesome during functionalization. If water is present in the PEG sample during the methacrylation procedure, the methacrylic anhydride will react preferentially with water to produce methacrylic acid, and poor functionalization of PEG will result.

Therefore, one of the most important steps that can be taken to ensure successful methacrylation of the PEG or peptide is to maintain anhydrous reaction conditions. The recommended step of drying all glassware before use is intended to prevent water contamination. The presence of water in the sample can be seen in the NMR analysis, as a broad peak at 1.7 ppm (Figure 4). If poor methacrylation is observed even after drying all glassware, chemicals may be dried over sodium sulfate or other drying agents (molecular sieves etc.) prior to use. Distillation can also be used to remove water and purify methacrylic anhydride prior to use, and azeotropic distillation can be used to dry PEG²³. In extreme cases, synthesis can be carried out in a glovebox to further ensure adequately anhydrous conditions. A second round of methacrylation, following the same procedure, can also be performed to increase functionalization. Because there is always a chance that additional rounds of functionalization will be required, care should be taken in step 1.7 and 1.9 to quickly collect PEGDM by vacuum filtration. Vacuum filtration for longer than is absolutely necessary increases exposure of PEG to air, increasing the opportunity for water adsorption.

Even though the percent excess of methacrylic anhydride to hydroxyl functional groups remains unchanged, increasing the PEG functionalization (e.g. arm #) on the PEG precursor is generally associated with decreases in percent functionalization achieved (unpublished results, Benoit lab). To preemptively address this reduction in functionalization efficiency, or if particular difficulties are encountered achieving sufficiently high functionalization, the duration of the microwave reaction may be increased, provided that the microwave interval is maintained at 30 sec. While 10 molar excess is typically sufficient, the amount of methacrylic anhydride used in the reaction can also be increased to increase the percent functionalization achieved¹².

It is important that the additional precipitation step (1.9) be performed to achieve good NMR signals. While it is tempting to perform the second precipitation the same day as synthesis, drying the sample overnight before reprecipitating has been found to aid the removal of excess methacrylic anhydride and methacrylic acid. Sample preparation is also important for achieving clean NMR spectra, and therefore samples should be prepared using recommended conditions. Figure 4 demonstrates representative ¹H-NMR results for correctly functionalized PEGDM. By analyzing the ratio of terminal methacrylate protons to central PEG protons, the PEGDM was determined to be adequately functionalized. MALDI sample preparation is similarly important for achieving a clear reading. MALDI is particularly sensitive to the presence of salts and high sample concentrations. If a clear MALDI reading (an intensity above 50 arbitrary units (a.u.) with a high signal:noise ratio) cannot be obtained, the sample solution should be diluted 1:100 in MALDI solvent before being combined with the matrix solution and reanalyzed. Figure 5 demonstrates representative MALDI-ToF results after correct peptide functionalization, cleavage, and sample preparation. Cleavage of a small sample of resin prior to functionalization (Figure 5A) shows correct synthesis of the peptide GKRGDSG, with correct methacrylamide functionalization of the peptide shown in Figure 5B.

While functionalization of on-resin peptides is a relatively robust procedure, the cleavage conditions required for each sequence often requires tuning. For long sequences where many amino acids have protected side chains (>30 amino acids long, or >15 amino acids with protecting groups), duration of cleavage should be increased by one hour. However, if cleavage time is extended too much, peptide bond cleavage may result due to long-term acidic exposure. MALDI analysis can be very helpful in revealing any errors which occurred in peptide synthesis or cleavage. An observed decrease below expected molecular weights can indicate that amino acid(s) did not properly couple, or that peptide fractionation occurred (see Table 2 for sources of commonly observed changes in molecular weight). If the observed molecular weight is higher than expected by the weight of a protecting group used, it is likely that cleavage and deprotection was insufficient and the peptide should be recleaved for additional time.

Table 2. Commonly observed changes in peptide molecular weight.

Macromers produced using microwave-assisted methacrylation methods can be used in a number of regenerative medicine or drug delivery applications. The functionalized peptides and PEGDM synthesized here can also be incorporated into polymers using Nitroxide-Mediated Polymerization (NMP), Atom Transfer Radical Polymerization (ATRP) or Reversible Addition-Fragmentation Transfer (RAFT) methods²⁴. Hydrogel networks can also be produced in the presence of cells, as previously demonstrated in the JoVE article by Khetan and Burdick²². This often requires the incorporation of cell adhesion peptides such as RGD or extracellular matrix molecules, as PEG alone does not provide cell-material interactions critical for survival and function of some cell types²⁵. Peptides, for example, can be synthesized using traditional solid-phase peptide synthesis and functionalized as described here to allow for incorporation into hydrogel networks. As seen in Figure 6, inclusion of the methacrylamide-functionalized cell adhesion peptide GKRGDSG in hydrogels (0.5 mM) facilitates adhesion of human mesenchymal stem cells (MSCs) to PEG hydrogel surfaces, increasing the number of attached and spreading cells (Figure 6B), compared to PEG hydrogels without the cell adhesion peptide (Figure 6A). However, previous work has demonstrated that cell-material interactions are further enhanced by inclusion of 3,400 Da PEG spacers between adhesive peptides and hydrogel networks, to reduce peptide-integrin steric hindrance. Without inclusion of the spacer, cells may interact with PEG hydrogels via nonspecific proteins that adsorb to the peptide, rather than through integrin-mediated interactions with peptides²⁶. To incorporate this PEG spacer and avoid nonspecific interactions, peptides can be conjugated to monofunctionalized PEG via N-hydroxysuccinimidyl-activated esters, as described by Hern and Hubbell²⁶.

Applications of hydrogel networks require tight control over material properties. A significant advantage to PEG hydrogels is a high degree of control over these properties. For example, the molecular weight, arm number, and the wt% of PEG used in the formation of hydrogel networks can be altered to fine-tune properties for specific applications. This allows tight control over hydrogel mesh size (ξ), which controls hydrogel swelling ratio (Q) and stiffness (modulus of elasticity, E). This is illustrated in Figure 7A and quantified in Figure 8, where increasing PEG macromer molecular weight results in an increase in hydrogel mesh size (Figure 8A) and a decrease in hydrogel stiffness (Figure 8B).

The underlying physical characteristic that controls bulk behavior in these hydrogel networks, mesh size, is calculated using the Flory-Rehner equation¹⁶. To perform this calculation, the volumetric swelling ratio (Q) is first calculated from equation 4:
(4)

where ρ_s is density of water (1 g/ml), ρ_p is density of PEG (1.12 g/ml), M_s is swollen mass of the hydrogel and M_D is dry mass of the hydrogel (often measured after freezing and lyophilization of hydrogels). The molecular weight between crosslinks (M_c , in g/mol) is then calculated from equation 5:
(5)

where M_N is the number-average MW of PEG (in g/mol), is the specific volume of the polymer , V₁ is the molar volume of water (18 ml/mol), V₂ is the equilibrium polymer volume fraction of the hydrogel
(), and X₁ is the polymer-solvent interaction parameter for PEG and water (0.426)¹⁶. The number of bonds between crosslinks (n) is then calculated from equation 6:
(6)

where N_b is the number of bonds in the PEG repeat (3) and M_r is the MW of the PEG repeat (44 g/mol)²⁷. This allows the root-mean-square end-to-end distance of the polymer chain (in nm) to be calculated from equation 7:
(7)

where l is the average bond length (0.146 nm, calculated based on C-C and C-O bond lengths) and C_n is the characteristic ratio of the polymer (4.0 for PEG)²⁸. Finally, mesh size of the hydrogel can be calculated from equation 8:
(8)

Hydrogel properties can similarly be tuned by adjusting the amount of PEG used in the formation of hydrogels. Decreasing the weight percentage of PEG macromer results in an increase in hydrogel mesh size, which subsequently reduces hydrogel stiffness. Figure 7B illustrates and Figure 9 quantifies how the weight percentage of PEG used in hydrogel formation can be used to control mesh size (Figure 9A) and resultant hydrogel stiffness (Figure 9B). As substrate stiffness has been shown to affect cell behaviors such as stem cell differentiation²⁹, the ability to tightly control stiffness is an important characteristic in hydrogel fabrication.

Hydrogels can also be used to control drug delivery. As illustrated in Figure 7A and demonstrated in Figure 10, increasing the molecular weight of PEG macromers increases mesh size of the hydrogel network, subsequently increasing the release of encapsulated model drug, bovine serum albumin (BSA). While hydrogel samples in this study were destroyed at t=195 hours to allow for measurement of hydrogel wet and dry masses for mesh size calculations, it is our experience that continued BSA release would occur had the samples been incubated for longer time periods. The incomplete release of BSA observed in Figure 10 is not unexpected, as other groups have also reported that BSA is resistant to diffusion within PEG hydrogel networks³⁰. Incomplete release of encapsulated protein can occur due to hydrogen bonding between proteins and PEG macromers, or covalent binding between the methacrylate group on the PEG and primary amine groups on lysine residues in BSA³¹. Additionally, BSA is prone to aggregation and disulfide bond formation over time, which can increase its effective Stokes radius and hinder its release from hydrogels. As chain-growth hydrogels, such as these PEGDM hydrogels, are prone to network nonidealities and heterogeneous hydrogel mesh size (Figure 2A), it is also possible that a fraction of the encapsulated BSA is contained in regions of the hydrogel which have significantly smaller mesh size than the overall average within the gel, preventing its release. While incomplete, nonFickian release (data not shown) of encapsulated BSA was observed in this case, controlled Fickian release of numerous other model drugs, including insulin and ovalbumin, has been demonstrated using similar PEGDM hydrogels³⁰. Additionally, Watkins and Anseth have used confocal laser scanning microscopy to demonstrate that release of fluorescent molecules from similar hydrogels is modeled acceptably with Fickian diffusion methods³².

While the hydrogels formed in this study are nondegradable, network degradation is another parameter that can be incorporated into and tuned within these networks. Providing for controlled hydrogel degradation can result in alterations in cell behavior³³, promotion of tissue growth or host tissue ingrowth, or elimination of the need for explantation³⁴. Degradable PEG hydrogels are commonly synthesized by ring-opening hydrolytically degradable d,l-lactide, glycolide, or ε-caprolactone groups onto hydroxyl groups within PEG prior to methacrylation³⁵. These three groups degrade by hydrolysis of ester functionalities, with the glycolide esters having the greatest susceptibility to degradation, followed by lactide, and caprolactone ester, due to their varying hydrophobicity. After incorporation of hydrolytically degradable groups, PEG can be further functionalized using the methacrylation procedure detailed in this article, enabling formation of hydrogel networks through subsequent radical-initiated chain polymerization^36,37. The rate of degradation of hydrogel networks can be controlled by varying the identity of the hydrolytically degradable group (glycolide, lactide, etc.) and by varying the number of degradable repeats incorporated in the structure^35,38.

Theoretically, the methods demonstrated here could be used for acrylation of PEG and peptides by replacing the methacrylic anhydride with acrylic anhydride in steps 1.3 and 3.3, respectively. However, acrylic anhydride is more than 20 times the cost of methacrylic anhydride^39,40, making microwave-assisted acrylation significantly less attractive than microwave-assisted methacrylation.

We have demonstrated a simple, rapid method to functionalize PEG and peptides, how to evaluate the efficiency of this procedure, and given resources for using the synthesized materials to form hydrogel networks. These synthetic tools are highly versatile in their applications, and should prove a staple in any number of drug delivery and materials research laboratories.