Glycidyl methacrylate and methacrylic anhydride characterization for silk fibroin methacrylation in tissue engineering

Abstract

The field of tissue engineering has been an ever-evolving discipline with a principal direction of creating artificial constructs to improve biological tissue types. Constructs used in tissue engineering arise from natural compounds, synthetic polymers, or a combination of the two to generate a hybrid biomaterial with optimized characteristics. In recent years, researchers have turned to silk fibroin (SF) as a natural source for its attractive physical characteristics and tunability. Using this platform, researchers have attempted to chemically modify SF through methacrylation to further improve its mechanical properties, thus making it a more appealing candidate for bioengineering applications. To date, the two most common methacrylating agents for synthesizing methacrylated SF across literature have been glycidyl methacrylate (GMA) and methacrylic anhydride (MA), which produce SFGMA and SFMA, respectively. However, the side-by-side characterization of SFGMA and SFMA has not been well compared with respect to their synthesis reactions and resulting degrees of methacrylation (DoM). To address this, our study developed a standardized protocol for SFGMA and SFMA synthesis in an effort to systematically compare the two NMR spectra. From this protocol, our results demonstrate GMA to be the superior methacrylating agent for its reactional consistency and DoM validity.

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1. Introduction

The field of tissue engineering has evolved significantly over the past decade with the advent of hybrid biomaterials that combine natural and synthetic constituents.¹ Hybrid biomaterials mimic the inherent biocompatibility of natural materials, while also utilizing the tunability of synthetic polymers’ superior mechanical properties.^2,3

One promising biomaterial is silk fibroin (SF), extracted from naturally-derived Bombyx mori (B. mori) cocoons.⁴B. mori silk is composed of 75% SF (semi-crystalline fibrous protein) as its structural component and 25% sericin (amorphous protein) as its adhesive component.⁵ The SF component has two subunits: a heavy chain (391 kDa) and a light chain (25 kDa), which are linked together via a covalent disulfide bond. An additional glycoprotein, P25 (30 kDa), is non-covalently attached to the heavy and light chain complex. Together, the heavy chain, light chain, and P25 form a 6 : 6 : 1 molar ratio in SF.⁶ The main amino acid residues of SF are as follows: arginine (0.3 mol%), asparagine (0.4 mol%), glutamine (0.2 mol%), and lysine (0.2 mol%), among other residues in small quantities, in the primary group, and histidine (0.1 mol%) and tryptophan (0.2 mol%) in the secondary group.^7,8

After a degumming process of boiling raw silk cocoons in a sodium carbonate (Na₂CO₃) buffer, the cocoons were washed to remove sericin and then dissolved, yielding pure SF.⁹ From the SF solution, methacrylating agents such as glycidyl methacrylate (GMA) or methacrylic anhydride (MA) may be added. Additional materials such as gelatin methacryloyl (GelMA), obtained from methacrylation of gelatin using methacrylic anhydride, and 2-isocyanatoethyl methacrylate (IEM), among others, have been attempted but not extensively characterized for tissue engineering applications beyond the hydrogel level.¹⁰ During the methacrylation reaction, previous work has proposed that methacrylate groups are added to the lysine groups via the primary amines of SF.¹¹ In particular, GMA has been reported to react with carboxyl and hydroxyl groups through either transesterification or epoxide ring-opening mechanisms, dependent on environmental conditions. Under acidic conditions (pH 3.5), the epoxide ring-opening mechanism dominates. Under basic conditions (pH 10.5), GMA undergoes hydrolysis and reacts with the SF's hydrogel groups through both mechanisms, though the ring-opening is preferred.¹² In contrast, when MA is used a methacrylating agent, previous groups have observed crystallization of SF due to a methacrylic acid byproduct. The lowered solution pH results in protonated and neutralized carboxyl groups, which reduces hydrophilicity, decreases charge repulsion, and accelerates hydrophobic interactions.¹³ Despite their differences in reaction stability, both methacrylating agents’ addition to SF's lysine side groups confers the protein with enhanced stability via chemical crosslinking bonds, thus improving the material's physical capabilities.¹⁴ In the field of tissue engineering, researchers have demonstrated the effect of methacrylating SF to improve the mechanical properties of SF-derived hydrogels.¹⁵ These improvements render SF more suitable for integration into tissue engineering applications, including as a potential biodegradable suture material for surgery.¹⁶

In our study, we proposed a standardized protocol for the synthesis of SF. Previous studies have reported a significant research gap in the variation of the degrees of methacrylation (DoM) between methacrylating agents like GMA and MA.¹¹ Specifically, varying DoM results in varying crosslinking abilities and thus variable physical properties. Previous research has proposed that GMA and MA have a twofold reactional difference with SF.¹⁷ Reports have also indicated a pH-associated inefficiency of MA during chemical synthesis, discussed in detail in a later section.¹³ Furthermore, in addition to pH, other studies have noted a need to standardize other factors in the methacrylation reaction to better characterize the material for tissue engineering applications.¹⁰ Among previous reports, unstandardized methacrylation methods have led to varied results and a difficult reproducibility.¹⁸ To address this, our group demonstrated the modification of SF with GMA and MA using a protocol that regulated dissolution and pH to produce SFGMA and SFMA solutions, respectively. While a wide range of methacrylating agents has been employed in previous studies,¹⁹ we chose to compare GMA and MA as they are the two foremost methacrylating agents across literature. Notably, differences between using GMA versus MA for silk fibroin methacrylation have not been extensively studied, aside from the acidic byproduct generated from MA.⁷ Herein, our group characterized the proton nuclear magnetic resonance (¹H NMR) spectra of SFGMA and SFMA to compare the respective DoM. From these results, SFGMA and SFMA were evaluated for their suitability in tissue engineering applications. Our work in comparing the DoM between SFGMA and SFMA and presenting a standardized protocol for methacrylation offers researchers an improved understanding of methacrylated silk fibroin. From our findings, we propose GMA to be a superior methacrylating agent over MA for its superior reaction and DoM.

2. Materials and methods

2.1. Synthesis of methacrylated silk fibroin

2 g of Bombyx mori SF lyophilized powder (catalog no. 5352, Advanced BioMatrix, Inc., San Diego, CA, USA) was sliced into small pieces and dissolved in 10 mL of 9.3 M LiBr (catalog no. 7550-35-8, Sigma-Aldrich, St. Louis, MO, USA) solution at 60 °C for 1 h in a 250 mL beaker according to a previous method,¹⁷ with a magnetic stirrer at a speed of 150 rpm. As shown in Table 1, for SFGMA synthesis, 0.2, 0.5, or 1.0 mL (147, 367, 733 mM) of GMA (catalog no. 106-91-2, Sigma-Aldrich, St. Louis, MO, USA) was added into the SF mixture, forming SFGMA147, SFGMA367, or SFGMA733, respectively. We divided the moles of GMA in the SFGMA samples by two for the corresponding SFMA samples, wherein 0.109, 0.273, or 0.546 mL (147, 367, 733 mM) of MA (catalog no. 760-93-0, Sigma-Aldrich, St. Louis, MO, USA) was added into the SF mixture, forming SFMA147, SFMA367, and SFMA733, respectively. Through this approach, we ensured that corresponding samples had double the number of molecules of GMA compared to MA, due to the twofold reactional difference as aforementioned. Both mixtures were dissolved at 60 °C for 3 h. The SFGMA mixture was dissolved at a stirring speed of 300 rpm, while the SFMA mixture was dissolved at 100–150 rpm (or lower as appropriate) due to increasing viscosity over time as well as foaming observed at higher stirring speeds. Beakers were sealed with parafilm to reduce evaporative loss.

Table 1 Volume of GMA and MA added to produce SFGMA and SFMA solutions of different molarities based on molar mass (GMA = 142.15 g mol⁻¹, MA = 154.16 g mol⁻¹) and density (GMA = 1.042 g mol⁻¹, MA = 1.035 g mol⁻¹)

	mL GMA	mL MA
147 mM	0.2	0.109
367 mM	0.5	0.273
733 mM	1.0	0.576

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The pH of the SFMA mixture was controlled by the addition of sodium hydroxide (NaOH) in 30-minute intervals as necessary to maintain a range (6 < pH < 9) consistent with the SFGMA mixture. For this experiment, the pH of the SFMA mixture was kept at 7.5 ± 0.5.

After fully dissolving, the SFGMA and SFMA solutions were poured into dialysis tubing (catalog no. 132754T, Fisher Scientific, Inc., Hampton, NH, USA) and clipped at both ends with dialysis tubing closures (catalog no. Z371017, MilliporeSigma, Burlington, MA, USA). The solutions were then dialyzed against deionized H₂O for at least 4 d at room temperature, with water changed twice daily.

Finally, the SFGMA and SFMA solutions were poured into conical tubes ¾ full (to accommodate expansion) and frozen in −80 °C for at least 12 h, before lyophilization in −50 °C for at least 48 h in a freeze dryer. The lyophilized SFGMA and SFMA had a final concentration of 4–5 wt%: a 2 g starting sample of lyophilized SF yielded 80–100 mg of lyophilized SFGMA and SFMA. This is consistent with available literature.⁸

Lyophilized samples were stored at −80 °C until use. For long-term storage, lyophilized samples can be stored for 1 year at high temperatures up to 37 °C and can be successfully reconstituted.²⁰ For short-term storage, lyophilized samples can be reconstituted in water into hydrogels, which can be stored for no longer than 2 weeks at low temperatures of 4 °C and in humid conditions to prevent the hydrogel from drying out.²¹ To prepare a 5% w/v hydrogel, add 5% w/v lyophilized SF-GMA or SFMA in diH₂O and 0.25% w/v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) to a syringe. Immediately after adding LAP, cover the syringe with tin foil to reduce light exposure which triggers photocrosslinking. Mix slowly across two syringes through a syringe coupler. If the solution remains inhomogeneous, sonicate for 30 s at a low speed to homogenize. Centrifuge, with the syringe tip facing up, at 200 g for 5 min, then remove air.

See Fig. 1 for an illustration of the SF methacrylation protocol.

Fig. 1 Synthesis of methacrylated SF by dissolving, dialyzing, and lyophilizing the solution.

2.2. NMR characterization

The success of methacrylation was characterized using ¹H NMR spectrometry, depicted in Fig. 2, the mechanism is shown in Fig. 3. Briefly, 15 mg of the lyophilized sample was sliced into fine pieces and added to a 5 mL scintillation vial. Next, 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (TMSP) (catalog no. 269913, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in a solvent of 0.6–1 mL D₂O (catalog no. 7789-20-0, Sigma-Aldrich, St. Louis, MO, USA) at 1 mg mL⁻¹. The TMSP and D₂O solution was then added to the scintillation vial and placed on heat (60 °C) for at least one hour or until mostly dissolved. Lastly, the fully homogenous solution was added to an NMR tube (catalog no. 6981A-7, Corning, Inc., Corning, NY, USA) for spectrometry. Undissolved sample was not added to the NMR tube.

Fig. 2 Preparation of methacrylated SF for NMR spectroscopy.

Fig. 3 Chemical reaction between SF and (A) GMA to produce SFGMA, (B) MA to produce SFMA. Created with BioRender.com.

¹H-NMR was analyzed with MestReNova. Spectra were normalized with the signal characteristic of SF's aromatic acids (δ = 7.5–6.5 ppm). DoM was calculated through integration of the lysine signals (δ = 2.95–2.8 ppm, in accordance with the widely used Habeeb method:²². Other relevant signals confirming the success of methacrylation, such as methacrylate vinyl²³ (δ = 6.2–5.6 ppm) indicating GMA, methylene²⁴ (δ = 5.8–5.2 ppm) indicating MA, and methyl¹⁷ (δ = 2–1.8 ppm), were also evaluated.

3. Results

The relevant peaks in the SFGMA and SFMA ¹H-NMR spectra are depicted in Fig. 4. The behavior of these signals upon the addition of a methacrylating agent is summarized in Table 2. The tyrosine (aromatic acid) signal of SF (δ  =  7.5–6.5 ppm) is characteristic of SF and can be used to normalize the ¹H-NMR spectra. Increasing concentrations of GMA and MA modifies an increasing number of SF lysine residues, resulting in a decrease in the lysine signal (δ = 2.95–2.8 ppm). An increase in methacrylating agent concentration corresponds to an increase in the methyl signal, in accordance with previous literature.¹⁷ The methacrylate vinyl signals similarly increase relative to the methacrylating agent concentration as proof of successful substitution. This can be observed through the integration of the two peaks in methacrylate vinyl (δ = 6.2–6 and 5.8–5.6 ppm) and methylene (δ = 5.8–5.6 and 5.4–5.2 ppm) for GMA and MA, respectively.²⁴

Fig. 4 Representative ¹H-NMR spectra of (A) SFGMA and (B) SFMA. Blue: aromatic acid (blue). Green: methacrylate vinyl (GMA) or methylene (MA). Yellow: lysine. Red: methyl.

Table 2 Peak ranges and behaviors of the characteristic signals of SF, GMA, and MA in ¹H NMR

	Characteristic signal	Peak range (ppm)	Behavior upon methacrylation
SF	Aromatic acid	7.5–6.5	Not affected
SF	Lysine	2.95–2.8	Decreases
SF	Methyl	2–1.8	Increases
GMA	Methacrylate vinyl	(1) 6.2–6, (2) 5.8–5.6	Increases
MA	Methylene	(1) 5.8–5.6, (2) 5.4–5.2	Increases

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Our results for NMR peak integrations and DoM are summarized in Tables 3 and 4, and presented in Fig. 5 and 6, for SFGMA and SFMA, respectively. For the SFGMA samples, the addition of GMA produced a significant effect on the signals of lysine, and thus DoM, as well as methacrylate vinyl, but not the methyl signal. Specifically, with increasing concentrations of GMA, the lysine signal decreased, and thus DoM increased, and the methacrylate vinyl signal increased. On the other hand, for the SFMA samples, the addition of MA yielded similar trends but did not produce significant effects on any signal. We hypothesize that this may be due to the small difference in molarities between the different samples. We chose to compare GMA molarities of 147, 367, and 733 which corresponded to 0.2-, 0.5-, and 1.0-mL volumes according to molar mass. Our 367 mM sample represents an ideal concentration, while our 147 mM and 733 mM samples represent nonideal concentrations for tissue engineering applications. To better characterize the effect of the methacrylating agent and DoM, future studies can consider using samples with even lower or even higher molarities.

Table 3 Peak integrations and degrees of methacrylation of SFGMA samples (n = 2). Degree of methacrylation was calculated according to the previously described Habeeb method. p values in this table were all obtained via the one-way analysis of variance test. p < 0.05 denotes statistical significance

	Lysine	Methyl	Methacrylate vinyl	DoM (%)
SF	0.24 ± 0.01	0.13 ± 0	0.06 ± 0.04	N/A
SFGMA147	0.20 ± 0.01	0.19 ± 0	0.14 ± 0.27	20.41 ± 8.66
SFGMA367	0.17 ± 0.01	0.26 ± 0.01	0.49 ± 0.16	30.61 ± 5.77
SFGMA733	0.17 ± 0.01	0.32 ± 0.05	0.50 ± 0.11	32.65 ± 2.89
p value	0.01	0.10	0.04	0.01

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Table 4 Peak integrations and degrees of methacrylation of SFMA samples (n = 2). Degree of methacrylation was calculated according to the previously described Habeeb method. p values in this table were all obtained via the one-way analysis of variance test. p < 0.05 denotes statistical significance

	Lysine	Methyl	Methylene	DoM (%)
SF	0.24 ± 0.01	0.13 ± 0	0.06 ± 0.04	N/A
SFMA147	0.23 ± 0.02	0.16 ± 0.01	0.10 ± 0.08	8.16 ± 8.66
SFMA367	0.22 ± 0.01	0.18 ± 0.04	0.38 ± 0.44	12.24 ± 2.89
SFMA733	0.21 ± 0.05	0.59 ± 0.47	0.77 ± 0.23	16.33 ± 20.20
p value	0.56	0.30	0.14	0.38

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Fig. 5 ¹H-NMR peak integrations of SF, SFGMA147, SFGMA367, and SFGMA733. For the lysine signal, integrations decreased with increasing GMA concentrations. For the methyl and methacrylate vinyl signals, integrations increased with increasing GMA concentrations.

Fig. 6 ¹H-NMR peak integrations of SF, SFMA147, SFMA367, and SFMA733. Changes in integration across MA concentrations were not significant.

A comparison of the samples with corresponding molarities is shown in Fig. 7. Our results reveal that the DoM of the SFGMA samples is approximately double that of SFMA.

Fig. 7 Degrees of methacrylation of SFGMA and SFMA at various concentrations (147, 367, and 733 mM).

Additional characterization of methacrylated SF, in addition to NMR, can be performed using circular dichroism (CD) spectroscopy. According to previous studies, for the unmodified protein, CD spectra revealed unordered conformation in a water solvent, β-sheet conformation in an aqueous methanol or dioxane solvent, and α-helices in an aqueous trifluoroethanol solvent.²⁵ In comparing the unmodified versus modified protein, studies have also shown that the unmodified protein exhibited a negative peak at 190–200 nm, suggesting an unordered conformation as aforementioned, while the modified protein exhibited a negative band at 210–220 nm, suggesting increased β-sheet conformation, which confirms the enhanced stability of the protein post-methacrylation.²⁶

4. Discussion

4.1. Methacrylation

Improving biomaterials’ structural and biological properties is a major focus in the field of tissue engineering. With these platforms, researchers can develop practical and economical strategies for repairing tissues.²⁷ In the past decade, methacrylation has become an increasingly used approach for enhancing biomaterials’ hydrogel properties. Specifically, the methacrylate group modification confers biomaterials with photocrosslinking abilities, which consequently impacts hydrogels’ pore size, porosity, and mechanical strength.²⁸ With respect to methacrylating SF, studies have demonstrated improved physical properties attributed to increasing DoM.²⁹

Our study used two methacrylating agents to modify SF, namely GMA and MA. Fig. 3 depicts the two synthetic chemical reactions of producing SFGMA and SFMA, respectively. In this reaction, both GMA and MA introduce methacryloyl groups to SF, wherein methacryloyl substitution then enables free-radical polymerizations at the amine, hydroxyl, and carboxyl groups of SF.³⁰ The crosslinking process may be accelerated with the addition of a photoinitiator such as LAP to better control the crosslinking rate.³¹ The crosslinked network imparts SF with improved structural stability, a key advantage for hydrogels, scaffolds, and other structures in tissue engineering applications.

Interestingly, recent research has shown different chemical effects of GMA and MA on SF. Unlike with GMA, the reaction with MA produces an acidic by-product, methacrylic acid, which reduces solution pH. As a result, the free amino groups in SF may become ionized, altogether inhibiting the reaction and resulting in crystallization of SF.^13,30 Additionally, Kim et al. proposed that, as opposed to two GMA molecules, only one MA molecule can react with each free amino group on the lysine residue of SF's vinyl groups. Kim et al. also argued that because GMA can react with SF's hydroxyl and carboxyl groups while MA cannot, GMA may yield greater methacrylation capabilities than MA.¹⁷ Combined, the observed and hypothetical differences between GMA and MA in reacting with SF suggest GMA to be a superior methacrylation agent.

Our study aimed to further characterize these distinctions between GMA and MA. Specifically, we proposed a novel synthesis protocol by regulating solution pH and standardizing GMA and MA molarities. The SFMA solution pH was regulated with NaOH during the synthesis process to match the measured SFGMA solution pH of 7.5 ± 0.5. Previous studies have similarly used NaOH to control the pH of methacrylated SF solutions.¹⁸ In theory, the regulation of pH reduced the formation of methacrylic acid, which facilitated the reaction and reduced SF crystallization. The molarities of GMA and MA in their respective solutions were standardized using their molar masses, detailed above in Table 1. This allowed for a direct comparison of the DoM between SFGMA and SFMA. With this approach of controlling pH and molarities, we hypothesized that the DoM in SFGMA would be approximately double that of SFMA due to the previously discussed reactional differences between GMA and MA with the lysine residue of SF's vinyl groups.

Our findings in SFGMA's doubled DoM compared to SFMA are in accordance with our previous discussion of the differences between GMA and MA in reacting with amino groups on SF lysine residues. Interestingly, despite our control of pH in the synthesis reactions, crystallization was still observed in the SFMA samples to a considerable extent. Crystallization suggests that our SFMA protocol is still imperfect in reducing the acidic by-product formation. Moving forward, standardizing other factors in addition to molarity and pH may be beneficial for researchers considering the use of GMA, MA, or other compounds to modify SF.

4.2. Tissue engineering applications

Unmodified SF exhibits a low concentration (6% w/v) after standard processing techniques, which is too low viscosity for bioprinting.³² To address this limitation, methacrylating SF with methacrylating agents, among other materials, has proven to increase viscosity to improve printability. To date, the concentration of SFGMA or SFMA used in tissue engineering ranged between 5% to 30% w/v, with specific concentrations largely application-dependent.^7,10,33 For example, 10% to 20% w/v hydrogels are favorable for enhancing vascular networks,³⁴ while 30% w/v hydrogels exhibit strength required for tracheal rings and other high strong, suturable sturctures.⁷

The chemical modification of SF with methacrylating agents represents a promising platform for the field of tissue engineering. Recently, this platform has been explored in terms of manufacturing hydrogels,³⁵ mats,³⁶ sponges,³⁷ and more. 3D bioprinting has also emerged as a popular approach to constructing anatomical structures in the bone,³⁸ cartilage,³⁹ skin,⁴⁰ and nerve systems.⁴¹ In this light, our proposed synthesis approach of SFGMA and SFMA is relevant to studies using GMA and MA for tissue engineering. Our findings with respect to the difference in DoM suggest the SFGMA platform to be superior than that of SFMA. Nevertheless, future research using these platforms may seek to combine GMA and MA with a blend of other biomaterials and seeded cell types.^{24,38,41–43} This is relevant to further optimize methacrylation, and thus improve physical properties, in an effort to simulate the natural tissue environment of various tissue types. Altogether, further experimentation with the methacrylated SF technique is necessary for advancing its applicability in biomedical research.

5. Conclusion

In this work, we proposed a novel approach to synthesize SFGMA and SFMA by controlling solution pH and molarity. Through this approach, we were able to quantitatively assess the two solutions’ respective DoM. Our results demonstrate GMA to be the optimal methacrylating agent over MA for its more consistent reaction and better-defined DoM. Unlike the GMA group, we found the relationship between MA and DoM to be insignificant, where increasing MA was weakly correlated with increasing DoM. Additionally, we observed crystallization in the SFMA samples, which was not evident in the SFGMA samples. As such, further studies may need to incorporate even higher or even lower molarities than our chosen samples to better characterize the relationship between MA and DoM, while also seeking to control factors besides molarity and pH during SFMA synthesis. Altogether, the methacrylation of SF is a promising technique and may be further optimized to improve its biological, chemical, and physical properties. These characteristics make SF a promising platform for applications across the tissue engineering, regeneration, and therapy fields.

Conflicts of interest

There are no conflicts to declare.

Data availability

The datasets generated and analyzed during the current study are not publicly available but are available from the authors on reasonable request.

Note added after first publication

This article replaces the version published on 27 November 2025, which contained errors in the figure captions for Fig 5. and Fig. 6.

Acknowledgements

This NMR was funded significantly by an NIH High End Instrumentation grant (1 S10 0D028697-01). We would also like to thank the generous donation from Mr. Kevin Taweel, who has made this research possible.

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