Hydrogels cross-linked by native chemical ligation

Abstract

We describe the use of native chemical ligation (NCL) reaction to covalently cross-link soluble polymers into hydrogels. Macromonomers consisting of a four-armed poly(ethylene glycol) (PEG) core end-functionalized with either thioester or N-terminal cysteine peptide were designed and synthesized. Upon mixing aqueous solutions of the thioester and N-terminal cysteine macromonomers, rigid hydrogels formed within minutes. The gelation time was affected by choice of buffer, pH, polymer concentration, reaction temperature, and chemical composition of the N-terminal cysteine conjugate. The kinetics of gel formation and the viscoelastic behavior of selected hydrogels were further studied by oscillatory rheology, which demonstrated a minimum gel formation time of approximately two minutes and the formation of an elastic cross-linked hydrogel via the NCL reaction. A useful feature of this hydrogel strategy is the regeneration of thiol functional groups as a result of the NCL reaction, thereby allowing functionalization of the polymer hydrogel with biomolecules. This was demonstrated by conjugation of a maleimide-GRGDSPG-NH(2) peptide to an NCL hydrogel, permitting the attachment of human mesenchymal stem cells (hMSCs) on the hydrogel. Due to the mild reaction conditions, chemoselectivity, and potential for biological functionalization, our approach may prove useful as a general method for hydrogel formation, including hydrogels intended for biomedical applications.

Figure 1

Oscillatory rheology of 10% macromonomer 2 in 100 mM NH₄HCO₃, pH 8.3. Storage (G′) and loss (G″) modulus vs time are shown (rheology method 2).

Figure 2

Oscillatory rheology of a mixture of 10% macromonomer 1 and 10% macromonomer 2 in 100 mM NH₄HCO₃, pH 8.3. Storage (G′) and loss (G″) modulus vs time are shown (rheology method 2).

Figure 3

Oscillatory rheology of a mixture of 20% macromonomer 1 and 20% macromonomer 3b in 100 mM NH₄HCO₃, pH 8.3. Storage (G′) modulus vs time is shown (rheology method 1).

Figure 4

Human mesenchymal stem cell culture on NCL hydrogel of 1 and 2 (10%) before (A) and after functionalization with maleimide-GRGDSPG-NH₂ peptide (B). Image analysis revealed cell adhesion on nonmodified NCL hydrogel to be <0.1% of available surface area (A), whereas cell adhesion increased to 68 ± 7.2% of available surface area on hydrogel functionalized with maleimide-GRGDSPG-NH₂ peptide (B). Only minimal cell adhesion was observed on hydrogel surface that was treated with nonthiol reactive Ac-GRGDSPG-NH₂ peptide (C).

Scheme 1

Chemistry of Native Chemical Ligation

Scheme 2

Structures of Thioester-Polymer 1 and N-Terminal Cysteine-Polymer Bioconjugates 2 and 3a–e

Scheme 3

Synthesis of Macromonomer 1

Scheme 4

Synthesis of N-Terminal Cysteine Macromonomers 3a–e