Click chemistry improved wet adhesion strength of mussel-inspired citrate-based antimicrobial bioadhesives

Abstract

For the first time, a convenient copper-catalyzed azide-alkyne cycloaddition (CuAAC, click chemistry) was successfully introduced into injectable citrate-based mussel-inspired bioadhesives (iCMBAs, iCs) to improve both cohesive and wet adhesive strengths and elongate the degradation time, providing numerous advantages in surgical applications. The major challenge in developing such adhesives was the mutual inhibition effect between the oxidant used for crosslinking catechol groups and the Cu(II) reductant used for CuAAC, which was successfully minimized by adding a biocompatible buffering agent typically used in cell culture, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), as a copper chelating agent. Among the investigated formulations, the highest adhesion strength achieved (223.11 ± 15.94 kPa) was around 13 times higher than that of a commercially available fibrin glue (15.4 ± 2.8 kPa). In addition, dual-crosslinked (i.e. click crosslinking and mussel-inspired crosslinking) iCMBAs still preserved considerable antibacterial and antifungal capabilities that are beneficial for the bioadhesives used as hemostatic adhesives or sealants for wound management.

Keywords: Antimicrobial; Bioadhesives; Citric acid; Click chemistry; Mussel.

Fig. 1

FTIR spectra of iC-3N₃ (A) and iC-2PL (B) pre-polymers; ¹H-NMR spectra of alkyne functionalized gelatin (Gelatin-Al, GL, C); and UV-vis spectra (D) of iC-3N₃ and iC-2PL pre-polymers.

Fig. 2

Gel times of iC-P₄-3N₃/2PL equal-weight mixture crosslinked by different formulations of CuSO₄-sodium L-ascorbate (NaLAc) (A); the effect of HEPES to the gel times of iC-P₄-3N3/2PL equal-weight mixture crosslinked by CuSO₄-NaLAc (B) and dual-crosslinked by CuSO₄-NaLAc and PI (C); gel times of dual-crosslinked iC-X-PI at 37°C (D, X = click).

Fig. 3

Rheological analysis on the gelation of iC-P₄-3N₃/2PL (1:1 weight ratio) solution (50 wt%) crosslinked by oxidant (8 wt% PI, black color), CuAAC (CuSO₄-NaLAc-HEPES, 5-50-20 mM, blue color), or dual-crosslinking (by CuSO₄-NaLAc -HEPES (5-50-20 mM) and 4 wt% PI together, red color) at 25°C.

Fig. 4

Evaluation of mechanical properties, showing stress-strain curves (A), degradation profiles (B), sol contents (leachable fractions) (C), and swelling ratios (D) of clickable iC mixture crosslinked by either sodium periodate (PI) or copper-catalyst, or both.

Fig. 5

Adhesion strength of fibrin glue, and normal iC and clickable iCs crosslinked by sodium periodate (PI), CuSO₄-NaLAc-HEPES or both, onto wet porcine small intestine submucosa, determined by lap shear strength test.

Fig. 6

Cytotoxicity evaluation of clickable iC family: MTT assay for hMSCs cultured with: iC-click pre-polymers and Gelatin-Al (GL) (A), leachable part (sol content) (B) and degradation product (C) of clickable iCs crosslinked through different routes for 24 hours. Live/Dead assay for hMSCs seeded on dual-crosslinked iC-X-PI casted glass slides 1, 4, and 7 days post cell seeding (D).

Fig. 7

Antibacterial and antifungal performance of clickable iCs (iC-X) hydrogels: Inhibition ratios kinetics curves of crosslinked iC-X series, iC-P₄-PI 4wt%, PEGDA/HEMA (w/w=1/1, as negative control), and Hydrofera Blue (as positive control) against S. aureus (A) and E. coli. (B); Fungal survival ratios after direct exposure to crosslinked iC-click series, iC-P4-PI 4wt%, PEGDA/HEMA, and Hydrofera Blue for 3 hrs (C) (**p<0.01). Long-term antimicrobial performance of iC-X series: Antifungal and antibacterial performance of degradation products at different dilutions (1×, 10×, and 100×) (D) and periodical release solutions (E) of crosslinked hydrogels: iC-X series, iC-P₄-PI 4wt%, PEGDA/HEMA (w/w=1/1, as negative control), and Hydrofera Blue (as positive control).

Scheme 1

Synthesis schemes of triazide (3N₃) (A) monomer, dipropiolate (2PL) (B) monomer, and clickable injectable citrate-based mussel-inspired bioadhesive pre-polymers (iC-3N₃ and iC-2PL) (C).

Scheme 2

Dual crosslinking (oxidant and click (CUAAC)) of clickable iC (mixture of iC-3N₃ and iC-2PL).