Fabrication of Quercetin-Functionalized Morpholine and Pyridine Motifs-Laden Silk Fibroin Nanofibers for Effective Wound Healing in Preclinical Study

Abstract

Choosing suitable wound dressings is crucial for effective wound healing. Spun scaffolds with bioactive molecule functionalization are gaining attention as a promising approach to expedite tissue repair and regeneration. Here, we present the synthesis of novel multifunctional quercetin with morpholine and pyridine functional motifs (QFM) embedded in silk fibroin (SF)-spun fibers (SF-QFM) for preclinical skin repair therapies. The verification of the novel QFM structural arrangement was characterized using ATR-FTIR, NMR, and ESI-MS spectroscopy analysis. Extensive characterization of the spun SF-QFM fibrous mats revealed their excellent antibacterial and antioxidant properties, biocompatibility, biodegradability, and remarkable mechanical and controlled drug release capabilities. SF-QFM mats were studied for drug release in pH 7.4 PBS over 72 h. The QFM-controlled release is mainly driven by diffusion and follows Fickian's law. Significant QFM release (40%) occurred within the first 6 h, with a total release of 79% at the end of 72 h, which is considered beneficial in effectively reducing bacterial load and helping expedite the healing process. Interestingly, the SF-QFM-spun mat demonstrated significantly improved NIH 3T3 cell proliferation and migration compared to the pure SF mat, as evidenced by the complete migration of NIH 3T3 cells within 24 h in the scratch assay. Furthermore, the in vivo outcome of SF-QFM was demonstrated by the regeneration of fresh fibroblasts and the realignment of collagen fibers deposition at 9 days post-operation in a preclinical rat full-thickness skin defect model. Our findings collectively indicate that the SF-QFM electrospun nanofiber scaffolds hold significant capability as a cost-effective and efficient bioactive spun architecture for use in wound healing applications.

Keywords: antibacterial; antioxidant; functionalized quercetin; rapid wound healing; spun silk fibroin; tissue engineering.

Scheme 1

Schematic illustration for the synthesis of quercetin pyridyl hydrazone (QFM).

Figure 1

(A) ATR-FTIR spectrum of synthesized QFM, SF, and SF-QFM fibrous scaffolds; (B) thermogravimetric analysis (TGA) of QFM, SF, and SF-QFM scaffolds.

Figure 2

(A–A2) SEM micrographs of the SF nanofibers; (B–B2) SF-QFM nanofibers; (C) fiber diameter distribution graph of the SF and SF-QFM electrospun nanofibers.

Figure 3

(A) In vitro water retention ability; (B) degradation profiles; (C) water contact angle; (D) cumulative drug release profiles of SF and SF-QFM nanofibers matrix.

Figure 4

(A) Antimicrobial activity of control, standard, and SF and SF-QFM nanofibers scaffolds samples against S. aureus, E. coli, P. aeruginosa, and E. faecalis. (B) ZOI difference in SF-QFM and standard. (C) DPPH free radical scavenging activity percentage against various concentrations of QFM. (D) Results of the in vitro cytotoxicity (MTT) assay for synthesized QFM.

Figure 5

(A) In vitro biocompatibility of SF and SF-QFM nanofibers scaffolds. (B) Quantification of NIH 3T3 fibroblast cells at various time points (scale bar: 100 μm). *** p < 0.001.

Figure 6

(A) In vitro wound healing (scratch assay) study of SF and SF-QFM fiber scaffolds. (B) Quantification of NIH 3T3 fibroblast cells at various time points (scale bar: 100 μm). *** p < 0.001.

Figure 7

(A) Representative photographs depict the progression of wound healing; (B) the wound closure rates; (C) quantification of length of wound area at day 9. The significant difference (** p < 0.01, *** p < 0. 001).

Figure 8

H&E staining image of the histological section of control, SF, and SF-QFM fibrous scaffolds on the fourth and eighth days (200× and 800×; scale bar 100 μm).

Figure 9

Masson’s trichrome staining images of control, SF, and SF-QFM fibrous scaffolds on the fourth and eighth days (200× and 800×; scale bar 100 μm).