One potent sponge based on plant-protein-polyphenol assemblies for coagulopathic hemostasis

Abstract

Commercially available gelatin sponges are widely used in coagulation-dependent bleeding wounds due to its porous structure that concentrates blood cells, and coagulation factors. However, effective hemostasis cannot currently be achieved under coagulopathic conditions. In this study, we designed a procoagulant zein-polyphenol conjugate (ZC) nanoassemblies prepared using an anti-solvent strategy, were directly applied to the surface functionalization of commercially available gelatin sponges. Polyphenols give ZC coatings excellent adhesion and enhance the gelatin sponge's procoagulant properties. Leveraging their distinctive secondary structure, the optimized ZC nanoparticle-coated sponges demonstrated enhanced in vitro hemostatic properties compared to unmodified commercial gelatin sponges and exhibited superior red blood cell and platelet adhesion characteristics. Additionally, the sponges enhanced with the ZC coating exhibited superior hemostatic potential in a femoral-artery-injury model, under both coagulation-dependent and coagulopathic conditions. The current study presents a promising approach for the utilisation of zein-based procoagulant materials for versatile hemostatic materials engineering applications.

Keywords: Coating; Hemostasis; Polyphenol; Sponge; Zein.

Graphical abstract

Fig. 1

Synthesis route of ZC, schematic illustration of the preparation of Z@GS and ZC@GS sponge for hemostatic applications.

Fig. 2

(a) ¹H NMR spectra, (b) conditions of dissolution in different solvent, (c) elemental analysis results (N/C ratio) and (d) fluorescence spectra of Z and ZC. (e) Secondary structure content by FTIR analysis and (f) SEM images and corresponding size distribution (inset) of Z and ZC nanoassemblies (n ≥ 200).

Fig. 3

(a) Photograph, (b) SEM images of the top-surface view of GS, Z₆₅@GS and ZC₆₅@GS. (c) Porosity (data are presented as the mean ± SD, n = 3), (d) swelling ratio in PBS at 30 min (data are presented as the mean ± SD, n = 3), and (e) the uniaxial compression stress-strain curves of GS, Z@GS and ZC@GS sponge.

Fig. 4

(a) L929 cell viability (data are presented as the mean ± SD, n = 3, one-way ANOVA), (b) hemolysis rate (data are presented as the mean ± SD, n = 3, one-way ANOVA), (c) APTT (data are presented as the mean ± SD, n = 3, one-way ANOVA), (d) BCI value (data are presented as the mean ± SD, n = 3, one-way ANOVA), (e) RBC-adhesion ratio (with plasma proteins) (data are presented as the mean ± SD, n = 3, one-way ANOVA), (f) platelet-adhesion ratio (with plasma proteins) (data are presented as the mean ± SD, n = 3, one-way ANOVA) and (g) whole-protein adhesion ratio under PPP conditions of GS, Z@GS and ZC@GS (data are presented as the mean ± SD, n = 3, one-way ANOVA). The scavenging efficiency of GS, Z@GS and ZC@GS for (h) DPPH free radical (data are presented as the mean ± SD, n = 3, one-way ANOVA) and (i) superoxide free radical (data are presented as the mean ± SD, n = 3, one-way ANOVA). (ns denotes no significant difference, while ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 represent statistically significant differences, one-way ANOVA).

Fig. 5

(a) Schematic illustration of the application, (b) pre-treatment blood loss (data are presented as the mean ± SD, n ≥ 3, one-way ANOVA), (c) post-treatment blood loss (data are presented as the mean ± SD, n ≥ 3, one-way ANOVA), (d) bleeding time (data are presented as the mean ± SD, n ≥ 3, one-way ANOVA) and (e) representative photographs of GS, Z₆₅@GS and ZC₆₅@GS in a rat femoral-artery-injury model. (ns denotes no significant difference, while ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 represent statistically significant differences, one-way ANOVA).

Fig. 6

(a) Schematic illustration of the establishment of heparinized rats and corresponding in vitro/in vivo assays. (b) PT (data are presented as the mean ± SD, n = 3), (c) APTT values of whole blood drawn from heparinized SD rats (data are presented as the mean ± SD, n = 3). (d) BCI value of GS, Z@GS and ZC@GS adopting heparinized blood (data are presented as the mean ± SD, n = 3, one-way ANOVA). (e) Pre-treatment blood loss (data are presented as the mean ± SD, n = 3, Student's t-tests), (f) post-treatment blood loss (data are presented as the mean ± SD, n = 3, Student's t-tests), (g) bleeding time (data are presented as the mean ± SD, n = 3, Student's t-tests) and (h) representative photographs of GS and ZC@GS groups in femoral-artery-injury model of heparinized SD rats. (ns denotes no significant difference, while ∗ p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 represent statistically significant differences).

Fig. 7

(a) In vitro degradation rate (data are presented as the mean ± SD, n = 3), (b) in vivo photographs of degradation of GS and ZC₆₅@GS (n = 3). (c) Quantitative analysis of positive regions (data are presented as the mean ± SD, n = 3, one-way ANOVA), (d) staining images of tissues surrounding of control, GS and ZC₆₅@GS for TNF-α in a subcutaneous implantation model of healthy rats (n = 3). (ns denotes no significant difference, while ∗ p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 represent statistically significant differences).