Neutrophil extracellular traps-inspired DNA hydrogel for wound hemostatic adjuvant

Abstract

Severe traumatic bleeding may lead to extremely high mortality rates, and early intervention to stop bleeding plays as a critical role in saving lives. However, rapid hemostasis in deep non-compressible trauma using a highly water-absorbent hydrogel, combined with strong tissue adhesion and bionic procoagulant mechanism, remains a challenge. In this study, a DNA hydrogel (DNAgel) network composed of natural nucleic acids with rapid water absorption, high swelling and instant tissue adhesion is reported, like a band-aid to physically stop bleeding. The excellent swelling behavior and robust mechanical performance, meanwhile, enable the DNAgel band-aid to fill the defect cavity and exert pressure on the bleeding vessels, thereby achieving compression hemostasis for deep tissue bleeding sites. The neutrophil extracellular traps (NETs)-inspired DNAgel network also acts as an artificial DNA scaffold for erythrocytes to adhere and aggregate, and activates platelets, promoting coagulation cascade in a bionic way. The DNAgel achieves lower blood loss than commercial gelatin sponge (GS) in male rat trauma models. In vivo evaluation in a full-thickness skin incision model also demonstrates the ability of DNAgel for promoting wound healing. Overall, the DNAgel band-aid with great hemostatic capacity is a promising candidate for rapid hemostasis and wound healing.

Fig. 1. Scheme of DNA hydrogel band-aid for accelerated hemostasis that can fulfill the clinical requirements of stabilizing a profuse bleeding wound.

Physiologically, neutrophils at the wound site released extracellular DNA to form traps (NETs) that promote fast and stable thrombosis. Similarly, our form factor conforming DNA hydrogel was able to form NETs-like thrombosis. Besides the conformational advantage, DNAgel is also highly absorptive of blood at the wound edge and high adhesive to the wetted wound surfaces, thus forming both a physical blockade and biological clot to stop the bleeding.

Fig. 2. Preparation and expansion properties of DNAgel.

a Schematic of the synthesis process. b Photographs of the DNA solution before and after crosslinking. Scale bar: 1 cm. c SEM image of DNAgel. Scale bar: 100 μm. d G′ and G″ of DNAgel on strain amplitude sweep. e ATR-FTIR analysis of pure salmon sperm DNA and DNAgel. f Images of water droplet absorbed by DNAgel. Scale bar: 1 mm. g Representative photographs of DNAgel in 2D and 3D shape during swelling process. Scale bar: 2 cm. h Mask Cutting, i macromolding for macro manufacturing (Scale bar: 2 cm) and j micromolding for micro-manufacturing (Scale bar: 2 mm) to obtain DNAgel in diverse shapes. Representative images are shown from three (b, c, f–j) independent experiments with similar results. Source data are provided as a Source Data file.

Fig. 3. DNAgel exhibited excellent wet adhesion to tissue.

a Bending shape of the DNAgel. Scale bar: 5 mm. b Schematic of adhesion formation between DNAgel and wet tissue. c Lap shear tests and d 90-degree peel tests of DNAgel with rat dorsal skin (1 cm*2 cm). Scale bar: 5 cm. Data were presented as mean ± SD (n = 3). e Adhesion of a DNAgel on rat liver in vivo. Scale bar: 1 cm. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons (c, d). Representative images are shown from three (a, c–e) independent experiments with similar results. Source data are provided as a Source Data file.

Fig. 4. DNAgel exhibited excellent in vitro procoagulant ability and biocompatibility.

a Representative SEM images of erythrocytes (red) and platelet (yellow) adhesion to the gelatin sponge (GS) with porous cavity and DNAgel with dense surface. Scale bar: 10 μm. b CLSM images of erythrocytes (red) and platelets (green) aggerated and adhered to the surface of DNA network (blue). Scale bar: 200 μm (left); Scale bar: 100 μm (right, enlarged). c Absorbance of blood clotting samples at different time points measured at 540 nm with UV–vis spectrophotometer and the representative pictures at 30 min. Scale bar: 1 cm. d Blood clotting index (BCI) of DNAgel and representative pictures, according to BCI(%) = $\frac{A_{\mathrm{Experimental}}}{A_{\mathrm{Negative}}}\times 100\%$. Scale bar: 1 cm. Data were presented as mean ± SD (n = 3 biologically independent samples). e Hemolysis ratio of DNAgel, according to Hemolysis ratio (%) = $\frac{A_p}{A_t}\times 100\%$. f Schematic illustration of adhesion and activation of erythrocytes and platelets on DNA gel. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons (c, d). Representative images are shown from three (a–d) independent experiments with similar results. Source data are provided as a Source Data file.

Fig. 5. DNAgel showed hemostatic performance in both superficial and deep bleeding models in vivo.

a–c Established SD rat tail amputation, liver puncture and femoral artery injury model, and the dynamic hemostatic ability using either gelatin sponge (GS) or DNAgel. Scale bar: 1 cm. Data were presented as mean ± SD (n = 3). Panels a–c was in part created with Biorender.com. d Peritoneal lavage fluid samples after femoral artery and liver hemostasis. Scale bar: 5 mm. Error bar, SDs (n = 5). (*p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons (a–d). Representative images are shown from three (a–d) independent experiments with similar results. Source data are provided as a Source Data file.

Fig. 6. DNAgel promoted platelet activation and accelerated the clotting process by upregulating related genes.

a Signaling pathway of clotting cascade triggered by DNAgel. Panel a was in part created with Biorender.com. b Statistical results of platelet activation and c representative flow cytometry plots under different treatment conditions. Data were presented as mean ± SD (n = 3). d RNA-seq analysis of whole blood cells, and differential expressed genes presented in a volcano map. Cutoff line: Log₂ fold change (Log₂FC) > |2| and adjusted p value < 0.01. e Heatmap of differential expressed genes related to platelet activation and f GSEA of KEGG pathway analysis related to platelet. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons (b). Source data are provided as a Source Data file. RNAseq data generated in this study have been deposited in the GEO DataSets under accession code GSE268849.

Fig. 7. DNAgel promoted wound healing and completed skin regeneration.

Six full-thickness skin defects, 1 cm in diameter, were created symmetrically along the spine on the back of each rat using a hole punch and were treated according to the group. a Representative wound images during healing process on Day 0, 3, 5, 7, 10. Scale bar: 2 mm. b Wound traces at different days. c Wound size and d healing rate at different days for each group, according to Extent of healing (%) = $\left(1-\frac{A_t}{A_0}\right)\times 100\%$. Data were presented as mean ± SD (n = 3). e Representative images of immunohistochemistry staining with CK14 (red) and DAPI (blue) on Day 7 (Wound closure time points in DNAgel group) and Day 10 (Total skin regeneration time point in DNAgel group) for each group. Scale bar: 100 μm. f Hematoxylin and Eosin staining, Masson’s Trichrome staining and Sirius Red staining on Day 7 and 10 of the newly regenerated skin tissues for each group. The purple dotted line marks the initial trauma margin. Scale bar: 1 mm (top); Scale bar: 100 μm (bottom, enlarged). Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons (c, d). Representative images are shown from two (a, e, f) independent experiments with similar results. Source data are provided as a Source Data file.