Co-delivery of deferoxamine and hydroxysafflor yellow A to accelerate diabetic wound healing via enhanced angiogenesis

Abstract

Nonhealing chronic wounds on foot induced by diabetes is a complicated pathologic process. They are mainly caused by impaired neovascularization, neuropathy, and excessive inflammation. A strategy, which can accelerate the vessel network formation as well as inhibit inflammatory response at the same time, makes it possible for effective diabetic ulcers treatment. Co-delivery of multiple drugs with complementary bioactivity offers a strategy to properly treat diabetic wound. We previously demonstrated that hydroxysafflor yellow A (HSYA) could accelerate diabetic wound healing through promoting angiogenesis and reducing inflammatory response. In order to further enhance blood vessel formation, a pro-angiogenic molecular called deferoxamine (DFO) was topically co-administrated with HSYA. The in vitro results showed that the combination of DFO and HSYA exerted synergistic effect on enhancing angiogenesis by upregulation of hypoxia inducible factor-1 alpha (HIF-1α) expression. The interpenetrating polymer networks hydrogels, characterized by good breathability and water absorption, were designed for co-loading of DFO and HSYA aiming to recruit angiogenesis relative cells and upgrade wound healing in vivo. Both DFO and HSYA in hydrogel have achieved sustained release. The in vivo studies indicated that HSYA/DFO hydrogel could accelerate diabetic wound healing. With a high expression of Hif-1α which is similar to that of normal tissue. The noninvasive US/PA imaging revealed that the wound could be recovered completely with abundant blood perfusion in dermis after given HSYA/DFO hydrogel for 28 days. In conclusion, combination of pro-angiogenic small molecule DFO and HSYA in hydrogel provides a promising strategy to productively promote diabetic wound healing as well as better the repair quality.

Keywords: Hydroxysafflor yellow A; angiogenesis; deferoxamine; diabetic wound healing; hydrogel.

Graphical abstract

Figure 1.

(A) Effects of DFO/HSYA on migration of HaCats were assessed by an IBIDI wound healing assay from 0 to 24h. (B) Wound closure of HaCaTs were expressed as a percentage of the initial wound. *p<.05, #p<.01 versus control (n = 9; mean ± SD). (C) Fluorescent images of tube formation assay in three-dimensional Matrigel after treatment of HSYA/DFO for 6h (scale bars: 500um, magnification: 10x). (D) Quantitative analysis of the total tube length in HSYA-treated HUVECs using the ImageJ software. (E) Cellular immunofluorescence staining of HIF-1α in human dermal fibroblasts (scale bars: 200um). (F) Quantification of the cellular immunofluorescence. *p<.05 and **p<.01 versus control; #p<.01 versus HSYA alone (n = 3; mean ± SD).

Figure 2.

(A) Schematic diagram of IPN porous structure using chitosan and gelatin. (B) SEM images of HSYA hydrogels produced at a variety of GL/Chi ratio; Scale bars: 200um; (a) 3:7, (b) 5:5, (c) 7:3, and (d) 7:3. (120×). (C) Cumulative release profiles of HSYA-loaded hydrogels. (n = 3; mean ± SD). (D) Cumulative release profiles of HSYA-DFO hydrogels. (GL/Chi ratio = 5:5; n = 3; mean ± SD). (E) Rheology test of HSYA-DFO hydrogels. (G’: storage modulus, G”: loss modulus).

Figure 3.

(A) Representative images of wound healing a particular time point. (B) Quantitative wound closure percentage at a particular time point using the ImageJ software. (C) Representative images of wound healing a particular time point. (D) Quantitative wound closure percentage at a particular time point using the ImageJ software. *p < .05 and **p < .01 versus control (n = 6; mean ± SD).

Figure 4.

(A) HE stained wound sections at day 28. Scale bars: 200 um. E: epidermis; GT: granulation tissue; D: dermal. (B) High power field (HPF) (40×) imaging of the center of wound granulation tissues. Scale bars, 50 um. Black arrows highlight the newly formed vascular. (C) Immuno fluorescence images for HIF-1α and CD31. Scale bars, 200um. Green: HIF-1α; Red: CD31; Blue: DAPI. (D) Granulation tissue thickness in the center of wounds. (E) Epidermis thickness in the center of wounds. *p < .05 and **p < .01 versus control (n = 6; mean ± SD).

Figure 5.

Cross-sectional combined ultrasound and photoacoustic images of diabetic wound for blood flow detection at day 28.