Transdermal deferoxamine prevents pressure-induced diabetic ulcers

Abstract

There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.

Keywords: angiogenesis; diabetes; drug delivery; small molecule; wound healing.

Fig. 1.

Development of a transdermal drug delivery system for DFO. DFO aggregates with PVP and surfactants to form reverse micelles (RMs). RMs are dispersed in the polymer ethyl cellulose. After release from the polymer matrix the RMs enter the stratum corneum and disintegrate. PVP dissolves and DFO is delivered to the dermis.

Fig. 2.

Encapsulation and controlled release of DFO by a TDDS. (A) SEM images of the TDDS at time 0 (Left) and 48 h post skin application (Right). Porous structure remains within the polymer after the drug is released to murine skin (Right). (Scale bars, 100 and 20 µm.) (B) AFM showing the topography of formed RM. (C) AFM phase imaging demonstrating DFO particles inside the RM. (D) Raman spectroscopy showing the lipid shell of the RM. (E) Raman imaging specific for DFO. (Scale bars, 2 µm.) (F) DFO TDDS delivery demonstrated a sustained drug release in vitro (n = 3). (G) In vitro penetration profile showing the concentration and location of DFO in full-thickness human skin after 24 h TDDS application (n = 3). (H) Application of different TDDS formulations on the intact skin of diabetic mice revealed an increase in HIF-1α stabilization in a dose-dependent manner. n = 3. All values represent mean ± SEM; *P < 0.05.

Fig. 3.

DFO TDDS improves healing of diabetic ulcers. (A) Full-thickness ulcer wounds of diabetic mice treated with a transdermal DFO TDDS formulation or vehicle control (n = 10). TDDS were replaced every 48 h. (B) Wound-healing kinetics (wound area as a function of time). Wound closure occurred significantly faster at day 27 in the DFO-treated group versus day 39 in the vehicle-treated controls (n = 10). (C) VEGF protein levels in ulcers of diabetic mice after transdermal DFO treatment for 1 and 2 d, respectively (n = 3). (D) Evaluation of VEGF protein expression in skin directly underneath the TDDS, adjacent to it, and 5 mm distant (n = 3). *P < 0.01.

Fig. 4.

Localized DFO treatment enhances neovascularization and dermal thickness. (A) Upon complete healing, immunohistochemistry was performed for the capillary endothelial cell marker CD31 (red). Increased vascularity was seen in transdermal DFO-treated diabetic mice. Blue indicates DAPI staining (Scale bars, 10 µm.) (B) Quantification of CD31-positive pixels per high-power field (HPF) (n = 10). (C) Dermal thickness of completely healed wounds was assessed by polarized light microscopy after picrosirius red staining. (Scale bars, 10 µm.) (D) Quantification of picrosirius red-positive pixels per HPF (n = 10). *P < 0.01.

Fig. 5.

Transdermal DFO treatment prevents ulcer formation in diabetic mice. (A) Representative photographs of skin after ulcer induction in diabetic mice pretreated with either DFO or control TDDS. No severe ulcer formation in the DFO-treated group. (B) Quantification of control and DFO-treated necrotic area (n = 10). (C) Representative histological H&E-stained tissue sections showing ulcer formation in the vehicle control group (n = 10). *P < 0.01. (Scale bars, 10 µm.)

Fig. 6.

DFO TDDS attenuates tissue necrosis by decreasing apoptosis and ROS stress. (A) Western blot analysis of Cleaved Caspase-3 (Cl Casp.3) and Bax proteins. DFO-pretreated mice show a significant reduction of both apoptotic markers. (B and C) Quantification of Western blot (n = 3). (D) DHE immunofluorescent stain for oxidative stress reveals decreased ROS accumulation (red) in DFO-treated wounds (n = 3). *P < 0.05.