In situ sprayed NIR-responsive, analgesic black phosphorus-based gel for diabetic ulcer treatment

Abstract

The treatment of diabetic ulcer (DU) remains a major clinical challenge due to the complex wound-healing milieu that features chronic wounds, impaired angiogenesis, persistent pain, bacterial infection, and exacerbated inflammation. A strategy that effectively targets all these issues has proven elusive. Herein, we use a smart black phosphorus (BP)-based gel with the characteristics of rapid formation and near-infrared light (NIR) responsiveness to address these problems. The in situ sprayed BP-based gel could act as 1) a temporary, biomimetic "skin" to temporarily shield the tissue from the external environment and accelerate chronic wound healing by promoting the proliferation of endothelial cells, vascularization, and angiogenesis and 2) a drug "reservoir" to store therapeutic BP and pain-relieving lidocaine hydrochloride (Lid). Within several minutes of NIR laser irradiation, the BP-based gel generates local heat to accelerate microcirculatory blood flow, mediate the release of loaded Lid for "on-demand" pain relief, eliminate bacteria, and reduce inflammation. Therefore, our study not only introduces a concept of in situ sprayed, NIR-responsive pain relief gel targeting the challenging wound-healing milieu in diabetes but also provides a proof-of-concept application of BP-based materials in DU treatment.

Keywords: analgesic; black phosphorus; diabetic ulcer; fibrin gel; wound healing.

Fig. 1.

Schematic illustration of in situ sprayed NIR-responsive, pain-relieving gel for accelerated wound healing in DU.

Fig. 2.

Characterizations of BP-based gel. (A) TEM image of BP NSs. (B) AFM image of BP NSs. (C) Thickness profile of BP NSs in B. (D) Raman spectrum of bulk BP and BP NSs. (E) SEM image of Gel. (F) Pseudocolor SEM image of BP@Gel. (G) EDX profile of BP@Gel. (H) Digital photograph of Gel and BP@Gel. (I) The gelation time of fibrinogen/thrombin gel with different BP NSs concentrations: (1) 0 μg⋅mL⁻¹, (2) 50 μg⋅mL⁻¹, and (3) 100 μg⋅mL⁻¹.

Fig. 3.

BP-based gel promotes the proliferation and vascularization of ECs. Cell proliferation after HUVECs were incubated on the surface of Gel and BP@Gel for (A) 1 d and (B) 3 d. (C) Fluorescent images of EdU-labeled proliferating HUVECs incubated on Gel and BP@Gel for 72 h. Green, proliferating cells; blue, total cells. (Scale bar, 40 µm.) (D) Quantitative measurement of the EdU+ HUVECs. (E) Effects of Gel and BP@Gel on HUVEC migration. (Scale bar, 200 µm.) (F) Quantitative analysis of cell migration after HUVECs were treated with Gel and BP@Gel. (G) Formation of capillary-like sprouts differentiated by HUVECs in Gel and BP@Gel and their growth at 2, 4, and 7 d. (Scale bar, 100 µm.) (H) FITC-phalloidin and DAPI staining of formed capillary after 7-d incubation of HUVECs. (Scale bar, 100 µm.) **P < 0.01, and ***P < 0.001.

Fig. 4.

BP-based gel eliminates bacteria through photothermal effects and provides on-demand pain relief in diabetic mice. (A) Photothermal curves of BP@Gel with different concentrations of BP NSs. (B) Growth profiles of SA bacteria with different treatments: (1) control, (2) NIR, (3) BP@Gel, and (4) BP@Gel + NIR. (C) Digital photographs of SA bacterial colonies grown on LB agar plates with different treatments and (D) corresponding quantitative measurement. (E) Bacterial membrane integrity was tested through calcein-AM/PI staining with different treatments. (Scale bar, 20 μm.) (F) SEM images of SA bacteria with or without PTT of BP@Gel. (G) Illustration of using von Frey filament to test the anesthetic effect of BP@Gel@Lid on mice. (H) Photothermal image of mice feet with different treatments of (1) BP@Gel@Lid, (2) NIR, and (3) BP@Gel@Lid + NIR. (I) The anesthetic effect of mice feet through mechanical allodynia caused by touching with von Frey filaments after different treatments. Data are means ± SE; n = 5. ***P < 0.001.

Fig. 5.

BP-based gel accelerates wound healing and reduces inflammation in diabetic mice. (A) Photothermal image of mice after treatment with BP@Gel, NIR, and BP@Gel@Lid + NIR. (B) Temperature changes of mice wound sites after different treatments. (C) Images of wound healing in mice skin at different times after different treatments: G1, control; G2, Gel; G3, BP@Gel; and G4, BP@Gel@Lid + NIR. (D) Relative wound area curves of mice after different treatments. (E) Time of wound healing to half of the wound area. (F) The relative average wound area of each group relative to the G4 group on day 10. (G) Representative H&E staining image of mice wound healing at different times: (#1) day 4 and (#2) endpoint. The left side of the black line represents normal tissue, and the right side of the black line represents the wound. (Scale bar, 200 μm.) (H) Quantitative analysis of inflammatory cells in G. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not statistically significant.

Fig. 6.

BP-based gel promotes angiogenesis and eliminates bacteria in diabetic mice. (A) Representative Masson staining image of mice wound healing at different times: (#1) day 4 and (#2) endpoint. (Scale bar, 200 μm.) (B) Quantitation of collagen deposition area in A. (C) Immunofluorescence staining for CD31 in wound tissues at different times after treatments: (#1) day 8 and (#2) endpoint. (Scale bar, 200 μm.) (D) Quantification of blood vessels in C. (E) Giemsa staining of wound tissues after 4-d treatment. (Scale bar, 25 μm). (F) Quantification of residual bacteria in E. *P < 0.05, **P < 0.01, and ***P < 0.001.