Preparation of W/O Hypaphorine-Chitosan Nanoparticles and Its Application on Promoting Chronic Wound Healing via Alleviating Inflammation Block

Abstract

Chronic wound repair is a common complication in patients with diabetes mellitus, which causes a heavy burden on social medical resources and the economy. Hypaphorine (HYP) has good anti-inflammatory effect, and chitosan (CS) is used in the treatment of wounds because of its good antibacterial effect. The purpose of this research was to investigate the role and mechanism of HYP-nano-microspheres in the treatment of wounds for diabetic rats. The morphology of HYP-NPS was observed by transmission electron microscopy (TEM). RAW 264.7 macrophages were used to assess the bio-compatibility of HYP-NPS. A full-thickness dermal wound in a diabetic rat model was performed to evaluate the wound healing function of HYP-NPS. The results revealed that HYP-NPS nanoparticles were spherical with an average diameter of approximately 50 nm. The cell experiments hinted that HYP-NPS had the potential as a trauma material. The wound test in diabetic rats indicated that HYP-NPS fostered the healing of chronic wounds. The mechanism was through down-regulating the expression of pro-inflammatory cytokines IL-1β and TNF-α in the skin of the wound, and accelerating the transition of chronic wound from inflammation to tissue regeneration. These results indicate that HYP-NPS has a good application prospect in the treatment of chronic wounds.

Keywords: HYP-NPS; IL-1β; TNF-α; chronic wound; inflammation.

Figure 1

Physicochemical characteristics of HYP-NPS. (A). Molecular structure of hypaphorine. (B). TEM of HYP-NPS. (C,D). Particle size distribution data and zeta potential of HYP-NPS. (E) HPLC detection of hypaphorine. (F). HPLC detection after centrifugation. (G,H) Viscosity properties of HYP-NPS hydrogels under 25 °C (G) and 37 °C (H) (n = 3).

Figure 2

(A) The FT-IR spectra of HYP-NPS nanoparticles. (B) Sustained release curves of HYP from HYP-NPS solution (n = 3).

Figure 3

Biocompatibility assessment of NPS, HYP, and HYP-NPS.

Figure 4

Effects of HYP (5 μg/mL), LPS + NPS (25 μg/mL), and LPS + NPS-HYP (25 μg/mL) on the mRNA expressions of IL-1β (A) and TNF-α (B), responding to LPS (1 μg/mL)-treated RAW264.7 cells for 24 h in vitro. Values are mean ± SD. ^## p < 0.01 vs. control, ** p < 0.01 vs. LPS. n = 6 for each group.

Figure 5

In vivo wound-healing results: (A) Histogram comparing the wound closure on the 3rd, 6th, 9th, and 12th days post-wounding. (B) Photographs of the macroscopic appearances of wounds treated on the 3rd, 6th, 9th, and 12th days post-wounding. * p < 0.05 and ** p < 0.01.

Figure 6

HYP-NPS inhibits the inflammatory response and accelerates re-epithelialization. H&E-stained microscopic sections of healed incisions in rats on the 6th (A), 9th (B), and 12th (C) days. I represent inflammatory cells, B represents blood vessels, and F represents fibroblasts.

Figure 7

HYP-NPS can down-regulate IL-1β and TNF-α expression levels. (A) IHC staining of skin sections on the 6th, 9th, and 12ths day post-wounding (magnification 400×). (B) Western blotting showing protein expression levels of TNF-α and IL-1β. Values are mean ± SD. * p < 0.05 and ** p < 0.01 compared to the CON group. The arrows represent IL-1β expression.