1α,25-dihydroxyvitamin D3-eluting nanofibrous dressings induce endogenous antimicrobial peptide expression

Abstract

Aim: The aim of this study was to develop a nanofiber-based dressing capable of local sustained delivery of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) and augmenting human CAMP induction.

Materials & methods: Nanofibrous wound dressings containing 1,25(OH)₂D₃ were successfully prepared by electrospinning, which were examined in vitro, in vivo and ex vivo.

Results: 1,25(OH)₂D₃ was successfully loaded into nanofibers with encapsulation efficiency larger than 90%. 1,25(OH)₂D₃ showed a sustained release from nanofibers over 4 weeks. Treatment of U937 and HaCaT cells with 1,25(OH)₂D₃-loaded poly(ϵ-caprolactone) nanofibers significantly induced hCAP18/LL37 expression in monocytes and keratinocytes, skin wounds of humanized transgenic mice and artificial wounds of human skin explants.

Conclusion: 1,25(OH)₂D₃ containing nanofibrous dressings could enhance innate immunity by inducing antimicrobial peptide production.

Keywords: 1α,25-dihydroxyvitamin D3; antimicrobial peptides LL-37; electrospun nanofibers; endogenous production; innate immunity; sustained delivery.

Figure 1.. Morphology characterization.

(A) Photograph shows a 1,25(OH)₂D₃-loaded PCL fiber membrane with a diameter of 5 mm. (B & C) SEM images of (B) PCL fibers, (C) 1,25(OH)₂D₃-loaded PCL fibers and (D) 1,25(OH)₂D₃-loaded PCL/pluronic F127 fibers. PCL: Poly(ϵ-caprolactone); SEM: Scanning electron microscope.

Figure 2.. In vitro release kinetics of 1,25(OH)₂D₃.

Release profiles of VD from VD-PCL membranes and VD-PCL/pluronic F127 nanofiber (VD-PCL/F) membranes. Each data point represents the arithmetic mean ± standard deviation values from three samples. F: Plutonic F127; PCL: Poly(ϵ-caprolactone); VD-PCL: 1,25(OH)₂D₃-loaded PCL nanofiber.

Figure 3.. 1,25(OH)₂D₃-loaded poly(ϵ-caprolactone) nanofibers induce hCAP18/LL37 protein expression.

Immunofluorescent staining with anti-hCAP18 antibody shows the expression of cathelicidin in U937 cells that were incubated in the absence (Ctr: without any treatment) and presence of pristine 1 mg/ml PCL fibers, 0.52% DMSO, 2.0 × 10^-7 M VD or VD-PCL fibers for 1, 3 and 5 days. Expression levels were determined by flow cytometry (lower panels). Ctr: Control; DMSO: Dimethyl sulfoxide; PCL: Poly(ϵ-caprolactone); VD: 1,25(OH)₂D₃.

Figure 4.. hCAP18/LL37 expression is induced in HaCaT cells after treatment with VD-PCL fibers.

Fluorescence microscopy images show hCAP18/LL37 expression in HaCaT cells that were incubated in the presence of 0.52% DMSO (Ctr: treatment with 0.52% DMSO), pristine 1 mg/ml PCL fibers, 2.0 × 10^-7 M VD and 1 mg/ml VD-PCL fibers for 5 days. Ctr: Control; DMSO: Dimethyl sulfoxide; PCL: Poly(ϵ-caprolactone); VD: 1,25(OH)₂D₃.

Figure 5.. Quantification of hCAP18/LL-37 expressed in U937 cells after treatment with different formulations.

Cells were incubated in the absence (Ctr: without any treatment) and presence of 1 mg/ml pristine PCL fibers, 0.52% DMSO, 2.0 × 10^-7 M VD or 1 mg/ml VD-PCL fibers for 1, 3 and 5 days. The levels of hCAP18/LL-37 were measured by ELISA. Each data point represents the arithmetic mean ± standard deviation values from three samples. Statistical significance was evaluated by one-way analysis of variance (*p < 0.05). Ctr: Control; DMSO: Dimethyl sulfoxide; PCL: Poly(ϵ-caprolactone); VD: 1,25(OH)₂D₃.

Figure 6.. 1,25(OH)₂D₃-loaded poly(ϵ-caprolactone)/pluronic F127 nanofiber (VD-PCL/F) dressings induce CAMP gene expression in full-thickness excisional wounds in human CAMP^Tg+/Tg+; murine Camp^KO/KO mice 3 days postwounding.

(A) Schematic showing location of nanofiber dressings in the wounds on each flank, the day 0 skin taken from the wound and the skin distal from the wound collected on day 3. (B) Cyp24a1 mRNA expression in the kidneys of mice treated with PCL or VD-PCL/F dressings collected on day 3. (C) CAMP mRNA expression in the day 0 skin, wound and skin distal to the wounds in mice treated with VD-PCL/F dressings. (D) CAMP mRNA expression in wounds containing PCL nanofibers (PCL) versus expression in wounds containing VD-PCL/F. Data are means ± SEM of two independent experiments (n = 6) and are normalized to mouse Ywhaz and expressed as a ratio. Panels (B) and (D), p < 0.05 by paired t-test. Panel (C), p < 0.01 by one-way analysis of variance and Tukey's multiple comparisons test. Statistical significance indicated by *p < 0.05. F: Pluronic F127; PCL: Poly(ϵ-caprolactone); SEM: Scanning electron microscopy; VD: 1,25(OH)₂D₃.

Figure 7.. Induction of hCAP18/LL-37 protein in the dermis postwounding of the human CAMP transgenic mouse.

Immunofluorescence staining of hCAP18/LL-37 protein (in red) on day 3 samples in the presence of PCL fibers with or without 1,25(OH)₂D₃. Nuclei were counterstained with DAPI (in blue). The yellow-dotted lines indicate the separation of the epidermis (E) from the dermis (D). Insets indicate higher magnification (40×) images of hCAP18/LL-37 protein expression in the absence (left) or presence (right) of 1,25(OH)₂D₃. Original magnifications: 10×. DAPI: 4′,6-diamidino-2-phenylindole; PCL: Poly(ϵ-caprolactone).

Figure 8.. 1,25(OH)₂D₃-loaded poly(ϵ-caprolactone) nanofibers induce hCAP18/LL37 production in human skin explants.

(A) An artificial wound (epidermal and partial dermal layer 1-mm thick) with diameter of 8 mm was created in each skin explant (Ctr: without any treatment). (B) Nanofibrous dressings containing either VD-PCL or vehicle (PCL) were placed in the wound. (C) Appearance of the skin tissue and nanofiber dressing after 5 days of culture. (D) Quantification of hCAP18/LL-37 production by ELISA after treatment for 1, 3 and 5 days. *p < 0.05. Ctr: Control; PCL: Poly(ϵ-caprolactone); VD: 1,25(OH)₂D₃.