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. 2022 Mar 7;19(3):974-984.
doi: 10.1021/acs.molpharmaceut.1c00944. Epub 2022 Feb 18.

Codelivery of 1α,25-Dihydroxyvitamin D3 and CYP24A1 Inhibitor VID400 by Nanofiber Dressings Promotes Endogenous Antimicrobial Peptide LL-37 Induction

Yajuan Su  1 , Gitali Ganguli-Indra  2   3 , Nilika Bhattacharya  2 , Isabelle E Logan  4 , Arup K Indra  2   3   4   5 , Adrian F Gombart  4 , Shannon L Wong  6 , Jingwei Xie  1   7
Affiliations

Codelivery of 1α,25-Dihydroxyvitamin D3 and CYP24A1 Inhibitor VID400 by Nanofiber Dressings Promotes Endogenous Antimicrobial Peptide LL-37 Induction

Yajuan Su et al. Mol Pharm. .

Abstract

Surgical site infections represent a significant clinical problem. Herein, we report a nanofiber dressing for topical codelivery of immunomodulating compounds including 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and VID400, a CYP24A1 inhibitor in a sustained manner, for inducing the expression of the endogenous cathelicidin antimicrobial peptide (CAMP) gene encoding the hCAP18 protein, which is processed into the LL-37 peptide. Nanofiber wound dressings with coencapsulation of 1,25(OH)2D3 and VID400 were generated by electrospinning. Both 1,25(OH)2D3 and VID400 were coencapsulated into nanofibers with loading efficiencies higher than 90% and exhibited a prolonged release from nanofiber membranes longer than 28 days. Incubation with 1,25(OH)2D3/VID400-coencapsulated poly(ϵ-caprolactone) nanofiber membranes greatly induced the hCAP18/LL-37 gene expression in monocytes, neutrophils, and keratinocytes in vitro. Moreover, the administration of 1,25(OH)2D3/VID400-coencapsulated nanofiber membranes dramatically promoted the hCAP18/LL-37 expression in dermal wounds created in both human CAMP transgenic mice and human skin tissues. The 1,25(OH)2D3- and VID400-coencapsulated nanofiber dressings enhanced innate immunity via the more effective induction of antimicrobial peptide than the free drug alone or 1,25(OH)2D3-loaded nanofibers. Together, 1,25(OH)2D3/VID400-embedded nanofiber dressings presented in this study show potential in preventing surgical site infections.

Keywords: 1α,25-dihydroxyvitamin D3; CYP24A1 inhibitor; antimicrobial peptide LL-37; codelivery; nanofiber dressings.

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Figures

Figure 1.
Figure 1.
Schematic showing antimicrobial peptide production is vitamin D dependent. CYP24A1 inhibitor can reduce the conversion of 1,25(OH)2D3 to 1,24R,25(OH)2D3 and 1,23S,25R-(OH)2D3.
Figure 2.
Figure 2.
Morphology characterization. (A) Photograph shows a 1,25(OH)2D3/VID400-loaded PCL nanofiber membrane with a diameter of 6 mm. (B-D) SEM images of (B) PCL/pluronic F127 nanofibers, (C) 1,25(OH)2D3-loaded PCL/pluronic F127 nanofibers, and (D) 1,25(OH)2D3/VID400-loaded PCL/pluronic F127 nanofibers.
Figure 3.
Figure 3.
In vitro release profiles of 1,25(OH)2D3 and VID400 from nanofibers over 28 days (A) and the daily release of 1,25(OH)2D3 (B) and VID400 (C).
Figure 4.
Figure 4.
CAMP gene relative expression of (A) HL60, (B) U937, (C) HaCaT cells after treatment with 200 nM 1,25(OH)2D3 and 0-2000 nM VID400 for 1, 3, and 5 days.
Figure 5.
Figure 5.
LL-37 expression level of (A) HL60, (B) U937, (C) HaCaT cells after treatment with 200 nM 1,25(OH)2D3 and 0-2000 nM VID400 for 1, 3, and 5 days.
Figure 6.
Figure 6.
In vitro cell toxicity of different nanofiber formulations for 1, 3, and 5 days. (A) HL60, (B) U937, (C) HaCaT, and (D) HDF-α. TCPS: tissue culture polystyrene. F127/PCL: PCL/pluronic F-127 blend nanofibers. F127/PCL/VD3: 1,25(OH)2D3 loaded PCL/pluronic F-127 blend nanofibers. F127/PCL/VD3/VID400: 1,25(OH)2D3 and VID400 coloaded PCL/pluronic F-127 blend nanofibers.
Figure 7.
Figure 7.
CAMP gene relative expression of (A) HL60, (B) U937, (C) HaCaT cells after treatment with different nanofiber formulations for 1, 3, and 5 days. Blank control: no treatment. VD3: free 1,25(OH)2D3. VD3/VID400: free 1,25(OH)2D3 and VID400. F127/PCL: PCL/pluronic F-127 blend nanofibers. F127/PCL/VD3: 1,25(OH)2D3 loaded PCL/pluronic F-127 blend nanofibers. F127/PCL/VD3/VID400: 1,25(OH)2D3 and VID400 co-loaded PCL/pluronic F-127 blend nanofibers. (*p<0.05.)
Figure 8.
Figure 8.
LL-37 expression of (A) HL60, (B) U937, (C) HaCaT cells after treatment with different nanofiber formulations for 1, 3, and 5 days. Blank control: no treatment. VD3: free 1,25(OH)2D3. VD3/VID400: free 1,25(OH)2D3 and VID400. F127/PCL: PCL/pluronic F-127 blend nanofibers. F127/PCL/VD3: 1,25(OH)2D3 loaded PCL/pluronic F-127 blend nanofibers. F127/PCL/VD3/VID400: 1,25(OH)2D3 and VID400 coloaded PCL/pluronic F-127 blend nanofibers. (*p<0.05)
Figure 9.
Figure 9.
Cathelicidin gene relative expression (A) and LL-37 expression (B) via treating fresh ex vivo human skin tissue with different nanofibers in Day 1, 3 and 5. Blank control: no treatment. F127/PCL: PCL/pluronic F-127 blend nanofibers. F127/PCL/VD3: 1,25(OH)2D3 loaded PCL/pluronic F-127 blend nanofibers. F127/PCL/VD3/VID400: 1,25(OH)2D3 and VID400 coloaded PCL/pluronic F-127 blend nanofibers. (*p<0.05)
Figure 10.
Figure 10.
1,25(OH)2D3 and 1,25(OH)2D3/VID400 nanofiber membranes promote LL-37 expression in the CAMPTg/Tg:KO/KO transgenic mouse wound model. (A) Induction of hCAP18 expression in Day 3 skin wounds post treatment with nanofibers containing 1,25(OH)2D3 and 1,25(OH)2D3/VID400. Protein samples were extracted from day 3 skin wounds of CAMPTg/Tg:KO/KO mice treated with F127/PCL only, F127/PCL/VD3 and F127/PCL/VD3/VID400. Western blots were performed using specific anti-hCAP18 antibody. Actin was used as a loading control. (B) Induction of hCAP18 expression in day 3 skin wounds post treatment with nanofibers containing 1,25(OH)2D3 and 1,25(OH)2D3/VID400. Significant induction of hCAP18 was observed in 1,25(OH)2D3 and 1,25(OH)2D3/VID400 loaded nanofibers compared to the PCL control. (C) Immunofluorescence staining of hCAP18/LL-37 protein (in red) and F4/80 (green) on day 3 samples post wounding in the presence of F127/PCL fibers alone and with 1,25(OH)2D3, as well as 1,25(OH)2D3/VID400. Nuclei were counterstained with DAPI (in blue). (a) F127/PCL; (b) F127/PCL/VD3 and (c) F127/PCL/VD3/VID400. Increased number of hCAP18+ cells were detected in the wound bed of F127/PCL/VD3 treated skin wounds, which was further increased in the skin wounds with F127/PCL/VD3/VID400 nanofibers. (D) Quantitative analysis of the number of hCAP18+ cells detected in the wound bed in day 3 for skin wounds with post-treatment of nanofibers containing 1,25(OH)2D3 and 1,25(OH)2D3/VID400. F127/PCL: PCL/pluronic F-127 blend nanofibers. F127/PCL/VD3: 1,25(OH)2D3 loaded PCL/pluronic F-127 blend nanofibers. F127/PCL/VD3/VID400: 1,25(OH)2D3 and VID400 coloaded PCL/pluronic F-127 blend nanofibers. (*p<0.05)
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