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

Abstract

Surgical site infections represent a significant clinical problem. Herein, we report a nanofiber dressing for topical codelivery of immunomodulating compounds including 1α,25dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) 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) 2 D 3 and VID400 were generated by electrospinning. Both 1,25(OH) 2 D 3 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) 2 D 3 /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) 2 D 3 /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) 2 D 3 -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) 2 D 3 -loaded nanofibers. Together, 1,25(OH) 2 D 3 /VID400-embedded nanofiber dressings presented in this study show potential in preventing surgical site infections.

Engineered Exosomes Containing Cathelicidin/LL‐37 Exhibit Multiple Biological Functions

“…[ 32,33 ] In our previous studies, we demonstrated the sustained delivery of immunomodulating compounds (1,25(OH) 2 D 3 and/or VID400) promotes the expression of LL‐37 in monocytic cells suggesting potential for preventing surgical site infection. [ 20 ] In this study, we demonstrated exosomes secreted by monocytic cells after treatment with VD 3 and VD 3 /VID400‐loaded PCL NF contained significantly elevated levels of hCAP18/LL‐37 which enhances their biological activities when compared to untreated cell exosomes. The main advantage of the present method of increasing hCAP18/LL‐37 in natural exosomes using immunomodulatory compounds is that one avoids direct encapsulation of LL‐37 peptide into exosomes using physical or chemical approaches as these processes may destabilize the peptide and compromise its biological activities.…”

“…This result was consistent with the trend we found for cellular mRNA from treated U937 cells. [20] To determine whether the induced cathelicidin mRNA levels correlated with an increase in protein expression, we quantified cathelicidin protein levels in total exosome lysates using an ELISA. Figure 2B shows increased levels of hCAP18/LL-37 in exosomes derived from U937 cells after treatment with VD 3 and VD 3 /VID400 free drugs and their nanofiber formulations on days 3 and 5.…”

“…These nanofiber formulations were produced following our recently established protocols. [ 20 ] The U937‐secreted exosomes were harvested by differential centrifugation of the conditioned media. Transmission electron microscopy (TEM) revealed exosomes derived from U937 cells primed with different nanofiber formulations exhibited a cup‐ or round‐shaped morphology with sizes of around 200 nm ( Figure A).…”

“…This result was consistent with the trend we found for cellular mRNA from treated U937 cells. [ 20 ]…”

“…This work was based on the previously reported findings: i) TLR activation of immune cells upregulates the expression of the vitamin D receptor, resulting in the induction of antimicrobial peptide cathelicidin [ 19 ] ; and ii) U937 cells express higher CAMP gene and hCAP/LL‐37 when treated with electrospun poly( ε ‐caprolactone) (PCL) nanofibers co‐loaded with 1,25(OH) 2 D 3 and the CYP24A1 inhibitor VID400 (VD 3 /VID400‐loaded PCL NF). [ 20 ] We hypothesized U937 cells primed with VD 3 /VID400‐loaded PCL NF could secrete exosomes containing more LL‐37 and such exosomes would possess increase antimicrobial and angiogenetic activity, and more effectively promote skin cell proliferation and migration.…”