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. 2018 Sep 5;26(9):2178-2188.
doi: 10.1016/j.ymthe.2018.04.021. Epub 2018 Apr 27.

Topical Lyophilized Targeted Lipid Nanoparticles in the Restoration of Skin Barrier Function following Burn Wound

Jilong Li  1 , Subhadip Ghatak  2 , Mohamed S El Masry  3 , Amitava Das  2 , Yang Liu  4 , Sashwati Roy  2 , Robert J Lee  5 , Chandan K Sen  6
Affiliations

Topical Lyophilized Targeted Lipid Nanoparticles in the Restoration of Skin Barrier Function following Burn Wound

Jilong Li et al. Mol Ther. .

Abstract

Lyophilized keratinocyte-targeted nanocarriers (TLNκ) loaded with locked nucleic acid (LNA) modified anti-miR were developed for topical application to full thickness burn injury. TLNκ were designed to selectively deliver LNA-anti-miR-107 to keratinocytes using the peptide sequence ASKAIQVFLLAG. TLNκ employed DOTAP/DODAP combination pH-responsive lipid components to improve endosomal escape. To minimize interference of clearance by non-targeted cells, especially immune cells in the acute wound microenvironment, surface charge was neutralized. Lyophilization was performed to extend the shelf life of the lipid nanoparticles (LNPs). Encapsulation efficiency of anti-miR in lyophilized TLNκ was estimated to be 96.54%. Cargo stability of lyophilized TLNκ was tested. After 9 days of loading with anti-miR-210, TLNκ was effective in lowering abundance of the hypoxamiR miR-210 in keratinocytes challenged with hypoxia. Keratinocyte uptake of DiD-labeled TLNκ was selective and exceeded 90% within 4 hr. Topical application of hydrogel-dispersed lyophilized TLNκ encapsulating LNA anti-miR-107 twice a week significantly accelerated wound closure and restoration of skin barrier function. TLNκ/anti-miR-107 application depleted miR-107 and upregulated dicer expression, which accelerated differentiation of keratinocytes. Expression of junctional proteins such as claudin-1, loricrin, filaggrin, ZO-1, and ZO-2 were significantly upregulated following TLNκ/anti-miR-107 treatment. These LNPs are promising as topical therapeutic agents in the management of burn injury.

Keywords: burn wound; drug targeting; microRNAs; nanoparticles; wound healing.

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Figures

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Graphical abstract
Figure 1
Figure 1
Characterization of the Keratinocytes Targeting Lyophilized Lipid Nanoparticles (A) Schematic representation of keratinocytes targeting lipid nanoparticles (TLNκ). (B) Change of TLNκ size due to aqueous storage condition under 4°C over a period of 7 days. (C) Representative photograph of the lyophilized keratinocytes targeting lipid nanoparticles. (D) Representative nanoparticle tracking analysis (NanoSight) showing particle size and concentration of freshly prepared TLNκ and reconstituted lyophilized TLNκ after 9 days of storage under 4°C (n = 4). (E) The zeta potential of the TLNκ at different pH. (F) miR-210 expression in HaCaT cells exposed to normoxia and hypoxia 24 hr after delivery of TLNκ/anti-miR-210. Data expressed as mean ± SD (n = 4). §p < 0.05, *p < 0.001 compared to hypoxia, ANOVA.
Figure 2
Figure 2
In Vitro Targeting Efficiency of TLNκ (A) Overlay of phase contrast and fluorescence microscopic images showing uptake of DiD-labeled (red) TLNκ in keratinocytes (HaCaT cells), endothelial cells (HMEC), fibroblasts (BJ), and differentiated monocytes (THP-1) over a period of 4 hr. DiD-labeled (red) nTLNκ served as control. Scale bars, 50 μm. (B) Flow cytometric analysis showing uptake of DiD-labeled (red) TLNκ in keratinocytes (HaCaT cells), endothelial cells (HMEC), fibroblasts (BJ), and differentiated monocytes (THP-1) over a period of 4 hr. The percentage of cells showing red fluorescence of DiD were plotted graphically. Data expressed as mean ± SD (n = 4). §p < 0.05, *p < 0.001 compared to nTLNκ, ANOVA.
Figure 3
Figure 3
In Vivo Targeting Efficiency of TLNκ (A) Digital photomicrographs showing (I) application of lyophilized TLNκ powder on the dorsal skin of mice post-burn; (II) application of 3M hydrogel to cover and reconstitute the dry powder; (III) application of 3M Tegaderm to cover and strap the dressing from falling off. (B) miR-107 expression from laser microdissected epidermis of murine skin and wound-edge tissue 24 hr after application of lyophilized TLNκ containing anti-miR-107. Data expressed as mean ± SEM (n = 3), §p < 0.05, †p < 0.01, ANOVA compared to nTLNκ/anti-miR-107. (C) Confocal microscopic images showing localization of the DiD-labeled nanoparticles (red) in the epidermis 24 hr after application of nTLNκ/anti-miR-107 and TLNκ/anti-miR-107. The sections were counterstained with K14 (green) and DAPI (blue). (D) Confocal microscopic images showing colocalization (white dots) of the DiD-labeled nanoparticles (red) in the burn wound-edge epidermis stained with K14 (green) at days 1, 3, and 7. The sections were counterstained with DAPI (blue). nTLNκ/anti-miR-107 and TLNκ/anti-miR-107 were applied as mentioned in the Materials and Methods. Scale bars, 50 μm.
Figure 4
Figure 4
TLNκ/anti-miR-107 Accelerates Wound Closure and Facilitates in Re-establishing Skin Barrier Function (A) Digital photographs of the full thickness burn wound at day 0, 6, 12, 18, and 24 days after topical application of TLNκ/scramble, nTLNκ/anti-miR-107, and TLNκ/anti-miR-107. The white dashed lines indicate the wound area. Scale bars, 1 cm. (B) Wound closure after topical application of TLNκ/scramble, nTLNκ/anti-miR-107, and TLNκ/anti-miR-107 was quantified by digital planimetry. Data expressed as mean ± SEM (n = 4), §p < 0.05, †p < 0.01 compared to day 0 (d0), ANOVA. (C) Transepidermal water loss at day 0 and at day 24 after delivery of TLNκ/scramble, nTLNκ/anti-miR-107, and TLNκ/anti-miR-107 was plotted graphically. Data expressed as mean ± SEM (n = 4), §p < 0.05 compared to d0, ANOVA.
Figure 5
Figure 5
TLNκ/anti-miR-210 Upregulates Epidermal Junctional Proteins TLNκ/anti-miR-107 increased the expression of claudin-1 (red), loricrin (green), filaggrin (red), ZO-1 (green), and ZO-2 (red) in murine skin at day 24. Sections were counterstained with DAPI (blue). Dermal-epidermal junction is indicated by dashed white line. Scale bars, 50 μm. Abundance of junctional proteins were quantified and expressed graphically as mean ± SEM (n = 4). §p < 0.05, †p < 0.01, ANOVA.
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