Figure 8: Delivery of keratinocyte-targeted lipid nanoparticles encapsulating si-hnRNPA2B1 compromised the quality of wound closure.
(A) Western blot analysis of hnRNPA2B1 in murine keratinocytes 48 h after treatment with TLNPk encapsulating either si-control or si-hnRNPA2B1. β-actin was used as loading control. Quantification of hnRNPA2B1 expression from immunoblots. (n=6) (B) Schematic diagram showing excisional wounding (6mm stented wound), application of TLNPk encapsulating either si-control or si-hnRNPA2B1 and tissue harvesting time points in C57BL/6 (wild type) mice. (C) Confocal microscopic image showing localization of DID-labeled TLNPk (red) in the epidermis (green) at 24h after post-treatment with DID-labeled TLNPk by subcutaneous injection. Scale, 50 μm. (D) Representative coimmunofluorescence images showing hnRNPA2B1 (green) and DAPI counterstaining in C57BL/6 mice at day 6 post wounding. White dashed lines indicate the dermal-epidermal junction. Scale, 50μm. (E) Quantification of excisional stented punch wounds (6mm) at different days by digital planimetry following delivery of TLNPk encapsulating either si-control or si-hnRNPA2B1. (n=6,8) (F) Representative Hematoxylin and Eosin (H&E) staining of day 10 murine wound tissue treated with TLNPk encapsulating either si-control or si-hnRNPA2B1. Scale, 200μm (for mosaic images) and 20μm (for inset images). (G) Representative coimmunofluorescence staining of F4–80 (red) with DAPI counterstaining in wound-edge tissue at day 10 post-wounding in C57BL/6 mice treated with either scramble or si-hnRNPA2B1 encapsulated keratinocyte targeted lipid nanoparticles. Scale, 50μm. Quantification of F4–80 intensity in wound-edge tissue at day 10 post-wounding. Each dot corresponds to one quantified ROI, except the blue and red dots, which correspond to the mean of each mouse. At least 5 ROI per mouse. (n = 4) (H) Representative coimmunofluorescence staining of F4–80 (red) and Arginase (green; proresolution macrophage marker) with DAPI counterstaining in day 10 wound-edge tissue of C57BL/6 mice treated with TLNPk encapsulating either si-control or si-hnRNPA2B1. The colocalization of red and green are shown as white dots. Scale, 20μm. (I) Representative coimmunofluorescence staining of F4–80 (red) and iNOS (green; proinflammatory macrophage marker) with DAPI counterstaining in day 10 wound-edge tissue of C57BL/6 mice treated with TLNPκ encapsulating either si-control or si-hnRNPA2B1. The colocalization of red and green are shown as white dots. Scale, 20μm. Data in A, G and E were shown as mean ± SEM and were analyzed by two-tailed unpaired Student’s t-test.