Neurogenic tissue nanotransfection in the management of cutaneous diabetic polyneuropathy

Abstract

This work rests on our recent report on the successful use of tissue nanotransfection (TNT) delivery of Ascl1, Brn2, and Myt1l (TNT_ABM) to directly convert skin fibroblasts into electrophysiologically active induced neuronal cells (iN) in vivo. Here we report that in addition to successful neurogenic conversion of cells, TNT_ABM caused neurotrophic enrichment of the skin stroma. Thus, we asked whether such neurotrophic milieu of the skin can be leveraged to rescue pre-existing nerve fibers under chronic diabetic conditions. Topical cutaneous TNT_ABM caused elevation of endogenous NGF and other co-regulated neurotrophic factors such as Nt3. TNT_ABM spared loss of cutaneous PGP9.5⁺ mature nerve fibers in db/db diabetic mice. This is the first study demonstrating that under conditions of in vivo reprogramming, changes in the tissue microenvironment can be leveraged for therapeutic purposes such as the rescue of pre-existing nerve fibers from its predictable path of loss under conditions of diabetes.

Keywords: Diabetic peripheral neuropathy; Nanochannel electroporation; Tissue nanotransfection.

Figure 1.. NEP_ABM transfection induced neurotrophic factors in MEF cells.

(A) Schematic diagram of NEP. (B) Delivery of Ascl1, Brn2 and Myt1l in MEF cells by NEP. Phenotypic characterization of induced neuron-like cells 2 weeks post-NEP (C) or 4 weeks post-NEP (D). Molecular markers are indicated on the top of each panel. Scale, 50 μM. (E) Ngf expression at weeks 1 and 4 post-NEP. NGF ELISA from differentiated MEF media at 4 weeks post-NEP (n = 10). RT-qPCR analysis of neurotrophin mRNA at (F) 1 week (n = 4) and (G) 4 weeks (n = 6) post-NEP. Data expressed as mean ± SEM, *P < 0.05.

Figure 2.. TNT_ABM into the dorsal skin of C57Bl/6 mice resulted in stromal reprogramming.

(A) Schematic diagram of TNT. (B) Confocal microscopic images showing three-plex in situ hybridization of Ascl1, Brn2, Myt1l, counterstained with DAPI. (C) RT–qPCR analysis of ABM gene expression in skin 24 h post-TNT. (n = 4), (D) Immunostaining showed TuJ1 fibers in skin. White dashed lines indicate epidermal and dermal junction. (E) Quantification of TuJ1⁺ fiber length per mm epidermis length. (n = 6) (F) Confocal microscopic images of skin showing co-localization (white) of FSP and TuJ1. (G) Quantification of TuJ1 and FSP positive cells per field of view. Data expressed as mean ± SEM (n = 3–4), *P < 0.05.

Figure 3.. TNT_ABM increased neurotrophic factor in skin of C57Bl/6 mice.

(A) RT-qPCR analysis of Ngf (n = 6) (B) NGF expression quantified by ELISA (n = 8), *P < 0.01. (C-D) Quantification and confocal microscopic images showing NGF in epidermis (n = 4). White dashed lines indicate epidermal and dermal junction. (E) Bdnf, Nt3 or Nt4/Nt5 expression in skin. Data expressed as mean ± SEM (n = 6), *P < 0.05.

Figure 4.. TNT_ABM increased NGF production and PGP9.5⁺ nerve fibers in skin of db/db mice.

(A) Immunostaining of TuJ1⁺ fibers in skin. (B) Quantitation of TuJ1⁺ fiber length per mm epidermis. (n = 6) (C) Tissue NGF was quantified by ELISA. (n = 9,10), *P < 0.01. Immunostaining of NGF in epidermis 4 weeks (D) or 9 weeks (F) post-TNT_ABM. Quantification of the IHC images (E, 4 weeks & G, 9 weeks). (n = 5–6), *P < 0.05. (H) Immunostaining indicated increased number of PGP9.5⁺ fibers (white arrowheads) in skin. (I) Quantification of the number of PGP9.5⁺ fibers per mm epidermis length. Data are mean ± SE (n = 4), *P < 0.01. (J) Isotype control and (K) no primary antibody yielded no signal. White dashed lines indicate epidermal and dermal junction.