Targeted self-assembled anti-NFκB AuNCs-aptamer nanoplatform for precise theranostics via tailored follicle regeneration

Abstract

NFκB is a vital transcription factor for the regulation of hair follicle cycle. As a therapeutic target, NFκB is specifically blocked by RNA aptamer with negligible side effects, but the targeted transmembrane transport of anti-NFκB aptamer remains a challenge due to its negative charge under physiological conditions. In this study, taking advantage of the depilation-induced oxidative stress microenvironment (OSM), it was confirmed for the first time that self-assembled gold nanoclusters and aptamer (AuNCs-Aptamer) complexes formed in the skin and enhanced the therapeutic effect of anti-NFκB aptamer drugs, effectively blocking the NFκB-mediated inflammatory response and inhibiting hair follicle regeneration. The hematoxylin-eosin (HE) staining of tissue section and hematology analysis demonstrated that OSM-responsive self-assembled AuNCs-Aptamer caused no toxicity to the living organism. Moreover, self-assembly occurred only in the oxidative stress-injured skin cells rather than the normal cells, which revealed that this self-assembly was a targeted, safe and effective therapy for hypertrichosis.

Keywords: AuNCs-Aptamer; Hypertrichosis; Oxidative stress microenvironment; Self-assembly; anti-NFκB aptamer.

Graphical abstract

Scheme 1

Schematic diagram of self-assembled AuNCs-Aptamer in the depilation-induced injured skin after the administration of the aptamer and HAuCl₄ coordination precursor complexes, and AuNCs-Aptamer inhibiting hair follicle regeneration by negatively regulating the NFκB(p50/p65)-mediated inflammatory response.

Fig. 1

(a) In vivo fluorescence imaging at the different time points after the first administration. (b) The right histogram showed the quantitative analysis statistics of the relative fluorescence signal intensity in the drug-administrated skin sites from 4 different groups of PBS, Au, Apt, and Au + Apt. The unit of average radiant efficiency was [p/s/cm²/sr]/[μW/cm²] × 10⁸. The fluorescence signal intensity of the dorsal skin at the dosed site from PBS group mice at 4 h was standardized to 1. (∗P < 0.05, and NS: no significance). (c) Camera photographs of PBS, Au, Apt and Au + Apt group mice on the 10^th day post the first dose. (d) HE staining of the skin tissue sections from PBS, Au, Apt and Au + Apt group mice on the 10^th day post the first dose. The dashed circle indicated the site of dose and supression of the hair follicle regeneration. The HE staining image of each group was made up of the four or five photos taken in succession and connected by front and back. (e) Local magnified picture of the dorsal skin of Au + Apt group mice.

Fig. 2

The characterization of the extracted AuNCs-Aptamer. (a) TEM image and the diameter distribution histogram of AuNCs. The average particle size was 2.18 nm. (b) HR-TEM image. The lattice fringe spacing was 0.22 nm, corresponding to the Au (111) planes. XPS and EDS were respectively exhibited in (c) and (d). (e) The typical AFM images of the OSM-responsively self-assembled AuNCs-Aptamer in the damaged skin. The coordination precursor complexes of HAuCl₄ (1 mM) and aptamer (10 μM) were injected into the depilated dorsal skin. (f) The local enlarged image in (e). The bright areas represented AuNCs-Aptamer. The maximum superposition height of AuNCs-Aptamer was about 4.72 nm. (g) and (h) showed the three-dimensional model diagrams corresponding to (e) and (f), respectively. (i) and (j) were the rigidity diagrams corresponding to (e) and (f). (k) The height map of AuNCs-Aptamer along the marked red line (I→II) in the insert from (e). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

The uptake of Cy3-labeled anti-NFκB aptamers in mechanically-damaged JB6 cells from the fluorescence microscope. (a) Cy3-labeled anti-NFκB aptamers were found in the injured cells around the scratch after administration of the HAuCl₄ and aptamer coordination precursor complexes, and there was no uptake of anti-NFkb aptamers after dose of the aptamer or HAuCl₄ alone. The merged pictures represented the overlap of the fluorescent and bright field. (b) The fluorescence intensity along the marked line (I→II) was analyzed by Image J software and (c) the corresponding fluorescence peak intensity statistics (∗P < 0.05). The concentrations of HAuCl₄ and aptamers were 10 μM and 1 μM, respectively.

Fig. 4

The expression level of (a) TNFα, (b) IL1 beta and (c) CCL2 in the whole layer skin respectively at the different time-points after administration by ELISA, and (d) the expression value of TNFα, IL1 beta and CCL2 corresponding to the 8^th, 4^th and 4^th day, denoting as mean ± standard deviation. The unit was pg/mg, representing the amount of the interested protein factor contained in total skin protein. (∗P < 0.05).

Fig. 5

The paraffin-embedded skin tissue section and immunofluorescence staining for (a) proliferation marker Ki67 and (b) differentiation marker AE15 to investigate the effects of AuNCs-Aptamer on proliferation and differentiation of hair follicle cells.