ISX9 loaded thermoresponsive nanoparticles for hair follicle regrowth

Abstract

There is a high demand for an optimal drug delivery system to treat androgenetic alopecia. Topical application of ISX9, which is a neurogenesis inducer, has been found to stimulate hair follicle (HF) regrowth by upregulating the Wnt/β-catenin signaling pathway, an essential pathway involved in initiating HF growth and development. In the present study, a temperature-sensitive, biopolymer-based, biocompatible, and eco-friendly drug-delivery system was synthesized. This system comprised chitosan-grafted poly(glycidyl methacrylate-co-N-isopropyl acrylamide) (Poly(GMA-co-NIPAAm)@CS-PGNCS) as the shell component and PF127 as the core polymer. The hydrophobic nature of the PF127 block copolymer efficiently dissolved the partially water-soluble drug, ISX9, and the thermos-responsive shell polymer effectively released the drug at a definite skin temperature. The optimized spherical nanoparticles demonstrated the lowest critical solution temperature (LCST) at 32 ± 2 °C with a diameter of 100-250 nm, which delivered encapsulated ISX9 with greater precision than topical ISX9. In a series of in vivo experiments, we demonstrated that ISX9-coated TBNPs upregulated the expression of β-catenin, active β-catenin, Wnt target genes, stemness marker genes, proliferating cell nuclear antigen, HF stem cell markers, and HF markers including VEGF, TGF, and IGF-1 more effectively than topical ISX9. These results suggest that TBNPs could be employed as a platform for effective transdermal delivery of various hydrophobic drugs.

Keywords: Androgenetic alopecia; ISX9; Nanoparticles; Transdermal drug delivery; Wnt/β-catenin pathway.

Graphical abstract

Fig. 1

Schematic representation of thermos-responsive bi-polymeric nanoparticles (TBNPs) synthesis; (a). Preparation of TBNPs, (b). Chemical structures of raw materials, and (c). A schematic representation of action of ISX9@TBNPs for hair regeneration via the Wnt/β-catenin pathway. ISX9 beleaguered Axin1 and strengthen the LRP6-Axin1 interaction, in this manner causing the stabilization of β-catenin and raised up expression of Wnt, hair and stemmness marker genes for hair regeneration.

Fig. 2

TEM images of nanoparticles- (a) PF127, (b). TBNPs-1, (c) TBNPs-2; Physicochemical properties of TBNPs having different amount of PGNCs-1 polymer as shell material with respect to the PF127 amount (i.e., 20 mg–50 mg), (d) hydrodynamic diameters, (e) polydispersity index (PDI), (f) zeta potential; and physicochemical properties of TBNPS-1 having different amount of ISX9 (i.e., 0.25 % wt to 1 % wt), (g) hydrodynamic diameters, (h) polydispersity index (PDI), and (i) zeta potential.

Fig. 3

ISX9 retains its activity and biocompatibility in TBNPs-encapsulated form. (a) HEK293T cells carrying a Wnt responsive luciferase reporter (SuperTOPFlash) were treated with topical control, topical ISX9, TBNPs, ISX9@TBNPs, DMSO and ISX9. The cells were lysed and luciferase activity was quantified. Values are means ± SD. *P < 0.05, significantly different from the topical control and TBNPs. (b) Apoptosis analysis of HaCat and NIH3T3 cells after topical control, topical ISX9, ISX9@TBNPs and TBNPs treatments via Flow cytometry analysis (FACS). (c) Cumulative release profiles of ISX9 from ISX9@TBNPs nanoparticles at 25 ± 2 °C and 32 ± 2 °C (skin temperature). Data were expressed as mean ± SD, n = 6.

Fig. 4

In vivo effectiveness of ISX9 loaded nanoparticles in C57BL/6 mice. C57BL/6J mice in the telogen phase (7 weeks old, female) were depilated. TBNPs or ISX9@TBNPs, were topically applied thrice a week to the dorsal skin for 24 days (n = 5 per group). (a) Representative photos of mice showed hair regrowth on 0, 8, 16, 24th day treated with different combinations: TBNPs or ISX9@TBNPs. (b) Quantitative measurements of hair coat recovery at the designated area at 24th day. (c) Gross analyses of weight of regrown hair in different groups treated at 24th day. Data shown were representative of five independent experiments (n = 5). Values are means ± SD. *P < 0.05, significantly different from the TBNPs. (d) H&E stained dorsal skin at 24th day in different groups (a, b). Also c, & d were the enlargements of the framed area in a, and b respectively. Scale bar = 100 μm. (e) Quantitative assessment of the relative integer of HF relative to H&E stained dorsal skin (n = 5). (f) Relative dermal thickness among mentioned groups at 24th day. Data are expressed as mean ± SD (n = 5). *P < 0.05 significantly different to control.

Fig. 5

ISX9@TBNPs promotes the expression of hair regrowth-related markers in C57BL/6J mice.C57BL/6J mice in the telogen phase were depilated. Topical control, topical ISX9, TBNPs and ISX9@TBNPs were topically applied thrice a week to the dorsal skin for 24 days (n = 5 per group). (a) Immunohistochemistry (IHC) staining for active and total β-catenin, and Ki67 using the dorsal skin of mice treated with topical control, topical ISX9, TBNPs and ISX9@TBNPs at 24th day. Scale bars = 50 μm. (b) Immunoblotting analyses for pLRP6, LRP6, GSK3β, pGSK3β, active β-catenin, β-catenin, Axin2, LEF1, survivin, SOX2, LGR5, Keratin 17 and Ki67 on 24th day. (c) Protein bands for pLRP6, LRP6, GSK3β, pGSK3β, active β-catenin, β-catenin, Axin2, LEF1, survivin, SOX2, LGR5, Keratin 17 and Ki67 were quantified by densitometry and graphically presented. (d) The expression of the indicated genes, Axin2, LEF1, survivin, Fibronectin, LGR5, SOX2, Twist1, OCT4, VEGF, HGF and IGF-1 in each treatment group was measured using real-time PCR analysis on 24th day. The relative expression levels of mentioned genes were quantified after normalization to GAPDH. Data shown were representative of five independent experiments (n = 5). Values are means ± SD. *P < 0.05, significantly different from the topical control and TBNPs; one-way ANOVA followed by Dunnett's t-test.