Biomimetic polydopamine loaded with janus kinase inhibitor for synergistic vitiligo therapy via hydrogel microneedles

Abstract

Background: Both oxidative stress and autoimmune responses play crucial roles in the development of vitiligo. Under oxidative stress, the apoptotic melanocytes expose self-antigens and release high mobility group box 1 (HMGB1), triggering autoimmune activation and recruiting CD8⁺ T cells. This process further leads to the destruction of melanocytes, resulting in the lack of melanin granules. Additionally, the accumulated CD8⁺ T cells release interferon-γ (IFN-γ) to activate janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in keratinocytes. Both oxidative stress and IFN-γ-JAK-STAT activation induce keratinocytes to express and release T cell chemotactic factors, exacerbating the process of vitiligo. Reducing the accumulation of CD8⁺ T cells by safeguarding melanocytes and keratinocytes from oxidative stress may be contemplated as a promising approach for vitiligo therapy.

Results: In this study, we introduce a novel therapeutic agent called PDA-JAKi, which is capable of both eliminating oxidative stress and inhibiting T cell activation. Specifically, we have incorporated the janus kinase inhibitor (JAKi) tofacitinib into antioxidant polydopamine (PDA) nanoparticles, resulting in the formation of uniform PDA-JAKi nanodrug. PDA-JAKi effectively mitigates oxidative stress-induced apoptosis in melanocytes, reducing the antigen presentation and release of HMGB1. In addition, PDA-JAKi simultaneously attenuates oxidative stress and blocks the IFN-γ-JAK-STAT pathway to reduce the expression of C-X-C motif chemokine ligand 9/10/16 (CXCL9/10/16) in keratinocytes. We precisely deliver this therapeutic agent to the dermis using microneedle (MN) patches, aiming to enhance therapeutic efficacy compared to traditional drug administration methods. After PDA-JAKi MN treatment, the symptoms of vitiligo in mice are alleviated, and the affected areas regain pigmentation. Enhancements have been observed in the dermal thickness, the numbers of melanocytes and the content of melanin within the treated skin area. Moreover, there is a notable reduction in reactive oxygen species (ROS) level. Concurrently, substantial decreases were noted in CD8⁺ T cell infiltration, as well as the levels of IFN-γ and chemotactic factors CXCL9/10/16.

Conclusions: In summary, PDA-JAKi MN patches emerge as a promising therapeutic agent for vitiligo treatment.

Keywords: JAK inhibitor; Microneedles; Oxidative stress; Polydopamine; Vitiligo.

Fig. 1

(a) The synthesis process of PDA-JAKi MN. (b) The mechanism mediated by PDA-JAKi MN (created with BioRender.com)

Fig. 2

(a) Representative TEM image and (b) hydrodynamic size distribution of PDA-JAKi (JAKi: PDA = 0.75:1, w/w). (c) UV/Vis absorbance of JAKi and PDA-JAKi. (d) The change of size and PDI of PDA-JAKi within 72 h. (e) The images of PDA/PDA-JAKi without or with H₂O₂ (1 M) after 24 h co-incubation. (f) The change of H₂O₂ concentration after treatment with PDA and PDA-JAKi. (g) Changes in absorbance of the DPPH solution after co-incubation with different concentrations of PDA-JAKi. (h) Changes in absorbance of ABST⁺ radical over time in the presence of PDA-JAKi. (i) After UV irradiation, the absorbance of the mixed solution including various concentrations of PDA-JAKi were measured

Fig. 3

Representative morphology images of (a) HA MN and (b) PDA-JAKi MN were captured by a stereomicroscope. (c) The representative SEM image of PDA-JAKi MN. (d) Mechanical properties of HA MN, PDA MN, and PDA-JAKi MN. (e) MB staining and (f) H&E staining images of mice skin after application of PDA-JAKi MN. (g) 3D reconstruction image of RhB-loaded HA MN. (h) FL images of mouse skin taken at different depths after application of RhB-loaded HA MN for 10 min

Fig. 4

(a) Cell viabilities of HaCaT and PIG1 cells co-incubated with different concentration of PDA-JAKi for 24 h. (b) Cell viabilities of HaCaT and (c) PIG1 cells co-incubated with various concentrations of PDA-JAKi and H₂O₂ (1 mM) for 24 h. (d) The content of MDA after different treatments. The enzyme activities of (e) SOD and (f) CAT after indicated treatments. (g) The apoptosis assay of PIG1 cells after different treatments. (h) Quantitative analysis of cell apoptosis by flow cytometric assay. (i) Fluorescence images of JC-1 (red/green) changes in PIG1 cells after the indicated treatments. mean ± SD, n = 3, *P < 0.5 **P < 0.01, ***P < 0.001, ns, no significance

Fig. 5

PDA-JAKi mimicked natural melanosome and overcame oxidative stress in keratinocytes. (a) Bio-TEM images of HaCaT cells after co-incubation with PDA-JAKi at different time points. Melanosomes are indicated by red arrows. keratin fibers are indicated by red triangles. (b) The mitochondrial integrity of HaCaT cells treated by 1 mM H₂O₂ with or without PDA and PDA-JAKi and then stained with mitochondrial probe (green) and DAPI (blue). Cells were stained with DCFH-DA for acquisition of (c) fluorescence images and (d) flow cytometric analysis. (e) The quantitative analysis of flow cytometric analysis. mean ± SD, n = 3, ****P < 0.0001, ns, no significance

Fig. 6

PDA-JAKi inhibited the release of HMGB1 and secretion of CXCL9/10/16. (a) Immunofluorescence photos of Hoechst (blue) and HMGB1 (red) in PIG1 cells after various treatments for 24 h. Relative expression of mRNA level of (b) CXCL9, (c) CXCL10 and (d) CXCL16 in HaCaT cells after H₂O₂ treatment. Secreted protein of (e) CXCL9, (f) CXCL10 and (g) CXCL16 after H₂O₂ treatment. (h) The protein expression of p-STAT1 and p-STAT3. Semi-quantitative analysis of (i) p-STAT1/GAPDH and (j) p-STAT3/GAPDH. Relative expression of mRNA level of (k) CXCL9 and (l) CXCL10 in HaCaT cells after INF-γ treatments. Secreted protein of (m) CXCL9 and (n) CXCL10 after INF-γ treatment of HaCaT cells supernatants. mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significance

Fig. 7

PDA-JAKi MN improved hair follicle growth in vitiligo mice. (a) Dermal thickness in the dissected mice skin after various treatments. (b) The quantitative analysis of dermal thickness at different groups. (c) MelanA staining after different treatments. Red fields represent hair follicles. (d) The quantitative analysis of the number of MelanA positive cells at different groups. (e) Masson-Fontana staining images of skin and (f) the quantitative analysis of melanin in hair follicles at different groups. (g) Masson-Fontana staining images of epidermal melanin and (h) the quantitative analysis of melanin epidermis at different groups. Red shape indicates melanin. Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significance

Fig. 8

The PDA-JAKi MN treated vitiligo mice depigmentation area had recovered through inhibition of CD8⁺ T cells recruitment and activation. (a) ROS immunofluorescence staining images after different treatments. (b) Immunofluorescence staining images of CD8⁺ T cells of the dissected mice skin after various treatments. ELISA assays were performed to determine the levels of (c) IFN-γ, (d) CXCL9, (e) CXCL10 and (f) CXCL16. Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significance