Effects And Mechanisms Of Bioinspired Melanin-like Nanoparticles Promote Repigmentation In Vitiligo

Background:Vitiligo is a common depigmentation disorder caused by selective destruction of melanocytes in the epidermis/mucosa,with a global incidence of about 0.5%to 2%.Vitiligo lesions occurred in exposed areas such as the face,neck and limbs.Melanin deficiency in epidermal not only affects the physical and mental health of patients,but also increases the morbidity of skin cancer.As research progresses,it is believed that the pathogenesis of vitiligo involves genetic factors,oxidative stress,autoimmune and melanocyte destruction.Existing treatments such as drug,ultraviolet light therapy and surgical transplantation are still unsatisfactory.Therefore,there is an urgent need to develop a new treatment target the pathogenesis of vitiligo to effectively promote repigment.Although the exact pathogenesis of vitiligo remains unclear,increasing evidence suggests that reactive oxygen species（ROS）may be a key cause of the entire inflammatory cascade of vitiligo.Therefore,targeted elimination of ROS and supplementation of exogenous melanin would be an effective means to promote repigment.Bioinspired melanin-like nanoparticle is a black-brown insoluble high molecular weight nano-polymer including polydopamine（PDA）and sepia melanin.Polydopamine（PDA）is a biopolymer material produced by the spontaneous polymerization of dopamine that possesses inherent biocompatibility and biodegradability.PDA can be taken up by keratinocytes and transported intracellularly,achieving the biological function mimicking the melanosomes.Studies have shown that PDA has a variety of biological effects,such as photoprotection,antioxidant,anti-inflammatory and immunosuppression.PDA can be well applied to UV protection,anti-inflammatory,antioxidant and some autoimmune diseases.It is still not clear that bioinspired melanin-like nanoparticles can be taken up by keratinocytes as exogenous supplement of melanin,thus play an effective anti-inflammatory and antioxidant role.Therefore,we propose the following scientific hypothesis:PDA modified by polyethylene glycol（PEG）can be effectively taken up by keratinocytes to supplement exogenous melanin,and to further promote repigment by eliminating ROS,playing a role in photoprotection,anti-inflammatory,antioxidant,and immunosuppression.Objective:1.The anti-inflammatory,antioxidant,photoprotective and immunosuppressive effects of bioinspired melanin-like nanoparticles with different structures were compared and analyzed.2.Elucidate the molecular mechanism of PDA@PEG exerting antioxidant effects in Ha Ca T cells.3.To observe the effort of microneedles-contained-PDA Gel MA/PDA@PEG MN on the repigment of vitiligo mice.Methods:1.PDA was prepared by the spontaneous polymerization of dopamine.Mesoporous polydopamine（MPDA）was prepared by templating method.PEG modifications were performed on the two nanoparticles to preparate PDA@PEG and MPDA@PEG respectively.Natural melanin nanoparticles derived from cuttlefish ink（CINP）were obtained by ultrasound-assisted enzymatic method.Transmission electron microscope was used to observe morphology and particle size of the five particles above.2.The dispersion stability of bioinspired melanin-like nanoparticles with different structures in PBS was studied,and the bioinspired melanin-like nanoparticles with different structures were co-cultured with Ha Ca T cells.The biocompatibility was detected by CCK-8 method,and the uptake and degradation of different bioinspired melanin-like nanoparticles by Ha Ca T cells was observed by immunofluorescence confocal microscopy.3.Ha Ca T cells were treated with different bioinspired melanin-like nanoparticles.After 24 h of H₂O₂ stimulation,the viability of Ha Ca T cells was detected by CCK-8 method,intracellular ROS levels were detected by flow cytometry and confocal microscopy,and the expression levels of IL-6,IL-8 and TNF-αin the supernatant of cell medium were detected by ELISA in order to clarify the anti-inflammatory and antioxidant effects of bioinspired melanin-like nanoparticles.4.Ha Ca T cells were treated with different bioinspired melanin-like nanoparticles.After UV irradiation,the viability of Ha Ca T cells was detected by CCK-8 method,intracellular ROS levels were detected by flow cytometry and confocal microscopy,so as to clarify the photoprotective effects of different bioinspired melanin-like nanoparticles.5.PBMC was activated in vitro and treated with PDA@PEG.The proliferation and activation of CD8⁺T cells and effector molecules IFN-γand Granzyme B were detected by CFSE proliferation assay and flow cytometry.6.PDA@PEG pretreated Ha Ca T cells for 24 h and stimulated by H₂O₂.Western Blot was used to detect the Nrf2 and HO-1 protein expression levels.To elucidate the probable molecular mechanism of the antioxidant of PDA@PEG,ML385 was used to inhibit Nrf2expression,intracellular ROS levels were detected.7.The hydrogel microneedle patch Gel MA/PDA@PEG MN loaded with PDA@PEG was constructed and applied to the vitiligo mice.The pigmentation degree,PDA@PEG distribution and melanin content were observed by gross observation,HE staining and melanin staining.The number of melanocytes,CD8⁺T cells and IFN-γproduction were observed by immunofluorescence and flow cytometry,so as to clarify the promoting repigment effect of PDA@PEG on the recolor of white spot in vitiligo mice.Results:1.Bioinspired melanin-like nanoparticles with different structures have uniform particle size,good biocompatibility,safety and reliability.The average particle sizes of PDA,MPDA,PDA@PEG and MPDA@PEG were 119.8±21.22 nm,347.2±25.19 nm,126.3±17.92 nm,354.7±21.68 nm,respectively.PDA and PDA@PEG were equivalent to CINP（120.6±25.47 nm）.2.The biocompatibility of PDA was improved after PEG modification.The cell viability remained above 90%when 200μg/ml PDA@PEG and MPDA@PEG were co-cultured with Heat for 24 h.PEG modification also improved its dispersion stability,and there was no aggregation and sedimentation at PDA@PEG and MPDA@PEG when it was placed at 4℃for 24 h.PDA@PEG and MPDA@PEG can be effectively ingested and degraded by Ha Ca T cells.3.All three bioinspired melanin-like nanoparticles can remove ROS,reduce the oxidative stress level of Ha Ca T cells,and play an antioxidant role,among which PDA@PEG has the best antioxidant effect.PDA@PEG treatment can significantly reduce the expression levels of IL-6,IL-8 and TNF-α,and play an anti-inflammatory role,while CINP can only reduce the level of IL-8.4.PDA@PEG can cover the nucleus and reduce UV irradiation induced ROS level to play a photoprotective role in Ha Ca T cells.This effort is significantly better than CINP.5.PDA@PEG can inhibit the proliferation of CD8⁺T cells in normal peripheral blood PBMC,reduce the activation level of CD8⁺T cells,reduce the level of effector molecular granzyme B and IFN-γ,play an immunosuppressive role.6.PDA@PEG Pretreated Ha Ca T cells for 24 h and stimulated by H₂O₂.Western Blot was used to detect the changes in Nrf2 and HO-1 protein expression levels,and Nrf2inhibitor ML385 was used to inhibit Nrf2 expression,and intracellular ROS levels were detected.To clarify the molecular mechanism that PDA@PEG plays an anti-oxidative stress role.7.The hydrogel microneedle patch Gel MA/PDA@PEG MN loaded with PDA@PEG was constructed and applied to the mouse model of vitiligo.The white spot recolor,PDA@PEG distribution and melanin content were observed by gross observation,HE staining and melanin staining.The number of melanocytes,CD8+T and IFN-γproduction were observed by immunofluorescence and flow cytometry,so as to clarify the promoting repigment effect of PDA@PEG in vitiligo mice.Conclusion:Bioinspired melanin-like nanoparticles PDA,MPDA,PDA@PEG,MPDA@PEG and CINP have good biocompatibility and disperse stability,and can be effectively taken up and degraded by Ha Ca T cells.Among them,PDA@PEG has stronger ROS scavenging ability,perform superior anti-inflammatory,antioxidant,photoprotection and immunosuppression function.PDA@PEG inhibits local oxidative stress by activating the Nrf2/HO-1 pathway.Animal studies have shown that Gel MA/PDA@PEG MN can effectively supplement exogenous melanin,significantly reduce skin depigmentation,promote repigment in vitiligo mice.This project for the first time clarified the therapeutic potential of bioinspired melanin-like nanoparticles PDA@PEG for vitiligo disease and explored its molecular mechanism,providing a new idea and a new method for the clinical treatment of vitiligo.