KPV-Modified Methylprednisolone And Polydopamine Co-loaded Liposomes For Vitiligo Therapy

Vitiligo,a common acquired skin pigmentation disorder characterized by the appearance of well-defined milky white patches of the skin on the face,neck and arms,is mainly caused by the decrease,loss or dysfunction of functional melanocytes in the skin or hair follicles.At present,the pathological mechanism of vitiligo is still unclear.The mainstream theories consider skin oxidative stress,inflammatory and their interaction as the possible pathogenesis of vitiligo.Current clinical treatments of vitiligo are mainly aimed at anti-oxidative stress,anti-inflammatory and delaying the progression of leukoplakia but fail to fundamentally improve the activity and functions of melanocytes.Therefore,ameliorating the microenvironment of melanocytes and restoring the functions of melanocytes may be keys to the successful treatment of vitiligo.Polydopamine（PDA）is the main component of natural melanin,which has a strong capacity of scavenging reactive oxygen species（ROS）and has the potential as the exogenous artificial bionic melanin to supplement melanin deficiency of melanocytes.In this research,we prepared PDA and methylprednisolone（MPS）co-loaded flexible liposomes and modified the minimum bioactive fragment ofα-melanocyte-stimulating hormone—Lys-Pro-Val（KPV）on the surface of flexible liposomes for the treatment of vitiligo.Utilizing the oxidizing and self-polymerizing reaction of dopamine hydrochloride under alkaline conditions,polydopamine nanoparticle with a nanoparticle of 6.82 nm was successfully prepared.KPV-polyethylene glycol-stearic amine conjugates were synthesized as the surfactant to prepare PDA and MPS co-loaded flexible.The particle size of liposomes was around 42 nm.The drug load efficiency and encapsulation efficiency of MPS were 2.68%and 95.53%respectively.The drug load efficiency and encapsulation efficiency of PDA were 1.25%and 24.95%respectively.In vitro drug release tests showed that MPS released comparatively fast at four hours and almost complete release at 10 hours.In vitro cytotoxicity assay indicated that liposomes possessed well biocompatibility as hardly any cytotoxicity was observed at a concentration of 2 to 500μg/m L.In vitro transdermal penetration experiments via Franz diffusion cells suggested that the specific binding of KPV and MC1R enabled liposomes to accumulate in the basal layer where melanocytes locate in.Human epidermal melanocytes（PIG1）were used as model cells to investigate the cellular uptake behavior of liposomes.The experimental results showed that PIG1 had a better cell internalization effect on KPV-modified flexible liposomes compared with blank liposomes.Melanocytes oxidative stress model was established by adding the proper concentration of H₂O₂ to the cell culture medium.After treating with liposomes,the SOD activity,CAT activity,MDA content and IL-6 level are detected via commercial kits.As a result,the SOD activity and CAT activity increased while MDA content and IL-6 level showed downward trends,indicating that the treatment by KPV-Lipo（PDA+MPS）could scavenge ROS,alleviate oxidative stress and exert anti-inflammatory effects.Also,cell proliferation experiments,carried out by using bromodeoxyuridine（Brd U）labeling regenerated cells,indicated that KPV-Lipo had a significant effect on promoting cell proliferation.To investigate the effect of preparation on melanosome transport,PIG1 cells were co-cultured with Human Immortalized Keratinocytes（HaCaT）,whose results showed that KPV-Lipo could considerably increase the number of melanosomes and the amount of melanin inside HaCaT cells.Female C57 mice were employed as model animals to establish the vitiligo model by applying 5%hydrogen peroxide on the back skin of mice.After 14 days of treatment,the SOD activity,CAT activity,MDA content,TNF-αlevel and IL-6 level were determined.Results showed that the SOD activity and CAT activity experienced upward trends while MDA content,TNF-αlevel and IL-6 level dropped significantly,manifesting that KPV-Lipo（PDA+MPS）was effective in alleviating oxidative stress and exerting anti-inflammatory effects.Finally,the dermoscopy,as well as Mosson-Fontana immunohistochemical,was employed to observe the repigmentation of the skin.Results that the number of melanosomes in hair follicles rase and skin color restored.In this research,the designed transdermal nano-drug delivery system was able to load MPS and PDA and targeted delivery to melanocytes through the KPV and MC1R receptor-ligand binding effect.Firstly,flexible liposomes could enhance the transdermal penetration of drugs for their excellent transdermal permeability.Secondly,the bioactivity of KPV which was descended fromα-MSH enabled the liposome to accumulate around melanocytes and thus enhanced drug retention.Thirdly,MPS encapsulated in liposomes had a potent anti-inflammatory effect and the depigmentation of vitiligo could be reversed by the supplement of bionic melanin.Besides,PDA could also help restore the ability of local skin melanin synthesis and ameliorates oxidative stress in diseased areas.Finally,the flexible liposomes remaining in the cortex after drug release would further promote melanocyte proliferation,promote melanin synthesis and protect melanocytes,and finally achieve the synergistic treatment of drugs,bionic melanin,and carrier.