Study On Anti-Pigmentation NPs Targeted To Melanocyte By Microneedles

In many regions of the world, having a light and even skin color is highly valued. But the skin-lightening products were usually not satisfied by the users. The type and amount of melanin determines the actual color of the skin. The melanin is synthesized by the melanocyte and distributes in the surrounding keratinocytes. Melanin forms through a series of oxidative reactions, and the first and rate-limiting step of melanin formation is mediated by tyrosinase. The melanization process was biologically regulated by many factors. Keratinocytes produce pro-opiomelanocortin(POMC)-derived peptides including the a-melanocyte-stimulating (a-MSH) and adrenocorticotropic hormone (ACTH). Both of them can activate melanogenesis after their binding to their receptor MC1R located at the melanocyte surface. This G-protein-coupled receptor activates the adenylate cyclase to produce cAMP which plays a key role in melanodenesis. After inflammation or being irradiated by UVB, the production of many of the above factors may increase and then act on the melanin synthesis. The melanocyte density and pigmentation increase, and then disorders of melanogenesis may appear. The melanin distribution would be inhomogeneous. The drug of anti-tyrosinase was always chosen to inhibit the melanogenesis.The purpose of this study was to prepare and evaluate the arbutin-loaded nanoparticles (NPs) modified by the agouti single protein (ASIP) which has double effects on the inhibition of the melanogenesis. The microneedle arrays were used to puncture the targeting location of the hypermelanosis skin, and overcome the stratum corneum (SC) barrier to facilitate the transdermal delivery of NPs (targeted to skin layer). Arbutin-loaded NPs were prepared and then the NPs were covalently conjugated with ASIP which was high specifically targeting to MC1R (targeted to the melanocyte). The ASIP conjugated to the arbutin-NPs is the antagonist ofα-MSH, which can decrease melanin synthesis. After the arbutin-NPs were endocytosised into the melanocyte, the arbutin was controlled released into the cells to decrease the melanin synthesis too. Based on the acts of microneedles on the skin, an active targeting drug delivery system which has double targeted effects and double function of anti-pigmentation was constructed.The penetration and the distribution of NPs in the hareless mouse skin and the human skin treated with microneedles were studied using NPs containing coumarin 6 and R-phycoerythrin (R-PE) as fluorescent probe. Confocal laser scanning microscopy (CLSM) was used to visualize the distribution of nanoparticles and the high performance liquid chromatography (HPLC) was utilized to quantified the amount of the nanoparticles. The CLSM images revealed that NPs could penetrate into the skin through the microconduits created with microneedles. Permeation study in vitro demonstrated that (i) the permeation of NPs into the skin was enhanced by microneedles; (ii) much more NPs deposited in the epidermis than those in the dermis, but no NP reached the receptor solution;(iii) the permeation was in a particle size-dependent manner. It was also found that the enhancement degree by microneedles in the human skin was more significant than that in the hareless mouse skin.B-16 melanoma cells were treated with skin-lightener. The tyrosinase activity, the melanin content and cell viability were respectively measured to optimize the drug. The results showed that the arbutin was the best drug with high anti-melanogenesis effect and low toxicity. The arbutin-loaed NPs preparation was optimized by the orthogonal design. The pharmaceutical characteristics of NPs made with the optimized prescription were evaluated. The results showed that the NPs were quite round and homogenous with the average size of about 200nm.ASIP was conjugated to the NPs by EDC and NHS to prepare the active targeting NPs. The resultant arbutin-NPs-ASIP was stable. The ASIP labeled with fluorescent R-phycoerythrin (R-PE) was used to modifiy the NPs. The fluorescent NPs were detected by the CLSM and the flow cytometry (FCM) to determine the conjugation of the ASIP to the NPs. The binding of the targeted NPs to the melanocyte was observed by CLSM. It was found that the NPs were binded to the membrane in 10 minutes and internalized in the cytoplasm of cells after 2h incubation. The recognition to the targeted cells was studied using the FCM in vitro experiment. The results showed that the ASIP conjugated to the NPs could recognize and bind to the targeted cell.In vitro experiment showed that the active targeting drug delivery system (arbutin-NPs-ASIP) could speci?cally target to MC1R high-expressing MC cells and be antagonistic to theα-MSH. Compared with the single drug, the arbutin-NPs-ASIP significantly decreased the melanin content and the cell proliferation.In vivo experiment showed that after the C57BL mouse was irradiated under UVB, the skin brightness (△L* value) in the arbutin-NPs-ASIP group was significantly higher than that in the control group. In addition, the amount of melanin and the number of melanocytes in the arbutin-NPs-ASP group were significantly lower than that in the control group. Compared to the other groups (arbutin, ASIP), the anti-pigmentation effect of the arbutin-NPs-ASIP group was more significant. Also, the nanoparticles had no irritation to the skin.