负载酪氨酸酶抑制肽的牛奶外泌体抑制酪氨酸酶活性及黑色素生成研究

目的:探讨酪氨酸酶抑制肽（YRS）功能化的牛奶外泌体（mEXOYRS），对酪氨酸酶活性及黑色素生成的抑制作用。方法:通过超速离心法获取牛奶外泌体（mEXO）；利用外泌体特异锚定肽CP05将YRS修饰在mEXO表面，获得mEXOYRS，随后通过流式细胞仪分析YRS的负载效率；在黑色素瘤细胞B16-F10中，检测细胞对mEXOYRS的摄取效率以及YRS与酪氨酸酶的共定位效率，评估mEXOYRS对B16-F10黑色素瘤细胞酪氨酸酶活性的抑制效果及对黑色素合成的抑制作用；进一步将mEXOYRS均匀涂抹于Nude BALB/c小鼠黑色素瘤细胞B16-F10所成皮下瘤模型上，评估其在体内对黑色素合成的抑制作用；通过对C57BL/6小鼠表面皮肤涂抹mEXOYRS，评估其对小鼠毛囊的着色程度及小鼠皮肤的美白效果。结果:YRS通过外泌体特异锚定肽CP05高效负载至mEXO，并且不影响mEXO的形态结构及标志性蛋白的表达；在B16-F10黑色素瘤细胞摄取实验中，mEXO能够增强YRS与细胞内酪氨酸酶共定位的效率，在1周、2周及6周时均显著抑制细胞产生黑色素（F=56.117、48.954、560.006，均P＜0.05）；在Nude BALB/c小鼠黑色素皮下瘤模型中，与YRS组相比，mEXOYRS更好的抑制了黑色素的产生；在C57BL/6小鼠模型中，mEXOYRS降低了黑色素的产生，并显著降低了黑色素含量（F=173.083，P＜0.05）及酪氨酸酶活性指标（F=34.156，P＜0.05），取得了美白效果。结论:YRS修饰的mEXO可通过内体转运途径，靶向运输到酪氨酸酶处，降低酪氨酸酶活性，并有效抑制黑色素生成，提高YRS的美白效果。

Inhibition of tyrosinase activity and melanin synthesis by milk exosomes loaded with tyrosinase inhibitory peptide

Objective: To investigate the inhibitory effect of milk-derived exosomes functionalized with tyrosinase inhibitory peptide YRS （mEXOYRS） on tyrosinase activity and melanin synthesis. Methods:Milk exosomes（mEXO） were isolated and purified via ultracentrifugation. The YRS peptide was loaded on mEXO surface with exosomal anchor peptide CP05 to form mEXOYRS. The loading efficiency of YRS peptide was analyzed by flow cytometer. In B16-F10 melanoma cells，the uptake efficiency of mEXOYRS，co-localization of YRS peptide and tyrosinase were detected. The inhibitory effect of mEXOYRS on tyrosinase activity and melanin synthesis were evaluated in melanoma cells B16-F10. mEXOYRS was uniformly applied to subcutaneous melanoma model of Nude BALB/c melanoma cell B16-F10 to evaluate its inhibitory effect on melanin synthesis in vivo. mEXOYRS was applied to the skin of C57BL/6 mice to evaluate the whitening effect of mEXOYRS and the color degree of hair follicles. Results: The YRS peptide was efficiently loaded on mEXO via CP05 without altering mEXO morphological structure and exosomal biomarker expression. In B16-F10 melanoma cell uptake experiments，mEXO could improve the efficiency of YRS uptake，and enhance the efficiency of YRS co-localization with tyrosinase. At 1，2 and 6 weeks，melanin synthesis was significantly inhibited（F=56.117，48.954，560.006，all P＜0.05）. In the Nude BALB/c mouse model of melanoma subcutaneous tumor，mEXOYRS significantly inhibited melanin synthesis compared to YRS group. In C57BL/6 mouse models，mEXOYRS reduced melanin synthesis and significantly inhibited the amout of melanin（F=173.083，P＜0.05） and tyrosinase activity（F=34.156，P＜0.05）. Conclusion: mEXOYRS can be transported to tyrosinase through endosomal transport pathway，reduce tyrosinase activity，resulting in reduction of melanin production and improvement of whitening effect of YRS in vivo.