Broering Milena Fronza, Leão Matheus de Castro, da Rocha Gustavo Henrique Oliveira, Scharf Pablo, Xavier Luana Fillipi, Alves Aline de Cristo Soares, Castro Inar, Reutelingsperger Chris, Uchiyama Mayara Klimuk, Araki Koiti, Guterres Sílvia Stanisçuaski, Pohlmann Adriana Raffin, Farsky Sandra Helena Poliselli
Department of Clinical & Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of Sao Paulo, SP, Brazil.
Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of Sao Paulo, SP, Brazil.
Eur J Pharm Biopharm. 2022 Dec;181:49-59. doi: 10.1016/j.ejpb.2022.10.022. Epub 2022 Nov 7.
Annexin A1 (AnxA1), a 37KDa protein, is secreted by inflammatory and epithelial cells and displays anti-inflammatory and wound healing activities in intestinal bowel diseases. Herein, we aimed to functionalize recombinant AnxA1 (AnxA1) on multi-wall lipid core nanocapsules (MLNC) and investigate its effectiveness on experimental colitis. MLNC were prepared by covering lipid core nanocapsules (LNC) with chitosan, which coordinates metals to specific protein chemisorption sites. Therefore, MLNC were linked to Zn and AnxA1 was added to form MLNC-AnxA1. LNC, MLNC and MLNC-AnxA1 presented average size of 129, 152 and 163 nm, respectively, and similar polydispersity indexes (0.xx); incorporation of chitosan inverted the negative potential zeta; the coordination efficiency of AnxA1 was 92.22 %, and transmission electron microscope photomicrograph showed MLNC-AnxA1 had a spherical shape. The effectiveness of MLNC-AnxA1 was measured in Dextran Sulfate Sodium (DSS)-induced colitis in male C57BL/6 mice. DSS (2 % solution) was administered from days 1-6; saline, LNC, MLNC, MLNC-AnxA1 or AnxA1 were administered, once a day, by oral or intraperitoneal (i.p.) routes, from days 6-9. Clinical parameters of the disease were measured from day 0-10 and gut tissues were collected for histopathology, immunohistochemistry and flow cytometry analyses. Only i.p. treatment with MLNC-AnxA1 reduced weight loss, diarrhea and disease activity index, and prevented loss of colonic structure integrity; induced the switch of macrophages into M2 phenotype in the lamina propria; recovered the colonic histoarchitecture by decreasing dysplasia of crypts, inflammation and ulcerations; restored the expression of claudin-1 Zonna-occludens-1 tight junctions in the inflamed gut; and induced stem cell proliferation in intestinal crypts. Associated, data highlight the functionalization of MLNC with AnxA1 as a tool to improve the local actions of such protein in the inflamed gut by inducing resolution of inflammation and tissue repair.
膜联蛋白A1（AnxA1）是一种37千道尔顿的蛋白质，由炎症细胞和上皮细胞分泌，在肠道疾病中具有抗炎和促进伤口愈合的活性。在此，我们旨在将重组AnxA1（AnxA1）功能化到多壁脂质核纳米胶囊（MLNC）上，并研究其对实验性结肠炎的有效性。MLNC是通过用壳聚糖覆盖脂质核纳米胶囊（LNC）制备的，壳聚糖将金属配位到特定的蛋白质化学吸附位点。因此，MLNC与锌连接，并添加AnxA1形成MLNC-AnxA1。LNC、MLNC和MLNC-AnxA1的平均粒径分别为129、152和163纳米，且多分散指数相似（0.xx）；壳聚糖的加入使负的zeta电位反转；AnxA1的配位效率为92.22%，透射电子显微镜照片显示MLNC-AnxA1呈球形。在雄性C57BL/6小鼠中，用硫酸葡聚糖钠（DSS）诱导结肠炎来检测MLNC-AnxA1的有效性。从第1 - 6天给予DSS（2%溶液）；从第6 - 9天，每天通过口服或腹腔注射（i.p.）途径给予生理盐水、LNC、MLNC、MLNC-AnxA1或AnxA1。从第0 - 10天测量疾病的临床参数，并收集肠道组织进行组织病理学、免疫组织化学和流式细胞术分析。只有腹腔注射MLNC-AnxA1能减轻体重减轻、腹泻和疾病活动指数，并防止结肠结构完整性丧失；诱导固有层巨噬细胞转变为M2表型；通过减少隐窝发育异常、炎症和溃疡来恢复结肠组织结构；恢复炎症肠道中claudin-1和紧密连接蛋白-1紧密连接的表达；并诱导肠道隐窝中的干细胞增殖。相关数据突出了用AnxA1对MLNC进行功能化作为一种工具，通过诱导炎症消退和组织修复来改善这种蛋白质在炎症肠道中的局部作用。
