Yan Le-Ping, Castaño Irene Mencía, Sridharan Rukmani, Kelly Domhnall, Lemoine Mark, Cavanagh Brenton L, Dunne Nicholas J, McCarthy Helen O, O'Brien Fergal J
Tissue Engineering Research Group, Department of Anatomy & Regenerative Medicine, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland; Scientific Research Center, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, China; Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin (TCD), Dublin, Ireland; Advanced Materials and Bioengineering Research Centre (AMBER), RCSI, Dublin, Ireland.
Tissue Engineering Research Group, Department of Anatomy & Regenerative Medicine, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland; Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin (TCD), Dublin, Ireland; Advanced Materials and Bioengineering Research Centre (AMBER), RCSI, Dublin, Ireland.
Mater Sci Eng C Mater Biol Appl. 2020 Sep;114:111022. doi: 10.1016/j.msec.2020.111022. Epub 2020 Apr 30.
Impaired wound healing of diabetic foot ulcers has been linked to high MMP-9 levels at the wound site. Strategies aimed at the simultaneous downregulation of the MMP-9 level in situ and the regeneration of impaired tissue are critical for improved diabetic foot ulcer (DFU) healing. To fulfil this aim, collagen/GAG (Col/GAG) scaffolds activated by MMP-9-targeting siRNA (siMMP-9) were developed in this study. The siMMP-9 complexes were successfully formed by mixing the RALA cell penetrating peptide with siMMP-9. The complexes formulated at N:P ratios of 6 to 15 had a diameter around 100 nm and a positive zeta potential about 40 mV, making them ideal for cellular uptake. In 2 dimensional (2D) culture of human fibroblasts, the cellular uptake of the complexes surpassed 60% and corresponded to a 60% reduction in MMP-9 gene expression in low glucose culture. In high glucose culture, which induces over-expression of MMP-9 and therefore serves as an in vitro model mimicking conditions in DFU, the MMP-9 gene could be downregulated by around 90%. In the 3D culture of fibroblasts, the siMMP-9 activated Col/GAG scaffolds displayed excellent cytocompatibility and ~60% and 40% MMP-9 gene downregulation in low and high glucose culture, respectively. When the siMMP-9 complexes were applied to THP-1 macrophages, the primary cell type producing MMP-9 in DFU, MMP-9 gene expression was significantly reduced by 70% and 50% for M0 and M1 subsets, in 2D culture. In the scaffolds, the MMP-9 gene and protein level of M1 macrophages decreased by around 50% and 30% respectively. Taken together, this study demonstrates that the RALA-siMMP-9 activated Col/GAG scaffolds possess high potential as a promising regenerative platform for improved DFU healing.
糖尿病足溃疡伤口愈合受损与伤口部位MMP - 9水平升高有关。旨在同时原位下调MMP - 9水平和修复受损组织的策略对于改善糖尿病足溃疡（DFU）愈合至关重要。为实现这一目标，本研究开发了由靶向MMP - 9的小干扰RNA（siMMP - 9）激活的胶原蛋白/糖胺聚糖（Col/GAG）支架。通过将RALA细胞穿透肽与siMMP - 9混合成功形成了siMMP - 9复合物。以6至15的N:P比配制的复合物直径约为100 nm，正ζ电位约为40 mV，使其成为细胞摄取的理想选择。在人成纤维细胞的二维（2D）培养中，复合物的细胞摄取率超过60%，在低糖培养中对应MMP - 9基因表达降低60%。在高糖培养中，高糖会诱导MMP - 9过度表达，因此可作为模拟DFU条件的体外模型，MMP - 9基因可下调约90%。在成纤维细胞的三维（3D）培养中，siMMP - 9激活的Col/GAG支架表现出优异的细胞相容性，在低糖和高糖培养中MMP - 9基因分别下调约60%和40%。当将siMMP - 9复合物应用于THP - 1巨噬细胞（DFU中产生MMP - 9的主要细胞类型）时，在二维培养中，M0和M1亚群的MMP - 9基因表达分别显著降低70%和50%。在支架中，M1巨噬细胞的MMP - 9基因和蛋白水平分别下降约50%和30%。综上所述，本研究表明RALA - siMMP - 9激活的Col/GAG支架作为改善DFU愈合的有前景的再生平台具有很高的潜力。
