Collagen/GAG scaffolds activated by RALA-siMMP-9 complexes with potential for improved diabetic foot ulcer healing

• DOI: 10.1016/j.msec.2020.111022
• Publication Date: 2020-04-30T17:09:34Z
• Authors: Le-Ping Yan; Irene Mencía Castaño; Rukmani Sridharan; Domhnall Kelly; Mark Lemoine; Brenton L. Cavanagh; Nicholas J. Dunne; Helen O. McCarthy; Fergal J. O'Brien
• Keywords: siRNA delivery; 610; Diabetic foot ulcer; Gene-activated scaffold; name=Mechanical Engineering; 03 medical and health sciences; Diabetes Mellitus; Humans; /dk/atira/pure/subjectarea/asjc/2500/2500; RNA, Small Interfering; name=General Materials Science; name=Mechanics of Materials; Cell penetrating peptide; Wound Healing; 0303 health sciences; /dk/atira/pure/subjectarea/asjc/3100/3104; 540; Diabetic Foot; name=SDG 3 - Good Health and Well-being; Matrix Metalloproteinase 9; name=Condensed Matter Physics; /dk/atira/pure/sustainabledevelopmentgoals/good_health_and_well_being; /dk/atira/pure/subjectarea/asjc/2200/2210; ral GTP-Binding Proteins; Collagen; MMP-9; /dk/atira/pure/subjectarea/asjc/2200/2211

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.