Efficiency and Safety of Dextran-PAMAM/siMMP-9 Complexes for Decreasing Matrix Metalloproteinase-9 Expression and Promoting Wound Healing in Diabetic Rats.

Difficult healing of diabetic foot ulcers is associated with overexpression of matrix metalloproteinase 9 (MMP-9) in the local wound. Therefore, strategies aimed at downregulation of MMP-9 levels in ulcer sites may promote tissue regeneration and accelerate healing of diabetic foot ulcers (DFU). To fulfill this aim, we exploited dextran conjugated with poly(amidoamine) (Dextran-PAMAM) as a gene carrier to deliver MMP-9 targeted siRNA (siMMP-9). The prepared complexes could be efficiently endocytosed with low cytotoxicity to HaCat cells. Dextran-PAMAM could efficiently deliver siMMP-9 and significantly inhibit MMP-9 expression in vitro. Diabetic rats wound models showed that topical application of the Dextran-PAMAM/siMMP-9 complex effectively knocked down MMP-9 expression in skin wound tissue, thus accelerating wound healing. Taken together, this study demonstrates that the Dextran-PAMAM/siMMP-9 complex possesses high potential for wound healing and could serve as a promising regenerative platform for improving DFU healing.

Efficiency and Safety of ß-CD-(D3)7 as siRNA Carrier for Decreasing Matrix Metalloproteinase-9 Expression and Improving Wound Healing in Diabetic Rats.

Overexpression of matrix metalloproteinase-9 (MMP-9) is critical for diabetic chronic wounds involved in the refractory wound healing process. We aimed to develop a strategy through RNAi to decrease MMP-9 expression and improve diabetic wound healing. We had explored ß-CD-(D3)7 as a gene carrier to take siRNA and effectively interfere with MMP-9 expression. It has been proven that ß-CD-(D3)7 could be used as an effective siRNA delivery system. In this study, we want to know about the efficiency and safety of ß-CD-(D3)7/MMP-9 siRNA for improving wound healing in diabetic rats. ß-CD-(D3)7/MMP-9 siRNA treated animals show lower levels of MMP-9 expression, which induce faster wound-close rates. Histological evaluation indicates that ß-CD-(D3)7/MMP-9 siRNA significantly increases the content of collagen around the injured tissues. The number of neutrophilic ganulocytes was significantly decreased through treatment of ß-CD-(D3)7/MMP-9 siRNA. In vivo fluorescence imaging assessment shows that ß-CD-(D3)7/MMP-9 siRNA could not cause organ damage and organ accumulation. The results suggest that ß-CD-(D3)7/MMP-9 siRNA might be developed as a novel topical agent for the diabetic wounds treatment.

Comparison of the cellular transport mechanism of cationic, star-shaped polymers and liposomes in HaCat cells.

Several biological barriers must be overcome to achieve efficient nonviral gene delivery. These barriers include target cell uptake, lysosomal degradation, and dissociation from the carrier. In this study, we compared the differences in the uptake mechanism of cationic, star-shaped polymer/MMP-9siRNA complexes (ß-CD-(D3)7/MMP-9siRNA complexes: polyplexes) and commercial liposome/MMP-9siRNA complexes (Lipofectamine® 2000/MMP-9siRNA complexes: liposomes). The uptake pathway and transfection efficiency of the polyplexes and liposomes were determined by fluorescence microscopy, flow cytometry, and reverse transcriptase-polymerase chain reaction. The occurrence of intracellular processing was assessed by confocal laser scanning microscopy. Endosomal acidification inhibitors were used to explore the endosomal escape mechanisms of the polyplexes and lysosomes. We concluded that the polyplexes were internalized by non-caveolae- and non-clathrin-mediated pathways, with no lysosomal trafficking, thereby inducing successful transfection, while the majority of liposomes were internalized by clathrin-dependent endocytosis (CDE), caveolae-mediated endocytosis, and macropinocytosis, and only CDE induced successful transfection. Liposomes might escape more quickly than polyplexes, and the digestion effect of acidic organelles on liposomes was faint compared to the polyplexes, although both complexes escaped from endolysosomes via the proton sponge mechanism. This may be the key aspect that leads to the lower transfection efficiency of the ß-CD-(D3)7/MMP-9siRNA complexes. The present study may offer some insights for the rational design of novel delivery systems with increased transfection efficiency but decreased toxicity.

α-ketoglutarate is associated with delayed wound healing in diabetes.

AIM: A high level of matrix metalloproteinase 9 (MMP-9) is a predictor of poor wound healing in diabetic foot ulcers. In skin keratinocytes, site-specific DNA demethylation plays an important role in MMP-9 expression. Ten-eleven translocation enzyme 2 (TET2) protein, one member of TET family, could rely on α-ketoglutarate (α-KG) as cosubstrate to exhibit catalytic activity of DNA demethylation. Here, we aimed to explore the changes of α-KG and its relationship with MMP-9 and TET2 during diabetic wound healing. METHODS: Seventy-one cases of patients with diabetic foot ulcers and 53 cases of nondiabetic ulcers were enrolled. Serum, urine and wound fluids were collected for measurement of α-KG levels and MMP-9 expression. Skin tissues were collected for the measurement of TET2 and MMP-9 expression. Clinical parameters were collected, and transcutaneous oxygen pressure (TcPO2) levels of feet were detected. RESULTS: The levels of α-KG, TET2 and MMP-9 were significantly increased in diabetic wound compared with nondiabetic wound (P = 0·010, 0·016 and 0·025). There was a significant correlation between a low TcPO2 and a high α-KG level of wound fluids (r = -0·395, P = 0·002). Further analysis showed that α-KG concentration had a positive correlation with both haemoglobin A1c (HbA1C) and 2 h postprandial blood glucose (PBG) (r = 0·393, P = 0·005; r = 0·320, P = 0·025, respectively). CONCLUSIONS: The levels of α-KG, TET2 and MMP-9 were significantly increased in diabetic wound compared with nondiabetic wound. Elevated α-KG was related to local hypoxia ischaemia status and systematic poor glycaemic control.

Cationic star-shaped polymer as an siRNA carrier for reducing MMP-9 expression in skin fibroblast cells and promoting wound healing in diabetic rats.

BACKGROUND: Excessive expression of matrix metalloproteinase-9 (MMP-9) is deleterious to the cutaneous wound-healing process in the context of diabetes. The aim of the present study was to explore whether a cationic star-shaped polymer consisting of ß-cyclodextrin (ß-CD) core and poly(amidoamine) dendron arms (ß-CD-[D3]7) could be used as the gene carrier of small interfering RNA (siRNA) to reduce MMP-9 expression for enhanced diabetic wound healing. METHODS: The cytotoxicity of ß-CD-(D3)7 was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MMT) method in the rat CRL1213 skin fibroblast cell line. The transfection efficiency of ß-CD-(D3)7/MMP-9-small interfering RNA (siRNA) complexes was determined by confocal microscopy and flow cytometry. Quantitative real time (RT) polymerase chain reaction was performed to measure the gene expression of MMP-9 after the transfection by ß-CD-(D3)7/MMP-9-siRNA complexes. The ß-CD-(D3)7/MMP-9-siRNA complexes were injected on the wounds of streptozocin-induced diabetic rats. Wound closure was measured on days 4 and 7 post-wounding. RESULTS: ß-CD-(D3)7 exhibited low cytotoxicity in fibroblast cells, and easily formed the complexes with MMP-9-siRNA. The ß-CD-(D3)7/MMP-9-siRNA complexes were readily taken up by fibroblast cells, resulting in the downregulation of MMP-9 gene expression (P<0.01). Animal experiments revealed that the treatment by ß-CD-(D3)7/MMP-9-siRNA complexes enhanced wound closure in diabetic rats on day 7 post-wounding (P<0.05). CONCLUSION: ß-CD-(D3)7 may be used as an efficient carrier for the delivery of MMP-9-siRNA to reduce MMP-9 expression in skin fibroblast cells and promote wound healing in diabetic rats.