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.