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.

Dendronized chitosan derivative as a biocompatible gene delivery carrier.

To improve the transfection efficiency of chitosan as a nonviral gene delivery vector, a dendronized chitosan derivative was prepared by a copper-catalyzed azide alkyne cyclization reaction of propargyl focal point poly(amidoamine) dendron with 6-azido-6-deoxy-chitosan. Its structure was characterized by (1)H NMR and FTIR analyses and its buffering capacity was evaluated by acid-base titration. In particular, its complexation with plasmid DNA was investigated by agarose gel electrophoresis, zeta potential, and particle size analyses as well as transmission electron microscopy observation. Compared to unmodified chitosan, such a chitosan derivative has better water solubility and buffering capacity. Compared to commonly used polyethyleneimine (PEI, 25 kDa), it could exhibit enhanced transfection efficiency in some cases and lower cell toxicity, as confirmed by in vitro transfection and cytotoxicity tests in human kidney 293T and human nasopharyngeal carcinoma CNE2 cell lines. In addition, the effect of serum on its transfection efficiency was also studied.

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.

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.

Cationic dendronization of amylose via click chemistry for complexation and transfection of plasmid DNA.

For the development of effective and safe gene carrier based on starch, the amylose from potato starch was azidized by reacting with 3-azidopropylamine in the presence of N, N'-carbonyldiimidazole and then conjugated with propargyl focal point poly(amidoamine) (PAMAM) dendrons by a Cu(I)-catalyzed azide-alkyne cycloaddition. Such a cationic dendronization was verified by Fourier transform infrared spectroscopy and proton nuclear magnetic resonance analyses. For the resultant amylose conjugates with various contents and generations of PAMAM dendron, their buffering capacity, binding ability with plasmid DNA and in vitro cytotoxicity were investigated. These amylose conjugates were found to exhibit good buffering capacity and biocompatibility. In particular, they could condense effectively plasmid DNA into the nanocomplexes, as confirmed by agarose gel electrophoresis, zeta potential, and particle size analyses as well as transmission electron microscopy observation. For their nanocomplexes with plasmid DNA, the in vitro transfection properties in human embryonic kidney 293T cells were studied by fluorescence microscopy and flow cytometry. It was found that the transfection efficiency could be optimized by the dendronization extent of amylose and the complexation extent of dendronized amylose with plasmid DNA.

Water soluble cationic dextran derivatives containing poly(amidoamine) dendrons for efficient gene delivery.

To develop new dextran derivatives for efficient gene delivery, the conjugation of poly(amidoamine) dendrons onto biocompatible dextran was carried out by a Cu(I)-catalyzed azide-alkyne cycloaddition, as confirmed by FTIR and (1)H NMR analyses. For resultant dextran conjugates with various generations of poly(amidoamine) dendrons, their buffering capacity and in vitro cytotoxicity were evaluated by acid-base titration and MTT tests, respectively. In particular, their physicochemical characteristics for the complexation with plasmid DNA were investigated by the combined analyses from agarose gel electrophoresis, zeta potential, particle size, transmission electron microscopy and fluorescence emission spectra. Moreover, their complexes with plasmid DNA were studied with respect to their transfection efficiency in human embryonic kidney (HEK293) cell lines. In the case of a higher generation of poly(amidoamine) dendrons, such a dextran conjugate was found to have much lower cytotoxicity and better gene delivery capability when compared to branched polyethylenimine (bPEI, 25kDa), a commonly used gene vector.

Click modification of helical amylose by poly(l-lysine) dendrons for non-viral gene delivery.

Although amylose as a naturally-occurring helical polysaccharide has been widely used for biomedical applications, few studies have dealt with its chemical modification for non-viral gene delivery. In this work, the click modification of amylose by poly(l-lysine) dendrons was carried out and then characterized by Fourier transform infrared spectroscopy, wide-angle X-ray diffraction and elemental analyses. Such a modified polysaccharide exhibited excellent ability to condense plasmid pMSCV-GFP-PARK2 into compact and spherical nanoparticles. Moreover, it displayed much lower cytotoxicity when compared to branched polyethylenimine (bPEI, 25kDa), a commercially available gene vector. Similar to bPEI, it had a dose-dependent gene transfection activity in human embryonic kidney 293T cells, as observed by confocal laser scanning microscopy and flow cytometry. At each optimized N/P ratio, the percentage of transfected cells by this modified polysaccharide was found to be comparable to that by bPEI. Western blot and cell apoptosis analyses confirmed its effectiveness for the delivery of plasmid pMSCV-GFP-PARK2 to 293T cells.