부산대학교 도서관

Introduction: Biological approaches to augment angiogenesis are promising for treatment of peripheral arterial disease (PAD). We propose the use of scaffold-based modified mRNA (mmRNA) delivery as a favorable approach for transient, localized gene delivery.Hypothesis: We hypothesized that hepatocyte growth factor (HGF) mmRNA-seeded nanofibrillar scaffolds will enable localized and temporally controlled delivery of mmRNA, leading to augmentation of angiogenesis in a murine model of PAD.Methods: To establish the efficacy of mmRNA therapy, mmRNA encoding green fluorescence protein (GFP) was used as a fluorescent reporter for quantification of transfection efficiency. Aligned nanofibrillar collagen scaffolds were loaded with mmRNA and lipofectamine transfection agent. The temporal kinetics of mmRNA release into media was measured by ribogreen assay. To determine the transfection efficiency, human fibroblasts were cultured on the aligned nanofibrillar scaffolds, or on tissue culture plastic, and the efficiency of transfection was measured for up to 7 days and assayed for GFP expression.Results: Based on ribogreen assay, the cumulative release of GFP mmRNA over the course of 14 days was 235 ng/cm scaffold. In vitro transfection efficiency on aligned scaffolds (75%) was markedly higher than on tissue culture plastic (45%) after 24h. The persistence of cellular transfection as quantified by western blotting showed GFP expression >5 days post-transfection. Next, scaffolds releasing HGF or GFP mmRNA were transplanted to the site of the murine ischemic hindlimb. At the end of the 14 day experiment, laser Doppler spectroscopy showed that HGF mmRNA scaffold group had a higher mean perfusion ratio (0.32 ±0.10) than the GFP mmRNA scaffold group (0.23±0.14), suggesting that HGF-scaffolds improved blood perfusion (Figure 1).Conclusions: In summary, these data suggest that HGF mmRNA-releasing scaffolds marked improved blood perfusion in a murine model of PAD.