Biomaterial constructs for delivery of multiple therapeutic genes: a spatiotemporal evaluation of efficacy using molecular beacons

• Published in: PloS one 2013-06, Vol.8 (6), p.e65749-e65749
• Main Authors: Alexander, Jennifer C, Browne, Shane, Pandit, Abhay, Rochev, Yury
• Format: Article
• Language: English
• Subjects: Angiogenesis; Arthritis; Beacons; Biology; Biomedical materials; Cardiovascular diseases; Care and treatment; Cells, Cultured; Collagen; Collagen - chemistry; Cytokines; Delayed-Action Preparations - chemistry; Deoxyribonucleic acid; Diffusion; DNA; Drug Carriers - chemistry; Drug Compounding; Effectiveness; Gene Expression; Gene therapy; Genes; Genetic aspects; Genetic engineering; Human Umbilical Vein Endothelial Cells - cytology; Human Umbilical Vein Endothelial Cells - metabolism; Humans; Inflammation; Inflammatory response; Interleukin 10; Interleukin-10 - genetics; Interleukin-10 - metabolism; Kinetics; Mesenchymal Stem Cells - cytology; Mesenchymal Stem Cells - metabolism; Methods; Microspheres; Molecular Probes - chemistry; Neovascularization, Physiologic; Nitric oxide; Nitric Oxide Synthase Type III - genetics; Nitric Oxide Synthase Type III - metabolism; Nitric-oxide synthase; Pathogenesis; Physiology; Plasmids; Plasmids - chemistry; Plasmids - metabolism; Polymerase chain reaction; Scaffolding; Scaffolds; Stem cells; Three dimensional models; Tissue Engineering; Tissue Scaffolds; Transfection; Vectors (Biology)
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0065749

Gene therapy is emerging as a potential therapeutic approach for cardiovascular pathogenesis. An appropriate therapy may require multiple genes to enhance therapeutic outcome by modulating inflammatory response and angiogenesis in a controlled and time-dependent manner. Thus, the aim of this research was to assess the spatiotemporal efficacy of a dual-gene therapy model based on 3D collagen scaffolds loaded with the therapeutic genes interleukin 10 (IL-10), a potent anti-inflammatory cytokine, and endothelial nitric oxide synthase (eNOS), a promoter of angiogenesis. A collagen-based scaffold loaded with plasmid IL-10 polyplexes and plasmid eNOS polyplexes encapsulated into microspheres was used to transfect HUVECs and HMSCs cells.The therapeutic efficacy of the system was monitored at 2, 7 and 14 days for eNOS and IL-10 mRNA expression using RT-PCR and live cell imaging molecular beacon technology. The dual gene releasing collagen-based scaffold provided both sustained and delayed release of functional polyplexes in vitro over a 14 day period which was corroborated with variation in expression levels seen using RT-PCR and MB imaging. Maximum fold increases in IL-10 mRNA and eNOS mRNA expression levels occurred at day 7 in HMSCs and HUVECs. However, IL-10 mRNA expression levels seemed dependent on frequency of media changes and/or ease of transfection of the cell type. It was demonstrated that molecular beacons are able to monitor changes in mRNA levels at various time points, in the presence of a 3D scaffolding gene carrier system and the results complemented those of RT-PCR.