title: Biomaterial constructs for delivery of multiple therapeutic genes: a spatiotemporal evaluation of efficacy using molecular beacons.
abstract: 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.
authors.affiliation: Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Ireland.
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Alexander
authors.forename: Shane
authors.initials: S
authors.lastname: Browne
authors.forename: Abhay
authors.initials: A
authors.lastname: Pandit
authors.forename: Yury
authors.initials: Y
authors.lastname: Rochev
mesh_terms: D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry; D003692:Delayed-Action Preparations / Q000737:chemistry; D004058:Diffusion; D004337:Drug Carriers / Q000737:chemistry*; D004339:Drug Compounding; D015870:Gene Expression; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D008863:Microspheres; D015335:Molecular Probes / Q000737:chemistry*; D018919:Neovascularization, Physiologic; D052250:Nitric Oxide Synthase Type III / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D023822:Tissue Engineering; D054457:Tissue Scaffolds; D014162:Transfection
chemical_list: D003692:Delayed-Action Preparations; D004337:Drug Carriers; C508609:IL10 protein, human; D015335:Molecular Probes; D016753:Interleukin-10; D003094:Collagen; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III