Biomaterial Constructs for Delivery of Multiple Therapeutic Genes: A Spatiotemporal Evaluation of Efficacy Using Molecular Beacons Jennifer C. Alexander 1 , Shane Browne 1 , Abhay Pandit 1 , Yury Rochev 1,2 * 1 Network of Excellence for Functional Biomaterials (NFB), National University of Ireland, Galway, Ireland, 2 National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland 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. Citation: Alexander JC, Browne S, Pandit A, Rochev Y (2013) Biomaterial Constructs for Delivery of Multiple Therapeutic Genes: A Spatiotemporal Evaluation of Efficacy Using Molecular Beacons. PLoS ONE 8(6): e65749. doi:10.1371/journal.pone.0065749 Editor: Yves St-Pierre, INRS, Canada Received January 9, 2013; Accepted April 29, 2013; Published June 3, 2013 Copyright: ß 2013 Alexander et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This material is based upon works supported by the Science Foundation Ireland and the European Research Development Fund [Grant no. 07/SRC/ B1163].The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Abhay Pandit is an academic editor for the PLOS ONE journal. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: yury.rochev@nuigalway.ie Introduction Cardiovascular diseases are the leading cause of death in the Western world and account for more than 17 million deaths globally (WHO 2012) [1]. Cell-based therapies have been investigated to promote tissue regeneration, but have proven to be challenging due to cell death, low retention of cells at the site, and poor integration of cells with the native tissue [2]. Gene therapy is emerging as a potential therapeutic approach to address the challenges of cell-based strategies [3], with local gene transfer being a more effective therapy [4]. In addition, combination therapies are becoming increasingly important strategies to improve the efficacy of therapeutics [5,6]. An appropriate gene therapy approach for cardiovascular pathogenesis may require multiple genes to enhance therapeutic outcome by modulating inflammatory response and angiogenesis in a controlled and time-dependent manner. Interleukin 10 (IL-10) is a potent multifunctional cytokine produced by a variety of cells [7]. It plays a crucial role in vivo in the attenuation of immune and inflammatory responses [8]. On the other hand, endothelial nitric oxide synthase is an inducible gene expressed in vascular endothelial cells (e.g. HUVECs) and few other cells [9,10]. It is an enzyme that catalyses the conversion of the amino acid L- arginine to L-citrulline to produce nitric oxide (NO), a potent vasodilator and mediator of angiogenesis and arteriogenesis [11]. NO has multiple biological functions and plays an important role in cardiovascular homeostasis [12,13]. Intracellular delivery of the genetic materials is the main challenge to specific and efficient gene therapy. There are two types of delivery systems available for gene transfer, viral and non- viral. Generally, non-viral vectors do not transfer gene material as efficiently as viral vectors [14]. However, non-viral vectors, typically plasmids, are considered safer as they generally exhibit lower toxicity, lower immune responses and do not integrate into the genome [15]. Among the agents used to form complexes with plasmid DNA and facilitate cellular uptake and transfection, SuperFectH, formulated from partially degraded (fractured) dendrimers, is one of the optimal [16]. Partially degraded dendrimers rupture endosomes to allow the escape of the plasmid DNA from degradation. Dendrimers are a class of polymers consisting of highly branched 3D macromolecules usually presenting well defined sizes and structures. The terminal groups exhibit high surface area presenting multiple sites for attachment of plasmids. These properties make dendrimers potential carrier candidates for gene delivery. PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e65749