Gene therapy represents a new technique for the treatment of cancers and diseases due to genomic defects or alterations. Although different approaches have been investigated, successful transport of foreign genetic materials through the cell membrane remains a major hurdle. Viral vehicles are the most commonly used carriers for DNA delivery but they need a precise controlled concentration to reduce toxicity and pathogenic effects. The micro-scaled environment of in vitro models could significantly improve gene transfection by eliminating diffusion issues associated with standard cell culture system but, due to long time experimental setting, it could result in cell nutrient deprivation potentially damaging cells. The development of a rapid and safe delivery technique maximizing efficiency, cell viability and reducing time, is crucial. Ultrasound and micro bubbles (USMB) together with microfluidic and carrier-based techniques can be combined to obtain the desired effect in shorter time. The main goal of this project is to investigate MBs stable cavitation effects on transfection efficiency, evoked by low-intensity US exposure, in a microfluidic platform. The insect baculovirus modified to vehicle actin-Green Fluorescent Protein (actin-GFP) is used in live cells for real time fluorescence imaging to visualize cellular cytoskeleton rearrangements. A flow containing growth media and high concentration of baculovirus is injected in the device seeded with Human Umbilical Vein Endothelial Cells (HUVECs). 2-hours break every 10 minutes is needed to let the virus explain its effect. The change in GFP expression over time is evaluated with a confocal microscope through a dedicated set-up in presence or not of US-activated MBs driven by 1MHz unfocused transducer. An increase of GFP transfection is expected with reduced viral-exposure time.
Recent advances in microfluidics have created new prospects for gene delivery and therapy using viral vectors. The micro-scaled environment within microfluidics system enables precise control and optimization of multiple processes and techniques used in gene transfection. Advantages of this in vitro system rely on number of few cells, small reagents quantities, optimization of surface-to-volume ratio (for a better contact between virus and cells) and tight control on mechanical stimuli [1]. Unlike conventional culture system, microfuidics brings the virus closer to the target cells avoiding waste of materials [2]. Nevertheless, some limitations are present. Due to reduced size, fluid flow through microfluidic channels experiences high pressure [3]. This in turn creates shear stress on cells interfering with endocytotic mechanism for virus uptake or physically removing the virus from cell membrane [4-6]. Furthermore, the small cell culture volume could impact the effective virus concentration and the optimal transfection conditions in cell culture dishes might not be applicable to microfluidic devices. A promising physical approach to improve intracellular molecule uptake is to induce reversible membrane-disruption by the mechanical action of USMB [7-8]. Indeed, it is shown that stable cavitation of gas-filled micro bubbles can induce pore formation and enhanced endocytotic uptake [9-10]. This project aims at investigating the effects of micro bubbles cavitation on transfection efficiency in combination with microfluidic and carrier-based techniques to synergies the overall efforts to overcome limitations coming from individual technique.
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