| Abstract |
Around 4000 human diseases have been traced to gene disorders so far (1). Most of them are life-threatening and affect all aspects of patient’s life in a daily basis. The only way to eliminate a genetic disease is to treat it at its roots. Gene therapy is an experimental technique that works in this direction by using different approaches including the replacement or the "silencing" of a mutated gene and the introduction of a new gene. The intracellular delivery of genetic material is well known as "transfection" and it is either viral or non-viral.
The delivery of genes to mammalian cells using non-viral methods has become a very promising approach for gene therapy in the last few years (2). One of the current impediments of successful gene therapy is the inefficient delivery of the corrective nucleic acid code into target cells. New methods are required to deliver nucleic acid reagents into diverse cell types effectively and with high yields.
In this study, we report on the development of a novel delivery platform for cell transfection studies in vitro, comprising of vertically aligned silicon nanowire (VA-SiN) arrays with a tapered profile, termed as "silicon micro-conical tips (Si MC tips)". Fabrication starts with self-assembly of a hexagonal close-packed (hcp) 2D array of polystyrene nanospheres (PSNS) over a large area of a Si wafer via convective assembly. The resulting hcp monolayer array is then converted into non-close-packed monolayer arrays using O2 plasma etching. The etched PSNS then serve as a mask for the Deep Reactive Ion Etching (DRIE) of silicon using the "Bosch" process. The final step in the fabrication of the micro-conical arrays is the formation of a smoother and sharper morphology using a combination of thermal oxidation and wet etching.
These arrays were classified in three different categories, regarding the height of the tips, exhibiting a range of architectures (Table 4.2) and together with flat Si wafers have been applied to in vitro cell cultures using HEK293 cells as a model cell line. After the functionalization of the Si MC tip arrays’ surface with Green Fluorescent Protein (GFP)-plasmid, the delivery was performed by mechanical penetration using centrifugation force, with the Si MC tips facing towards the cells (Delivery Method 1). A delivery system like this can offer us two main advantages: fast delivery and parallel delivery of the plasmid to a huge amount of cells. Before proceeding to cell transfection studies, we examined the viability of the cells during their contact with the Si MC tips as well as the existence or not of penetration through the cell membrane into the cytoplasm by FIB-SEM.
The viability studies performed demonstrated that a prolonged incubation (for a couple of hours) of the aforementioned setup leads progressively to the death of the cells in both cases. At the footprint that the Si MC tips leave, after their removal, on the well plate and at the Si MC tips themselves. This is probably attributed to the lack of oxygen and nutrition available to the cells during the contact. However, a short incubation of maximum 30 m time leaves all the cells alive. In addition, there are still remaining cells attached on the well plate, fact that gives us the potential of achieving transfection on the well plate as well. As far as the FIB-SEM imaging is concerned, the fluorescent images taken revealed the successful cytoplasm penetration of the Si MC tips of all different characteristics within 15 m and 30 m incubation time respectively. This is a good but not an adequate indicator that we will have transfection as well.
Finally, we proceeded to transfection studies in order to test the efficacy of our plasmid DNA delivery system. For comparative reasons, cell transfection studies were performed employing an additional strategy (cells on top of the Si MC tips – Delivery Method 2) and following a similar protocol. Our results showed that cell transfection was achieved using both delivery methods. Transfected cells were detected on the surface of the Si MC tip arrays and especially on those comprising of higher and pointed tips in the same time. However, no transfected cells were detected on the well plate using Delivery Method 1 (Si MC tips on top of the cells) within 20 m of incubation.
To sum up, in this study we presented at first a novel method for the fabrication of vertically aligned silicon NW arrays with a tapered profile using a combination of nanosphere lithography and deep reactive ion etching. At a second step, we used these SiNW arrays as a cell transfection platform in vitro in order to deliver plasmid DNA into HEK293 cells. The delivery of the plasmid is achieved by mechanical penetration of the NWs into the cytoplasm with the aid of centrifugation force using a very short experimental protocol. We suggest that a delivery system like this could be a future tool in facilitating gene therapy practices.
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Processing of biomaterials surfaces for gene delivery applications
