Abstract
Access to the inside of the cell remains one of the long-standing hurdles for therapeutics and diagnostics. A diversity of physical, biological and chemical methodologies have emerged to achieve the delivery of membrane-impermeable exogenous materials across the cell membrane. Striving for spatial-temporal control and enhanced in vitro intracellular delivery efficiencies, optoporation has emerged as a promising technique. This physical method involves using a high-intensity laser pulse to induce transient pores within the cell membrane. Specifically, the high selectivity of optical transfection enables precise cellular manipulation for a deeper understanding of biomolecular mechanisms and opens new avenues, for example, in in vitro studies on stem cell differentiation and reprogramming dynamics. To improve the optoporation efficiency, nanomaterials such as gold and carbon-based nanoparticles are often coupled with the laser, however, these are not biodegradable and can be genotoxic, limiting their applicability in advanced therapies and modelling.This work presents an alternative photothermal nanomaterial to the conventional, frequently used nanoparticles. Herein, I fabricated and characterised biodegradable porous silicon nanoparticles through metal-assisted chemical etching and electrochemical etching to produce rod-like and discoid-like particles, respectively. Viability assays have shown that a 24-hour interaction between the nanoparticles and the cells had no significant influence on cell death or proliferation. The two different geometries, both approximately 300 nm in size, were coupled with a femtosecond laser in the near-infrared region (800 nm). The coupling excited the porous silicon nanoparticles and achieved spatial-selective control over the delivery of propidium iodide only in the individually laser-scanned MCF-7 breast cancer cells. Maintaining cell viability after delivery is crucial, therefore, the short-term cell viability (30 minutes) post-optoporation was confirmed in 2D cell culture through calcein-AM retention as a sign of reversible membrane permeability. Through further optimisations of this nano-sensitised optoporation technique, propidium iodide was also delivered in selected areas within the 3D MCF-7 cancerous spheroid model. Furthermore, this optoporation system led to the spatial-selective transfection of the cells with eGFP mRNA and expression of the fluorescent GFP protein for both the 2D and the 3D systems. Thus, confirming selective cellular manipulation of target cells. These data demonstrate efficacy of porous silicon nanoparticles combined with a femtosecond laser in the near-infrared region for the delivery and expression of genetic material. The successful outcome observed in both the 2D and 3D models establishes the groundwork for spatial-temporal nucleic acid delivery, enabling applications in cell engineering and advancing our understanding of cellular biological mechanisms.
| Date of Award | 1 May 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Ciro Chiappini (Supervisor) & Khuloud Al-Jamal (Supervisor) |