Proc. SPIE 12131, Nanophotonics IX
Type: Zeitschriftenaufsatz (non-reviewed)
Molecular transfer across cellular membranes into living cells represents a fundamental technical challenge for the biological sciences. Within the biomedical field, a variety of laser-based transfer methods have been explored and successfully applied. In the most abundant approach, a NIR fs laser is tightly focused on the cellular membrane in order to achieve transient permeabilization and a diffusion-driven volume exchange. However, this approach is limited by the single-cell throughput. Plasmonic nanoparticles have proven as viable mediator to dramatically improve the throughput of laser-based molecular delivery within a process termed gold nanoparticle mediated (GNOME) laser transfection: membrane bound nanoparticles are illuminated by 532 nm, 850 ps laser pulses, leading to a confined nanoheater effect. With careful selection of the process parameters, the effect is localized to tens of nanometers around the nanoparticles and can achieve efficient and gentle transient permeabilization of the cellular membrane using a scanning laser setup. This approach has been successfully utilized to deliver a variety of molecules into different mammalian cell types. Herein, we investigate for the first time GNOME laser transfection in the context of plant cells. Plant cells are a promising target for manipulation via genome editing for breeding purposes, but delivery efficiencies for genome editing tools like CRISPR/Cas9 ribonucleotide complexes are still limited. For RNOME laser transfection, as well as for other laser based delivery approaches, the plant cell architecture yields several hurdles. The cells wall represents a quit rigid barrier for molecules above the size exclusion limit of about 40 to 60 kDa. Permeabilization of the cell wall requires high laser energy, which could raise concerns regarding the viability of the cells. In addition, the turgor would induce an outwards driven volume flow upon permeabilization. The presented approach therefore includes the formation of protoplasts from climate chamber cultivated Nicotiana benthamiana plant in isotonic solution before the incubation with the gold nanoparticles. Nanoparticles and protoplasts are brought into close vicinity by a centrifugation approach, before laser illumination of the sample. We investigate the impact of different process parameters (e.g. incubation conditions, laser fluence, scanning speed, …) on the viability and delivery efficiency of marker molecules. The presented approach provides the basis for future vector free genome editing of plant cells.