Plasmonic-induced molecular transfer and its perspectives in plant Science

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. 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) photoinjection: 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. Herein, we investigate GNOME laser transfection in the context of plant cells, which are a promising target for manipulation via genome editing for breeding purposes. For GNOME laser transfection, as well as for other laser-based delivery approaches, the plant's cell wall represents a major 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. The presented approach therefore includes the formation of protoplasts from climate chamber cultivated Nicotiana benthamiana plants in isotonic solution before incubation with the gold nanoparticles and laser illumination of the sample. We investigate the impact of different process parameters on the viability and delivery efficiency of marker molecules. The presented approach provides the basis for future vector free genome editing of plant cells.