A switching role of hard-uptake nanoparticles in microalgae cell electroporation

The microalgal cell wall is a natural barrier that limits the efficiency of gene delivery in algae genetic engineering. Here, we report the role of hard-uptake nanoparticles (huNPs) in microalgae cell electroporation to enhance the delivery of genes in Chlamydomonas reinhardtii. This role can be divided into two categories: (i) a 'transient state' for short-term behavior under confocal visualization and (ii) a 'steady state' for long-term behavior in cell culture. First, the 'transient' role of gene-huNP complexes was investigated after washing for clear confocal imaging to observe the location of huNPs after electroporation. Second, the 'steady-state' role of the gene-huNP complexes was examined after electroporation by transferring cells to a fresh, medium-rich culture environment without washing to obtain a stable cell culture. For selection of the huNPs, we used two types of nanoparticles (NPs, 250 nm and 530 nm) larger than the threshold size of electroporation uptake to avoid unwanted endocytic uptake of NPs. In the transient state, the visualization results indicate that gene-NP (250 nm) complexes were positioned on the cells and helped to deliver more genes than did the 530 nm NPs. In the steady state, the gene-NP (530 nm) complexes helped stably deliver more genes to the cells by precipitation of NPs due to gravity. We believe that these findings illustrate how gene-NP complexes function in microalgae cell electroporation and could help set up a protocol for enhanced microalgae applications associated with NPs such as environmental waste removal and biofuel production.