Prussian blue nanoparticles delivery and internalization in Coffea arabica living protoplasts

Nanoparticle cargo in animal cells, in particular Prussian blue nanoparticles even of 70 nm size, can enter inside and provide therapeutic action for cancer therapy. However, passive transport of macromolecules is not feasible challenging in plants, as plant cells are enclosed by a cell wall due to the cell wall barrier, which restricts the size exclusion limit of particles. To overcome this limitation, we developed a high-density protoplast preparation from embryogenic calli of Coffea arabica cv. Caturra. The ability of these protoplasts to uptake 70 nm aminoanthracene-functionalized Prussian blue nanoparticles (1@AA) was investigated using luminescence tracking at 504 nm. Under electroporation conditions (220V for 70 μs), we observed the highest uptake of 1@AA nanoparticles into the protoplasts. Transmission electron microscopy (TEM) and multiphoton spectral imaging confirmed the internalization of nanoparticles within the cytoplasm. A viability test indicated a 77-90% survival rate of protoplasts after 24-h treatment, demonstrating that the electroporation-assisted uptake of large nanoparticles (ca. 70 nm) is feasible in C. arabica protoplasts without compromising cell viability. These results represent the first demonstration of large nanoparticle internalization in Coffea arabica protoplasts under controlled electroporation conditions, offering a potential platform for future nanobiotechnological applications in plant cells.