Nanoharvesting of Polyphenolic Flavonoids from Solidago Nemoralis Hairy Root Cultures Using Functionalized Mesoporous Silica Nanoparticles

Plant secondary metabolites are produced in response to the environment of certain plants as a defense mechanism against predators and other stressors. Sometimes, these metabolites also bind to human receptors to induce favorable responses to diseases and pathogens, which can be enriched in a particular plant cell cultures by genetic and environmental manipulations. The conventional method of recovery of natural products from these genetically engineered plants is to grow the plants to a certain degree of maturity and then to harvest whole tissues, macerate the tissues to gain access to the compounds, and extract them. Nanoharvesting offers an alternative for continuous production of secondary metabolites from plant cell cultures, in which nanoparticles are designed to bind and carry molecules out of living cells. The carriers are to be designed so that they enter into plant cells and are released without inducing significant toxicity, and with specific binding of compounds of interest.

Here, nanoharvesting of polyphenolic flavonoids, enriched in genetically modified S. nemoralis hairy root cultures, is carried out using mesoporous silica nanoparticles (MSNPs) functionalized with both amines and titanium dioxide (TiO₂). Amine functionalization was performed to facilitate uptake of nanoparticles into plant cells, and TiO₂ functionalization to provide coordination binding sites for metabolites. Non-magnetic MSNPs (170 nm diameter) and magnetic MSNPs with Fe₃O₄ cores (70 nm diameter) with highly porous structures were synthesized and functionalized with amines and TiO₂. Both particles were shown to be taken up in S. nemoralis hairy roots, and recovered using external centrifugal and magnetic forces, respectively. Intracellular uptake and localization of the nanoparticles (0-1000 µg/ml in Murashige and Skoog media) in hairy roots were visualized by fluorescent imaging, after tagging the nanoparticles with rhodamine B isothiocyanate. Quenching of fluorescence in bulk solution using trypan blue was used to confirm intracellular localization of the tagged particles. Post-uptake viability of hairy roots was demonstrated by a concentration-dependent growth study. Proof of the flavonoid nanoharvesting concept was inferred from the color change (white to orange) of the recovered nanoparticles after exposure to hairy roots. Quantitative determination and analysis of nanoharvested flavonoids will be reported based on antiradical activity of metabolites bound to particles and HPLC.