(496g) Nanoharvesting of Therapeutics from Living Plant Cultures By Engineered Mesoporous Silica Nanoparticles

Plant secondary metabolites often bind to human receptors to induce favorable responses to diseases and pathogens, and as a result are valuable therapeutics. Recent advances allow enrichment of these biomolecules in a particular plant cell cultures by genetic and environmental manipulations, but the conventional method of recovery of natural products from these genetically engineered plants is destructive to the tissues, involving macerating the whole tissues to gain access to the compounds. 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 designed so that they enter into plant cells and are released without inducing significant toxicity, and with specific binding of compounds of interest. An understanding of time and concentration dependent interaction of engineered nanoparticle with plant cells by tracking the particles in their routes in and out of the cells is necessary in order to design optimum particle properties.

Here, nanoharvesting of polyphenolic flavonoids, a model class of plant produced therapeutics, enriched in genetically modified S. nemoralis hairy root cultures, is performed 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. MSNPs (165 nm diameter) with highly porous structures were synthesized and functionalized with amines and TiO₂. Particles were shown to be taken up in S. nemoralis hairy roots, and recovered using external centrifugal forces. 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 (RITC). Quenching of fluorescence in bulk solution using trypan blue was used to confirm intracellular localization of the tagged particles. Post-uptake viability and flavonoid reproduction potential of hairy roots was demonstrated by a concentration and functionalization dependent growth study. Proof of the flavonoid nanoharvesting was demonstrated from increased antiradical activity against 2, 2-diphenyl-1-picrylhydrazyl and displacement of specific ligand [3H]-methyllycaconitine by centrifugally recovered particles. Quantification of the TiO₂ functionalized and fluorescent tagged nanoparticle uptake and recovery mechanisms after uptake will be reported by analyzing TiO₂ content of the particle exposed roots and the fluorescence intensity of recovered solution, respectively. This application of engineered mesoporous silica nanoparticles is broadly applicable as advanced separation process for the broad classes of biomolecules from living and functioning plant cultures.