Harvesting specific plant metabolites from hairy root cultures using magnetized nanoparticles

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Abstract: Plant cell cultures are becoming a commercially valuable source of pharmaceuticals, particularly those that are too complex for economical chemical synthesis. For example Phyton Biotech, in Germany, has achieved great commercial success by generating taxoids for Paclitaxel production in sterile plant cell bioreactors. However, the efficiency of these systems is limited by the loss in viability of the slow-growing plant cells associated with conventional extraction procedures. The objective here is to develop a system that allows plant cells to be harvested repeatedly for high value pharmaceutical products without losing viability. Phase I demonstrated that nanoparticles can be functionalized to enter plant cells and bind specific bioactive flavonoid metabolites before being extruded, and these metabolites recovered, all without loss of plant cell viability. Phase II now aims to demonstrate that a similar, but more selective, approach can be used to harvest higher value pharmaceuticals from plant cells (i.e. proof of application). The most valuable types of metabolite currently produced from plants include isoflavones, alkaloids and monoclonal antibodies (the latter from transgenic plants). Phase II aims to show that each of these types of product can be harvested from plant cells by their selective binding to nanoparticles on which specific oligopeptides have been conjugated. Each product example is relevant to anti-cancer therapeutics. The first is the phytoestrogen, liquiritigenin, which is a selective agonist of the estrogen receptor (ER)beta that should reduce risk of breast cancer post-menopause. This flavanone will be harvested from overproducing mutant cultures of licorice root by selective binding to the ERbeta ligand-binding oligopeptide conjugated to nanoparticles. The second example is to nanoharvest the chemotherapeutic vinca alkaloids (currently extracted from intact plant material by Eli Lilly) from overproducing mutant cultures of Catharanthus roseus. These alkaloids will be harvested by affinity to nanoparticles bearing oligopeptides representing their binding sites on human tubulin. These two examples are natural metabolites, but the most commercially important application of this technology may be to harvest foreign polypeptides, i.e. “biologics”, such as antibodies, from transgenic plant cells. Here the example will be the harvesting from transgenic tobacco cell cultures of a monoclonal antibody (mAbH10) directed against tumor cells. Selective binding will be achieved using nanoparticles in which an oligopeptide mimicking the antibody-binding site on the antigen has been conjugated to the surface. In all of these examples the objective is to show that nanoparticles can repeatedly remove the desired commercial product without loss of plant cell viability. This will reduce “down time” and could also reduce “response time”, for example the urgent requirement for antibodies or vaccines in an outbreak of disease. In addition, separation of product by affinity to an oligopeptide binding site means that the harvested products will be simultaneously semi-purified. Phase II should demonstrate proof of application for the nanoparticle harvesting technology as applied to high value anti-cancer pharmaceuticals. The applicants will then move toward commercialization in partnership with identified pharmaceutical and biotechnology companies in the US and Europe (see Commercialization Plan).