SBIR-STTR Award

Plants produce a wide variety of valuable bioactive metabolites, but these are commonly present in low concentrations in the wild-type plant. This makes the separation and purification of these compounds complicated and expensive. The applicant company, Naprogenix Inc, has developed a technology for increasing the yields of specific bioactive metabolites in mutant plant cell cultures, which promises to lessen this problem. However, the value of this, or any other approach using genetically-modified plant cells, is limited by the necessity of the wasteful destruction of the plant cells in order to extract the required products. The process would be more efficient if products could be harvested from cells in continuous culture. Nanoparticles are taken up by cells by endocytosis, and subsequently exocytosed. They can also be engineered to adsorb specific chemicals. This suggests that specifically-engineered (functionalized and magnetized) nanoparticles could be used to repeatedly "harvest" specific metabolites from living plant cell cultures. One of the company's major projects is designed to generate novel flavonoids in goldenrod hairy root cultures. The applicants have shown that functionalized silica nanoparticles adsorb similar flavonoids and that they are taken up and removed from these plant cell cultures without compromising the viability of the cultures. The objective is now to engineer optimally-functionalized magnetized silica nanoparticles (by collaborators at the University of Kentucky) to harvest specific flavonoids from these hairy roots. The specific aims of phase I are to show that (a) exposure to these nanoparticles translocate flavonoids from the intracellular compartment of hairy roots to the extracellular medium (b) these extracellular magnetized nanoparticles can be concentrated in a magnetic field (c) significant amounts of flavonoids can be eluted and collected from these nanoparticles following their passage through plant cells. If this is successful, then, in phase II, the applicants will design ad test nanoparticles for harvesting other bioactive metabolites from optimized plant cell cultures. These will include very high value chemotherapeutic agents, such as the taxoids and vinca alkaloids. If successful this approach should make a major impact on the use of plant cell cultures for the production and isolation of high value natural products. The nanoparticles, and the harvesting technologies as applied to plant cells, are patentable, and have major commercial implications for the applicant company and the University.

Public Health Relevance Statement:


Public Health Relevance:
Many valuable plant compounds can be produced in tissue culture, but their isolation requires destruction of the plant tissue before chemically separating the required compounds from others. This project aims to develop magnetized nanoparticle devices that can be used to remove specific types of compounds from living plant tissue. This is predicted to greatly increase the efficiency of producing medicines or nutrients in plant tissue culture. These devices will also increase the commercial value of the proprietary biotechnology used by the applicant company, which is designed to optimize plant cell cultures for their ability to produce specific natural products.

Project Terms:
Address; Area; Binding (Molecular Function); Biological Factors; Biotechnology; Cell Culture Techniques; Cell Survival; Cells; Characteristics; Chemical Engineering; Chemicals; chemotherapeutic agent; Collaborations; commercialization; Complex Mixtures; cytotoxicity; design; Devices; Drug Industry; Endocytosis; Engineering; Exposure to; extracellular; Faculty; Fermentation; Flavonoids; Funding; Genetically Modified Plants; Harvest; High Pressure Liquid Chromatography; improved; in vitro testing; Individual; Industry; Kentucky; Licensing; Life; magnetic field; Mammalian Cell; Marketing; Measures; Medicine; Methods; microbial; mutant; nanoparticle; novel; Nutrient; One-Step dentin bonding system; Pharmacologic Substance; Phase; Plant Roots; Plants; Preparation; Process; Production; Protocols documentation; prototype; public health relevance; Quercetin; radioligand; Research; Research Personnel; response; Schedule; Silicon Dioxide; Small Business Technology Transfer Research; Solidago; Specificity; Surface; System; Taxoids; Technology; Testing; Time; tissue culture; Tissues; Universities; Vinca Alkaloids

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).

Public Health Relevance Statement:
Project narrative Drugs can be produced in plant cell cultures as an alternative to extracting them from intact plants. This has some advantages, but a major disadvantage is that “harvesting” the drugs requires the destruction of the slow growing and expensive plant cell cultures. This phase II proposal is to further develop a continuous harvesting system in which nanoparticles can be used to extract drugs from plant cells without damaging the cells.

Project Terms:
Affect; Affinity; Agonist; Agreement; Alkaloids; Antibodies; Antibody Binding Sites; antigen binding; Antigens; Artificial nanoparticles; Binding; Binding Sites; Bioreactors; Biotechnology; Catharanthus roseus; Cell Culture Techniques; cell injury; Cell Survival; Cells; Characteristics; chemical synthesis; Chemicals; chemotherapy; commercialization; Complex; crystallinity; Development; Disadvantaged; Disease Outbreaks; Engineering; Enteral; Equilibrium; Estrogen Receptor beta; Estrogen Receptors; Europe; Excision; experimental study; Exposure to; Flavanones; Flavonoids; Fluorescence; Germany; Harvest; Human; Immobilization; In Vitro; indexing; Individual; International; Isoflavones; kartocid; Legal patent; Licensing; Licorice; Ligand Binding; malignant breast neoplasm; Malignant Neoplasms; Monoclonal Antibodies; mutant; Mutation; nanoparticle; neoplastic cell; Nicotiana tabacum; Oligopeptides; Paclitaxel; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Phytoestrogens; Plant Roots; Plants; polypeptide; Postmenopause; Procedures; Production; protein aminoacid sequence; Proteins; Quartz; radioligand; Reaction Time; Risk; S-Adenosylmethionine; solvent extraction; Source; Sterility; success; Surface; System; Taxoids; Technology; Testing; Therapeutic Effect; Time; Tissues; Tobacco; Transgenic Organisms; Transgenic Plants; Tubulin; Vaccines; Vinca Alkaloids