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Harvesting specific plant metabolites from hairy root cultures using magnetized nanoparticles

使用磁化纳米颗粒从毛状根培养物中收获特定的植物代谢物

基本信息

批准号：
9343261
负责人：
JOHN M. LITTLETON
金额：
$ 53.03万
依托单位：
NAPROGENIX, INC
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9343261
关键词：
Affect Affinity Agonist Agreement Alkaloids Antibodies Antibody Binding Sites Antigens Artificial nanoparticles Binding Binding Sites Bioreactors Biotechnology Catharanthus roseus Cell Culture Techniques Cell Survival Cells Characteristics Chemicals Complex Development Disadvantaged Disease Outbreaks Engineering Equilibrium Estrogen Receptor beta Europe Excision Exposure to Flavanones Flavonoids Fluorescence Germany Harvest Human Immobilization In Vitro Individual International Isoflavones Legal patent Licensing Licorice Ligand Binding Malignant Neoplasms Monoclonal Antibodies Mutation Nicotiana tabacum Oligopeptides Paclitaxel Peptides Pharmaceutical Preparations Pharmacologic Substance Pharmacology Phase Phytoestrogens Plant Roots Plants Postmenopause Procedures Production Proteins Quartz Reaction Time Risk Source Sterility Surface System Taxoids Technology Testing Therapeutic Effect Time Tissues Tobacco Transgenic Organisms Transgenic Plants Tubulin Vaccines Vinca Alkaloids anti-cancer therapeutic antigen binding base cell injury chemical synthesis chemotherapy commercialization crystallinity experimental study indexing kartocid malignant breast neoplasm mutant nanoparticle neoplastic cell polypeptide protein aminoacid sequence radioligand solvent extraction success

项目摘要

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).

摘要：植物细胞培养物正在成为具有商业价值的药物来源，特别是那些对于经济化学合成而言过于复杂的物质。例如，德国的 Phyton Biotech 已通过在无菌植物细胞中生成紫杉醇用于紫杉醇生产，取得了巨大的商业成功生物反应器。然而，这些系统的效率受到生长缓慢的植物活力丧失的限制与传统提取程序相关的细胞。这里的目标是开发一个系统允许重复收获植物细胞以生产高价值的药品，而不会失去活力。第一阶段证明纳米颗粒可以功能化进入植物细胞并结合特定的生物活性挤压前的类黄酮代谢物，以及这些代谢物的回收，均不损失植物细胞生存能力。第二阶段现在的目标是证明可以使用类似但更具选择性的方法来收获来自植物细胞的更高价值的药物（即应用证明）。最有价值的代谢物类型目前从植物中生产的化合物包括异黄酮、生物碱和单克隆抗体（后者来自转基因植物）。第二阶段旨在证明每种类型的产品都可以从植物细胞中收获通过它们选择性地结合到已缀合有特定寡肽的纳米颗粒上。每个产品例子与抗癌疗法有关。第一种是植物雌激素甘草素，它是一种选择性雌激素。雌激素受体 (ER)β 激动剂，可降低绝经后患乳腺癌的风险。这黄烷酮将从甘草根的过量生产突变体培养物中通过选择性结合来收获 ERbeta 配体结合寡肽与纳米颗粒缀合。第二个例子是纳米收获化疗用长春花生物碱（目前由礼来公司从完整的植物材料中提取）来自过度生产长春花的突变培养物。这些生物碱将通过与带有纳米粒子的亲和力来收获代表其在人微管蛋白上的结合位点的寡肽。这两个例子是天然代谢物，但该技术最重要的商业应用可能是收获外源多肽，即来自转基因植物细胞的“生物制品”，例如抗体。这里的例子是从针对肿瘤细胞的单克隆抗体 (mAbH10) 的转基因烟草细胞培养物。选择性结合将使用纳米颗粒来实现，其中寡肽模仿抗体结合位点抗原已缀合至表面。在所有这些例子中，目的是表明纳米粒子可以反复去除所需的商业产品，而不损失植物细胞的活力。这将减少“停机时间”，也可以减少“响应时间”，例如紧急需求疾病爆发时的抗体或疫苗。此外，通过对产物的亲和力进行分离寡肽结合位点意味着收获的产物将同时被半纯化。第二阶段应证明纳米颗粒收获技术应用于高价值抗癌药物。然后，申请人将与以下公司合作走向商业化：确定了美国和欧洲的制药和生物技术公司（参见商业化计划）。