Harvesting specific plant metabolites from hairy root cultures using magnetized nanoparticles

摘要：植物细胞培养正在成为一种有商业价值的药物来源，尤其是 那些对于经济的化学合成来说太复杂的东西。例如，德国的Phyton Biotech拥有 在无菌植物细胞中生产紫杉醇，获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生存能力的丧失的限制。 与常规提取程序相关的细胞。这里的目标是开发一种 允许植物细胞被重复收获，以生产高价值的制药产品，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。 黄酮类代谢物在挤压前被挤压，这些代谢物被回收，全部不损失植物细胞 生存能力。第二阶段现在的目标是证明可以使用类似的、但更有选择性的方法来收获 来自植物细胞的价值更高的药物(即应用证明)。最有价值的代谢物类型 目前从植物中生产的包括异黄酮类、生物碱和单抗(后者来自 转基因植物)。第二阶段的目的是证明每一种类型的产品都可以从植物细胞中获得。 通过它们选择性地结合到其上连接了特定寡肽的纳米颗粒。每种产品 这个例子与抗癌疗法有关。第一种是植物雌激素甘草素，它是一种选择性的 雌激素受体(ER)β激动剂，应可降低绝经后患乳腺癌的风险。这 黄烷酮将通过选择性地与甘草根结合，从高产的甘草根突变菌种中获得 结合ERbeta配体的寡肽结合到纳米颗粒上。第二个例子是纳米收获 化学治疗长春花碱(目前由礼来公司从完整的植物材料中提取)防止过度生产 长春花突变体培养。这些生物碱将通过亲和力作用于含有 代表其与人微管蛋白结合部位的寡肽。这两个例子是天然的代谢物， 但这项技术最重要的商业应用可能是收获外来多肽，即 来自转基因植物细胞的“生物制品”，如抗体。这里的示例将是从 针对肿瘤细胞的单抗(MAbH10)的转基因烟草细胞培养。有选择性的 结合将使用纳米颗粒，其中的寡肽模拟抗体结合部位 抗原已结合到表面上。在所有这些例子中，目标都是要表明 纳米颗粒可以反复去除所需的商业产品，而不会损失植物细胞的活力。这 将减少“停机时间”，还可能减少“响应时间”，例如，紧急要求 疾病暴发中的抗体或疫苗。此外，按亲和力将产品分离 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的应用证明适用于高价值 抗癌药物。然后，申请者将与以下公司合作走向商业化 确定美国和欧洲的制药和生物技术公司(见商业化计划)。

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).