Empty virus-like particles (eVLPs) as bio-compatible targeted drug-delivery vehicles

药理学的一个主要挑战是设计方法，使药物能够特异性地递送到目标组织。这在抗癌药物中是一个特别的问题，因为抗癌药物通常根据癌细胞分裂更快的事实来区分癌细胞和正常细胞。然而，抗癌药物对所有细胞都是有毒的，因此往往有严重的副作用。为了避免这种情况，将药物分子特异性地靶向癌变组织显然是可取的。实现这一目标的一种潜在方法是将药物分子包装或封装在一种颗粒中，这种颗粒被设计成只与癌组织结合。这样的封装还有一个额外的好处，就是可以防止药物在血浆中分解。为了使这成为现实，有必要开发出可以在其外表面进行修饰以达到预期目标的颗粒，并且可以包含药物分子。这些颗粒必须足够小，能够在血液中移动，没有毒性，并且能够进入细胞。一种具有所有这些特征的颗粒是植物病毒，豇豆花叶病毒（CPMV），它可以通过感染植物大量产生。先前的研究表明，可以将分子“粘”在CPMV颗粒的表面，使它们能够靶向特定的细胞。尽管有这些优点，CPMV颗粒迄今尚未被开发为药物递送载体。其原因是植物感染产生的颗粒已经充满了病毒自身的遗传物质。这意味着几乎没有空间可以放置其他东西，如药物分子，在颗粒内。即使这样的分子能以某种方式被挤进去，人们仍然会担心管理含有病毒遗传物质的颗粒，即使它来自一种不能感染动物的植物病毒。该项目通过利用John Innes中心最近发现的一种在植物中生产大量纯空（缺乏遗传物质）CPMV病毒样颗粒（evlp）的方法，解决了病毒颗粒内遗传物质的问题。该方法包括使用最近开发的高效植物瞬时表达系统同时表达编码病毒外壳蛋白前体的基因和用于处理它的酶。应用这种方法，我们将生产出经过修饰的颗粒，使它们能够特异性地与癌细胞（而非正常细胞）表面表达的蛋白质结合。这种修饰可以通过化学方法或与病毒外壳蛋白进行基因融合来完成。我们将研究用抗癌药物吉西他滨装载靶向颗粒的最佳方法。为了做到这一点，我们将利用病毒颗粒中的孔，这些孔允许小分子在特定条件下进入。研究人员将通过测试目标颗粒在培养物中与癌细胞结合并杀死癌细胞的能力，来研究它们将药物输送到癌细胞的能力。这将是开发全新治疗人类癌症药物的第一步。

A major challenge in pharmacology is to devise methods whereby drugs can be delivered specifically to target tissues. This is a particular issue in the case of anti-cancer drugs which usually discriminate between cancerous and normal cells by the fact that the cancer cells are dividing more rapidly. However anti-cancer drugs are toxic to all cells and thus often have severe side-effects. To avoid this, it would clearly be desirable to target the drug molecule specifically to the cancerous tissue. A potential means of achieving this would be to package or encapsulate the drug molecules inside a particle which is designed to bind solely to the cancerous tissue. Such encapsulation would have the additional advantage of protecting the drug from breakdown in blood plasma. For this to become a reality it will be necessary to develop particles which can be modified on their outer surface to achieve the desired targeting and which can contain drug molecules. The particles need to be small enough to be able to move in the bloodstream, are not toxic and to be able to enter cells. One type of particle which has all these characteristics is the plant virus, cowpea mosaic virus (CPMV), which can be produced in large quantities by infecting plants. Previous research has shown that it is possible to 'stick' molecules on the surface of CPMV particles which enable them to be targeted to specific cells. Despite these advantages, CPMV particles have not, to date, been exploited as a drug-delivery vehicle. The reason for this is that particles produced by the infection of plants are already filled with the virus' own genetic material. This means that there is little or no room to put anything else, such as drug molecules, inside the particles. Even if such molecules could somehow be squeezed in, there would still be concerns about administering particles containing viral genetic material, even though it is from a plant virus that cannot infect animals. This project addresses the issue of the genetic material within the virus particles by exploiting the recent discovery at the John Innes Centre of a method of producing large quantities of pure empty (lacking genetic material) virus-like particles (eVLPs) of CPMV in plants. The method involves simultaneously expressing genes coding for a precursor of the viral coat proteins and the enzyme used to process it using a recently developed highly efficient plant transient expression system. Applying this approach we will produce particles which are modified so that they will specifically bind to proteins expressed on the surface of cancerous, but not normal, cells. This modification will be done either chemically or by making genetic fusions to the virus coat protein. We will investigate the best way of loading the targeted particles with the anti-cancer drug, gemcitabine. To do this we will make use of pores in the virus particles which allow small molecules to enter under certain conditions. The ability of the targeted particles to deliver the drug to cancer cells will be investigated by testing their ability to bind to and kill cancers in culture. This will be the first important step in potentially developing brand new therapeutic agents to tackle human cancer.