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  1. 1. Plastic Antibodies: Recent Advances in Synthetic Receptors for Biological Macromolecules Biology with Psychology C1C8 Jananan Nandrakumar 090137918
  2. 2. Jananan Nandakumar (090137918) Biology with Psychology C1C8 New Scientist Article 2 27th September 2011, Professor Shea Plastic Antibodies: Recent Advances in Synthetic Receptors for Biological Macromolecules Antibodies are primary products produced by the immune system particular by a group of specific cells called B-cells. They function in helping destroy and killing harmful and invasive pathogens. These pathogens can be in the form of viruses and bacteria, but each antibody has a unique design specific for each antigen (a substance that triggers immune system to produce antibodies). The design and establishment of synthetic materials has greatly been enhanced over the past few years, and one uses of these synthetic materials is in manipulation of antibodies. The use of nanoparticles is beginning to be used and functionalised to transport artificial antibodies to target site. However, this process is widely becoming a cornerstone of pharmaceutical science and is rapidly evolving. The problem lies though in targeting specific sites. Professor Shea and his team have developed a new means to target specific molecule. This process requires a different strategy of one which involves a protein- protein complex. These protein-protein complexes have really strong bonds (hydrogen bonds). The idea is to create a synthetic molecule which has complementary “weak bonds” that can interact with the protein to form a protein- polymer bond for the intended target of pathogen. The important key requirements for these synthetic antibodies are the control of the sequence and functional groups on the polypeptide chain. Also an element of flexibility is required, so the polymers can adapt to the complementary site. This being said there are many approaches to create synthetic and semi-synthetic proteins. The use of aptamers (specific binding molecule) for example or the combination of small molecule synthesis can be used in this regards. However, Professor Shea’s group have shown that using nanoparticles to create synthetic molecules is a novel means to create functional valid groups of binding sites whilst promoting a technique called molecular imprinting. The technique of molecular imprinting heightens the affinity and selectivity of the synthetic polymer. This occurs by adding a small amount of an epitope (part of an antigen recognised by the immune system), of the intended target into the polymerisation reaction. The importance of the polymerisation is to allow the imprint to organise the functional groups to create a three-dimensional binding site. Once an unstructured domain (epitope) is found and targeted, it is then synthesised via polymerisation, and the epitope is removed to produce a synthetic polymer. The synthetic polymer is then integrated to capture the protein target. First tested molecule was cytochrome c (cyt c), and its C-terminal epitope. The epitope synthesised was –AYLKKATNE-CO2H. From this a synthetic polymer film was
  3. 3. created, then added to a solution to five different proteins, where it was incubated, extracted, and evaluated to show functioning protein on the target site. This was tested by checking whether the synthetic molecule has a high affinity for certain polymers. The results however, showed sensitivity when using imprinting to change the protein structure. Problems lie in the fact that the film technique is technology carries a low capacity, i.e. the amount of proteins processed is quite small. So higher surface area is required, and hence the reason why nanoparticles are so useful. Nanoparticles were then created by techniques called micro-emulsion and precipitation. However, these nanoparticles required optimisation against the target peptide or protein domain. Minimisation of non-specific protein binding needs to occur whilst the addition of the epitope has to result in polymerisation. Once, the nanoparticles were successfully created the peptide melittin was used in experiments. Melittin is a protein which is found as venom in a honey bee. It is amphiphilic and is located in the membrane of targeted cells. So Professor Shea and his colleagues searched a nanoparticle library for a monomer that was able to polymerise in order to produce the correct nanoparticle. Nanoparticles are then evaluated by a technique called atomic force microscopy (AFM). Tests were then carried out by micro-balancing which uses an oscillating crystal with a gold surface (with melittin attached). Nanoparticles then flow through the melittin surface, where frequency is recorded to see if the nanoparticle has affinity. Results showed many nanoparticles to have no affinity however, there were some that did. The nanoparticles that did have affinity were seen to possess hydrophobic domains and negative charge (the same as melittin). Affinity was also tested by a red blood cell test (melittin lyses cells), where nanoparticles were shown to inhibit melittin’s ability to supress red blood cells being lysed. It was found that nanoparticles that were hydrophobic and negatively charged were those that were able to supress melittin. Optimisation then showed while binding melittin to nanoparticles through imprinting, selectivity of high affinity molecules was achieved. In vivo studies in mice were showed no immunogenic response or toxicity issues. Injection of a lethal dose of melittin was administered, followed by an injection of nanoparticles. The problem was that naturally nanoparticles in the biological system have difficulties, as they get coated by serum proteins that alter or supress nanoparticles. When tested these nanoparticles did show little interaction with serum proteins. Results from in vivo studies showed a larger dose of nanoparticles improved survival rate. Real- time tracking experiments by tagging the nanoparticles, with fluorescent markers, showed nanoparticles did in fact neutralised melittin. An autopsy carried out on the mice showed the nanoparticles and melittin were bound together in macrophages in the liver (Figure 1). Figure 1: Toxin Melittin present in the liver accumulated with nanoparticles and macrophages (shown in light blue) (Shea et al, 2008)
  4. 4. These experiments are vital in showing real usage of synthetic polymer nanoparticles as materials that have different sizes and affinities and can be used in a variety of methods in science. However, due to their extremely small size, they are present other problems such as they get separated on affinity, just like other large proteins, and therefore sequencing them could be difficult. A process called thermal catch has been proposed to help in the collection of higher affinity nanoparticles. Nevertheless, it must not be forgotten the importance of implementation of Nano-science in life technologies such as the production of polymer antibodies and as is it a low cost method, highly targeted to specific antigen and can be integrated into existing assays. The stability and storage of these particles is also an advantage, where animals and live cells are not required for production; therefore having a greater ethical approval by the public. However, the creation and research into their use can importantly be used in venom antidote production as well as a range of other tasks. Words; 1055