Aptamers are selected nucleic acid binding species with affinities and specificities for protein targets that rival those of monoclonal antibodies. Furthermore, aptamers have definite advantages over antibodies, in that they can be chemically synthesized and modifications can be introduced that improve their stabilities and pharmacokinetic properties. To date most approaches selectively targeting nanoparticles have used antibodies or antibody derived molecules. In this project we are investigating the potential of aptamers to
(i) target specific cancer cells and
(ii) develop strategies for aptamer nanoparticle coupling, testing and separation.
Long term objectives
Depending upon future funding and the results of pilot experiments there are a number of areas which could be investigated
Aptamer targeting of drugs targeted to reduce cancer cell invasion. Dr Elizabeth Rapa & Dr Sophie Hill identified a number of genes involved in cell invasion. Using an aptamer targeting approach cancer cells could be targeted in the circulation with nanoparticles containing siRNA which down regulates cell invasion genes. Thus reducing cell invasion and hopefully metastatic disease. In addition to cell targeting, release of the siRNA once inside the cell and selecting the appropriate nano vector are key areas to address.
Aptamer targeting of diagnostics to monitor treatment responses in vivo. Our aim is target specific angiogenic markers associated with new tumour vasculature as a way of rapidly monitoring the effectiveness of new cancer therapies. Allowing new drugs to be assessed in days rather than weeks. We will be screening aptamers for our target over the next few months. A number of companies are developing nanoparticle MRI contrast agents which could be very useful. In addition our experience in nanoparticle coupling will be very valuable in this project.
We are looking nanoparticles as a means of delivering small molecules selectively to cancer cells. The initial work is being carried out by a “sandwich” student as there is some initial work to demonstrate the proof of principle.
Elizabeth Rapa, in her PhD and immediate post-doctoral work, demonstrated that it was possible to inhibit the spread of rhabdomyosaroma cells in a laboratory test system by using a small RNA molecule. The nanoparticle system might be a good way to deliver the inhibitory molecule selectively to the cancer cells in a patient, so that it had no effect on the normal functions of non-cancerous cells.
We have just finish writing up Elizabeth’s work and will very shortly be submitting it for publication. Elizabeth was awarded her PhD in September 2008, and was in part supported by William’s fund.
Over the next year we hope to develop the nanoparticle system further and fund a PhD student to work on it for a three year period. We hope that the student will be the “William Dodd Fellow”, and that person will be very largely funded by William’s Fund.
Current progress and future work
Target specific cancer cells. An aptamer library was prepared containing 1018 different aptamer molecules. Optimal conditions have been determined to exponentially amplify the library and we are currently in the process of enriching for binding to the neuroblastoma line SKN-SH. Enrichment for aptamers is a lengthy process with 20-30 phases of binding of the aptamer library to the cell line followed by amplification of the bound aptamers to enrich for the high efficiency binding molecules. A relatively inexperienced 12 month sandwich placement project student is currently working on this project. Much of the period so far has been taken up with training in the various procedures required and setting up new protocols (tissue culture, PCR etc).
Next 5 months: Over the next 5 months we will identify aptamers binding neuroblastoma cells under different conditions of cell harvesting (i.e trypsin, acutase and scraping). A negative selection protocol will be developed to remove aptamers which bind non-specifically. The final aptamer panel will be tested for binding to other cancer and non-cancer cell lines to assess specificity.
Develop strategies for aptamer nanoparticle coupling, testing and separation. Cells readily take up nanoparticles giving high levels of non-specific uptake which is both a problem in the laboratory and in a clinical setting. In collaboration with groups in the Department of Engineering Science at Oxford and Professor Jeung Sang Go currently on sabbatical from Pusan National University (PNU), Korea we are developing approaches to separate nanoparticles when coupled to aptamers/antibodies
CCRFMagnetic Separation of antibody coupled nanoparticles – manufacture of the device is depending on a £7k application for funding support from the University of Oxford, John Fell Fund .
We currently have a 2 year MSc student who is developing separation approaches for aptamer bound nanoparticles. The project involves the coupling of the well characterised aptamer to the cancer marker MUC1 to fluorescently labelled nanoparticles.
Progress to date: MUC1 aptamers have been coupled to FITC labelled nanoparticles. Coupled particles currently have aggregation problems which
are being addressing. pH changes and coating particles with anti-aggregation agents will be assessed.