Schematic representation of a layer of PAMAM-G4 (red) and DLPA (blue) adsorbed at the silica–water interface interacting with single stranded nucleic acids by means of selective pairing to one of the four nitrogenous bases (blue, orange, green and purple). Reproduced by permission of The Royal Society of Chemistry.
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Genetic disorders can be caused by mutations within the individual’s genetic code, resulting in the formation of non-functioning proteins. Normal cellular processes are therefore inhibited. It may be possible to treat these disorders using what is called ‘gene therapy’. Gene therapy involves the insertion of a healthy gene into the individual, allowing a normal, functioning protein to be expressed, or the suppression of a defective protein. Research carried out at STFC’s ISIS facility may have found one of the first steps in advancing the use of gene therapy by studying the molecular interactions between DNA and RNA, and potential gene delivery systems.
The required gene needs to be transported to the intended destination within the body, and requires a ‘vehicle’ which must be non-toxic. Traditionally, viruses have been used to do this as they have evolved specialised molecular mechanisms to efficiently transport their genomes inside the cells they infect. However, viral vectors often provoke an immune response; therefore a safer vehicle must be produced.
Vehicles with non-viral agents, such as dendrimers, can be used. These reduce the likelihood of the nucleic acid being destructed by enzymes within the individual, and also reduce an immune response. A dendrimer is a repetitively branched macromolecule and is typically symmetric around the core, with internal branches and surface groups. The symmetric and hyper-branched structure of a dendrimer has been proposed as an excellent candidate for pharmaceutical applications which include drug delivery. Poly(amidoamine) (PAMAM) is one of the best dendrimers for this, and has been widely investigated for this purpose. Its hyper-branched and exact structure means that PAMAM dendrimers have particularly well-defined nano-structures on surfaces.
PAMAM dendrimers, which are positively charged, condensate negatively charged nucleic acids such as DNA and RNA since they have opposite charges. This means that they are electrostatically attracted, which is vital for transporting and protecting nucleic acids when being delivered to a specific target within the body. However, PAMAM dendrimers lack chemical specificity, meaning they are able to bind many negatively charged surfaces, such as cell membranes. For PAMAM dendrimers to be efficiently used for drug delivery, they must be selective for nucleic acids, and research carried out by scientists at ISIS, Lund University, Sweden, and University of Florence, Italy, may have found one of the first steps towards overcoming this. This can be achieved by functionalising surface groups of the PAMAM dendrimer with molecules called nucleolipids. Nucleolipids are a type of lipid with a nucleoside head group - one of the four nitrogenous bases found in nucleic acids attached to a 5-carbon sugar - with a net negative charge. They have the ability to interact with nucleic acids, which is favoured by selective base pairing, i.e., hydrogen bonds between complimentary base pairs. Scientists using the ISIS facility have shown that the nucleolipid shields the electrostatic attraction of the positive charges of the dendrimer towards negatively charged molecules that do not have nucleotides, providing the dendrimer with chemical specificity for nucleic acids. The work was conducted also in close collaboration with scientists at the ILL and MLZ.
The experiment conducted at ISIS investigated the non-covalent attachment of PAMAM with the oppositely charged nucleolipid 1,2-dilauroyl-sn-glycero-3 phosphatidyladenosine (DLPA). This allowed molecular recognition of DNA and RNA to occur. Neutron reflectivity (NR) experiments on the INTER instrument were carried out. Dr Marianna Yanez from Lund University said “The combination of the neutron flux at ISIS and the resolution of this reflectometer makes possible to record a large number of measurements in just a few days. This can only be achieved currently with very few neutron reflectometers at large scale facilities.”
Attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy was also used. The technique is new to ISIS, and this research is one of the first that utilised the ATR FT-IR spectrophotometer at ISIS. ATR FT-IR refers to the use of a beam of infrared light which is passed through a crystal in contact with a sample, and internal reflectance measurements are taken. The two techniques used complemented each other very well for this investigation. Marianna explains “ATR FT-IR spectroscopy can give you information about chemical bonds of the molecules close to an interface and, in the case of studies with nucleic acids, the conformation of DNA and RNA and the interaction of these molecules with nucleolipids. However, it cannot resolve the structure of the film, i.e. it cannot tell you where the dendrimer, the nucleolipid or the nucleic acids are located. Neutron reflectometry does not have the ability to look at specific chemical bonds but it gives the location and amount of each component in the film.”
It may also be possible for dendrimer/nucleolipid layers to form at solid interfaces, giving way to biomedical applications including biosensors for DNA. A biosensor is a device used for the detection of a specific biological molecule; in this case DNA. Biosensors are becoming an increasingly useful analytical tool, allowing tissue matching and forensic examinations to be carried out. Marianna explains “Dendrimers have been proposed as biosensors since they can immobilize single strands of nucleic acid due to their electrostatic attraction. These nucleic acid strands are used as a target to match complementary strands with specific sequences, which can be e.g. a type of contaminant in the sample to analyse. However, this type of biosensor is sensitive to detect unwanted impurities since there is no chemical specificity to electrostatic attraction. We propose that the functionalization of PAMAM dendrimers with DLPA can avoid such effects and improve the selective molecular recognition of nucleic acids.”
This is a promising new area of research, with a number of avenues for further investigation available. Marianna says “Currently we are evaluating the behaviour of dendrimer/nucleolipid complexes in bulk solution, which is fundamental if one wishes to employ them as gene delivery vehicles. Additionally, the biocompatibility of the dendrimer/nucleolipid system should be investigated. There are many other variables that can be explored in order to understand better these delivery vehicles e.g. the dendrimer generation (i.e. size and number of surface groups of the dendrimer) and the type of nucleolipid.”
Research date: March 2014
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