Improving the biological activity of CpG ODN by linking it to carbon nanotubes

Preventative immunotherapeutic treatments have been an area of great interest to combat infectious disease because of the ability to stimulate the host’s immune system which prepares the host to fight pathogenic microbes. The immunotherapeutic approach requires the use of an immune stimulating molecule that is able to boost the host’s immune response. A major problem exists that these immune stimulating molecules are often very expensive and require a large dose to be effective. To reduce the cost of using these molecules, a delivery system can be used which is able to lower the effective dose of the immune stimulant while not causing any toxic effects towards the host’s health. In this study, the immune stimulating molecules synthetic unmethylated cytidine-phosphate-guanosine oligodeoxynucleotides were attached non-covalently to multi-walled carbon nanotubes. The use of carbon nanotubes as a delivery mechanism could result in a lower effective dose able to stimulate a protective immune response in a chicken model. In this study, we first assessed which of the non-covalant linkages was ideal for linking the immune stimulant to the carbon nanotubes. This was conducted by looking at which method of linkage would allow the best cellular proliferation and transcriptional activation of selected innate immune genes. Once an appropriate linkage method had been selected, cellular uptake studies were conducted to establish that cytidine-phosphate-guanosine oligodeoxynucleotides were delivered to intracellular target receptors. After cellular uptake was demonstrated, it was important that the carbon nanotubes linked to the immune stimulant do not cause toxicity towards the host. To measure toxicity, in vitro studies were conducted to observe cell viability post treatment with carbon nanotube linked immune stimulant. Further studies were conducted on any alterations to the immune stimulants’ ability to activate immune cells by studying the pathway of macrophage activation. The protective ability of the molecules was then measured by the ability to protect chickens from a lethal challenge with S. typhimurium. Once the protective nature of the molecules was established, the mechanism of immune stimulation was examined by in vivo cell recruitment and in vitro cytokine production. These studies indicate that linking cytidine-phosphate-guanosine oligodeoxynucleotides to carbon nanotubes can lower the effective dose of the immune stimulant without altering the biological function of the molecule