Carbon Dots for Gene Therapy

Carbon Dots (CDs) are extremely promising fluorescent nanoparticles. These particles are frequently used in Nanomedicine as biosensors, drug carriers and imaging probes. In this project CDs are synthesized to interact with nucleic acids to promote gene delivery and therapy. Is convenient to use nanoparticles as DNA/RNA vectors because they can be better engineered, tuned and it’s possible to control collateral effects. Folic Acid, Citric Acid, o-Phenylenediamine and Polyethylenimine 25 kDa were used as precursors. After a purification step, the particles were characterized in term of: UV-Vis spectrum, Fluorescence spectrum, AFM images, TEM images, Zeta Potential, TGA, FTIR and Mass spectrum. Following these preliminary characterizations, made to evaluate features and chemical structure of the particles, DNA affinity tests were performed. The complex CDs-calf thymus DNA in different ratios was studied in term of UV-Vis spectrum, fluorescence spectrum, Circular Dichroism and electrophoresis. In the end, cytotoxicity tests were performed on different cancer and healthy cell lines. Two different healthy cell lines were used with only CDs and CDs in combination with DNA. The viability of cell lines was evaluated using different concentrations of particles. The formed nanoparticles showed a very narrow distribution size (around 1 nm), a positive surface charge and a good excitation independent fluorescence spectrum. Due to the large amount of PEI 25 kDa, the particles could be identified as random polymeric structures with the presence of organized benzene like domains and folic acid domains. In addition, the particles showed a good stability in deionized water solution and in solid state. The DNA binding tests revealed a good interaction between CDs and DNA. Furthermore, the DNA is able to quench the fluorescence of the particles. Cellular cytotoxicity tests confirm a good biocompatibility of the complex nanoparticle-nucleic acid, in this way, the compound can safely deliver genes to the cells. Besides passive targeting of cancer cells, active targeting can be achieved due to the presence of Folic Acid in the particles. Next steps in this research may include linking a particular DNA or RNA sequence to treat a specific type of cancer. More importantly, cellular and nucleus internalization studies must be performed. The aim of the studies would be to ensure to have a good and efficient nano-carrier that is able to efficiently deliver nucleic acids to tumor cells.