AMINO ACID FUNCTIONALIZED NANODIAMONDS AS GENE DELIVERY VECTORS: SYNTHESIS, PHYSICOCHEMICAL CHARACTERIZATION AND CELLULAR INTERACTION STUDIES
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Nanodiamonds (NDs) are the most biocompatible member of the carbon nanofamily which are widely researched for diagnostic and therapeutic applications. Unlike other carbon nanomaterials, the surface of NDs is innately reactive, hence capable of conjugating various chemical moieties for targeted actions. This work focuses on utilizing the surface reactivity of NDs for gene therapeutics and addressing the challenges associated with its application in the biological environment. Pristine carboxylated NDs were functionalized with basic amino acids (lysine and lysyl-histidine) through covalent conjugation via a three carbon chain linker. Amino acid functionalized NDs were characterized by infrared spectroscopy, thermogravimetry and size and zeta potential measurements. Lysine conjugation was evident through a marked change in the zeta potential of ND dispersion from negative to a high positive value (-54.6 mV to +26.3 mV). The thermogram of lysine functionalized NDs (Lys-NDs) revealed a significant weight loss from 150ᵒC to 700ᵒC confirming the functionalization through loss of amino acid conjugates from the surface and total loading was calculated as 1.97 mmols/g. Lys-NDs also showed optimum binding with pDNA and siRNA at weight ratios of 1:1 and 1:20 (pDNA/siRNA:ND), respectively. Functionalization of NDs with lysine contributed to limiting aggregation and enhancing the colloidal stability of ND dispersions in biological milieu. The aqueous dispersion of lys-NDs showed minimum sedimentation and remained stable over a period of 25 days. Average sizes under 100 nm and zeta potentials higher than +20 mV indicate a preservation of the cationic surface throughout the testing period. Moreover, size distributions and zeta potentials changed significantly upon incubation of lys-NDs with blood serum suggesting an interaction with biomolecules, mainly proteins and a possible formation of a protein corona. Cellular internalization of bare lys-NDs and their diamoplexes (i.e. complexes of NDs with nucleic acids) was assessed through scanning transmission X-ray microscopy and flow cytometry. Functional efficiency of lysine NDs was determined by flow cytometry monitoring the GFP knockdown through anti-GFP siRNA delivery. Results reveal a promising GFP knockdown of ~17% upon treating the cells with NDs/siRNA diamoplexes at a ratio of 20:1. Subsequent analyses regarding the effect of NDs to prevent cellular proliferation and to cause cellular apoptosis confirmed that they are innately biocompatible at a wide range of concentrations. Unlike lysine NDs, lysyl-histidine functionalization was limited and the surface loading of this conjugate on NDs was very low. Therefore, they were unable to bind pDNA and siRNA even at high weight ratios and hence demand design modifications. Overall this work demonstrates a novel approach of functionalizing NDs with basic amino acids capable of enhancing colloidal stability and delivering of therapeutic genes into mammalian cells. It represents an important step in the development of safe and efficient gene therapy for inherited and acquired diseases.
DegreeMaster of Science (M.Sc.)
DepartmentPharmacy and Nutrition
CommitteeChitanda, Jackon M.; El-Aneed, Anas
Copyright DateSeptember 2015