Development of novel inorganic nanocrystals for delivery of pDNA and siRNA to breast cancer cells
thesisposted on 02.03.2017, 03:12 by Bakhtiar, Athirah
Genetic manipulation of human cells through the delivery of functional genes such as plasmid DNA (pDNA) and short-interfering RNA (siRNA) is an attractive approach to treat many critical diseases with single or multiple gene defects, including carcinoma; precisely and efficiently. Despite their potential effect, naked therapeutic genes are rapidly degraded by nucleases, non-specific to the target cells; in addition to exhibiting low cellular uptake, and poor transfection efficiency. Hence, the development of safe and efficient gene carriers is undeniably crucial for the success of gene therapy. Recent studies have been focused on developing smart nanoparticles for excellent delivery of transgenes and siRNAs into cancerous cells of an animal model through active and passive targeting. The precipitation reaction is one of the facile and convenient ways to synthesize nanoparticles, which an insoluble salt is formed upon the mixture of two water-soluble salts. In this study, we aim to develop the potential salt crystals with nano-size diameters having the capacity of adsorbing negatively charged plasmid DNA and siRNA, effectively carrying them across the plasma membrane and finally leading to efficient gene expression and silencing of the target (reporter as well as endogenous) gene(s), respectively, in mammary carcinoma of mammalian cells. The generated insoluble salts have been subjected to a rigorous screening process based on observation of particle morphology under optical microscope, determination of growth kinetics, particle diameters and electrostatic affinity towards the negatively charged pDNA/siRNA, qualitative and quantitative estimation of cellular endocytosis rate and finally assessment of transfection efficacy in case of transgene expression and knockdown by target siRNAs. Among the screened precipitates, strontium sulfite, strontium fluoride, and magnesium sulfite have shown the best potency in aiding cellular delivery of reporter gene/siRNA, in addition to proficient transgene expression and silencing effect into both mice and human mammary carcinoma cells. Our in vivo discoveries revealed efficiency of nanocrystals with the ability to efficiently transport pDNA as well as siRNA into 4T1-induced tumor model through biodistribution assays and tumor regression activities. Strontium sulfite, strontium fluoride, and magnesium sulfite improve the genetic material delivery, demonstrated through regression of tumor growth activity. Protein coating enhances the nanocarrier activity through the involvement of active targeting via ligand-receptor interactions. It is hoped that the potential nanoparticles can be applied for conceivable nano-vector application in the clinical setting for cancer treatment in the future.