374186 Lactobionic Acid Functionalized Chitosan-Graft- Beta Cyclodextrin-p53 Nanoparticles As Efficient Carriers for Targeted Gene Delivery
The p53 is a well-known tumor suppressor protein that induces apoptosis upon cellular stress (e.g. DNA damages) or regulates cell-cycle progression via activation of transcriptional factors such as p21. In normal cells, the p53 activities are regulated by expression of negative regulators, viz., murine double minute 2 (MDM2) and MDM4 proteins. However, previous studies demonstrated that p53 is the most frequently mutated or deleted transcriptional factor in human tumors. Therefore, restoration of the activity of p53 in cancer cells by delivering fresh and healthy p53 plasmid DNA can reactivate the cellular apoptosis pathway and increase cell sensitivity to chemotherapeutic agents.
This study aims to prepare a chitosan-based nano-carrier for the delivery of p53-plasmid DNA to liver cancer cells in order to reactivate the p53-dependent apoptosis pathway as well as to increase sensitivity of the cells to anti-cancer drugs. To this end, chitosan-graft-ß-cyclodextrin at four different ß-cyclodextrin/chitosan molar ratios were successfully prepared and characterized using Fourier transform infrared spectroscopy (FTIR), H1NMR, elemental analysis, and X-ray diffraction. The cytoxicity of the prepared compounds was examined in three different cell lines (MCF7, C6, and HepG2), where chitosan and PEI (25 kDa) were used as negative controls. Next, adamantine- poly-ethylene-glycol 5000-disulfide bond- lactobionic acid (Ada-PEG5000-SS-LA, (a targeting ligand for HepG2 cells)) was successfully synthesized and used for functionalizing chitosan-graft-ß cyclodextrin nanoparticles encapsulating p53. Ada-PEG5000-LA functionalized nanoparticles were synthesized as control to test the effect of the S-S group on gene transfection efficiency. The nanoparticles were characterized using TEM and zeta-potential measurements. The ability of the functionalized nanoparticle to encapsulate and protect p53-pDNA was tested through gel retardation and PicoGreen assays. The in vitro transfection efficiency was carried out in HepG2 and Hela cell lines using pRL-CMV as reporter gene. The luciferase gene expression was quantified using commercial kits.
The results demonstrated that functionalized chitosan-graft-β cyclodextrin nanoparticles had no cytoxicity and exhibited higher transfection efficiency on HepG2 cells compared to chitosan.
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