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Title: Development of novel biodegradable core-shell nanoparticles as carriers for gene delivery
Authors: Hetti, Mimi
Keywords: Hong Kong Polytechnic University -- Dissertations
Gene therapy
Genetic vectors
Issue Date: 2010
Publisher: The Hong Kong Polytechnic University
Abstract: Highly uniform gelatin/poly(ethyleneimine) (gelatin/PEI) core-shell nanoparticles that are composed of biodegradable gelatin cores and cationic branched polyethylenimine (PEI) shells were designed and successfully synthesized. The highly uniform gelatin microgels were firstly prepared through thermal denaturation, followed by covalent linkage of the branched PEI polymer (Mw 25 kD) onto the preformed gelatin microgels via coupling reaction using N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC). The gelatin core-to-PEI shells weight ratio was varied by adding different amount of PEI and changing the reaction pH. After purification by ultrafiltration, the gelatin/PEI nanoparticles were treated with an appropriate mixture of ethanol/water solution to generate gelatin/PEI nanoparticles with well-defined gelatin cores and PEI shells. The resultant nanoparticles had hydrodynamic sizes ranging from 196 to 424 nm in diameter with surface charges between +47 to +64 mV depending on the PEI content and ethanol concentration. By varying the reaction pH and amount of PEI added, the PEI contents of the nanoparticles could be controlled between 25 and 65%. Transmission electron microscopy images revealed that the nanoparticles had a well-defined core-shell nanostructure. Application of the gelatin/PEI core-shell nanoparticle as a potential gene carrier was explored. It was found that the nanoparticles were able to completely condense plasmid DNA and siRNA at N/P ratios of 2 and 5, respectively. The condensed gene molecules could be effectively released from the nanoparticles through polyelectrolyte exchange reaction with poly(aspartic acid). The integrity of the gene molecules was preserved during these complexation and release processes. In addition, the nanoparticles were able to protect the condensed pDNA and siRNA from enzymatic degradations. In vitro studies revealed that the gelatin/PEI nanoparticles could deliver argininosuccinate synthetase (ASS) gene targeting siRNA to the HeLa cells to perform gene silencing. After transfection of the siRNA/nanoparticle complexes to the cells for about one day, the percentage knock-down of ASS-mRNA level was as high as 70%. The confocal laser scanning microcopic studies indicated that the core-shell nanoparticles were effective in delivering siRNA to both the cytoplasm and nuclei of HeLa cells. The flow cytometry studies also suggested that the percentage of cellular uptake of the complexed siRNA was as high as 94.7% while the native siRNA were failed to enter to the cells. The results of MTT assay showed that cytotoxicity of the nanoparticles was lower than that of the native PEI polymer. Results from these studies show that our gelatin/PEI core-shell nanoparticle is a promising candidate for non-viral gene delivery.
Description: xvii, 200 p. : ill. (some col.) ; 30 cm.
PolyU Library Call No.: [THS] LG51 .H577M ABCT 2010 Hetti
Rights: All rights reserved.
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