Cationic Polymers/Superparamagnetic Iron Oxide Hybrid Nanoparticles As Gene Delivery Vectors

Gene therapy is a potential method for the treatment of human genetic and acquired diseases. Viral and non-viral vectors have been applied to protect and deliver nucleic acid drugs used in gene therapy. Although viral vectors exhibit high transfection efficiencies when delivering nucleic acid drugs to numerous cell lines, researchers express a major concern on their safety. Non-viral vectors, including cationic lipids and polymers, are another kind of available vehicle for efficient gene transfection. They are attractive due to their biocompatibility, high gene carrying capacity and potential for large-scale production. However, non-viral vectors exhibit low transfection efficiencies compared to viral vectors because they are limited by numerous extra-and intracellular barriers. Ongoing efforts have been directed toward overcoming barriers both in vitro and in vivo by designing various cationic compounds to optimize DNA condensation, membrane fusion, endosomal escape or nuclear targeting. However, there is still a simple physical barrier, slow vector accumulation and consequently low DNA concentration on the cell surface, which reduces the trasfection efficiencies of non-viral vectors. To overcome this barrier, a new technology termed "magnetofection" has been developed. In magnetofection, nucleic acid drugs are associated with magnetic nanoparticles to form magnetoplexes, and thus they can be rapidly concentrated on the target cells with an additional magnetic field. As a result, high-level transgene expression can be achieved with relatively short incubation time and low DNA dose. In this paper, we prepared a series of cationic polymers/superparamagnetic iron oxide hybrid nanoparticles as gene delivery vectors.In chapter 1, the current significant non-viral gene delivery system were reviewed. The main barriers for nonviral gene delivery and the current strategies for overcoming these barriers were also discussed.In chapter 2, we developed a novel strategy to enhance the transfection efficiency of branched PEI (25 kDa) with a magnetic field using dendrimer modified magnetic iron oxide nanoparticle/DNA/PEI ternary magnetoplexes. We prepared DMSPION-G6/DNA/PEI ternary magnetoplexes by precondensing DMSPION-G6 with DNA at low mass ratio to yield DMSPION-G6/DNA magnetoplexes with negative surface charge, followed by further coating with branched PEI (25 kDa) via electrostatic interactions. The magnetoplexes exhibited appropriate positive surface charge and nanocsale particle size. We measured the transfection efficiencies of DMSPION-G6/DNA/PEI ternary magnetoplexes in COS-7,293T and HeLa cells in the presence or absence of a magnetic field. Compared with PEI/DNA polyplexes, DMSPION-G6/DNA/PEI ternary magnetoplexes exhibited enhanced transfection efficiencies in all the three cell lines when a magnetic field was applied, especially in the presence of 10% FBS. Time-resolved and dose-resolved transfection indicated that high-level transgene expression was achievable with relatively short incubation time and low DNA dose when magnetofection was employed. Further evidences from Prussian blue staining, quantification of cellular iron concentration and cellular uptake of Cy-3 labelled DNA demonstrated that the magnetic field could quickly gather the magnetoplexes to the surface of target cells and consequently enhanced the uptake of magnetoplexes by the cells. This represents a novel strategy for polycation-based in vitro gene delivery enhanced by a magnetic field.In our previous work, we prepared a series of biodegradable poly(disulfide amine)s with high buffer capacity, good DNA condensation capacity and low cytotoxicity. Unfortunately, their transfection efficiencies were relative low. In chapter 3, we empoly the strategy described in charpter 2 to enhanced the transfection efficiencies of these poly(disulfide amine)s. We prepared DMSPION-G6/DNA/polymer ternary magnetoplexes by precondensing DMSPION-G6 with DNA at the mass ratio of 2 to yield DMSPION-G6/DNA magnetoplexes with negative surface charge, followed by further coating with polymers via electrostatic interactions. The introduction of DMSPION-G6 to polymer/DNA polyplexes did not have any effect on their cytotoxicity against 293T and COS-7 cells. Compared with polymer/DNA polyplexes, DMSPION-G6/DNA/polymer ternary magnetoplexes exhibited enhanced transfection efficiencies in 293T and COS-7 cells when a magnetic field was applied, especially in the presence of 10% FBS. Further evidences from Prussian blue staining, ICP-AES and cellular uptake of Cy-3 labelled DNA demonstrated that the magnetic field could quickly gather the magnetoplexes to the surface of target cells and consequently enhanced the uptake of magnetoplexes by the cells. Thus, high level of transgene expression was achievable.In chapter 4, amphiphilic triblock copolymers methoxy poly(ethylene glycol)-poly(ε-caprolactone)-poly(aminoethyl methacrylate) (mPEG-PCL-PAEMAs) was synthesized by ATRP for encapsulation of SPION to form micelles with SPION in their hydrophobic core. The SPION-containing micelles were nanosized with diameters of 150-200 nm. MTT assay illustrated that the cytotoxicity of micelle 1-3 increased with the increasing repeating unit of AEMA. The positively charged shell of the micelles could condense DNA to form magnetoplexes with appropriate positive surface charge and nanocsale particle size. The micelle/DNA magnetoplexes exhibited enhanced transfection efficiencies when a magnet field was applied, which were 5-500 fold higher than that without magnetical application. Further evidences from Prussian blue staining, ICP-AES, TEM and cellular uptake of Cy-3 labelled DNA demonstrated that more magnetoplexes could be taken up by the cells when a magnet field was applied. Thus, high level of transgene expression was achievable. The SPION-containing micelle and micelle/DNA complexes had relatively higher T2 relaxivities of 174 mM-1S-1 and 171 mM-1S-1, respectively. These SPION-containing micelles could be used as contrast agents for MR imaging, which provided a benefit for monitoring gene delivery.In chapter 5, cationic amphiphilic polymers, which consisted of bromohexadecane side chains and disulfide containing main chains, were synthesized and used for encapsulation of SPION to form bioreducible micelles with nanoscaled size, positive surface charge and low cytotoxicity by a self-assembly process in aqueous solution. The cationic shell of the nanoparticles could condense DNA to form magnetoplexes with nanoscaled size and positive surface charge. The dissociation of magnetoplexes was achievable at DTT conditions. The luciferase and GFP expression level induced by these micelles was significantly enhanced in the presence of a magnet field. More Fe was detected in the cells, which indicated more manetoplexes were taken up by the cells when a magnet field was applied. The relaxivities of micelle and micelle/DNA complexes were similar (229 mM-1 S-1 for micelle versus 209 mM-1 S-1 for micelle/DNA complexes), confirming no appreciable change in magnetism after DNA complexing. We could use MR imaging to monitor gene delivery.