To enter the realm of human gene therapy, a novel drug delivery system is required for efficient delivery of small molecules with high safety for clinical usage. We have developed a unique vector "HVJ-E (hemagglutinating virus of Japan-envelope)" that can rapidly transfer plasmid DNA, oligonucleotide, and protein into cells by cell-fusion. In this study, we associated HVJ-E with magnetic nanoparticles, which can potentially enhance its transfection efficiency in the presence of a magnetic force. Magnetic nanoparticles, such as maghemite, with an average size of 29 nm, can be regulated by a magnetic force and basically consist of oxidized Fe which is commonly used as a supplement for the treatment of anemia. A mixture of magnetite particles with protamine sulfate, which gives a cationic surface charge on the maghemite particles, significantly enhanced the transfection efficiency in an in vitro cell culture system based on HVJ-E technology, resulting in a reduction in the required titer of HVJ. Addition of magnetic nanoparticles would enhance the association of HVJ-E with the cell membrane with a magnetic force. However, maghemite particles surface-coated with heparin, but not protamine sulfate, enhanced the transfection efficiency in the analysis of direct injection into the mouse liver in an in vivo model. The size and surface chemistry of magnetic particles could be tailored accordingly to meet specific demands of physical and biological characteristics. Overall, magnetic nanoparticles with different surface modifications can enhance HVJ-E-based gene transfer by modification of the size or charge, which could potentially help to overcome fundamental limitations to gene therapy in vivo.
The development of a 3-dimensional (3-D) navigation system of ferromagnetic particles in a flow system was performed. In order to improve the practice of using externally-applied magnetic fields for targeting the magnetic particles to a circumscribed body region, we tested the feasibility of a novel 3-D navigation system, made by applying a strong external (magnetic) field through a GdBaCuO bulk superconductor. A 3-D theoretical model is proposed and used in order to evaluate the efficiency of the navigation/retention of magnetic particles in the flow system. Furthermore, an experimental model system was made and the efficiency of a prototype system was examined. Comparisons of experimental and the corresponding calculation results were made to examine the theoretical model system.
We have been developing the device for magnetically targeted drug delivery system (MT-DDS), which can allow us to navigate and to accumulate the drug at the local diseased part inside the body by controlling to magnetic field strength and/or gradient generated by the superconducting magnets. In considering an application of this technique to the network of the blood vessel, a number of magnets should be placed within a small region. The magnetic field at the branching point is superimposed the fields from the magnets. The optimal arrangement of magnetic field for MT-DDS was calculated and experimental verification was made using several different sizes of Y-shaped glass tubes with multiple branching points as a model system of blood vessel. Ferromagnetic particles were injected into the Y-shape glass tube and were navigated by the magnetic field which was generated by the magnet optimally arranged based on the calculation. The ratio of the amount of the navigated ferromagnetic particle to the dosed amount was measured as magnetic navigation efficiency. It is found that experimental result of magnetic navigation efficiency agreed with the calculation results, which shows that magnetic navigation by the superconducting magnet can be a promising way for realization of MT-DDS.
Development of a simple method for converting the lipid envelope of an inactivated virus to a gene transfer vector was achieved a couple years ago in the medical school of Osaka University. Hemagglutinating virus of Japan (HVJ; Sendai virus) envelope (HVJ-E) vector was constructed by incorporating plasmid DNA into inactivated HVJ particles. This HVJ envelope vector introduced plasmid DNA efficiently and rapidly into various cell lines, including cancer cells and several types of primary cell culture. In the present study, efficiency of gene transfer was found to be greatly enhanced by application of a magnetic field. Therefore, we developed a new type of magnet for magnetically enhancing and targeting gene transfection system by using vectors associated with ferromagnetic particles coated with positively/negatively charged biopolymers, which can help to enhance and target gene delivery with higher efficiency. For the transfection experiment in vitro, the HVJ-E vector was mixed with ferromagnetic particles coated with biopolymer and this mixture was added to cultured cells which were set up under the permanent magnet. The effect of the dose of the ferromagnetic particles on the transfection efficiency was discussed. In order to clarify the effect of magnetic field gradient on the accumulation possibility of the magnetic particles and the accuracy of the targeted site in the blood vessels, calculation of the applied magnetic force for the ferromagnetic particles inside the blood vessel was also performed.
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