Study on the nucleic acid transfection technology using superparamagnetic nanoparticles

• Main Authors: Chao-Bin Chen, 陳昭斌
• Other Authors: Wen-Chien Lee
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/47720765963618669824

博士 === 國立中正大學 === 化學工程所 === 97 === Superparamagnetic iron oxide nanoparticles (SPIONs) with appropriate surface modification can be used for various biomedical applications, such as magnetic resonance imaging, hyperthermia, drug delivery, tissue repair, cell separation, and magnetofection. In this study, SPIONs were used as the vectors for the delivery of DNA to target cell mediated by the pulsed magnetic field. SPIONs were prepared by co-precipitating di- and trivalent Fe ions (in the molar ratio of 1:2) in alkaline solution and treating under hydrothermal conditions. After the surface modification with polyethyleneimine (PEI) and poly-L-lysine (PLL), the positively charged nanoparticles were complexed with negatively charged plasmid pEGFP-C1. To mediate the cellular uptake, cells were mixed with complexes of DNA/PLL-bounded SPIONs or DNA/PEI-coated SPIONs and exposed once or several times to pulsed magnetic field. Results from the transfection of adherent HeLa cells indicated that the transfection efficiency was influenced by the strength of pulsed magnetic field, number of pulsing, and mass of SPIONs complexed with DNA. A transfection yield of 60.7±1.8% could be achieved by pulsing the mixture of cells and DNA complexes to a magnetic field of 0.6 Tesla seven times. In comparison with no magnetic pulsing, the enhancement in transfection efficiency was about two fold on average by pulsing in magnetic field. Besides the adherent HeLa, this method could apply to other cell types such as HepG2/C3A, CHO, COS-7 and MSCs. Suspended cells could also be effectively transfected by the mediation of pulsed magnetic field. Disregarding the cytotocixity of internalized PEI, the loss of cell viability by magnetic pulsing was not evidenced. To bypass the cytotocixity of PEI, we prepared magnetic nanoparticles by co-precipitating heat-induced single-stranded DNA with di and trivalent Fe ions. These special SPIONs could associate with double-stranded DNA via triple-helix binding. Whenever the the external magnetic field were applied or not, the transfection efficiency of HeLa cells was about 40% by using the triple-helix DNA/SPIONs. Results from the MTT assay suggested that the triple-helix DNA/SPIONs had no cytotocixity. The conjugate of fluorescent protein and surface-modified SPIONs was prepared and exposed to the pulsed magnetic field with cells for the investigation of transfection mechanism. In addition to endocytosis, the cellular internalization of DNA/SPIONs complex could be caused by the pulsed magnetic field that accelerated the DNA/SPIONs to bombard cells. In summary, the present work proposed a novel, high-efficiency technique of gene transfer mediated by the action of a pulsed magnetic field.