利用正價膽固醇為基底的奈米載體進行短鏈核苷酸遞送之效率探討

Abstract

Gene delivery has been an indispensable tool in biomedical researches. With the development of molecular biology, different materials and methods for gene delivery have long been developed and continued to be modified. We can overexpress or inhibit particular genes by delivering plasmids, antisense oligonucleotides or RNA interference (RNAi) to analyze specific gene functions. There have been two major systems for gene delivery used currently: the viral vector system and the non-viral vector system. Since the instability of viral vectors and the requirements of clinical applications, most studies still use the non-viral vectors for gene delivery. The purpose of this study is to develop non-viral cholesterol-based nanocarriers as transfection reagent for delivering nucleic acids into cells efficiently and not affecting normal cell viability. Such platform is going to benefit the genetic manipulation of primary cells and even stem cells. A novel cationic cholesterol, 3-β-[N-(2-guanidinoethyl)carbamoyl]-cholesterol (GEC-Chol), was mixed with cholesterol to form the GEC-Chol/ Chol lipoplexes (GCC) and then incorporated with hydrophobic superparamagnetic iron oxide (SPIO) nanoparticles to form as GCC-Fe3O4 nanocarriers. The GCC-Fe3O4 nanocarriers were transfected together with fluorescent-labeled oligonucleotides (ODN) into mouse CT-26 colon cancer cells in order to evaluate the transfection efficiency and cytotoxicity by fluorescent microscopy and further confirmed with confocal microscopy as well as flow cytometry analysis. Other cell types such as the rat acinar cells, and human embryonic stem cells were also used as primary cell and stem cell models. The results indicated that GCC-Fe3O4 nanocarriers have high physical stability with size less than 135 nm. These GCC-Fe3O4 nanocarriers also showed great transfection efficiency as well as low cytotoxicity in both serum-reducing condition and normal serum-containing medium. We also compared GCC-Fe3O4 nanocarriers with common commercialized transfection vectors and found the GCC-Fe3O4 nanocarriers had better transfection efficiency under serum condition. GCC-Fe3O4 nanocarriers not only worked well on cancer cell line, but also good for primary acinar cells and even embryonic stem cells for further tissue remodeling. Moreover, the magnetic nanoparticles may also have the potential to work as contrast agent for tracking engineered cells in vivo and can be manipulated by magnetic force. This novel developed GCC-Fe3O4 nanocarriers had the capability for high efficiency of gene delivery and molecular imaging simultaneously, and provide a better choice for future clinical applications.