Developing a novel cholesterol-based nanocarrier with high transfection efficiency and serum compatibility for gene therapy.

Background/purpose: Primary cells are sensitive to culture conditions, which can be more difficult to get efficient transfection. The purpose of this study is to develop a serum-compatible cholesterol-based nanocarrier for delivering therapeutic nucleic acids into cells efficiently for future clinical gene therapy.

Methods: A novel cationic 3-β-[N-(2-guanidinoethyl)carbamoyl]-cholesterol (GEC-Chol) was mixed with cholesterol and superparamagnetic iron oxide (SPIO) nanoparticles to form GCC-FeO nanocarrier. Transfection efficiency and cytotoxicity in serum and non-serum conditions were evaluated. Florescent-labeled oligonucleotides (ODNs) were transfected as indicators. Fluorescent microscopy, confocal microscopy, and flow cytometry analysis were used for evaluations. Besides, we also delivered functional antisense c-myc ODNs as surrogates for specific gene manipulation in vitro.

Results: Results indicated that GCC-FeO nanocarrier could have size down to less than 135 nm, which structure was highly stable and consistent over time. It also showed great transfection efficiency and low cytotoxicity in both serum and non-serum conditions. Our results demonstrated that GCC-FeO nanocarrier had exceeded 90% transfection efficiency, which was much better than common commercialized transfection reagents under same conditions. Such nanocarrier not only worked well in cell lines, but also ideal for gene delivery in primary cells.

Conclusion: With high transfection efficiency and serum compatibility, this novel biocompatible cholesterol-based nanocarrier provides an ideal platform especially for RNAi-based gene manipulation. It also opens a wide range of biomedical applications for in vivo cell tracking and gene therapeutics for clinical usage.