Lipoplexes for Gene Delivery Characterized by Fluorescence Correlation Spectroscopy

Abstract

Liposomes composed of cationic lipids have been extensively investigated as drug delivery systems [1] and carrier for gene therapy and regenerative medicine as an alternative to viral approaches [2]. One exemplary regenerative medicine concept is the use of induced pluripotent stem cells (iPS) reprogrammed through the introduction of pluripotency genes such as OCT4, Sox2, KLF4 and c-Myc [3]. In this work, we study the complexation of adequate liposome formulations with these four genes through Fluorescence Cross-Correlation Spectroscopy (FCCS). [4]Liposomes stained with the carbocyanine dye 1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine (DiD) and fluorescently labelled plasmids carrying the OCT4, Sox2, KLF4 and c-Myc genes are characterized via single photon FCCS studies with a confocal microscope (LSM780, Zeiss, Oberkochen, Germany). Using CX-rhodamine and fluorescein as fluorescent labelling for the genes, we create two complementary wavelength windows to the far red wavelength window necessary to observe DiD fluorescence. Thereby, we create the possibility to monitor liposome loading with more than one gene.[1] Allen, T. M. & Cullis, P. R. Liposomal drug delivery systems: From concept to clinical applications. Adv. Drug Deliv. Rev. 65, 36-48 (2013).[2] Niidome, T. & Huang, L. Gene Therapy Progress and Prospects: Nonviral vectors. Gene Ther. 9, 1647-1652 (2002).[3] Takahashi, K. & Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 126, 663-676 (2006).[4] Merkle, D., Lees-Miller, S. P. & Cramb, D. T. Structure and Dynamics of Lipoplex Formation Examined Using Two-Photon Fluorescence Cross-Correlation Spectroscopy. Biochemistry 43, 7263-7272 (2004).