HUMAN GENE THERAPY 13:1991–2004 (November 1, 2002) © Mary Ann Liebert, Inc. Transferrin-Facilitated Lipofection Gene Delivery Strategy: Characterization of the Transfection Complexes and Intracellular Trafficking NIRMAL JOSHEE, DHUNDY R. BASTOLA, and PI-WAN CHENG ABSTRACT We previously showed that mixing transferrin with a cationic liposome prior to the addition of DNA, greatly enhanced the lipofection efficiency. Here, we report characterization of the transfection complexes in formu- lations prepared with transferrin, lipofectin, and DNA (pCMVlacZ) in various formulations. DNA in all the formulations that contain lipofectin was resistant to DNase I treatment. Transfection experiments performed in Panc 1 cells showed that the standard formulation, which was prepared by adding DNA to a mixture of transferrin and lipofectin, yielded highest transfection efficiency. There was no apparent difference in zeta potential among these formulations, but the most efficient formulation contained complexes with a mean di- ameter of three to four times that of liposome and the complexes in other gene delivery formulations. Trans- mission electron microscopic examination of the standard transfection complexes formulated using gold-la- beled transferrin showed extended circular DNA decorated with transferrin as compared to extensively condensed DNA found in lipofectin-DNA complexes and heterogeneous structures in other formulations. By confocal microscopy, DNA and transferrin were found to colocalize at the perinuclear space and in the nu- cleus, suggesting cotransportation intracellularly, including nuclear transport. We propose that transferrin enhances the transfection efficiency of the standard lipofection formulation by preventing DNA condensation, and facilitating endocytosis and nuclear targeting. 1991 OVERVIEW SUMMARY We showed that the lipofection formulation prepared by mixing transferrin with lipofectin prior to addition of DNA greatly enhanced lipofection efficiency in Panc 1 cells. DNA in this and other formulations that contain lipofectin was resistant to DNase I treatment. There was no apparent dif- ference in Zeta potential for all formulations, but the size of the transfection complex in the most efficient formula- tion was approximately three to four times that of the com- plexes in other gene delivery formulations. Transmission electron microscopic analysis of the most efficient formula- tion prepared with gold-labeled transferrin revealed circu- lar DNA decorated with gold particles, which was different from the condensed DNA found in Lipofectin plus DNA for- mulation and heterogeneous structures in other formula- tions. DNA and transferrin were colocalized at the perinu- clear space and in the nucleus, suggesting intracellular co- transportation. We propose that transferrin enhances the lipofection efficiency of the most efficient lipofection for- mulation by preventing condensation of DNA and facilitat- ing endocytosis and nuclear targeting. INTRODUCTION L IPOSOME-MEDIATED GENE TRANSFER is the most extensively studied nonviral gene delivery strategy (Tseng and Huang, 1998; Ropert, 1999; Li and Huang, 2000). This strategy utilizes the cationic property of the liposome to bind plasmids and de- liver the gene to the cells in culture and the organs in vivo (Brigham et al., 1989; Lu et al., 1989). Liposome-based gene transfer vectors have several advantages over viral vectors. Li- posomes and plasmids can be easily produced and purified in Department of Biochemistry and Molecular Biology, College of Medicine, and Eppley Cancer Center, University of Nebraska Medical Cen- ter, Omaha, NE 68198-4525.