The Insertion Timing of PEGylated Lipids to Galactosylated Lipoplexes Is Important for Liver-Selective Transfection in Mice
International Journal of Genetics and Genomics
Volume 4, Issue 6, December 2016, Pages: 68-78
Received: Mar. 10, 2016; Accepted: Mar. 22, 2016; Published: Mar. 22, 2017
Views 3437      Downloads 300
Shintaro Fumoto, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
Naoki Taniguchi, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
Yuri Ikai, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
Naoki Yoshikawa, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
Hirotaka Miyamoto, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
Hitoshi Sasaki, Department of Hospital Pharmacy, Nagasaki University Hospital, Nagasaki, Japan
Mitsuru Hashida, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan
Shigeru Kawakami, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
Koyo Nishida, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
Article Tools
Follow on us
In the present study, we demonstrated the importance of PEGylation timing of galactosylated liposome/plasmid DNA (pDNA) complexes (lipoplexes) for liver-selective transfection in mice. Because a fenestrated endothelium can be a barrier for penetration of lipoplexes though sinusoids, the particle size of lipoplexes is one of the determining factors for in vivo liver parenchymal cell (hepatocyte, PC)-selective transfection. Here, we found that syn-insertion, as a novel PEGylation timing, was useful to control the particle size of galactosylated lipoplexes. Syn-insertion of PEGylated lipids was performed by simple mixing of pDNA solution containing PEGylated lipids and dispersion of the cationic liposomes. Both syn- and pre-insertion of PEGylated lipids decreased the particle size of lipoplexes, whereas post-insertion did not. Moreover, syn-insertion of PEGylated lipids to galactosylated lipoplexes improved liver selectivity and the PC/non-parenchymal cell ratio of transgene expression after intravenous injection in mice. Hence, these data will be valuable for the design and preparation of PEGylated lipoplexes for gene targeting.
Gene Therapy, PEGylation, Galactosylated Liposomes, Targeting, Hepatocyte
To cite this article
Shintaro Fumoto, Naoki Taniguchi, Yuri Ikai, Naoki Yoshikawa, Hirotaka Miyamoto, Hitoshi Sasaki, Mitsuru Hashida, Shigeru Kawakami, Koyo Nishida, The Insertion Timing of PEGylated Lipids to Galactosylated Lipoplexes Is Important for Liver-Selective Transfection in Mice, International Journal of Genetics and Genomics. Vol. 4, No. 6, 2016, pp. 68-78. doi: 10.11648/j.ijgg.20160406.15
Copyright © 2016 Authors retain the copyright of this article.
This article is an open access article distributed under the Creative Commons Attribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
K. Domvri, P. Zarogoulidis, K. Porpodis, M. Koffa, M. Lambropoulou, S. Kakolyris, G. Kolios, K. Zarogoulidis, and E. Chatzaki, “Gene therapy in liver diseases: state-of-the-art and future perspectives,” Curr. Gene Ther., vol. 12, pp. 463-483, December 2012.
E. van Craeyveld, F. Jacobs, S. C. Gordts, and B. de Geest, “Gene therapy for familial hypercholesterolemia,” Curr. Pharm. Des., vol. 17, pp. 2575-2591, 2011.
G. P. Gao, M. R. Alvira, L. Wang, R. Calcedo, J. Johnston, and J. M. Wilson, “Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy,” Proc. Natl. Acad. Sci. USA, vol. 99, pp. 11854-11859, September 2002.
M. Johnson, S. Huyn, J. Burton, M. Sato, and L. Wu, “Differential biodistribution of adenoviral vector in vivo as monitored by bioluminescence imaging and quantitative polymerase chain reaction,” Hum. Gene Ther., vol. 17, pp. 1262-1269, December 2006.
F. Park, K. Ohashi, and M. A. Kay, “Therapeutic levels of human factor VIII and IX using HIV-1-based lentiviral vectors in mouse liver,” Blood, vol. 96, pp. 1173-1176, August 2000.
S. Kawakami, S. Fumoto, M. Nishikawa, F. Yamashita, and M. Hashida, “In vivo gene delivery to the liver using novel galactosylated cationic liposomes,” Pharm. Res., vol. 17, pp. 306-313, March 2000.
S. Fumoto, S. Kawakami, Y. Ito, K. Shigeta, F. Yamashita, and M. Hashida, “Enhanced hepatocyte-selective in vivo gene expression by stabilized galactosylated liposome/plasmid DNA complex using sodium chloride for complex formation,” Mol. Ther., vo. 10, pp. 719-729, October 2004.
K. Morimoto, M. Nishikawa, S. Kawakami, T. Nakano, Y. Hattori, S. Fumoto, F. Yamashita, and M. Hashida, “Molecular weight-dependent gene transfection activity of unmodified and galactosylated polyethyleneimine on hepatoma cells and mouse liver,” Mol. Ther., vol. 7, pp. 254-261, February 2003.
Y. Hu, M. T. Haynes, Y. Wang, F. Liu, and L. Huang, “A highly efficient synthetic vector: nonhydrodynamic delivery of DNA to hepatocyte nuclei in vivo,” ACS Nano, vol. 7, pp. 5376-5384, June 2013.
S. E. Raper, N. Chirmule, F. S. Lee, N. A. Wivel, A. Bagg, G. P. Gao, J. M. Wilson, and M. L. Batshaw, “Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer,” Mol. Genet. Metab., vol. 80, pp. 148-158, September-October 2003.
J. C. Nault, S. Datta, S. Imbeaud, A. Franconi, M. Mallet, G. Couchy, E. Letouzé, C. Pilati, B. Verret, J. F. Blanc, C. Balabaud, J. Calderaro, A. Laurent, M. Letexier, P. Bioulac-Sage, F. Calvo, and J. Zucman-Rossi, “Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas,” Nat. Genet., vol. 47, pp. 1187-1193, October 2015.
S. Fumoto, S. Kawakami, M. Hashida, and K. Nishida, “Section 1 Approached to Gene Therapy, Chapter 1 Targeted Gene Delivery: Importance of Administration Routes,” INTECH "Novel Gene Therapy Approaches" Editor, Ming Wei; Co-editor, David Good, pp. 3-31, February 2013.
S. Kawakami, and M. Hashida, “Glycosylation-mediated targeting of carriers,” J. Control. Release, vol. 190, pp. 542-555, September 2014.
M. A. Hickman, R. W. Malone, K. Lehmann-Bruinsma, T. R. Sih, D. Knoell, F. C. Szoka, R. Walzem, D. M. Carlson, and J. S. Powell, “Gene expression following direct injection of DNA into liver,” Hum. Gene Ther., vol. 5, pp. 1477-1483, December 1994.
K. Kawabata, Y. Takakura, and M. Hashida, “The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake,” Pharm. Res., vol. 12, pp. 825-30, June 1995.
F. Liu, Y. Song, and D. Liu, “Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA,” Gene Ther., vol. 6, pp. 1258-1266, July 1999.
G. Zhang, V. Budker, and J. A. Wolff, “High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA,” Hum. Gene Ther., vol. 10, pp. 1735-1737, July 1999.
H. Hatakeyama, H. Akita, and H. Harashima, “The polyethyleneglycol dilemma: advantage and disadvantage of PEGylation of liposomes for systemic genes and nucleic acids delivery to tumors,” Biol. Pharm. Bull., vol. 36, pp. 892-899, June 2013.
J. Shin, P. Shum, and D. H. Thompson, “Acid-triggered release via dePEGylation of DOPE liposomes containing acid-labile vinyl ether PEG-lipids,” J. Control. Release, vol. 91, pp. 187-200, August 2003.
H. Hatakeyama, H. Akita, K. Kogure, M. Oishi, Y. Nagasaki, Y. Kihira, M. Ueno, H. Kobayashi, H. Kikuchi, and H. Harashima, “Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid,” Gene Ther., vol. 14, pp. 68-77, January 2006.
F. Shi, L. Wasungu, A. Nomden, M. C. Stuart, E. Polushkin, J. B. Engberts, and D. Hoekstra, “Interference of poly(ethylene glycol)-lipid analogues with cationic-lipid-mediated delivery of oligonucleotides; role of lipid exchangeability and non-lamellar transitions,” Biochem. J., vol. 366, pp. 333-341, August 2002.
Y. P. Zhang, L. Sekirov, E. G. Saravolac, J. J. Wheeler, P. Tardi, K. Clow, E. Leng, R. Sun, P. R. Cullis, and P. Scherrer, “Stabilized plasmid-lipid particles for regional gene therapy: formulation and transfection properties,” Gene Ther., vol. 6, pp. 1438-1447, August 1999.
K. W. Mok, A. M. Lam, and P. R. Cullis, “Stabilized plasmid-lipid particles: factors influencing plasmid entrapment and transfection properties,” Biochim. Biophys. Acta, vol. 1419, pp. 137-150, July 1999.
L. Peeters, N. N. Sanders, A. Jones, J. Demeester, and S. C. de Smedt, “Post-pegylated lipoplexes are promising vehicles for gene delivery in RPE cells,” J. Control. Release, vol. 121, pp. 208-217, August 2007.
S. Kawakami, R. Hirayama, K. Shoji, R. Kawanami, K. Nishida, M. Nakashima, H. Sasaki, T. Sakaeda, and J. Nakamura, “Liver- and lobe-selective gene transfection following the instillation of plasmid DNA to the liver surface in mice,” Biochem. Biophys. Res. Commun., vol. 294, pp. 46-50, May 2002.
Y. C. Lee, S. P. Stowell, and M. J. Krantz, “2-Imino-2-methoxyethyl 1-thioglycosides: new reagents for attaching sugars to proteins,” Biochemistry, vol. 15, pp. 3956-3963, September 1976.
F. Sakurai, T. Nishioka, H. Saito, T. Baba, A. Okuda, O. Matsumoto, T. Taga, F. Yamashita, Y. Takakura, and M. Hashida, “Interaction between DNA-cationic liposome complexes and erythrocytes is an important factor in systemic gene transfer via the intravenous route in mice: the role of the neutral helper lipid,” Gene Ther., vol. 8, pp. 677-686, May 2001.
H. Eliyahu, N. Servel, A. J. Domb, and Y. Barenholz, “Lipoplex-induced hemagglutination: potential involvement in intravenous gene delivery,” Gene Ther., vol. 9, pp. 850-858, July 2002.
B. M. Tandia, C. Lonez, M. Vandenbranden, J. M. Ruysschaert, and A. Elouahabi, “Lipid mixing between lipoplexes and plasma lipoproteins is a major barrier for intravenous transfection mediated by cationic lipids,” J. Biol. Chem., vol. 280, pp. 12255-12261, April 2005.
N. Yoshikawa, S. Fumoto, M. Nakashima, K. Shimokawa, H. Miyamoto, and K. Nishida. “The role of fibronectin in pulmonary gene transfer following intravenous administration of lipoplex in mice,” Biol. Pharm. Bull., vol. 36, pp. 1807-1813, November 2013.
K. Kusumoto, H. Akita, A. El-Sayed, and H. Harashima, “Effect of the anchor in polyethylene glycol-lipids on the transfection activity of PEGylated cationic liposomes encapsulating DNA,” Biol. Pharm. Bull., vol. 35, pp. 445-448, April 2012.
K. Remaut, B. Lucas, K. Braeckmans, J. Demeester, S. C. de Smedt, “Pegylation of liposomes favours the endosomal degradation of the delivered phosphodiester oligonucleotides,” J. Control. Release, vol. 117, pp. 256-266, February 2007.
M. C. Deshpande, M. C. Davies, M. C. Garnett, P. M. Williams, D. Armitage, L. Bailey, M. Vamvakaki, S. P. Armes, and S. Stolnik, “The effect of poly(ethylene glycol) molecular architecture on cellular interaction and uptake of DNA complexes,” J. Control. Release, vol. 97, pp. 143-156, May 2004.
S. A. Johnstone, D. Masin, L. Mayer, and M. B. Bally, “Surface-associated serum proteins inhibit the uptake of phosphatidylserine and poly (ethylene glycol) liposomes by mouse macrophages,” Biochim. Biophys. Acta, vol. 1513, pp. 25-37, July 2001.
C. R. Miller, B. Bondurant, S. D. McLean, K. A. McGovern, and D. F. O'Brien, “Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes,” Biochemistry, vol. 37, pp. 12875-12883, September 1998.
Y. Higuchi, S. Kawakami, S. Fumoto, F. Yamashita, and M. Hashida, “Effect of the particle size of galactosylated lipoplex on hepatocyte-selective gene transfection after intraportal administration,” Biol. Pharm. Bull., vol. 29, pp. 1521-1523, July 2006.
G. R. Harper, S. S. Davis, M. C. Davies, M. E. Norman, T. F. Tadros, D. C. Taylor, M. P. Irving, J. A. Waters, and J. F. Watts, “Influence of surface coverage with poly(ethylene oxide) on attachment of sterically stabilized microspheres to rat Kupffer cells in vitro,” Biomaterials, vol. 16, pp. 427-439, April 1995.
E. Wisse, “An electron microscopic study of the fenestrated endothelial lining of rat liver sinusoids,” J. Ultrastruct. Res., vol. 31, pp. 125-150, April 1970.
Z. Gatmaitan, L. Varticovski, L. Ling, R. Mikkelsen, A. M. Steffan, and I. M. Arias, “Studies on fenestral contraction in rat liver endothelial cells in culture,” Am. J. Pathol., vol. 148, pp. 2027-2041, June 1996.
E. Wisse, R. B. de Zanger, K. Charels, P. van der Smissen, and R. S. McCuskey, “The liver sieve: considerations concerning the structure and function of endothelial fenestrae, the sinusoidal wall and the space of Disse,” Hepatology, vol. 5, pp. 683-692, July-August 1985.
S. Fumoto, F. Nakadori, S. Kawakami, M. Nishikawa, F. Yamashita, and M. Hashida, “Analysis of hepatic disposition of galactosylated cationic liposome/plasmid DNA complexes in perfused rat liver,” Pharm. Res., vol. 20, pp. 1452-1459, September 2003.
S. Fumoto, Kawakami S, M. Ishizuka, M. Nishikawa, F. Yamashita, and M. Hashida, “Analysis of hepatic disposition of native and galactosylated polyethylenimine complexed with plasmid DNA in perfused rat liver,” Drug Metab. Pharmacokinet., vol. 18, pp. 230-237, August 2003.
M. A. Arangoa, N. Düzgüneş, C. Tros de Ilarduya, “Increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes,” Gene Ther., vol. 10, pp. 5-14, January 2003.
J. G. Seol, D. S. Heo, H. K. Kim, J. H. Yoon, B. I. Choi, H. S. Lee, N. K. Kim, and C. Y. Kim, “Selective gene expression in hepatic tumor with trans-arterial delivery of DNA/liposome/transferrin complex,” In Vivo, vol. 14, pp. 513-517, July-August 2000.
M. Ukawa, H. Akita, Y. Hayashi, R. Ishiba, K. Tange, M. Arai, K. Kubo, Y. Higuchi, K. Shimizu, S. Konishi, M. Hashida, and H. Harashima, “Neutralized nanoparticle composed of SS-cleavable and pH-activated lipid-like material as a long-lasting and liver-specific gene delivery system,” Adv. Healthc. Mater. vol. 3, pp. 1222-1229, August 2014.
Science Publishing Group
1 Rockefeller Plaza,
10th and 11th Floors,
New York, NY 10020
Tel: (001)347-983-5186