Interaction with Blood Components Plays a Crucial Role in Asialoglycoprotein Receptor-Mediated in Vivo Gene Transfer by Galactosylated Lipoplex

Abstract

In this study, we evaluated the effect of blood components (whole blood and serum) on asialoglycoprotein receptor-mediated in vivo gene transfer. The hepatic transfection activity of galactosylated lipoplex preincubated with serum was approximately 10 times higher than that without incubation after intraportal injection in mice. However, preincubation with whole blood significantly reduced hepatic transfection activity. Fluorescent resonance energy transfer analysis and agarose gel electrophoresis revealed that preincubation with serum reduced the degree of destabilization of the galactosylated lipoplex in blood, partially supporting enhanced hepatic transfection activity by preincubation with serum. Inhibition of hepatic transfection activity by predosing galactosylated bovine serum albumin indicated that the galactosylated lipoplex exposed to serum is recognized by asialoglycoprotein-receptors on hepatocytes. Inactivation of serum prior to mixing with galactosylated lipoplex reduced liver accumulation and completely abolished enhancement of hepatic transfection activity by preincubation with active serum, suggesting that not only the stability of the lipoplex in blood but also the serum opsonin activity plays important roles. Alternatively, preincubation with inactivated serum reduced the lung accumulation and inflammatory cytokine production of galactosylated lipoplex. The information provided by this study will be valuable for the future use, design, and development of galactosylated lipoplex for in vivo asialoglycoprotein receptor-mediated gene transfer.

The Role of Fibronectin in Pulmonary Gene Transfer Following Intravenous Administration of Lipoplex in Mice

“…Furthermore, Fumoto et al reported that heat inactivation of serum reduced the pulmonary accumulation of lipoplex compared with that of native serum after intraportal injection. 6) Hence, it was suggested that the interaction between lipoplex and serum component is required for the pulmonary accumulation of lipoplex after intravenous injection. Both supplementation of calcium ions to divalent cation-depleted serum and supplementation of fibronectin to QF simultaneously increased the pulmonary accumulation and transgene expression after the intravenous injection of lipoplex (Figs.…”

“…6,8,9) DOTAP and Chol were dissolved in chloroform at a molar ratio of 1 : 1, vacuumdesiccated, and resuspended in sterile 5% glucose solution at a concentration of 4 mg of total lipid per mL to form liposomes. Liposomes were extruded 11 times through a polycarbonate membrane filter (100 nm pore size) using a commercially available instrument (Mini-Extruder, Avanti Polar Lipids, Inc.).…”

“…5) Fumoto et al reported that pre-incubation of galactosylated lipoplex with serum enhanced hepatic transgene expression after intraportal injection. 6) Therefore, it was suggested that the role of serum in gene transfer differs between in vitro and in vivo conditions. While macroscopic information regarding the effects of serum on in vivo gene transfer has been obtained, the serum components that contribute to gene transfer by intravascular injection of lipoplex have not been fully elucidated.…”

Effect of the Particle Size of Galactosylated Lipoplex on Hepatocyte-Selective Gene Transfection after Intraportal Administration

“…7,8) Recently, we have developed Gal-liposomes containing cholesten-5-yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)amino)butyl) formamide (Gal-C4-Chol) for hepatocyte-selective gene transfection after intraportal administration to mice. 7,9,10) However, the level of in vivo gene expression was not as high as that expected from the in vitro results. 7,11) This phenomenon could explain the several barriers associated intrinsically with in vivo situations; therefore, these in vivo barriers need to be investigated to allow the successful development of an effective gene vector.…”

“…Preparation of Gal-liposome Gal-liposomes were prepared as reported previously. 7,10) The mixtures of DOTMA, Chol, and Gal-C4-Chol were dissolved in chloroform at a molar ratio of 2 : 1 : 1 for Gal-liposomes, vacuum-desiccated, and resuspended in sterile 5% dextrose solution at a concentration of 4 mg total lipids per ml. For small sized liposomes (about 49.6 nm, see Fig.…”