Selective extraction of biologically active F‐glycoprotein from dithiothreitol reduced sendai virus particles

Lee

et al. 2008

Cancer Gene Ther

By means of a simple mixing procedure, we have constructed cationic Sendai virosomes consisting of fusogenic viral F/HN proteins and cationic lipids. Sendai virus F/HN proteins were purified by Triton X-100 treatment and sequential centrifugation, and then quantitatively added to cationic liposomes. The presence of HN proteins is essential for hemolytic activity of Sendai virus as well as efficient gene transfection. The amount of detergent added for purification of F/HN proteins was also crucial for hemolytic activity. The relevance of F/HN proteins in the gene-transfer capability of the cationic Sendai F/HN virosomes (CSVs) was verified by heat inactivation of the F/HN proteins, and cell-binding competition. DNA condensation by protamine sulfate was able to further enhance the transfection efficiency and serum resistance of CSV. The enhanced transfection efficiency of protamine-condensed DNA-encapsulating cationic Sendai F/HN virosomes (PCSVs) may result from specific and efficient cell binding mediated by F/HN proteins and efficient DNA encapsulation by protamine. The DNA condensation by protamine was crucial for systemic in vivo gene transfer by CSVs. The PCSVs exhibited a higher gene expression in various organs, especially the liver, compared to DOTAP/Chol lipoplexes. These results demonstrate the potential for the use of PCSV as gene delivery vehicles for systemic gene transfer.

“…16,17 A suspension of 10 mg of Sendai virus in PBS was centrifuged at 100 000 g for 1 h at 4 1C. The pellet was resuspended in 2 ml of PBS containing 1% Triton X-100.…”

Section: Sendai Virus Preparationmentioning

confidence: 99%

An efficient liposomal gene delivery vehicle using Sendai F/HN proteins and protamine

Lee

et al. 2008

Cancer Gene Ther

“…Sendai F-proteins were puri®ed as described by Tomasi and Loyter (1981) with minor modi®cations. The Sendai virus pellet was suspended in 4 ml of bu er A (150 mm NaCl, 20 mm Tris, pH 8.4) containing 3 mm DTT.…”

Section: Preparation Of F-virosomesmentioning

confidence: 99%

“…Regarding target-speci®c delivery of liposome drugs, there have been remarkable progresses, which are development of various types of immunoliposomes speci®c to unique antigens on cell surface (Vingerhoeds et al 1996) and various types of ligand-coupled liposomes speci®c to certain receptors (Shimada et al 1997) or cell adhesion molecules (Bloemen et al 1995) on cell membranes. Meanwhile, there has not been much progress in the area of e cient transfer of liposomal drugs into cytosol.…”

mentioning

confidence: 99%

Enhanced cytotoxicity of doxorubicin encapsulated in liposomes with reconstituted Sendai F-proteins

Cho

Journal of Microencapsulation

Ahn³

et al. 2001

Sendai F-virosomes, a novel type of liposome with reconstituted Sendai F-proteins, have been tested as a delivery system for various bioactive materials. However, encapsulation limitations and difficulties in controlling their constituents were drawbacks for further application to therapeutic purposes. We have tried to control virosomal constituents and have enhanced drug encapsulation efficiency into the virosomes. In vitro cytotoxicity of doxorubicin encapsulated in the F-virosomes were compared with free doxorubicin and doxorubicin in conventional liposomes. The F-virosomes were spontaneously prepared by detergent dialysis, a reconstitution process of Sendai F-proteins into liposomes. The reconstitution density of F-proteins affected the vesicle size of virosomes prepared by detergent dialysis; the larger amount of F-proteins made a smaller size of virosomes. There was little variation of size with time at physiological conditions, whilst the vesicle size of virosomes increased at acidic storage conditions (pH 5.5). Doxorubicin encapsulated in the F-virosomes exhibited a lower IC50 against B16BL6 mouse melanoma cells and Chang human hepatocarcinoma cells than that in conventional liposomes. The F-virosomes also exhibited higher cellular uptake than conventional liposomes. Addition of dioleoylphophatidylethanolamine, a fusogenic phospholipid, into the F-virosome further increased the cellular uptake as well as in vitro cytotoxicity. These types of virosome formulations can be clinically applicable as versatile vesicles for the efficient delivery of various therapeutic drugs, including genetic materials.

“…Sendai F-and HN-proteins were purified, as described by a previous report (Tomasi and Loyter, 1981) with minor modifications. A suspension of the Sendai virus in PBS was centrifuged at 100,000 g at 4 o C for 1 h. The pellet that was obtained was suspended in 2 ml of buffer B (150 mM NaCl, 10 mM TrisHCl, 2 mM Ca 2+ , 2 mM Mg 2+ , pH 7.4) that contained 50 mM octylglucoside (OG).…”

Section: Preparation and Purification Of F-proteins And Hn-proteinsmentioning

confidence: 99%

Effect of Lipid Compositions on Gene Transfer into 293 Cells Using Sendai F/HN-virosomes

Park²

2002

BMB Reports