Bovine serum albumin modified the intracellular distribution and improved the antiviral activity of an oligonucleotide

被引:8
作者
Arnedo, A
Irache, JM
Gaitano, GG
Valgañón, M
Espuelas, S [1 ]
机构
[1] Univ Navarra, Fac Farm, Dept Farm & Tecnol Farmaceut, Ctr Galen, E-31080 Pamplona, Spain
[2] Univ Navarra, Dept Quim & Edafol, E-31080 Pamplona, Spain
[3] Univ Navarra, Dept Genet, E-31080 Pamplona, Spain
关键词
albumin; antisense oligonucleotide; complexes; fusogenic; cytomegalovirus; antiviral;
D O I
10.1080/10611860310001595238
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
In the present work, we describe the ability of bovine serum albumin (BSA) to improve the cytoplasmatic delivery of a phosphodiester oligonucleotide (PO), whose phosphorotioate analogue is ISIS 2922 (Vitravene (R) ). The changes in intensity and lambda max in fluorescence spectroscopy and the increase in fluorescence anisotropy upon complex formation between the oligonucleotide and the protein (PO-BSA) were used to determine the dissociation constant, K m =6.3x10 -8 M. The stoichiometry of the complex is mainly 1:1, although 2 PO per BSA have been detected at high PO/BSA ratios. The complexation did not protect PO against enzymatic degradation by snake venom phosphodiesterase (0.1 mg/mL). Fluorescence microscopy revealed that PO-BSA complexes showed a decreased uptake and modified pattern of intracellular distribution with a rapid nuclear accumulation (rather than vesicular localization in the cytoplasm observed for free oligonucleotide). The effect was only observed over a certain threshold of BSA concentration (higher than found in media supplemented with 10% serum). By this way, the interaction with albumin increased the antiviral activity of the oligonucleotide, tested by a plaque reduction assay in MRC-5 fibroblasts infected with human cytomegalovirus (strain RC 256) at a MOI of 0.0035. At 10 muM PO concentration, free PO decreased virus plaques to 85% of the control, while PO-BSA complexes reduced to 60%, in the same order than ISIS 2922. The phosphorotioate analogue complexed by BSA (ISIS 2922-BSA) showed the highest activity with 45% of the control plaques.
引用
收藏
页码:197 / 204
页数:8
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