Enhanced Extravasation, Stability and in Vivo Cardiac Gene Silencing via in Situ siRNA鈥揂lbumin Conjugation
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- 作者：Shannen Lau ; Bim Graham ; Nga Cao ; Ben J. Boyd ; Colin W. Pouton ; Paul J. White
- 刊名：Molecular Pharmaceutics
- 出版年：2012
- 出版时间：January 1, 2012
- 年：2012
- 卷：9
- 期：1
- 页码：71-80
- 全文大小：434K
- 年卷期：v.9,no.1(January 1, 2012)
- ISSN：1543-8392
文摘
A potential barrier to progression of siRNA therapeutics to the clinic is the ability of these agents to cross the vascular endothelium to reach target cells. This study aimed to bypass the endothelial barrier by harnessing the extravasation capability of the serum protein albumin to allow siRNA to reach cardiomyocytes. A strategy for conjugating siRNA to albumin in vivo was developed that involved activating 3鈥?amine, 2鈥?O-methyl, phosphorothioate modified siRNA with succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to yield maleimide-functionalized siRNA (鈥渁ctivated siRNA鈥?; this thiol-reactive species can then irreversibly link to the single surface-exposed cysteine residue of endogenous albumin following intravenous administration. An IGF-I-receptor (IGF-IR) siRNA sequence which was effective in vitro was used to test the ability of the siRNA鈥揳lbumin conjugate to bypass the endothelial barrier in Balb/C mice and produce silencing. In situ conjugation of maleimide-functionalized siRNA to albumin in mouse serum occurred within minutes of addition, and the resulting conjugate was found to be nuclease stable by SDS鈥揚AGE analysis. In Sprague鈥揇awley rats, activated siRNA showed a significantly enhanced elimination half-life (75.9 卤 18.2 min) compared to unactivated siRNA (5.1 卤 0.2 min). Intravenous injection of this activated siRNA (1 mg/kg daily for four days) significantly reduced left ventricle IGF-IR mRNA to 64.1 卤 4.1% of that in vehicle-treated animals (mean 卤 SEM), while the control siRNA (unconjugated) had no effect (n = 4, P > 0.05). Imaging of microvessels from mice treated with fluorescein-labeled activated siRNA showed clear evidence of extravasation and cellular uptake in capillary endothelial cells, cardiomyocytes and vascular smooth muscle cells for mice treated with the activated siRNA but not mice treated with the unactivated siRNA. siRNA鈥揳lbumin constructs are therefore capable of extravasation to the myocardium resulting in silencing in this otherwise silencing-resistant organ.