An efficient biodelivery system for antisense polyamide nucleic acid (PNA)
An efficient biodelivery system for antisense polyamide nucleic acid (PNA)
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DOI：
10.1089/oli.2008.0126
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发表时间：
2008-09-01
期刊：
影响因子：
--
通讯作者：
Patino, Nadia
中科院分区：
文献类型：
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作者：
Mehiri, Mohamed;Upert, Gregory;Patino, Nadia
为了开发一种用于细胞内递送裸肽核酸（PNA）的通用且简单的程序，我们设计了一种基于细胞内可生物降解的三苯基磷（TPP）阳离子的转运系统。在该系统中，TPP 通过生物不稳定的二硫桥连接至活化的巯基乙氧基羰基部分，使其能够通过氨基甲酸酯键直接偶联至游离 PNA 的 N 末端。我们发现这种 TPP-PNA-氨基甲酸酯缀合物在含有胎牛血清的细胞培养基中高度稳定。在模拟细胞质的含谷胱甘肽的培养基中，缀合物迅速降解成不稳定的中间体，该中间体自发分解，释放出游离的 PNA。使用荧光标记的 PNA-TPP 缀合物，我们证明缀合物被细胞吸收。通过针对 HIV-1 基因组 TAR 区域的 16 聚体 PNA 的 TPP 缀合物测量的抗 HIV 活性，进一步证实了 PNA 的有效细胞摄取和释放到细胞质中。该缀合物表现出1μM的IC50值，而游离的16聚体PNA在相同的细胞测试中不抑制HIV的复制。
With the aim of developing a general and straightforward procedure for the intracellular delivery of naked peptide nucleic acids (PNAs), we designed an intracellularly biodegradable triphenylphosphonium (TPP) cation based transporter system. In this system, TPP is linked, via a biolabile disulfide bridge, to an activated mercaptoethoxycarbonyl moiety, allowing its direct coupling to the N-terminal extremity of a free PNA through a carbamate bond. We found that such TPP-PNA-carbamate conjugates were highly stable in a cell culture medium containing fetal calf serum. In a glutathione-containing medium mimicking the cytosol, the conjugates were rapidly degraded into an unstable intermediate, which spontaneously decomposed, releasing the free PNA. Using a fluorescence-labeled PNA-TPP conjugate, we demonstrated that conjugates were taken up by cells. Efficient cellular uptake and release of the PNA into the cytosol was further confirmed by the anti-HIV activity measured for the TPP-conjugate of a 16-mer PNA targeting the TAR region of the HIV-1 genome. This conjugate exhibited an IC50 value of 1 mu M, while the free 16-mer PNA did not inhibit replication of HIV in the same cellular test.