Simplified preparation via streptavidin of antisense oligomers/carriers nanoparticles showing improved cellular delivery in culture
Simplified preparation via streptavidin of antisense oligomers/carriers nanoparticles showing improved cellular delivery in culture
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DOI：
10.1021/bc070032c
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发表时间：
2007-07-01
影响因子：
4.7
通讯作者：
Hnatowich, Donald J.
中科院分区：
文献类型：
--
作者：
Wang, Yi;Nakamura, Kayoko;Hnatowich, Donald J.
目的：在反义肿瘤靶向和其他需要跨膜递送低聚物的应用中，载体越来越被认为是提高细胞摄取的帮助甚至是必不可少的。对许多大量可用的和潜在有用的载体的评价仅受到通过共价偶联制备低聚物/载体的复杂性的限制。然而，使用链霉亲和素作为生物素化载体和生物素化反义寡聚物之间的连接物，只需要简单的混合即可制备。本研究的目的是评价载体/链亲和素纳米颗粒反义磷二胺morpholino （MORF）低聚物的制备及其在培养中的细胞积累。方法：模型载体是胆固醇，一种10聚体hiv肽，和一种10聚体聚精氨酸，每一种都在其他地方报道过，以改善低聚物的细胞递送。模型反义寡聚物为靶向survivin mRNA的25 mer MORF。在表达survivin的MCF-7细胞中，将反义MORF/载体纳米颗粒的积累量与有义MORF/载体、无载体纳米颗粒和裸反义MORF进行比较。morf和多肽是购买的生物素化，而胆固醇是内部生物素化。在所有情况下，Tc-99m放射性标签都放在低聚物上。细胞研究是在低nM浓度下进行的，这是反义成像应用所需的，在37℃下进行的，主要是在1%的FBS中进行的。结果：以1:1的摩尔比仔细混合，成功制备了放射性标记低聚物/链霉亲和素/载体纳米颗粒。作为载体参与的证据，放射性标记的MORF在作为纳米颗粒孵育时，与无载体的纳米颗粒相比，在细胞中的积累增加了11倍。反义MORF/链亲和素/tat纳米颗粒的积累量显著高于义MORF/链亲和素/tat纳米颗粒的积累量，这是特异性反义靶向的证据。结论：以链霉亲和素为连接剂制备低聚物/载体纳米颗粒比共价偶联制备工艺简单。此外，我们的研究结果表明，链霉亲和素的加入不会干扰tat、聚精氨酸或胆固醇携带者的细胞递送功能，也不会干扰MORF低聚物的特异性反义mRNA结合功能。
Objective: Carriers are increasingly now viewed as helpful or even essential to improve cellular uptake in connection with antisense tumor targeting and other applications requiring transmembrane delivery of oligomers. Evaluation of many of the large number of available and potentially useful carriers is limited only by the complexities of preparing the oligomer/carriers by covalent conjugation. However, using streptavidin as a linker between biotinylated carriers and biotinylated antisense oligomers would require only simple mixing for preparation. The goal of this study was to evaluate the preparation and cell accumulation in culture of carrier/streptavidin nanoparticle of an antisense phosphorodiamide morpholino (MORF) oligomer. Methods: The model carriers were cholesterol, a 10 mer HIV-tat peptide, and a 10 mer polyarginine, each having been reported elsewhere to improve cellular delivery of oligomers. The model antisense oligomer was the 25 mer MORF targeting the survivin mRNA. The accumulations of the antisense MORF/carrier nanoparticle were compared to the sense MORF/carrier, to the carrier-free nanoparticles, and to the naked antisense MORF in the survivin-expressing MCF-7 cells. The MORFs and peptides were purchased biotinylated, while the cholesterol was biotinylated in-house. In all cases, the Tc-99m radiolabel was placed on the oligomers. Cell studies were performed at low nM concentration as required for antisense imaging applications and at 37 degrees C primarily in 1% FBS. Results: Each radiolabeled oligomer/streptavidin/carrier nanoparticle was successfully prepared by careful mixing at a 1:1 molar ratio. As evidence of carrier participation, the radiolabeled MORF showed increased accumulation in cells when incubated as the nanoparticle compared to the carrier-free nanoparticle and by as much as a factor of 11. Accumulation of the antisense MORF/streptavidin/tat nanoparticle was significantly higher than the sense MORF/streptavidin/tat nanoparticle as evidence of specific antisense targeting. Conclusions: The preparation of oligomer/carrier nanoparticles was greatly simplified over covalent conjugations by using streptavidin as a linker. Furthermore, our results suggest that the addition of streptavidin did not interfere with the cellular delivery function of the tat, polyarginine, or cholesterol carriers nor with the specific antisense mRNA binding function of the MORF oligomer.