Fluorocarbons Enhance Intracellular Delivery of Short STAT3-sensors and Enable Specific Imaging.
Fluorocarbons Enhance Intracellular Delivery of Short STAT3-sensors and Enable Specific Imaging.
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DOI：
10.7150/thno.19704
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发表时间：
2017
期刊：
影响因子：
12.4
通讯作者：
Bogdanov A Jr
中科院分区：
文献类型：
--
作者：
Metelev V;Zhang S;Zheng S;Kumar ATN;Bogdanov A Jr
目前正在广泛研究短寡核苷酸序列的潜在治疗特性。用短氟化残基修饰寡核苷酸末端能够极大地改变它们在复杂的体外和体内系统中的行为，因此可以大大增强它们的治疗潜力。我们工作的主要目标是探索：1）用一两个短碳氟化合物（FC）残基修饰 STAT3 转录因子结合寡脱氧核苷酸（ODN）双链体（ODND）如何改变它们在体外和体内的特性，如果是这样，这将如何影响癌细胞对它们的细胞内摄取，以及 2）这种修饰的 ODND 与 FC 修饰的载体大分子形成非共价复合物的能力。后者具有产生 19F 特异性磁共振 (MR) 成像特征的固有优势。因此，我们还测试了此类共聚物产生 19F-MR 信号的能力。材料和方法。通过使用自动合成或通过 ODN 5' 端上的活化酯将氟化核酸残基掺入 ODN 中。为了量化细胞对 ODND 的摄取并跟踪其稳定性，我们使用核苷间连接技术用荧光团共价标记 ODN；通过聚乙二醇化聚赖氨酸接枝共聚物与全氟十一烷酸的酰化合成了FC修饰载体(M5-gPLL-PFUDA)。结果。与对照未修饰的 ODN 相比，具有单个 FC 基团的 ODN 表现出形成具有更高熔点的双链体的倾向，并且具有更高的抗降解稳定性。正如分子动力学模拟预测的那样，携带氟化残基的 ODND 显示与 M5-gPLL-PFUDA 形成复合物。此外，FC 组调节 ODND 与 STAT3 靶标结合的特异性。最后，通过 A431 和 INS-1 细胞的定量共焦荧光成像测定，与未修饰的 ODND 的摄取相比，FC 修饰导致更高的细胞摄取（高 2 至 4 倍）。结论。 FC 残基的 ODND 修饰能够微调蛋白质对双链结合基序的结合特异性，并导致培养物中 A431 和 INS-1 细胞的内化增加。我们的结果表明，用 FC 残基修饰 ODN 末端是开发更有效的基于核酸的疗法的可行且强大的策略，并且具有允许 ODND 治疗靶向和反应的非侵入性 MR 成像的额外好处。
Short oligonucleotide sequences are now being widely investigated for their potential therapeutic properties. The modification of oligonucleotide termini with short fluorinated residues is capable of drastically altering their behavior in complex in vitro and in vivo systems, and thus may serve to greatly enhance their therapeutic potential. The main goals of our work were to explore: 1) how modification of STAT3 transcription factor-binding oligodeoxynucleotide (ODN) duplexes (ODND) with one or two short fluorocarbon (FC)-based residues would change their properties in vitro and in vivo, and if so, how this would affect their intracellular uptake by cancer cells, and 2) the ability of such modified ODND to form non-covalent complexes with FC-modified carrier macromolecule. The latter has an inherent advantage of producing a 19F-specific magnetic resonance (MR) imaging signature. Thus, we also tested the ability of such copolymers to generate 19F-MR signals. Materials and Methods. Fluorinated nucleic acid residues were incorporated into ODN by using automated synthesis or via activated esters on ODN 5'-ends. To quantify ODND uptake by the cells and to track their stability, we covalently labeled ODN with fluorophores using internucleoside linker technology; the FC-modified carrier was synthesized by acylation of pegylated polylysine graft copolymer with perfluoroundecanoic acid (M5-gPLL-PFUDA). Results. ODN with a single FC group exhibited a tendency to form duplexes with higher melting points and with increased stability against degradation when compared to control non-modified ODNs. ODND carrying fluorinated residues showed complex formation with M5-gPLL-PFUDA as predicted by molecular dynamics simulations. Moreover, FC groups modulated the specificity of ODND binding to the STAT3 target. Finally, FC modification resulted in greater cell uptake (2 to 4 fold higher) when compared to the uptake of non-modified ODND as determined by quantitative confocal fluorescence imaging of A431 and INS-1 cells. Conclusion. ODND modification with FC residues enables fine-tuning of protein binding specificity to double-strand binding motifs and results in an increased internalization by A431 and INS-1 cells in culture. Our results show that modification of ODN termini with FC residues is both a feasible and powerful strategy for developing more efficient nucleic acid-based therapies with the added benefit of allowing for non-invasive MR imaging of ODND therapeutic targeting and response.