Department of Chemistry and Macromolecules and Interfaces Institute, Virginia Tech , Blacksburg, Virginia 24060, United States.
J Am Chem Soc. 2013 Oct 16;135(41):15417-24. doi: 10.1021/ja404941p. Epub 2013 Oct 1.
When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly(methacrylamidotrehalose) or "poly(trehalose)" and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (n = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers [PEG-P(AEMA) and P(AEMA)]. First, we demonstrate that the poly(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC50 of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications.
当纳米粒子与环境相互作用时，这种相互作用的性质主要由其最外层的性质决定。在这里，我们利用一种常见二糖海藻糖的特殊性质，海藻糖因其独特的生物稳定作用而广为人知。为此，我们开发了一种简便的合成方法，可得到这种二糖的聚合物，即聚（甲基丙烯酰胺海藻糖）或“聚（海藻糖）”，以及含有这种聚合物的二嵌段共聚物，其中聚合物的 51 个重复单元链用氨乙基甲基丙烯酰胺（AEMA）延伸，聚合度为 3（n = 34、65 和 84）。进行了两项实验系列来详细研究这些二嵌段共聚物，并将其性质与两种对照聚合物[PEG-P（AEMA）和 P（AEMA）]进行比较。首先，我们证明聚（海藻糖）涂层可确保含有 siRNA 的聚阳离子在高盐浓度和血清蛋白存在下的胶体稳定性。聚（海藻糖）保留了海藻糖降低与水冻结相关的相变能的能力，并可在冷冻干燥过程中保护 siRNA 聚阳离子，使纳米颗粒完全再悬浮而不损失生物功能。其次，我们表明，用聚（海藻糖）涂层的 siRNA 聚阳离子具有进入神经胶质瘤细胞的非凡细胞内化能力，其进入过程呈零级动力学。此外，通过控制细胞培养介质中的 siRNA 浓度，可以控制聚（海藻糖）嵌段共聚物递送的 siRNA 量。使用共聚焦显微镜，我们表明，在用海藻糖包被的聚阳离子内化后，它们在细胞质中进行主动转运，并且在 siRNA 诱导的靶基因下调方面取得了显著效果，IC50 为 19 nM。这些发现加上微不足道的细胞毒性表明，聚（海藻糖）有可能成为核酸治疗性纳米粒子制剂的重要组成部分，并有望扩展为其他基于纳米的技术和大分子（特别是与纳米医学应用相关的技术和大分子）的新型涂层。
