Department of Chemistry, University of California, Irvine, California, USA.
1] Department of Chemistry, University of California, Irvine, California, USA. [2] Department of Medical Biochemistry, Graduate Division of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
Nat Protoc. 2015 Apr;10(4):595-604. doi: 10.1038/nprot.2015.032. Epub 2015 Mar 19.
Synthetic polymer nanoparticles (NPs) with intrinsic affinity for target biomacromolecules hold great promise in the development of novel tools for biological and biomedical research. We recently reported the design and synthesis of abiotic, synthetic polymer NPs with high intrinsic affinity for a peptide toxin melittin. The NP was selected by screening a small library of NPs (∼100 nm) composed of various ratios of monomers that contain functional groups complementary to the peptide melittin. The selected polymer NP, a co-polymer of acrylic acid (AAc), N-tert-butylacrylamide (TBAm), N-isopropylacrylamide (NIPAm) and N,N'-methylenebisacrylamide (BIS), effectively captures and neutralizes the toxicity of the peptide through a combination of electrostatic and hydrophobic interactions. This protocol describes a step-by-step procedure for the preparation and evaluation of synthetic polymer NPs for sequestration and neutralization of the target peptide toxin. The polymer NPs can be synthesized in a one-step polymerization reaction using commercially available reagents. The polymerization reaction for the synthesis of polymer NPs takes several hours, and the total protocol including subsequent purification and characterization by dynamic light scattering, NMR and toxicity neutralization assays takes 1-2 weeks in total.
具有与靶生物大分子固有亲和力的合成聚合物纳米颗粒 (NPs) 在开发用于生物和生物医学研究的新型工具方面具有巨大的潜力。我们最近报道了具有高固有亲和力的非生物、合成聚合物 NPs 的设计和合成，用于一种称为蜂毒素的肽毒素。通过筛选由各种单体组成的小 NP 文库（~100nm）来选择 NP，这些单体包含与肽蜂毒素互补的功能基团。所选聚合物 NP 是由丙烯酸 (AAc)、叔丁基丙烯酰胺 (TBAm)、N-异丙基丙烯酰胺 (NIPAm) 和 N,N'-亚甲基双丙烯酰胺 (BIS) 组成的共聚物，通过静电和疏水相互作用的结合，有效地捕获和中和肽的毒性。本方案描述了一种用于制备和评估用于捕获和中和目标肽毒素的合成聚合物 NPs 的逐步程序。聚合物 NPs 可以使用市售试剂通过一步聚合反应合成。用于合成聚合物 NPs 的聚合反应需要几个小时，包括随后通过动态光散射、NMR 和毒性中和测定进行的纯化和表征，总共需要 1-2 周的时间。
