Zhang Aiwen, Nakanishi Jun
Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan.
Research Center for Functional Materials, National Institute for Materials Science (NIMS), Tsukuba, Japan.
Sci Technol Adv Mater. 2021 Sep 6;22(1):616-626. doi: 10.1080/14686996.2021.1944783. eCollection 2021.
Epidermal growth factor (EGF)-nanoparticle conjugates have the potential for cancer therapeutics due to the unique cytotoxic activity in cancer cells with EGF receptor (EGFR) overexpression. To gain its maximum activity, the EGF molecule should be immobilized on the nanoparticle surface in a defined orientation so as the bulky nanoparticle will not interfere EGF-EGFR interaction. Herein, we demonstrate successful enhancement of the anti-cancer activity of EGF-gold nanoparticle conjugates (EGF-GNPs) by controlling the EGF orientation on the surface of the nanoparticle through site-specific mutagenesis. Three lysine-free EGF variants (RR, RS, and SR) were designed, where two endogenous lysine residues were replaced with either arginine (R) or serine (S). The EGF mutants can be conjugated to the GNPs in a controlled orientation through the single amino group at the N-terminus. The ability of the mutants to induce extracellular signal-regulated kinase (ERK) phosphorylation was no different from wild type EGF (WT) in soluble form, rather lowered for one mutant (RR). However, after conjugated to GNPs, the SR mutants exhibited an enhanced biological activity than WT, in terms of ERK phosphorylation and growth inhibition of cancer cells. Further analysis of the binding constant of each mutant indicated the emergent enhanced activity of the GNP conjugates of the SR mutant was not solely contributed to the orientation, but to its higher binding activity to EGFR. These results validate the present genetic recombination strategy to improve the anticancer efficiency of EGF-GNPs.
表皮生长因子（EGF）-纳米颗粒缀合物由于在表皮生长因子受体（EGFR）过表达的癌细胞中具有独特的细胞毒性活性，因而具有癌症治疗潜力。为了发挥其最大活性，EGF分子应以特定的方向固定在纳米颗粒表面，以使庞大的纳米颗粒不会干扰EGF与EGFR的相互作用。在此，我们通过位点特异性诱变控制纳米颗粒表面EGF的方向，成功增强了EGF-金纳米颗粒缀合物（EGF-GNPs）的抗癌活性。设计了三种无赖氨酸的EGF变体（RR、RS和SR），其中两个内源性赖氨酸残基被精氨酸（R）或丝氨酸（S）取代。EGF突变体可以通过N端的单个氨基以可控的方向与GNPs缀合。突变体诱导细胞外信号调节激酶（ERK）磷酸化的能力与可溶性形式的野生型EGF（WT）没有差异，其中一个突变体（RR）的能力有所降低。然而，与GNPs缀合后，就ERK磷酸化和癌细胞生长抑制而言，SR突变体表现出比WT更强的生物活性。对每个突变体结合常数的进一步分析表明，SR突变体的GNP缀合物新出现的增强活性并非仅归因于其方向，还归因于其与EGFR更高的结合活性。这些结果验证了当前用于提高EGF-GNPs抗癌效率的基因重组策略。
