Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia.
Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia.
J Nanobiotechnology. 2020 Jul 13;18(1):95. doi: 10.1186/s12951-020-00654-x.
Currently, the diagnosis and treatment of neuroblastomas-the most frequent solid tumors in children-exploit the norepinephrine transporter (hNET) via radiolabeled norepinephrine analogs. We aim to develop a nanomedicine-based strategy towards precision therapy by targeting hNET cell-surface protein with hNET-derived homing peptides.
The peptides (seq. GASNGINAYL and SLWERLAYGI) were shown to bind high-resolution homology models of hNET in silico. In particular, one unique binding site has marked the sequence and structural similarities of both peptides, while most of the contribution to the interaction was attributed to the electrostatic energy of Asn and Arg (< - 228 kJ/mol). The peptides were comprehensively characterized by computational and spectroscopic methods showing ~ 21% β-sheets/aggregation for GASNGINAYL and ~ 27% α-helix for SLWERLAYGI. After decorating 12-nm ferritin-based nanovehicles with cysteinated peptides, both peptides exhibited high potential for use in actively targeted neuroblastoma nanotherapy with exceptional in vitro biocompatibility and stability, showing minor yet distinct influences of the peptides on the global expression profiles. Upon binding to hNET with fast binding kinetics, GASNGINAYLC peptides enabled rapid endocytosis of ferritins into neuroblastoma cells, leading to apoptosis due to increased selective cytotoxicity of transported payload ellipticine. Peptide-coated nanovehicles significantly showed higher levels of early apoptosis after 6 h than non-coated nanovehicles (11% and 7.3%, respectively). Furthermore, targeting with the GASNGINAYLC peptide led to significantly higher degree of late apoptosis compared to the SLWERLAYGIC peptide (9.3% and 4.4%, respectively). These findings were supported by increased formation of reactive oxygen species, down-regulation of survivin and Bcl-2 and up-regulated p53.
This novel homing nanovehicle employing GASNGINAYLC peptide was shown to induce rapid endocytosis of ellipticine-loaded ferritins into neuroblastoma cells in selective fashion and with successful payload. Future homing peptide development via lead optimization and functional analysis can pave the way towards efficient peptide-based active delivery of nanomedicines to neuroblastoma cells.
目前，神经母细胞瘤（儿童最常见的实体肿瘤）的诊断和治疗利用放射性标记的去甲肾上腺素类似物靶向去甲肾上腺素转运体（hNET）。我们旨在通过用 hNET 衍生的归巢肽靶向 hNET 细胞表面蛋白，开发一种基于纳米医学的精准治疗策略。
肽（序列为 GASNGINAYL 和 SLWERLAYGI）被证明可以在计算机上结合 hNET 的高分辨率同源模型。特别是，一个独特的结合位点标记了这两种肽的序列和结构相似性，而相互作用的大部分贡献归因于天冬酰胺和精氨酸的静电能（< -228 kJ/mol）。通过计算和光谱方法对肽进行了全面表征，结果表明 GASNGINAYL 约有 21%的β-折叠/聚集，SLWERLAYGI 约有 27%的α-螺旋。用半胱氨酸化肽修饰 12nm 铁蛋白基纳米载体后，两种肽都表现出用于主动靶向神经母细胞瘤纳米治疗的高潜力，具有出色的体外生物相容性和稳定性，对全局表达谱的影响很小但很明显。GASNGINAYL 肽与 hNET 快速结合后，铁蛋白迅速内吞到神经母细胞瘤细胞中，由于转运的有效载荷椭圆碱的选择性细胞毒性增加，导致细胞凋亡。肽包被的纳米载体在 6 小时后显示出比非包被的纳米载体更高水平的早期凋亡（分别为 11%和 7.3%）。此外，与 SLWERLAYGIC 肽相比，GASNGINAYLC 肽靶向治疗导致晚期凋亡的程度显著更高（分别为 9.3%和 4.4%）。这些发现得到了活性氧形成增加、survivin 和 Bcl-2 下调以及 p53 上调的支持。
本研究开发的新型归巢纳米载体采用 GASNGINAYLC 肽，能够以选择性方式将载有椭圆碱的铁蛋白快速内吞到神经母细胞瘤细胞中，并成功输送有效载荷。通过先导优化和功能分析进一步开发归巢肽，可以为基于肽的神经母细胞瘤细胞的高效主动递药铺平道路。
