Collado Camps Estel, van Lith Sanne A M, Frielink Cathelijne, Lankhof Jordi, Dijkgraaf Ingrid, Gotthardt Martin, Brock Roland
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboudumc, 6525 GA Nijmegen, The Netherlands.
Department of Medical Imaging, Nuclear Medicine, Radboudumc, 6525 GA Nijmegen, The Netherlands.
Pharmaceuticals (Basel). 2021 Jun 23;14(7):602. doi: 10.3390/ph14070602.
Nanobodies are well-established targeting ligands for molecular imaging and therapy. Their short circulation time enables early imaging and reduces systemic radiation exposure. However, shorter circulation time leads to lower tracer accumulation in the target tissue. Cell-penetrating peptides (CPPs) improve cellular uptake of various cargoes, including nanobodies. CPPs could enhance tissue retention without compromising rapid clearance. However, systematic investigations on how the functionalities of nanobody and CPP combine with each other at the level of 2D and 3D cell cultures and in vivo are lacking. Here, we demonstrate that conjugates of the epidermal growth factor receptor (EGFR)-binding nanobody 7D12 with different CPPs (nonaarginine, penetratin, Tat and hLF) differ with respect to cell binding and induction of endocytosis. For nonaarginine and penetratin we compared the competition of EGF binding and performance of L- and D-peptide stereoisomers, and tested the D-peptide conjugates in tumor cell spheroids and in vivo. The D-peptide conjugates showed better penetration into spheroids than the unconjugated 7D12. Both in vivo and in vitro, the behavior of the agent reflects the combination of both functionalities. Although CPPs cause promising increases in in vitro uptake and 3D penetration, the dominant effect of the CPP in the control of biodistribution warrants further investigation.
纳米抗体是用于分子成像和治疗的成熟靶向配体。它们较短的循环时间有利于早期成像并减少全身辐射暴露。然而，较短的循环时间会导致示踪剂在靶组织中的积累减少。细胞穿透肽（CPPs）可改善包括纳米抗体在内的各种货物的细胞摄取。CPPs可以增强组织滞留，同时不影响快速清除。然而，目前缺乏关于纳米抗体和CPPs的功能如何在二维和三维细胞培养水平以及体内相互结合的系统研究。在这里，我们证明了表皮生长因子受体（EGFR）结合纳米抗体7D12与不同CPPs（九聚精氨酸、穿膜肽、Tat和人乳铁蛋白）的缀合物在细胞结合和内吞作用诱导方面存在差异。对于九聚精氨酸和穿膜肽，我们比较了表皮生长因子（EGF）结合的竞争性以及L-和D-肽立体异构体的性能，并在肿瘤细胞球体和体内测试了D-肽缀合物。D-肽缀合物比未缀合的7D12表现出更好的穿透球体的能力。在体内和体外，该制剂的行为反映了两种功能的结合。尽管CPPs在体外摄取和三维穿透方面有令人期待的增加，但CPPs在生物分布控制中的主导作用值得进一步研究。
