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Updated: Oct 30, 2025
Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy
Published on: April 6, 2015
CPPs to the Test: Effects on Binding, Uptake and Biodistribution of a Tumor Targeting Nanobody
Estel Collado Camps1,2, Sanne A M van Lith2, Cathelijne Frielink2
1Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboudumc, 6525 GA Nijmegen, The Netherlands.
Abstract:
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.
Insights
Cell-penetrating peptides (CPPs) enhance nanobody uptake in tissues, improving molecular imaging and therapy. Conjugates show better tumor penetration, but CPPs significantly influence biodistribution, requiring further study.
Area of Science:
- Bioconjugation Chemistry
- Molecular Imaging
- Nanomedicine
Background:
- Nanobodies are effective targeting ligands for molecular imaging and therapy due to rapid clearance.
- Short circulation times limit nanobody accumulation in target tissues.
- Cell-penetrating peptides (CPPs) enhance cellular uptake of various molecules, including nanobodies.
Purpose of the Study:
- To investigate the combined functionalities of nanobody-CPP conjugates for molecular imaging.
- To evaluate how different CPPs affect nanobody cell binding, endocytosis, and tissue penetration.
- To assess the in vitro and in vivo performance of nanobody-CPP conjugates.
Main Methods:
- Conjugation of epidermal growth factor receptor (EGFR)-binding nanobody 7D12 with various CPPs (nonaarginine, penetratin, Tat, hLF).
- Assessment of cell binding, endocytosis, and EGF binding competition in 2D cell cultures.
- Evaluation of D-peptide conjugates' penetration in 3D tumor spheroids and in vivo biodistribution.
Main Results:
- Nanobody-CPP conjugates exhibited varying cell binding and endocytosis induction.
- D-peptide conjugates demonstrated superior penetration into tumor spheroids compared to unconjugated nanobody.
- In vivo and in vitro behavior reflected a combination of nanobody and CPP functionalities.
Conclusions:
- CPP conjugation enhances nanobody in vitro uptake and 3D spheroid penetration.
- The biodistribution of nanobody-CPP conjugates is significantly influenced by the CPP moiety.
- Further investigation is needed to understand and optimize CPP effects on nanobody biodistribution for imaging and therapy.
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