Abstract
Poly-histidine peptides such as H6 (HHHHHH) are used in protein biotechnologies as purification tags, protein-assembling agents and endosomal-escape entities. The pleiotropic properties of such peptides make them appealing to design protein-based smart materials or nanoparticles for imaging or drug delivery to be produced in form of recombinant proteins. However, the clinical applicability of H6-tagged proteins is restricted by the potential immunogenicity of these segments. In this study, we have explored several humanized histidine-rich peptides in tumor-targeted modular proteins, which can specifically bind and be internalized by the target cells through the tumoral marker CXCR4. We were particularly interested in exploring how protein purification, self-assembling and endosomal escape perform in proteins containing the variant histidine-rich tags. Among the tested candidates, the peptide H5E (HEHEHEHEH) is promising as a good promoter of endosomal escape of the associated full-length protein upon endosomal internalization. The numerical modelling of cell penetration and endosomal escape of the tested proteins has revealed a negative relationship between the amount of protein internalized into target cells and the efficiency of cytoplasmic release. This fact demonstrates that the His-mediated, proton sponge-based endosomal escape saturates at moderate amounts of internalized protein, a fact that might be critical for the design of protein materials for cytosolic molecular delivery.
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Acknowledgements
Acknowledgements We are indebted to Agencia Estatal de Investigación (AEI) and to Fondo Europeo de Desarrollo Regional (FEDER) (BIO2016-76063-R, AEI/FEDER, UE) to Villaverde A, AGAUR (2017SGR-229) to Villaverde A and 2017SGR-865 GRC to Mangues R; CIBER-BBN (NANOPROTHER) granted to Villaverde A and CIBER-BBN project 4NanoMets to Mangues R; ISCIII (PI15/00272 co-founding FEDER) to Vazquez E and ISCIII (Co-founding FEDER) PIE15//00028 and PI18/00650 to Mangues R, and to EU COST Action CA 17140. We are also indebted to the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) that is initiatively funded by the VI National R&D&I Plan 2008–2011, In-iciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III, with assistance from the European Regional Development Fund. Protein production has been partially performed by the ICTS “NANBIOSIS”, more specifically by the Protein Production Platform of CIBER in Bioengineering, Biomaterials & Na-nomedicine (CIBER-BBN)/IBB, at the UAB sePBioEs scientific-technical service (http://www.nanbiosis.es/portfolio/u1-protein-production-plat-form-ppp/) and the nanoparticle size analysis by the Biomaterial Processing and Nanostructuring Unit. Biodistribution studies were performed by the ICTS “NANBIOSIS”, Nantoxicology Unit (http://www.nanbiosis.es/portfolio/u18-nanotoxicology-unit/). Electron microscopy studies were performed by the Servei de Microscòpia at the UAB. AV received an ICREA ACADEMIA award. HLL was supported by a predoctoral fellowship from AGAUR (2019 FI_B 00352) and UU by PERIS program from the Health Department of the Generalitat de Catalunya.
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López-Laguna H performed the experiments and prepared the corresponding figures, under the supervision of Un-zueta U; Cubarsi R performed the mathematical modelling and prepared the corresponding figures. Mangues R interpreted data relative to CXCR4, and Vazquez E conceived the study. The manuscript was mainly written by Villaverde A, and all authors have revised and edited it.
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Mangues R, Vázquez E and Villaverde A are cofounders of NANOLIGENT SL, devoted to developing protein-based antitumoral drugs.
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Hèctor López-Laguna graduated in biochemistry and obtained his Master’s degree in advanced nanoscience and nanotechnology from the Autonomous University of Barcelona. He is studying for a PhD thesis at the Nanobiotechnology group headed by Prof. Antonio Villaverde at the Institute of Biotechnology and Biomedicine (IBB), working on the rational design of protein-based nanomaterials with plethora of applications in nanomedicine.
Esther Vázquez is a Senior Researcher at the Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Spain, and Associate Professor at the Department de Genètica i de Microbiologia, of the same university. She is also member of the CIBER of Bioengineering, Biomaterials and Nanomedicine (BBN). Being formed at the State University of New York, USA, and after working in different Universities on molecular medicine, she is now interested in protein drug design, recombinant protein production, nanobiotechnology and protein nanoparticles for targeted therapies.
Antonio Villaverde graduated in biological sciences in 1982 and got his PhD in 1985. He has been scientifically formed in Barcelona, Madrid, London, Lausanne and Braunschweig. Since 1987, he has been Professor of Microbiology at the Universitat Autònoma de Barcelona, Spain, where he got a Full Professorship in 2002. He leads the Nanobiotechnology group in this university and in the CIBER-BBN, focusing on the design of protein-based materials for biomedical applications. He founded the journal Microbial Cell Factories in 2002, being its Editor-in-Chief for 15 years.
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López-Laguna, H., Cubarsi, R., Unzueta, U. et al. Endosomal escape of protein nanoparticles engineered through humanized histidine-rich peptides. Sci. China Mater. 63, 644–653 (2020). https://doi.org/10.1007/s40843-019-1231-y
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DOI: https://doi.org/10.1007/s40843-019-1231-y