Controlling self-assembling and tumor cell-targeting of protein-only nanoparticles through modular protein engineering

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  • ReceivedJul 24, 2019
  • AcceptedAug 11, 2019
  • PublishedSep 19, 2019


Modular protein engineering is suited to recruit complex and multiple functionalities in single-chain polypeptides. Although still unexplored in a systematic way, it is anticipated that the positioning of functional domains would impact and refine these activities, including the ability to organize as supramolecular entities and to generate multifunctional protein materials. To explore this concept, we have repositioned functional segments in the modular protein T22-GFP-H6 and characterized the resulting alternative fusions. In T22-GFP-H6, the combination of T22 and H6 promotes self-assembling as regular nanoparticles and selective binding and internalization of this material in CXCR4-overexpressing tumor cells, making them appealing as vehicles for selective drug delivery. The results show that the pleiotropic activities are dramatically affected in module-swapped constructs, proving the need of a carboxy terminal positioning of H6 for protein self-assembling, and the accommodation of T22 at the amino terminus as a requisite for CXCR4+ cell binding and internalization. Furthermore, the failure of self-assembling as regular oligomers reduces cellular penetrability of the fusions while keeping the specificity of the T22-CXCR4 interaction. All these data instruct how multifunctional nanoscale protein carriers can be designed for smart, protein-driven drug delivery, not only for the treatment of CXCR4+ human neoplasias, but also for the development of anti-HIV drugs and other pathologies in which CXCR4 is a relevant homing marker.


We are indebted to Agencia Estatal de Investigación and to Fondo Europeo de Desarrollo Regional (grant BIO2016-76063-R, AEI/FEDER, UE) to Villaverde A, AGAUR (2017SGR-229) to Villaverde A and 2017SGR-865 GRC to Mangues R; CIBER-BBN (project NANOPROTHER) granted to Villaverde A and CIBER-BBN project 4NanoMets to Mangues R; ISCIII (PI15/00272 co-founding FEDER) to Vázquez 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 an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa 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 & Nanomedicine (CIBER-BBN)/IBB, at the UAB sePBioEs scientific-technical service (http://www.nanbiosis.es/portfolio/u1-protein-production-platform-ppp/) and the nanoparticle size analysis by the Biomaterial Processing and Nanostructuring Unit. Confocal and electron microscopy studies were performed at the Servei de Microscòpia and cell culture experiments at the SCAC, both located at the UAB. Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311. Villaverde A received an ICREA ACADEMIA award. Sánchez-García L and López-Laguna H were supported by a predoctoral fellowship from AGAUR (2018FI_B2_00051 and 2019FI_B_00352) respectively and Unzueta U by PERIS program from the Health Department of la Generalitat de Catalunya.

Interest statement

Mangues R, Vázquez E and Villaverde A are co-founders of Nanoligent SL, a company devoted to the design of anticancer drugs based on T22.

Contributions statement

Voltà-Durán E performed most of the experiments and figures, assisted by Cano-Garrido O, Serna N, López-Laguna H and Sánchez-García L. Pesarrodona P designed and produced one of the proteins used here. Sánchez-Chardi A performed the electron microscopy and some statistics. Unzueta U supervised the study. Mangues R, Villaverde A and Vázquez E conceived the whole study, which was mostly written by Villaverde A. All authors gave approval to the final version of the manuscript.

Author information

Eric Voltà-Durán graduated in biotechnology in 2018 and developed his Master thesis in Advanced Nanoscience and Nanotechnology at the Nanobiotechnology group led by Prof. Villaverde at the Autonomous University of Barcelona. He is currently starting a PhD in biotechnology in the same group, working on the design and production of proteins with biomedical interest. At present, he is focused on the rational engineering of CXCR4-targeted protein nanoparticles, with great potential in cancer therapy.

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. 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 a 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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  • Figure 1

    Properties of building blocks and protein nanoparticles. (a) Modular organization of the multifunctional proteins used in the study. Segment sizes are indicative. Protein names are according to the order of modules in the constructs. T22-GFP-H6(LOOP) refers to the insertion of H6 into a solvent-exposed permissive loop of GFP. Theoretical molecular masses are indicated in kDa, as well as the specific fluorescence (FU) relative to GFP-H6. (b) Protein purity upon purification, shown by SDS-PAGE, demonstrating their proteolytic stability as single molecular mass species. M in the image indicates the molecular marker lane, showing the molecular masses in kDa. (c) Mass spectrometry of the same protein samples.

  • Figure 2

    Protein assembly as oligomeric nanoparticles. (a) Representative FESEM images of the GFP-based recombinant proteins upon purification. Bars indicate 30 nm. (b) Hydrodynamic size of unassembled proteins and protein nanoparticles, indicating the peak size of DLS plots and the polydispersion index (PDI). (c) Controlled disassembly of T22-GFP-H6 nanoparticles mediated by 3% SDS or by EDTA at 1:1 molar ratio related to His residues. (d) Molecular modelling of native GFP showing both the monomeric and dimeric forms and their larger atomic distances. (e) MTT cell viability analysis of HeLa cells exposed to pure proteins (at 1000 nmol L−1) for 48 h. Data are referred to the viability of non-exposed cells cultured under the same conditions.

  • Figure 3

    Efficacy and specificity of cell penetrability of protein nanoparticles. (a) Internalization of T22-GFP-H6, T22-GFP-H6(LOOP), H6-GFP-T22 and GFP-H6 in CXCR4+ HeLa cells, recorded after 2 and 24 h of exposure, at three different concentrations (25, 100 and 1000 nmol L−1). Crude fluorescence values were normalized by the specific fluorescence emission of each protein to allow molar-based comparison. Y-axis in arbitrary units of fluorescence (a.u.). Significant differences with T22-GFP-H6 internalization under the same conditions (included as a control) are represented by * (p<0.001). (b) Relative intracellular fluorescence values (a.u.) for each protein under prior treatment of CXCR4+ HeLa cells with AMD3100, a specific inhibitor of T22-CXCR4 binding, compared with those obtained under the same conditions in absence of this compound. The analysis was done 2 h after exposure. Significant difference with protein internalization under the same conditions but without AMD3100 are represented by * (p<0.001). The standard error is represented by grey lines at each sample.

  • Figure 4

    Intracellular localization of proteins in protein-exposed cells. 2D confocal imaging of protein-exposed HeLa cells. Red signal indicates labelled membranes, blue signal labelled nuclear nucleic acids and the green signal is the natural green fluorescence of proteins. Cells cultured in absence of proteins are also shown for a clear visualization of blue and red signals in absence of protein. At the bottom, 3D Imaris reconstruction of confocal stacks along the z axis of the same experiments. Analysis was performed after 24 h of protein exposure at 1 μmol L−1. Bars indicate 10 μm.

  • Figure 5

    Intracellular and membrane localization of proteins in protein-exposed cells. Fine details of 3D Imaris reconstructions of confocal stacks along the z axis, showing the green fluorescent material. Cells cultured in absence of proteins are also shown. Red signal indicates labelled membranes and blue signal labelled nuclear nucleic acids. The analysis was performed after 24 h of protein exposure at 1 μmol L−1. Bars indicate 10 μm.

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