Engineering a recombinant chlorotoxin as cell-targeted cytotoxic nanoparticles

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  • ReceivedNov 26, 2018
  • AcceptedJan 3, 2019
  • PublishedJan 16, 2019



the Agencia Estatal de Investigación(AEI)

Fondo Europeo de Desarrollo Regional(FEDER)





This study has been funded by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (BIO2016-76063-R, AEI/FEDER, UE), AGAUR (2017SGR-229) and CIBER-BBN (project VENOM4CANCER) granted to Villaverde A, ISCIII (PI15/00272 co-founding FEDER) to Vázquez E. Protein production and DLS have been partially performed by the ICTS “NANBIOSIS”, more specifically by the Protein Production Platform of CIBER-BBN/IBB (http://www.nanbiosis.es/unit/u1-protein-production-platform-ppp/) and the Biomaterial Processing and Nanostructuring Unit (http://www.nanbiosis.es/portfolio/u6-biomaterial-processing-and-nanostructuring-unit/). Cytometry and cell culture experiments were performed at the Cytometry and Cell Culture Unit of the UAB (SCAC). Díaz R received an overseas predoctoral fellowship from Conacyt (Gobierno de México, 2016). Sánchez-Garcia L was supported by predoctoral fellowship from AGAUR (2018FI_B2_00051), Serna N was supported by a predoctoral fellowship from the Government of Navarra, and Unzueta U is supported by PERIS program from the health department of la Generalitat de Cataluña. Villaverde A received an ICREA ACADEMIA award.

Interest statement

The authors declare no conflict of interest.

Contributions statement

Díaz R performed most of the protein production and characterization experiments assisted by Sánchez-Garcia L, Serna N, Cano-Garrido O and Sánchez JM. Serna N, Díaz R and Sánchez-garcía L designed the fusion proteins and Sánchez-Chardi A performed the electron microscopy studies. All authors have discussed the data and prepared the figures and methods. Unzueta U, Vazquez E and Villaverde A conceived the study, supervised the experiments and organized the figures. The manuscript was mainly written by Villaverde A.

Author information

Raquel Díaz studied chemical engineering at the University of Sonora, (Mexico, 2008) and achieved a one-year academic exchange at the University of British Columbia (Canada, 2007-2008). Later she fulfilled her Master’s study in materials science at the University of Sonora (Mexico, 2012) and is currently studying her PhD in biotechnology at the Autonomous University of Barcelona (Spain, 2019), particularly in the cancer research investigation line.

Ugutz Unzueta developed his PhD in biotechnology at the Nanobiotechnology group led by Prof. Villaverde at the Autonomous University of Barcelona and he is currently a post-doctoral researcher at Oncogenesis and Antitumoral drugs group at Sant Pau Biomedical Research Institute in Barcelona. His research line is mainly focused on the design, production and characterization of self-assembling protein nanoparticles and nanoconjugates for targeted cancer nanomedicines.

Antonio Villaverde graduated in biological sciences in 1982 and got his PhD in 1985. Since 1987, he is Professor of Microbiology at the Universitat Autònoma de Barcelona in Spain, where he got a Full Professorship in 2002. He leads the Nanobiotechnology group in this university and in the CIBER-BBN, and he is devoted to 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.

Supplementary data

Supplementary information

Experimental details are available in the online version of the paper.


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  • Figure 1

    Modular organization of CTX-based building blocks and nanoparticle characterization. (a) Schematic representation of the fusion proteins showing the amino acid sequences, where CTX (green) is placed at the amino termini and a hexahistidine tail (H6, blue) at the carboxy termini. Linker regions (purple) were placed in both cases between CTX and GFP (grey), to ensure fluorescence emission of the fusion protein. A cationic (red) region was inserted in CTX-KRKRK-GFP-H6 downstream the CTX. Siding amino acid sequences, we show the Comassie blue staining of proteins upon elution from affinity chromatography and polyacrylamide gel electrophoresis. Relevant molecular weight markers are indicated. At the bottom, the molecular weights of the whole constructs as determined by matrix-assisted laser desorption/ ionization time of flight mass spectrometry. (b) Field emission scanning electron microscopy images of purified protein, showing their nanoarchitecture. Particles were diluted in two buffers, in which nanoparticles were tested for stability, namely carbonate buffer (C) and carbonate buffer plus 333 mmol L−1 NaCl (C+S). Bar size is 20 nm in all panels. (c) Dynamic light scattering plots showing the hydrodynamic size of nanoparticles. The peak value and the polydispersion index (Pdi) are indicated. Determinations were done on the material dissolved in buffer C and C+S. (d) The hydrodynamic size of the particles in these buffers was also determined in presence of 10% BSA and in Optipro cell culture medium. SDS (at 1%), that promotes the disassembling of protein-only nanoparticles was alternatively added to the buffer to identify the size of the building blocks. The size of the parental GFP-H6 is also indicated in nm. Untreated nanoparticles are shown by coloured plots. All the experiments were performed at pH 8. The peak value of the samples in SDS, BSA and Optipro are specified over the respective plots.

  • Figure 2

    Cell penetrability of CTX-based nanoparticles. (a) Internalized nanoparticles in two alternative cell lines, namely HeLa and U87MG cells, 24 h after exposure to different protein amounts. Intracellular fluorescence was corrected by the specific emission to result in data representative of protein amounts. Cells were submitted to a harsh trypsin treatment before measurements to remove externally attached protein as described [26]. Nanoparticles were administered as dissolved in either C or C+S buffer. Y axis scales might be not precisely comparable. (b) Selective antibody-mediated inhibition of nanoparticle uptake in HeLa cells, by an anti-annexin-2 monoclonal antibody (mAb) and polyclonal antibody (pAb). The statistical analysis was performed using an ANOVA Tukey’s multiple comparisons test (*p<0.05; **p<0.01). Normality was confirmed by Shapiro-Wilk W where p ˃ 0.05. Comparisons were done always with samples without antibody. (c) Internalization of the related CXCR4-binding T22-GFP-H6 nanoparticles in (CXCR4+) HeLa cells, and inhibition by the CXCR4 antagonist AMD3100 at an excess molar ratio 10:1. The parental GFP-H6 protein is unable to enter cultured cells. All the experiments using HeLa cells were performed at pH 7.0–7.4, and those using U87MG cells at pH 6.8–7.2.

  • Figure 3

    Cell viability upon exposure to CTX-based nanoparticles. HeLa cells (a) and U87MG cells (b) were exposed to protein nanoparticles for 72 h. Nanoparticles were administered as dissolved in either C or C+S buffer. The statistical analysis was performed using an ANOVA Tukey’s multiple comparisons test (*p<0.05; **p<0.01). Normality was confirmed by Shapiro-Wilk W where p˃0.05. Symbols at the top of the bars indicate the comparison with the control (100%). Symbols at the left of the bars indicate comparisons between protein pairs, indicated by white linkers. (c) HeLa cell viability upon exposure to control, non-toxic GFP-H6 protein and cytotoxic T22-PE24-H6 nanoparticles. All the experiments using HeLa cells were performed at pH 7.0–7.4, and those using U87MG cells at pH 6.8–7.2.


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