the Agencia Estatal de Investigación(AEI)
Fondo Europeo de Desarrollo Regional(FEDER)
AGAUR(2017SGR-229)
CIBER-BBN(project,VENOM4CANCER)
ISCIII(PI15/00272,co-founding,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 (
The authors declare no conflict of interest.
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.
Supplementary information Experimental details are available in the online version of the paper.
[1] Serna N, Sánchez-García L, Unzueta U, et al. Protein-based therapeutic killing for cancer therapies. Trends Biotech, 2018, 36: 318-335 CrossRef PubMed Google Scholar
[2] DeBin JA, Maggio JE, Strichartz GR. Purification and characterization of chlorotoxin, a chloride channel ligand from the venom of the scorpion. Am J Physiol-Cell Physiol, 1993, 264: C361-C369 CrossRef PubMed Google Scholar
[3] DeBin JA, Strichartz GR. Chloride channel inhibition by the venom of the scorpion Leiurus quinquestriatus. Toxicon, 1991, 29: 1403-1408 CrossRef Google Scholar
[4] Veiseh M, Gabikian P, Bahrami SB, et al. Tumor paint: a chlorotoxin:Cy5.5 bioconjugate for intraoperative visualization of cancer foci. Cancer Res, 2007, 67: 6882-6888 CrossRef PubMed Google Scholar
[5]
Deshane
J,
Garner
CC,
Sontheimer
H.
Chlorotoxin inhibits glioma cell invasion
[6] Xu T, Fan Z, Li W, et al. Identification of two novel chlorotoxin derivatives CA4 and CTX-23 with chemotherapeutic and anti-angiogenic potential. Sci Rep, 2016, 6: 19799 CrossRef PubMed ADS Google Scholar
[7] Ojeda PG, Henriques ST, Pan Y, et al. Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake. Biopolymers, 2017, 108: e23025 CrossRef PubMed Google Scholar
[8] Mamelak AN, Jacoby DB. Targeted delivery of antitumoral therapy to glioma and other malignancies with synthetic chlorotoxin (TM-601). Expert Opin Drug Deliver, 2007, 4: 175-186 CrossRef PubMed Google Scholar
[9]
Kasai
T,
Nakamura
K,
Vaidyanath
A, et al.
Chlorotoxin fused to IgG-Fc inhibits glioblastoma cell motility
[10] Vazquez E, Mangues R, Villaverde A. Functional recruitment for drug delivery through protein-based nanotechnologies. Nanomedicine, 2016, 11: 1333-1336 CrossRef PubMed Google Scholar
[11] Rueda F, Céspedes MV, Conchillo-Solé O, et al. Bottom-up instructive quality control in the biofabrication of smart protein materials. Adv Mater, 2015, 27: 7816-7822 CrossRef PubMed Google Scholar
[12] Serna N, Céspedes MV, Sánchez-García L, et al. Peptide-based nanostructured materials with intrinsic proapoptotic activities in CXCR4+ solid tumors. Adv Funct Mater, 2017, 27: 1700919 CrossRef Google Scholar
[13] Sánchez-García L, Serna N, Álamo P, et al. Self-assembling toxin-based nanoparticles as self-delivered antitumoral drugs. J Control Release, 2018, 274: 81-92 CrossRef PubMed Google Scholar
[14] Díaz R, Pallarès V, Cano-Garrido O, et al. Selective CXCR4+ cancer cell targeting and potent antineoplastic effect by a nanostructured version of recombinant ricin. Small, 2018, 14: 1800665 CrossRef PubMed Google Scholar
[15] Céspedes MV, Unzueta U, Aviñó A, et al. Selective depletion of metastatic stem cells as therapy for human colorectal cancer. EMBO Mol Med, 2018, 10: e8772 CrossRef PubMed Google Scholar
[16] Unzueta U, Ferrer-Miralles N, Cedano J, et al. Non-amyloidogenic peptide tags for the regulatable self-assembling of protein-only nanoparticles. Biomaterials, 2012, 33: 8714-8722 CrossRef PubMed Google Scholar
[17] Sánchez JM, Sánchez-García L, Pesarrodona M, et al. Conformational conversion during controlled oligomerization into nonamylogenic protein nanoparticles. Biomacromolecules, 2018, 19: 3788-3797 CrossRef PubMed Google Scholar
[18]
Céspedes
MV,
Unzueta
U,
Tatkiewicz
W, et al.
[19] Unzueta U, Céspedes MV, Ferrer-Miralles N, et al. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles. Int J Nanomedicine, 2012, 7: 4533-4544 CrossRef PubMed Google Scholar
[20] Serna N, Céspedes MV, Saccardo P, et al. Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles. NanoMed-Nanotechnol Biol Med, 2016, 12: 1241-1251 CrossRef PubMed Google Scholar
[21]
Locatelli
E,
Naddaka
M,
Uboldi
C, et al.
Targeted delivery of silver nanoparticles and alisertib:
[22] Graf N, Mokhtari TE, Papayannopoulos IA, et al. Platinum(IV)-chlorotoxin (CTX) conjugates for targeting cancer cells. J Inorg Biochem, 2012, 110: 58-63 CrossRef PubMed Google Scholar
[23] Asphahani F, Wang K, Thein M, et al. Single-cell bioelectrical impedance platform for monitoring cellular response to drug treatment. Phys Biol, 2011, 8: 015006 CrossRef PubMed ADS Google Scholar
[24] Locatelli E, Broggi F, Ponti J, et al. Lipophilic silver nanoparticles and their polymeric entrapment into targeted-PEG-based micelles for the treatment of glioblastoma. Adv Healthcare Mater, 2012, 1: 342-347 CrossRef PubMed Google Scholar
[25] Poty S, Désogère P, Goze C, et al. New AMD3100 derivatives for CXCR4 chemokine receptor targeted molecular imaging studies: synthesis, anti-HIV-1 evaluation and binding affinities. Dalton Trans, 2015, 44: 5004-5016 CrossRef PubMed Google Scholar
[26] Richard JP, Melikov K, Vives E, et al. Cell-penetrating peptides. J Biol Chem, 2003, 278: 585-590 CrossRef PubMed Google Scholar
[27] Dardevet L, Rani D, Aziz TAE, et al. Chlorotoxin: a helpful natural scorpion peptide to diagnose glioma and fight tumor invasion. Toxins, 2015, 7: 1079-1101 CrossRef PubMed Google Scholar
[28] Allen M, Bjerke M, Edlund H, et al. Origin of the U87MG glioma cell line: Good news and bad news. Sci Transl Med, 2016, 8: 354re3 CrossRef PubMed Google Scholar
[29] Shen J, Wolfram J, Ferrari M, et al. Taking the vehicle out of drug delivery. Mater Today, 2017, 20: 95-97 CrossRef PubMed Google Scholar
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
Figure 2
Cell penetrability of CTX-based nanoparticles. (a) Internalized nanoparticles in two alternative cell lines, namely HeLa and U87MG cells,
Figure 3
Cell viability upon exposure to CTX-based nanoparticles. HeLa cells (a) and U87MG cells (b) were exposed to protein nanoparticles for