Engineering a recombinant chlorotoxin as cell-targeted cytotoxic nanoparticles 1, 2, 3 1, 2, 3 1, 2, 3 Raquel Díaz , Laura Sánchez-Garcia , Naroa Serna , Alejandro Sánchez- Chardi 4, Olivia Cano-Garrido 1, 2, 3 §, Julieta M. Sánchez 1, 2, 5, Ugutz Unzueta 2, 6 *, Esther Vázquez 1, 2, 3, Antonio Villaverde 1, 2, 3 * 1 Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain 2 Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain 3 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193 Barcelona, Spain 4 Servei de Microscòpia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain 5 Universidad Nacional de Córdoba, Facultad de Ciencias Exactas, Físicas y Naturales. ICTA and Departamento de Química, Cátedra de Química Biológica. Córdoba. Argentina. CONICET, Instituto de Investigaciones Biológicas y Tecnológicas (IIBYT), Córdoba, Argentina. Av. Velez Sarsfield 1611, X5016GCA Córdoba, Argentina 6 Biomedical Research InstituteSant Pau (IIB-Sant Pau) and Josep Carreras Research Institute, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain § Present address: Biomedical Research InstituteSant Pau (IIB-Sant Pau) and Josep Carreras Research Institute, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain * Corresponding authors: UU uunzueta@santpau.cat; AV antoni.villaverde@uab.cat Material and methods Protein design and gene cloning Two closely related fusion proteins (Figure 1) were designed to expose CTX at the N- terminus, siding the enhanced fluorescent protein GFP and with a hexahistidine tail at the C-terminus. CTX-GFP-H6 was based on a plain CTX. In CTX-KRKRK-GFP-H6, five intercalated lysines and arginines were intersected between CTX and GFP to enhance the cationic nature of the fusion protein at its N-terminus. A high cationic load in the N- terminal end, in combination with a histidine-rich C-terminus, promotes spontaneously self-assembling of multidomain polypeptides in form of stable, nanoscale oligomers [1]. The genes were inserted in pET22b-derived plasmids and generated by Gene Art. The unassembled parental protein GFP-H6, and the self-assembling CXCR4-targeted T22- GFP-H6 versions have been described elsewhere [2, 3] and have been used as controls. T22-PE24-H6 is a potent cytotoxic protein nanoparticle targeted to CXCR4 [4], that has been also included in some experiments. Bacterial cell transformation and testing of culture conditions Recombinant plasmids were dissolved in type 1 ultrapure water (Millipore) at a concentration of 100 ng/µL. Escherichi coli Origami B was chosen for protein production as it promotes formation of disulphide bridges in recombinant proteins, as the ones contained in CTX. A 0.5 µg of each plasmid (coding CTX-GFP-H6 and CTX-KRKRK- GFP-K-H6 respectively) was used to transform competent cells. After incubation under ice for 1 h, the mixtures received a thermal shock at 42 ºC for 45 s. and were placed on ice for 30 s. Immediately, 850 µL of Lysogenic broth (LB) media were added, and transformed cells were incubated at 37 °C for 1 h. Finally, cells were plated on LB-agar plates with ampicillin, kanamycin and tetracycline according to standard procedures [5]. Four different protein production conditions were tested, regarding cell growth temperature, gene expression time, shaking rate and concentration of isopropyl-β- thiogalactopyronaside (IPTG). They were 37 ºC, 3 h, 0.1 mM IPTG; 37 ºC, 3 h, 1 mM IPTG; 20 ºC, overnight, 0.1 mM IPTG; 20 ºC, overnight, 1 mM IPTG, all of them at shaking rate of 250 rpm. After gene expression during the set times, cultures were centrifuged at 5000 g for 15 min. at 4 ºC, and the cell pellets stored at -80 ºC until disruption by sonication in a Branson ultrasonics sonifier (2 rounds at 10 % amplitude, 1 round at 15 % amplitude; 10 min. per round with intervals of 0.5 s each). Cell extracts were dissolved in phosphate buffered saline (PBS) to equal bacterial concentration determined by the optical density (O D550) and further submitted to SDS electrophoresis on TGX Stain-Free gels (Bio-Rad). Recombinant CTXs were immunodetected by Western blotting using an anti-His monoclonal antibody (Santa Cruz Biotechnology). Protein production and purification Inoculums of transformed bacteria were grown overnight in 100 mL of LB medium with 100 µg/mL of ampicillin, 15 µg/mL of kanamycin and 12.5 µg/mL of tetracycline, at 37 ºC and at 250 rpm. Then, flasks of 500 mL of the same medium were re-inoculated to an OD between 0.05 and 0.1 and further incubated at 37 ºC and 250 rpm. 0.1 mM IPTG was added to cultures when an optical density (OD550) between 0.5 and 0.7 was reached to induce the recombinant gene expression. Then, flasks were incubated overnight at 20 ºC and 250 rpm. Cells were harvested and centrifuged (5000 g, 15 min, 4 ºC). The cell pellets were resuspended in Wash Buffer (20 mM Tris-HCl, 500 mM NaCl and 10 mM imidazole, pH 8) in the presence of protease inhibitor cocktail Complete EDTA-Free (Roche). Bacterial cells were sonicated and the extracts centrifuged (15000 g, 45 min, 4 ºC). Recombinant proteins in the soluble cell fraction were purified by His-tag affinity chromatography with a 1 mL HiTrap Chelating column in an AKTA purifier FPLC (GE Healthcare). After the samples were filtered (0.22 µm) and injected into the column, the fractions to be collected for both proteins were eluted from 20-25 % Elution Buffer (20 mM Tris-HCl, 500 mM NaCl and 500 mM imidazole) onwards. Then, purified proteins were dialyzed using a molecular porous membrane tubing (12-14 kDa, Spectrumlabs) in two alternative buffers; Carbonate Buffer (166 mM NaCO3H) and Carbonate-salt buffer (166 mM NaCO3H, 333 mM NaCl). Buffer exchange was done in three steps; 2 h at room temperature followed by a further dialysis overnight at 4 ºC and 2 h at room temperature. The amount of protein was determined by Bradford’s assay, and the integrity of the protein was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blot analysis and Mass Spectrometry as described below. After dialyzing in both buffers, each protein was recovered, filtered through 0.22 µm filters under sterile conditions and frozen at -80 ºC. Western Blot Proteins were analyzed by SDS-PAGE on TGX Stain-Free Gels (Bio-Rad). The samples were mixed in denaturing buffer (0.53 M Tris Base, 5.52 M glycerol, 0.27 M sodium dodecyl sulphate, 2.84 M β-mercaptoethanol, 7.99 M urea) at a ratio 3:1 and boiled at 96 ºC for 10 min. For Western Blot, protein was transferred to a polyvinylidene difluoride immune-blot membrane (Bio-Rad) and blocked overnight with 5 % skim milk in PBS at 4 ºC. An anti-His monoclonal antibody was used (1:500, Santa Cruz Biotechnology) followed by incubation with the goat anti mouse IgG (H+L)-HRP secondary antibody conjugate (1:1000, Bio-Rad). Protein bands were observed using the clarity ECL western blotting substrates (Bio-Rad) in a ChemiDoc Touch Imaging System (Bio-Rad). Mass Spectrometry A volume of 5 µL of the purified protein samples was dialyzed against type 1 ultrapure water in Millipore membranes for 20 min. Other 0.5 μL of the dialyzed samples were spotted on a MALDI plate with sinapinic acid (SA) and analyzed using a MALDI- TOF/TOF mass spectrometer (Bruker Daltonics Ultraflex TOF/TOF) in lineal mode, in the range 20-80 kDa. Particle size and fluorescence Volume size distribution of nanoparticles was measured by dynamic light scattering (DLS) at 633 nm (Zetasizer Nano ZS, Malvern Instruments Limited). Protein concentration was 1 mg/ml as determined by a conventional Bradford’s colorimetric assay. The specific fluorescence of the particles was measured by triplicate at an excitation wavelength of 488 nm and emission wavelength range between 500 and 550 nm in a Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies). The chamber excite-slit was set on 2.5 nm while the emit-slit on 5 nm. Data measurements were recovered at 510 nm. Field Emission Scanning Electron Microscopy Geometry (size and shape) of nanoparticles was characterized at ultrastructural level with a rapid method using field emission scanning electron microscopy (FESEM), to obtain a high resolution imaging of protein nanoconstructs at a nearly native state. Drops of 3 µL of each sample of nanoparticles diluted at 0.25 mg/mL were directly deposited on silicon wafers (Ted Pella Inc) for 30 s, excess of liquid was blotted with Whatman filter paper number 1 (GE Healthcare), air dried for few min, and immediately observed without coating in a FESEM Zeiss Merlin (Zeiss) operating at 1 kV and equipped with a high resolution in-lens secondary electron detector. For each sample, representative images of different fields were captured at high magnifications (from 200,000x to 700,000x). Cell viability The cytotoxicity of the recombinant CTX was tested in HeLa cell line (ATCC-CCL-2, as a model of annexin-2-overexpressing cells) and in U87MG cell line (as a model of MMP- 2-overexpressing cells) at two different doses (100 nM and 1 µM). Cells were cultured at 37 ºC in a 5 % CO2 for HeLa cells and in a 10 % CO2 for U87MG cells humidified atmosphere in MEM-Alpha media supplemented with 10% foetal calf serum (Gibco). When a confluence of 70 % was reached, cells were seeded in an opaque 96-well plate (3x104 cells/well) for 24 h. The next day, CTX versions were added to a final volume of 100 µL/well, and cells were incubated during 48 h and 72 h. Cell viability was determined by CellTiterGlo Luminescent Cell Viability Assay (Promega) in a Multilabel Plater Reader Victor3 (Perkin Elmer). Cell internalization Uptake of recombinant CTX proteins was monitored in both HeLa and U87MG cells, both targeted by CTX [6-8]. HeLa and U87MG cells were cultured at 37 °C for 24 h and plated (3x104cells/well and 6.5x104 cells/well respectively) in two treated 24 well plates (Nunclon surface, Nunc 150628) with MEM-Alpha media supplemented with 10 % foetal bovine serum and 2 mM Glutamax (Gibco). Later, medium was removed and the cells were washed with Dulbecco's phosphate-buffered saline (DPBS). Then, CTX proteins in 250 µL Optipro supplemented with 2 mM L-glutamine were added. After incubation at 37 °C for 2 or 24 h, cells were harshly treated with trypsin in DPBS (1 mg/mL) for 15 min. and centrifuged at 1200 rpm for 15 min. Samples were analyzed by flow cytometry using a FACSCanto system (Becton–Dickinson) with a 15 W air-cooled argon-ion laser at 488 nm excitation for GFP. The cell uptake of T22-GFP-H6 was inhibited by the CXCR4 antagonist AMD3100, upon 1 h incubation at a molar excess ratio of 10:1 as described [9]. Specificity in cell binding Protein internalization over cultured HeLa cells, expressing annexin-2, was inhibited with the anti-annexin-A2 (D11G2, rabbit, Cell signal) monoclonal antibody at a 0.1025 µg/well and with the anti-annexin-A2 (AA 180-230) polyclonal antibody at 0.06 µg/well, both incubated 1h before protein incorporation. Then, CTX proteins in Optipro supplemented with 2 mM L-glutamine were added at 2 µM final concentration. After incubation at 37 °C for 2 or 24 h, cells were treated with trypsin in DPBS (1 mg/mL) for 15 min, centrifuged at 1200 rpm for 15 min and resuspended in 300 µL DPBS. Samples were analyzed by flow cytometry using a FACSCanto system (Becton–Dickinson) with a 15 W air-cooled argon-ion laser at 488 nm excitation for GFP. Statistical analysis Quantitative data are expressed as mean ± standard error (SE). Previously to perform statistical analyses, all variables were tested for normality and homogeneity of variances employing the Shapiro–Wilk and the Levene test, respectively. Pairwise comparisons were made with Tukey’s test. Significance was accepted at p < 0.05 and relevant differences were expressed as * p < 0.05 or ** p < 0.01. References [1] Unzueta U, Ferrer-Miralles N, Cedano J, Zikung X, Pesarrodona M, Saccardo P, et al. Non-amyloidogenic peptide tags for the regulatable self-assembling of protein-only nanoparticles. Biomaterials. 2012;33:8714-22. [2] Unzueta U, Cespedes MV, Ferrer-Miralles N, Casanova I, Cedano J, Corchero JL, et al. Intracellular CXCR4(+) cell targeting with T22-empowered protein-only nanoparticles. International journal of nanomedicine. 2012;7:4533-44. [3] Cespedes MV, Unzueta U, Avino A, Gallardo A, Alamo P, Sala R, et al. Selective depletion of metastatic stem cells as therapy for human colorectal cancer. EMBO molecular medicine. 2018. [4] Sanchez-Garcia L, Serna N, Alamo P, Sala R, Cespedes MV, Roldan M, et al. Self- assembling toxin-based nanoparticles as self-delivered antitumoral drugs. Journal of controlled release : official journal of the Controlled Release Society. 2018;274:81-92. [5] J. Sambrook EFF, T. Maniatis. Molecular cloning : a laboratory manual Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory Press; 1989. [6] Asphahani F, Wang K, Thein M, Veiseh O, Yung S, Xu J, et al. Single-cell bioelectrical impedance platform for monitoring cellular response to drug treatment. Physical biology. 2011;8:015006. [7] Locatelli E, Broggi F, Ponti J, Marmorato P, Franchini F, Lena S, et al. Lipophilic silver nanoparticles and their polymeric entrapment into targeted-PEG-based micelles for the treatment of glioblastoma. Advanced healthcare materials. 2012;1:342-7. [8] Locatelli E, Naddaka M, Uboldi C, Loudos G, Fragogeorgi E, Molinari V, et al. Targeted delivery of silver nanoparticles and alisertib: in vitro and in vivo synergistic effect against glioblastoma. Nanomedicine. 2014;9:839-49. [9] Sanchez-Garcia L, Serna N, Mattanovich M, Cazzanelli P, Sanchez-Chardi A, Conchillo-Sole O, et al. The fusogenic peptide HA2 impairs selectivity of CXCR4- targeted protein nanoparticles. Chemical communications. 2017;53:4565-8.