SUPPORTING INFORMATION




   Engineering a Nanostructured Nucleolin-
Binding Peptide for Intracellular Drug Delivery
 in Triple-Negative Breast Cancer Stem Cells
    Mireia Pesarrodona∞#‡, Laura Sánchez-García#∞ƒ‡, Joaquin Seras-Franzoso¥,
Alejandro Sánchez-Chardi§, Ricardo Baltà-Foix¥†, Patricia Cámara-Sánchez#¥∂, Petra
  Gener#¥, José Juan Jara#+, Daniel Pulido#+∆, Naroa Serna∞#, Simó Schwartz Jr#¥,
  Miriam Royo#+∆, Antonio Villaverde∞# ƒ*, Ibane Abasolo#¥∂*, Esther Vázquez∞†#ƒ.

  ∞ Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona,
                          Bellaterra, 08193 Barcelona, Spain

# CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), C/ Monforte
                        de Lemos 3-5, 28029 Madrid, Spain

 ƒ Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona,
                       Bellaterra, 08193 Barcelona, Spain

   ¥ Drug Delivery & Targeting CIBBIM-Nanomedicine, Vall d'Hebron Institut de
    Recerca (VHIR), Universitat Autònoma de Barcelona, 08035 Barcelona, Spain



  § Departament de Biologia Evolut iva, Ecologia i Ciències Ambientals, Facultat de
            Biologia, Universitat de Barcelona, 08028 Barcelona, Spain

∂ Functional Validation & Preclinical Research, CIBBIM-Nanomedicine, Vall d'Hebron
  Institut de Recerca (VHIR), Universitat Autònoma de Barcelona, 08035 Barcelona,
                                        Spain

 + Combinatorial Chemistry Unit, Barcelona Science Park, Universitat de Barcelona,
                            08028 Barcelona, Spain

    ∆ Multivalent Systems for Nanomedicine, Institute for Advanced Chemistry of
                  Catalonia (IQAC-CSIC), Barcelona, 08034, Spain



Corresponding Author

* AV antoni.villaverde@uab.cat; IA ibane.abasolo@vhir.org




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Figure S1. Fluorochrome labeling of peptides. A) An Ahx spacer was added between
the fluorophore (5(6)-carboxyfluorescein, CF) and the peptide, whenever the
internalization assays showed that the fluorescence obtained was higher than with the
direct linkage. B) Examples in the differences in cell internalization results between
peptides with or without Ahx spacer. In detail, Hv6pep showed no difference between
the versions with our without Ahx, whereas sCH9 and P17 rendered higher number of
positive cells for CF in flow cytometry experiments. Therefore, for testing the capability
of different versions, Ahx was only included when these preliminary assays showed
better internalization. Assays were conducted in low attachment conditions in MDA-
MB-231 cells.




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Figure S2. Matrix-Assisted Laser Desorption/Ionization (MALDI) of all the recombinant
proteins targeted towards breast cancer produced and characterized in this work.




                                                                                  S3
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Figure S3. F3RK-GFP-H6 internalization in MDA-MB-231 cells. Three µM of F3RK-GFP-
H6 were added and incubated ON. Cell medium was removed and cells were stained
with 5 µg/mL Cell MaskTM (Invitrogen) and DAPI and fixed in 4% paraformaldehyde
(PFA). Image acquisition was carried out in a spectral confocal microscopy FV1000
(Olympus) with a PLAPON 60XO objective. Z projections sections were acquired with a
step size between 0.4 and 0.6 µm and further reconstructed using Image J. Green dots
in the perinuclear area correspond to F3RK-GFP-H6 peptide constructs, clearly
localized inside the cells as shown by cross sections (bottom and right).




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