Research Article
Switching cell penetrating and CXCR4-binding activities of nanoscale-organized arginine-rich peptides

https://doi.org/10.1016/j.nano.2018.05.002Get rights and content

Abstract

Arginine-rich protein motifs have been described as potent cell-penetrating peptides (CPPs) but also as rather specific ligands of the cell surface chemokine receptor CXCR4, involved in the infection by the human immunodeficiency virus (HIV). Polyarginines are commonly used to functionalize nanoscale vehicles for gene therapy and drug delivery, aimed to enhance cell penetrability of the therapeutic cargo. However, under which conditions these peptides do act as either unspecific or specific ligands is unknown. We have here explored the cell penetrability of differently charged polyarginines in two alternative presentations, namely as unassembled fusion proteins or assembled in multimeric protein nanoparticles. By this, we have observed that arginine-rich peptides switch between receptor-mediated and receptor-independent mechanisms of cell penetration. The relative weight of these activities is determined by the electrostatic charge of the construct and the oligomerization status of the nanoscale material, both regulatable by conventional protein engineering approaches.

Graphical Abstract

Polyarginines exhibit dual and alternating CXCR4-dependent and CXCR4-independent cell penetrating activities when displayed on nanoscale entities.

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Section snippets

Proteins and protein production

Four GFP-derived fusion proteins, namely R3-GFP-H6, R6-GFP-H6, R7-GFP-H6 and R9-GFP-H611 were used in the present study upon recombinant production in bacteria. As previously described, R9-GFP-H6 was modified by directed mutagenesis to generate the other constructions, replacing arginines by glycines and alanines, in order to maintain the same length of peptide tag with different charges.11 All of the fusion proteins are based on the same modular scheme (Figure 1, A), in which the cationic

Results

R3-GFP-H6, R6-GFP-H6, R7-GFP-H6 and R9-GFP-H6 (Figure 1, A) were successfully bio-produced in E. coli (Figure 1B) and stored in either Tris Dextrose buffer (named as Dextrose in the Figures) or Tris NaCl buffer (named as NaCl in the Figures), depending on their solubility. R3 and R6 derivatives were preferentially soluble in Tris NaCl buffer, R9 in Tris Dextrose buffer and R7 was found to be soluble in both (data not shown). All produced proteins were fluorescent, although with important

Discussion

The delivery of therapeutic molecules into cells requires the smart engineering of fusogenic agents, mainly lipids22, 23 and proteins,24, 25 that act as unspecific but highly efficient cell penetrating agents. Alternatively, cell-targeting tools, such as antibodies or peptidic ligands, provide selectivity in the cell binding of drug vehicles,26, 27 although they are generically less competent than unspecific CPP tools in promoting internalization.28, 29 The still poorly explored combination of

Acknowledgment

Protein production has been partially performed by the ICTS “NANBIOSIS”, more specifically by the Protein Production Platform of CIBER-BBN/ IBB, at the UAB SepBioES scientific-technical service (http://www.nanbiosis.es/unit/u1-protein-production-platform-ppp/). Nanoparticle size determination was performed at the NANBIOSIS Biomaterial Processing and Nanostructuring Unit of CIBER-BBN (http://www.nanbiosis.es/portfolio/u6-biomaterial-processing-and-nanostructuring-unit). We appreciate the generic

References (42)

  • J. Ye et al.

    15 years of ATTEMPTS: a macromolecular drug delivery system based on the CPP-mediated intracellular drug delivery and antibody targeting

    J Control Release

    (2015)
  • E. Vazquez et al.

    Peptide-assisted traffic engineering for nonviral gene therapy

    Drug Discov Today

    (2008)
  • N. Ferrer-Miralles et al.

    Membrane-active peptides for non-viral gene therapy: making the safest easier

    Trends Biotechnol

    (2008)
  • G. Guidotti et al.

    Cell-penetrating peptides: from basic research to clinics

    Trends Pharmacol Sci

    (2017)
  • Z. Niu et al.

    Rational design of polyarginine nanocapsules intended to help peptides overcoming intestinal barriers

    J Control Release

    (2017)
  • Y. Kawaguchi et al.

    Syndecan-4 is a receptor for Clathrin-mediated endocytosis of arginine-rich cell-penetrating peptides

    Bioconjug Chem

    (2016)
  • M.V. Cespedes et al.

    Cancer-specific uptake of a liganded protein nanocarrier targeting aggressive CXCR4+ colorectal cancer models

    Nanomedicine

    (2016)
  • T. Murakami et al.

    Chemokine receptors and melanoma metastasis

    J Dermatol Sci

    (2004)
  • R.E. Kontermann et al.

    Bispecific antibodies

    Drug Discov Today

    (2015)
  • U.H. Weidle et al.

    Tumor-antigen-binding bispecific antibodies for cancer treatment

    Semin Oncol

    (2014)
  • F. Barbieri et al.

    Role of chemokine network in the development and progression of ovarian cancer: a potential novel pharmacological target

    J Oncol

    (2010)
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    We are indebted to Agencia Estatal de Investigación (AEI) and to Fondo Europeo de Desarrollo Regional (FEDER) (grant BIO2016-76063-R, AEI/FEDER, UE), AGAUR (2017 SGR-229) and CIBER de Bioingeniería, Biomateriales y Nanomedicina (project NANOPROTHER) for funding AV. Also, to Marató de TV3 foundation (TV32013-3930) and ISCIII (PI15/00272 co-founding FEDER) granted to EV and ISCIII (PI15/00378 and PIE15/00028, co-founding FEDER), Marató TV3 (2013-2030) and AGAUR 2014-PROD0005 granted to RM, for funding research on targeted, protein-based drug delivery. MTPF received a fellowship from Fundação de Amparo a Pesquisa do Estado de São Paulo, Brazil (2015/20193-3), LSG was supported by AGAUR (2017FI_B100063), NS by a predoctoral fellowship from the Government of Navarra and UU received a Sara Borrell postdoctoral fellowship from ISCIII. AV received an Icrea Academia Award.

    The authors do not appreciate any conflict of interest.

    1

    Equally contributed.

    2

    Present address: Hospital Sant Joan de Déu, Passeig de Sant Joan de Déu, 2, 08950 Esplugues de Llobregat, Barcelona.

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