Research ArticleSwitching cell penetrating and CXCR4-binding activities of nanoscale-organized arginine-rich peptides
Graphical Abstract
Polyarginines exhibit dual and alternating CXCR4-dependent and CXCR4-independent cell penetrating activities when displayed on nanoscale entities.
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
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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.