Oliver Sandre¹, Gauvin Hémery¹, Emmanuel Ibarboure¹, Elisabeth Garanger¹, Sébastien Lecommandoux¹, Pauline Jeanjean², Coralie Genevois², Franck Couillaud², Sabrina Lacomme³, Etienne Gontier³, Ashutosh Chilkoti⁴

1 LCPO UMR5629 Univ Bordeaux, CNRS, Bordeaux INP, ENSCBP, Pessac, France

² IMOTION EA7435 Univ Bordeaux, Bordeaux, France.

³ BIC UMS3420 Univ Bordeaux, CNRS, Inserm, Bordeaux, France.

⁴ Biomedical Engineering, Duke University, Durham, NC, United States.

This communication reports the grafting onto iron oxide nanoparticles (IONPs) of recombinant polypeptides made of di-block elastin-like peptide (ELP_40-60) and cell-penetrating peptide (Tat) sequence.[1]The ELP₄₀ block is thermosensitive and undergoes a water de-swelling transition at a critical temperature around 42 °C in solution, the ELP₆₀ block is hydrophilic and provides colloidal stability to the resulting γ-Fe₂O₃@ELP_40-60-Tat core-shell IONPs. Magnetic IONPs were synthesized by a polyol pathway with either monocore (nanospheres) or multi-core (nanoflowers) morphology, narrow size-dispersity and suitable heating efficiency under an alternating magnetic field (AMF).[2] The bio-functionalization of these IONPs with the di-block ELP_40-60-Tat was achieved by a convergent strategy through strong coordination bonding of a phosphonate group introduced near the N-terminus of the polypeptide. To the best of our knowledge, this is the first report on a thermosensitive ELP_m-n polypeptide brush grafting onto magnetic IONPs. Large temperature variations of the sample (up to 30 °C) could be obtained in a few minutes by applying an AMF. Fast size changes of the magnetic core-thermosensitive shell nanoparticles were measured by in situ dynamic light-scattering (DLS) while the AMF was on. Variations of the hydrodynamic size were compared to the classical polymer brush model revised for the highly curved surface of nanoparticles. Cellular internalization and toxicity assays were performed on a glioblastoma (U87) human cancer cell line in view of applications for drug delivery activated magnetically. Superior cellular uptake was observed in vitro for multicore IONPs compared to monocore IONPs (for the same PEG coating),[3] and for IONPs@ELP_40-60-Tat peptide-grafted nanoparticles compared to IONPs@PEG controls prepared from the same (spherical) cores. The internalization pathway in lysosomes was monitored by electron microscopy on microtomes and confocal optical microscopy on live cells. Cellular toxicity after AMF application with these core-shell IONPs was ascribed to lysosomal membrane rupture and leakage into the cytosol. The intra-cellular fate of such IONPs, from their internalization to the effect of an AMF application, validates the use of thermosensitive peptide brushes on IONPs as drug delivery systems, addressing lysosomal compartments and triggering leakage of their content by external AMF application. Preliminary in vivo experiments evidenced the positive effect of the Tat peptide end-sequence compared to the PEG brush control on the bio-distribution, with similar contents in the liver and in U87 model tumor in mice. Long term fate (after 48 h) is discussed in view of the cell division with equal sharing of the magnetically loaded lysosomes among daughter cells, possibly envisioning the successive application of magnetic hyperthermia on time scales superior to the cellular life cycle

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[1] E Garanger, S MacEwan, O Sandre, A Brûlet, L Bataille, A Chilkoti, S Lecommandoux, Macromol. 2015, 48, 6617

[2] G Hemery, A Keyes, E Garaio, I Rodrigo, J A Garcia, F Plazaola, E Garanger, O Sandre, Inorg. Chem. 2017, 56, 8232

[3] G Hemery, C Genevois, F Couillaud, S Lacomme, E Gontier, E Ibarboure, S Lecommandoux, E Garanger, O Sandre, Molecular Systems Design & Engineering 2017, 2 629