Quelques réflexions sur la typochronologie de la sculpture anthropomorphe de l’âge du Fer à partir des têtes de Arzheim et Freinsheim (Allemagne/Rhénanie-Palatinat)

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Nanostructures for delivery of therapeutic oligonucleotides

Fluoropyrimidines, such as 5-fluorouracil (5-FU) and related prodrugs are considered one of the most-successful agents in the treatment of colorectal cancer, yet poor specificity and tumor cell resistance remain major limiting bottlenecks. In this work, we exploited the ability of two DNA nanostructures, DNA tetrahedron (Td) and rectangle DNA origami, to incorporate 5-fluoro-2¿-deoxyuridine (FdUn) oligomers. DNA nanostructures bearing FdUn revealed to be able to circumvent 5-FU low sensitivity of colorectal drug-resistant cancer cells. Both DNA nanostructures attained comparable cytotoxic effect yet Td displays higher antiproliferative action. DNA nanoscaffolds functionalized with FdUn exhibited an enhanced cytotoxicity and higher ability to trigger apoptosis in colorectal cancer cells relative to conventional 5-FU and FdU drugs, especially having cholesterol as internalization helper. In addition, we will describe the preparation of a protein nanoparticle carrying FdUn oligomers with high affinity for cancer stem cells preventing the formation of metastasis in mice. The present work shows that DNA and protein nanoparticles are privileged carriers for delivering fluoropyrimidines, opening new avenues to the development of promising therapeutics for cancer treatment

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

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.Peer ReviewedPostprint (author's final draft

Formulating tumor-homing peptides as regular nanoparticles enhances receptor-mediated cell penetrability

The authors acknowledge the financial support granted to E.V. (PI12/00327) and R.M. (PI12/01861) from FIS, to E.V. (TV32013-133930) and to R.M. and A.V. (TV32013-132031) from La Marató de TV3 (416/C/2013), to A.V. from MINECO (Grant BIO2013-41019-P) and from the Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina (NANOPROTHER and NANOCOMETS projects). We are grateful to the Protein Production Platform (CIBER-BBN-UAB) for protein production and purification services (http://www.ciber-bbn.es/en/programas/89-plataforma-de-produccion-de-proteinas-ppp), to the Servei de Cultius Cel·lulars, Producció d׳Anticossos i Citometria (SCAC), to the Servei de Microscòpia, both at the UAB and to the Soft Materials Service (ICMAB-CSIC/CIBER-BBN). Z.X. received a fellowship grant from China Scholarship Council (Grant no. 2011630065) and U.U. from ISCIII. AV received an ICREA ACADEMIA award.Homing peptides are exploited in nanomedicine to functionalize either free drugs or nanostructured materials used as drug carriers. However, the influence of multivalent versus monovalent peptide presentation on the interaction with the receptor and on the consequent intracellular delivery of the associated cargo remains poorly explored. By using a tumor-homing peptide (T22) with regulatable self-assembling properties we have investigated here if its display in a either a monomeric form or as multimeric, self-assembled protein nanoparticles might determine the efficacy of receptor-mediated penetrability into target cells. This has been monitored by using a fluorescent cargo protein (iRFP), which when fused to the homing peptide acts as convenient reporter. The results indicate that the nanoparticulate protein versions are significantly more efficient in mediating receptor-dependent uptake than their unassembled counterparts. These finding stresses an additional benefit of nanostructured materials based on repetitive building blocks, regarding the multivalent presentation of cell ligands that facilitate cell penetration in drug delivery applications

Targeting low-density lipoprotein receptors with protein-only nanoparticles

We are grateful to the Protein Production Platform (CIBER-BBN-UAB) for helpful technical assistance and for protein production and purification services (http://​www.​ciber-bbn.​es/​en/​programas/​89-plataforma-de-produccion-de-proteinas-ppp). We are indebted to FIS PI12/01861, Marató 416/C/2013-2030 and NanoMets to RM, MINECO BIO2013-41019-P to AV, AGAUR (2014SGR-132), and CIBER de Bioingeniería, Biomateriales y Nanomedicina (project NANOPROTHER) for funding our research on protein-based therapeutics. We thank the CIBER-BBN Nanotoxicology Unit for fluorescent in vivo follow-up using the IVIS equipment. We are also indebted to the Cell Culture and Citometry Units of the Servei de Cultius Cel·lulars, Producció d'Anticossos i Citometria (SCAC), and to the Servei de Microscòpia, both at the UAB, and to the Soft Materials Service (ICMAB-CSIC/CIBER-BBN). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Z. X. and M. P. acknowledge financial support from China Scholarship Council and Universitat Autònoma de Barcelona through pre-doctoral fellowships, respectively. AV received an ICREA ACADEMIA award.Low-density lipoprotein receptors (LDLR) are appealing cell surface targets in drug delivery, as they are expressed in the blood-brain barrier (BBB) endothelium and are able to mediate transcytosis of functionalized drugs for molecular therapies of the central nervous system (CNS). On the other hand, brain-targeted drug delivery is currently limited, among others, by the poor availability of biocompatible vehicles, as most of the nanoparticles under development as drug carriers pose severe toxicity issues. In this context, protein nanoparticles offer functional versatility, easy and cost-effective bioproduction, and full biocompatibility. In this study, we have designed and characterized several chimerical proteins containing different LDLR ligands, regarding their ability to bind and internalize target cells and to self-organize as viral mimetic nanoparticles of about 18 nm in diameter. While the self-assembling of LDLR-binding proteins as nanoparticles positively influences cell penetration in vitro, the nanoparticulate architecture might be not favoring BBB crossing in vivo. These findings are discussed in the context of the use of nanostructured materials as vehicles for the systemic treatment of CNS diseases

A multivalent Ara-C-prodrug nanoconjugate achieves selective ablation of leukemic cells in an acute myeloid leukemia mouse model

Current therapy in acute myeloid leukemia (AML) is based on chemotherapeutic drugs administered at high doses, lacking targeting selectivity and displaying poor therapeutic index because of severe adverse effects. Here, we develop a novel nanoconjugate that combines a self-assembled, multivalent protein nanoparticle, targeting the CXCR4 receptor, with an Oligo-Ara-C prodrug, a pentameric form of Ara-C, to highly increase the delivered payload to target cells. This 13.4 nm T22-GFP-H6-Ara-C nanoconjugate selectively eliminates CXCR4+ AML cells, which are protected by its anchoring to the bone marrow (BM) niche, being involved in AML progression and chemotherapy resistance. This nanoconjugate shows CXCR4-dependent internalization and antineoplastic activity in CXCR4+ AML cells in vitro. Moreover, repeated T22-GFP-H6-Ara-C administration selectively eliminates CXCR4+ leukemic cells in BM, spleen and liver. The leukemic dissemination blockage induced by T22-GFP-H6-Ara-C is significantly more potent than buffer or Oligo-Ara-C-treated mice, showing no associated on-target or off-target toxicity and, therefore, reaching a highly therapeutic window. In conclusion, T22-GFP-H6-Ara-C exploits its 11 ligands-multivalency to enhance target selectivity, while the Oligo-Ara-C prodrug multimeric form increases 5-fold its payload. This feature combination offers an alternative nanomedicine with higher activity and greater tolerability than current intensive or non-intensive chemotherapy for AML patients.This work was supported by Instituto de Salud Carlos III (ISCIII, Co-funding from FEDER) [PI18/00650, PIE15/00028, PI15/00378 and EU COST Action CA 17 140 to R.M.; FIS PI17/01246 and RD16/0011/0028 to J.S.; and PI20/00400 to U·U.]; Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (grant BIO2016-76063-R, AEI/FEDER, UE) to A.V.; (grant PID2019-105416RB-I00) to E.V.; CIBER-BBN [CB06/01/1031 and 4NanoMets to R.M., VENOM4CANCER to A.V., NANOREMOTE to E.V. and NANOLINK to U·U.]; AGAUR [2017 FI_B 00680 to A.F., 2018 FI_B2_00051 to L.S.G.; 2017-SGR-865 to R.M., 2017-SGR-1395 to J.S. and 2017SGR-229 to A.V.]; Josep Carreras Leukemia Research Institute [P/AG to R.M.]; La Marató TV3 [201 941-30-31-32 to J.S. and A.V.]; a grant from the Cellex Foundation, Barcelona [to J.S.]; a grant from La Generalitat de Catalunya (PERIS) [SLT002/16/00433to J.S.]; a grant from the Generalitat de Catalunya CERCA Programme. U.U. is supported by Miguel Servet fellowship (CP19/00028) from Instituto de Salud Carlos III co-funded by Fondo Social Europeo (ESF investing in your future). Finally, A.V. received an ICREA ACADEMIA Award supported by the Catalan Government. We are also indebted to the ISCIII Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) and its ICTS Nanbiosis Platform for their funding. The bioluminescent follow-up of cancer cells and toxicity studies has been performed in the ICTS-141007 Nanbiosis Platform, using its CIBER-BBN Nanotoxicology Unit (http://www.nanbiosis.es/portfolio/u18-nanotoxicology-unit/). Protein production has been partially performed by the ICTS “NANBIOSIS”, more specifically by the Protein Production Platform of CIBER-BBN/IBB (http://www.nanbiosis.es/unit/u1-protein-production-platform-ppp/). Finally, we are very grateful to Servei de Microscopia from UAB for their excellent confocal and electron microscopy services and especially to Alejandro Sánchez-Chardi for its excellent support and contribution in TEM and FESEM images.Peer reviewe

Rational engineering of a human GFP-like protein scaffold for humanized targeted nanomedicines

Green fluorescent protein (GFP) is a widely used scaffold for protein-based targeted nanomedicines because of its high biocompatibility, biological neutrality and outstanding structural stability. However, being immunogenicity a major concern in the development of drug carriers, the use of exogenous proteins such as GFP in clinics might be inadequate. Here we report a human nidogen-derived protein (HSNBT), rationally designed to mimic the structural and functional properties of GFP as a scaffold for nanomedicine. For that, a GFP-like β-barrel, containing the G2 domain of the human nidogen, has been rationally engineered to obtain a biologically neutral protein that self-assembles as 10nm-nanoparticles. This scaffold is the basis of a humanized nanoconjugate, where GFP, from the well-characterized protein T22-GFP-H6, has been substituted by the nidogen-derived GFP-like HSNBT protein. The resulting construct T22-HSNBT-H6, is a humanized CXCR4-targeted nanoparticle that selectively delivers conjugated genotoxic Floxuridine into cancer CXCR4+ cells. Indeed, the administration of T22-HSNBT-H6-FdU in a CXCR4-overexpressing colorectal cancer mouse model results in an even more efficient selective antitumoral effect than that shown by its GFP-counterpart, in absence of systemic toxicity. Therefore, the newly developed GFP-like protein scaffold appears as an ideal candidate for the development of humanized protein nanomaterials and successfully supports the tumor-targeted nanoscale drug T22-HSNBT-H6-FdU.Patricia Álamo and Juan Cedano contributed equally to this work. The authors are indebted to Agencia Estatal de Investigación (AEI) and to Fondo Europeo de Desarrollo Regional (FEDER) (grant BIO2016-76063-R, AEI/FEDER, UE), to AGAUR (2017SGR-229) and CIBER-BBN (project NANOPROTHER), granted to AV, to CIBER-BBN (project NANOSCAPE and NANOLINK) and ISCIII (PI20/00400 co-funding FEDER) granted to UU, to ISCIII (PI15/00272 co-founding FEDER) granted to EV and to ISCIII (PIE15/00028 and PI18/00650, co-funding FEDER) and AGAUR (2017 SGR 865 GRC) granted to RM. We are also indebted to CERCA programme (Generalitat de Catalunya) and to the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) that is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III, with assistance from the European Regional Development Fund. We also appreciate the support from the COST-Action Nano2Clinics. Protein production has been partially performed by the ICTS “NANBIOSIS”, more specifically by the Protein Production Platform of CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN)/ IBB, at the UAB sePBioEs scientific-technical service (http://www.nanbiosis.es/portfolio/u1-protein-production-platform-ppp/), and the nanoparticle size analysis by the Biomaterial Processing and Nanostructuring Unit. Synthesis of thiolated oligo-FdU was performed by the ICTS NANBIOSIS Oligonucleotide Synthesis Platform (CIBER-BBN). The in vivo work was performed by the ICTS NANBIOSIS of the CIBER-BBN Nanotoxicology Unit (http://www.nanbiosis.es/portfolio/u18-nanotoxicology-unit/). We are indebted to Servei de Microscopia from UAB for their excellent confocal and electronic microscopy services. We are also indebted to Servei de Cultius Celulars i Anticossos (SCAC) form UAB for their excellent cell culture and flow cytometry facilities and especially to Fran Cortes for his excellent technical support. We are thankful to Dra. Marta Taulés from CCiT-UB for her help in SPR experiments and analysis. We are also thankful to Luis Carlos Navas from Institut d'Investigacions biomèdiques Sant Pau (IIB Sant Pau) for his technical support in immunohistochemistry experiments. UU and LMCD were supported by Miguel Servet (CP19/00028) and PFIS (FI19/00148) contracts respectively from ISCIII co-funded by European Social Fund (ESF investing in your future). NS was supported by a pre-doctoral fellowship from the Government of Navarra and LAC was supported by AECC Scientific Foundation grant postdoctoral fellow. AV received an ICREA ACADEMIA award.Peer reviewe