Chromo-fluorogenic probes for beta-galactosidase detection

[EN] beta-Galactosidase (beta-Gal) is a widely used enzyme as a reporter gene in the field of molecular biology which hydrolyzes the beta-galactosides into monosaccharides. beta-Gal is an essential enzyme in humans and its deficiency or its overexpression results in several rare diseases. Cellular senescence is probably one of the most relevant physiological disorders that involve beta-Gal enzyme. In this review, we assess the progress made to date in the design of molecular-based probes for the detection of beta-Gal both in vitro and in vivo. Most of the reported molecular probes for the detection of beta-Gal consist of a galactopyranoside residue attached to a signalling unit through glycosidic bonds. The beta-Gal-induced hydrolysis of the glycosidic bonds released the signalling unit with remarkable changes in color and/or emission. Additional examples based on other approaches are also described. The wide applicability of these probes for the rapid and in situ detection of de-regulation beta-Gal-related diseases has boosted the research in this fertile fieldR.M laboratory members received the financial support from the Spanish Government (project RTI2018-100910-B-C41) and the Generalitat Valenciana (project PROMETEO 2018/024). B.L-T. received support from the Spanish Ministry of Economy for their PhD grants (FPU15/02707). J. F.-B received fellowship (CD19/00038)Lozano-Torres, B.; Blandez, JF.; Sancenón Galarza, F.; Martínez-Máñez, R. (2021). Chromo-fluorogenic probes for beta-galactosidase detection. Analytical and Bioanalytical Chemistry. 413(9):2361-2388. https://doi.org/10.1007/s00216-020-03111-8S236123884139Fernandes P. Enzymes in food processing: a condensed overview on strategies for better biocatalysts. Enzyme Res. 2010;2010:86253–73.Likidlilid A, Patchanans N, Peerapatdit T, Sriratanasathavorn C. 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Glycosyltransferase and glycosidase activities in ovarian cancer

Influence of the organic linker substituent on the catalytic activity of MIL-101(Cr) for the oxidative coupling of benzylamines to imines

[EN] MIL-101(Cr) having substituents at the terephthalate linker (X = H, NO2, SO3H, Cl, CH3 and NH2) promotes the aerobic oxidation of benzylamines to the corresponding N-benzylidene benzylamines at different rates. MIL-101(Cr)¿NO2 was the most active catalyst, about 6-fold more active than the parent MIL-101(Cr). MIL-101(Cr)¿NO2 does not deactivate significantly upon five consecutive reuses, does not leach the metal to the solution and maintains its crystallinity. MIL-101(Cr)¿NO2 is active for a wide range of benzylamines including para-substituted, heterocyclic benzylamines and di- and tribenzylamines.Financial support by the Spanish Ministry of Economy and Competitiveness (CTQ 2015-69153-CO2-1, CTQ2014-53292-R, Severo Ochoa) and Generalitat Valenciana (Prometeo 2013014) is gratefully acknowledged.Santiago-Portillo, A.; Blandez, JF.; Navalón Oltra, S.; Alvaro Rodríguez, MM.; García Gómez, H. (2017). Influence of the organic linker substituent on the catalytic activity of MIL-101(Cr) for the oxidative coupling of benzylamines to imines. Catalysis Science & Technology. 7(6):1351-1362. https://doi.org/10.1039/c6cy02577cS135113627

Real-Time In Vivo Detection of Cellular Senescence through the Controlled Release of the NIR Fluorescent Dye Nile Blue

This is the peer reviewed version of the following article: B. Lozano-Torres, J. F. Blandez, I. Galiana, A. García-Fernández, M. Alfonso, M. D. Marcos, M. Orzáez, F. Sancenón, R. Martínez-Máñez, Angew. Chem. Int. Ed. 2020, 59, 15152., which has been published in final form at https://doi.org/10.1002/anie.202004142. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] In vivo detection of cellular senescence is accomplished by using mesoporous silica nanoparticles loaded with the NIR-FDA approved Nile blue (NB) dye and capped with a galactohexasaccharide (S3). NB emission at 672 nm is highly quenched inside S3, yet a remarkable emission enhancement is observed upon cap hydrolysis in the presence of beta-galactosidase and dye release. The efficacy of the probe to detect cellular senescence is tested in vitro in melanoma SK-Mel-103 and breast cancer 4T1 cells and in vivo in palbociclib-treated BALB/cByJ mice bearing breast cancer tumor.R.M. thanks financial support from the Spanish Government (RTI2018-100910-B-C41 and RTI2018-101599-B-C22 (MCUI/AEI/FEDER, UE)) and the Generalitat Valenciana (PROMETEO 2018/024). M.O. thanks the financial support from SAF2017-84689-R project and MINECO/AEI/FEDER, UE and the Generalitat Valenciana (PROMETEO/2019/065). B.L.-T. is grateful to the Spanish Ministry of Economy for her PhD grant. I.G. thanks her contract from IDM. J.F.-B. and M.A. thank the UPV for their postdoctoral fellowship.Lozano-Torres, B.; Blandez, JF.; Galiana, I.; García-Fernández, A.; Alfonso-Navarro, M.; Marcos Martínez, MD.; Orzáez, M.... (2020). Real-Time In Vivo Detection of Cellular Senescence through the Controlled Release of the NIR Fluorescent Dye Nile Blue. 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Electro-responsive films containing voltage responsive gated mesoporous silica nanoparticles grafted onto PEDOT-based conducting polymer

[EN] The characteristics and electromechanical properties of conductive polymers together to their biocompatibility have boosted their application as a suitable tool in regenerative medicine and tissue engineering. However, conducting polymers as drug release materials are far from being ideal. A possibility to overcome this drawback is to combine conducting polymers with on-command delivery particles with inherent high-loading capacity. In this scenario, we report here the preparation of conduction polymers containing gated mesoporous silica nanoparticles (MSN) loaded with a cargo that is delivered on command by electro-chemical stimuli increasing the potential use of conducting polymers as controlled delivery systems. MSNs are loaded with Rhodamine B (Rh B), anchored to the conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)], functionalized with a bipyridinium derivative and pores are capped with heparin (P3) by electrostatic interactions. P3 releases the entrapped cargo after the application of ¿640 mV voltage versus the saturated calomel electrode (SCE). Pore opening in the nanoparticles and dye delivery is ascribed to both (i) the reduction of the grafted bipyridinium derivative and (ii) the polarization of the conducting polymer electrode to negative potentials that induce detachment of positively charged heparin from the surface of the nanoparticles. Biocompatibility and cargo release studies were carried out in HeLa cells cultures.Alba Garcia-Fernandez, Beatriz Lozano-Torres contributed equally to this work. A. Garcia-Fernandez and B. Lozano-Torres are grateful to the "Ministerio de Economia y Competitividad" of the Spanish Government for her PhD fellowships. J. F. Blandez thanks the "Universitat Politecnica de Valencia" for his postdoctoral fellowship (PAID-10-17). The authors thank to the Spanish Government (Projects RTI2018-100910-B-C41 and RTI2018-101599-B-C22 (MCUI/AEI/FEDER, EU)) and the Generalitat Valencia (Project PROMETEO2018-024) for support.García-Fernández, A.; Lozano-Torres, B.; Blandez, JF.; Monreal-Trigo, J.; Soto Camino, J.; Collazos-Castro, JE.; Alcañiz Fillol, M.... (2020). Electro-responsive films containing voltage responsive gated mesoporous silica nanoparticles grafted onto PEDOT-based conducting polymer. Journal of Controlled Release. 323:421-430. https://doi.org/10.1016/j.jconrel.2020.04.048S421430323Aznar, E., Oroval, M., Pascual, L., Murguía, J. R., Martínez-Máñez, R., & Sancenón, F. (2016). Gated Materials for On-Command Release of Guest Molecules. 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A Two-Photon Probe Based on Naphthalimide-Styrene Fluorophore for the In Vivo Tracking of Cellular Senescence

Cellular senescence is a state of stable cell cycle arrest that can negatively affect the regenerative capacities of tissues and can contribute to inflammation and the progression of various aging-related diseases. Advances in the in vivo detection of cellular senescence are still crucial to monitor the action of senolytic drugs and to assess the early onset or accumulation of senescent cells. Here, we describe a naphthalimide-styrene-based probe (HeckGal) for the detection of cellular senescence both in vitro and in vivo. HeckGal is hydrolyzed by the increased lysosomal β-galactosidase activity of senescent cells, resulting in fluorescence emission. The probe was validated in vitro using normal human fibroblasts and various cancer cell lines undergoing senescence induced by different stress stimuli. Remarkably, HeckGal was also validated in vivo in an orthotopic breast cancer mouse model treated with senescence-inducing chemotherapy and in a renal fibrosis mouse model. In all cases, HeckGal allowed the unambiguous detection of senescence in vitro as well as in tissues and tumors in vivo. This work is expected to provide a potential technology for senescence detection in aged or damaged tissues

Galacto-conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity.

Pharmacologically active compounds with preferential cytotoxic activity for senescent cells, known as senolytics, can ameliorate or even revert pathological manifestations of senescence in numerous preclinical mouse disease models, including cancer models. However, translation of senolytic therapies to human disease is hampered by their suboptimal specificity for senescent cells and important toxicities that narrow their therapeutic windows. We have previously shown that the high levels of senescence-associated lysosomal β-galactosidase (SA-β-gal) found within senescent cells can be exploited to specifically release tracers and cytotoxic cargoes from galactose-encapsulated nanoparticles within these cells. Here, we show that galacto-conjugation of the BCL-2 family inhibitor Navitoclax results in a potent senolytic prodrug (Nav-Gal), that can be preferentially activated by SA-β-gal activity in a wide range of cell types. Nav-Gal selectively induces senescent cell apoptosis and has a higher senolytic index than Navitoclax (through reduced activation in nonsenescent cells). Nav-Gal enhances the cytotoxicity of standard senescence-inducing chemotherapy (cisplatin) in human A549 lung cancer cells. Concomitant treatment with cisplatin and Nav-Gal in vivo results in the eradication of senescent lung cancer cells and significantly reduces tumour growth. Importantly, galacto-conjugation reduces Navitoclax-induced platelet apoptosis in human and murine blood samples treated ex vivo, and thrombocytopenia at therapeutically effective concentrations in murine lung cancer models. Taken together, we provide a potentially versatile strategy for generating effective senolytic prodrugs with reduced toxicities

Influence of functionalization of terephthalate linker on the catalytic activity of UiO-66 for epoxide ring opening

[EN] A series of five isostructural zirconium terephthalate UiO-66 metal organic frameworks bearing different functional groups on the terephthalate linker (UiO-66-X; X = H, NH2, NO2, Br, Cl,) have been successfully prepared and characterized. UiO-66-X materials were evaluated as heterogeneous catalysts for the epoxide ring opening of styrene oxide by methanol, observing an increase in the initial reaction rate from UiO-66-H to UiO-66-Br, over one order of magnitude. The reactivity order, however, does not follow a linear relationship between the Hammett constant value of the substituent and the initial reaction rate. UiO-66-Br exhibits a wide scope, its activity depending on the structure of epoxide and nucleophile. The absence of Zr leaching to the solution together with the preservation of the UiO-66-X crystallinity confirms the stability of the framework under the reaction conditions. Nevertheless, UiO-66 undergoes a progressive deactivation upon reuse that was attributed to a strong adsorption of the reaction product.Financial support by the Spanish Ministry of Economy and Competitiveness (Severo Ochoa and CTQ2015-69153 and CTQ2014-53292-R and Generalitat Valenciana (Prometeo 2013/14) and is grateful acknowledged. We also thank EU under the Being Energy contract for partial funding. J.F.B. thanks the Universitat Politecnica de Valencia for a postgraduate scholarship. S.N. thanks the Spanish Ministerio de Educacion, Cultura y Deporte for Jose Castillejo mobility programme (CAS14/00067).Blandez, JF.; Santiago-Portillo, A.; Navalón Oltra, S.; Gimenez Marques, M.; Alvaro Rodríguez, MM.; Horcajada, P.; García Gómez, H. (2016). Influence of functionalization of terephthalate linker on the catalytic activity of UiO-66 for epoxide ring opening. Journal of Molecular Catalysis A Chemical. 425:332-339. https://doi.org/10.1016/j.molcata.2016.10.022S33233942

Lipofuscin labeling through biorthogonal strain-promoted azide-alkyne cycloaddition for the detection of senescent cells

[EN] A new method for senescent cell detection is described, which is based on lipofuscin labeling with a fluorescent reporter through a biorthogonal strain-promoted azide-alkyne cycloaddition. The sensing protocol involves a first step where the interaction of lipofuscin with a Sudan Black B derivative containing an azide moiety (SBB-N3 ) is carried out. In the final step, the azide moiety reacts with a fluorophore containing a cyclooctene ring (BODIPY). The efficacy of this two-step protocol is assessed in senescent melanoma SK-MEL-103 cells, senescent triple-negative breast cancer MDA-MB-231 cells and senescent WI-38 fibroblasts. In all cases, a clear fluorescence pattern was observed in senescent cells, compared to proliferative cells, only when the SBB-N3 -BODIPY probe was formed. Our results provide an alternative tool for the detection of senescent cells, based on an in situ bio-orthogonal reaction for lipofuscin labelingRM laboratory members are grateful for financial support from the FEDER foundation of European Union (IDIFEDER/2021/044), the Spanish Government (projects RTI2018-100910-B-C41 and RTI2018-101599-B-C22) and the Generalitat Valenciana (project PROMETEO 2018/024). BL-T and AG-F acknowledge their current Margarita Salas postdoctoral fellowship from UPV-MIU and 'Next Generation EU' program. JF-B is grateful for his Sara Borrell postdoctoral fellowship (CD19/00038).Lozano-Torres, B.; Blandez, JF.; García-Fernández, A.; Sancenón Galarza, F.; Martínez-Máñez, R. (2023). Lipofuscin labeling through biorthogonal strain-promoted azide-alkyne cycloaddition for the detection of senescent cells. FEBS Journal. 290(5):1314-1325. https://doi.org/10.1111/febs.1647713141325290

Nickel nanoparticles supported on graphene as catalysts for aldehyde hydrosilylation

[EN] Nickel nanoparticles (NPs) supported on different undoped or doped with N or B graphenes (Gs) have been tested as catalyst for the hydrosilylation of aldehydes to obtain the corresponding siloxanes with high conversion and good selectivity in short reaction time. The different Gs employed were obtained by pyrolysis under inert atmosphere of alginate or chitosan, modified or not with boric acid. Then the metal NPs obtained by polyol reduction method using ethylene glycol were adsorbed on Gs. The Ni-containing G catalysts were characterized by electron microscopy, XPS and Raman spectroscopy. The scope of the Ni/G catalyst includes aliphatic and aromatic aldehydes as well as a variety of hydrosilanes. (C) 2015 Elsevier B.V. All rights reserved.Financial support by the Spanish Ministry of Economy and Competitiveness (Severo Ochoa and CTQ2012-32315) and Generalitat Valenciana (Prometeo 2013-019) in gratefully acknowledged. J.F.B. and I.E.A. thanks to the Technical University of Valencia and the Spanish Ministry of Economy and Competitiveness for postgraduate scholarships, respectively.Blandez, JF.; Esteve-Adell, I.; Primo Arnau, AM.; Alvaro Rodríguez, MM.; García Gómez, H. (2016). Nickel nanoparticles supported on graphene as catalysts for aldehyde hydrosilylation. Journal of Molecular Catalysis A Chemical. 412:13-19. https://doi.org/10.1016/j.molcata.2015.11.011S131941

Synthesis, Structural Characterization, and Catalytic Activity of IPrNi(styrene)2 in the Amination of Aryl Tosylates

[EN] A novel bis-styrene IPrNi0 derivative has been synthesized from the reaction of Ni(COD)2 and free 1,3-bis(2,6-diisopropylphenyl)imidazolidene (IPr) ligand in the presence of styrene. The complex has been characterized by spectroscopic data as well as by X-ray crystallography. Its catalytic performance in the amination reaction of aryl tosylates is also reported. The catalytic reactions proceed in a very selective manner, affording moderate to high yields of cross-coupling products in short reaction times at 110 °C.This research was financially supported by the MINECO (Proyecto CTQ2011-24502 and Consolider Ingenio 2010, Grant No. CSD2006-003) and the Junta de Andalucia (Proyecto P07-FQM-02745). M.J.I. thanks the Junta de Andalucia for a research fellowship.Iglesia, MJ.; Blandez, JF.; Fructos, MR.; Prieto, A.; Alvarez, E.; Belderrain, TR.; Nicasio, MC. (2012). Synthesis, Structural Characterization, and Catalytic Activity of IPrNi(styrene)2 in the Amination of Aryl Tosylates. Organometallics. 31(17):6312-6316. https://doi.org/10.1021/om300566mS63126316311