Synthesis of heterogeneous enzyme-metal nanoparticle biohybrids in aqueous media and their applications in C-C bond formation and tandem catalysis

The straightforward synthesis of novel enzyme-metalNP nanobiohybrids in aqueous medium was developed. These new nanobiohybrids were excellent multivalent catalysts combining both activities in various sets of synthetic reactions even at ultra-low concentrations (ppb amount). © 2013 The Royal Society of Chemistry.This research was supported by The Spanish National Research Council (CSIC). Authors thank European Community (FP7-MULTIFUN) for the contract of M.M.Peer Reviewe

In-situ particles reorientation during magnetic hyperthermia application: Shape matters twice

Promising advances in nanomedicine such as magnetic hyperthermia rely on a precise control of the nanoparticle performance in the cellular environment. This constitutes a huge research challenge due to difficulties for achieving a remote control within the human body. Here we report on the significant double role of the shape of ellipsoidal magnetic nanoparticles (nanorods) subjected to an external AC magnetic field: first, the heat release is increased due to the additional shape anisotropy; second, the rods dynamically reorientate in the orthogonal direction to the AC field direction. Importantly, the heating performance and the directional orientation occur in synergy and can be easily controlled by changing the AC field treatment duration, thus opening the pathway to combined hyperthermic/ mechanical nanoactuators for biomedicine. Preliminary studies demonstrate the high accumulation of nanorods into HeLa cells whereas viability analysis supports their low toxicity and the absence of apoptotic or necrotic cell death after 24 or 48 h of incubationThis work was partially supported by the EC FP-7 grant “NanoMag” (grant agreement no. 604448), the Spanish Ministry of Economy and Competitiveness (MAT2013-47078-C2-2-P, MAT2014-52069-R, MAT2013-47395-C4-3-R, MAT2015- 67557-C2-1-P-MICINN, CONSOLIDER CSD2007-00041, CTQ2013-48767-C3-3-R), and Gobierno de la Comunidad de Madrid (NANOFRONTMAG, S2013/MIT-2850). D.S. acknowledges financial support from Xunta de Galicia (I2C Postdoctoral Plan). A.T. thanks UAM for a predoctoral contrac

Synthesis of a theranostic platform based on fibrous silica nanoparticles for the enhanced treatment of triple-negative breast cancer promoted by a combination of chemotherapeutic agents.

A new series of theranostic silica materials based on fibrous silica particles acting as nanocarriers of two different cytotoxic agents, namely, chlorambucil and an organotin metallodrug have been prepared and structurally characterized. Besides the combined therapeutic activity, these platforms have been decorated with a targeting molecule (folic acid, to selectively target triple negative breast cancer) and a molecular imaging agent (Alexa Fluor 647, to enable their tracking both in vitro and in vivo). The in vitro behaviour of the multifunctional silica systems showed a synergistic activity of the two chemotherapeutic agents in the form of an enhanced cytotoxicity against MDA-MB-231 cells (triple negative breast cancer) as well as by a higher cell migration inhibition. Subsequently, the in vivo applicability of the siliceous nanotheranostics was successfully assessed by observing with in vivo optical imaging techniques a selective tumour accumulation (targeting ability), a marked inhibition of tumour growth paired to a marked antiangiogenic ability after 13 days of systemic administration, thus, confirming the enhanced theranostic activity. The systemic nanotoxicity was also evaluated by analyzing specific biochemical markers. The results showed a positive effect in form of reduced cytotoxicity when both chemotherapeutics are administered in combination thanks to the fibrous silica nanoparticles. Overall, our results confirm the promising applicability of these novel silica-based nanoplatforms as advanced drug-delivery systems for the synergistic theranosis of triple negative breast cancer.We would like to thank the funding of the Ministerio de Ciencia e Innovación of Spain (former Ministerio de Ciencia Innovación y Universidades of Spain) and FEDER, Una manera de hacer Europa for the grant number RTI2018-094322-B-I00. We would also like to thank Comunidad de Madrid for the predoctoral grant PEJD-2017-PRE/BMD3512 (I.M.-P.). M.M, Y.L.M., and M.F. are grateful to the Comunidad Autónoma de Madrid and FEDER for the I + D collaborative Programme in Biomedicine NIETO-CM (Project reference B2017-BMD3731). M.F. and K.O.P. thank the Comunidad Autonoma ´ de Madrid for research project No. 2017-T1/BIO-4992 (“Atraccion ´ de Talento” Action) cofunded by Universidad Complutense de Madrid. M.F is grateful to Instituto de Salud Carlos III (ISCIII) for project No DTS20/00109 (AES-ISCIII). M. M., M.F. and L.L.C would also like to thank Comunidad de Madrid for the predoctoral grant IND2020/BIO-17523. M.F. and K.O.P. acknowledge the support of Microscopy & Dynamic Imaging Unit of CNIC, Madrid, Spain. The Unit is part of the ReDiB-ICTS and has the support of FEDER, “Una manera de hacer Europa.” The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovacion ´ (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/ 501100011033).S

Understanding the Influence of a Bifunctional Polyethylene Glycol Derivative in Protein Corona Formation around Iron Oxide Nanoparticles

Superparamagnetic iron oxide nanoparticles are one of the most prominent agents used in theranostic applications, with MRI imaging the main application assessed. The biomolecular interface formed on the surface of a nanoparticle in a biological medium determines its behaviour in vitro and in vivo. In this study, we have compared the formation of the protein corona on highly monodisperse iron oxide nanoparticles with two different coatings, dimercaptosuccinic acid (DMSA), and after conjugation, with a bifunctional polyethylene glycol (PEG)-derived molecule (2000 Da) in the presence of Wistar rat plasma. The protein fingerprints around the nanoparticles were analysed in an extensive proteomic study. The results presented in this work indicate that the composition of the protein corona is very difficult to predict. Proteins from different functional categories—cell components, lipoproteins, complement, coagulation, immunoglobulins, enzymes and transport proteins—were identified in all samples with very small variability. Although both types of nanoparticles have similar amounts of bonded proteins, very slight differences in the composition of the corona might explain the variation observed in the uptake and biotransformation of these nanoparticles in Caco-2 and RAW 264.7 cells. Cytotoxicity was also studied using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Controlling nanoparticles’ reactivity to the biological environment by deciding on its surface functionalization may suggest new routes in the control of the biodistribution, biodegradation and clearance of multifunctional nanomedicines

Versatile Graphene-Based Platform for Robust Nanobiohybrid Interfaces

Technologically useful and robust graphene-based interfaces for devices require the introduction of highly selective, stable, and covalently bonded functionalities on the graphene surface, whilst essentially retaining the electronic properties of the pristine layer. This work demonstrates that highly controlled, ultrahigh vacuum covalent chemical functionalization of graphene sheets with a thiol-terminated molecule provides a robust and tunable platform for the development of hybrid nanostructures in different environments. We employ this facile strategy to covalently couple two representative systems of broad interest: metal nanoparticles, via S-metal bonds, and thiol-modified DNA aptamers, via disulfide bridges. Both systems, which have been characterized by a multi-technique approach, remain firmly anchored to the graphene surface even after several washing cycles. Atomic force microscopy images demonstrate that the conjugated aptamer retains the functionality required to recognize a target protein. This methodology opens a new route to the integration of high-quality graphene layers into diverse technological platforms, including plasmonics, optoelectronics, or biosensing. With respect to the latter, the viability of a thiol-functionalized chemical vapor deposition graphene-based solution-gated field-effect transistor array was assessed

Broad virus inactivation using inorganic micro/nano-particulate materials

Inorganic materials can provide a set of tools to decontaminate solid, liquid or air containing viral particles. The use of disinfectants can be limited or not practical in scenarios where continuous cleaning is not feasible. Physicochemical differences between viruses raise the need for effective formulations for all kind of viruses. In the present work we describe two types of antimicrobial inorganic materials: i) a novel soda-lime glass (G3), and ii) kaolin containing metals nanoparticles (Ag or CuO), as materials to disable virus infectivity. Strong antiviral properties can be observed in G3 glass, and kaolin-containing nanoparticle materials showing a reduction of viral infectivity close to 99%. in the first 10 ​min of contact of vesicular stomatitis virus (VSV). A potent virucidal activity is also present in G3 and kaolin containing Ag or CuO nanoparticles against all kinds of viruses tested, reducing more than 99% the amount of HSV-1, Adenovirus, VSV, Influenza virus and SARS-CoV-2 exposed to them. Virucidal properties could be explained by a direct interaction of materials with viruses as well as inactivation by the presence of virucidal elements in the material lixiviates. Kaolin-based materials guarantee a controlled release of active nanoparticles with antiviral activity. Current coronavirus crisis highlights the need for new strategies to remove viruses from contaminated areas. We propose these low-cost inorganic materials as useful disinfecting antivirals in the actual or future pandemic threats.This research was performed with support from The Spanish National Research Council (CSIC) (Project No 202060E109). M.F. is grateful to the Comunidad Autonoma de Madrid for research project No. 2017-T1/BIO-4992 (“Atracción de Talento” Action) cofunded by Universidad Complutense de Madrid. This publication was also supported by the European Virus Archive GLOBAL (EVA-GLOBAL) project that has received funding from the European Union's Horizon 2020 research and innovation program ​under grant agreement 871029. S. R.-R. was supported by the the FPI fellowship funded by Universidad San Pablo CEU. J.A-H. was supported by the PFIS fellowship co-funded by the FEDER/FSE and the ISCIII

Attomolar detection of hepatitis C virus core protein powered by molecular antenna-like effect in a graphene field-effect aptasensor

Biosensors based on graphene field-effect transistors have become a promising tool for detecting a broad range of analytes. However, their performance is substantially affected by the functionalization protocol. In this work, we use a controlled in-vacuum physical method for the covalent functionalization of graphene to construct ultrasensitive aptamer-based biosensors (aptasensors) able to detect hepatitis C virus core protein. These devices are highly specific and robust, achieving attomolar detection of the viral protein in human blood plasma. Such an improved sensitivity is rationalized by theoretical calculations showing that induced polarization at the graphene interface, caused by the proximity of covalently bound molecular probe, modulates the charge balance at the graphene/aptamer interface. This charge balance causes a net shift of the Dirac cone providing enhanced sensitivity for the attomolar detection of the target proteins. Such an unexpected effect paves the way for using this kind of graphene-based functionalized platforms for ultrasensitive and real-time diagnostics of different diseases.EU Graphene Flagship funding (Grant Graphene Core3 881603), the Ministerio de Ciencia e Innovación of Spain: PID2020-113142RB-C21, the European Structural Funds via FotoArt-CM project (P2018/NMT-4367) and the Portuguese Foundation for Science and Technology (FCT) via the Strategic Funding UIDB/04650/2020. Work at CAB was funded by the Spanish Ministerio de Ciencia e Innovación (MICINN) grant no. PID2019-104903RB-I00 and the Spanish Agencia Estatal de Investigación (AEI) Project no. MDM-2017-0737 - Unidad de Excelencia “María de Maeztu,” and it also benefits from the interdisciplinary framework provided by CSIC through “LifeHUB.CSIC” initiative (PIE 202120E047-CONEXIONES-LIFE). CIBERehd is funded by Instituto de Salud Carlos III (ISCIII). A.N. is supported by the predoctoral fellowship PRE-CAB-BIOMOLECULAS 2 from INTA. B.T-V. is supported by the predoctoral fellowship TS17/16 from INTA and by the CSIC “Garantía Juvenil” contract CAM19_PRE_CAB_001 funded by Comunidad de Madrid (CAM). FCT supports T.D. and P.C. under Ph.D. grants SFRH/BD/08181/2020 and SFRH/BD/128579/2017. M.M. would like to thank Comunidad de Madrid for the predoctoral grant IND2020/BIO-17523. P.A. and C.B. also acknowledge the support provided by La Caixa Foundation through Project LCF/PR/HR21/52410023. L. V. would like to thank Comunidad de Madrid (TRANSNANOAVANSENS program: S2018-NMT-4349) and E.V. García-Frutos for her assistance during the AFM experiments

Attomolar detection of hepatitis C virus core protein powered by molecular antenna-like effect in a graphene field-effect aptasensor

This study presents the development of a lab-on-a-chip (LoC) by integrating a graphene field-effect transistor (FET) chip with a programmable microfluidic device for DNA detection. The real-time biochemical events on the graphene FET chip were monitored through Dirac voltage shift data from the portable graphene curve reader with changes dependent on the fluidic flow into the sensing interface by a fully automated programmable microfluidic system. High sensitivity with high reliability can be obtained with a nine-graphene sensor layout on a single chip. The portable graphene curve reader also provides a tunable electrical parameter setup and straightforward data acquisition. Fluidic control was performed through a multi-position valve, allowing sequential commands for liquid injection into the polydimethylsiloxane (PDMS) flow cell mounted on the sensing chip. The flow cell design with impinging jet geometry and the microfluidic system packaging offer high precision and portability as a less laborious and low-cost sensing setup. The merged system allows for various functionalities, including probe DNA (pDNA) immobilization, a blocking step, and DNA hybridization with stable signal output autonomously, even in a long-run experimental setup. As a DNA sensor, the proposed prototype has demonstrated a high sensitivity of ~44 mV/decade of target DNA concentration, with an outstanding limit of detection (LoD) of ~0.642 aM, making it one of the most sensitive sensors reported up to date. The programmable device has demonstrated essential versatilities for biomolecular detection in a fully portable and automated platform.This research is supported by PORTGRAPHE-Control of Port and Douro Wines authenticity using graphene DNA sensors project co-funded by Fundação para a Ciência e a Tecnologia (FCT) Portugal (PTDC/BIA-MOL/31069/2017) and the ERDF through COMPETE2020 (POCI-01–0145-FEDER-031069). One of the authors (Telma Domingues) acknowledges a Ph.D. grant from Fundação para a Ciência e a Tecnologia (FCT) Portugal (SFRH/BD/08181/2020). FCT partially supported University of Minho´s research in the Strategic Funding UIDB/04650/2020

Innovative systems for mucosal and transmucosal drug administration

Negli ultimi anni è stata rivolta grande attenzione, in campo farmaceutico, alla messa a punto di sistemi di rilascio di farmaci da somministrare per vie alternative a quella orale, quali le vie buccale e vaginale, attraverso cui è possibile realizzare una somministrazione mucosale o transmucosale di farmaci. Una prima fase della ricerca è stata dedicata allo studio di nuovi sali di polimeri di origine naturale (chitosano e acido ialuronico) con migliori proprietà di mucoadesione e/o promozione dell’assorbimento rispetto ad altri sali già descritti in letteratura. In particolare sono state valutate le proprietà di mucoadesione, di promozione dell’assorbimento e di biocompatibilità del chitosano ascorbato e dell’acido ialuronico in combinazione con aminoacidi basici. La seconda fase della ricerca ha avuto come obiettivi l’allestimento e la valutazione “in vitro” ed “ex vivo” di formulazioni ad applicazione buccale e vaginale a base dei polimeri sopra citati e di derivati del chitosano per la veicolazione di farmaci ad azione locale o sistemica. In particolare sono stati sviluppati: film ad applicazione buccale per la somministrazione di diidroergotamina mesilato; medicazioni “sponge-like” per la somministrazione vaginale di farmaci; sistemi nanoparticellari per la somministrazione vaginale di farmaci peptidici; sistemi gelificabili “in situ” per la somministrazione buccale di benzidamina.In the last years a great deal of attention has been devoted, in the pharmaceutical field, to the development of drug delivery systems for administration through routes other than the oral and parenteral ones. Among alternative routes, buccal and vaginal ones are gaining an increasing interest. Through such routes it is possible to realize a mucosal (local effect) and a transmucosal (systemic effect) administration of drugs. A first phase of the research dealt with the investigations of new salts of polymers of natural origin (such as chitosan and hyaluronic acid) with improved mucoadhesion and/or penetration enhancer properties with respect to other salts already described in literature. In particular the mucoadhesion, penetration enhancement properties and biocompatibility of chitosan ascorbate and hyaluronic acid in combination with basic aminoacids have been studied. A second phase of the research had as goals the preparation and in vivo and ex vivo evaluation of formulations based on the above mentioned polymers or chitosan derivatives to be applied on buccal and vaginal mucosae. In particular were developed films for buccal administration of dihydroergotamine mesylate; polymeric sponges for vaginal administration of drugs; nanoparticle systems for vaginal administration of peptidic drugs; in situ gelling systems for buccal administration of benzidamine