Nanocomposite Scaffolds for Monitoring of Drug Diffusion in Three-Dimensional Cell Environments by Surface-Enhanced Raman Spectroscopy

[EN]Monitoring dynamic processes in complex cellular environments requires the integration of uniformly distributed detectors within such three-dimensional (3D) networks, to an extent that the sensor could provide real-time information on nearby perturbations in a non-invasive manner. In this context, the development of 3D-printed structures that can function as both sensors and cell culture platforms emerges as a promising strategy, not only for mimicking a specific cell niche but also toward identifying its characteristic physicochemical conditions, such as concentration gradients. We present herein a 3D cancer model that incorporates a hydrogel-based scaffold containing gold nanorods. In addition to sustaining cell growth, the printed nanocomposite inks display the ability to uncover drug diffusion profiles by surface-enhanced Raman scattering, with high spatiotemporal resolution. We additionally demonstrate that the acquired information could pave the way to designing novel strategies for drug discovery in cancer therapy, through correlation of drug diffusion with cell death.J.P. acknowledges an FPU fellowship from the Spanish Ministry of Science, Innovation and Universities. L.M.L.-M. acknowledges funding from the European Research Council (Grants ERC AdG 787510, 4DbioSERS) and the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant MDM-2017-0720). A.C. was funded by MICINN (Grant PID2019-108787RB-I00 (FEDER/EU)) and the European Research Council (ERC Consolidator Grant 819242)

Multiplex SERS Detection of Metabolic Alterations in Tumor Extracellular Media

The composition and intercellular interactions of tumor cells in the tissues dictate the biochemical and metabolic properties of the tumor microenvironment. The metabolic rewiring has a profound impact on the properties of the microenvironment, to an extent that monitoring such perturbations could harbor diagnostic and therapeutic relevance. A growing interest in these phenomena has inspired the development of novel technologies with sufficient sensitivity and resolution to monitor metabolic alterations in the tumor microenvironment. In this context, surface-enhanced Raman scattering (SERS) can be used for the label-free detection and imaging of diverse molecules of interest among extracellular components. Herein, the application of nanostructured plasmonic substrates comprising Au nanoparticles, self-assembled as ordered superlattices, to the precise SERS detection of selected tumor metabolites, is presented. The potential of this technology is first demonstrated through the analysis of kynurenine, a secreted immunomodulatory derivative of the tumor metabolism and the related molecules tryptophan and purine derivatives. SERS facilitates the unambiguous identification of trace metabolites and allows the multiplex detection of their characteristic fingerprints under different conditions. Finally, the effective plasmonic SERS substrate is combined with a hydrogel-based three-dimensional cancer model, which recreates the tumor microenvironment, for the real-time imaging of metabolite alterations and cytotoxic effects on tumor cells.J.P. acknowledges an FPU fellowship from the Spanish Ministry of Science, Innovation and Universities. L.M.L.-M. acknowledges funding from the European Research Council (ERC AdG 787510, 4DbioSERS) and the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant No. MDM-2017-0720). C.G.-A. acknowledges a Juan de la Cierva Fellowship from the Spanish Ministry of Science, Innovation and Universities (FJCI-2016-28887). The authors thank Dr. J. Calvo and Dr. D. Otaegui at CIC biomaGUNE for support with LC/ESI-HRMS measurements. The work of A.C. was supported by the Basque Department of Industry, Tourism and Trade (Elkartek), and the department of education (IKERTALDE IT1106-16, also participated by A. Gomez-Munoz), the BBVA foundation, the MINECO (SAF2016-79381-R (FEDER/EU); Severo Ochoa Excellence Program SEV-2016-0644-18-1; Excellence Networks SAF2016-81975-REDT), European Training Networks Project (H2020-MSCA-ITN-308 2016 721532), the AECC (IDEAS175CARR, GCTRA18006CARR), La Caixa Foundation (HR17-00094), and the European Research Council (starting Grant 336343, PoC 754627). CIBERONC was co-funded with FEDER funds and funded by ISCIII. A.M. acknowledges funding from the European Research Council (Consolidator Grant 819242) and the Spanish Ministry of Science, Innovation and Universities for the excellence program SEV-2015-049

Remodeling arteries: studying the mechanical properties of 3D-bioprinted hybrid photoresponsive materials.

3D-printed cell models are currently in the spotlight of medical research. Whilst significant advances have been made, there are still aspects that require attention to achieve more realistic models which faithfully represent the in vivo environment. In this work we describe the production of an artery model with cyclic expansive properties, capable of mimicking the different physical forces and stress factors that cells experience in physiological conditions. The artery wall components are reproduced using 3D printing of thermoresponsive polymers with inorganic nanoparticles (NPs) representing the outer tunica adventitia, smooth muscle cells embedded in extracellular matrix representing the tunica media, and finally a monolayer of endothelial cells as the tunica intima. Cyclic expansion can be induced thanks to the inclusion of photo-responsive plasmonic NPs embedded within the thermoresponsive ink composition, resulting in changes in the thermoresponsive polymer hydration state and hence volume, in a stimulated on-off manner. By changing the thermoresponsive polymer composition, the transition temperature and pulsatility can be efficiently tuned. We show the direct effect of cyclic expansion and contraction on the overlying cell layers by analyzing transcriptional changes in mechanoresponsive mesenchymal genes associated with such microenvironmental physical cues. The technique described herein involving stimuli-responsive 3D printed tissue constructs, also described as four- dimensional (4D) printing, offers a novel approach for the production of dynamic biomodels.Financial support is acknowledged from the MCIN/AEI/ 10.13039/501100011033 through grant # PID2019-108854RAI00. C. G. A. thanks to the Ministerio de Ciencia e Innovacio´n (MCIN) for a Juan de la Cierva Incorporacio´n Fellowship (IJC2019-040827-I). M. S.-A. is recipient of a Ramo´n y Cajal contract and a ‘‘Generacio´n de Conocimiento’’ grant from the Ministerio de Ciencia e Innovacio´n (RYC2020-029690-I and PID2021-128106NA-I00). MAdP is coordinator and PL of ‘‘AtheroConvergence’’ La Caixa Foundation Health Research consortium (HR20-00075). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MCIN and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S). We acknowledge ALBA for provision of synchrotron radiation facilities. We would like to thank Dr Marc Malfois for assistance in using BL11-NCD beamline, and Unai Cossı´o and Daniel Padro for help with image analysis.S

Carbonaceous filler type and content dependence of the physical-chemical and electromechanical properties of thermoplastic elastomer polymer composites

Graphene, carbon nanotubes (CNT), and carbon nanofibers (CNF) are the most studied nanocarbonaceous fillers for polymer-based composite fabrication due to their excellent overall properties. The combination of thermoplastic elastomers with excellent mechanical properties (e.g., styrene-b-(ethylene-co-butylene)-b-styrene (SEBS)) and conductive nanofillers such as those mentioned previously opens the way to the preparation of multifunctional materials for large-strain (up to 10% or even above) sensor applications. This work reports on the influence of different nanofillers (CNT, CNF, and graphene) on the properties of a SEBS matrix. It is shown that the overall properties of the composites depend on filler type and content, with special influence on the electrical properties. CNT/SEBS composites presented a percolation threshold near 1 wt.% filler content, whereas CNF and graphene-based composites showed a percolation threshold above 5 wt.%. Maximum strain remained similar for most filler types and contents, except for the largest filler contents (1 wt.% or more) in graphene (G)/SEBS composites, showing a reduction from 600% for SEBS to 150% for 5G/SEBS. Electromechanical properties of CNT/SEBS composite for strains up to 10% showed a gauge factor (GF) varying from 2 to 2.5 for different applied strains. The electrical conductivity of the G and CNF composites at up to 5 wt.% filler content was not suitable for the development of piezoresistive sensing materials. We performed thermal ageing at 120 °C for 1, 24, and 72 h for SEBS and its composites with 5 wt.% nanofiller content in order to evaluate the stability of the material properties for high-temperature applications. The mechanical, thermal, and chemical properties of SEBS and the composites were identical to those of pristine composites, but the electrical conductivity decreased by near one order of magnitude and the GF decreased to values between 0.5 and 1 in aged CNT/SEBS composites. Thus, the materials can still be used as large-deformation sensors, but the reduction of both electrical and electromechanical response has to be considered.ThisworkwassupportedbythePortugueseFoundationforScienceandTechnology(FCT)intheframework of the StrategicFunding UID/FIS/04650/2013 and UID/CTM/50025/2013. Financial support was also provided by ERDF funds through the Portuguese Operational Programme for Competitiveness and Internationalization-COMPETE 2020, and national funds through FCT, under projects PTDC/EEI-SII/5582/2014 and PTDC/CTM-ENE/5387/2014. PC thanks to FCT by financial support for the SFRH/BPD/110914/2015 grant. Financial support from the Spanish Ministry of Economy and Competitiveness (MINECO) through the project MAT2016-76039-C4-3-R (AEI/FEDER, UE) (including the FEDER financial support) and from the Basque Government Industry Department under the ELKARTEK (ACTIMAT project) and HAZITEK programare also acknowledged.info:eu-repo/semantics/publishedVersio

Multiplex SERS Detection of Metabolic Alterations in Tumor Extracellular Media

The composition and intercellular interactions of tumor cells in the tissues dictate the biochemical and metabolic properties of the tumor microenvironment. The metabolic rewiring has a profound impact on the properties of the microenvironment, to an extent that monitoring such perturbations could harbor diagnostic and therapeutic relevance. A growing interest in these phenomena has inspired the development of novel technologies with sufficient sensitivity and resolution to monitor metabolic alterations in the tumor microenvironment. In this context, surface-enhanced Raman scattering (SERS) can be used for the label-free detection and imaging of diverse molecules of interest among extracellular components. Herein, the application of nanostructured plasmonic substrates comprising Au nanoparticles, self-assembled as ordered superlattices, to the precise SERS detection of selected tumor metabolites, is presented. The potential of this technology is first demonstrated through the analysis of kynurenine, a secreted immunomodulatory derivative of the tumor metabolism and the related molecules tryptophan and purine derivatives. SERS facilitates the unambiguous identification of trace metabolites and allows the multiplex detection of their characteristic fingerprints under different conditions. Finally, the effective plasmonic SERS substrate is combined with a hydrogel-based three-dimensional cancer model, which recreates the tumor microenvironment, for the real-time imaging of metabolite alterations and cytotoxic effects on tumor cells.J.P. acknowledges an FPU fellowship from the Spanish Ministry of Science, Innovation and Universities. L.M.L.-M. acknowledges funding from the European Research Council (ERC AdG 787510, 4DbioSERS) and the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant No. MDM-2017-0720). C.G.-A. acknowledges a Juan de la Cierva Fellowship from the Spanish Ministry of Science, Innovation and Universities (FJCI-2016-28887). The authors thank Dr. J. Calvo and Dr. D. Otaegui at CIC biomaGUNE for support with LC/ESI-HRMS measurements. The work of A.C. was supported by the Basque Department of Industry, Tourism and Trade (Elkartek), and the department of education (IKERTALDE IT1106-16, also participated by A. Gomez-Munoz), the BBVA foundation, the MINECO (SAF2016-79381-R (FEDER/EU); Severo Ochoa Excellence Program SEV-2016-0644-18-1; Excellence Networks SAF2016-81975-REDT), European Training Networks Project (H2020-MSCA-ITN-308 2016 721532), the AECC (IDEAS175CARR, GCTRA18006CARR), La Caixa Foundation (HR17-00094), and the European Research Council (starting Grant 336343, PoC 754627). CIBERONC was co-funded with FEDER funds and funded by ISCIII. A.M. acknowledges funding from the European Research Council (Consolidator Grant 819242) and the Spanish Ministry of Science, Innovation and Universities for the excellence program SEV-2015-049

Multiplex SERS Detection of Metabolic Alterations in Tumor Extracellular Media

The composition and intercellular interactions of tumor cells in the tissues dictate the biochemical and metabolic properties of the tumor microenvironment. The metabolic rewiring has a profound impact on the properties of the microenvironment, to an extent that monitoring such perturbations could harbor diagnostic and therapeutic relevance. A growing interest in these phenomena has inspired the development of novel technologies with sufficient sensitivity and resolution to monitor metabolic alterations in the tumor microenvironment. In this context, surface‐enhanced Raman scattering (SERS) can be used for the label‐free detection and imaging of diverse molecules of interest among extracellular components. Herein, the application of nanostructured plasmonic substrates comprising Au nanoparticles, self‐assembled as ordered superlattices, to the precise SERS detection of selected tumor metabolites, is presented. The potential of this technology is first demonstrated through the analysis of kynurenine, a secreted immunomodulatory derivative of the tumor metabolism and the related molecules tryptophan and purine derivatives. SERS facilitates the unambiguous identification of trace metabolites and allows the multiplex detection of their characteristic fingerprints under different conditions. Finally, the effective plasmonic SERS substrate is combined with a hydrogel‐based three‐dimensional cancer model, which recreates the tumor microenvironment, for the real‐time imaging of metabolite alterations and cytotoxic effects on tumor cells.J.P. acknowledges an FPU fellowship from the Spanish Ministry of Science, Innovation and Universities. L.M.L.‐M. acknowledges funding from the European Research Council (ERC AdG 787510, 4DbioSERS) and the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant No. MDM‐2017‐0720). C.G.‐A. acknowledges a Juan de la Cierva Fellowship from the Spanish Ministry of Science, Innovation and Universities (FJCI‐2016‐28887). The authors thank Dr. J. Calvo and Dr. D. Otaegui at CIC biomaGUNE for support with LC/ESI‐HRMS measurements. The work of A.C. was supported by the Basque Department of Industry, Tourism and Trade (Elkartek), and the department of education (IKERTALDE IT1106‐16, also participated by A. Gomez‐Muñoz), the BBVA foundation, the MINECO (SAF2016‐79381‐R (FEDER/EU); Severo Ochoa Excellence Program SEV‐2016‐0644‐18‐1; Excellence Networks SAF2016‐81975‐REDT), European Training Networks Project (H2020‐MSCA‐ITN‐308 2016 721532), the AECC (IDEAS175CARR, GCTRA18006CARR), La Caixa Foundation (HR17‐00094), and the European Research Council (starting Grant 336343, PoC 754627). CIBERONC was co‐funded with FEDER funds and funded by ISCIII. A.M. acknowledges funding from the European Research Council (Consolidator Grant 819242) and the Spanish Ministry of Science, Innovation and Universities for the excellence program SEV‐2015‐0496.J.P. acknowledges an FPU fellowship from the Spanish Ministry of Science, Innovation and Universities. L.M.L.‐M. acknowledges funding from the European Research Council (ERC AdG 787510, 4DbioSERS) and the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant No. MDM‐2017‐0720). C.G.‐A. acknowledges a Juan de la Cierva Fellowship from the Spanish Ministry of Science, Innovation and Universities (FJCI‐2016‐28887). The authors thank Dr. J. Calvo and Dr. D. Otaegui at CIC biomaGUNE for support with LC/ESI‐HRMS measurements. The work of A.C. was supported by the Basque Department of Industry, Tourism and Trade (Elkartek), and the department of education (IKERTALDE IT1106‐16, also participated by A. Gomez‐Muñoz), the BBVA foundation, the MINECO (SAF2016‐79381‐R (FEDER/EU); Severo Ochoa Excellence Program SEV‐2016‐0644‐18‐1; Excellence Networks SAF2016‐81975‐REDT), European Training Networks Project (H2020‐MSCA‐ITN‐308 2016 721532), the AECC (IDEAS175CARR, GCTRA18006CARR), La Caixa Foundation (HR17‐00094), and the European Research Council (starting Grant 336343, PoC 754627). CIBERONC was co‐funded with FEDER funds and funded by ISCIII. A.M. acknowledges funding from the European Research Council (Consolidator Grant 819242) and the Spanish Ministry of Science, Innovation and Universities for the excellence program SEV‐2015‐0496.Peer reviewe