Optical visualization of ultrathin mica flakes on semitransparent gold substrates

We show that optical visualization of ultrathin mica flakes on metallic substrates is viable using semitransparent gold as substrates. This enables to easily localize mica flakes and rapidly estimate their thickness directly on gold substrates by conventional optical reflection microscopy. We experimentally demonstrate it by comparing optical images with atomic force microscopy images of mica flakes on semitransparent gold. Present results open the possibility for simple and rapid characterization of thin mica flakes as well as other thin sheets directly on metallic substrates

Nanoscale measurement of the dielectric constant of supported lipid bilayers in aqueous solutions with electrostatic force microscopy

We present what is, to our knowledge, the first experimental demonstration of dielectric constant measurement and quantification of supported lipid bilayers in electrolyte solutions with nanoscale spatial resolution. The dielectric constant was quantitatively reconstructed with finite element calculations by combining thickness information and local polarization forces which were measured using an electrostatic force microscope adapted to work in a liquid environment. Measurements of submicrometric dipalmitoylphosphatidylcholine lipid bilayer patches gave dielectric constants of εr ∼ 3, which are higher than the values typically reported for the hydrophobic part of lipid membranes (εr ∼ 2) and suggest a large contribution of the polar headgroup region to the dielectric response of the lipid bilayer. This work opens apparently new possibilities in the study of biomembrane electrostatics and other bioelectric phenomena

Dielectric properties and lamellarity of single liposomes measured by in-liquid scanning dielectric microscopy

Liposomes are widely used as drug delivery carriers and as cell model systems. Here, we measure the dielectric properties of individual liposomes adsorbed on a metal electrode by in-liquid scanning dielectric microscopy in force detection mode. From the measurements the lamellarity of the liposomes, the separation between the lamellae and the specifc capacitance of the lipid bilayer can be obtained. As application we considered the case of non-extruded DOPC liposomes with radii in the range ~100-800 nm. Uni-, bi- and tri-lamellar liposomes have been identifed, with the largest population corresponding to bi-lamellar liposomes. The interlamellar separation in the bi-lamellar liposomes is found to be below ~10 nm in most instances. The specifc capacitance of the DOPC lipid bilayer is found to be ~0.75 µF/cm2 in excellent agreement with the value determined on solid supported planar lipid bilayers. The lamellarity of the DOPC liposomes shows the usual correlation with the liposome's size. No correlation is found, instead, with the shape of the adsorbed liposomes. The proposed approach ofers a powerful label-free and non invasive method to determine the lamellarity and dielectric properties of single liposom

High IGKC-Expressing Intratumoral Plasma Cells Predict Response to Immune Checkpoint Blockade.

Este artículo ha sido publicado en la revista International Journal of Molecular Sciences. Esta versión tiene Licencia Creative Commons CC-BYResistance to Immune Checkpoint Blockade (ICB) constitutes the current limiting factor for the optimal implementation of this novel therapy, which otherwise demonstrates durable responses with acceptable toxicity scores. This limitation is exacerbated by a lack of robust biomarkers. In this study, we have dissected the basal TME composition at the gene expression and cellular levels that predict response to Nivolumab and prognosis. BCR, TCR and HLA profiling were employed for further characterization of the molecular variables associated with response. The findings were validated using a single-cell RNA-seq data of metastatic melanoma patients treated with ICB, and by multispectral immunofluorescence. Finally, machine learning was employed to construct a prediction algorithm that was validated across eight metastatic melanoma cohorts treated with ICB. Using this strategy, we have unmasked a major role played by basal intratumoral Plasma cells expressing high levels of IGKC in efficacy. IGKC, differentially expressed in good responders, was also identified within the Top response-related BCR clonotypes, together with IGK variants. These results were validated at gene, cellular and protein levels; CD138+ Plasma-like and Plasma cells were more abundant in good responders and correlated with the same RNA-seq-defined fraction. Finally, we generated a 15-gene prediction model that outperformed the current reference score in eight ICB-treated metastatic melanoma cohorts. The evidenced major contribution of basal intratumoral IGKC and Plasma cells in good response and outcome in ICB in metastatic melanoma is a groundbreaking finding in the field beyond the role of T lymphocytes

Propietats estructurals i mecàniques a la nanoescala de les bicapes lipídiques en aire

[eng] Cell membranes are 2-D heterogeneous fluid systems that show nanoscale structures of great interest because of its importance in membrane functions. Due to the inherent complexity of natural membranes, the study of model membranes is essential to obtain important information about membranes. The small lateral size and tiny variation of the height of the lipid structures require the use of nanoscale characterization techniques. However, the requirement of liquid environment to preserve the integrity of the membrane limits the number of techniques that can be applied in the study of the physical and chemical properties of biomembranes. The objective of the present thesis was to develop a procedure to prepare model lipid bilayers stable in air environment and showing physicochemical properties as close as possible to their equivalent in liquid media. For the first experiments DOPC was selected as model to adapt the existing protocols for hydrated spin-coated samples to the new protocol for dried samples. In the structural study it was demonstrated that the dewetting pattern described by previous authors not only depends on the proximity of the layer to the substrate because also depends on the lipid concentration on the coating solution. Apart from that the presence of a continuous monolayer in contact with the mica substrate was demonstrated contrary to previous results with other lipids. The force spectroscopy measurements represented the first study on single bilayers in air and surprisingly demonstrated that the lipid layers in air presented similar mechanical properties than hydrated samples. Secondly the preparation protocol was adapted for phospholipids with different characteristics, the saturated phosphocholines. Contrary to unsaturated lipids, which present in general a high fluidity at ambient temperature, this is not always true for saturated lipids. For phosphocholines with short hidrocarbonated chains the melting temperature is low and then they are fluid, but for long hidrocarbonated chains the melting temperature is high and they can present a non-fluid behavior. For this reason different saturated lipids with different chain lengths were studied (DLPC, DMPC, DPPC and DSPC). The results with these lipids demonstrate that the conventional protocol of spin-coating induces the interdigitation of certain areas of the samples for the cases of DPPC and DSPC. The effect of the presence of alcohols and lateral tension were studied, being the rotation speed determined as causative of this phenomenon. Finally, we studied the more important case of multicomponent samples. In this study, ternary samples made of DOPC, Sphingomyelin and Cholesterol, relevant for lipid raft models, were selected. The corresponding binary samples with Cholesterol were also studied to determine separately the effect of Cholesterol in each of the components (DOPC and SM). Results unambiguously showed that air stable multicomponent lipid bilayers can be prepared by the spin coating technique with structural and mechanical properties remarkably resembling those of the equivalent systems in liquid media, specially on what concerns phase segregation phenomena. In particular, and more importantly, we showed that the ternary mixtures of DOPC/Chol/SM under dry conditions showed also the presence of lipid rafts. In summary, the present thesis has showed that it is possible to prepare lipid bilayer model systems morphologically stable in dry air conditions that present similar topography and mechanical properties than hydrated samples. Therefore it opens the possibility to characterize these systems with nanoscale techniques that until now have not been applied to them, thus offering the possibility to clarify physicochemical properties of lipid bilayer model systems that still today remain unexplored in spite of the vast literature of lipid bilayer model systems.[cat] Les membranes cel•lulars són fluids en 2D heterogenis que presenten estructures a la nanoescala (dominis) de gran interès degut a la seva importància en les funcions de membrana. Com a conseqüència de la inherent complexitat de les membranes naturals, els estudis amb membranes naturals són essencials per tal d’obtenir informació sobre les propietats fisicoquímiques de les membranes. No obstant això, el requeriment de medi líquid, per preservar la integritat de la bicapa lipídica, limita el nombre de tècniques que es poden aplicar en l'estudi de les propietats físiques i químiques de les biomembranes. L'objectiu d'aquesta tesi és desenvolupar un procediment per preparar bicapes lipídiques model estables en aire i que mostrin propietats fisicoquímiques similars a la dels seus equivalents en medi líquid. Per aquest fi es va utilitzar la tècnica d’spin-coating per preparar les mostres, tant monocomponent (fosfolípids insaturats i saturats) com multicomponent (barreges binàries i models de lipid raft). Les mostres varen ser caracteritzades amb microscòpia de forces atòmiques (AFM) i es va demostrar la seva similitud amb les mostres hidratades tant mecànicament com estructuralment. En resum, enquesta tesi ha demostra que és possible preparar sistemes l model de bicapa lipídica morfològicament estables en aire sec que presenten topografia similar i les propietats mecàniques que les mostres hidratades. Per tant, s'obre la possibilitat de caracteritzar aquests sistemes a nanoescala amb tècniques que fins ara no s'han aplicat a ells, oferint així la possibilitat d'aclarir les propietats fisicoquímiques de sistemes lipídics model bicapa que encara avui romanen inexplorades tot i la vasta literatura del model de bicapa lipídica sistemes

Nanoscale structural and mechanical properties of lipid bilayers in air environment / Propietats estructurals i mecàniques a la nanoescala de les bicapes lipídiques en aire

Cell membranes are 2-D heterogeneous fluid systems that show nanoscale structures of great interest because of its importance in membrane functions. Due to the inherent complexity of natural membranes, the study of model membranes is essential to obtain important information about membranes. The small lateral size and tiny variation of the height of the lipid structures require the use of nanoscale characterization techniques. However, the requirement of liquid environment to preserve the integrity of the membrane limits the number of techniques that can be applied in the study of the physical and chemical properties of biomembranes. The objective of the present thesis was to develop a procedure to prepare model lipid bilayers stable in air environment and showing physicochemical properties as close as possible to their equivalent in liquid media. For the first experiments DOPC was selected as model to adapt the existing protocols for hydrated spin-coated samples to the new protocol for dried samples. In the structural study it was demonstrated that the dewetting pattern described by previous authors not only depends on the proximity of the layer to the substrate because also depends on the lipid concentration on the coating solution. Apart from that the presence of a continuous monolayer in contact with the mica substrate was demonstrated contrary to previous results with other lipids. The force spectroscopy measurements represented the first study on single bilayers in air and surprisingly demonstrated that the lipid layers in air presented similar mechanical properties than hydrated samples. Secondly the preparation protocol was adapted for phospholipids with different characteristics, the saturated phosphocholines. Contrary to unsaturated lipids, which present in general a high fluidity at ambient temperature, this is not always true for saturated lipids. For phosphocholines with short hidrocarbonated chains the melting temperature is low and then they are fluid, but for long hidrocarbonated chains the melting temperature is high and they can present a non-fluid behavior. For this reason different saturated lipids with different chain lengths were studied (DLPC, DMPC, DPPC and DSPC). The results with these lipids demonstrate that the conventional protocol of spin-coating induces the interdigitation of certain areas of the samples for the cases of DPPC and DSPC. The effect of the presence of alcohols and lateral tension were studied, being the rotation speed determined as causative of this phenomenon. Finally, we studied the more important case of multicomponent samples. In this study, ternary samples made of DOPC, Sphingomyelin and Cholesterol, relevant for lipid raft models, were selected. The corresponding binary samples with Cholesterol were also studied to determine separately the effect of Cholesterol in each of the components (DOPC and SM). Results unambiguously showed that air stable multicomponent lipid bilayers can be prepared by the spin coating technique with structural and mechanical properties remarkably resembling those of the equivalent systems in liquid media, specially on what concerns phase segregation phenomena. In particular, and more importantly, we showed that the ternary mixtures of DOPC/Chol/SM under dry conditions showed also the presence of lipid rafts. In summary, the present thesis has showed that it is possible to prepare lipid bilayer model systems morphologically stable in dry air conditions that present similar topography and mechanical properties than hydrated samples. Therefore it opens the possibility to characterize these systems with nanoscale techniques that until now have not been applied to them, thus offering the possibility to clarify physicochemical properties of lipid bilayer model systems that still today remain unexplored in spite of the vast literature of lipid bilayer model systems.Les membranes cel•lulars són fluids en 2D heterogenis que presenten estructures a la nanoescala (dominis) de gran interès degut a la seva importància en les funcions de membrana. Com a conseqüència de la inherent complexitat de les membranes naturals, els estudis amb membranes naturals són essencials per tal d’obtenir informació sobre les propietats fisicoquímiques de les membranes. No obstant això, el requeriment de medi líquid, per preservar la integritat de la bicapa lipídica, limita el nombre de tècniques que es poden aplicar en l'estudi de les propietats físiques i químiques de les biomembranes. L'objectiu d'aquesta tesi és desenvolupar un procediment per preparar bicapes lipídiques model estables en aire i que mostrin propietats fisicoquímiques similars a la dels seus equivalents en medi líquid. Per aquest fi es va utilitzar la tècnica d’spin-coating per preparar les mostres, tant monocomponent (fosfolípids insaturats i saturats) com multicomponent (barreges binàries i models de lipid raft). Les mostres varen ser caracteritzades amb microscòpia de forces atòmiques (AFM) i es va demostrar la seva similitud amb les mostres hidratades tant mecànicament com estructuralment. En resum, enquesta tesi ha demostra que és possible preparar sistemes l model de bicapa lipídica morfològicament estables en aire sec que presenten topografia similar i les propietats mecàniques que les mostres hidratades. Per tant, s'obre la possibilitat de caracteritzar aquests sistemes a nanoescala amb tècniques que fins ara no s'han aplicat a ells, oferint així la possibilitat d'aclarir les propietats fisicoquímiques de sistemes lipídics model bicapa que encara avui romanen inexplorades tot i la vasta literatura del model de bicapa lipídica sistemes

Polymeric nanoparticles from nano-emulsion templating: Plenty of room of applications

Resumen del trabajo presentado en las 44º Jornadas del CED-Annual Meeting CED, organizadas por el Comité Español de la Detergencia, Tensoactivos y Afines (CED), celebradas en Barcelona los días 12 y 13 de marzo de 2014.Polymeric nanoparticles focus a great deal of interest because of their high specific surface and versatility. In the recent years their use is spreading from the basic research to the industry, with applications from environment to nanomedicine. Among the different existing approaches for their preparation, our group has focused to the fabrication of polymeric nanoparticles using nano-emulsions as templates. Nano-emulsions are emulsions with a droplet size typically in the range 20-200nm. Due to the small droplet size they are stable against sedimentation or creaming and their aspect is transparent to translucent [1]. Nano-emulsions are generally prepared by high-energy methods (e.g. high-shear stirring) but, in the lasts years, low-energy methods are the focus of increasing attention [2]. Low-energy emulsification methods are based on the use of the chemical energy stored in the system which is released during emulsification. These methods allow obtaining droplets with smaller size and lower polydispersity than high-energy methods. In addition, the energy input is considerably reduced. The characteristic properties of nano-emulsions (size, stability, safety), make them appropriate candidates as templates for nanoparticle fabrication [3,4]. Using this approach, different materials can be prepared depending on the final application. This presentation will focus on how the components and composition, the size, the inner-core and the surface of the nanoparticle can be tuned to obtain polymeric nanoparticles with given properties. This wide range of tunable characteristics open the door, at the same time, to a wide range of applications. In this context, biomedical applications of the systems prepared by our group (e.g. polymeric nanoparticle dispersions of Poly(lactic-co-glycolic acid) (PLGA), ethyl cellulose (EC) with controlled size and stability) will be described. Furthermore, drug encapsulation and nanoparticle functionalization (with monoclonal antibodies, dendrons, etc.) to fine-tune their properties for their final target or application will be also described

Polymeric nanoparticles by nano-emulsion templating for biomedical applications

Trabajo presentado en el V Workshop CBN 2013, 5ª Jornada del Departamento de Nanotecnología Química y Biomolecular del IQAC, celebrado en Barcelona el 17 de octubre de 2013

Design of parenteral MNP-loaded PLGA nanoparticles by a low-energy emulsification approach as theragnostic platforms for intravenous or intratumoral administration

Encapsulation of magnetic nanoparticles (MNP) into PLGA nanoparticles has been achieved by nano-emulsion templating using for the first time both, a low-energy emulsification method as well as biocompatible components accepted for pharmaceuticals intended for human use. The incorporation of MNP by nano-emulsion templating method proposed in this work has been investigated in two different systems applying mild process conditions and is shown to be simple and versatile, providing stable MNP-loaded PLGA nanoparticles with tunable size and MNP concentration. MNP-loaded PLGA nanoparticles showed sizes below 200nm by DLS and 50nm by TEM, and mean MNP loading per PLGA nanoparticle of 1 to 4, depending on the nanoparticle dispersion composition. Physical-chemical features suggest that the MNP-loaded PLGA nanoparticles obtained are good candidates for intravenous or intratumoral administration.Financial support from MINECO (grant CTQ2014-52687-C3-1-P); Generalitat de Catalunya (grant 2014-SGR-1655), and CIBER-BBN are acknowledged. 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. Cristina Fornaguera is grateful to AGAUR for their Predoctoral Fellowship (grant FI-DGR 2012).Peer reviewe

Design and characterization of polymeric nanoparticles with encapsulated drugs for neurodegenerative disease treatment

Trabajo presentado en el VI Workshop CBN 2014, celebrado en Barcelona el 16 de octubre de 2014.Nanoparticles (NPs) are solid materials with sizes between 20 – 500nm, which can be obtained from a large diversity of materials and methods. Properties and applications can be achieved by choosing the right type of materials and methods. The use of polymeric NPs improves drug properties such as half-life in blood, specific targeting and enhanced intracellular penetration, showing potential in pharmaceutical applications (e.g. neurodegenerative disease treatments). Neurodegenerative diseases generally present axonal degeneration that leads to neuronal death, and are responsible of neurologic disability and cognitive or motor problems. Their pathogenic mechanisms are related with accumulation, aggregation and modification of certain proteins or molecules, homeostasis deregulations, immunological dysfunctions or viral infections