Introducción. Este trabajo se centró en la síntesis, caracterización físicoquímica, evalua- ción de su bioactividad y aplicaciones biomédicas de nuevos nanomateriales biohíbridos. La sepiolita es un silicato natural nanofibroso que presenta atractivas características como nanoportador para la liberación de ácidos nucleicos en células, entre otras aplicaciones in- novadoras en el área de las ciencias de la vida y de la biotecnología. Métodos. La caracteri- zación físico-química y evaluación biológica de los biohíbridos fue realizada usando proce- dimientos y técnicas novedosas, incluyendo entre otras AFM, TEM, microscopía confocal, TLVM, FTIR, Potencial Zeta, UV-vis, FACS, Western-blot, RT- qPCR. Resultados. Se obtuvieron nuevos biohíbridos mediante ensamblado de ácidos nucleicos con nanofibras de sepiolita. Se propusieron sus mecanismos de internalización y respuesta celular. Demostramos que la sepiolita es un nanoportador prometedor para la transferencia estable no-viral de ADN plasmídico en bacterias, células de mamíferos y humanas, con eficiencia optimizada. Debido a su capacidad de anclar varias biomoléculas, la sepiolita constituye también un nanoporta- dor para la vectorización simultánea de diferentes moléculas biológicas, incluyendo proteí- nas, nucleasas y anticuerpos. Adicionalmente, obtuvimos nuevos protocolos para aumentar significativamente la eficiencia de transformación bacterial, y para la extracción de ADN de bacterias, mediante métodos rápidos, seguros y económicos que no requieren la preparación de células competentes, representando una ventajosa alternativa a los costosos kits comerciales. En conclusión, la sepiolita es un producto de bajo costo, baja toxicidad y fluorescencia natural, con facilidad y conveniencia en métodos de síntesis de biohíbridos sepiolita/ADN, con posibilidad de aumentar su eficiencia de transfección, que junto a potenciales desarrollos futuros, representa una atractiva nanoplataforma para aplicaciones en nanomedicina y nanobiotecnología.

Oral presentation given at the XVI International Clay Conference, held in Granada (Spain) on July 17-21, 2017.

Sepiolite is a nanofibrous natural silicate that can be used as a nanocarrier because it can be naturally internalized into mammalian cells, due to its nano-size dimension. Therefore, deciphering the mechanisms of sepiolite cell internalization constitutes a question interesting biotechnology, for the use of sepiolite as nanocarrier, as well as environmental and public health concerns. Though it is low, the perfectly stable and natural intrinsic fluorescence of sepiolite nanofibers allows to follow their fate into cells by specifically sensitive technics. By combining fluorescence microscopy (including confocal analysis), time-lapse video microscopy, fluorescence activated cell sorting and transmission electron microscopy, we show that sepiolite can be spontaneously internalized into mammalian cells through both non-endocytic and endocytic pathways, macropinocytosis being one of the main pathways. Interestingly, exposure of the cells to endocytosis inhibitors, such as chloroquine, two-fold increase the efficiency of sepiolite-mediated gene transfer, in addition to the 100-fold increased resulting from sepiolite sonomechanical treatment. As sepiolite is able to bind various biological molecules, this nanoparticulate silicate could be a good candidate as a nanocarrier for simultaneous vectorization of diverse biological molecules.

This work was supported by La Ligue Nationale Contre le Cancer, ANR (Agence Nationale de la recherche, ANR-14-CE10-0010-02), AFM-Telethon and INCa (Institut National du Cancer, 2011-1-RT-01, 2011-1-PLBIO-09, 2013-1-PLBIO-14), the MINECO in Spain (projects MAT2012-31759 and MAT2015-71117-R), and the EU COST Action MP1202. Thanks are due to the French Embassy in Cuba and the Campus France for their contribution in partial financial support.

In this study, we present a new application of the transmission electron microscopy dark field mode for cell imaging. We have applied this imaging mode to two types of cellular systems: human HeLa cells to analyze molecular membrane systems and HC11 mouse mammary cells containing lipid molecule droplets. We have also studied a third macromolecular system, copolymer nanoparticles for the characterization of core-shell structures. We want to show the effective use of diffraction contrast, even on amorphous systems for increasing the image contrast and the signal/noise ratio. We discuss the TEM dark field advantages for the analysis of polymers and other macromolecular systems, including biological, systems compared to the bright field mode.

Nanofibers of sepiolite, a natural silicate belonging to the clay minerals family, might constitute a potential promising nanocarrier for the non-viral transfer of bio-molecules. We show here that sepiolite nanofibers efficiently bind different types of DNA molecules through electrostatic interactions, hydrogen bonding, cation bridges, and van der Waals forces. Moreover, Fourier-transform infrared spectroscopy identified the external silanol groups as the main sites of interaction with the DNA. Furthermore, as a proof of concept, we show that sepiolite is able to stably transfer plasmid DNA into mammalian cells and that the efficiency can be optimized. Indeed, sonication of sepiolite 100-fold stimulated DNA transfection efficiency. These results open the way to the use of sepiolite-based biohybrids as a novel class of nanoplatform for gene transfer with potential clinical applications.

Presentation given at the Fourth International Conference on Multifunctional, Hybrid and Nanomaterials, held in Barcelona (Spain) on March 9-13 March, 2015.

The synthesis and detailed characterization of gold nanoparticles (AuNPs) inside human hair has been achieved by treatment of hair with HAuCl4 in alkaline medium. The AuNPs, which show a strong red fluorescence under blue light, are generated inside the fiber and are arranged in the cortex in a remarkably regular pattern of whorls based on concentric circles, like a fingerprint. It opens an area of genuine nanocomposites with novel properties due to AuNPs inside the hair shaft.

The second volume of the “Sample Preparation Handbook for Transmission Electron Microscopy” contains descriptions of 14 preliminary and/or complementary sample preparation techniques and 21 thin slice preparation techniques for the transmission electron microscope (TEM).

This technique is used to produce a thin slice (measuring 3 mm in diameter and between 50- and 100-μm thick) without strain hardening, by thinning until a perforation is made in the center. The resulting hole has electron-transparent thin edges.

Given the different nature of the artifacts and drawbacks induced by mechanical, chemical, or ionic techniques, or even those involving changes in physical state, it is important to combine several techniques in order to confirm the intrinsic structure of a given material. The combination of techniques can vary, depending on the different properties of materials, their physical or chemical state, and their organization.