32 research outputs found
Wear behavior of PAEK, poly(aryl-ether-ketone), under physiological conditions, outlooks for performing these materials in the field of hip prosthesis
International audienceThis study is focused on performing tribological tests on new materials for orthopaedic implants applications, PAEK (poly aryl ether ketone) polymer group. The experiments were performed in physiological liquid, at 37 °C, for simulating the human body fluid. PAEK's tribological properties that are wear rate of polymers and wear mechanisms on common metallic alloys used as orthopaedic implants: Co-Cr, 316L SS and Ti-6Al-4V are compared to the gold standard used for hip joint prosthesis, the UHMWPE (ultra high molecular weight polyethylene) on the same metal alloys. PEEK (poly ether ether ketone) and PEKK (poly ether ketone ketone)/CF (carbon fibers) show the lowest wear rate on every counter metallic material; the system UHMWPE on any metal alloys exhibit the highest wear rate although having the lowest friction coefficient. From microscopic images and the evolution of the friction coefficient, a wear mechanism was suggested for each polymeric material
Characterising a human endogenous retrovirus(HERV)-derived tumour-associated antigen: enriched RNA-Seq analysis of HERV-K(HML-2) in mantle cell lymphoma cell lines.
BACKGROUND: The cell-surface attachment protein (Env) of the HERV-K(HML-2) lineage of endogenous retroviruses is a potentially attractive tumour-associated antigen for anti-cancer immunotherapy. The human genome contains around 100 integrated copies (called proviruses or loci) of the HERV-K(HML-2) virus and we argue that it is important for therapy development to know which and how many of these contribute to protein expression, and how this varies across tissues. We measured relative provirus expression in HERV-K(HML-2), using enriched RNA-Seq analysis with both short- and long-read sequencing, in three Mantle Cell Lymphoma cell lines (JVM2, Granta519 and REC1). We also confirmed expression of the Env protein in two of our cell lines using Western blotting, and analysed provirus expression data from all other relevant published studies. RESULTS: Firstly, in both our and other reanalysed studies, approximately 10% of the transcripts mapping to HERV-K(HML-2) came from Env-encoding proviruses. Secondly, in one cell line the majority of the protein expression appears to come from one provirus (12q14.1). Thirdly, we find a strong tissue-specific pattern of provirus expression. CONCLUSIONS: A possible dependency of Env expression on a single provirus, combined with the earlier observation that this provirus is not present in all individuals and a general pattern of tissue-specific expression among proviruses, has serious implications for future HERV-K(HML-2)-targeted immunotherapy. Further research into HERV-K(HML-2) as a possible tumour-associated antigen in blood cancers requires a more targeted, proteome-based, screening protocol that will consider these polymorphisms within HERV-K(HML-2). We include a plan (and necessary alignments) for such work
Functional protein-based nanomaterial produced in GRAS microorganism : a new platform for biotechnology
Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest
Integrating mechanical and biological control of cell proliferation through bioinspired multi-effector materials
In nature, cells respond to complex mechanical and biological stimuli whose understanding is required for tissue construction in regenerative medicine. However, the full replication of such bimodal effector networks is far to be reached. Engineering substrate roughness and architecture allows regulating cell adhesion, positioning, proliferation, differentiation and survival, and the external supply of soluble protein factors (mainly growth factors and hormones) has been long applied to promote growth and differentiation. Further, bioinspired scaffolds are progressively engineered as reservoirs for the in situ sustained release of soluble protein factors from functional topographies. We review here how research progresses toward the design of integrative, holistic scaffold platforms based on the exploration of individual mechanical and biological effectors and their further combination
Rapid quantification of 4-ethylphenol in wine using high-performance liquid chromatography with a fluorimetric detector
A versatile evaporation-assisted methodology based on the coffee-drop effect is described to deposit nanoparticles on surfaces, obtaining for the first time patterned gradients of protein nanoparticles (pNPs) by using a simple custom-made device. Fully controllable patterns with specific periodicities consisting of stripes with different widths and distinct nanoparticle concentration as well as gradients can be produced over large areas (∼10 cm²) in a fast (up to 10 mm²/min), reproducible, and cost-effective manner using an operational protocol optimized by an evolutionary algorithm. The developed method opens the possibility to decorate surfaces "a-la-carte" with pNPs enabling different categories of high-throughput studies on cell motility
Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles
CD44 is a multifunctional cell surface protein involved in proliferation and differentiation, angiogenesis and signaling. The expression of CD44 is up-regulated in several types of human tumors and particularly in cancer stem cells, representing an appealing target for drug delivery in the treatment of cancer. We have explored here several protein ligands of CD44 for the construction of self-assembling modular proteins designed to bind and internalize target cells. Among five tested ligands, two of them (A5G27 and FNI/II/V) drive the formation of protein-only, ring-shaped nanoparticles of about 14 nm that efficiently bind and penetrate CD44(+) cells by an endosomal route. The potential of these newly designed nanoparticles is evaluated regarding the need of biocompatible nanostructured materials for drug delivery in CD44-linked conditions
Stable nanovesicles formed by intrinsically planar bilayers
Quatsome nanovesicles, formed through the self-assembly of cholesterol (CHOL) and cetyltrimethylammonium bromide (CTAB) in water, have shown long-term stability in terms of size and morphology, while at the same time exhibiting high CHOL-CTAB intermolecular binding energies. We hypothesize that CHOL/CTAB quatsomes are indeed thermodynamically stable nanovesicles, and investigate the mechanism underlying their formation.This work was supported by funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 712949 (TECNIOspring PLUS) and from the Agency for Business Competitiveness of the Government of Catalonia. The production of quatsomes and part of their characterization has been performed by the ICTS “NANBIOSIS”, more specifically by the Biomaterial Processing and Nanostructuring Unit (U6), Unit of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) located at the Institute of Materials Science of Barcelona (ICMAB-CSIC). ICMAB-CSIC acknowledges support from the MINECO through the Severo Ochoa Programme for Centres of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S). Authors acknowledge financial support from the Spanish Ministry of Science and Innovation through grants “MOL4BIO” (PID2019-105622RB-I00), “SimBioSoft” (PID2021-124297NB-C33) and the FUNFUTURE-FIP-2020 Severo Ochoa project, from Generalitat de Catalunya through grant 2017-SGR-918, from CSIC through grant 2019AEP133, and from the European Commission through the H2020 PHOENIX project (contract no. 953110). We acknowledge the support of the Israel scienceIsrael science Foundation, grant 1117/2016, and thank Dr. Inbal Ionita for her professional assistance in the cryo-TEM analysis. We thank Jannik Nedergaard Pedersen and Beatrice Plazzotta for help with the SAXS measurements. The simulations reported here were performed using the Cori Supercomputing facility of the National Energy Research Scientific Computing Center (NERSC), a U.S. Department of Energy Office of Science User Facility operated under Contract No. DE-AC02-05CH11231.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe
In vivo architectonic stability of fully de novo designed protein-only nanoparticles
The fully de novo design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein-protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the in vivo stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids
Two-dimensional engineering of molecular nanoparticles for biological applications
El trabajo realizado en esta tesis se ha centrado en dos sistemas de nanopartículas moleculares que tienen un uso potencial en el campo de la nanomedicina: i) vesículas lipídicas – entidades supramoleculares que se proponen como sistemas de liberación de fármacos y ii) cuerpos de inclusión (Inclusion Bodies, IBs) – nanopartículas formadas por agregados proteicos.
La primiera parte del trabajo se ha centrado en el estudio comparativo de sistemas vesiculares preparados por i) diferentes metodologías así como ii) diferentes composiciones. Los métodos comparados son DELOS-susp, un método de una sola etapa basado en fluidos comprimidos y procesos convencionales que constan de varias etapas, como hidratación de película delgada (thin film hydration, TFH) y tratamiento con ultrasonidos seguido de extrusión (ultrasound, US). La influencia de la estructura interna se ha investigado con vesículas con diferentes composiciones mixtas: CTAB-Colesterol y DOPC-Colesterol. Para este estudio hemos utilizado métodos que monitorizan la interacción de nanomaterials con superficies como la resonancia de plasmones superficiales (surface plasmon resonance, SPR) y la microbalanza de cristal de cuarzo con disipación (quartz crystal microbalance with dissipation, QCM-D). A partir del análisis de datos hemos demostrado que todos los sistemas investigados forman monocapas de vesículas enteras tras la interacción con superficies de oro. Ha sido posible calcular la absorción de masa, espesor, densidad y propiedades mecánicas de los diferentes sistemas vesiculares estudiados. Hemos concluido, que la influencia del método de preparación es muy. Es decir, en este tipo de sistemas, la arquitectura interna, una vez lograda, es la que determina las propiedades mecánicas de las entidades supramoleculares. Por otro lado, se ha encontrado una influencia de la composición interna de las vesículas en sus propiedades mecánicas. Así, las vesículas formadas por colesterol y CTAB han demostrado ser más rígidas que las formadas por colesterol y DOPC.
Por otro lado, esta tesis se ha centrado en el uso de IBs para la funcionalización de superficies con diferentes patrones utilizando técnicas de litografía blanda. Posteriormente, se han caracterizado y evaluado las propiedades de estas superficies decoradas con IBs como soportes para el cultivo y guiaje de células.
En el marco de esta tesis se han conseguido preparar con éxito patrones geométricos de IBs de alta resolución. Un análisis estadístico de los datos obtenidos a partir de imágenes de microscopía óptica y confocal ha permitido sacar demostrar, que la orientación, la morfología, y el posicionamiento de las células dependen de la geometría de los patrones.
La siguiente etapa de la tesis estuvo centrada en la desarrollo de un dispositivo que permitiera la deposición de gradientes de IBs en superficies a partir de suspensiones coloidales. La técnica está basada en el fenómeno ampliamente conocido del “efecto de la gota de café”. Así, un dispositivo ha sido diseñado, construido, calibrado y utilizado con éxito para preparar sustratos funcionalizados con patrones, muy fáciles de modificar y con bajo coste. Con este dispositivo se ha propuesto e implementado un protocolo de la deposición de gradientes de nanopartículas. Los gradientes obtenidos se han caracterizado en cada caso confirmando la presencia de cambios lineales de concentración de IB sobre grandes áreas (ca 500 micras) tal y como se necesita para realizar estudios de motilidad celular.
En la última parte de la tesis hemos utilizado los gradientes de IBs para estudiar la motilidad celular. El método de deposición permitió preparación de sustrato complejo con más de 80 diferentes entornos para el cultivo celular. Como conclusión general de esta parte podemos confirmar que existe un control claro de la motilidad celular provocada por los patrones de gradientes de IBs ingeniados.
En general, con el trabajo realizado hemos demostrado que los IBs pueden considerarse una nueva herramienta muy interesante y útil para el guiaje celular, lo cual puede tener implicaciones muy interesantes en el campo de la medicina regenerativa y la ingeniería de tejidos.This Thesis is focused on two systems of molecular nanoparticles that have a prospective use in nanomedicine. These systems are: (i) lipidic vesicles – supramolecular entities that are already used as drug delivery systems and (ii) Inclusion Bodies (IBs) - proteic nanoaggregates, that are emerging as a new tool in the light of tissue engineering
The first part of this work is focused on the comparison of a vesicular system prepared by DELOS ‐ susp, a compressed fluid‐based single-step method previously developed in our group, and conventional multi‐step processes that are usually employed for vesicle production, like thin film hydratation or sonication followed by extrusion. We have compared also two different vesicle compositions: one system is based on DOPC phospholipid and cholesterol (liposome) and the other contain a quaternary ammonia amphiphile, CTAB, and cholesterol (quatsomes). To study the structural characteristics of both systems we have used the combination of two non-labelling methods: Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance with Dissipation (QCM-D) in order to obtain complementary data. We conclude that both investigated vesicular systems form layers of vesicles when interacting with gold surfaces. We have calculated the mass uptake, thickness, density and mechanical properties of the studied vesicular systems. We conclude, that the influence of the preparation method is negligible in the case of quatsomes. That is, the internal architecture, once achieved, determines the mechanical properties of these supramolecular entities. On the other hand, vesicles formed by quaternary salts and cholesterol have demonstrated to be more rigid than the liposomes based on phospholipid and cholesterol.
The work developed in following three Chapters has been focused on the use IBs for surface engineering. In this part we have characterized and evaluated IBs decorated surfaces as supports for cell cultivation and guidance.
The second Chapter is centred on the formation of two-dimensional microscale patterns of IBs using a soft lithography technique and evaluation of cell behaviour when cultivated on them. We have successfully prepared high resolution geometrical IBs patterns and cultivated cell on them. Basing on a deep, statistical analysis of data derived from optical and confocal microscopy images we derived conclusions about the influence of the IBs pattern geometry on cell´s behaviour. The orientation, morphology and positioning of cells clearly depends on the geometry of IBs patterns proving the usefulness of IBs for cell guidance. Moreover, the synergy between biological activity and topographical stimuli of cells by IBs has been confirmed.
In the third Chapter, in order to study cell motility induced by IBs, we have focused on the design and engineering of a device allowing deposition of surface-bound IBs gradients from colloidal suspensions. The developed technique is based on the widely known coffee drop effect. A deposition device was constructed, calibrated and successfully used to prepare substrates with the desired patterns, which can be obtained in a fast (up to 1 mm per 1 min) and cost-effective manner. Also, a robust protocol for gradient deposition was proposed and implemented. The obtained gradients were characterized, confirming the presence of linear changes of IBs concentration over broad areas (c.a. 500 μm) as needed to perform cell motility studies.
The last Chapter describes the use of the IBs gradients to study cell motility. A complex substrate with 80 different zones for high-throughput cell culture study was successfully produced. Individual cell movement assay were carried out using confocal microscopy time-lapse acquisition. Cell movement descriptors such as average travelled distance, directionality, etc. were quantified, enabling us to investigate in detail how factors such as the gradient slope or the concentration of IBs influence cell motility. As an overall conclusion of this part we can confirm the control over cell motility by the IBs gradient patterns.
In general we have proved that IBs as well as the different two-dimensional engineering methods used are interesting and useful approaches with a prospective use in the control of cell guidance as well as a promising tools in regenerative medicine and tissue engineering