by Keyword: adsorption
Elyaderani AK, De Lama-Odría MDC, Valle LJD, Puiggalí J, (2022). Multifunctional Scaffolds Based on Emulsion and Coaxial Electrospinning Incorporation of Hydroxyapatite for Bone Tissue Regeneration International Journal Of Molecular Sciences 23, 15016
Tissue engineering is nowadays a powerful tool to restore damaged tissues and recover their normal functionality. Advantages over other current methods are well established, although a continuous evolution is still necessary to improve the final performance and the range of applications. Trends are nowadays focused on the development of multifunctional scaffolds with hierarchical structures and the capability to render a sustained delivery of bioactive molecules under an appropriate stimulus. Nanocomposites incorporating hydroxyapatite nanoparticles (HAp NPs) have a predominant role in bone tissue regeneration due to their high capacity to enhance osteoinduction, osteoconduction, and osteointegration, as well as their encapsulation efficiency and protection capability of bioactive agents. Selection of appropriated polymeric matrices is fundamental and consequently great efforts have been invested to increase the range of properties of available materials through copolymerization, blending, or combining structures constituted by different materials. Scaffolds can be obtained from different processes that differ in characteristics, such as texture or porosity. Probably, electrospinning has the greater relevance, since the obtained nanofiber membranes have a great similarity with the extracellular matrix and, in addition, they can easily incorporate functional and bioactive compounds. Coaxial and emulsion electrospinning processes appear ideal to generate complex systems able to incorporate highly different agents. The present review is mainly focused on the recent works performed with Hap-loaded scaffolds having at least one structural layer composed of core/shell nanofibers.
JTD Keywords: bone tissue, coaxial electrospinning, composite nanofibers, drug-release behavior, emulsion electrospinning, hydroxyapatite, in-vitro evaluation, mechanical-properties, osteogenic differentiation, pickering emulsions, protein adsorption, structured scaffolds, surface-initiated polymerization, tissue regeneration, Bone tissue, Coaxial electrospinning, Emulsion electrospinning, Hydroxyapatite, Multifunctional scaffolds, Poly(3-hydroxybutyrate) phb patches, Tissue regeneration
Bonany M, Pérez-Berná AJ, Dučić T, Pereiro E, Martin-Gómez H, Mas-Moruno C, van Rijt S, Zhao Z, Espanol M, Ginebra MP, (2022). Hydroxyapatite nanoparticles-cell interaction: New approaches to disclose the fate of membrane-bound and internalised nanoparticles Biomaterials Advances 142, 213148
Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form.Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.
JTD Keywords: adsorption, cryo-soft x-ray tomography, cytotoxicity, expression, flow cytometry, internalisation, intracellular calcium, magnesium, nano, nanomaterials, nanoparticles, proliferation, protein corona, ultrastructure, Calcium-phosphate nanoparticles, Cryo-soft x-ray tomography, Flow cytometry, Hydroxyapatite, Internalisation, Intracellular calcium, Nanoparticles
De Lama-Odría, María del Carmen, del Valle, Luis J., Puiggalí, Jordi, (2022). Hydroxyapatite Biobased Materials for Treatment and Diagnosis of Cancer International Journal Of Molecular Sciences 23, 11352
Great advances in cancer treatment have been undertaken in the last years as a consequence of the development of new antitumoral drugs able to target cancer cells with decreasing side effects and a better understanding of the behavior of neoplastic cells during invasion and metastasis. Specifically, drug delivery systems (DDS) based on the use of hydroxyapatite nanoparticles (HAp NPs) are gaining attention and merit a comprehensive review focused on their potential applications. These are derived from the intrinsic properties of HAp (e.g., biocompatibility and biodegradability), together with the easy functionalization and easy control of porosity, crystallinity and morphology of HAp NPs. The capacity to tailor the properties of DLS based on HAp NPs has well-recognized advantages for the control of both drug loading and release. Furthermore, the functionalization of NPs allows a targeted uptake in tumoral cells while their rapid elimination by the reticuloendothelial system (RES) can be avoided. Advances in HAp NPs involve not only their use as drug nanocarriers but also their employment as nanosystems for magnetic hyperthermia therapy, gene delivery systems, adjuvants for cancer immunotherapy and nanoparticles for cell imaging.
JTD Keywords: antitumoral, cell imaging, controlled-release, drug-carrier, efficient drug-delivery, fatty-acid-metabolism, fe3o4 nanoparticles, gene delivery, hydroxyapatite, hyperthermia, immunotherapy, in-vitro, magnetic hydroxyapatite, nano-hydroxyapatite, protein adsorption, tumor-growth, Calcium-phosphate nanoparticles, Cancer
Riedelová, Zuzana, de los Santos Pereira, Andres, Svoboda, Jan, Pop‐Georgievski, Ognen, Májek, Pavel, Pečánková, Klára, Dyčka, Filip, Rodriguez‐Emmenegger, Cesar, Riedel, Tomáš, (2022). The Relation Between Protein Adsorption and Hemocompatibility of Antifouling Polymer Brushes Macromolecular Bioscience 22, 2200247
Whenever an artificial surface comes into contact with blood, proteins are rapidly adsorbed onto its surface. This phenomenon, termed fouling, is then followed by a series of undesired reactions involving activation of complement or the coagulation cascade and adhesion of leukocytes and platelets leading to thrombus formation. Thus, considerable efforts are directed towards the preparation of fouling-resistant surfaces with the best possible hemocompatibility. Herein, a comprehensive hemocompatibility study after heparinized blood contact with seven polymer brushes prepared by surface-initiated atom transfer radical polymerization is reported. The resistance to fouling is quantified and thrombus formation and deposition of blood cellular components on the coatings are analyzed. Moreover, identification of the remaining adsorbed proteins is performed via mass spectroscopy to elucidate their influence on the surface hemocompatibility. Compared with an unmodified glass surface, the grafting of polymer brushes minimizes the adhesion of platelets and leukocytes and prevents the thrombus formation. The fouling from undiluted blood plasma is reduced by up to 99%. Most of the identified proteins are connected with the initial events of foreign body reaction towards biomaterial (coagulation cascade proteins, complement component, and inflammatory proteins). In addition, several proteins that are not previously linked with blood-biomaterial interaction are presented and discussed.
JTD Keywords: biosensor, blood-plasma, coagulation, coatings, compatibility, glycoprotein, hemocompatibility, identification, methacrylate), ms identification, polymer brushes, protein adsorption, surface-chemistry, Antifouling surfaces, High-density-lipoprotein
Quandt, Jonas, GaraySarmiento, Manuela, Witzdam, Lena, Englert, Jenny, Rutsch, Yannik, Stöcker, Cornelia, Obstals, Fabian, Grottke, Oliver, RodriguezEmmenegger, Cesar, (2022). Interactive Hemocompatible Nanocoating to Prevent SurfaceInduced Coagulation in Medical Devices Advanced Materials Interfaces 9, 2201055
JTD Keywords: anti-fxiia antibody, artificial surfaces, blood compatibility, complement activation, factor xii, fibrinolytic system, hemocompatible coatings, interactive hemocompatibility, poly(2-methacryloyloxyethyl phosphorylcholine), polyethylene oxide, polymer brushes, radical polymerization, sequential coimmobilization, Antifouling polymer brushes, Protein adsorption
Martí D, Martín-Martínez E, Torras J, Betran O, Turon P, Alemán C, (2022). In silico study of substrate chemistry effect on the tethering of engineered antibodies for SARS-CoV-2 detection: Amorphous silica vs gold Colloids And Surfaces B-Biointerfaces 213, 112400
The influence of the properties of different solid substrates on the tethering of two antibodies, IgG1-CR3022 and IgG1-S309, which were specifically engineered for the detection of SARS-CoV-2, has been examined at the molecular level using conventional and accelerated Molecular Dynamics (cMD and aMD, respectively). Two surfaces with very different properties and widely used in immunosensors for diagnosis, amorphous silica and the most stable facet of the face-centered cubic gold structure, have been considered. The effects of such surfaces on the structure and orientation of the immobilized antibodies have been determined by quantifying the tilt and hinge angles that describe the orientation and shape of the antibody, respectively, and the dihedrals that measure the relative position of the antibody arms with respect to the surface. Results show that the interactions with amorphous silica, which are mainly electrostatic due to the charged nature of the surface, help to preserve the orientation and structure of the antibodies, especially of the IgG1-CR3022, indicating that the primary sequence of those antibodies also plays some role. Instead, short-range van der Waals interactions with the inert gold surface cause a higher degree tilting and fraying of the antibodies with respect to amorphous silica. The interactions between the antibodies and the surface also affect the correlation among the different angles and dihedrals, which increases with their strength. Overall, results explain why amorphous silica substrates are frequently used to immobilize antibodies in immunosensors. © 2022 The Authors
JTD Keywords: amorphous silica, enzyme, gol d, immobilization, immunosensor, molecu l a r dynamics, protein adsorption, sars-cov-2 immunosensor, simulations, spike protein, surface interactions, target, vaccine, Amorphous silica, Antibodies, Antibody engineering, Antibody immobilization, Antibody structure, Article, Chemical detection, Computer model, Controlled study, Dihedral angle, Gold, In-silico, Molecular dynamics, Molecular levels, Molecular-dynamics, Nonhuman, Property, Sars, Sars-cov-2 immunosensor, Severe acute respiratory syndrome coronavirus 2, Silica, Silico studies, Silicon dioxide, Solid substrates, Structure analysis, Substrate chemistry, Substrates, Van der waals forces, Virus detection
Bar L, Perissinotto F, Redondo-Morata L, Giannotti MI, Goole J, Losada-Pérez P, (2022). Interactions of hydrophilic quantum dots with defect-free and defect containing supported lipid membranes Colloids And Surfaces B-Biointerfaces 210, 112239
Quantum dots (QDs) are semiconductor nanoparticles with unique optical and electronic properties, whose interest as potential nano-theranostic platforms for imaging and sensing is increasing. The design and use of QDs requires the understanding of cell-nanoparticle interactions at a microscopic and nanoscale level. Model systems such as supported lipid bilayers (SLBs) are useful, less complex platforms mimicking physico-chemical properties of cell membranes. In this work, we investigated the effect of topographical homogeneity of SLBs bearing different surface charge in the adsorption of hydrophilic QDs. Using quartz-crystal microbalance, a label-free surface sensitive technique, we show significant differences in the interactions of QDs onto homogeneous and inhomogeneous SLBs formed following different strategies. Within short time scales, QDs adsorb onto topographically homogeneous, defect-free SLBs is driven by electrostatic interactions, leading to no layer disruption. After prolonged QD exposure, the nanomechanical stability of the SLB decreases suggesting nanoparticle insertion. In the case of inhomogeneous, defect containing layers, QDs target preferentially membrane defects, driven by a subtle interplay of electrostatic and entropic effects, inducing local vesicle rupture and QD insertion at membrane edges. © 2021
JTD Keywords: adsorption, atomic force microscopy, bilayer formation, gold nanoparticles, hydrophilic quantum dots, lipid membrane defects, model, nanomechanics, quartz crystal microbalance with dissipation, size, supported lipid bilayers, surfaces, Atomic force microscopy, Atomic-force-microscopy, Cytology, Defect-free, Electronic properties, Electrostatics, Hydrophilic quantum dot, Hydrophilic quantum dots, Hydrophilicity, Hydrophilics, Lipid bilayers, Lipid membrane defect, Lipid membrane defects, Lipid membranes, Lipids, Nanocrystals, Nanomechanics, Optical and electronic properties, Quartz, Quartz crystal microbalance with dissipation, Quartz crystal microbalances, Quartz-crystal microbalance, Semiconductor nanoparticles, Semiconductor quantum dots, Supported lipid bilayers
Konka, J, Espanol, M, Bosch, BM, de Oliveira, E, Ginebra, MP, (2021). Maturation of biomimetic hydroxyapatite in physiological fluids: a physicochemical and proteomic study Materials Today Bio 12,
Biomimetic calcium-deficient hydroxyapatite (CDHA) as a bioactive material exhibits exceptional intrinsic osteoinductive and osteogenic properties because of its nanostructure and composition, which promote a favorable microenvironment. Its high reactivity has been hypothesized to play a relevant role in the in vivo performance, mediated by the interaction with the biological fluids, which is amplified by its high specific surface area. Paradoxically, this high reactivity is also behind the in vitro cytotoxicity of this material, especially pro-nounced in static conditions. The present work explores the structural and physicochemical changes that CDHA undergoes in contact with physiological fluids and to investigate its interaction with proteins. Calcium-deficient hydroxyapatite discs with different micro/nanostructures, coarse (C) and fine (F), were exposed to cell-free complete culture medium over extended periods of time: 1, 7, 14, 21, 28, and 50 days. Precipitate formation was not observed in any of the materials in contact with the physiological fluid, which would indicate that the ionic exchanges were linked to incorporation into the crystal structure of CDHA or in the hydrated layer. In fact, CDHA experienced a maturation process, with a progressive increase in crystallinity and the Ca/P ratio, accompanied by an uptake of Mg and a B-type carbonation process, with a gradual propagation into the core of the samples. However, the reactivity of biomimetic hydroxyapatite was highly dependent on the specific surface area and was amplified in nanosized needle-like crystal structures (F), whereas in coarse specimens the ionic exchanges were restricted to the surface, with low penetration in the material bulk. In addition to showing a higher protein adsorption on F substrates, the proteomics study revealed the existence of protein selectivity to-ward F or C microstructures, as well as the capability of CDHA, and more remarkably of F-CDHA, to concentrate specific proteins from the culture medium. Finally, a substantial improvement in the material's ability to support cell proliferation was observed after the CDHA maturation process.
JTD Keywords: calcium phosphates, ion exchange, nanostructure, protein adsorption, Biological-systems, Biomaterials, Biomimetic hydroxyapatites, Biomimetics, Bone-formation, Calcium deficient hydroxyapatite, Calcium phosphate, Calcium phosphates, Cell proliferation, Crystal structure, Crystallinity, Crystals structures, Culture medium, Growth, High reactivity, Hydroxyapatite, In-vitro, Ion exchange, Ionic exchange, Molecular biology, Nanocrystalline apatites, Nanostructure, Nanostructures, Octacalcium phosphate, Physicochemical studies, Physiological fluids, Physiology, Protein adsorption, Proteins, Proteomic studies, Raman spectroscopy, Serum-albumin, Specific surface area
González-García, C., Cantini, M., Ballester-Beltrán, J., Altankov, G., Salmerón-Sánchez, M., (2018). The strength of the protein-material interaction determines cell fate Acta Biomaterialia 77, 74-84
Extracellular matrix (ECM) proteins are key mediators of cell/material interactions. The surface density and conformation of these proteins adsorbed on the material surface influence cell adhesion and the cellular response. We have previously shown that subtle variations in surface chemistry lead to drastic changes in the conformation of adsorbed fibronectin (FN). On poly(ethyl acrylate) (PEA), FN unfolds and displays domains for cell adhesion and FN-FN interaction, whereas on poly(methyl acrylate) (PMA) â€“ with only one methyl group less â€“ FN remains globular as it is in solution. The effect of the strength of the protein/material interaction in cell response, and its relation to protein density and conformation, has received limited attention so far. In this work, we used FN-functionalized AFM cantilevers to evaluate, via force spectroscopy, the strength of interaction between fibronectin and the underlying polymer which controls FN conformation (PEA and PMA). We found that the strength of FN/PEA interaction is significantly higher than FN/PMA, which limits the mobility of FN layer on PEA, reduces the ability of cells to mechanically reorganize FN and then leads to enhanced proteolysis and degradation of the surrounding matrix with compromised cell viability. By contrast, both PEA and PMA support cell adhesion when FN density is increased and also in the presence of serum or other serum proteins, including vitronectin (VN) and bovine serum albumin (BSA), which provide a higher degree of mobility to the matrix. Statement of Significance: The identification of parameters influencing cell response is of paramount importance for the design of biomaterials that will act as synthetic scaffolds for cells to anchor, grow and, eventually, become specialised tissues. Cells interact with materials through an intermediate layer of proteins adsorbed on the material surface. It is known that the density and conformation of these proteins determine cell behaviour. Here we show that the strength of protein/material interactions, which has received very limited attention so far, is key to understand the cellular response to biomaterials. Very strong protein/material interactions reduce the ability of cells to mechanically reorganize proteins at the material interface which results in enhanced matrix degradation, leading ultimately to compromised cell viability.
JTD Keywords: Fibronectin adsorption, Fibronectin remodeling, Protein mobility, Protein-material interaction strength
Coelho, N. M., Llopis-Hernández, V., Salmerón-Sánchez, M., Altankov, G., (2016). Dynamic reorganization and enzymatic remodeling of type IV collagen at cell–biomaterial interface Advances in Protein Chemistry and Structural Biology (ed. Christo, Z. Christov), Academic Press (San Diego, USA) 105, 81-104
Abstract Vascular basement membrane remodeling involves assembly and degradation of its main constituents, type IV collagen (Col IV) and laminin, which is critical during development, angiogenesis, and tissue repair. Remodeling can also occur at cellâ€“biomaterials interface altering significantly the biocompatibility of implants. Here we describe the fate of adsorbed Col IV in contact with endothelial cells adhering on positively charged NH2 or hydrophobic CH3 substrata, both based on self-assembly monolayers (SAMs) and studied alone or mixed in different proportions. AFM studies revealed distinct pattern of adsorbed Col IV, varying from single molecular deposition on pure NH2 to network-like assembly on mixed SAMs, turning to big globular aggregates on bare CH3. Human umbilical endothelial cells (HUVECs) interact better with Col IV adsorbed as single molecules on NH2 surface and readily rearrange it in fibril-like pattern that coincide with secreted fibronectin fibrils. The cells show flattened morphology and well-developed focal adhesion complexes that are rich on phosphorylated FAK while expressing markedly low pericellular proteolytic activity. Conversely, on hydrophobic CH3 substrata HUVECs showed abrogated spreading and FAK phosphorylation, combined with less reorganization of the aggregated Col IV and significantly increased proteolytic activity. The later involves both MMP-2 and MMP-9, as measured by zymography and FITC-Col IV release. The mixed SAMs support intermediate remodeling activity. Taken together these results show that chemical functionalization combined with Col IV preadsorption provides a tool for guiding the endothelial cells behavior and pericellular proteolytic activity, events that strongly affect the fate of cardiovascular implants.
JTD Keywords: Type IV collagen, Adsorption, Remodeling, Pericellular proteolysis, Reorganization, Substratum chemistry, CH3 and NH2 groups, Self-assembly monolayers
Pérez-Madrigal, M. M., Giannotti, M. I., Del Valle, L. J., Franco, L., Armelin, E., Puiggalí, J., Sanz, F., Alemán, C., (2014). Thermoplastic polyurethane:polythiophene nanomembranes for biomedical and biotechnological applications ACS Applied Materials & Interfaces 6, (12), 9719-9732
Nanomembranes have been prepared by spin-coating mixtures of a polythiophene (P3TMA) derivative and thermoplastic polyurethane (TPU) using 20:80, 40:60, and 60:40 TPU:P3TMA weight ratios. After structural, topographical, electrochemical, and thermal characterization, properties typically related with biomedical applications have been investigated: swelling, resistance to both hydrolytic and enzymatic degradation, biocompatibility, and adsorption of type I collagen, which is an extra cellular matrix protein that binds fibronectin favoring cell adhesion processes. The swelling ability and the hydrolytic and enzymatic degradability of TPU:P3TMA membranes increases with the concentration of P3TMA. Moreover, the degradation of the blends is considerably promoted by the presence of enzymes in the hydrolytic medium, TPU:P3TMA blends behaving as biodegradable materials. On the other hand, TPU:P3TMA nanomembranes behave as bioactive platforms stimulating cell adhesion and, especially, cell viability. Type I collagen adsorption largely depends on the substrate employed to support the nanomembrane, whereas it is practically independent of the chemical nature of the polymeric material used to fabricate the nanomembrane. However, detailed microscopy study of the morphology and topography of adsorbed collagen evidence the formation of different organizations, which range from fibrils to pseudoregular honeycomb networks depending on the composition of the nanomembrane that is in contact with the protein. Scaffolds made of electroactive TPU:P3TMA nanomembranes are potential candidates for tissue engineering biomedical applications.
JTD Keywords: Bioactive platform, Biodegradable blend, Collaged adsorption, Scaffolds, Tissue engineering, Ultrathin films
Marques, J., Moles, E., Urbán, P., Prohens, R., Busquets, M. A., Sevrin, C., Grandfils, C., Fernàndez-Busquets, X., (2014). Application of heparin as a dual agent with antimalarial and liposome targeting activities toward Plasmodium-infected red blood cells Nanomedicine: Nanotechnology, Biology, and Medicine 10, (8), 1719-1728
Heparin had been demonstrated to have antimalarial activity and specific binding affinity for Plasmodium-infected red blood cells (pRBCs) vs. non-infected erythrocytes. Here we have explored if both properties could be joined into a drug delivery strategy where heparin would have a dual role as antimalarial and as a targeting element of drug-loaded nanoparticles. Confocal fluorescence and transmission electron microscopy data show that after 30. min of being added to living pRBCs fluorescein-labeled heparin colocalizes with the intracellular parasites. Heparin electrostatically adsorbed onto positively charged liposomes containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane and loaded with the antimalarial drug primaquine was capable of increasing three-fold the activity of encapsulated drug in Plasmodium falciparum cultures. At concentrations below those inducing anticoagulation of mouse blood in vivo, parasiticidal activity was found to be the additive result of the separate activities of free heparin as antimalarial and of liposome-bound heparin as targeting element for encapsulated primaquine. From the Clinical Editor: Malaria remains an enormous global public health concern. In this study, a novel functionalized heparin formulation used as drug delivery agent for primaquine was demonstrated to result in threefold increased drug activity in cell cultures, and in a murine model it was able to provide these benefits in concentrations below what would be required for anticoagulation. Further studies are needed determine if this approach is applicable in the human disease as well.
JTD Keywords: Heparin, Liposomes, Malaria, Plasmodium, Targeted drug delivery, Heparin, Malaria, Plasmodium, Red blood cell, Targeted drug delivery, Liposomes, 1,2 dioleoyl 3 trimethylammoniopropane, fluorescein, heparin, liposome, nanoparticle, primaquine, adsorption, animal experiment, anticoagulation, antimalarial activity, Article, binding affinity, confocal microscopy, controlled study, drug targeting, encapsulation, erythrocyte, female, fluorescence microscopy, human, human cell, in vivo study, liposomal delivery, mouse, nonhuman, Plasmodium falciparum, transmission electron microscopy
Miranda Coelho, Nuno, Gonzalez-Garcia, Cristina, Salmeron-Sanchez, Manuel, Altankov, George, (2011). Arrangement of type IV collagen and laminin on substrates with controlled density of -OH groups Tissue Engineering Part A , 17, (17-18), 2245-2257
Collagen IV (Col IV) and laminin (Lam) are the main structural components of the basement membrane where they form two overlapping polymeric networks. We studied the adsorption pattern of these proteins on five model surfaces with tailored density of -OH groups obtained by copolymerization of different ratios ethyl acrylate (EA) and hydroxyl EA (HEA): X(OH) = 0, X(OH) = 0.3, X(OH) = 0.5, X(OH) = 0.7, and X(OH) = 1 (where X refers the ratio of HEA). Atomic force microscopy revealed substratum-specific adsorption patterns of Col IV and Lam, ranging from single molecules deposition on more hydrophilic substrata to the formation of complex networks on hydrophobic ones. Human umbilical endothelial cells were used to study the biological performance of adsorbed proteins, following the overall cell morphology, the quantities for cell adhesion and spreading, and the development of focal adhesion complexes and actin cytoskeleton. Surprisingly, two optima in the cellular interaction were observed-one on the most hydrophilic X(OH) = 1 and other on the relatively hydrophobic X(OH) = 0.3 substrate-valid for both Col IV and Lam. When the proteins were adsorbed consecutively, a hydrophobic shift to X(OH) = 0 substratum was obtained. Collectively, these data suggest that varying with the density of -OH groups one can tailor the conformation and the functional activity of adsorbed basement membrane proteins.
JTD Keywords: Atomic-force microscopy, Fibronectin adsorption, Basement-membranes, Polymer surfaces, Cell-adhesion, Biomaterials, Wettability, Fibrinogen
Hristova, K., Pecheva, E., Pramatarova, L., Altankov, G., (2011). Improved interaction of osteoblast-like cells with apatite-nanodiamond coatings depends on fibronectin Journal of Materials Science: Materials in Medicine , 22, (8), 1891-1900
New apatite (AP)/nanodiamond (ND) coating has been developed to improve physical and biological properties of stainless steel (SS) versus single AP coating. Homogeneously electrodeposited AP-ND layer demonstrates increased mechanical strength, interlayer cohesion and ductility. In the absence of serum, osteoblast-like MG63 cells attach well but poorly spread on both AP and AP-ND substrata. Pre-adsorption with serum or fibronectin (FN) improves the cellular interaction-an effect that is better pronounced on the AP-ND coating. In single protein adsorption study fluorescein isothiocyanate-labeled FN (FITC-FN) shows enhanced deposition on the AP-ND layer consistent with the significantly improved cell adhesion, spreading and focal adhesions formation (in comparison to SS and AP), particularly at low FN adsorption concentrations (1 mu g/ml). Higher FN concentrations (20 mu g/ml) abolish this difference suggesting that the promoted cellular interaction of serum (where FN is low) is caused by the greater affinity for FN. Moreover, it is found that MG63 cells tend to rearrange both adsorbed and secreted FN on the AP-ND layer suggesting facilitated FN matrix formation.
JTD Keywords: Extracellular-matrix, Protein adsorption, Integrins, Adhesion, Biomaterials, Surfaces, Polymerization, Composite, Implants, Titanium
Coelho, N. M., Gonzalez-Garcia, C., Planell, J. A., Salmeron-Sanchez, M., Altankov, G., (2010). Different assembly of type iv collagen on hydrophilic and hydrophobic substrata alters endothelial cells interaction European Cells & Materials , 19, 262-272
Considering the structural role of type IV collagen (Col IV) in the assembly of the basement membrane (BM) and the perspective of mimicking its organization for vascular tissue engineering purposes, we studied the adsorption pattern of this protein on model hydrophilic (clean glass) and hydrophobic trichloro(octadecyl) silane (ODS) surfaces known to strongly affect the behavior of other matrix proteins. The amount of fluorescently labeled Col IV was quantified showing saturation of the surface for concentration of the adsorbing solution of about 50 mu g/ml, but with approximately twice more adsorbed protein on ODS. AFM studies revealed a fine-nearly single molecular size-network arrangement of Col IV on hydrophilic glass, which turns into a prominent and growing polygonal network consisting of molecular aggregates on hydrophobic ODS. The protein layer forms within minutes in a concentration-dependent manner. We further found that human umbilical vein endothelial cells (HUVEC) attach less efficiently to the aggregated Col IV (on ODS), as judged by the significantly altered cell spreading, focal adhesions formation and the development of actin cytoskeleton. Conversely, the immunofluorescence studies for integrins revealed that the fine Col IV network formed on hydrophilic substrata is better recognized by the cells via both alpha 1 and alpha 2 heterodimers which support cellular interaction, apart from these on hydrophobic ODS where almost no clustering of integrins was observed.
JTD Keywords: Collagen type IV, Adsorption, Assembly, Hydrophilic, Hydrophobic, Surfaces
Toromanov, Georgi, González-García, Cristina, Altankov, George, Salmerón-Sánchez, Manuel, (2010). Vitronectin activity on polymer substrates with controlled -OH density Polymer 51, (11), 2329-2336
Vitronectin (VN) adsorption on a family of model substrates consisting of copolymers of ethyl acrylate and hydroxyl ethylacrylate in different ratios (to obtain a controlled surface density of -OH groups) was investigated by Atomic Force Microscopy (AFM). It is shown that the fraction of the substrate covered by the protein depends strongly on the amount of hydroxyl groups in the sample and it monotonically decreases as the -OH density increases. Isolated globular-like VN molecules are observed on the surfaces with the higher OH density. As the fraction of hydroxyl groups decreases, aggregates of 3-5 VN molecules are observed on the sample. Overall cell morphology, focal adhesion formation and actin cytoskeleton development are investigated to assess the biological activity of the adsorbed VN on the different surfaces. Dermal fibroblast cells show excellent material interaction on the more hydrophobic samples (OH contents lower than 0.5), which reveals enhanced VN activity on this family of substrates as compared with other extracellular matrix proteins (e.g., fibronectin and fibrinogen).
JTD Keywords: Copolymers, Vitronectin, AFM, Self-assembled monolayers, Cell-adhesion, Thermal transitions, Protein adsorption, Surfaces, Fibronectin, Biomaterials, Attachment, Fibrinogen
Gugutkov, Dencho, Gonzalez-Garcia, Cristina, Rodriguez Hernandez, Jose Carlos, Altankov, George, Salmeron-Sanchez, Manuel, (2009). Biological activity of the substrate-induced fibronectin network: insight into the third dimension through electrospun fibers Langmuir 25, (18), 10893-10900
Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that results in conformational changes of FN molecules and the organization of actin cytoskeleton. A similar process can be induced by some chemistries in the absence of cells, e.g., poly(ethyl acrylate) (PEA), which enhance FN-FN interactions leading to the formation of a biologically active network. Atomic force microscopy images of single FN molecules, at the early stages of adsorption on plane PEA, allow one to rationalize the process. Further, the role of the spatial organization of the FN network on the cellular response is investigated through its adsorption on electrospun fibers. Randomly oriented and aligned PEA fibers were prepared to mimic the three-dimensional organization of the extracellular matrix. The formation of the FN network on the PEA fibers but not on the supporting coverglass was confirmed. Fibroblasts aligned with oriented fibers, displayed extended morphology, developed linearly organized focal adhesion complexes, and matured actin filaments. Conversely, on random PEA fibers, cells acquired polygonal morphology with altered actin cytoskeleton but well-developed focal adhesions. Late FN matrix formation was also influenced: spatially organized FN matrix fibrils along the oriented PEA fibers and an altered arrangement on random ones.
JTD Keywords: AFM, Cell-adhesion, Dependent conformations, Hydrophobic surfaces, Extracellular-matrix, Bound fibronectin, Polymer surfaces, Integrin binding, Biocompatibility, Adsorption
Rico, P., Rodriguez Hernandez, J. C., Moratal, D., Altankov, G., Monleon Pradas, M., Salmeron-Sanchez, M., (2009). Substrate-induced assembly of fibronectin into networks. Influence of surface chemistry and effect on osteoblast adhesion Tissue Engineering Part A , 15, (00), 1-11
The influence of surface chemistry -substrates with controlled surface density of -OH groups- on fibronectin conformation and distribution is directly observed by Atomic Force Microscopy (AFM). FN fibrillogenesis, which is known to be a process triggered by interaction with integrins, is shown in our case to be induced by the substrate (in absence of cells), which is able to enhance FN-FN interactions leading to the formation of a protein network on the material surface. This phenomenon depends both on surface chemistry and protein concentration. The level of the FN fibrillogenesis was quantified by calculating the fractal dimension of the adsorbed protein from image analysis of the AFM results. The total amount of adsorbed FN is obtained by making use of a methodology which employs western-blotting combined with image analysis of the corresponding protein bands, with the lowest sensitivity threshold equal to 15 ng of adsorbed protein. Furthermore, FN adsorption is correlated to human osteoblast adhesion through morphology and actin cytoskeleton formation. Actin polymerization is in need of the formation of the protein network on the substrate's surface. Cell morphology is more rounded (as quantified by calculating the circularity of the cells by image analysis) the lower the degree of FN fibrillogenesis on the substrate.
JTD Keywords: Cell-adhesion, Conformational-changes, Electron-microscopy, Protein adsorption, Fractal dimension, Integrin binding, Biocompatibility, Monolayers, Matrix, Fibrillogenesis
Olmedo, Ivonne, Araya, Eyleen, Sanz, Fausto, Medina, Elias, Arbiol, Jordi, Toledo, Pedro, Àlvarez-Lueje, Alejandro, Giralt, Ernest, Kogan, Marcelo J., (2008). How changes in the sequence of the peptide CLPFFD-NH2 can modify the conjugation and stability of gold nanoparticles and their affinity for beta-amyloid fibrils Bioconjugate Chemistry , 19, (6), 1154-1163
In a previous work, we studied the interaction of β-amyloid fibrils (Aβ) with gold nanoparticles (AuNP) conjugated with the peptide CLPFFD-NH2. Here, we studied the effect of changing the residue sequence of the peptide CLPFFD-NH2 on the efficiency of conjugation to AuNP, the stability of the conjugates, and the affinity of the conjugates to the Aβ fibrils. We conjugated the AuNP with CLPFFD-NH2 isomeric peptides (CDLPFF-NH2 and CLPDFF-NH2) and characterized the resulting conjugates with different techniques including UV−Vis, TEM, EELS, XPS, analysis of amino acids, agarose gel electrophoresis, and CD. In addition, we determined the proportion of AuNP bonded to the Aβ fibrils by ICP-MS. AuNP-CLPFFD-NH2 was the most stable of the conjugates and presented more affinity for Aβ fibrils with respect to the other conjugates and bare AuNP. These findings help to better understand the way peptide sequences affect conjugation and stability of AuNP and their interaction with Aβ fibrils. The peptide sequence, the steric effects, and the charge and disposition of hydrophilic and hydrophobic residues are crucial parameters when considering the design of AuNP peptide conjugates for biomedical applications.
JTD Keywords: Self-assembled monolayers, Aggregation, Dispersions, Adsorption, Particles, Design, Size
Gustavsson, J., Altankov, G., Errachid, A., Samitier, J., Planell, J. A., Engel, E., (2008). Surface modifications of silicon nitride for cellular biosensor applications Journal of Materials Science-Materials in Medicine , 19, (4), 1839-1850
Thin films of silicon nitride (Si3N4) can be used in several kinds of micro-sized biosensors as a material to monitor fine environmental changes related to the process of bone formation in vitro. We found however that Si3N4 does not provide optimal conditions for osseointegration as osteoblast-like MG-63 cells tend to detach from the surface when cultured over confluence. Therefore Si3N4 was modified with self-assembled monolayers bearing functional end groups of primary amine (NH2) and carboxyl (COOH) respectively. Both these modifications enhanced the interaction with confluent cell layers and thus improve osseointegration over Si3N4. Furthermore it was observed that the NH2 functionality increased the adsorption of fibronectin (FN), promoted cell proliferation, but delayed the differentiation. We also studied the fate of pre-adsorbed and secreted FN from cells to learn more about the impact of above functionalities for the development of provisional extracellular matrix on materials interface. Taken together our data supports that Si3N4 has low tissue integration but good cellular biocompatibility and thus is appropriate in cellular biosensor applications such as the ion-sensitive field effect transistor (ISFET). COOH and NH2 chemistries generally improve the interfacial tissue interaction with the sensor and they are therefore suitable substrates for monitoring cellular growth or matrix deposition using electrical impedance spectroscopy.
JTD Keywords: Adsorption, Amines/chemistry, Biocompatible Materials/ chemistry, Biosensing Techniques, Cell Differentiation, Cell Line, Cell Proliferation, Electric Impedance, Extracellular Matrix/metabolism, Fibronectins/chemistry, Humans, Materials Testing, Osteoblasts/ cytology, Silicon Compounds/ chemistry, Surface Properties
Casuso, I., Pla, M., Gomila, G., Samitier, J., Minic, J., Persuy, M. A., Salesse, R., Pajot-Augy, E., (2008). Immobilization of olfactory receptors onto gold electrodes for electrical biosensor Materials Science & Engineering C 5th Maghreb-Europe Meeting on Materials and their Applicatons for Devices and Physical, Chemical and Biological Sensors , Elsevier Science (Mahdia, TUNISIA) 28, (5-6), 686-691
We investigate the immobilization of native nanovesicles containing functional olfactory receptors onto gold electrodes by means of atomic force microscopy in liquid. We show that nanovesicles can be adsorbed without disrupting them presenting sizes once immobilized ranging from 50 run to 200 nm in diameter. The size of the nanovesicles shows no dependence on the electrode hydrophobicity being constant in a height/width ratio close to 1:3. Nevertheless, electrode hydrophobicity does affect the surface coverage, the surface coverage is five times higher in hydrophilic electrodes than on hydrophobic ones. Surface coverage is also affected by nanovesicles dimensions in suspension, the size homogenization to around 50 nm yields a further five fold increment in surface coverage achieving a coverage of about 50% close to the hard spheres jamming limit (54.7%). A single layer of nanovesicles is always formed with no particle overlap. Present results provide insights into the immobilization on electrodes of olfactory receptors for further olfactory electrical biosensor development.
JTD Keywords: AFM, Adsorption, Odorant, Taste