RESUMEN
Thermal decomposition is a very efficient synthesis strategy to obtain nanosized metal oxides with controlled structures and properties. For the iron oxide nanoparticle synthesis, it allows an easy tuning of the nanoparticle's size, shape, and composition, which is often explained by the LaMer theory involving a clear separation between nucleation and growth steps. Here, the events before the nucleation of iron oxide nanocrystals are investigated by combining different complementary in situ characterization techniques. These characterizations are carried out not only on powdered iron stearate precursors but also on a preheated liquid reaction mixture. They reveal a new nucleation mechanism for the thermal decomposition method: instead of a homogeneous nucleation, the nucleation occurs within vesicle-like-nanoreactors confining the reactants. The different steps are: 1) the melting and coalescence of iron stearate particles, leading to "droplet-shaped nanostructures" acting as nanoreactors; 2) the formation of a hitherto unobserved iron stearate crystalline phase within the nucleation temperature range, simultaneously with stearate chains loss and Fe(III) to Fe(II) reduction; 3) the formation of iron oxide nuclei inside the nanoreactors, which are then ejected from them. This mechanism paves the way toward a better mastering of the metal oxide nanoparticles synthesis and the control of their properties.
Asunto(s)
Nanopartículas del Metal , Óxidos , Medios de Cultivo , Compuestos Férricos/química , Hierro , Nanopartículas del Metal/química , Óxidos/química , EstearatosRESUMEN
Polymersomes are multicompartmental vesicular nano-objects obtained by self-assembly of amphiphilic copolymers. When prepared in the aqueous phase, they are composed of a hydrophobic bilayer enclosing water. Although such fascinating polymeric nano-objects have been widely reported with synthetic block copolymers, their formation from polysaccharide-based copolymers remains a significant challenge. In the present study, the powerful platform technology known as polymerization-induced self-assembly was used to prepare in situ pure vesicles from a polysaccharide-grafted copolymer: dextran-g-poly(2-hydroxypropyl methacrylate) (Dex-g-PHPMA). The growth of the PHPMA grafts was performed with a dextran-based macromolecular chain transfer agent in water at 20 °C using photomediated reversible addition fragmentation chain transfer polymerization at 405 nm. Transmission electron microscopy, cryogenic electron microscopy, small-angle X-ray scattering, atomic force microscopy, and dynamic light scattering revealed that amphiphilic Dex-g-PHPMAX = 100-300 (X is the targeted average degree of polymerization, XnÌ , of each graft at full conversion) exhibit remarkable self-assembly behavior. On the one hand, vesicles were obtained over a wide range of solid concentrations (from 2.5% to 13.5% w/w), which can facilitate posterior targeting of such rare morphology. On the other hand, the extension of XnÌ induces an increase in the vesicle membrane thickness, rather than a morphological evolution (spherical micelles to cylinders to vesicles).
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Micelas , Polímeros , Interacciones Hidrofóbicas e Hidrofílicas , Polimerizacion , PolisacáridosRESUMEN
This study explores the production of polysaccharides (PS) in the strain Pf2289 of the food species Propionibacterium freudenreichii. Pf2289 presents characteristics atypical of the species: a molar-shaped morphotype upon plating, and cells strongly aggregative in liquid medium. When plating Pf2289, another morphotype was observed with a 4% frequency of appearance: round-shaped colonies, typical of the species. A clone was isolated, designated Pf456. No reversibility of Pf456 towards the molar-shaped morphotype was observed. Pf2289 was shown to produce a surface polysaccharide (PS) bound to the cell wall, mainly during the stationary growth phase. Meanwhile, Pf456 had lost the ability to produce the PS. AFM images of Pf2289 showed that entangled filaments spread over the whole surface of the bacteria, whereas Pf456 exhibited a smooth surface. Adhesion force maps, performed with concanavalin-A grafted probes, revealed twice as much adhesion of Pf2289 to concanavalin-A compared to Pf456. Furthermore, the length of PS molecules surrounding Pf2289 measured at least 7 µm, whereas it only reached 1 µm in Pf456. Finally, the presence of PS had a strong impact on adhesion properties: Pf2289 did not adhere to hydrophobic surfaces, whereas Pf456 showed strong adhesion.
Asunto(s)
Polisacáridos/metabolismo , Propionibacterium freudenreichii/química , Propionibacterium freudenreichii/metabolismo , Adhesión Bacteriana , Pared Celular/química , Pared Celular/metabolismo , Propionibacterium freudenreichii/crecimiento & desarrollo , Propiedades de SuperficieRESUMEN
Breast cancer is the leading cause of death from cancer among women. Higher consumption of dietary marine n-3 long-chain polyunsaturated fatty acids (LC-PUFAs) is associated with a lower risk of breast cancer. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are two n-3 LC-PUFAs found in fish and exert anticancer effects. In this study, natural marine- derived lecithin that is rich in various polyunsaturated fatty acids (PUFAs) was extracted from salmon heads and transformed into nanoliposomes. These nanoliposomes were characterized and cultured with two breast cancer lines (MCF-7 and MDA-MB- 231). The nanoliposomes decreased the proliferation and the stiffness of both cancer cell types. These results suggest that marine-derived lecithin possesses anticancer properties, which may have an impact on developing new liposomal delivery strategies for breast cancer treatment.
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Antineoplásicos/química , Ácidos Docosahexaenoicos/química , Ácido Eicosapentaenoico/química , Peces , Liposomas/química , Animales , Antineoplásicos/farmacología , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Femenino , Humanos , Liposomas/farmacología , Células MCF-7/efectos de los fármacosRESUMEN
Curcumin is a hydrophobic drug gaining growing attention because of its high availability, its innocuity, and its anticancer, antitumoral, and antioxidative activity. However, its poor bioavailability in the human body, caused by its low aqueous solubility and fast degradation, presents a big hurdle for its oral administration. Here, we used nano-vesicles made of phospholipids to carry and protect curcumin in its membrane. Various curcumin amounts were encapsulated in the produced phospholipid system to form drug-loaded liposomes. Curcumin's concentration was evaluated using UV-visible measurements. The maximal amount of curcumin that could be added to liposomes was assessed. Nuclear magnetic resonance (NMR) analyses were used to determine curcumin's interactions and localization within the phospholipid membrane of the liposomes. X-ray scattering (SAXS) and atomic force microscopy (AFM) experiments were performed to characterize the membrane structure and organization, as well as its mechanical properties at the nanoscale. Conservation of the membrane's properties is found with the addition of curcumin in various amounts before saturation, allowing the preparation of a defined nanocarrier with desired amounts of the drug.
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Antineoplásicos Fitogénicos/química , Curcumina/química , Liposomas/química , Fosfatidilcolinas/química , Composición de Medicamentos/métodos , Sistemas de Liberación de Medicamentos , Soluciones , Agua/químicaRESUMEN
Against the increase of bacterial resistance to traditional antibiotics, antimicrobial peptides (AMP) are considered as promising alternatives. Bacterial biofilms are more resistant to antibiotics that their planktonic counterpart. The purpose of this study was to investigate the action of an AMP against a nascent bacterial biofilm. The activity of dermaseptin S4 derivative S4(1-16)M4Ka against 6 h-old Pseudomonas fluorescens biofilms was assessed by using a combination of Attenuated Total Reflectance-Fourier Transform InfraRed (ATR-FTIR) spectroscopy in situ and in real time, fluorescence microscopy using the Baclight™ kit, and Atomic Force Microscopy (AFM, imaging and force spectroscopy). After exposure to the peptide at three concentrations, different dramatic and fast changes over time were observed in the ATR-FTIR fingerprints reflecting a concentration-dependent action of the AMP. The ATR-FTIR spectra revealed major biochemical and physiological changes, adsorption/accumulation of the AMP on the bacteria, loss of membrane lipids, bacterial detachment, bacterial regrowth, or inhibition of biofilm growth. AFM allowed estimating at the nanoscale the effect of the AMP on the nanomechanical properties of the sessile bacteria. The bacterial membrane elasticity data measured by force spectroscopy were consistent with ATR-FTIR spectra, and they allowed suggesting a mechanism of action of this AMP on sessile P. fluorescens. The combination of these three techniques is a powerful tool for in situ and in real time monitoring the activity of AMPs against bacteria in a biofilm.
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Proteínas Anfibias/farmacología , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Biopelículas/efectos de los fármacos , Pseudomonas fluorescens/efectos de los fármacos , Proteínas Anfibias/síntesis química , Antibacterianos/síntesis química , Péptidos Catiónicos Antimicrobianos/síntesis química , Adhesión Bacteriana/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Módulo de Elasticidad/efectos de los fármacos , Lípidos de la Membrana/química , Pruebas de Sensibilidad Microbiana , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Pseudomonas fluorescens/química , Pseudomonas fluorescens/crecimiento & desarrollo , Pseudomonas fluorescens/ultraestructura , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Colistin (Polymyxin E), an antimicrobial peptide, is increasingly put forward as salvage for severe multidrug-resistant infections. Unfortunately, colistin is potentially toxic to mammalian cells. A better understanding of the interaction with specific components of the cell membranes may be helpful in controlling the factors that may enhance toxicity. Here, we report a physico-chemical study of model phospholipid (PL) mono- and bilayers exposed to colistin at different concentrations by Langmuir technique, atomic force microscopy (AFM) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The effect of colistin on chosen PL monolayers was examined. Insights into the topographical and elastic changes in the PL bilayers within time after peptide injection are presented via AFM imaging and force spectra. Finally, changes in the PL bilayers' ATR-FTIR spectra as a function of time within three bilayer compositions, and the influence of colistin on their spectral fingerprint are examined together with the time-evolution of the Amide II and νCO band integrated intensity ratios. Our study reveals a great importance in the role of the PL composition as well as the peptide concentration on the action of colistin on PL model membranes.
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Antibacterianos/química , Colistina/química , Membrana Dobles de Lípidos/química , Liposomas Unilamelares/química , 1,2-Dipalmitoilfosfatidilcolina/química , Elasticidad , Microscopía de Fuerza Atómica , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
IgG films are widely used in the field of immunoassays, especially in (double) antibody-sandwich ELISA tests where capture antibodies are coated on surfaces like polystyrene or hydrophobic self-assembled monolayers (h-SAMs). It is critical to analyze-at a molecular scale and under liquid conditions-the structure of the deposited IgG film in order to quantitatively address the efficiency of the ELISA test in terms of antigen detection. In this communication, we report an atomic force microscopy (AFM) analysis evidencing a strong relationship between immunological activities of mouse monoclonal anti-human interleukin-2 (IL-2) and 6 (IL-6) antibodies, thickness and roughness of the IgG monolayer adsorbed onto h-SAMs, and surface concentration of IgG molecules. Indirect information may be further obtained on antibody orientation. Collating the results obtained by AFM and those from ELISA tests leads us to conclude that antibodies like anti-IL-6 forming flat monolayers should be more efficient under ELISA detection conditions. In addition, the concentration of IgG in the coating suspension should be optimized to obtain a monolayer heavily populated by "end-on" adsorbed molecules, an orientation that is desirable for enhancing ELISA tests performance.
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Anticuerpos/ultraestructura , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G/química , Adsorción , Animales , Anticuerpos/química , Anticuerpos/inmunología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/inmunología , Inmunoglobulina G/ultraestructura , Interleucina-6/inmunología , Ratones , Microscopía de Fuerza Atómica , Propiedades de SuperficieRESUMEN
This study aims to design an optimal polyelectrolyte multilayer film of poly-l-lysine (PLL) and hyaluronic acid (HA) as an anti-inflammatory cytokine release system in order to decrease the implant failure due to any immune reactions. The chemical modification of the HA with aldehyde moieties allows self-cross-linking of the film and an improvement in the mechanical properties of the film. The cross-linking of the film and the release of immunomodulatory cytokine (IL-4) stimulate the differentiation of primary human monocytes seeded on the films into pro-healing macrophages phenotype. This induces the production of anti-inflammatory cytokines (IL1-RA and CCL18) and the decrease of pro-inflammatory cytokines secreted (IL-12, TNF-α, and IL-1ß). Moreover, we demonstrate that cross-linking PLL/HA film using HA-aldehyde is already effective by itself to limit inflammatory processes. Finally, this functionalized self-cross-linked PLL/HA-aldehyde films constitutes an innovative and efficient candidate for immunomodulation of any kind of implants of various architecture and properties.
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Reactivos de Enlaces Cruzados/química , Citocinas/administración & dosificación , Ácido Hialurónico/química , Inmunomodulación/efectos de los fármacos , Inflamación/tratamiento farmacológico , Polielectrolitos/química , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/química , Humanos , Inflamación/inmunología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Propiedades de SuperficieRESUMEN
The short-term kinetics of bacterial repopulation were evaluated after chlorination of high-density polyethylene (HDPE) colonized with drinking water biofilms and compared with bare HDPE surfaces. The effect of chlorination was partial as a residual biofilm persisted and was time-limited as repopulation occurred immediately after water resupply. The total number of bacteria reached the same levels on both the bare and chlorinated biofilm-fouled HDPE after a seven-day exposure to drinking water. Due to the presence of a residual biofilm, the hydrophobicity of chlorinated biofilm-fouled surface exhibited much lower adhesion forces (2.1 nN) compared to bare surfaces (8.9 nN). This could explain the rapid repopulation after chlorination, with a twofold faster bacterial accumulation rate on the bare HDPE surface. γ-Proteobacteria dominated the early stages of repopulation of both surfaces and a shift in the dominance occurred over the colonization time. Such observations define a timescale for cleaning frequency in industrial environments and guidelines for a rinsing procedure using drinking water.
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Biopelículas/efectos de los fármacos , Cloro/farmacología , Agua Potable/microbiología , Gammaproteobacteria/crecimiento & desarrollo , Polietileno/química , Abastecimiento de Agua/normas , Carga Bacteriana , Gammaproteobacteria/aislamiento & purificación , Gammaproteobacteria/fisiología , Halogenación , Cinética , Microbiología del Agua/normasRESUMEN
The physicochemical determinants governing the temperature-dependent adhesion of Streptococcus thermophilus to abiotic surfaces are identified under physiological condition for cells either lacking or not the Rgg0182 transcriptional regulator involved in their thermal adaptation. For that purpose, the wild type LMG18311 strain and Δrgg0182 mutant were imaged using highly resolved atomic force microscopy (AFM) at various cell growth temperatures (42 to 55 °C). The corresponding hydrophobic/hydrophilic balance of the cells was quantitatively addressed via the measurement by chemical force microcopy of their adhesion to a reference hydrophobic surface. Analysis of force-separation distance curves further allowed us to discriminate cell surfaces according to the presence or absence of biopolymers. These results were interpreted in relation to the measured adhesion of the Δrgg0182 mutant onto the hydrophobic wall of microwells in the temperature range from 46 to 52 °C. It is evidenced that the viscoelastic Δrgg0182 cell envelop behaves as a thermo-responsive film whose hydrophobicity increases with increasing temperature, thereby favoring cell attachment to hydrophobic surfaces. Regardless cell growth temperature, wild-type cells do not attach to hydrophobic surfaces and the presence of the Rgg0182 transcriptional regulator is associated with the synthesis of hydrophilic cell surface biopolymers. Throughout, the impact of electrostatics on bioadhesion is ruled out upon examination of electrohydrodynamic cell properties at 50 °C.
Asunto(s)
Streptococcus thermophilus/química , Temperatura , Adhesión Bacteriana , Proliferación Celular , Interacciones Hidrofóbicas e Hidrofílicas , Streptococcus thermophilus/citología , Streptococcus thermophilus/genética , Propiedades de SuperficieRESUMEN
Despite the exceptional progress in breast cancer pathogenesis, prognosis, diagnosis, and treatment strategies, it remains a prominent cause of female mortality worldwide. Additionally, although chemotherapies are effective, they are associated with critical limitations, most notably their lack of specificity resulting in systemic toxicity and the eventual development of multi-drug resistance (MDR) cancer cells. Liposomes have proven to be an invaluable drug delivery system but of the multitudes of liposomal systems developed every year only a few have been approved for clinical use, none of which employ active targeting. In this review, we summarize the most recent strategies in development for actively targeted liposomal drug delivery systems for surface, transmembrane and internal cell receptors, enzymes, direct cell targeting and dual-targeting of breast cancer and breast cancer-associated cells, e.g., cancer stem cells, cells associated with the tumor microenvironment, etc.
RESUMEN
Deciphering the mechanism of Alzheimer's disease is a key element for designing an efficient therapeutic strategy. Molecular dynamics (MD) calculations, atomic force microscopy, and infrared spectroscopy were combined to investigate ß-amyloid (Aß1-42) peptide interactions with supported lipid bilayers (SLBs). The MD simulations showed that nascent Aß1-42 monomers remain anchored within a model phospholipid bilayer's hydrophobic core, which suggests their stability in their native environment. We tested this prediction experimentally by studying the behavior of Aß1-42 monomers and oligomers when interacting with SLBs. When Aß1-42 monomers and oligomers were self-assembled with a lipid bilayer and deposited as an SLB, they remain within the bilayers. Their presence in the bilayers induces destabilization of the model membranes. No specific interactions between Aß1-42 and the SLBs were detected when SLBs free of Aß1-42 were exposed to Aß1-42. This study suggests that Aß can remain in the membrane after cleavage by γ-secretase and cause severe damage to the membrane.
Asunto(s)
Enfermedad de Alzheimer , Humanos , Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , Membrana Dobles de Lípidos/químicaRESUMEN
Glycogen is mainly found as the principal storage form of glucose in cells. Many bacteria are able to synthesize large amounts of glycogen under unfavorable life conditions. By combining infrared spectroscopy, single molecule force spectroscopy (SMFS) and immuno-staining technique, we evidenced that planktonic P. fluorescens (Pf) cells are also able to produce glycogen as an extracellular polymeric substance. For this purpose, Pf suspensions were examined at 3 and 21 h of growth in nutritive medium (LB, 0.5 g/L). The conformation of the extracellular glycogen, revealed through its infrared spectral signature, has been investigated by SMFS measurements using Freely Jointed Chain model. The analysis of force versus distance curves showed over growth time that the increase of glycogen production was accompanied by an increase in glycogen contour lengths and ramifications. These results demonstrated that the production of extracellular bacterial glycogen can occur even if the cells are not subjected to unfavorable life conditions.
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Glucógeno/biosíntesis , Pseudomonas fluorescens/metabolismo , Adhesión Bacteriana , Conformación de Carbohidratos , Pared Celular/metabolismo , Pared Celular/fisiología , Técnicas de Cultivo , Elasticidad , Glucógeno/química , Glucógeno/metabolismo , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Pseudomonas fluorescens/ultraestructura , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de SuperficieRESUMEN
Proteins are great therapeutic candidates as endogenous biomolecules providing a wide range of applications. However, their delivery suffers from some limitations and specifically designed delivery systems having an efficient protein anchoring and delivery strategy are still needed. In this work, we propose to combine large pore stellate mesoporous silica (STMS) with isobutyramide (IBAM), as a "glue" molecule which has been shown promising for immobilization of various biomacromolecules at silica surface. We address here for the first time the ability of such IBAM-modified NPs to sustainably deliver proteins over a prolonged time. In this work, a quantitative loading study of proteins (serum albumin (HSA), peroxidase (HRP), immunoglobulin (IgG) and polylysine (PLL)) on STMS@IBAM is first presented using three complementary detection techniques to ensure precision and avoid protein quantification issues. The results demonstrated a high loading capacity for HSA and HRP (≥ ca. 350 µg.mg-1) but a moderate one for IgG and PLL. After evaluating the physicochemical properties of the loaded particles and their stability over scaling-up and washings, the ability of STMS@IBAM to release proteins over prolonged time was evaluated in equilibrium (static) and flow mimicking (dynamic) conditions and at different temperatures (25, 37, 45 °C). Results show not only the potential of such "glue" functionalized STMS to release proteins in a sustained way, but also the retention of the biological activity of immobilized and released HRP, used as an enzyme model. Finally, an AFM-force spectroscopy study was conducted to decipher the interactions between IBAM and proteins, showing the involvement of different interactions in the adsorption and release processes.
RESUMEN
The extracellular matrix (ECM) offers the opportunity to create a biomaterial consisting of a microenvironment with interesting biological and biophysical properties for improving and regulating cell functions. Animal-derived ECM are the most widely used as an alternative to human tissues that are of very limited availability. However, incomplete decellularization of these tissues presents a high risk of immune rejection and disease transmission. In this study, we present an innovative method to extract human ECM derived from the Wharton's jelly (WJ-ECMaa) of umbilical cords as a novel biomaterial to be used in tissue engineering. WJ-ECMaa was very efficiently decellularized, suggesting its possible use in allogeneic conditions. Characterization of its content allowed the identification of type I collagen as its main component. Various other matrix proteins, playing an important role in cell adhesion and proliferation, were also detected. WJ-ECMaa applied as a surface coating was analyzed by fluorescent labeling and atomic force microscopy. The results revealed a particular arrangement of collagen fibers not previously described in the literature. This biomaterial also presented better cytocompatibility compared to the conventional collagen coating. Moreover, it showed adequate hemocompatibility, allowing its use as a surface with direct contact with blood. Application of WJ-ECMaa as a coating of the luminal surface of umbilical arteries for a use in vascular tissue engineering, has improved significantly the cellularization of this surface by allowing a full and homogeneous cell coverage. Taking these results together, our novel extraction method of human ECM offers a very promising biomaterial with many potential applications in tissue engineering such as the one presented direct in vascular tissue engineering. Further characterization of the composition and functionality will help explore the ways it can be used in tissue engineering applications, especially as a scaffold or a surface coating.
RESUMEN
Metal-based complexes are well-established cancer chemotherapeutic drug candidates. Although our knowledge regarding their exact activity vs. toxicity profile is incomplete, changes in cell membrane biophysical properties and cytoskeletal structures have been implicated as part of the mechanism of action. Thus, in this work, we characterised the effects of iron(II)-based complexes on the structural and morphological properties of epithelial non-tumorigenic (MCF 10A) and tumorigenic (MDA-MB-231) breast cell lines using atomic force microscopy (AFM), flow cytometry and immunofluorescence microscopy. At 24 h of exposure, both the MCF 10A and MDA-MB-231 cells experienced a cell softening, and an increase in size followed by a re-stiffening at 96 h. In addition, the triple negative breast cancer cell line, MDA-MB-231, sustained a notable cytoskeletal and mitochondrial reorganization with increased actin stress fibers and cell-to-cell communication structures. An extensive all-atom molecular dynamic simulation suggests a possible direct and unassisted internalization of the metallodrug candidate, and confirmed that the cellular effects could not be ascribed to the simple physical interaction of the iron-based complexes with the biological membrane. These observations provide an insight into a link between the mechanisms of action of such iron-based complexes as anti-cancer treatment and cytoskeletal architecture.
Asunto(s)
Antineoplásicos , Neoplasias de la Mama , Neoplasias de la Mama Triple Negativas , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Compuestos Ferrosos , Humanos , Hierro , Células MCF-7 , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
Atomic force microscope techniques and multi-staining fluorescence microscopy were employed to study the steps in drinking water biofilm formation. During the formation of a conditioning layer, surface hydrophobic forces increased and the range of characteristic hydrophobic forces diversified with time, becoming progressively complex in macromolecular composition, which in return triggered irreversible cellular adhesion. AFM visualization of 1 to 8 week drinking water biofilms showed a spatially discontinuous and heterogeneous distribution comprising an extensive network of filamentous fungi in which biofilm aggregates were embedded. The elastic modulus of 40-day-old biofilms ranged from 200 to 9000 kPa, and the biofilm deposits with a height >0.5 µm had an elastic modulus <600 kPa, suggesting that the drinking water biofilms were composed of a soft top layer and a basal layer with significantly higher elastic modulus values falling in the range of fungal elasticity.
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Biopelículas/crecimiento & desarrollo , Módulo de Elasticidad , Microbiología del Agua , Adhesión Celular , Hongos/crecimiento & desarrollo , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía de Fuerza Atómica , Agua/análisisRESUMEN
The elaboration of efficient hydrogel-based materials with antimicrobial properties requires a refined control of defining their physicochemical features, which includes mechanical stiffness, so as to properly mediate their antibacterial activity. In this work, we design hydrogels consisting of polyelectrolyte multilayer films for the loading of T4 and φX174 bacteria-killing viruses, also called bacteriophages. We investigate the antiadhesion and bactericidal performances of this biomaterial against Escherichia coli, with a specific focus on the effects of chemical cross-linking of the hydrogel matrix, which, in turn, mediates the hydrogel stiffness. Depending on the latter and on phage replication features, it is found that the hydrogels loaded with the bacteria-killing viruses make both contact killing (targeted bacteria are those adhered at the hydrogel surface) and release killing (planktonic bacteria are the targets) possible with ca. 20-80% efficiency after only 4 h of incubation at 25 °C as compared to cases where hydrogels are free of viruses. We further demonstrate the lack of dependence of virus diffusion within the hydrogel and of the maximal viral storage capacity on the hydrogel mechanical properties. In addition to the evidenced bacteriolytic activity of the phages loaded in the hydrogels, the antimicrobial property of the phage-loaded materials is shown to be partly controlled by the chemistry of the hydrogel skeleton and, more specifically, by the mobility of the peripheral free polycationic components, known for their ability to weaken and permeabilize membranes of bacteria, the latter then becoming "easier" targets for the viruses.
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Antibacterianos/farmacología , Bacteriófagos/química , Materiales Biocompatibles/farmacología , Escherichia coli/efectos de los fármacos , Hidrogeles/farmacología , Antibacterianos/química , Materiales Biocompatibles/química , Hidrogeles/química , Ensayo de Materiales , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Tamaño de la Partícula , Estrés MecánicoRESUMEN
Yad fimbriae are currently viewed as versatile bacterial adhesins able to significantly mediate host or plant-pathogen recognition and contribute to the persistence of Escherichia coli in both the environment and within hosts. To date, however, the underlying adhesion process of Yad fimbriae on surfaces defined by controlled coating chemistries has not been evaluated on the relevant molecular scale. In this work, the interaction forces operational between Yad fimbriae expressed by genetically modified E. coli and self-assembled monolayers (SAM) differing in terms of charge, hydrophobicity or the nature of decorating sugar units are quantified by Single Molecule Force Spectroscopy (SMFS) on the nanoscale. It is found that the adhesion of Yad fimbriae onto probes functionalized with xylose is as strong as that measured with probes decorated with anti-Yad antibodies (ca. 80 to 300 pN). In contrast, the interactions of Yad with galactose, lactose, mannose, -OH, -NH2, -COOH and -CH3 terminated SAMs are clearly non-specific. Interpretation of SMFS measurements on the basis of worm-like-chain modeling for polypeptide nanomechanics further leads to the estimates of the maximal extension of Yad fimbriae upon stretching, of their persistence length and of their polydispersity. Finally, we show for the first time a strong correlation between the adhesion properties of Yad-decorated bacteria determined from conventional macroscopic counting methods and the molecular adhesion capacity of Yad fimbriae. This demonstration advocates the effort that should be made to understand on the nanoscale level the interactions between fimbriae and their cognate ligands. The results could further help the design of potential anti-adhesive molecules or surfaces to better fight against the virulence of bacterial pathogens.