ABSTRACT
Silver nanoparticles (AgNPs) may be synthesized by many different methods, with those based on the thermal reduction of silver salts by citric acid or citric acid/tannic acid being amongst the most commonly used. These methods, although widely used and technically simple, can produce particles in which the size, polydispersivity and morphology can vary greatly. In this work nearly mono-dispersed spherical AgNPs have been synthesized via a one-step reduction method by using sodium citrate and varying quantities of Tannic Acid (TA), which was thermally conditioned prior to use in the growth process. It was found that the final size can be further tailored by controlling the amount of TA and the thermal conditioning of the TA at 60 °C at different time points, which changes the size and polydispersivity of AgNPs. To better understand the origin of this effect, optical spectroscopic analysis and 1H NMR of the TA following mild thermal conditioning of the solution have been done. Comparison of thermally conditioned TA and TA exposed to basic pH shows that similar chemical modifications occur and consequently produce similar effects on growth when used in the synthesis of AgNPs. It is proposed that thermal preconditioning of the TA introduces either chemical or structural changes, which decrease the final particle size under a given total silver content.
ABSTRACT
Multi-functionalized nanoparticles are of great interest in biotechnology and biomedicine, especially for diagnostic and therapeutic purposes. However, at the moment the characterization of complex, multi-functional nanoparticles is still challenging and this hampers the development of advanced nanomaterials for biological applications. In this work, we have designed a model system consisting of gold nanoparticles functionalized with two differentially-terminated poly(ethylene oxide) ligands, providing both "stealth" properties and protein-binding capabilities to the nanoparticles. We use a combination of techniques (Centrifugal Liquid Sedimentation, Dynamic Light Scattering, Flow Field Flow Fractionation, Transmission Electron Microscopy, and Circular Dichroism) to: (i) monitor and quantify the ratios of ligand molecules per nanoparticle; (ii) determine the effect of coating density on non-specific protein adsorption; (iii) to assess the number and structure of the covalently-bound proteins. This article aims at comparing the complementary outcomes from typical and orthogonal techniques used in nanoparticle characterization by employing a versatile nanoparticle-ligands-biomolecule model system.
Subject(s)
Gold , Metal Nanoparticles/chemistry , Proteins/chemistry , Adsorption , Circular Dichroism , Dynamic Light Scattering , Fractionation, Field Flow , Microscopy, Electron, Transmission , Particle Size , Polyethylene GlycolsABSTRACT
Characterisation of engineered nanomaterials (NMs) is of outmost importance for the assessment of the potential risks arising from their extensive use. NMs display indeed a large variety of physico-chemical properties that drastically affect their interaction with biological systems. Among them, hydrophobicity is an important property that is nevertheless only slightly covered by the current physico-chemical characterisation techniques. In this work, we developed a method for the direct characterisation of NM hydrophobicity. The determination of the nanomaterial hydrophobic character is carried out by the direct measurement of the affinity of the NMs for different collectors. Each collector is an engineered surface designed in order to present specific surface charge and hydrophobicity degrees. Being thus characterised by a combination of surface energy components, the collectors enable the NM immobilisation with surface coverage in relation to their hydrophobicity. The experimental results are explained by using the extended DLVO theory, which takes into account the hydrophobic forces acting between NMs and collectors. Graphical abstractDetermination of hydrophobicity character of nanomaterials by measuring their affinity to engineered surfaces.
ABSTRACT
Analytical ultracentrifugation (AUC) is a powerful tool for the study of particle size distributions and interactions with high accuracy and resolution. In this work, we show how the analysis of sedimentation velocity data from the AUC can be used to characterize nanocarrier drug delivery systems used in nanomedicine. Nanocarrier size distribution and the ratio of free versus nanoparticle-encapsulated drug in a commercially available liposomal doxorubicin formulation are determined using interference and absorbance based AUC measurements and compared with results generated with conventional techniques. Additionally, the potential of AUC in measuring particle density and the detection of nanocarrier sub-populations is discussed as well. The unique capability of AUC in providing reliable data for size and composition in a single measurement and without complex sample preparation makes this characterization technique a promising tool both in nanomedicine product development and quality control.
Subject(s)
Antibiotics, Antineoplastic/analysis , Doxorubicin/analogs & derivatives , Doxorubicin/analysis , Nanomedicine , Particle Size , Polyethylene Glycols/analysis , UltracentrifugationABSTRACT
pH-sensitive nonionic surfactant vesicles (niosomes) by polysorbate-20 (Tween-20) or polysorbate-20 derivatized by glycine (added as pH sensitive agent), were developed to deliver Ibuprofen (IBU) and Lidocaine (LID). For the physical-chemical characterization of vesicles (mean size, size distribution, zeta potential, vesicle morphology, bilayer properties and stability) dynamic light scattering (DLS), small angle X-ray scattering and fluorescence studies were performed. Potential cytotoxicity was evaluated on immortalized human keratinocyte cells (HaCaT) and on immortalized mouse fibroblasts Balb/3T3. In vivo antinociceptive activity (formalin test) and anti-inflammatory activity tests (paw edema induced by zymosan) in murine models were performed on drug-loaded niosomes. pH-sensitive niosomes were stable in the presence of 0 and 10% fetal bovine serum, non-cytotoxic and able to modify IBU or LID pharmacological activity in vivo. The synthesis of stimuli responsive surfactant, as an alternative to add pH-sensitive molecules to niosomes, could represent a promising delivery strategy for anesthetic and anti-inflammatory drugs.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Disease Models, Animal , Ibuprofen/pharmacology , Inflammation/drug therapy , Lidocaine/pharmacology , Liposomes/chemistry , Pain/drug therapy , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , BALB 3T3 Cells , Cells, Cultured , Fibroblasts/drug effects , Humans , Hydrogen-Ion Concentration , Ibuprofen/administration & dosage , Lidocaine/administration & dosage , Liposomes/administration & dosage , Liposomes/pharmacology , Mice , Pain Measurement , Surface-Active Agents/chemistry , Surface-Active Agents/pharmacologyABSTRACT
Development of reliable cell-based nanotoxicology assays is important for evaluation of potentially hazardous engineered nanomaterials. Challenges to producing a reliable assay protocol include working with nanoparticle dispersions and living cell lines, and the potential for nano-related interference effects. Here we demonstrate the use of a 96-well plate design with several measurement controls and an interlaboratory comparison study involving five laboratories to characterize the robustness of a nanocytotoxicity MTS cell viability assay based on the A549 cell line. The consensus EC50 values were 22.1 mg/L (95% confidence intervals 16.9 mg/L to 27.2 mg/L) and 52.6 mg/L (44.1 mg/L to 62.6 mg/L) for positively charged polystyrene nanoparticles for the serum-free and serum conditions, respectively, and 49.7 µmol/L (47.5 µmol/L to 51.5 µmol/L) and 77.0 µmol/L (54.3 µmol/L to 99.4 µmol/L) for positive chemical control cadmium sulfate for the serum-free and serum conditions, respectively. Results from the measurement controls can be used to evaluate the sources of variability and their relative magnitudes within and between laboratories. This information revealed steps of the protocol that may need to be modified to improve the overall robustness and precision. The results suggest that protocol details such as cell line ID, media exchange, cell handling, and nanoparticle dispersion are critical to ensure protocol robustness and comparability of nanocytotoxicity assay results. The combination of system control measurements and interlaboratory comparison data yielded insights that would not have been available by either approach by itself.
Subject(s)
Hazardous Substances/toxicity , Laboratories/statistics & numerical data , Nanoparticles/toxicity , Polystyrenes/toxicity , Toxicity Tests/statistics & numerical data , A549 Cells , Humans , Laboratories/standards , Reproducibility of Results , Toxicity Tests/standardsABSTRACT
Peptide-lipid interactions support a variety of biological functions. Of particular interest are those that underpin fundamental mechanisms of innate immunity that are programmed in host defense or antimicrobial peptide sequences found virtually in all multicellular organisms. Here we synthetically modulate antimicrobial peptide-lipid interactions using an archetypal helical antimicrobial peptide and synthetic membranes mimicking bacterial and mammalian membranes in solution. We probe these interactions as a function of membrane-induced folding, membrane stability and peptide-lipid ratios using a correlative approach encompassing light scattering and spectroscopy measurements such as circular dichroism spectroscopy, fluorescence and nuclear magnetic resonance spectroscopy. The peptide behavior is assessed against that of its anionic counterpart having similar propensities for α-helical folding. The results indicate strong correlations between peptide folding and membrane type, supporting folding-responsive binding of antimicrobial peptides to bacterial membranes. The study provides a straightforward approach for modulating structure-activity relationships in the context of membrane-induced antimicrobial action, thus holding promise for the rational design of potent antimicrobial agents.
Subject(s)
Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/metabolism , Protein Folding , Unilamellar Liposomes/metabolism , Amino Acid Sequence , Cell Membrane/metabolism , Protein BindingABSTRACT
Surfaces with high water-adhesion properties are promising materials for different applications in the field of water treatment and management, such as for water-harvesting systems or oil/water separation membranes. Herein, we developed rose-petal-like substrates that demonstrate interesting parahydrophobic character. This bioinspired material mimics the natural substrate thanks to a combination of two fabrication steps: (1)â micropatterning to create a microstructured gold-coated substrate consisting of square pillars and (2)â an electropolymerization process generating nanostructures over the micropillars. Judicious choice of the micropatterning specifications (pillar diameter and pitch), the type of electropolymerizable monomer, and the electrochemical parameters produces a material with both extremely high water contact angles (up to 160°), while retaining a remarkably high water-adhesion level. Our study suggests that a composite interface is expressed by the existence of the Wenzel state on the micropillars and the Cassie-Baxter state between the pillars ("Cassie-filled nanostructure"), as observed during our contact-angle measurements. Indeed, we show that the pitch should be small to obtain the optimal micropillar surface density. Moreover, a relatively low deposition charge of approximately 50â mC cm-2 is preferable for coating the square pillars exclusively with nanostructures.
ABSTRACT
Invited for this month's cover are the collaborating groups of Dr. Thierry Darmanin at Université Côte d'Azur, France and Dr. François Rossi at JRC European Commission, Italy. The cover picture shows a novel strategy for preparing substrates having a rose-petal effect (high water adhesion). The micropatterning specifications (pillar diameter and pitch) and the electropolymerization parameters are key to obtaining both high water apparent contact angles and a high hysteresis. Read the full text of the article at 10.1002/cplu.201600387.
ABSTRACT
The detection and quantification of nanoparticles is a complex issue due to the need to combine "classical" identification and quantification of the constituent material, with the accurate determination of the size of submicrometer objects, usually well below the optical diffraction limit. In this work, the authors show that one of the most used analytical methods for silver nanoparticles, asymmetric flow field-flow fractionation, can be strongly influenced by the presence of dissolved organic matter (such as alginate) and lead to potentially misleading results. The authors explain the anomalies in the separation process and show a very general way forward based on the combination of size separation and size measurement techniques. This combination of techniques results in more robust AF4-based methods for the sizing of silver nanoparticles in environmental conditions and could be generally applied to the sizing of nanoparticles in complex matrices.
Subject(s)
Alginates , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Particle Size , Silver , Fractionation, Field Flow , Glucuronic Acid , Hexuronic AcidsABSTRACT
Ultraviolet (UV) radiation, temperature, and time can degrade proteins. Here, the authors show that gold nanoparticles significantly protect human serum albumin from denaturation when exposed to "stressing" conditions such as UV irradiation and sustained exposure in suboptimal conditions. In particular, the authors show that gold nanoparticles significantly reduce the decrease in secondary structure induced by UV irradiation or extended exposure to ambient temperature.
Subject(s)
Gold , Nanoparticles/chemistry , Protein Denaturation/radiation effects , Serum Albumin/chemistry , Temperature , Ultraviolet Rays , Circular Dichroism , Humans , Protein Conformation/radiation effects , Protein Stability , Serum Albumin, Human , Time FactorsABSTRACT
Microarray technology was developed in the early 1990s to measure the transcription levels of thousands of genes in parallel. The basic premise of high-density arraying has since been expanded to create cell microarrays. Cells on chip are powerful experimental tools for high-throughput and multiplex screening of samples or cellular functions. Miniaturization increases assay throughput while reducing both reagent consumption and cell population heterogeneity effect, making these systems attractive for a wide range of assays, from drug discovery to toxicology, stem cell research and therapy. It is usual to functionalize the surface of a substrate to design cell microarrays. One form of cell microarrays, the transfected cell microarray, wherein plasmid DNA or siRNA spotted on the surface of a substrate is reverse-transfected locally into adherent cells, has become a standard tool for parallel cell-based analysis. With the advent of technology, cells can also be directly spotted onto functionalized surfaces using robotic fluid-dispensing devices or printed directly on bio-ink material. We are providing herein an overview of the latest developments in optical cell microarrays allowing high-throughput and high-content analysis.
Subject(s)
High-Throughput Screening Assays/methods , Tissue Array Analysis/methods , Animals , Flow Cytometry , High-Throughput Screening Assays/instrumentation , Humans , Single-Cell Analysis , Tissue Array Analysis/instrumentationABSTRACT
BACKGROUND: The constant increase of the use of nanomaterials in consumer products is making increasingly urgent that standardized and reliable in vitro test methods for toxicity screening be made available to the scientific community. For this purpose, the determination of the cellular dose, i.e. the amount of nanomaterials effectively in contact with the cells is fundamental for a trustworthy determination of nanomaterial dose responses. This has often been overlooked in the literature making it difficult to undertake a comparison of datasets from different studies. Characterization of the mechanisms involved in nanomaterial transport and the determination of the cellular dose is essential for the development of predictive numerical models and reliable in vitro screening methods. RESULTS: This work aims to relate key physico-chemical properties of gold nanoparticles (NPs) to the kinetics of their deposition on the cellular monolayer. Firstly, an extensive characterization of NPs in complete culture cell medium was performed to determine the diameter and the apparent mass density of the formed NP-serum protein complexes. Subsequently, the kinetics of deposition were studied by UV-vis absorbance measurements in the presence or absence of cells. The fraction of NPs deposited on the cellular layer was found to be highly dependent on NP size and apparent density because these two parameters influence the NP transport. The NP deposition occurred in two phases: phase 1, which consists of cellular uptake driven by the NP-cell affinity, and phase 2 consisting mainly of NP deposition onto the cellular membrane. CONCLUSION: The fraction of deposited NPs is very different from the initial concentration applied in the in vitro assay, and is highly dependent of the size and density of the NPs, on the associated transport rate and on the exposure duration. This study shows that an accurate characterization is needed and suitable experimental conditions such as initial concentration of NPs and liquid height in the wells has to be considered since they strongly influence the cellular dose and the nature of interactions of NPs with the cells.
Subject(s)
Nanoparticles/toxicity , Dose-Response Relationship, Drug , Humans , In Vitro Techniques , Spectrophotometry, UltravioletABSTRACT
In this work, Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS), Principal Component Analysis (PCA) and X-ray Photoelectron Spectroscopy (XPS) have been used to characterize the surface chemistry of gold substrates before and after functionalization with thiol-modified glucose self-assembled monolayers and subsequent biochemical specific recognition of maltose binding protein (MBP). The results indicate that the surface functionalization is achieved both on flat and nanoparticles gold substrates thus showing the potential of the developed system as biodetection platform. Moreover, the method presented here has been found to be a sound and valid approach to characterize the surface chemistry of nanoparticles functionalized with large molecules. Both techniques were proved to be very useful tools for monitoring all the functionalization steps, including the investigation of the biological behavior of the glucose-modified particles in the presence of the maltose binding protein.
ABSTRACT
The following work presents a simple, reliable and scalable seeding-growth methodology to prepare silica nanoparticles (SiO2 NPs) (20, 30, 50 and 80 nm) directly in aqueous phase, both as plain- as well as fluorescent-labeled silica. The amount of fluorescent label per particle remained constant regardless of size, which facilitates measurements in terms of number-based concentrations. SiO2 NPs in dispersion were functionalized with an epoxysilane, thus providing a flexible platform for the covalent linkage of wide variety of molecules under mild experimental conditions. This approach was validated with ethylenediamine, two different amino acids and three akylamines to generate a variety of surface modifications. Accurate characterization of particle size, size distributions, morphology and surface chemistry is provided, both for as-synthesized particles and after incubation in cell culture medium. The impact of physicochemical properties of SiO2 NPs was investigated with human alveolar basal epithelial cells (A549) such as the effect in cytotoxicity, cell internalization and membrane interaction.
Subject(s)
Cell Survival/drug effects , Epithelial Cells/drug effects , Nanoparticles/chemistry , Silicon Dioxide/chemistry , Cell Line , Culture Media/chemistry , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/chemistry , Humans , Nanoparticles/administration & dosage , Particle Size , Silicon Dioxide/administration & dosage , Surface PropertiesABSTRACT
The wide use of titanium dioxide nanoparticles (TiO2 NPs) in industrial applications requires the investigation of their effects on human health. In this context, we investigated the effects of nanosized and bulk titania in two different crystalline forms (anatase and rutile) in vitro. By colony forming efficiency assay, a dose-dependent reduction of the clonogenic activity of Balb/3T3 mouse fibroblasts was detected in the presence of rutile, but not in the case of anatase NPs. Similarly, the cell transformation assay and the micronucleus test showed that rutile TiO2 NPs were able to induce type-III foci formation in Balb/3T3 cells and appeared to be slightly genotoxic, whereas anatase TiO2 NPs did not induce any significant neoplastic or genotoxic effect. Additionally, we investigated the interaction of TiO2 NPs with Balb/3T3 cells and quantified the in vitro uptake of titania using mass spectrometry. Results showed that the internalization was independent of the crystalline form of TiO2 NPs but size-dependent, as nano-titania were taken up more than their respective bulk materials. In conclusion, we demonstrated that the cytotoxic, neoplastic and genotoxic effects triggered in Balb/3T3 cells by TiO2 NPs depend on the crystalline form of the nanomaterial, whereas the internalization is regulated by the particle size.
Subject(s)
Metal Nanoparticles/toxicity , Mutagens/toxicity , Titanium/toxicity , Animals , BALB 3T3 Cells , Biological Transport , Cell Survival/drug effects , Cell Transformation, Neoplastic/drug effects , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Mice , Micronucleus Tests , Microscopy, Electron, Transmission , Mutagens/chemistry , Titanium/chemistry , Titanium/pharmacologyABSTRACT
This paper charts the almost ten years of history of OECD's work on nanosafety, during which the programme of the OECD on the Testing and Assessment of Manufactured Nanomaterials covered the testing of eleven nanomaterials for about 59 end-points addressing physical-chemical properties, mammalian and environmental toxicity, environmental fate and material safety. An overview of the materials tested, the test methods applied and the discussions regarding the applicability of the OECD test guidelines, which are recognised methods for regulatory testing of chemicals, are given. The results indicate that many existing OECD test guidelines are suitable for nanomaterials and consequently, hazard data collected using such guidelines will fall under OECD's system of Mutual Acceptance of Data (MAD) which is a legally binding instrument to facilitate the international acceptance of information for the regulatory safety assessment of chemicals. At the same time, some OECD test guidelines and guidance documents need to be adapted to address nanomaterials while new test guidelines and guidance documents may be needed to address endpoints that are more relevant to nanomaterials. This paper presents examples of areas where test guidelines or guidance for nanomaterials are under development.
Subject(s)
Guidelines as Topic , Nanostructures/adverse effects , Nanotechnology , Toxicity Tests , Animals , Consensus , Guidelines as Topic/standards , History, 21st Century , Humans , Nanostructures/history , Nanostructures/standards , Nanotechnology/history , Nanotechnology/standards , Policy Making , Program Development , Risk Assessment , Toxicity Tests/history , Toxicity Tests/standardsABSTRACT
Amorphous carbon films exhibit attractive optical and surface properties. In this work, modified amorphous carbon films incorporating nitroxide groups (α-CNO) have been obtained by searching for a condensed analogue to classical soft antifouling materials. Thin films deposited by reactive magnetron sputtering in air discharges at varying power conditions were characterized by ellipsometry, atomic force microscopy, and water contact angle. Plasma power was observed to activate the densification and roughness of nanograined films. Most hydrophilic films deposited at 30 W exhibited the lowest refractive index, negligible optical absorption in the vis-IR, and presented a close to stoichiometric C2NO composition, as derived from X-ray photoelectron spectroscopy. Micropatterns prepared by photolithography validated the transparency-hydrophilicity of the α-CNO, as observed by water condensation contrast imaging. An albumin adsorption experiment evaluated through fluorescence revealed that α-CNO behaves as antifouling with respect to Si. Such thin antifouling films are of interest for the initiation of immobilization cascades in imaging surface plasmon resonance, where they have confirmed their antifouling contrast enhancement role. These results illustrate that the combination of a nanorough surface with nitroxide chemistry induces an antifouling behavior. In association with the optical transparency, the results invite the exploration of the bioengineering dimension of α-CNO films.
ABSTRACT
Asymmetric Flow Field-Flow Fractionation (AF4) combined with multidetector analysis form a promising technique in the field of nanoparticle characterization. This system is able to measure the dimensions and physicochemical properties of nanoparticles with unprecedented accuracy and precision. Here, for the first time, this technique is optimized to characterize the interaction between an archetypal antimicrobial peptide and synthetic membranes. By using charged and neutral liposomes it is possible to mimic some of the charge characteristics of biological membranes. The use of AF4 system allows determining, in a single analysis, information regarding the selectivity of the peptides, the quantity of peptides bound to each liposome, the induced change in the size distribution and morphology of the liposomes. The results obtained provide relevant information for the study of structure-activity relationships in the context of membrane-induced antimicrobial action. This information will contribute to the rational design of potent antimicrobial agents in the future. Moreover, the application of this method to other liposome systems is straightforward and would be extremely useful for a comprehensive characterization with regard to size distribution and protein interaction in the nanomedicine field.