Surface chemistry of metal oxide nanoparticles: NMR and TGA quantification.

Surface functionalization is widely used to control the behavior of nanomaterials for a range of applications. However, methods to accurately quantify surface functional groups and coatings are not yet routinely applied to nanomaterial characterization. We have employed a combination of quantitative NMR (qNMR) and thermogravimetric analysis (TGA) to address this problem for commercial cerium, nickel, and iron oxide nanoparticles (NPs) that have been modified to add functional coatings with (3-aminopropyl)triethoxysilane (APTES), stearic acid, and polyvinylpyrrolidone (PVP). The qNMR method involves quantification of material that is released from the NPs and quantified in the supernatant after removal of NPs. Removal of aminopropylsilanes was accomplished by basic hydrolysis whereas PVP and stearic acid were removed by ligand exchange using sodium hexametaphosphate and pentadecafluorooctanoic acid, respectively. The method accuracy was confirmed by analysis of NPs with a known content of surface groups. Complementary TGA studies were carried out in both air and argon atmosphere with FT-IR of evolved gases in argon to confirm the identity of the functional groups. TGA measurements for some unfunctionalized samples show mass loss due to unidentified components which makes quantification of functional groups in surface-modified samples less reliable. XPS provides information on the presence of surface contaminants and the level of surface hydroxylation for selected samples. Despite the issues associated with accurate quantification using TGA, the TGA estimates agree reasonably well with the qNMR data for samples with high surface loading. This study highlights the issues in analysis of commercial nanomaterials and is an advance towards the development of generally applicable methods for quantifying surface functional groups.

Interlaboratory Comparison on the Quantification of Total and Accessible Amine Groups on Silica Nanoparticles with qNMR and Optical Assays.

Risk assessment of nanomaterials requires not only standardized toxicity studies but also validated methods for nanomaterial surface characterization with known uncertainties. In this context, a first bilateral interlaboratory comparison on surface group quantification of nanomaterials is presented that assesses different reporter-free and labeling methods for the quantification of the total and accessible number of amine functionalities on commercially available silica nanoparticles that are widely used in the life sciences. The overall goal of this comparison is the identification of optimum methods as well as achievable measurement uncertainties and the comparability of the results across laboratories. We also examined the robustness and ease of implementation of the applied analytical methods and discussed method-inherent limitations. In summary, this comparison presents a first step toward the eventually required standardization of methods for surface group quantification.

Acellular oxidative potential assay for screening of amorphous silica nanoparticles.

Silica nanoparticles (SiNPs) are used in a wide range of consumer products, engineering and medical applications, with likelihood of human exposure and potential health concerns. It is essential to generate toxicity information on SiNP forms and associated physicochemical determinants to conduct risk assessment on these new materials. To address this knowledge gap, we screened a panel of custom synthesized, well-characterized amorphous SiNPs pristine and surface-modified (-C3-COOH, -C11-COOH, -NH2, -PEG) of 5 different sizes: (15, 30, 50, 75, 100 nm) for their oxidative potential using an acellular assay. The assay is based on oxidation of dithiothreitol (DTT) by reactive oxygen species and can serve as a surrogate test for oxidative stress. These materials were characterized for size distribution, aggregation, crystallinity, surface area, surface modification, surface charge and metal content. Tests for association between oxidative potential of SiNPs and their physicochemical properties were carried out using analysis of variance and correlation analyses. These test results suggest that the size of amorphous SiNPs influenced their oxidative potential irrespective of the surface modification, with 15 nm exhibiting relatively higher oxidative potential compared to the other sizes. Furthermore, SiNP surface area, surface modification and agglomeration in solution also appeared to affect oxidative potential of these SiNPs. These findings indicate that physicochemical properties are critical in influencing the oxidative behaviour of amorphous SiNPs, with potential to trigger cellular oxidative stress and thus toxicity, when exposed. This information advances our understanding of potential toxicities of these amorphous SiNPs and supports risk assessment efforts and the design of safer forms of silica nanomaterials.

Polycarboxylated Dextran as a Multivalent Linker: Synthesis and Target Recognition of the Antibody-Nanoparticle Bioconjugates in PBS and Serum.

Nanoparticles (NPs) functionalized with antibodies on their surface are used in a wide range of research applications. However, the bioconjugation chemistry between the antibodies and the surface of nanoparticles can be very challenging, often accompanied by several undesired effects such as nanoparticle aggregation, antibody denaturation, or poor target recognition of the surface-bound antibodies. Here, we report on a synthesis of fluorescent silica nanoparticle-antibody (NP-Ab) conjugates, in which polycarboxylated dextran is used as the multivalent linker. First, we present a synthetic methodology to prepare polycarboxylated dextrans with molecular weights of 6, 40, and 70 kDa. Second, we used water-soluble, polycarboxylated dextrans as a multivalent spacers/linkers to immobilize antibodies onto fluorescent silica nanoparticles. The prepared NP-Ab conjugates were tested in a direct binding assay format in both phosphate-buffered saline buffer and whole serum to investigate the role of the spacer/linker in the capacity of the NP-Ab to specifically recognize their target in "clean" and also in complex media. We have compared the dextran conjugates with two standards: (a) NP-Ab with antibodies attached on the surface of nanoparticles through the classical physical adsorption method and (b) NP-Ab where an established poly(amidoamine) (PAMAM) dendrimer was used as the linker. Our results showed that the polycarboxylated 6 kDa dextran facilitates antibody immobilization efficiency of nearly 92%. This was directly translated into the improved molecular recognition of the NP-Ab, which was measured by a direct binding assay. The signal-to-noise ratio in buffered solution for the 6 kDa dextran NP-Ab conjugates was 81, nearly 3 times higher than that of PAMAM G4.5 conjugates and 9 times higher than the physically adsorbed NP-Ab sample. In whole serum, the effect of 6 kDa dextran was more hindered due to the formation of protein corona but the signal-to-noise ratio was at least double that of the physically adsorbed NP-Ab conjugates.

Quantification of surface functional groups on silica nanoparticles: comparison of thermogravimetric analysis and quantitative NMR.

Thermogravimetric analysis (TGA) coupled with evolved gas analysis-FT-IR has been examined as a potential method to study the functional group content for surface modified silica nanoparticles. A comparison with a quantitative solution NMR method based on analysis of groups released after dissolution of the silica matrix is used to provide benchmark data for comparison and to assess the utility and limitations of TGA. This study focused primarily on commercially available silicas and tested whether it was possible to use a correction based on bare silica to account for the significant mass loss that occurs due to condensation of surface hydroxyl groups and loss of matrix-entrapped components at temperatures above ∼200 °C. Although this approach has been used successfully in the literature for in-house prepared samples, it was problematic for commercial silicas prepared by the Stöber method. For these materials the agreement between estimates from qNMR and TGA mass loss was poor in many cases. However much better agreement was observed for samples for which the mass loss above 200 °C is relatively low, such as non-porous silica, or samples for which the mass fraction of functional group is large (e.g., high molecule weight groups or multilayers). FT-IR was useful in identifying the likely structure of the components lost from the surface at various temperatures and in some cases provided evidence of contaminants in the sample. Nevertheless, in other cases correlation of thermograms and FT-IR with NMR data was necessary, particularly for samples where multi-step modification of the silica surface results in incomplete functionalization that gives a mixture of products. Overall the results indicate that TGA provides reliable results for silicas of low porosity or those for which the functional group accounts for a significant fraction of the total sample mass. It is also suitable as a supplementary or screening technique to indicate the presence of coatings or covalent surface modification, prior to applying other techniques or for routine analyses where sensitivity is not critical.

Quantification and Stability Determination of Surface Amine Groups on Silica Nanoparticles Using Solution NMR.

Surface chemistry is a critical factor for determining the behavior of a nanomaterial after incorporation in composites, devices, and biomedical products, and is also important for nanotoxicology studies. We have developed an optimized protocol for dissolution of aminated silicas and determination of functional-group contents by quantitative 1H NMR (qNMR) analysis of the released amines. A number of variables were optimized for the dissolution protocol, including the base concentration, mass of silica, time, temperature, and method of sample agitation, in order to achieve adequate NMR signals for quantification. The protocol was tested using nanoparticles from a single commercial supplier with sizes ranging from 20 to 120 nm that were functionalized with 3-aminopropyl groups. Interestingly the batch-to-batch variability for some sizes of these aminated silicas was as high as 50%. Amine contents measured by a ninhydrin colorimetric assay were typically ∼20% lower than those measured by qNMR, consistent with measurement of only ninhydrin-reagent accessible amines. The dissolution-qNMR protocol was compatible with aminated silicas from other commercial suppliers, and in these cases, an even larger variability in surface coverage was observed. Silica nanoparticles with longer-chain amines and variable amine loadings were synthesized to demonstrate the ability to quantify amines with more complex structures and to assess the limit of quantification for the dissolution-qNMR method. Finally, the stability of the aminated nanoparticles was examined. Loss of 3-aminopropyl groups occurred in water at room temperature and was significantly more rapid at higher temperatures. Amine loss increased with increasing surface coverage and was slower for long-chain amines, consistent with studies of amine stability on planar silica. Overall, this work highlights the importance of developing methods for quantifying surface functionalization, particularly given the variability in surface coverage for commercial samples, and for ensuring that the amine group is stable under its usage conditions.

Physical Characterization and Cellular Toxicity Studies of Commercial NiO Nanoparticles.

Nickel oxide (NiO) nanoparticles from several manufacturers with different reported sizes and surface coatings were characterized prior to assessing their cellular toxicity. The physical characterization of these particles revealed that sizes often varied from those reported by the supplier, and that particles were heavily agglomerated when dispersed in water, resulting in a smaller surface area and larger hydrodynamic diameter upon dispersion. Cytotoxicity testing of these materials showed differences between samples; however, correlation of these differences with the physical properties of the materials was not conclusive. Generally, particles with higher surface area and smaller hydrodynamic diameter were more cytotoxic. While all samples produced an increase in reactive oxygen species (ROS), there was no correlation between the magnitude of the increase in ROS and the difference in cytotoxicity between different materials.

Salicylanilide carbamates: antitubercular agents active against multidrug-resistant Mycobacterium tuberculosis strains.

A series of 27 salicylanilide-based carbamates was prepared as a part of our ongoing search for new antituberculosis drugs. These compounds exhibited very good in vitro activity against Mycobacterium tuberculosis, Mycobacterium kansasii and Mycobacterium avium and, in particular, against five multidrug-resistant strains, with MIC values between 0.5-2 micromol/L. Moreover, they displayed moderate toxicity against intestinal cells with the selectivity index being up to 96. Furthermore, acid stability and a half-life of 43h at pH 7.4 were shown. Thus, these novel salicylanilide derivatives are drug candidates which should be seriously consider for further screening.

A Multi-Method Approach for Quantification of Surface Coatings on Commercial Zinc Oxide Nanomaterials.

Surface functionalization is a key factor for determining the performance of nanomaterials in a range of applications and their fate when released to the environment. Nevertheless, it is still relatively rare that surface groups or coatings are quantified using methods that have been carefully optimized and validated with a multi-method approach. We have quantified the surface groups on a set of commercial ZnO nanoparticles modified with three different reagents ((3-aminopropyl)-triethoxysilane, caprylsilane and stearic acid). This study used thermogravimetric analysis (TGA) with Fourier transform infrared spectroscopy (FT-IR) of evolved gases and quantitative solution 1H nuclear magnetic resonance (NMR) for quantification purposes with 13C-solid state NMR and X-ray photoelectron spectroscopy to confirm assignments. Unmodified materials from the same suppliers were examined to assess possible impurities and corrections. The results demonstrate that there are significant mass losses from the unmodified samples which are attributed to surface carbonates or residual materials from the synthetic procedure used. The surface modified materials show a characteristic loss of functional group between 300-600 °C as confirmed by analysis of FT-IR spectra and comparison to NMR data obtained after quantitative release/extraction of the functional group from the surface. The agreement between NMR and TGA estimates for surface loading is reasonably good for cases where the functional group accounts for a relatively large fraction of the sample mass (e.g., large groups or high loading). In other cases TGA does not have sufficient sensitivity for quantitative analysis, particularly when contaminants contribute to the TGA mass loss. X-ray photoelectron spectroscopy and solid state NMR for selected samples provide support for the assignment of both the functional groups and some impurities. The level of surface group loading varies significantly with supplier and even for different batches or sizes of nanoparticles from the same supplier. These results highlight the importance of developing reliable methods to detect and quantify surface functional groups and the importance of a multi-method approach.

Characterization of Commercial Metal Oxide Nanomaterials: Crystalline Phase, Particle Size and Specific Surface Area.

Physical chemical characterization of nanomaterials is critical to assessing quality control during production, evaluating the impact of material properties on human health and the environment, and developing regulatory frameworks for their use. We have investigated a set of 29 nanomaterials from four metal oxide families (aluminum, copper, titanium and zinc) with a focus on the measurands that are important for the basic characterization of dry nanomaterials and the determination of the dose metrics for nanotoxicology. These include crystalline phase and crystallite size, measured by powder X-ray diffraction, particle shape and size distributions from transmission electron microscopy, and specific surface area, measured by gas adsorption. The results are compared to the nominal data provided by the manufacturer, where available. While the crystalline phase data are generally reliable, data on minor components that may impact toxicity is often lacking. The crystal and particle size data highlight the issues in obtaining size measurements of materials with broad size distributions and significant levels of aggregation, and indicate that reliance on nominal values provided by the manufacturer is frequently inadequate for toxicological studies aimed at identifying differences between nanoforms. The data will be used for the development of models and strategies for grouping and read-across to support regulatory human health and environmental assessments of metal oxide nanomaterials.

COX-1 is coupled with mPGES-1 and ABCC4 in human cervix cancer cells.

Cyclooxygenases are key enzymes in the arachidonic acid metabolism. Their unstable intermediate, prostaglandin H(2), is further metabolized to bioactive lipids by various downstream enzymes. In this study, utilizing short hairpin RNAs, we prepared a cell line of human cervix carcinoma with stable down-regulated cyclooxygenase-1 (COX-1) to assess the impact of COX-1 reduction on the downstream enzymes. We found a significant microsomal prostaglandin E synthase-1 (mPGES-1) suppression. In addition, mRNA expression of multidrug resistance protein 4 (MRP4, ABCC4), supposed to take part in antiviral and anticancer drug transport from cells, was up-regulated after COX-1 down-regulation. Our findings indicate that mPGES-1, believed to be coexpressed preferentially with cyclooxygenase-2, may be coupled to COX-1. ABCC4 up-regulation further supports the assumption of its involvement in prostanoid transport.

New antituberculotics originated from salicylanilides with promising in vitro activity against atypical mycobacterial strains.

A new series of 30 N-protected amino acid esters were prepared as a part of ongoing search for new anti-tuberculosis active salicylanilides. The esters possess high in vitro activity against Mycobacterium tuberculosis, Mycobacterium avium, and two strains of Mycobacterium kansasii, where one is an isolate from the patient, with MIC in the range 1-32 micromol/L for all tested strains. The prepared esters can be considered as prodrugs with better bio-availability and as more efficient transport forms through the mycobacterial cell membranes due to the higher lipophilicity. The experimental and calculated lipophilicity, stability, antituberculotic activity, cytotoxicity as well as the quantitative structure-activity relationships (QSARs) explored by the Intelligent Problem Solver (IPS) in Trajan Neural Network Simulator 6.0 are presented.

Quantification of amine functional groups on silica nanoparticles: a multi-method approach.

Surface chemistry is an important factor for quality control during production of nanomaterials and for controlling their behavior in applications and when released into the environment. Here we report a comparison of four methods for quantifying amine functional groups on silica nanoparticles (NPs). Two colorimetric assays are examined, ninhydrin and 4-nitrobenzaldehyde, which are convenient for routine analysis and report on reagent accessible amines. Results from the study of a range of commercial NPs with different sizes and surface loadings show that the assays account for 50-100% of the total amine content, as determined by dissolution of NPs under basic conditions and quantification by solution-state 1H NMR. To validate the surface quantification by the colorimetric assays, the NPs are modified with a trifluoromethylated benzaldehyde probe to enhance sensitivity for quantitative 19F solid state NMR and X-ray photoelectron spectroscopy (XPS). Good agreement between the assays and the determination from solid-state NMR is reinforced by elemental ratios from XPS, which indicate that in most cases the difference between total and accessible amine content reflects amines that are outside the depth probed by XPS. Overall the combined results serve to validate the relatively simple colorimetric assays and indicate that the reactions are efficient at quantifying surface amines, by contrast to some other covalent modifications that have been employed for functional group quantification.

A2A adenosine-receptor-mediated facilitation of noradrenaline release in rat tail artery involves protein kinase C activation and betagamma subunits formed after alpha2-adrenoceptor activation.

This work aimed to investigate the molecular mechanisms involved in the interaction of alpha2-adrenoceptors and adenosine A2A-receptor-mediated facilitation of noradrenaline release in rat tail artery, namely the type of G-protein involved in this effect and the step or steps where the signalling cascades triggered by alpha2-adrenoceptors and A2A-receptors interact. The selective adenosine A2A-receptor agonist 2-p-(2-carboxy ethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 100 nM) enhanced tritium overflow evoked by trains of 100 pulses at 5 Hz. This effect was abolished by the selective adenosine A2A-receptor antagonist 5-amino-7-(2-phenyl ethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine (SCH 58261; 20 nM) and by yohimbine (1 microM). CGS 21680-mediated effects were also abolished by drugs that disrupted G(i/o)-protein coupling with receptors, PTX (2 microg/ml) or NEM (40 microM), by the anti-G(salpha) peptide (2 microg/ml) anti-G(betagamma) peptide (10 microg/ml) indicating coupling of A2A-receptors to G(salpha) and suggesting a crucial role for G(betagamma) subunits in the A(2A)-receptor-mediated enhancement of tritium overflow. Furthermore, phorbol 12-myristate 13-acetate (PMA; 1 microM) or forskolin (1 microM), direct activators of protein kinase C and of adenylyl cyclase, respectively, also enhanced tritium overflow. In addition, PMA-mediated effects were not observed in the presence of either yohimbine or PTX. Results indicate that facilitatory adenosine A2A-receptors couple to G(salpha) subunits which is essential, but not sufficient, for the release facilitation to occur, requiring the involvement of G(i/o)-protein coupling (it disappears after disruption of G(i/o)-protein coupling, PTX or NEM) and/or G(betagamma) subunits (anti-G(betagamma)). We propose a mechanism for the interaction in study suggesting group 2 AC isoforms as a plausible candidate for the interaction site, as these isoforms can integrate inputs from G(salpha) subunits (released after adenosine A2A-receptor activation; prime-activation), G(betagamma) subunits (released after activation of G(i/o)-protein coupled receptors) which can directly synergistically stimulate the prime-activated AC or indirectly via G(betagamma) activation of the PLC-PKC pathway.