Assessment of Protein Binding Using Asymmetric-Flow Field-Flow Fractionation Combined with Multi-angle Light Scattering and Dynamic Light Scattering.

Asymmetric-flow field-flow fractionation (AF4) is a valuable tool to separate and assess different size populations in nanotherapeutics. When coupled with both static light scattering and dynamic light scattering, it can be used to qualitatively assess protein binding to nanoparticles by comparing the shape factors for both non-plasma-incubated samples and plasma-incubated samples. The shape factor is defined as the ratio of the derived root mean square radius (by static light scattering) to the measured hydrodynamic radius (by dynamic light scattering). The shape factor gives an idea of where the center of mass lies in a nanoparticle, and any shift in the shape factor to larger values is indicative of a mass addition to the periphery of the nanoparticle and suggests the presence of protein binding. This protocol will discuss how to set up an experiment to assess protein binding in nanoparticles using AF4, multi-angle light scattering (MALS), and dynamic light scattering (DLS).

Nanoparticle Size Distribution and Stability Assessment Using Asymmetric-Flow Field-Flow Fractionation.

Nanomaterials are inherently polydisperse. Traditional techniques, such as the widely used batch-mode dynamic light-scattering (DLS) analysis, are not ideal nor thoroughly descriptive enough to define the full complexity of these materials. Asymmetric-flow field-flow fractionation (AF4) with various in-line detectors, such as ultraviolet-visible (UV-vis), multi-angle light scattering (MALS), refractive index (RI), and DLS, is an alternative technique that can provide flow-mode analysis of not only size distribution but also shape, drug release/stability, and protein binding.

Assessment of the Effects of Structural Modification of Gastrodia elata Polysaccharide on Anti-Breast Cancer Activity Using Asymmetrical Flow Field-Flow Fractionation.

Gastrodia elata ("Tian Ma" in Chinese) is used as a food and medical ingredient in traditional Chinese medicine. In this study, to enhance the anti-breast cancer activity of Gastrodia elata polysaccharide (GEP), GEPs were modified via sulfidation (SGEP) and acetylation (AcGEP). The physicochemical properties (such as solubility and substitution degree) and structural information (such as molecular weight Mw and radius of gyration Rg) of GEP derivatives were determined by Fourier transformed infrared (FTIR) spectroscopy and asymmetrical flow field-flow fractionation (AF4) coupled online with multiangle light scattering (MALS) and differential refractive index (dRI) detectors (AF4-MALS-dRI). The effects of the structural modification of GEP on the proliferation, apoptosis, and cell cycle of MCF-7 cell were studied systematically. The ability of MCF-7 cell for the uptake of GEP was studied by laser scanning confocal microscopy (LSCM). The results suggested that the solubility and anti-breast cancer activity of GEP were enhanced and the average Rg and Mw of GEP decreased after chemical modification. The AF4-MALS-dRI results showed that the chemical modification process simultaneously caused the degradation and aggregation of GEPs. The LSCM results revealed that more SGEP can enter the MCF-7 cell interior compared with AcGEP. The results indicated that the structure of AcGEP could play a dominating role in antitumor activity. The data obtained in this work can be used as a starting point for investigating the structure-bioactivity of GEPs.

Reliable assessment of carbon black nanomaterial of a variety of cell culture media for in vitro toxicity assays by asymmetrical flow field-flow fractionation.

Carbon black nanomaterial (CB-NM), as an industrial product with a large number of applications, poses a high risk of exposure, and its impact on health needs to be assessed. The most common testing platform for engineered (E)NMs is in vitro toxicity assessment, which requires prior ENM dispersion, stabilization, and characterization in cell culture media. Here, asymmetric flow field-flow fractionation (AF4) coupled to UV-Vis and dynamic light scattering (DLS) detectors in series was used for the study of CB dispersions in cell culture media, optimizing instrumental variables and working conditions. It was possible to disperse CB in a non-ionic surfactant aqueous solution due to the steric effect provided by surfactant molecules attached on the CB surface which prevented agglomeration. The protection provided by the surfactant or by culture media alone was insufficient to ensure good dispersion stability needed for carrying out in vitro toxicity studies. On the other hand, cell culture media in combination with the surfactant improved dispersion stability considerably, enabling the generation of shorter particles and a more favourable zeta potential magnitude, leading to greater stability due to electrostatic repulsion. It was demonstrated that the presence of amino acids in the culture media improved the monodisperse nature and stability of the CB dispersions, and resulted in a turn towards more negative zeta potential values when the pH was above the amino acid isoelectric point (IEP). Culture media used in real cell culture scenarios were also tested, and in vitro toxicity assays were developed optimizing the compatible amount of surfactant.

Assessment by a multi-technique approach of PtNPs' transformations in waters under relevant environmental concentrations and conditions.

Once released to the environment, platinum nanoparticles (PtNPs) can undergo different transformations and are affected by several environmental conditions. An only analytical technique cannot provide all the information required to understand those complex processes, so new analytical developments are demanded. In the present work, the potential of asymmetric flow field flow fractionation hyphenated to inductively coupled plasma mass spectrometry (AF4-ICP-MS) for these studies, has been investigated, and classical dynamic and electrophoretic light scattering (DLS & ELS) have been used as complementary techniques. The role of ionic strength, ionic water composition, and natural organic matter (NOM) in the behaviour of PtNPs of different sizes (5 and 50 nm) has been specifically studied. Dynamic and electrophoretic light scattering have been used to track changes in the hydrodynamic diameters (dh) and polydispersity index (PdI) for 50 nm PtNPs (5 nm cannot be studied by DLS) and Z-potential values (for all sizes) to monitor aggregation. AF4-ICP-MS has been also employed to have a solid insight of aggregation at low environmental concentrations for different sizes of PtNPs simultaneously. The information gathered with those techniques was useful to observe changes as the ionic strength increases, which induces aggregation. Also, it was observed that this aggregation process was attenuated in the presence of organic matter. This approach, based on complementary analytical techniques, is needed for a comprehensive study of such complex interactions of NPs in the environment. AF4-ICP-MS is still under-exploited but shows a great potential for this purpose, especially low size NPs and concentrations.

Rapid assessment of silver nanoparticle migration from food containers into food simulants using a qualitative method.

Migration of silver nanoparticles (AgNPs) from food containers (FCs) has been assessed for the first time using a screening method previously validated. Migration was evaluated using water and 3% acetic acid as food simulants (FSs), from 20 to 70 °C at contact times of 2 h and 10 days. Total and migrated Ag were determined by inductively coupled plasma-mass spectrometry (ICP-MS) in the FCs and FSs, respectively. Then, the screening method was validated, and probability of detection (POD) curves were constructed in both FSs to characterize the response to AgNPs. The results provided by the present screening method showed no release of AgNPs. The FSs in contact with FCs were spiked at levels above, inside and below the unreliability region, with a reliability rate (RLR) of 0.90. Asymmetric flow field flow fractionation coupled to inductively coupled plasma mass-spectrometry (AF4-ICP-MS) was used for confirmative analyses.

Quantitative Assessment of Individual Populations Present in Nanoparticle-Antibody Conjugate Mixtures Using AF4-ICP-MS/MS.

A current remaining challenge in nanotechnology is the fast and reliable determination of the ratios between engineered nanoparticles and the species attached to their surface after chemical functionalization. The approach proposed herein based on the online coupling of asymmetric flow field-flow fractionation (AF4) with inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS) allows for the first time the direct determination of such ratios in CdSe/ZnS core-shell quantum dot:rat monoclonal IgG2a antibody (QD:Ab) conjugate mixtures in a single run without any previous sample preparation (i.e., derivatization). AF4 provides full recovery and adequate resolution of the resulting bioconjugate from the excess of nanoparticles and proteins used in the different bioconjugation mixtures (1:1, 2:1, and 3:1 QD:Ab molar ratios were assessed). The point-by-point determination by ICP-MS/MS of the metal to sulfur ratios along the bioconjugate fractographic peak allowed disclosing the mixture of the different species in the bioconjugated sample, providing not only the limits of the range of QD:Ab ratios in the different bioconjugate species resulting after functionalization but also a good estimation of their individual relative abundance in the mixture. Interestingly, a wide variety of compositions were observed for the different bioconjugate mixtures studied (QD:Ab molar ratios ranging from 0.27 to 4.6). The resulting weighted QD:Ab ratio computed in this way for each bioconjugate peak matches well with both the global (average) QD:Ab ratio experimentally obtained by the simpler peak area ratio computation and the theoretical QD:Ab molar ratios assayed, which internally validates the procedure developed.

An assessment of retention behavior for gold nanorods in asymmetrical flow field-flow fractionation.

Applications of asymmetrical flow field-flow fractionation (AF4) continue to expand rapidly in the fields of nanotechnology and biotechnology. In particular, AF4 has proven valuable for the separation and analysis of particles, biomolecular species (e.g., proteins, bacteria) and polymers (natural and synthetic), ranging in size from a few nanometers to several micrometers. The separation of non-spheroidal structures (e.g., rods, tubes, etc.) with primary dimensions in the nanometer regime, is a particularly challenging application deserving of greater study and consideration. The goal of the present study was to advance current understanding of the mechanism of separation of rod-like nano-objects in the AF4 channel. To achieve this, we have systematically investigated a series of commercially available cetyltrimethylammonium bromide stabilized gold nanorods (AuNRs), with aspect ratios from 1.7 to 10. Results show clearly that the retention time is principally dependent on the translational diffusion coefficient of the AuNRs. Equations used to calculate translational and rotational diffusion coefficients (cylinder and prolate ellipsoid models) yield similarly good fits to experimental data. Well characterized gold nanorods (length and diameter by transmission electron microscopy) can be used as calibrants for AF4 measurements allowing one to determine the aspect ratio of nanorod samples based on their retention times. Graphical abstract ᅟ.

Assessment of the removal of side nanoparticulated populations generated during one-pot synthesis by asymmetric flow field-flow fractionation coupled to elemental mass spectrometry.

Coupling of asymmetric flow field-flow fractionation (AF4) to an on-line elemental detection (inductively coupled plasma-mass spectrometry, ICP-MS) has been recently proposed as a powerful diagnostic tool for characterization of the bioconjugation of CdSe/ZnS core-shell Quantum Dots (QDs) to antibodies. Such approach has been used herein to demonstrate that cap exchange of the native hydrophobic shell of core/shell QDs with the bidentate dihydrolipoic acid ligands directly removes completely the eventual side nanoparticulated populations generated during simple one-pot synthesis, which can ruin the subsequent final bioapplication. The critical assessment of the chemical and physical purity of the surface-modified QDs achieved allows to explain the transmission electron microscopy findings obtained for the different nanoparticle surface modification assayed.

Quantitative In Vitro Assessment of Liposome Stability and Drug Transfer Employing Asymmetrical Flow Field-Flow Fractionation (AF4).

PURPOSE: In the present study we introduce an efficient approach for a size-based separation of liposomes from plasma proteins employing AF4. We investigated vesicle stability and release behavior of the strongly lipophilic drug temoporfin from liposomes in human plasma for various incubation times at 37°C. METHODS: We used the radioactive tracer cholesteryl oleyl ether (COE) or dipalmitoyl-phosphocholine (DPPC) as lipid markers and (14)C-labeled temoporfin. First, both lipid labels were examined for their suitability as liposome markers. Furthermore, the influence of plasma origin on liposome stability and drug transfer was investigated. The effect of membrane fluidity and PEGylation on vesicle stability and drug release characteristics was also analyzed. RESULTS: Surprisingly, we observed an enzymatic transfer of (3)H-COE to lipoproteins due to the cholesterol ester transfer protein (CETP) in human plasma in dependence on membrane rigidity and were able to inhibit this transfer by plasma preincubation with the CETP inhibitor torcetrapib. This effect was not seen when liposomes were incubated in rat plasma. DPPC labels suffered from hydrolysis effects during preparation and/or storage. Fluid liposomes were less stable in human plasma than their PEGylated analogues or a rigid formulation. In contrast, the transfer of the incorporated drug to lipoproteins was higher for the rigid formulations. CONCLUSIONS: The observed effects render COE-labels questionable for in vivo studies using CEPT-rich species. Here, choline labelled (14)C-DPPC was found to be the most promising alternative. Bilayer composition has a high influence on stability and drug release of a liposomal formulation in human plasma.

Analytical assessment about the simultaneous quantification of releasable pharmaceutical relevant inorganic nanoparticles in tap water and domestic waste water.

For pharmaceutical applications, the use of inorganic engineered nanoparticles is of growing interest while silver (Ag) and gold (Au) are the most relevant elements. A few methods were developed recently but the validation and the application testing were quite limited. Therefore, a routinely suitable multi element method for the identification of nanoparticles of different sizes below 100 nm and elemental composition by applying asymmetric flow field flow fraction (AF4) - inductively coupled plasma mass spectrometry (ICPMS) is developed. A complete validation model of the quantification of releasable pharmaceutical relevant inorganic nanoparticles based on Ag and Au is presented for the most relevant aqueous matrices of tap water and domestic waste water. The samples are originated from locations in the Netherlands and it is of great interest to study the unwanted presence of Ag and Au as nanoparticle residues due to possible health and environmental risks. During method development, instability effects are observed for 60 nm and 70 nm Ag ENPs with different capping agents. These effects are studied more closely in relation to matrix effects. Besides the methodological aspects, the obtained analytical results and relevant performance characteristics (e.g. measuring range, limit of detection, repeatability, reproducibility, trueness, and expanded uncertainty of measurement) are determined and discussed. For the chosen aqueous matrices, the results of the performance characteristics are significantly better for Au ENPs in comparison to Ag ENPs; e.g. repeatability and reproducibility are below 10% for all Au ENPs respectively maximal 27% repeatability for larger Ag ENPs. The method is a promising tool for the simultaneous determination of releasable pharmaceutical relevant inorganic nanoparticles.

Association of ranibizumab (Lucentis®) or bevacizumab (Avastin®) with dexamethasone and triamcinolone acetonide: an in vitro stability assessment.

The in vitro stability of monoclonal antibodies used for age-related macular degeneration, ranibizumab and bevacizumab, was investigated. The aggregation profile of the antibodies was compared, alone and after association with dexamethasone sodium phosphate or triamcinolone acetonide. Commercial formulations of ranibizumab and bevacizumab were dialysed into three different buffers. After dialysis, samples were stored at 4°C, 25°C and 40°C during 35 days, alone and in combination with dexamethasone sodium phosphate, triamcinolone acetonide phosphate solution or triamcinolone acetonide suspension. Combined formulations based on both commercial formulations were investigated as well. The aggregation state of the antibodies was measured by multi-angle light scattering (MALS) after separation by asymmetrical flow field-flow fractionation (AFFF) or size-exclusion chromatography (SEC). Ranibizumab results to be more stable than bevacizumab, alone and in combination with dexamethasone sodium phosphate or triamcinolone acetonide. Elevation in concentration, pH and temperature causes a decrease in stability of both antibodies. The association of triamcinolone acetonide phosphate solution with either ranibizumab or bevacizumab is observed to be the least stable combination of all samples tested. Dexamethasone sodium phosphate was shown to have a stabilizing effect on bevacizumab, although this is not the case for its combination with the commercial formulation Avastin®. The results demonstrate that the in vitro association of either ranibizumab or bevacizumab with dexamethasone sodium phosphate or triamcinolone acetonide suspension does not decrease the stability of these antibodies. Although ranibizumab is more stable than bevacizumab in vitro, further research has to point out how this affects their mechanism of action in vivo.

Biological assessment of triazine dendrimer: toxicological profiles, solution behavior, biodistribution, drug release and efficacy in a PEGylated, paclitaxel construct.

The physicochemical characteristics, in vitro properties, and in vivo toxicity and efficacy of a third generation triazine dendrimer bearing approximately nine 2 kDa polyethylene glycol chains and twelve ester linked paclitaxel groups are reported. The hydrodynamic diameter of the neutral construct varies slightly with aqueous solvent ranging from 15.6 to 19.4 nm. Mass spectrometry and light scattering suggest radically different molecular weights with the former approximately 40 kDa mass consistent with expectation, and the latter 400 kDa mass consistent with a decameric structure and the observed hydrodynamic radii. HPLC can be used to assess purity as well as paclitaxel release, which is insignificant in organic solvents or aqueous solutions at neutral and low pH. Paclitaxel release occurs in vitro in human, rat, and mouse plasma and is nonlinear, ranging from 7 to 20% cumulative release over a 48 h incubation period. The construct is 2-3 orders of magnitude less toxic than Taxol by weight in human hepatocarcinoma (Hep G2), porcine renal proximal tubule (LLC-PK1), and human colon carcinoma (LS174T) cells, but shows similar cytotoxicity to Abraxane in LS174T cells. Both Taxol and the construct appear to induce caspase 3-dependent apoptosis. The construct shows a low level of endotoxin, is not hemolytic and does not induce platelet aggregation in vitro, but does appear to reduce collagen-induced platelet aggregation in vitro. Furthermore, the dendrimer formulation slightly activates the complement system in vitro due most likely to the presence of trace amounts (<1%) of free paclitaxel. An animal study provided insight into the maximum tolerated dose (MTD) wherein 10, 25, 50, and 100 mg of paclitaxel/kg of construct or Abraxane were administered once per week for three consecutive weeks to non tumor bearing athymic nude mice. The construct showed in vivo toxicity comparable to that of Abraxane. Both formulations were found to be nontoxic at the administered doses, and the dendrimer had an acute MTD greater than the highest dose administered. In a prostate tumor model (PC-3-h-luc), efficacy was observed over 70 days with an arrest of tumor growth and lack of luciferase activity observed in the twice treated cohort.

Nanoparticle analysis and characterization methodologies in environmental risk assessment of engineered nanoparticles.

Environmental risk assessments of engineered nanoparticles require thorough characterization of nanoparticles and their aggregates. Furthermore, quantitative analytical methods are required to determine environmental concentrations and enable both effect and exposure assessments. Many methods still need optimization and development, especially for new types of nanoparticles in water, but extensive experience can be gained from the fields of environmental chemistry of natural nanomaterials and from fundamental colloid chemistry. This review briefly describes most methods that are being exploited in nanoecotoxicology for analysis and characterization of nanomaterials. Methodological aspects are discussed in relation to the fields of nanometrology, particle size analysis and analytical chemistry. Differences in both the type of size measures (length, radius, aspect ratio, etc.), and the type of average or distributions afforded by the specific measures are compared. The strengths of single particle methods, such as electron microscopy and atomic force microscopy, with respect to imaging, shape determinations and application to particle process studies are discussed, together with their limitations in terms of counting statistics and sample preparation. Methods based on the measurement of particle populations are discussed in terms of their quantitative analyses, but the necessity of knowing their limitations in size range and concentration range is also considered. The advantage of combining complementary methods is highlighted.

Size characterization of barley starch granules by gravitational field-flow fractionation: a rapid, low-cost method to assess the brewing capability of different strains.

Cereal starch occurs as two types of micrometer-sized granules, large and small. Large starch granules are more susceptible to enzymatic hydrolysis. When cereal starch is used for fermentation processes, as in brewing of barley malt, the barley strains with the highest content of large starch granules should be preferred. Gravitational field-flow fractionation (GFFF) is a separation method able to fractionate starch samples at low cost and short analysis time. In this work, the search for the best GFFF conditions for the analytical separation of barley starch within an inter-laboratory approach is presented. For different barley strains cultivated under monitored conditions the size distributions of starch granules is here quickly monitored and characterized by GFFF. As a consequence, dimensional characterization of barley starch can allow for the selection of the most suitable strains with the lowest content of non-degradable starch.