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[Display omitted] • A novel characterization method for liposomes and lipid nanoparticles using CF3. • Particle size and drug-loading amounts alter the elution profiles of nanoparticles in CF3. • Density is one of the crucial properties of nanoparticles. • First evaluation method for lipid nanoparticles utilizing CF3. Nanomedicines comprise multiple components, and particle density is considered an important property that regulates the biodistribution of administered nanomedicines. The density of nanoparticles is characterized by centrifugal methods, such as analytical ultracentrifugation. Particle size and distribution are key physicochemical and quality attributes of nanomedicines. In this study, we developed a novel profiling method applicable to liposomes and lipid nanoparticles (LNPs), based on particle size and density, using centrifugal field-flow fractionation (CF3). We evaluated the elution profiles of PEGylated liposomes of different sizes with various doxorubicin (DOX)-loading amounts using CF3. This method was applied to evaluate the drug release of DOX-loaded liposomes, intra- and inter-batch variability, reconstitution reproducibility of AmBisome®, and elution characteristics of LNPs in COVID-19 vaccines (Comirnaty® and SpikevaxTM). The data obtained in the present study underscore the significance of the proposed methodology and highlight the importance of profiling and characterizing liposomes and LNPs using CF3 fractograms and a multi-angle light-scattering detector. [ABSTRACT FROM AUTHOR]

Liposomal formulations for the treatment of cancer and other diseases are the most common form of nanotechnology enabled pharmaceuticals (NEPs) submitted for market approval and in clinical application today. The accurate characterization of their physical-chemical properties is a key requirement; in particular, size, size distribution, shape, and physical-chemical stability are key among properties that regulators identify as critical quality attributes. Here we develop and validate an optimized method, based on multi-detector asymmetrical-flow field flow fractionation (MD-AF4) to accurately and reproducibly separate liposomal drug formulations into their component populations and to characterize their associated size and size distribution, whether monomodal or polymodal in nature. In addition, the results show that the method is suitable to measure liposomes in the presence of serum proteins and can yield information on the shape and physical stability of the structures. The optimized MD-AF4 based method has been validated across different instrument platforms, three laboratories, and multiple drug formulations following a comprehensive analysis of factors that influence the fractionation process and subsequent physical characterization. Interlaboratory reproducibility and intra-laboratory precision were evaluated, identifying sources of bias and establishing criteria for the acceptance of results. This method meets a documented high priority need in regulatory science as applied to NEPs such as Doxil and can be adapted to the measurement of other NEP forms (such as lipid nanoparticle therapeutics) with some modifications. Overall, this method will help speed up development of NEPS, and facilitate their regulatory review, ultimately leading to faster translation of innovative concepts from the bench to the clinic. Additionally, the approach used in this work (based on international collaboration between leading non-regulatory institutions) can be replicated to address other identified gaps in the analytical characterization of various classes of NEPs. Finally, a plan exists to pursue more extended interlaboratory validation studies to advance this method to a consensus international standard. Unlabelled Image [ABSTRACT FROM AUTHOR]

Regular algal blooms are occurring in Taihu lake, which may be triggered by resuspension of sediments containing relevant amounts of phosphorus. Therefore, our study aims at quantification of phosphorus concentrations bound to suspended particulate matter in Taihu water samples to investigate this hypothesis. A field flow fractionation (FFF) method online with ICP-MS detection was developed to achieve an overview on particulate fractions of phosphorus and related elements including Fe, Al and C from the low nanometer to the low micrometer size range. Mass balance of dissolved and particulate elemental contents was established for quality control purpose and indicated low recovery of Fe, Al and P. Complementary determination of volume based particle size distribution by dynamic imaging analysis showed a majority of particle volume and thus mass in particles with size >5 μm. In order to address this challenge, the upper particle size limit of FFF online with ICP-MS was for the first time investigated in detail using well characterised monodisperse latex particles as model for organic matter in the low micrometer size range including microalgae. The effect of pre-filtration of the sample as well as the contribution of sample introduction via three different interfaces including micromist nebuliser/spray chamber, direct injection nebulisation and APEX with heated spray chamber and solvent removal by condensation on the particulate carbon recovery was studied by ICP-MS detection. The same instrumental setup was also applied for the characterisation of particulate elemental contents in the Taihu water samples as far as possible. Significant improvement of the detected particulate fraction in Taihu water samples was achieved by increasing the membrane pore size for pre-filtration and by using the APEX for introduction of the eluate from FFF into ICP-MS. Image 1 • First study on systematic investigation of the upper particle size limit of flow-FFF online with ICP-MS. • Full mass balance for water samples including particulate and dissolved elemental species both with and without filtration. • Study of the permeability of 5 μm and 10 μm filters for latex particles using carbon detection by ICP-MS and for Taihu water. • First application of direct injection nebulisation and APEX for online hyphenation of flow-FFF to ICP-MS. [ABSTRACT FROM AUTHOR]

• In Ap horizons of arable Luvisols, colloids control the aggregation of small soil microaggregates (<20 μm) in wet-dry cycles. • The presence of <450 nm colloids favored the formation of 1–40 μm soil microaggregates. • In absence of <1 μm colloids, >40 μm soil micro- and macroaggregates were preferentially formed. • The particle size is decisive for the aggregate formation under the current experimental condition. In the hierarchical model of soil aggregates, small soil microaggregates (small SMA; <20 μm) are often considered to be fundamental building units at the micron scale. Below which, soil colloids (<1 µm) have recently been proposed as binding agents of (micro)aggregates. However, the way in which soil colloids contribute to the formation and stability of soil micro- and macroaggregates remains largely unknown. For clarification, we evaluated potential impacts of the colloidal content, particularly the <450 nm colloids, on the aggregation of small SMA. Free water stable small SMA and <450 nm colloids were isolated from Ap-horizons of Stagnic Luvisols under different management (cropped and bare fallow). The size-resolved elemental composition of the <450 nm colloids was analyzed by asymmetric flow field-flow fractionation in combination with an inductively coupled plasma mass spectrometer and an organic carbon detector. To vary the colloidal content in small SMA, (1) suspensions containing different amounts of <450 nm colloids were added in small SMA, or (2) <1 µm colloids were removed from small SMA by centrifugation. In the maximum colloidal addition treatment, the mass ratios of added colloids to small SMA were 3.0 and 5.1 wt% for the cropped and bare fallow soil samples, respectively. Aggregation of small SMA with different colloidal amounts was performed in three successive wet-dry cycles. Afterwards, the size distribution of the resulting aggregates was measured by laser diffraction. Our results indicated that, in wet-dry cycles, colloids were important binding agents for the formation of SMA. Their presence, especially those <450 nm, was likely to support the formation of solid bridges during drying at particle contacts of 1–10 µm small SMA, favoring hereby SMA build-up in a relatively small size range of 1–40 µm. In contrast, the absence of <1 μm colloids in small SMA led to a preferential generation of relatively large aggregates in wet-dry cycles, i.e., typically with sizes >40 μm up to 1700 μm in maximum. Our study on aggregation in wet-dry cycles revealed that the colloidal content has a controlling effect on the size distribution of resulting aggregates by acting as a binding agent and provides hereby new insights into the evolvement of aggregate hierarchy in soils. [ABSTRACT FROM AUTHOR]

Characterization of natural nanomaterial (NNM) physicochemical properties – such as size, size distribution, elemental composition and elemental ratios - is often hindered by lack of methods to disperse NNMs from environmental samples. This study evaluates the effect of extractant composition, pH, and ionic strength on soil NNM extraction in term of recovery and release of primary particles/small aggregate sizes (i.e. , <200 nm). The extracted NNMs were characterized for hydrodynamic diameter and zeta potential by dynamic light scattering and laser Doppler electrophoresis, natural organic matter desorption by UV–Vis spectroscopy, element composition by inductively coupled plasma-mass spectroscopy (ICP-MS), size based elemental distribution by field flow fractionation coupled to ICP-MS, and morphology by transmission electron microscopy. The extracted NNM concentrations increased following the order of NaOH ≤ Na 2 CO 3 < Na 2 C 2 O 4 < Na 4 P 2 O 7. Na 4 P 2 O 7 was the most efficient extractant and results in 2–12 folds higher NNM extraction than other extractants. The Na 4 P 2 O 7 extracted NNMs exhibited narrower size distribution with smaller modal size relative to NaOH, Na 2 CO 3 , Na 2 C 2 O 4 extracted NNMs. Thus, Na 4 P 2 O 7 enhances the extraction of primary NNMs and/or smaller NNM aggregates (i.e. , size <200 nm). Na 4 P 2 O 7 promote soil microaggregates breakup and release of NNMs by reducing free multivalent cation concentration in soil pore water by forming metal-phosphate complexes and by enhancing NNM surface charge via phosphate sorption on NNM surfaces. Additionally, the extracted NNM concentrations increased with the increase in extractant concentration and pH, except at 100 mM where the high ionic strength might have induced NNM aggregation. The improved NNM-extraction will improve the overall understanding of the physicochemical properties of NNMs in environmental systems. This study presents the key properties of NNMs that can be used as background information to differentiate engineered nanomaterials (ENMs) from NNMs in complex environmental media. Unlabelled Image • Efficient extraction of natural nanomaterials from complex environmental matrices • Multimethod approach for characterization of natural nanomaterials from soils • Sodium pyrophosphate to efficiently disperse nanomaterials from soil heteroaggregates • Key nanomaterial properties to discriminate engineered from natural nanomaterials in soils [ABSTRACT FROM AUTHOR]

Magnetic particles (MPs) and magnetic nanoparticles (MNPs) are appealing candidates for biomedical and analytical applications due to their unique physical and chemical properties. Given that magnetic fields can be readily used to control the motion and properties of M(N)Ps, their integration in analytical methods opens new avenues for sensing and quantitative analysis. There is a large body of literature related to their synthesis, with a relatively small number of methods reporting the analysis of M(N)Ps using separation methods, which provide information on their purity and monodispersity. This review discusses analytical separation methods of M(N)Ps published between 2013 and June 2018. The analytical separation methods evaluated in this work include (i) field flow fractionation, (ii) capillary electrophoresis, (iii) macroscale magnetophoresis and (iv) microchip magnetophoresis. Among the trends in analytical separations of M(N)Ps an inclination towards miniaturization is moving from conventional benchtop methods to rapid and low-cost methods based on microfluidic devices. • The importance of analytical separations of magnetic (nano)particles is highlighted. • Analytical separations of magnetic (nano)particles during the period between 2013 and 2018 include field flow fractionation, macroscale magnetophoresis, capillary electrophoresis and microchip magnetophoresis. • Challenges and successes of different approaches on magnetic (nano)particle analytical separations are discussed and the future trends are explored. [ABSTRACT FROM AUTHOR]

Highlights • Polymer adsorption on GPC columns is observed for partially hydrolyzed PVA. • No adsorption for fully hydrolyzed PVA on these GPC columns is found. • Slow desorption of adsorbed PVA from porous packing particles is non-exponential. • Desorption of adsorbed PVA follows stretched exponential kinetics with index 0.36. • AF4 can be used for the characterization of PVA, regardless of the degree of hydrolysis. Abstract Both fully (98%) and partially (88%) hydrolyzed poly(vinyl alcohol) (PVA) are characterized by aqueous gel permeation chromatography (GPC) coupled with multi-angle laser light scattering. Polymer adsorption on hydrophilic GPC columns is observed for partially hydrolyzed PVA but not for fully hydrolyzed PVA, which is driven by the hydrophobic interactions between the residual acetate groups on PVA and the stationary phase. Desorption of these partially hydrolyzed PVA chains from the packing materials is monitored by decay of the light scattering signal. It is found that the desorption of these polymer chains from porous particles is non-exponential and follows stretched exponential kinetics with index 0.36. It is also shown that asymmetric flow field flow fractionation can be successfully used for the molar mass characterization of PVA, irrespective of the degree of hydrolysis. [ABSTRACT FROM AUTHOR]

Abstract An acoustic field flow fractionation (FFF) device was developed to fractionate a micro-particle mixture on the basis of the particle diameter using an acoustic force field in a carrier liquid flow. In the acoustic FFF channel used in the device, ultrasound waves generated from piezoelectric transducers driven by a sinusoidal signal of 2.02 Mhz propagated into the carrier liquid flow and built up a quarter-wavelength ultrasound standing wave field across the channel height. It was experimentally demonstrated that the acoustic field with a pressure node plane at the bottom surface of the channel reduced the thickness of the particle diffusion layer in a stagnant liquid proportional to the applied voltage driving the piezoelectric transducer. In the size-dependent particle separation, the particle mixture flowing through the acoustic FFF channel experienced an acoustic radiation force in the gravitational direction. As a result, suppressing the diffusion of small particles, particles were transported along the bottom surface of the channel with the local velocity of the carrier liquid at the particle center. The developed acoustic FFF device successfully fractionated a fluorescent micro-particle mixture (1, 3, 5, and 10 μm diameter), whereas the 3 and 5 μm particles were not fractionated in the FFF device using only the gravitational force field due to the diffusion of 3 μm particles. Graphical abstract Image 1 Highlights • Develops a field flow fractionation technique using an acoustic radiation force. • Analyzes the induced acoustic force field across the height of the field flow fractionation channel. • Demonstrates the effect of the induced acoustic force field on the resolution of field flow fractionation. • Predicts the resolution and the relaxation time of an acoustic field flow fractionation. [ABSTRACT FROM AUTHOR]

Abstract For best photosensitizer activity phthalocyanine dyes used in photodynamic therapy should be molecularly dispersed. Polyethylene glycol- block -polylactide derivatives presenting benzyl side-groups were synthesized to encapsulate a highly lipophilic phthalocyanine dye (AlClPc) and evaluate the effect of π-π interactions on the nanocarrier colloidal stability and dye dispersion. Copolymers with 0, 1, 2 and 6 mol% of benzyl glycidyl ether (BGE) were obtained via polyethylene glycol initiated ring-opening copolymerization of D, l -lactide with BGE. The block copolymers formed stable, monodisperse nanospheres with low in vitro cytotoxicity. AlClPc loading increased the nanosphere size and affected their colloidal stability. The photo-physical properties of the encapsulated dye, studied in batch and after separation by field flow fractionation, demonstrated the superiority of plain PEG-PLA over BGE-containing copolymers in maintaining the dye in its monomeric (non-aggregated) form in aqueous suspension. High dye encapsulation and sustained dye release suggest that these nanocarriers are good candidates for photodynamic therapy. Graphical abstract Unlabelled Image Highlights • Polyethylene glycol- block -poly(lactide- co -benzyl glycidyl ether) were obtained. • The copolymers form stable non-cytotoxic nanospheres below 100 nm. • Less benzylated copolymer meant better chloroaluminum phthalocyanine dispersion. • Controlled release, stable and bright phthalocyanine nanocarriers were achieved. [ABSTRACT FROM AUTHOR]

Abstract The interaction of polymer nanocapsules (NC) prepared from four biodegradable polyesters with variable polymer hydrophobicity (PCL, PLA, PLGA and PLA-PEG) was investigated in the non-phagocytic Vero, Caco-2 and HepG2 cell lines. The NC, labeled with the highly lipophilic fluorescent indocarbocyanine dye DIL, had very similar sizes (approx. 140 nm) and negative zeta-potentials. Asymmetric flow field-flow fractionation evidenced NC colloidal stability and negligible transfer of the dye to serum proteins in the incubation medium. The cytotoxicity of the NC was evaluated via MTT assay over a large polymer concentration range (1–1000 μg/mL) and time of exposure (2, 24 and 48 h). The NC were safe in vitro up to a concentration of approx. 100 μg/mL or higher, depending on the cell line and nature of the polymer. Vero cells were more sensitive to the NC, in particular NC of the more hydrophobic polymer. The cells were exposed to endocytosis inhibitors, incubated with NC, and the cell-associated fluorescence was quantified by spectrofluorometry. HepG2 cells presented a 1.5–2-fold higher endocytic capacity than Caco-2 and Vero cells. The main mechanism of NC uptake was caveolin-mediated endocytosis in HepG2 and Vero cells, and macropinocytosis in Caco-2 cells. Polymer hydrophobicity had an effect on the level of NC associated to HepG2 cells and to a lesser extent on the endocytosis mechanisms in Vero and Caco-2 cells. The NC uptake levels and endocytosis mechanisms differed significantly between cell lines tested. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Basic and applied virus research requires specimens that are purified to high homogeneity. Thus, there is much interest in the efficient production and purification of viruses and their subassemblies. Advances in the production steps have shifted the bottle neck of the process to the purification. Nonetheless, the development of purification techniques for different viruses is challenging due to the complex biological nature of the infected cell cultures as well as the biophysical and -chemical differences in the virus particles. We used bacteriophage ϕ6 as a model virus in our attempts to provide a new purification method for enveloped viruses. We compared asymmetrical flow field-flow fractionation (AF4)-based virus purification method to the well-established ultracentrifugation-based purification of ϕ6. In addition, binding of ϕ6 virions to monolithic anion exchange columns was tested to evaluate their applicability in concentrating the AF4 purified specimens. Our results show that AF4 enables one-hour purification of infectious enveloped viruses with specific infectivity of ~1 × 10 13  PFU/mg of protein and ~65–95% yields. Obtained purity was comparable with that obtained using ultracentrifugation, but the yields from AF4 purification were 2–3-fold higher. Importantly, high quality virus preparations could be obtained directly from crude cell lysates. Furthermore, when used in combination with in-line light scattering detectors, AF4 purification could be coupled to simultaneous quality control of obtained virus specimen. [ABSTRACT FROM AUTHOR]

Silver nanoparticles (Ag-NP) are used in various consumer products and are one of the most prevalent metallic nanoparticle in commodities and are released into the environment. Transport behavior of Ag-NP in groundwater is one important aspect for the assessment of environmental impact and protection of drinking water resources in particular. Ag-NP transport processes in saturated single-fractured sandstones using triaxial flow cell experiments with different kind of sandstones is investigated. Ag-NP concentration and size are analyzed using flow field-flow fractionation and coupled SEM-EDX analysis. Results indicate that Ag-NP are more mobile and show generally lower attachment on rock surface compared to experiments in undisturbed sandstone matrix and partially fractured sandstones. Ag-NP transport is controlled by the characteristics of matrix porosity, time depending blocking of attachment sites and solute chemistry. Where Ag-NP attachment occur, it is heterogeneously distributed on the fracture surface. [ABSTRACT FROM AUTHOR]

The effect of polymeric nanocapsule dose on plasmatic and liver concentrations 20 min after intravenous administration in mice was evaluated. Nanocapsules were prepared with different polymers, namely, poly(D,L-lactide) (PLA), polyethylene glycol- block -poly(D,L-lactide) (PLA-PEG), and PLA with chitosan (PLA-Cs) and compared with a nanoemulsion. These formulations were labelled with a phthalocyanine dye for fluorescent detection. The nanostructures had narrow size distributions upon separation by asymmetric flow field flow fractionation with static and dynamic light scattering detection, with average hydrodynamic diameters in the 130–300 nm range, negative zeta potentials, except PLA-Cs nanocapsules, which had a positive zeta potential. Flow cytometry revealed uptake mostly by monocytes and neutrophils in mice and human blood. PLA nanocapsules and the nanoemulsion showed dose-dependent plasma concentrations, where the percentage of plasmatic fluorescence increased with increasing administered dose. In contrast, PLA-PEG nanocapsules led to a dose-independent plasmatic profile. PLA-Cs nanocapsules showed the lowest plasmatic and liver levels of fluorescence at all administered doses and significant intravenous toxicity in mice. This work demonstrates the importance of considering the nanocarrier dose when evaluating pharmacokinetic and biodistribution data and emphasizes the role of surface features in determining the plasmatic and liver concentrations of a poorly soluble lipophilic encapsulated compound. [ABSTRACT FROM AUTHOR]

Cell specific ligand molecules are attached to polyethylene glycol (PEG) modified nanoparticles to enhance the drug delivery efficiency. The tethered PEG would interfere in ligand recognition as well as providing biocompatibility to the nanoparticles. The denser PEG can give the greater biocompatibility, while should more hamper the ligand recognition. Therefor it is important to tune PEG density at an appropriate amount to compensate these two factors. In this study, we prepared a series of nanoparticles composed of α-mannose-bearing lipid, dioleoyltrimethyl ammoniumpropane (DOTAP) and 1,2-distearoyl- sn -glycero -3-phosphoethanolamine- N -[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000 ). The nanoparticles were characterized with dynamic light scattering (DLS), field flow fractionation (FFF), small angle X-ray scattering (SAXS), and quartz-crystal microbalance (QCM). Based on the structural parameter obtained from DLS, SAXS, and FFF, we determined the PEG crowding parameter ( σ ) quantitatively; when σ = 1.0 , the PEG chain occupies the same value on the surface as when the chain is present in the unperturbed state, and at σ = 1.0 , the PEG chains start to contact each other. We found that the recognition ability had the maximum around σ ∼ 0.75 and there was the critical composition at σ = 1.0 for which the recognition was drastically reduced. The present results demonstrated that quantitative characterization and controlling the PEG density are key to designing effective targeting delivery system. [ABSTRACT FROM AUTHOR]

We demonstrate the use of inverse supercritical carbon dioxide (scCO 2 ) extraction as a novel method of sample preparation for the analysis of complex nanoparticle-containing samples, in our case a model sunscreen agent with titanium dioxide nanoparticles. The sample was prepared for analysis in a simplified process using a lab scale supercritical fluid extraction system. The residual material was easily dispersed in an aqueous solution and analyzed by Asymmetrical Flow Field-Flow Fractionation (AF4) hyphenated with UV- and Multi-Angle Light Scattering detection. The obtained results allowed an unambiguous determination of the presence of nanoparticles within the sample, with almost no background from the matrix itself, and showed that the size distribution of the nanoparticles is essentially maintained. These results are especially relevant in view of recently introduced regulatory requirements concerning the labeling of nanoparticle-containing products. The novel sample preparation method is potentially applicable to commercial sunscreens or other emulsion-based cosmetic products and has important ecological advantages over currently used sample preparation techniques involving organic solvents. [ABSTRACT FROM AUTHOR]

Immunomagnetic isolation of biochemical and organic entities in a microfluidic environment is a popular tool for a wide range of bioMEMS applications, including biosensors. Numerical analyses of magnetophoretic separation and capture of magnetic microspheres in microfluidic field flow fractionation devices under the influence of line dipoles are presented in this work. Microchannel flow and magnetic beads are simulated for different values of dipole strength and position in the microchannel configurations. Magnetic beads based microfluidics offer great potential towards overcoming the major challenges involved with the design of lab-on-a-chip devices. Various bio-cells conjugated magnetic beads are separated at different outlet due to the different mobility by the field flow fraction technique. Two dipoles having different strength are placed near the bottom wall of micro-channel. Thus two different field is generated which leads to separation of magnetic particles. The objective of the device is to collect maximum number of particles in its designated outlet with as little as possible intermixing between particles. The device performance is designated in terms of capture efficiencies at outlet-1 and outlet-2. It is expected that only larger particles are captured at outlet-1 and smaller particles are captured at outlet-2 but there is possibility of mixing in both of outlets. That means that both types of particles are captured in both outlets. In that case two additional parameters such as separation index at outlet-1 and outlet-2 are required to measure the extent of separation of particles. Transport and field flow fractionation of magnetic microparticles subject to a magnetic field gradient in a microfluidic channel is investigated through a numerical simulation using an Eulerian-Lagrangian approach. [ABSTRACT FROM AUTHOR]

In situ measurements of labile metal species using diffusive gradients in thin films (DGT) passive samplers are based on the diffusion rates of individual species. Although most studies have dealt with chemically isolated humic substances, the diffusion of dissolved organic matter (DOM) across the hydrogel is not well understood. In this study, the diffusion coefficient ( D ) and molecular weight (MW) of 11 aquatic DOM and 4 humic substances (HS) were determined. Natural, unaltered aquatic DOM was capable of diffusing across the diffusive gel membrane with D values ranging from 2.48 × 10 −6 to 5.31 × 10 −6 cm 2 s −1 . Humic substances had diffusion coefficient values ranging from 3.48 × 10 −6 to 6.05 × 10 −6 cm 2 s −1 , congruent with previous studies. Molecular weight of aquatic DOM and HS samples (∼500–1750 Da) measured using asymmetrical flow field-flow fractionation (AF4) strongly influenced D, with larger molecular weight DOM having lower D values. No noticeable changes in DOM size properties were observed during the diffusion process, suggesting that DOM remains intact following diffusion across the diffusive gel. The influence of molecular weight on DOM mobility will assist in further understanding and development of the DGT technique and the uptake and mobility of contaminants associated with DOM in aquatic environments. [ABSTRACT FROM AUTHOR]

Compared to other sub-techniques of field flow fractionation (FFF), cyclical electrical field flow fractionation (CyElFFF) is a relatively new method with many opportunities remaining for improvement. One of the most important limitations of this method is the separation of particles smaller than 100 nm. For such small particles, the diffusion rate becomes very high, resulting in severe reductions in the CyElFFF separation efficiency. To address this limitation, we modified the electrical circuitry of the ElFFF system. In all earlier ElFFF reports, electrical power sources have been directly connected to the ElFFF channel electrodes, and no alteration has been made in the electrical circuitry of the system. In this work, by using discrete electrical components, such as resistors and diodes, we improved the effective electric field in the system to allow high resolution separations. By modifying the electrical circuitry of the ElFFF system, high resolution separations of 15 and 40 nm gold nanoparticles were achieved. The effects of applying different frequencies, amplitudes and voltage shapes have been investigated and analyzed through experiments. [ABSTRACT FROM AUTHOR]

Transport behavior and fate of engineered silver nanoparticles (AgNP) in the subsurface is of major interest concerning soil and groundwater protection in order to avoid groundwater contamination of vital resources. Sandstone aquifers are important groundwater resources which are frequently used for public water supply in many regions of the world. The objective of this study is to get a better understanding of AgNP transport behavior in partially fractured sandstones. We executed AgNP transport studies on partially fissured sandstone drilling cores in laboratory experiments. The AgNP concentration and AgNP size in the effluent were analyzed using flow field-flow fractionation mainly. We employed inverse mathematical models on the measured AgNP breakthrough curves to identify and quantify relevant transport processes. Physicochemical filtration, time-dependent blocking due to filling of favorable attachment sites and colloid-facilitated transport were identified as the major processes for AgNP mobility. Physicochemical filtration was found to depend on solute chemistry, mineralogy, pore size distribution and probably on physical and chemical heterogeneity. Compared to AgNP transport in undisturbed sandstone matrix reported in the literature, their mobility in partially fissured sandstone is enhanced probably due to larger void spaces and higher hydraulic conductivity. [ABSTRACT FROM AUTHOR]

Highlights: [•] Underlying theory and critical parameters are introduced. [•] A rational workflow is proposed to optimize and refine A4F methods. [•] Specific optimization steps and validation parameters are delineated. [•] Pedagogical examples are provided to demonstrate the process. [•] Use and relevance of different detection modalities is addressed. [Copyright &y& Elsevier]

Highlights: [•] Cationic polymers were characterized by asymmetric flow field-flow fractionation. [•] Different membranes and eluents were evaluated to find optimal separation conditions. [•] Analytical ultracentrifugation, DLS, SEC and 1H NMR spectroscopy were used to evaluate the results. [•] Asymmetric flow field-flow fractionation enables the fast and efficient determination of molar masses, polydispersity index values and conformational parameters of charged polymers. [ABSTRACT FROM AUTHOR]

Highlights: [•] FFF-MALS is a simple and reliable method that can determine the concentration of both live and inactivated influenza virus. [•] Simplified quantification is desirable as a quality control step throughput vaccine production. [•] FFF-MALS is a potentially useful technique that can increase speed and efficiency during the vaccine production pipeline. [ABSTRACT FROM AUTHOR]

Abstract: The principal objective of this work was to develop and demonstrate a new methodology for silver nanoparticle (AgNP) detection and characterization based on asymmetric-flow field flow fractionation (A4F) coupled on-line to multiple detectors and using stable isotopes of Ag. This analytical approach opens the door to address many relevant scientific challenges concerning the transport and fate of nanomaterials in natural systems. We show that A4F must be optimized in order to effectively fractionate AgNPs and larger colloidal Ag particles. With the optimized method one can accurately determine the size, stability and optical properties of AgNPs and their agglomerates under variable conditions. In this investigation, we couple A4F to optical absorbance (UV–vis spectrometer) and scattering detectors (static and dynamic) and to an inductively coupled plasma mass spectrometer. With this combination of detection modes it is possible to determine the mass isotopic signature of AgNPs as a function of their size and optical properties, providing specificity necessary for tracing and differentiating labeled AgNPs from their naturally occurring or anthropogenic analogs. The methodology was then applied to standard estuarine sediment by doping the suspension with a known quantity of isotopically enriched 109AgNPs stabilized by natural organic matter (standard humic and fulvic acids). The mass signature of the isotopically enriched AgNPs was recorded as a function of the measured particle size. We observed that AgNPs interact with different particulate components of the sediment, and also self-associate to form agglomerates in this model estuarine system. This work should have substantial ramifications for research concerning the environmental and biological fate of AgNPs. [Copyright &y& Elsevier]

Abstract: Characterization of nanomaterials must include analysis of both size and chemical composition. Many analytical techniques, such as dynamic light scattering (DLS), are capable of measuring the size of suspended nanometer-sized particles, yet provide no information on the composition of the particle. While field flow fractionation (FFF) is a powerful nanoparticle sizing technique, common detectors used in conjunction with the size separation, including UV, light-scattering, and fluorescence spectroscopy, do not provide the needed particle compositional information. Further, these detectors do not respond directly to the mass concentration of nanoparticles. The present work describes the advantages achieved when interfacing sensitive and elemental specific detectors, such as inductively coupled plasma atomic emission spectroscopy and mass spectrometry, to FFF separation analysis to provide high resolution nanoparticle sizing and compositional analysis at the μg/L concentration level, a detection at least 10–100-fold lower than DLS or FFF–UV techniques. The full benefits are only achieved by utilization of all detector capabilities, such as dynamic reaction cell (DRC) ICP–MS. Such low-level detection and characterization capability is critical to nanomaterial investigations at biologically and environmentally relevant concentrations. The techniques have been modified and applied to characterization of all four elemental constituents of cadmium selenide–zinc sulfide core–shell quantum dots, and silver nanoparticles with gold seed cores. Additionally, sulfide coatings on silver nanoparticles can be detected as a potential means to determine environmental aging of nanoparticles. [Copyright &y& Elsevier]

Abstract: Asymmetric flow field-flow fractionation (AF4) in combination with on-line optical detection and mass spectrometry is one of the most promising methods for separation and quantification of nanoparticles (NPs) in complex matrices including food. However, to obtain meaningful results regarding especially the NP size distribution a number of parameters influencing the separation need to be optimized. This paper describes the development of a separation method for polyvinylpyrrolidone-stabilized silver nanoparticles (AgNPs) in aqueous suspension. Carrier liquid composition, membrane material, cross flow rate and spacer height were shown to have a significant influence on the recoveries and retention times of the nanoparticles. Focus time and focus flow rate were optimized with regard to minimum elution of AgNPs in the void volume. The developed method was successfully tested for injected masses of AgNPs from 0.2 to 5.0μg. The on-line combination of AF4 with detection methods including ICP-MS, light absorbance and light scattering was helpful because each detector provided different types of information about the eluting NP fraction. Differences in the time-resolved appearance of the signals obtained by the three detection methods were explained based on the physical origin of the signal. Two different approaches for conversion of retention times of AgNPs to their corresponding sizes and size distributions were tested and compared, namely size calibration with polystyrene nanoparticles (PSNPs) and calculations of size based on AF4 theory. Fraction collection followed by transmission electron microscopy was performed to confirm the obtained size distributions and to obtain further information regarding the AgNP shape. Characteristics of the absorbance spectra were used to confirm the presence of non-spherical AgNP. [Copyright &y& Elsevier]

Abstract: Chemical speciation of Zn, Cu, Ni, Cd and Pb in River Rhine was studied by measuring free ion concentration and distribution in nanoparticles, and by comparing the measurement with speciation modeling. Concentrations of free metal ions were determined in situ using Donnan Membrane Technique (DMT). The percentage of free over total (filtered) metal concentration is 52%, 33%, 2.6%, 0.48% and 0.12% for respectively Zn, Cd, Ni, Pb and Cu, i.e. the degree of metal complexation in the river is the lowest for Zn and the highest for Cu. Metals in 1–300 nm particles were analyzed using Asymmetric Flow Field Flow Fractionation (AsF-FFF), but the overall recovery is quite low. The nano-sized Cu detected is mainly associated with DOM of 1–5 nm, whereas Pb and Zn are dominantly associated with particles of iron hydroxides and clay of larger size (30–100 nm). Free ion concentrations calculated with the speciation modeling are in good agreement with the measurements, except for Pb. Based on the model, DOM-bound is the most important complexed form for Cu and Cd, whereas formation of (bi)carbonate and EDTA complexes are more important for Ni and Zn. Adsorption of Pb to DOM is probably overestimated by the model, whereas Pb adsorption to iron hydroxides is underestimated. [Copyright &y& Elsevier]

Abstract: A diffusion Split-Flow Thin Cell (SPLITT) system was used to partially remove small peptides such as β2 microglobulin (β2M) and parathyroid hormone (PTH) in a continuous manner from an input flow stream while preserving most (over 97%) of the larger protein in the sample, such as albumin. To help determine the operating conditions for this work, a two-dimensional numerical model based on the Navier–Stokes equation and convection–diffusion equations was developed for diffusional SPLITT using COMSOL multiphysics software (COMSOL Inc., MA). These simulations were used to obtain the relationship between important operational parameters and the purification efficiency for proteins of interest. The diffusion-based SPLITT system was fabricated using xurography and was used to demonstrate protein purification based on the differences in size or diffusion coefficient of the sample. The results obtained from the experiments are compared with the mathematical model and show good agreement, while the variations between these results are discussed. The results show that significant portions of small peptides (>25%) can be removed while preserving larger proteins (up to 95%) in the carrier stream. A potential application of this technique is to be used as an additional step in kidney dialysis to remove toxins that are not effectively removed by current dialysis protocols. [Copyright &y& Elsevier]

Abstract: This paper presents results from the first analyses of the mesostructure of natural rubber (NR) by asymmetrical flow field flow fractionation (AF4). The results are compared with those obtained by size exclusion chromatography (SEC) in terms of average molar masses, radius of gyration and insoluble part (or gel quantity). Comparable results were obtained for the sample not containing gel. Conversely, for samples with gel, significant differences were found due to the presence of microaggregates. Contrary to SEC, AF4 fractionation enables partial fractionation of polyisoprene chains and microaggregates in a single run without preliminary treatment. The results presented here also highlight the special structure (very compact spheres) of microaggregates in NR compared to chemical crosslinked microaggregates in synthetic polyisoprene. The advantages and drawbacks of both techniques for analysing NR samples are also discussed. [Copyright &y& Elsevier]

Abstract: The elution behaviour of linear and branched polyethylene samples in SEC was studied. For the branched samples an abnormal late co-elution of large and small macromolecules manifests itself as an abnormal re-increase of the molar mass and the radius of gyration values detected with multi angle light scattering at high elution volumes in SEC. The late co-elution of small and large macromolecules cannot be explained by the SEC mechanism alone. The influence of several experimental parameters on the late co-elution was studied. It was found that the type of SEC column and the flow rate have a significant influence. The late eluting part of the sample was fractionated and separated by HT-SEC- and HT-AF4-MALS. The different results of both methods have been discussed with the aim to find possible explanations for the late elution. The experiments indicate that especially large branched structures show an increased tendency for the phenomenon. [Copyright &y& Elsevier]

Abstract: Various Be-containing micro-particle suspensions were equilibrated with simulated lung fluid (SLF) to examine their dissolution behavior as well as the potential generation of nanoparticles. The motivation for this study was to explore the relationship between dissolution/particle generation behaviors of Be-containing materials relevant to Be-ore processing, and their epidemiologically indicated inhalation toxicities. Limited data suggest that BeO is associated with higher rates of beryllium sensitization (BS) and chronic beryllium disease (CBD) relative to the other five relevant materials studied: bertrandite-containing ore, beryl-containing ore, frit (a processing intermediate), Be(OH)2 (a processing intermediate), and silica (control). These materials were equilibrated with SLF at two pH values (4.5 and 7.2) to reflect inter- and intra-cellular environments in lung tissue. Concentrations of Be, Al, and Si in SLF increased linearly during the first 20 days of equilibration, and then rose slowly, or in some cases reached a maximum, and subsequently decreased. Relative to the other materials, BeO produced relatively low Be concentration in solution at pH 7.2; and relatively high Be concentration in solution at pH 4.5 during the first 20 days of equilibration. For both pH values, however, the Be concentration in SLF normalized to Be content of the material was lowest for BeO, demonstrating that BeO was distinct among the four other Be-containing materials in terms of its persistence as a source of Be to the SLF solution. Following 149 days of equilibration, the SLF solutions were fractionated using flow-field flow fractionation (FlFFF) with detection via ICP-MS. For all materials, nanoparticles (which were formed during equilibration) were dominantly distributed in the 10–100nm size range. Notably, BeO produced the least nanoparticle-associated Be mass (other than silica) at both pH values. Furthermore, BeO produced the highest Be concentrations in the size range corresponding to <3kDa (determined via centrifugal ultrafiltration), indicating that in addition to persistence, the BeO produced the highest concentrations of truly dissolved (potentially ionic) Be relative to the other materials. Mass balance analysis showed reasonable sample recoveries during FFF fractionation (50–100%), whereas recoveries during ICP-MS (relative to acidified standards) were much lower (5–10%), likely due to inefficiencies in nebulizing and ionizing the nanoparticles. [Copyright &y& Elsevier]

Abstract: WO3 colloidal suspensions obtained through a simple sol–gel procedure were subjected to a controlled temperature aging process whose time evolution in terms of particle mass and size distribution was followed by sedimentation field flow fractionation (SdFFF) and flow field flow fractionation (FlFFF). The experiments performed at a temperature of 60°C showed that in a few hours the initially transparent sol of WO3 particles, whose size was less than 25nm, undergoes a progressive size increase allowing nanoparticles to reach a maximum equivalent spherical size of about 130nm after 5h. The observed shift in particle size distribution maxima (SdFFF), the broadening of the curves (FlFFF) and the SEM–TEM observations suggest a mixed mechanism of growth-aggregation of initial nanocrystals to form larger particles. The photoelectrochemical properties of thin WO3 films obtained from the aged suspensions at regular intervals, were tested in a biased photoelectrocatalytic cell with 1M H2SO4 under solar simulated irradiation. The current–voltage polarization curves recorded in the potential range 0–1.8V (vs. SCE) showed a diminution of the maximum photocurrent from 3.7mAcm−2 to 2.8mAcm−2 with aging times of 1h and 5h, respectively. This loss of performance was mainly attributed to the reduction of the electroactive surface area of the sintered particles as suggested by the satisfactory linear correlation between the integrated photocurrent and the cyclic voltammetry cathodic wave area of the W(VI)→W(V) process measured in the dark. [Copyright &y& Elsevier]

Abstract: The use of flow field flow fractionation (FlFFF) for the separation and characterization of natural colloids and nanoparticles has increased in the last few decades. More recently, it has become a popular method for the characterization of manufactured nanoparticles. Unlike conventional filtration methods, FlFFF provides a continuous and high-resolution separation of nanoparticles as a function of their diffusion coefficient, hence the interest for use in determining particle size distribution. Moreover, when coupled to other detectors such as inductively coupled plasma-mass spectroscopy, light scattering, UV-absorbance, fluorescence, transmission electron microscopy, and atomic force microscopy, FlFFF provides a wealth of information on particle properties including, size, shape, structural parameters, chemical composition and particle-contaminant association. This paper will critically review the application of FlFFF for the characterization of natural colloids and natural and manufactured nanoparticles. Emphasis will be given to the detection systems that can be used to characterize the nanoparticles eluted from the FlFFF system, the obtained information and advantages and limitation of FlFFF compared to other fractionation and particle sizing techniques. This review will help users understand (i) the theoretical principles and experimental consideration of the FlFFF, (ii) the range of analytical tools that can be used to further characterize the nanoparticles after fractionation by FlFFF, (iii) how FlFFF results are compared to other analytical techniques and (iv) the range of applications of FlFFF for natural and manufactured NPs. [Copyright &y& Elsevier]

Atomic force microscopy (AFM) was used to image and quantify natural nanoparticles (prefiltered <25 nm) from three different freshwater sites (Vale Lake, Bailey Brook and Tern Rivers). Four fractions were analysed by AFM; the prefiltered fraction (<25 nm) and three fractions collected after separation of this prefiltered sample by flow field-flow fractionation (FlFFF) which corresponds to material which has size ranges of <4.2 nm, 4.2–15.8 nm and 15.8–32.4 nm, as determined by FlFFF theory. The large majority of materials in all samples appeared as <3 nm nanoparticles, nearly spherical and rich in chromophores active at 254 nm UV, which thus correspond to natural organic matter. However, nanoparticles were also imaged up to slightly more than 25 nm in size, indicating a slight disagreement in sizing between filtration and FlFFF. In addition, some particles in certain fractions were found to be covered with a thin film of less than 0.5–1.0 nm. Substantial differences between sites were observed. [Copyright &y& Elsevier]

Abstract: Adequate biophysical characterization of influenza virions is important for vaccine development. The influenza virus vaccines are produced from the allantoic fluid of developing chicken embryos. The process of viral replication produces a heterogeneous mixture of infectious and non-infectious viral particles with varying states of aggregation. The study of the relative distribution and behavior of different subpopulations and their inter-correlation can assist in the development of a robust process for a live virus vaccine. This report describes a field flow fractionation and multiangle light scattering (FFF-MALS) method optimized for the analysis of size distribution and total particle counts. The FFF-MALS method was compared with several other methods such as transmission electron microscopy (TEM), atomic force microscopy (AFM), size exclusion chromatography followed by MALS (SEC-MALS), quantitative reverse transcription polymerase chain reaction (RT Q-PCR), median tissue culture dose (TCID50), and the fluorescent focus assay (FFA). The correlation between the various methods for determining total particle counts, infectivity and size distribution is reported. The pros and cons of each of the analytical methods are discussed. [Copyright &y& Elsevier]

Abstract: The efficiency and the effectiveness of wheat starch granules as a support for the immobilization of the alcohol resistant psychrophilic yeast Saccharomyces cerevisiae strain AXAZ-1 was studied. The growth rate of these cells in the presence or the absence of the support in the culture medium was investigated by the technique of sedimentation/steric field-flow fractionation (Sd/StFFF). An abrupt increase of biomass productivity in less required time was observed in the case of the presence of wheat starch granules in the culture medium. The results indicate that wheat starch granules might be a good medium for yeast cell culture and bioreactor formation. [Copyright &y& Elsevier]

Abstract: This paper examines geometric scaling models for field flow fractionation systems to understand how channel dimensions affect resolution and retention. Specifically, the changing contribution of the instrumental plate height during miniaturization of field flow fractionation (FFF) systems is reported. The work is directed towards determining the optimal geometrical parameters for miniaturization of field flow fractionation systems. The experimental relationship between channel height in FFF systems and instrumental plate heights is reported. FFF scaling models are modified to: (i) better clarify the dependence of plate height and resolution on channel height in FFF and (ii) include a more complete geometrical scaling analysis and model comparison in the low retention regime. Electrical field flow fractionation has been shown to benefit from miniaturization, so this paper focuses on that subtype, but surprisingly, the results also indicate the possibility of improvement in performance with miniaturization of other field flow fractionation systems including general FFF subtypes in which the applied field does not vary with channel height. This paper also discusses the potential role of more powerful microscale field flow fractionation systems as a new class of sample preparation units for micro-total-analysis systems (μ-TAS). [Copyright &y& Elsevier]

Abstract: A new sedimentation field flow fractionation (SdFFF) method is presented for the estimation of the total potential energy of interaction between colloidal particles and the channel wall. The method is based on the variation of the mean cloud thickness in SdFFF due to the variation of the suspension''s ionic strength. It requires only two SdFFF experiments at two different ionic strengths and at a constant acceleration field. The found values are compatible with those calculated from the various forms of equations of the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory. [Copyright &y& Elsevier]

Hyaluronic acid (HA)-based prodrugs bearing double-responsive (acid pH or oxidation) boronates of catechol-containing drugs were used to treat xenografted human prostate tumours (LNCaP) in SCID mice. The HA prodrugs accumulated significantly only in tumours (impressively, up to 40% of the injected dose after 24 h) and in liver, with negligible – actually anti-inflammatory - consequences in the latter. A quercetin-HA prodrug significantly slowed down tumour growth, in a dose-dependent fashion and with a much higher efficacy (up to 4 times) than equivalent doses of free quercetin. In short, boronated HA appears to be a very promising platform for targeted chemotherapy. Hyaluronic acid-based prodrugs bearing side-chain boronic esters show excellent tumour accumulation (targeting) and acid pH- or reactive oxygen species (ROS)-responsive release of polyphenolic drugs such as quercetin. The chemotherapeutic performance of the latter in xenografted mice is dramatically improved upon conjugation with HA. [Display omitted] • Boronate-linked payloads can be released from hyaluronic acid upon acidification or oxidation. • Boronate-based hyaluronic acid prodrugs accumulate in tumours up to 40–50% of the injected doses. • Boronate-based hyaluronic acid prodrugs efficiently treat human prostate tumours in SCID mice. [ABSTRACT FROM AUTHOR]

• The AF4 technique is an important tool to study the release, transfer, and partition of fluorescent dyes from polymeric nanoparticles. • Physically encapsulated dyes can be transferred from nanoparticles to proteins present in the medium. • The transfer is related to the dyes physicochemical properties such as lipophilicity. • AF4 elution conditions are mild enough to maintain dye-protein complexes stable. • Proteins mediate dye release from nanoparticles as evidenced by AF4. Fluorescent probes are used in drug nanocarrier pre-clinical studies or as active compounds in theranostics and photodynamic therapy. In the biological medium, nanoparticles interact with proteins, which can result in the off-target release of their cargo. The present study used asymmetric flow field-flow fractionation with online multi-angle laser light scattering and fluorescence detection (AF4-MALLS-FLD) to study the release, transfer, and partition of fluorescent dyes from polymeric nanoparticles (NP). NP formulations containing the dyes Rose Bengal, Rhodamine B, DiI, 3-(α-azidoacetyl)coumarin and its polymer conjugate, Nile Red, and IR780 and its polymer conjugate were prepared. NP suspensions were incubated in a medium with serum proteins and then analyzed by AF4. AF4 allowed efficient separation of proteins (< 10 nm) from fluorescently labeled NP (range of 54 – 180 nm in diameters). The AF4 analyses showed that some dyes, such as Rose Bengal, IR780, and Coumarin were transferred to a high extent (68-77%) from NP to proteins. By contrast, for DiI and dye-polymer conjugates, transfer occured to a lower extent. The studies of dye release kinetics showed that the transfer of IR780 from NP to proteins occurs at a high extent (~50%) and rate, while Nile Red was slowly released from the NP over time with reduced association with proteins (~20%). This experiment assesses the stability of fluorescence labeling of nanocarriers and probes the transfer of fluorescent dyes from NP to proteins, which is otherwise not accessible with commonly used techniques of separation, such as dialysis and ultrafiltration/centrifugation employed in drug encapsulation and release studies of nanocarriers. Determining the interaction and transfer of dyes to proteins is of utmost importance in the pre-clinical evaluation of drug nanocarriers for improved correlation between in vitro and in vivo studies. [ABSTRACT FROM AUTHOR]

• AF4 × AF4 method provides minimum number of user-defined peak to deconvolution for start. • Fraction fractograms were used for deconvolution to assess the overfitting issues. • Mw distribution and Se abundant were identified in the low Mw protein fractions in Se-SPI. • Significant differences were found in the ability of binding Se for low Mw Se-rich proteins in Se-SPI. An analytical challenge that arises in environmental and food analysis is to quantify heterogeneous nanoparticles especially in polydisperse and complex samples. The method stated herein based on the reinjection asymmetrical flow field-flow fractionation (AF4 × AF4) coupled with inductively coupled plasma-mass spectrometer (ICP-MS) and statistical deconvolution allowed for identifying the molecular weight (Mw) and selenium abundance of the low Mw protein fractions (ca. < 132 kDa) in an unknown and complex sample (e.g., selenium-rich soybean protein isolates (Se-SPI)). A non-linear decay crossflow program was also developed to get better resolution and shorter elution time for both low and high Mw components. The concept of the reinjection method was based on the excellent ability for reducing sample complexity regarding the size fractionation, and peak reproducibility under the identical conditions of AF4 system. The standard protein mixture was used as a proof-of-principle sample. The results showed the underlying peaks predicted by the reinjection method were agreed with the separation result using the standard mixture (the relative standard deviation of peak locations < 1%), which indicated the reinjection method could provide an accurate assessment of the underlying peak number and location, and was promising to minimize the overfitting problem for statistic deconvolution. Interestingly, significant differences of Se abundance in protein fractions were observed in the low Mw range for Se-SPI, ranging from 0.28 to 1.66 cps/V with the Mw ranging from 13.75 kDa to 104.17 kDa, which indicated significant differences in the ability of binding Se for these selenium-rich proteins in Se-SPI. [ABSTRACT FROM AUTHOR]

• Critical review for Dielectrophoresis-field flow fractionation (DEP-FFF) platforms. • Theory for DEP-FFF is detailed. • Operational modes of DEP-FFF are discussed. • Future directions in DEP-FFF are proposed. Dielectrophoresis-field flow fractionation (DEP-FFF) has emerged as an efficient in-vitro , non-invasive, and label-free mechanism to manipulate a variety of nano- and micro-scaled particles in a continuous-flow manner. The technique is mainly used to fractionate particles/cells based on differences in their sizes and/or dielectric properties by employing dielectrophoretic force as an external force field applied perpendicular to the flow direction. The dielectrophoretic force is the result of a spatially non-uniform electric field in the microchannel that can be generated either by exploiting microchannel geometry or using special arrangements of microelectrode arrays. Several two-dimensional (e.g., coplanar interdigitated, castellated) and three-dimensional (e.g., top-bottom, side-wall) microelectrode designs have been successfully utilized to perform fractionation of heterogeneous samples. Although originally introduced as a separation technique, DEP-FFF has attracted increasing interest in performing other important operations such as switching, focusing, dipping, and surface functionalization of target particles. Nonetheless, the technique still suffers from limitations such as low throughput and joule heating. By comparatively analyzing recent developments that address these shortcomings, this work is a step forward towards realizing the full potential of DEP-FFF as an ideal candidate for point-of-care (POC) devices with diverse applications in the fields of biomedical, chemical, and environmental engineering. [ABSTRACT FROM AUTHOR]

• Multi-detector AF4 for characterization of nanomedicines is reviewed • The development of high quality Standard Operating Procedures is presented • The process for developing validated standard test methods is discussed • Multi-detector AF4 is a robust technique for measuring quality attributes Asymmetric-flow field-flow fractionation (AF4) has been recognized as an invaluable tool for the characterisation of particle size, polydispersity, drug loading and stability of nanopharmaceuticals. However, the application of robust and high quality standard operating procedures (SOPs) is critical for accurate measurements, especially as these complex drug nanoformulations are most often inherently polydisperse. In this review we describe a unique international collaboration that lead to the development of a robust SOP for the measurement of physical-chemical properties of nanopharmaceuticals by multi-detector AF4 (MD-AF4) involving two state of the art infrastructures in the field of nanomedicine, the European Union Nanomedicine Characterization Laboratory (EUNCL) and the National Cancer Institute-Nanotechnology Characterisation Laboratory (NCI-NCL). We present examples of how MD-AF4 has been used for the analysis of key quality attributes, such as particle size, shape, drug loading and stability of complex nanomedicine formulations. The results highlight that MD-AF4 is a very versatile analytical technique to obtain critical information on a material particle size distribution, polydispersity and qualitative information on drug loading. The ability to conduct analysis in complex physiological matrices is an additional very important advantage of MD-AF4 over many other analytical techniques used in the field for stability studies. Overall, the joint NCI-NCL/EUNCL experience demonstrates the ability to implement a powerful and highly complex analytical technique such as MD-AF4 to the demanding quality standards set by the regulatory authorities for the pre-clinical safety characterization of nanomedicines. [ABSTRACT FROM AUTHOR]

• The theoretical background and limitations of AF4 techniques are outlined. • Hyphenated AF4 methods for analyzing natural and manufactured nanoparticles are reviewed. • Advances in investigating colloidal behavior, and colloid-metal interactions are reviewed. • Future needs for methodological advances are outlined. Aquatic ecosystems are facing multiple direct and indirect stressors from anthropogenic activities. Aquatic colloids, including natural and manufactured nanoparticles, have received global concern owing to their ability to transport metals, nutrients and pollutants, and their potential to pose ecological risks for wildlife and human health. Asymmetric flow-field flow fractionation (AF4) has become one of the most promising and powerful approaches to characterize colloidal particles over the last ten years. This review overviews the advances and applications of AF4 for the study of aquatic systems over the last decade. The analytical challenge of AF4 and known deviation from theoretical assumptions are also critically discussed, such as sample losses, membrane-analyte interactions, and overloading. Future needs for methodological advances are addressed. [ABSTRACT FROM AUTHOR]

• Soil colloids promote the stability of microaggregate occlusion in macroaggregates. • Microaggregate reaggregation is affected by colloidal iron and organic carbon. • Less microaggregate reaggregation with reduced organic carbon concentrations in Luvisols. • Smaller-sized microaggregate structures in presence of colloidal-sized iron. Several beneficial soil functions are linked to aggregates, but how the formation and stability depend on the presence of colloidal- and nanosized (1000–1 nm) bulding blocks is still understood poorly. Here, we sampled subsites from an arable toposequence with 190 and 340 g kg−1 clay, and isolated small soil microaggregates (SMA; <20 µm) from larger macroaggregate units (>250 µm) using an ultrasonic dispersion energy of 60, 250, and 440 J mL−1, respectively. We then allowed these small SMA to reaggregated after chemical removal of organic carbon (OC) as well as of Fe- and Al (hydr)oxides, respectively. The size distribution of the reaggregated small SMA and fine colloids (<0.45 µm) was analyzed via laser diffraction and asymmetric flow field-flow fractionation coupled to inductively coupled plasma mass spectrometry and organic carbon detection, respectively. We found elevated amounts of both finer colloids and stable SMA at subsites with higher clay contents. The size distribution of small SMA was composed of two distinctive fractions including colloids and larger microaggregates with an average size of 5 µm. The removal of Fe with Dithionite-Citrate-Bicarbonate (DCB) shifted the size of the small SMA to a larger equivalent diameter, while removal of OC with NaOCl reduced it. After three wetting and drying cycles, the concentration of colloids declined, whereas the small SMA without chemical pre-treatments reaggregated to particles with larger average diameters up to 10 µm, with the size depending on the clay content. Intriguingly, this gain in size was more pronounced after Fe removal, but it was not affected by OC removal. We suggest that Fe (hydr)oxides impacts the stability of small SMA primarily by being present in small-sized pores and thus cementing the aggregates to smaller size. In contrast, the effect of OC was restricted to the size of colloids, gluing them together to small SMAs within defined size ranges when OC was present but releasing these colloids when OC was absent. [ABSTRACT FROM AUTHOR]

• The origin of colloids in streams indicates elemental transport pathways. • Monthly analysis of stream & groundwater, rainwater, throughfall, soil leachate. • Field flow fractionation revealed three fractions in all sample types. • Groundwater derived colloids dominated the stream signal. • Water flux and particle reformation affect the seasonal dynamics of particles. Biogeochemical changes of whole catchments may, at least in part, be deduced from changes in stream water composition. We hypothesized that there are seasonal variations of natural nanoparticles (NNP; 1–100 nm) and fine colloids (<300 nm) in stream water, which differ in origin depending on catchment inflow parameters. To test this hypothesis, we assessed the annual dynamics of the elemental composition of NNP and fine colloids in multiple water compartments, namely in stream water, above and below canopy precipitation, groundwater and lateral subsurface flow from the Conventwald catchment, Germany. In doing so, we monitored meteorological and hydrological parameters, total element loads, and analyzed element concentrations of org C, Al, Si, P, Ca, Mn and Fe by Asymmetric Flow Field Flow Fractionation (AF4). The results showed that colloid element concentrations were < 5 µmol/L. Up to an average of 55% (Fe) of total element concentrations were not truly dissolved but bound to NNP and fine colloids. The colloid patterns showed seasonal variability with highest loads in winter. The presence of groundwater-derived colloidal Ca in stream water showed that groundwater mainly fed the streams throughout the whole year. Overall, the results showed that different water compartments vary in the NNP and fine colloidal composition making them a suitable tool to identify the streams NNP and fine colloid sources. Given the completeness of the dataset with respect to NNP and fine colloids in multiple water compartments of a single forest watershed this study adds to the hitherto underexplored role of NNP and fine colloids in natural forest watersheds. [ABSTRACT FROM AUTHOR]

Casein, the major milk protein fraction, is responsible for structure formation in fermented dairy products, and enzymatic cross-linking of caseins represents a possibility to modify texture and physical product properties. In this research, casein cross-linking by microbial transglutaminase (mTGase) was characterised from a colloid rheological perspective. Sodium caseinate (NaCn) at 50 g kg−1 was not (0 h), moderately (3 h) or excessively cross-linked (24 h) using 3 U mTGase per g protein, and subsequently freeze-dried and redissolved at 10–300 g kg−1. The resulting suspensions were characterised in steady and oscillatory shear rheology to obtain viscosity-concentration plots and to evaluate shear thinning and viscoelastic properties. Additional molecular analysis was done by dynamic light scattering and asymmetric flow field flow fractionation, showing that NaCn molecules were associated to hydrated nanoparticles with hydrodynamic radii of ~12 nm and cross-linked predominantly within the same particle, leading to an increased apparent density of the particles with ongoing incubation. This resulted in a transition from soft particles towards a more hard sphere like behaviour in the rheological experiments. While concentrated suspensions of uncross-linked NaCn (0 h) showed properties of a viscoelastic liquid in frequency sweeps over the tested temperature range (5–40 °C), a true liquid-to-solid transition upon cooling was observed for the cross-linked samples. The threshold for this transition was lower for moderately (3 h) than for excessively cross-linked NaCn (24 h), i.e., at lower concentration and higher temperature, presumably because interpenetration of the particles was still possible to some extent. Image 1 • Sodium caseinate (NaCn) particles were internally cross-linked by transglutaminase. • An increased apparent density was found by multi angle light scattering experiments. • A transition from soft particles towards more hard sphere like behaviour was noticed. • A true phase transition upon cooling was observed for cross-linked NaCn particles. [ABSTRACT FROM AUTHOR]

• The NIR probe IR780 was derivatized and covalently linked to polylactide. • The polymer-dye conjugate provides stable fluorescence labelling of nanocarriers. • Encapsulated IR780 leaks from the nanocarriers but not the polymer conjugate. • IR780 linked to PLA can be used for in vitro and in vivo fluorescence imaging. Near-infrared dyes are useful to monitor nanocarriers in vitro and in vivo and can serve as photosensitizers in cancer photodynamic therapy. However, strategies need to be developed to guarantee that the dye photophysical properties and loading within the drug delivery system remain stable for reliable tracking within biological systems. This work reports the facile chemical conjugation of the carbocyanine heptamethine near-infrared dye IR780 to polylactide for stable fluorescent labeling of biodegradable polyester nanocarriers. "Clickable" polylactide was synthesized via organocatalyzed ring opening polymerization of D , L -lactide with a cyclooctyne initiator. IR780 was derivatized and conjugated to polylactide via a one-pot copper-free azide-alkyne cycloaddition reaction. The synthetic strategy developed was effective to promote conjugation of the near-infrared fluorescent dye to polylactide, as confirmed by high performance liquid chromatography. Nanoparticles containing the dye–polymer conjugate were prepared by nanoprecipitation and characterized. Asymmetric flow field-flow fractionation with light scattering and fluorescence detection revealed that the near-infrared fluorescence of the nanoparticles remained stable and was not transferred to serum proteins. In contrast, significant transfer of the dye to serum proteins was evidenced when the dye was merely encapsulated in similar nanoparticles through physical entrapment. Confocal microscopy and fluorescence tomography imaging showed that the polymer-dye conjugate confers fluorescence properties to the NP suitable for further in vitro and in vivo pre-clinical studies. [ABSTRACT FROM AUTHOR]

Two different clay nanocomposites, as sepiolite-Ag and kaolinite-Ag, are studied as carriers for silver nanoparticles (AgNPs) oral administration as antimicrobial agent in additives for animal feed. A three-step digestibility assay, corresponding to stomach, small and large intestine simulations, has been followed. Ultrafiltration and asymmetrical flow field-flow fractionation (AF4) coupled to UV–Vis absorption and ICPMS detectors have been used for size characterisation of the silver species released during the in vitro digestibility assays. Less than 1% of the total silver is released in the stomach simulation step, probably due to the formation of silver chloride on the nanocomposite surface. In the case of the intestine simulation, silver released increases and tends to form complexes with the enzymes present in the media. A larger amount of silver was released from kaolinite-Ag compared to sepiolite-Ag (17 vs. 7%), probably due to a higher retention rate of silver shown by sepiolite, justified by its sorption capacity and fibrous structure. No evidences were found about a significant release of silver nanoparticles along the in vitro assay from any of the two nanocomposites studied. These results have been compared to the silver levels found in liver and faeces from weaned pigs fed with these supplements for 35 days and followed by basal diet free of silver for 4 weeks in an in vivo assay. Piglets fed with kaolinite-Ag retained more silver in liver than those receiving sepiolite-Ag, and the opposite was observed in faeces, although differences were not statistically significant. Silver levels found in muscles were below the limit of detection (0.009 μg Ag g−1) in all cases. • Two different clay nanocomposites based on silver nanoparticles as feed additives have been studied. • Characterization of different silver forms released during digestion has been carried out in in vitro studies by AF4-ICP-MS. • Silver content found in tissues of pigs show a tendency respect to the silver species determined in the in vitro assays. • These results are relevant to understand the processes affecting nanomaterials during digestion. [ABSTRACT FROM AUTHOR]