Development of complementary analytical methods to characterize extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) are involved in intercellular communication and various biological processes. They hold clinical promise for the diagnosis and management of a wide range of pathologies, including cancer, cardiovascular diseases and degenerative diseases, and are of interest as regenerative therapies. Understanding the complex structure of these EVs is essential to perceive the current challenges associated with their analysis and characterization. Today, challenges remain in terms of access to high-yield, high-purity isolation methods, as well as analytical methods for characterizing and controlling the quality of these products for clinical use. RESULTS: We isolated EVs from the same immortalized human cell culture supernatant using two commonly used approaches, namely differential ultracentrifugation and membrane affinity. Then we evaluated EV morphology, size, zeta potential, particle and protein content, as well as protein identity using cryogenic electron microscopy, nanoparticle tracking analysis, asymmetric field flow fractionation (AF4) and size exclusion chromatography (SEC) coupled to multi angle light scattering, bicinchoninic acid assay, electrophoretic light scattering, western blotting and high-resolution mass spectrometry. Compared to membrane affinity isolation, dUC is a more efficient isolation process for obtaining particles with the characteristics expected for EVs and more specifically for exosomes. To validate an isolation process, cryogenic electron microscopy is essential to confirm vesicles with membranes. High resolution mass spectrometry is powerful for understanding the mechanism of action of vesicles. Separative methods, such as AF4 and SEC, are interesting for separating vesicle subpopulations and contaminants. SIGNIFICANCE: This study provides a critical assessment of eight different techniques for analyzing EVs, some of which are mandatory for in-depth characterization and deciphering, while others are more appropriate for routine analysis, once the production and isolation process has been validated. The strengths and limitations of the different approaches used are highlighted.

Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles.

Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications.

Quantitative determination of the intracellular uptake of silica nanoparticles using asymmetric flow field flow fractionation coupled with ICP mass spectrometry and their cytotoxicity in HepG2 cells.

We established a size separation method for silica nanoparticles (SiNPs) measuring 10, 30, 50, 70, and 100 nm in diameter using asymmetric flow field flow fractionation hyphenated with inductively coupled plasma mass spectrometry (AF4-ICP-MS), and evaluated the cytotoxicity of SiNPs in human hepatoma HepG2 cells. Analysis of the mixture sample revealed that nanoparticles of different sizes were eluted at approximately 2-min intervals, with no effect on each elution time or percentage recovery. Compared with larger SiNPs, smaller SiNPs exhibited high cytotoxicity when the volume of SiNPs exposed to the cells was the same. We measured SiNPs in culture medium and inside cells by AF4-ICP-MS and found that approximately 17% of SiNPs in the mixture of five differently sized particles were absorbed by the cells. Transmission electron microscopy revealed that 10 nm SiNPs formed aggregates and accumulated in the cells. Based on AF4-ICP-MS analysis, there is no clear difference in the particle volume absorbed by the cells among different sizes. Therefore, the high toxicity of small SiNPs can be explained by the fact that their large surface area relative to particle volume efficiently induces toxicological influences. Indeed, the large surface area of 10 nm SiNPs significantly contributed to the production of reactive oxygen species.

Increasing the Resolution of Field-Flow Fractionation with Increasing Crossflow Gradients.

The resolution of flow field-flow fractionation (flow FFF) depends primarily on the crossflow rate and its change over time. In this work, we demonstrate a method for modulation of the crossflow rate during separation that increases the peak-to-peak resolution of the resulting fractograms. In classical FFF methods, the crossflow rate is either maintained constant or decreased during the separation of the different species. In this work, higher resolution between peaks was achieved by a novel gradient method in which the crossflow is increased briefly during separation to allow stronger retention of the later eluting peaks. We first outline the theoretical basis by which improved separation is achieved. We confirm our hypothesis by quantifying the impact of increasing crossflow on the resolution between a monoclonal antibody monomer and its high-molecular-weight aggregate. We then demonstrate that this method is applicable to two different FFF methods (AF4 and HF5) and various pharmaceutically relevant samples (monoclonal antibodies and adeno-associated viruses). Finally, we hypothesize that increasing the force perpendicular to the laminar flow as described here is broadly applicable to all FFF methods and improves the quality of FFF-based separations.

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.

Characterization of Virus Particles and Submicron-Sized Particulate Impurities in Recombinant Adeno-Associated Virus Drug Product.

Characterization of particulate impurities such as aggregates is necessary to develop safe and efficacious adeno-associated virus (AAV) drug products. Although aggregation of AAVs can reduce the bioavailability of the virus, only a limited number of studies focus on the analysis of aggregates. We explored three technologies for their capability to characterize AAV monomers and aggregates in the submicron (<1 µm) size range: (i) mass photometry (MP), (ii) asymmetric flow field flow fractionation coupled to a UV-detector (AF4-UV/Vis) and (iii) microfluidic resistive pulse sensing (MRPS). Although low counts for aggregates impeded a quantitative analysis, MP was affirmed as an accurate and rapid method for quantifying the genome content of empty/filled/double-filled capsids, consistent with sedimentation velocity analytical ultracentrifugation results. MRPS and AF4-UV/Vis enabled the detection and quantification of aggregate content. The developed AF4-UV/Vis method separated AAV monomers from smaller aggregates, thereby enabling a quantification of aggregates <200 nm. MRPS was experienced as a straightforward method to determine the particle concentration and size distribution between 250-2000 nm, provided that the samples do not block the microfluidic cartridge. Overall, within this study we explored the benefits and limitations of the complementary technologies for assessing aggregate content in AAV samples.

Characterizing Non-covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation On-Line Coupled to Native Mass Spectrometry.

We report an online analytical platform based on the coupling of asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry (nMS) in parallel with UV-absorbance, multi-angle light scattering (MALS), and differential-refractive-index (UV-MALS-dRI) detectors to elucidate labile higher-order structures (HOS) of protein biotherapeutics. The technical aspects of coupling AF4 with nMS and the UV-MALS-dRI multi-detection system are discussed. The "slot-outlet" technique was used to reduce sample dilution and split the AF4 effluent between the MS and UV-MALS-dRI detectors. The stability, HOS, and dissociation pathways of the tetrameric biotherapeutic enzyme (anticancer agent) l-asparaginase (ASNase) were studied. ASNase is a 140 kDa homo-tetramer, but the presence of intact octamers and degradation products with lower molecular weights was indicated by AF4-MALS/nMS. Exposing ASNase to 10 mM NaOH disturbed the equilibrium between the different non-covalent species and led to HOS dissociation. Correlation of the information obtained by AF4-MALS (liquid phase) and AF4-nMS (gas phase) revealed the formation of monomeric, tetrameric, and pentameric species. High-resolution MS revealed deamidation of the main intact tetramer upon exposure of ASNase to high pH (NaOH and ammonium bicarbonate). The particular information retrieved from ASNase with the developed platform in a single run demonstrates that the newly developed platform can be highly useful for aggregation and stability studies of protein biopharmaceuticals.

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.

Study on effects of preparation method on the structure and antioxidant activity of protein-Tremella fuciformis polysaccharide complexes by asymmetrical flow field-flow fractionation.

Oxidation is an essential biological process for human life. In this study, low density lipoprotein-Tremella fuciformis polysaccharide (LDL-TFP) complexes were prepared by electrostatic and covalent methods. The effects of preparation method on the structure and antioxidant activity of LDL-TFP complexes were investigated by asymmetrical flow field-flow fractionation (AF4) coupled with ultraviolet-visible (UV/Vis), multiangle light scattering (MALS), and differential refractive index (dRI) detectors. The results showed that the electrostatic LDL-TFP complexes had a spherical structure, while the covalent LDL-TFP complexes had a rod-like structure as indicated by the ratio of Rg (radius of gyration) to Rh (hydrodynamic radius). Moreover, the results revealed that the antioxidant activity of the LDL-TFP complexes on the HepG2 could be related to the structure of LDL-TFP complexes. The antioxidant activity of LDL-TFP complexes formed by LDL modified with phospholipase A2 was further enhanced. This study would help expand the application of TFP.

Study of the relationship between red wine colloidal fraction and astringency by asymmetrical flow field-flow fractionation coupled with multi-detection.

Macromolecules including condensed tannins and polysaccharides impact wine taste and especially astringency. Asymmetrical Flow-Field-Flow-Fractionation (AF4) coupled to UV detection (UV), multi-angle light scattering (MALS) and refractive index detection (dRI) has been proposed to separate red wine colloids. The present work aimed at relating AF4-mutidetection profiles with red wine astringency. Fifty commercial red wines characterized by a trained sensory panel were analysed by AF4-UV-MALS-dRI and UV-visible spectroscopy. The analytical data set was built by selecting the three variables most predictive of the astringency score from each table (UV, dRI, MALS, Mw distribution, and UV-visible spectra of whole wine, permeate and retentate A4F fractions) and analysed by principal component analysis. Red wine astringency was more related to variables extracted from the AF4 data than to UV- absorbance of the wine or permeate, confirming the relevance of AF4-multidetection for analysis of the colloidal fraction involved in this perception.

Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors.

Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (Rh) measured by online DLS with the Rh values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The Rg/Rh ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.

Release, transfer and partition of fluorescent dyes from polymeric nanocarriers to serum proteins monitored by asymmetric flow field-flow fractionation.

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.

Dielectrophoresis-field flow fractionation for separation of particles: A critical review.

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.

Asymmetric flow field-flow fractionation coupled to surface plasmon resonance detection for analysis of therapeutic proteins in blood serum.

Coupling of surface plasmon resonance (SPR) detection to asymmetric flow field-flow fractionation (AF4) offers the possibility to study active fractions of bio-separations on real samples, such as serum and saliva, including the assessment of activity of possibly aggregated species. The coupling of SPR with AF4 requires the possibility to select fractions from a fractogram and redirect them to the SPR. The combination of SPR with chromatography-like methods also requires a mechanism for regeneration of the receptor immobilised onto the SPR sensor surface. In recent work, the combination of size exclusion chromatography (SEC) with SPR was pioneered as a successful methodology for identification, characterisation and quantification of active biocomponents in biological samples. In this study, the approach using AF4 is evaluated for the antibody trastuzumab in buffer and serum. The particular object of this study was to test the feasibility of using AF4 in combination with SPR to detect and quantify proteins and aggregates in complex samples such as blood serum. Also, in the investigation, three different immobilisation methods for the receptor HER-2 were compared, which involved (1) direct binding via EDC/NHS, the standard approach; (2) immobilisation via NTA-Ni-Histag complexation; and (3) biotin/avidin-linked chemistry using a regenerable form of avidin. The highest specific activity was obtained for the biotin-avidin method, while the lowest specific activity was observed for the NTA-Ni-Histag linkage. The data show that AF4 can separate trastuzumab monomers and aggregates in blood serum and that SPR has the ability to selectively monitor the elution. This is an encouraging result for automated analysis of complex biological samples using AF4-SPR.

Perspectives on protein biopolymers: miniaturized flow field-flow fractionation-assisted characterization of a single-cysteine mutated phaseolin expressed in transplastomic tobacco plants.

The development of plant-based protein polymers to employ in biofilm production represents the promising intersection between material science and sustainability, and allows to obtain biodegradable materials that also possess excellent physicochemical properties. A possible candidate for protein biopolymer production is phaseolin, a storage protein highly abundant in P Vulgaris beans. We previously showed that transformed tobacco chloroplasts could be employed to express a mutated phaseolin carrying a signal peptide (directing it into the thylakoids) also enriched of a cysteine residue added to its C-terminal region. This modification allows for the formation of inter-chain disulfide bonds, as we previously demonstrated, and should promote polymerization. To verify the effect of the peptide modification and to quantify polymer formation, we employed hollow-fiber flow field-flow fractionation coupled to UV and multi-angle laser scattering detection (HF5-UV-MALS): HF5 allows for the selective size-based separation of phaseolin species, whereas MALS calculates molar mass and conformation state of each population. With the use of two different HF5 separation methods we first observed the native state of P.Vulgaris phaseolin, mainly assembled into trimers, and compared it to mutated phaseolin (P*) which instead resulted highly aggregated. Then we further characterized P* using a second separation method, discriminating between two and distinct high-molecular weight (HMW) species, one averaging 0.8 × 106 Da and the second reaching the tens of million Da. Insight on the conformation of these HMW species was offered from their conformation plots, which confirmed the positive impact of the Cys modification on polymerization.

Preparative field flow fractionation for complex environmental samples: online detection by inductively coupled plasma mass spectrometry and offline detection by gas chromatography with flame ionization.

Asymmetric flow field flow fractionation (AF4) in particular online with elemental detection via inductively coupled plasma mass spectrometry (ICP-MS) has been developed as powerful and flexible separation technique for suspensions of nano- and micro-particles covering a broad range of applications including environmental water samples and soil extracts. However, for challenging applications, such as particulate phosphorus determination in non-contaminated water samples at levels close to the limit of detection the throughput of the analytical field flow fractionation (FFF) is not sufficient. The same holds true for more specific identification and quantification of black carbon (BC) which needs a subsequent complex multi-step analysis using the well-established benzene polycarboxylic acids (BPCA) method. To overcome these limitations, the performance of a commercially available preparative AF4 channel, which has rarely been applied, yet, was investigated in this study. Using the example of an extract from charcoal spiked soil, method development for the preparative channel was performed and the results from six replicate fractionations with multi-element online detection by ICP-MS were compared to the results from the analytical channel for the same extracts. A similar fractionation pattern was achieved and the quantitative results agreed well for most of the particulate fractions (ratio 1.7 with standard deviation (SD) 0.2 for fraction 1, ratio 0.81 with SD 0.14 for fraction 2 and ratio 1.1 with SD 0.2 for fraction 3). Relative standard deviations were in the range of 9% to 18% for the preparative channel and between 3% and 17% for the analytical channel. Transferability of the separation parameters between both channels is discussed as well as the operational challenges of the preparative channel. As proof of principle, preparative fractionation of an extract from charcoal spiked soil was performed with fraction collection and subsequent quantification of BC via the BPCA method including derivatization, cation exchange pre-cleaning and finally gas chromatographic separation and quantification via flame ionization detection. The results indicated the majority of detected BC in the often so-called dissolved fraction was bound to nanoparticles (48%) and colloids (27%). Only 25% was detected in the cross flow (truly dissolved fraction). This successful example opens new possibilities for hyphenation of FFF separation with multiple detection techniques for improved characterization of particulate matter in challenging applications.

Fractionation and characterization of polyphenolic compounds and macromolecules in red wine by asymmetrical flow field-flow fractionation.

Red wine is a complex matrix containing macromolecules such as condensed tannins and polysaccharides. Wine macromolecular components and their interactions have been reported to impact taste properties such as astringency but the colloidal systems formed in wine are not well known. A key prerequisite to characterize these systems is the ability to work under analytical conditions as close as possible to the colloid environment, preserving the sample structure and limiting the denaturation of macromolecular complexes. A method of Asymmetric Flow Field-Flow Fractionation (AF4) coupled with UV detection, multi-angle light scattering (MALS), and differential refractometer index (dRI) (AF4-UV-MALS-dRI) has been developed to analyse macromolecules, including tannins and polysaccharides, and macromolecular complexes, in red wine. This method separates objects according to their hydrodynamic radius and does not require calibration to determine molecular weight (Mw). AF4 can provide native separation of wine colloidal matter while working with simulated wine as mobile phase. The channel was equipped with a 350-µm spacer and the membrane made in regenerated cellulose had a cut-off of 5kDa. Different parameters of crossflow rate were investigated using a generic red wine to optimize separation conditions. Then, purified fractions of polysaccharides and tannins were analysed using the selected AF4 parameters. The comparison of the peaks obtained for these fractions and for the wine sample allowed us to determine the retention time associated with these macromolecules. The AF4 fractogram of wine was divided into four fractions. The first three were assigned to higher Mw tannins coeluted with lower Mw polysaccharides such as rhamnogalacturonan II (F1), to intermediate Mw polysaccharides (F2), and to higher Mw mannoproteins (F3) whereas the last fraction (F4) was not identified. Furthermore, our results have shown that AF4-UV-MALS-dRI could be an efficient technique to separate large size tannins as well as polysaccharides and macromolecular complexes.

The presence of iron oxide nanoparticles in the food pigment E172.

Iron oxides used as food colorants are listed in the European Union with the number E172. However, there are no specifications concerning the fraction of nanoparticles in these pigments. Here, seven E172 products were thoroughly characterized. Samples of all colors were analyzed with a broad spectrum of methods to assess their physico-chemical properties. Small-Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), zeta-potential, Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), X-ray diffraction (XRD), Brunauer-Emmett-Teller analysis (BET), Asymmetric Flow Field-Flow Fractionation (AF4) and in vitro cell viability measurements were used. Nanoparticles were detected in all E172 samples by TEM or SAXS measurements. Quantitative results from both methods were comparable. Five pigments were evaluated by TEM, of which four had a size median below 100 nm, while SAXS showed a size median below 100 nm for six evaluated pigments. Therefore, consumers may be exposed to iron oxide nanoparticles through the consumption of food pigments.

Matrix-dependent size modifications of iron oxide nanoparticles (Ferumoxytol) spiked into rat blood cells and plasma: Characterisation with TEM, AF4-UV-MALS-ICP-MS/MS and spICP-MS.

Engineered nanoparticles such as iron oxide (Fe3O4) nanoparticles (IONPs) offer several benefits in nanomedicine, notably as contrast agents in magnetic resonance imaging (MRI). Ferumoxytol, a suspension of IONPs (with a manufacturer's reported particle diameter of 27 nm-30 nm) was characterized as a standard by spiking into rat blood plasma and cell fractions. Nanoparticle separation, and characterisation was investigated with asymmetric flow field-flow fractionation (AF4) coupled online to ultraviolet-visible spectroscopy (UV-VIS), multi-angle light scattering (MALS) and inductively coupled plasma mass spectrometry (ICP-MS) detectors; also with single particle inductively coupled plasma mass spectrometry (spICP-MS) and transmission electron microscopy (TEM). MALS signal of pristine Ferumoxytol indicated radii of gyration (Rg) between 15 and 28 nm for the Fe-containing fraction and 30-75 nm for the non-Fe fraction. IONPs spiked into blood plasma indicated a polydisperse distribution between 40 nm - 120 nm suggesting matrix-induced size alterations. Spiking of the IONPs into cells showed a shift in ICP-MS Fe signal to 15 min, however the MALS signal was undetected within the Fe containing fraction of the IONPs suggesting NP loss due to membrane-particle attraction. spICP-MS analysis of IONPs spiked in rat plasma suggested the release of Fe-containing colloids into plasma causing an increase in diameter of IONPs to 52 ±â€¯0.8 nm; whereas no major variation in particle size and distribution of the IONPs spiked in cell fractions was observed (33.2 ±â€¯2.0 nm) suggesting non-alteration of the NP Fe core. A complementary application of microscopic, light scattering, and mass spectrometry techniques for the characterisation of NPs in challenging biological matrices like blood has been demonstrated.

A New Label-Free Approach to Glioblastoma Cancer Stem Cell Sorting and Detection.

Cancer stem cells (CSCs) play critical roles in cancer, making them important targets for new diagnostic and therapeutic approaches. Since CSCs are heterogeneous and not abundant in tumors, and few specific markers for these cells currently exist, new methods to isolate and characterize them are required. To address this issue, we developed a new label-free methodology to isolate, enrich, and identify CSCs from an heterogeneous tumor cell subpopulation using a cell sorting method (sedimentation field flow fractionation, SdFFF) and a biosensor as a detector. Enrichment was optimized using an original protocol and U87-MG glioblastoma cells cultured in a normal (N) or defined (D) medium (± fetal bovine serum, FBS) under normoxic (N, pO2 = 20%) or hypoxic (H, pO2 < 2%) conditions to obtain four cell populations: NN, NH, DN, and DH. After elution of CSCs via SdFFF using the hyperlayer mode (inertial elution mode for micrometer-sized species), we isolated eight subpopulations with distinct CSC contents based on phenotypical and functional properties, ranging from NN F1 with a lower CSC content to DH F3 with a higher CSC content. Reflecting biological differences, the intrinsic intracellular dielectric permittivity increased from NN to DH conditions. The largest difference in electromagnetic signature was observed between NN F1 and DH F3, in which the CSC content was lowest and highest, respectively. The results demonstrate that microwave dielectric spectroscopy can be used to reliably and efficiently distinguish stem cell characteristics. This new instrumental and methodological approach is an important innovation that allows both enrichment and detection of CSCs, opening the door to novel diagnostic and therapeutic approaches.