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Epidemiological evidence has indicated a closely link between PM0.1 exposure and the incidence rate of cardiovascular diseases. This study explores the underlying communication roles of platelet-derived extracellular vesicles (PEVs) heterogeneous subpopulations in cardiovascular injury. PEVs and PMEVs which were extracted from platelet-rich plasma (PRP) un-exposure or exposure to PM0.1 by TIM4 affinity beads. By optimizing separation conditions, replacing pipelines, and resetting injection procedures, Asymmetric flow field-flow fractionation (AF4) was employed to separate, purify, characterize, and enrich PEVs and PMEVs heterogeneous subpopulations (small PEVs, PEVs-S/PMEVs-S: <100 nm; medium PEVs, PEVs-M/PMEVs-M: 100-200 nm; and large PEVs, PEVs-L/PMEVs-L: >200 nm). The results showed that the cargoes of PMEVs heterogeneous subpopulations which were released by PRP stimulated by PM0.1 were changed obviously. Moreover, compared with PEVs, PMEVs can lead to a decrease in the survival rate of Human Umbilical Vein Endothelial Cells (HUVECs). In PMEVs-S subpopulations, the alterations of lipids associated with membrane fusion and cell signaling transport (such as PC, Cer), as well as miRNAs related to inflammation, angiogenesis, and migration (miR-223, miR-22, miR-126, and miR-150), are similar to those in PMEVs-M subpopulations but distinct from PMEVs-L subpopulations. This study revealed the diverse communication mechanisms underlying PM0.1-induced cardiovascular injury, thereby offering potential avenues for the development of new biomarkers and therapeutic targets.

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Field-flow fractionation (FFF) is a family of single-phase separative techniques exploited to gently separate and characterize nano- and microsystems in suspension. These techniques cover an extremely wide dynamic range and are able to separate analytes in an interval between a few nm to 100 µm size-wise (over 15 orders of magnitude mass-wise). They are flexible in terms of mobile phase and can separate the analytes in native conditions, preserving their original structures/properties as much as possible. Molecular biology is the branch of biology that studies the molecular basis of biological activity, while biotechnology deals with the technological applications of biology. The areas where biotechnologies are required include industrial, agri-food, environmental, and pharmaceutical. Many species of biological interest belong to the operational range of FFF techniques, and their application to the analysis of such samples has steadily grown in the last 30 years. This work aims to summarize the main features, milestones, and results provided by the application of FFF in the field of molecular biology and biotechnology, with a focus on the years from 2000 to 2022. After a theoretical background overview of FFF and its methodologies, the results are reported based on the nature of the samples analyzed.

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Asymmetrical flow field-flow fractionation (AF4), a gentle tool for the separation and characterization of particles and macromolecules, has attracted increased interest in recent years owing to its broad dynamic size range and utilization of "open channel" voids in the packing or stationary phase. A steric transition phenomenon in which the sample elution mode change from the normal mode to the steric/hyperlayer mode occurs. Accurate characterization by AF4 requires the absence of steric transition, particularly when the sample has a broad size distribution, because the effect of the combination of different modes is difficult to interpret. In this study, the relative molecular mass (M), radius of gyration (Rg), and conformation of Gastrodia elata polysaccharides (GEPs) were characterized using AF4 coupled with online multi-angle light scattering (MALS) and differential refractive index (dRI) detection (AF4-MALS-dRI). Steric transition was observed during GEP separation by AF4 owing to the broad size distribution of the molecules. This phenomenon would result in the inaccurate characterization of the GEPs in terms of M and Rg because two GEP groups of different sizes may elute together. In this study, the effects of constant and exponentially decaying cross-flow rates, sample mass concentration, and spacer thickness on steric transition were systematically investigated. The results indicated that a high GEP mass concentration (i. e., 0.75 mg/mL) can lead to steric transition. The spacer thickness affected the resolution and retention time of the GEPs and changed the steric transition point (di). An exponentially decaying cross-flow rate not only adjusted the di of the polydisperse GEP samples but also improved the GEP resolution and shortened the analysis time. The influence of steric transition was solved under the following operating conditions: injected GEP mass concentration=0.5 mg/mL; injection volume=50 µL; spacer thickness=350 µm; detector flow rate=1.0 mL/min; and cross-flow rate exponentially decayed from 0.2 to 0.05 mL/min with a half-life of 2 min. Moreover, the influence of GEP origins and ultrasound treatment time on the M and Rg distributions and conformation of GEPs were investigated under the optimized operating conditions. The results showed that the M and Rg distributions of Yunnan and Sichuan GEPs decreased with increasing ultrasound time. When the ultrasound treatment time was 15 min, the Yunnan GEPs had a loosely hyperbranched chain conformation, whereas the Sichuan GEPs had a spherical conformation. When the ultrasound treatment time was increased to 30 or 60 min, the GEPs from both Yunnan and Sichuan had a hyperbranched chain conformation, indicating that ultrasound treatment resulted in GEP degradation. Under the same extraction conditions, GEPs from Yunnan had larger M and Rg values than those from Sichuan. AF4-MALS-dRI showed good repeatability for the characterization of GEPs under the optimized operating conditions. The relative standard deviations of Rg and M were 0.5% and 1.7%, respectively. The data presented in this study can be used as a starting point for in-depth studies on the structural bioactivity of GEPs.

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Nanogels are candidates for biomedical applications, and core-shell nanogels offer the potential to tune thermoresponsive behaviour with the capacity for extensive degradation. These properties were achieved by the combination of a core of poly(N-isopropylmethacrylamide) and a shell of poly(N-isopropylacrylamide), both crosslinked with the degradable crosslinker N,N'-bis(acryloyl)cystamine. In this work, the degradation behaviour of these nanogels was characterised using asymmetric flow field flow fractionation coupled with multi-angle and dynamic light scattering. By monitoring the degradation products of the nanogels in real-time, it was possible to identify three distinct stages of degradation: nanogel swelling, nanogel fragmentation, and nanogel fragment degradation. The results indicate that the core-shell nanogels degrade slower than their non-core-shell counterparts, possibly due to a higher degree of self-crosslinking reactions occurring in the shell. The majority of the degradation products had molecule weights below 10 kDa, which suggests that they may be cleared through the kidneys. This study provides important insights into the design and characterisation of degradable nanogels for biomedical applications, highlighting the need for accurate characterisation techniques to measure the potential biological impact of nanogel degradation products.

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High molar mass polyethylene oxide (HM-PEO) is commonly used to enhance the mechanical strength of solid oral opioid drug products to deter abuse. Because the properties of PEO depend on molar mass distribution, accurately determining the molar mass distribution is a necessary part of understanding PEO's role in abuse-deterrent formulations (ADF). In this study, an asymmetrical flow field-flow fractionation (AF4) analytical procedure was developed to characterize PEO polymers with nominal molar masses of 1, 4 or 7 MDa as well as those from in-house prepared placebo ADF. The placebo ADF were manufactured using direct compress or hot-melt-extrusion methods, and subjected to physical manipulation, such as heating and grinding before measurement by AF4 were performed. The molar mass distribution characterized by AF4 revealed that PEO was sensitive to thermal stress, exhibiting decreased molar mass with increased heat exposure. The optimized AF4 method was deemed suitable for characterizing HM-PEO, offering adequate dynamic separation range for PEO with molar mass from 100 kDa to approximately 10 MDa.

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Asymmetric-flow field-flow fractionation (AF4) is a gentle, flexible, and powerful separation technique that is widely utilized for fractionating nanometer-sized analytes, which extend to many emerging nanocarriers for drug delivery, including lipid-, virus-, and polymer-based nanoparticles. To ascertain quality attributes and suitability of these nanostructures as drug delivery systems, including particle size distributions, shape, morphology, composition, and stability, it is imperative that comprehensive analytical tools be used to characterize the native properties of these nanoparticles. The capacity for AF4 to be readily coupled to multiple online detectors (MD-AF4) or non-destructively fractionated and analyzed offline make this technique broadly compatible with a multitude of characterization strategies, which can provide insight on size, mass, shape, dispersity, and many other critical quality attributes. This review will critically investigate MD-AF4 reports for characterizing nanoparticles in drug delivery, especially those reported in the last 10-15 years that characterize multiple attributes simultaneously downstream from fractionation.

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Immunoaffinity chromatography (IAC) with selective antibodies immobilized on polymeric monolithic disk columns enables selective isolation of biomacromolecules from human plasma, while asymmetrical flow field-flow fractionation (AsFlFFF or AF4) can be used for further fractionation of relevant subpopulations of biomacromolecules (e.g., small dense low-density lipoproteins, exomeres, and exosomes) from the isolates. Here we describe how the isolation and fractionation of subpopulations of extracellular vesicles can be achieved without the presence of lipoproteins using on-line coupled IAC-AsFlFFF. With the developed methodology, it is possible to have fast, reliable, and reproducible automated isolation and fractionation of challenging biomacromolecules from human plasma with a high purity and high yields of subpopulations.

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Carbon black (CB) particles tend to aggregate in aqueous solutions, and finding an optimum dispersing condition (e.g., selection of the type of dispersant) is one of the important tasks in related industries. In the present study, three types of styrene maleic acid (SMA) copolymer dispersants were synthesized, labeled respectively 'SMA-1000', 'SMA-2000', and 'SMA-3000', which have 1, 2, and 3 styrene groups in their repeating units. Then, asymmetrical flow field-flow fractionation (AsFlFFF) was employed to measure the particle size distributions of the aqueous CB dispersions. For the particle size analysis of the CB dispersions, dynamic light scattering (DLS) showed relatively lower reproducibility than AsFlFFF. AsFlFFF showed that the use of SMA-3000 yielded a CB dispersion with the most uniform particle size distribution. When the SMA-3000 dispersant was used, the particle size tended to increase after 1 h of milling as the milling time increased, probably due to the re-agglomeration of the particles by excessive milling. The particle size distributions from AsFlFFF were consistent with the colorimetric observations. With the SMA-3000 dispersant, the lowest L∗ value was observed after 1 h of milling. The AsFlFFF and colorimetric analyses suggest that a stable CB dispersion can be obtained by either 3-h of milling with the SMA-2000 or 1-h of milling with the SMA-3000.

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Physiochemical degradation of therapeutic proteins in vivo during plasma circulation after administration can have a detrimental effect on their efficacy and safety profile. During drug product development, in vivo animal studies are necessary to explore in vivo protein behaviour. However, these studies are very demanding and expensive, and the industry is working to decrease the number of in vivo studies. Consequently, there is considerable interest in the development of methods to pre-screen the behaviour of therapeutic proteins in vivo using in vitro analysis. In this work, asymmetrical flow field-flow fractionation (AF4) and liquid chromatography-mass spectrometry (LC-MS) were combined to develop a novel analytical methodology for predicting the behaviour of therapeutic proteins in vivo. The method was tested with two proteins, a monoclonal antibody and a serum albumin binding affibody. After incubation of the proteins in plasma, the method was successfully used to investigate and quantify serum albumin binding, analyse changes in monoclonal antibody size, and identify and quantify monoclonal antibody aggregates.

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Infections by Streptococcus pneumoniae can cause serious pneumococcal diseases and other medical complications among patients. Polysaccharide-based vaccines have been successfully developed as prophylactic agents against such deadly bacterial infections. In the 1980s, PNEUMOVAX® 23 were introduced as the first pneumococcal polysaccharide vaccines (PPSV). Later, pneumococcal polysaccharides were conjugated to a carrier protein to improve immune responses. Pneumococcal conjugate vaccines (PCV) such as PREVNAR® and VAXNEUVANCE™ have been developed. Of the more than 90 pneumococcal bacteria serotypes, serotype 1 (ST-1) and serotype 4 (ST-4) are the two main types that cause invasive pneumococcal diseases (IPD) that could lead to morbidity and mortality. Development of a novel multi-valent PCV against these serotypes requires extensive biophysical and biochemical characterizations of each monovalent conjugate (MVC) in the vaccine. To understand and characterize these high molecular weight (Mw) polysaccharide protein conjugates, we employed the multi-angle light scattering (MALS) technique coupled with size-exclusion chromatography (SEC) separation and asymmetrical flow field flow fractionation (AF4). MALS analysis of MVCs from the two orthogonal separation mechanisms helps shed light on the heterogeneity in conformation and aggregation states of each conjugate.

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Asymmetrical flow field-flow fractionation (AF4) is a separation method based on hydrodynamic size of the sample components. It can separate a broad size range of components (~103 to 109 Da; particle diameter from ~1 nm to ~1 µm), but is especially well suited for high molecular weight samples such as virus-sized particles and extracellular vesicles. Separation takes place in an open channel where the flows control sample elution. Separation does not involve stationary phase, allowing gentle separation and good recoveries. The method is compatible with a wide variety of buffers. Coupling to various analytical detectors enables rapid assays on the molecular weight and size and their distribution, degradation, and aggregation of the sample components giving information on the sample quality. In addition to being an advanced analytical method, AF4 can be used in a semipreparative mode for purification. Here, we summarize archaeal virus production methods and virus purification by AF4 and provide examples on the steps that need optimization for obtaining good separation with the focus on halophilic archaeal viruses. Importantly, AF4 method is suitable for a variety of viruses and extracellular vesicles regardless of their host organism.

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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.

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Asymmetrical flow field-flow fractionation (AF4) is a powerful technique employed for the separation of macromolecules, nanoparticles, and their assemblages according to their hydrodynamic behavior. It is well known that at this size range, complex interactions can occur between components (e.g. surface adsorption, aggregation) controlling the fate of trace metals (TMs) bound to them. AF4 coupling to inductively coupled plasma mass spectrometry (ICP-MS) allows the quantification of metal-containing species at trace levels present in environmental and biological systems on a size-composition basis. The combination of AF4-ICP-MS with other online detectors provides additional information that allows the assessment of the origin of analytes present in mixtures and complex matrixes with minimal sample preparation, which is crucial for understanding the behavior of trace metal contaminants. Despite the increasing use of AF4-ICP-MS in environmental contexts, we acknowledge that the quantification of inorganic species using such combined techniques requires further development of standardized procedures and need certified reference materials. In this review, we also discuss critical endpoints within the ICP-MS instrument coupled to AF4 that need to be controlled before quantitative measurements can be validated. Then, we illustrate how the combination of different online detectors in addition to ICP-MS offers an integrated picture of natural components states, thus providing key information on the changes in behavior of trace metal species and metallic nanoparticles (MNPs) as observed in both environmental samples and biofluids.

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In this study, the starches were isolated from three botanical sources (i.e., rice, sweet potato, and lotus seed). The size distributions of starch granules and molecules were determined by asymmetrical flow field-flow fractionation (AF4), and compared with those measured from optical microscopy (OM) and dynamic light scattering (DLS). Furthermore, the starches were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). AF4 coupled online with UV-visible, multiangle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI) was employed for the investigation of the digestion and retrogradation properties of starches. Meanwhile, the relationships between the size of starch at nano- to microscale and its functional properties (i.e., digestibility, retrogradation, and thermal properties) were studied by Pearson correlation analysis. AF4-UV-MALS-dRI was proved to be a rapid and gentle method for the separation and size characterization of starches at both micro- and nano-molecule levels. Moreover, it was demonstrated that AF4-UV-MALS-dRI is a useful tool for the monitoring of the digestion and retrogradation properties of starches. The results suggested that the sizes of starch granules and molecules were to some extent correlated with their thermal properties and digestibility, but not with retrogradation property.

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Starch occurs naturally in the form of semicrystalline granules, and is composed of two types of carbohydrate molecules, amylose (AM) and amylopectin (AP). Starch granules and starch molecules have sizes in the range of 1-100 µm and 20-250 nm, respectively; these size ranges are among the key factors affecting the functional properties of starch. Asymmetrical flow field-flow fractionation (AF4) is a size-based separation technique. The major difference between AF4 and dynamic light scattering or microscopy techniques is that AF4 enables the separation of particles based on their size; consequently, the elution profile can be converted to the size distribution of the samples. In the last two decades, AF4 systems, when coupled online with multiangle light scattering (MALS) and differential refractive index (dRI) detectors (AF4-MALS-dRI), have demonstrated to be applicable for the size characterization of starch at the molecular level. Unlike size exclusion chromatography (SEC), AF4 systems use an open channel that does not require a stationary phase or packing materials. Thus, the shear scission of AP molecules during AF4 separation is minimized. The size detection range of a commercial AF4 system ranges from 1 nm to 10 µm, which is smaller than the size range of starch granules. In this study, a home-made AF4 system was developed, and its capability for the size characterization of starch granules extracted from sweet potato, lotus seed, and rice was investigated. The performance of the developed AF4 system was evaluated by running a mixture of polystyrene (PS) with diameter of 2, 6, 12, and 20 µm, respectively. Baseline separation of four PS samples was achieved, and the resolution for 6 µm PS and 12 µm PS was 1.40. The detection limit of the developed AF4 system was higher than that of commercial AF4 systems. Thus, the developed AF4 system is promising for the separation and characterization of starch granules. The effect of the composition of the carrier liquid on the AF4 separation of starch granules was also studied. Moreover, the accuracy of AF4 in terms of size characterization of the starch granules was evaluated by optical microscopy (OM). The results revealed that the type of dispersant and viscosity of the carrier liquid affect the accuracy of size characterization of the starch granules. The size distribution of rice starch granules obtained using a carrier liquid containing 0.01% (w/v) sodium dodecyl sulfate (used as a dispersant), 0.02% (w/v) NaN3 (used as a bactericide), and 0.001% (w/v) hydroxypropylmethylcellulose (used to adjust the viscosity of the carrier liquid) was in agreement with that obtained from OM. Furthermore, a commercial AF4 system coupled with MALS and dRI detectors was employed for the separation and characterization of starch molecules. A molecularly dispersed solution is necessary for the reliable molecular characterization of starch. The effect of the starch dissolution temperature on the AF4 characterization of starch was also investigated. The optimal dissolution temperature for lotus seed and rice starch granules was 75 ℃, while that for sweet potato starch granules was 78 ℃; this difference is mainly attributed to the different botanical origins of the granules. The results showed that the ratio of the radius of gyration (Rg) to the hydrodynamic radius (Rh) of rice starch and sweet potato starch is in the range of 0.9-1.1 over the molar mass range of 10 6-108 g/mol. For rice starch, the Rg/Rh ratio is between 1.2 and 1.4. Rice starch has the highest apparent density among the three starches, indicating that rice starch molecules have a dense structure. The results demonstrated that the AF4 system developed in this study is rapid and accurate for the size characterization of starch granules. The developed AF4 system, when combined with commercial AF4 systems coupled online with MALS and dRI detectors, can provide technical support to study the relationship between the size from the nanoscale to the microscale and functional properties of starch.

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Liposome size and in vitro release of the active substance belong to critical quality attributes of liposomal carriers. Here, we apply asymmetric flow field-flow fractionation (AF4) to characterize theranostic liposomes prepared by thin lipid film hydration/extrusion or microfluidics. The vesicles' size was derived from multi-angle laser light scattering following fractionation (AF4) and compared to sizes derived from dynamic light scattering measurements. Additionally, we adapted a previously developed AF4 method to study zinc phthalocyanine (ZnPc) release/transfer from theranostic liposomes. To this end, theranostic liposomes were incubated with large acceptor liposomes serving as a sink (mimicking biological sinks) and were subsequently separated by AF4. During incubation, ZnPc was transferred from donor to acceptor fraction until reaching equilibrium. The process followed first-order kinetics with half-lives between 119.5-277.3 min, depending on the formulation. The release mechanism was postulated to represent a combination of Fickian diffusion and liposome relaxation. The rate constant of the transfer was proportional to the liposome size and inversely proportional to the ZnPc/POPC molar ratio. Our results confirm the usefulness of AF4 based method to study in vitro release/transfer of lipophilic payload, which may be useful to estimate the unwanted loss of drug from the liposomal carrier in vivo.

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To better understand the structure-function relationship of Gastrodia elata polysaccharides (PGEs), PGEs were extracted by ultrasound-assisted extraction method and the effects of extraction time on the structure and conformation of PGEs were evaluated by asymmetrical flow field-flow fractionation (AF4) coupled online with multiangle light scattering (MALS) and differential refractive index (dRI) detectors (AF4-MALS-dRI). Besides separation, AF4-MALS-dRI can provide more information about PGEs, such as size and molecular weight (Mw) distributions, apparent density, and conformation. The effects of PGEs on the proliferation, apoptosis, and cell cycle of MCF-7 cells were investigated. The cell activity assay indicated that the PGEs can inhibit the growth of MCF-7 cells by inducing late apoptosis. The results indicated that PGEs with a spherical conformation and compact structure seem to be beneficial to inducing MCF-7 cells late apoptosis. Moreover, results demonstrated that the information obtained by AF4-MALS-dRI is valuable for better understanding of the relationship of structure-activity of PGEs.

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1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is widely used as a crosslinker for fluorescence labeling of protein in the fields of biochemistry and food analysis. Many natural polysaccharides often contain some proteins or peptides that are very low in content but play a vital role in their biological function as well as technical applications. Determination of these low-content proteinaceous matters requires a highly sensitive and selective method. In this study, a methodological approach for investigations of the presence of proteinaceous material over the molar mass distribution (MD) of polysaccharides was developed using gum acacia (GA) as a model polysaccharide. EDC fluorescence-labeling method was modified by changing the pH (7, 9, and 11) of the solution for the analysis of low-content protein in food materials. Fluorescence spectroscopy and asymmetrical flow field-flow fractionation (AF4) were employed for characterizing the labeling efficiency and physiochemical properties of unlabeled and fluorescence-labeled GA. AF4 provided molar mass (M) and the radius of gyration (rG) of arabinogalactan (AG) and arabinogalactan protein complex (AGP) and determined the presence of proteinaceous matter over the MD. The labeling efficiencies of GA at pH 7, 9, and 11 determined by fluorescence spectroscopy were 56.5, 68.4, and 72.0%, respectively, with an increment of 15.5% when pH was increased from 7 to 11. The modified EDC fluorescence-labeling method allows highly sensitive and selective analysis of low-content proteinaceous matters and their distribution in natural polysaccharides.

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Accurate determinations of particle size and particle size distribution (PSD) are essential to achieve the clinical translation of medical nanoparticles (NPs). Herein, dextran-based NPs produced via a water-in-oil emulsification/crosslinking process and developed as nanomedicines were studied. NPs were first characterized using traditional batch-mode techniques as dynamic light scattering (DLS) and laser diffraction. In a second step, their analysis by frit-inlet asymmetrical flow field-flow fractionation (FI-AF4) was explored. The major parameters of the AF4 procedure, namely, crossflow, detector flow, crossflow decay programming and relaxation time were set up. The sizes of the particle fractions eluted under optimized conditions were measured using DLS as an online detector. We demonstrate that FI-AF4 is a powerful method to characterize dextran-NPs in the 200 nm -1 µm range. It provided a more realistic and comprehensive picture of PSD, revealing its heterogenous character and clearly showing the ratio of different populations in the sample, while batch-mode light scattering techniques only detected the biggest particle sizes.

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Previously, a liposomal formulation of a chemotherapeutic agent melphalan (Mlph) incorporated in a fluid lipid bilayer of natural phospholipids in the form of dioleoylglyceride ester (MlphDG) was developed and the antitumor effect was confirmed in mouse models. The formulation composed of egg phosphatidylcholine (ePC), soybean phosphatidylinositol (PI), and MlphDG (8:1:1, by mol) showed stability in human serum for at least 4-5 h. On the contrary, replacing PI with pegylation of the liposomes, promoted fast dissociation of the components from the bilayer. In this work, interactions of MlphDG-liposomes with the most abundant plasma protein-albumin-in function of the presence of PI in the formulation were explored using Fourier transform infrared spectroscopy. The release of MlphDG from the liposomes was studied by asymmetrical flow field-flow fractionation (AF4) using micelles formed by a polyethylene glycol conjugate with phosphatidylethanolamine to mimic the physiological lipid sink like lipoproteins. Our results show that PI actually protects the membrane of MlphDG-liposomes from the protein penetration, presumably due to pairing between the positively charged MlphDG and negatively charged PI, which compensates for the heterogeneity of the lipid bilayer. The AF4 technique also evidences high stability of the formulation as a drug carrier.