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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.

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.

Trehalose is widely assumed to be the most effective sugar for protein stabilization, but exactly how unique the structure is and the mechanism by which it works are still debated. Herein, we use a polyion complex micelle approach to control the position of trehalose relative to the surface of glucose oxidase within cross-linked and non-cross-linked single-enzyme nanoparticles (SENs). The distribution and density of trehalose molecules in the shell can be tuned by changing the structure of the underlying polymer, poly(N-[3-(dimethylamino)propyl] acrylamide (PDMAPA). SENs in which the trehalose is replaced with sucrose and acrylamide are prepared as well for comparison. Isothermal titration calorimetry, dynamic light scattering, and asymmetric flow field-flow fraction in combination with multiangle light scattering reveal that two to six polymers bind to the enzyme. Binding either trehalose or sucrose close to the enzyme surface has very little effect on the thermal stability of the enzyme. By contrast, encapsulation of the enzyme within a cross-linked polymer shell significantly enhances its thermal stability and increases the unfolding temperature from 70.3 °C to 84.8 °C. Further improvements (up to 92.8 °C) can be seen when trehalose is built into this shell. Our data indicate that the structural confinement of the enzyme is a far more important driver in its thermal stability than the location of any sugar.

Lignin is the second most abundant biopolymer on earth and accrues in large amounts in the pulp- and paper industry. While currently lignins are mainly used for low-value applications such as energy production, recently the potential of laccases for upgrading lignins has been demonstrated. In this study, two laccases of fungal and bacterial origin were characterized regarding their potential to polymerize lignosulfonate. The laccases MaL1 from Melanocarpus albomyces, and SrLA from Streptomyces rochei were heterologously expressed and showed typical characteristics of laccases, like acidic pH optima for ABTS at pH 4 and 5, respectively, and temperature optima at 50 °C and 80 °C. Polymerization of lignosulfonate with MaL1 led to an almost two-fold increase of the molecular weight, according to size exclusion (SEC) multiangle laser light scattering (MALLS) analysis. In contrast, SrLA showed considerably less activity on lignosulfonates, as measured based on oxygen consumption and SEC-MALLS.

In this study, the effect of external treatment conditions [i.e., heat treatment conditions, storage conditions, pH, and high-intensity ultrasound (HIU)] on the stability of low-density lipoprotein (LDL) in egg yolk plasma modified by phospholipase A2 (PLA2) was studied. Asymmetrical flow field-flow fractionation (AF4) coupled with ultraviolet–visible (UV/Vis), multiangle light scattering (MALS) and differential refractive index (dRI) detectors was employed for the analysis of LDL modified with PLA2. The AF4 results show that PLA2 modification enhanced the stability of LDL in terms of heat treatment and −18 °C storage. However, the extent of aggregation of PLA2-modified egg yolk plasma with HIU treatment (400 W for 10 min) was larger than that of raw egg yolk plasma, and the stability decreased. Alkaline condition (i.e., pH 10) promoted the aggregation of egg yolk plasma regardless of modification with PLA2. The increased knowledge of how external factors studied in this work affect the stability of PLA2-modified LDL could increase the understanding and controlling of properties of the food made of the egg yolk.

Even the most advanced protein-polymer conjugate therapeutics do not eliminate antibody-protein and receptor-protein recognition. Next-generation bioconjugate drugs will need to replace stochastic selection with rational design to select desirable levels of protein-protein interaction while retaining function. The "Holy Grail" for rational design would be to generate functional enzymes that are fully catalytic with small molecule substrates while eliminating interaction between the protein surface and larger molecules. Using chymotrypsin, an important enzyme that is used to treat pancreatic insufficiency, we have designed a series of molecular chimeras with varied grafting densities and shapes. Guided by molecular dynamic simulations and next-generation molecular chimera characterization with asymmetric flow field-flow fractionation chromatography, we grew linear, branched, and comb-shaped architectures from the surface of the protein by atom-transfer radical polymerization. Comb-shaped polymers, grafted from the surface of chymotrypsin, completely prevented enzyme inhibition with protein inhibitors without sacrificing the ability of the enzyme to catalyze the hydrolysis of a peptide substrate. Asymmetric flow field-flow fractionation coupled with multiangle laser light scattering including dynamic light scattering showed that nanoarmor designed with comb-shaped polymers was particularly compact and spherical. The polymer structure significantly increased protein stability and reduced protein-protein interactions. Atomistic molecular dynamic simulations predicted that a dense nanoarmor with long-armed comb-shaped polymer would act as an almost perfect molecular sieve to filter large ligands from substrates. Surprisingly, a conjugate that was composed of 99% polymer was needed before the elimination of protein-protein interactions.

α-Synuclein (αSyn) is an intrinsically disordered protein that forms oligomers and fibrils associated with Parkinson’s disease. As such, the mechanism of its oligomerization and its possible links to neurotoxicity have been the focus of many studies. Out of the numerous oligomer types, dimers are the smallest oligomers of aSyn that have been reported. As such, αSyn dimers serve as the earliest steps in the nucleation of αSyn oligomers and later fibrils. Therefore, it is important to characterize αSyn dimers. The identification of αSyn dimers in ensemble-averaged measurements without the use of chemical modifications have been difficult, due to their apparent low abundance. Using analytical anion exchange chromatography coupled to multi angle light scattering as well as to dynamic light scattering, we show that recombinant αSyn is in equilibrium between different types of monomers and compact dimers, and that both are abundant. Additionally, bulk Forster resonance energy transfer (FRET), fluorescence cross-correlation spectroscopy (FCCS) of FRET and pulsed-interleaved excitation single-molecule FRET (PIE smFRET) measurements of mixtures of donor- and acceptor-labeled αSyn. These measurements indicated a dimer dissociation constant of 1.75 μM. We concluded that αSyn dimers exist as abundant species in equilibrium with monomers only if produced to reach concentrations of hundreds of nanomolar or above.

In this study, response surface methodology was employed to optimize the ultrasound-assisted extraction (UAE) process of perilla seed meal polysaccharides (PSMP). The optimal conditions for UAE of PSMP were: liquid-solid ratio of 26.00 mL/g, ultrasonic temperature of 43.00 °C, ultrasonic time of 52.00 min, and ultrasonic power of 229.00 W, the optimal conditions lead to an yield of 6.137 ± 0.062%. The structural characteristics of molecular weight, compositional monosaccharides, and glycosidic linkages were determined by size exclusion chromatography with multiangle light scattering, gas chromatography-mass spectrometry, Fourier-transfer infrared spectroscopy, and nuclear magnetic resonance detections. Scanning electron microscopy analysis showed that many holes were formed on the surface of PSM after UAE. The antioxidant activities of PSMP were investigated using various assays in vitro. The results suggested that PSMP is potential natural resource of antioxidants for medicine and functional foods. PRACTICAL APPLICATION: The selection of raw material perilla seed meal is conducive to the comprehensive utilization of edible resources. With consumer demands for newly developed foods with natural, wholesome ingredients are increasing nowadays. This study provides effective reference for in-depth research on other medicine-food dual-use resources. Ultrasound-assisted extraction (UAE) is a promising alternative method for hot water reflux extraction (HWRE) of polysaccharides for advantages of high efficiency and energy saving. In this work, the UAE process optimized by response surface methodology is more suitable for industrial application that can effectively decrease total cost of production by reducing the extraction temperature, shortening extraction time, and increasing raw material utilization.

To accurately understand the biological pollution level and toxicity of polydisperse nanoplastics, an effective solution is presented to separate polydisperse nanoplastics and detect their size, mass and number concentration in a biological matrix by asymmetrical flow field fractionation coupled with a diode array detector and a multiangle light scattering detector.

Interactions between histones and their binding partners are an important aspect of chromatin biology. Determining the stoichiometry of histone-containing complexes is an important pre-requisite for performing in vitro biochemical, biophysical and structural analyses. In this article, we detail how Size Exclusion Chromatography (SEC) coupled to Multi-Angle Light Scattering (MALS) can be used to study histone chaperones and their complexes. Our protocol details system setup, sample preparation, data collection, and data interpretation. We provide tips on designing an informative SEC-MALS experiment, using histone chaperones Nap1 and Vps75 as demonstrative examples. We outline recommendations to overcome specific challenges such as protein oligomerization, heterogeneity, and non-specific binding. We find SEC-MALS to be a robust and user-friendly approach for characterizing histone-binding proteins and their complexes.

We generated rod-like micelles of uniform length by living crystallization-driven self-assembly of a polyferrocenylsilane (PFS) block copolymer PFS26-b-POEGMA163 in a methanol-ethanol mixture and then transferred these micelles to water. The corona chains consisted of poly(oligoethyleneglycol methacrylate) that had a lower critical solution temperature (LCST) of 40.5 °C in water. We used a combination of static (SLS) and dynamic (DLS) multiangle light scattering to determine the dimensions of these cylindrical brush micelles in solution. Measurements carried out in dilute solution in water over a series of temperatures from 23 to 50 °C showed that the collapse transition was broad and continuous, both upon heating and cooling. This response is different from the collapse transition of POEGMA163 homopolymer in water, which occurs over a very narrow temperature range. Thus we show that the collapse transition of a cylindrical brush has important features in common with the collapse of a brush of thermoresponsive polymers on a planar surface.

Understanding the basic elements of the airway mucosal surfaces and how they form a functional barrier is essential in understanding disease initiation, progression, pathogenesis and ultimately treating chronic lung diseases. Using primary airway epithelial cell cultures, atomic force microscopy (AFM), multiangle light scattering and quartz crystal micro balance with dissipation monitoring techniques, here we report that the membrane bound mucins (MBMs) found in the periciliary layer (PCL) of the airway surface are densely decorated with keratan sulfate (KS). AFM and immunoblotting show that the KS sidechains can be removed enzymatically with keratanase II (KII) treatment, and the antibody accessibility for B2729 (MUC1), MUCH4 (MUC4) and OC125 (MUC16) was substantially enhanced. Light scattering analysis confirmed that KII treatment removed ~40% of the mass from the mucin fractions. Surface binding experiments indicated that MBMs were able to pack into a tighter conformation following KS removal, suggesting that negatively charged KS sidechains play a role in mucin–mucin repulsion and contribute to “space filling” in the PCL. We also observed that soluble filtrate from the common airway pathogen Pseudomonas aeruginosa is capable of stripping KS from MBMs. Altogether, our findings indicate that KS glycosylation of MBMs may play an important role in the integrity of the airway mucosal barrier and its compromise in disease.

納豆のネバネバの主因であるポリグルタミン酸(PGA)は、枯草菌の一種である納豆菌が分泌生産する高分子です。これまで、PGA生合成に必須なタンパク質は、PGA合成関連pgsオペロンから翻訳されるPgsB、PgsC、およびPgsAと報告されてきました。本研究では、pgsオペロンにおいてpgsAの下流に位置するpgsE遺伝子に着目し、PgsEタンパク質がPGAの生合成に与える影響を動的粘度法や多角度光散乱検出器による分子量解析を通して調べました。その結果、PgsB、PgsC、およびPgsAの3つのタンパク質で生産されたPGAは、ほとんど粘性を示さず、47,000の分子量でした。一方、PgsB、PgsC、PgsA、およびPgsEの4つのタンパク質で生産されたPGAは高粘性を示し、その分子量も2,900,000であることが判明しました。以上の結果より、PgsEは、PGAの分子量を大幅に向上させ、納豆をネバネバにさせる主因である可能性が示唆されました。, Poly(γ-glutamic acid) (PGA) is a biopolymer produced by Bacillus spp. via the γ-amide linkages of D- and/or L-glutamate. Although high-molecular-weight (HMW) PGA possesses many attractive properties, such as flocculating, wound healing, and immune-stimulating effects, no studies have reported factors useful for increasing the molecular weight of PGA during microbial production. PgsB, PgsC, and PgsA are the minimum protein sets required for PGA production in B. subtilis, and PgsE improves PGA productivity. Analysis by size-exclusion chromatography combined with multiangle laser light scattering revealed that the molecular weight of PGA was M_w = 2,900,000 g mol^−1 and predominantly M_w = 47,000 g mol^−1 in preparations derived from B. subtilis cells with and without pgsE, respectively. PgsE may be required to increase the molecular weight of PGA.

N-(2-Hydroxypropyl)methacrylamide (HPMA)-based statistical copolymers bearing anticancer drugs have attracted attention for their efficacy in cancer treatments. However, controlling the size and morphology of aggregates of this type of polymer has been challenging and is far from being understood. In this study, small-angle X-ray scattering and asymmetric-flow field-flow fractionation with multiangle light scattering were used to investigate the structure of aggregates formed in aqueous solutions of HPMA-based statistical copolymers of different molecular weights with the model drug pyrene borne in different amounts. The analysis revealed that spherical objects (flower micelles) were formed by the assembly of pyrene moieties in low-molecular-weight copolymers, and the flower micelles connected linearly to form string-of-pearls assemblies (flower necklaces) in high-molecular-weight copolymers. The number of pyrene moieties per polymer chain likely dominates the size and morphology of the copolymer micelles. This study shows how to alter the aggregate structure by changing the molecular weight and composition of copolymers.

Exosomes are extracellular vesicles that mediate intercellular communication, immune response, and tumour metastasis. However, exosome isolation from the blood is complicated because their size and density are similar to those of blood lipoproteins. Here, we employed field programming frit-inlet asymmetrical flow field-flow fractionation (FIAF4) coupled with multiangle light scattering (MALS) for the effective separation of exosomes from free unbound proteins and lipoproteins present in serum samples using different pre-treatment methods, namely, a commercial exosome isolation kit, ultracentrifugation (UC), and a simple centrifugation followed by ultrafiltration (UF). Sizes of the eluted exosomes, as calculated by MALS signals, approximated well with the results of batch dynamic light scattering of the collected fractions and with the sizes of polystyrene particles. Exosome separation from lipoproteins was validated by western blotting with several markers of exosomes and lipoproteins, followed by proteomic analysis using nanoflow ultrahigh-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry. UC requires relatively large amounts of serum samples (at least 2 mL) but is more efficient at removing lipoproteins. The UF method with a centrifugal concentrator (300 kDa) was found to be more effective in retrieving exosomes with low serum volumes (50 μL). Altogether, this study demonstrates the application of field programming FIAF4 for the isolation/purification of exosomes from proteins and lipoproteins in the serum.

The potential of lignosulfonates as widely underutilized byproducts of the pulp and paper industry for the synthesis of a biodegradable pesticide carrier system was assessed in this study. Design of experiment software MODDE Pro was for the first time applied to optimize lignosulfonate granule production using Myceliophthora thermophila laccase as a biocatalyst. Enzymatic cross-linking was monitored using size exclusion chromatography coupled online to multiangle laser light scattering, viscosity measurement, and enzyme activity. The determined optimal and experimentally confirmed incubation conditions were: 33 °C, 30 cm3/min O2 supply, and 190 min reaction time. The granules were thereafter loaded with 2 g/kg 3,6-dichloro-2-methoxybenzoic acid (Dicamba), a broad-spectrum herbicide. According to the HPLC analysis, complete release of Dicamba was achieved after 48 h of release. This study showed the green production of a 100% lignosulfonate-based biodegradable solid carrier with potential application in agriculture.

Fast quantitative characterization of a rectangular groove with sizes from sub-micrometers to micrometers on a surface has been demanding in quality control of optical elements. We report a numerical procedure for retrieval of the geometrical parameters of a rectangular groove, including width and depth, from the angular distribution of the scattered light generated by launching plane waves on the groove on a metallic surface from different propagation directions. The far-field intensity of scattering light is first obtained by numerically solving the electromagnetic problem with the modal theory. Then, a two-stage inverse modeling approach is developed to retrieve the groove parameters based on the lookup table and repulsive particle swarm optimization from multi-angle scattering light distribution that is highly sensitive to the groove parameters. Noise influences are considered in the simulation. Results suggest that our method can precisely obtain the groove parameters for noisy signals. As the measurement of light scattering can be conducted with high efficiency, it should be a powerful tool to characterize rectangle grooves on metallic surfaces.

The native reversible self-association of monoclonal antibodies has been associated with high viscosity, liquid-liquid, and liquid-solid phase separation. We investigated the native reversible self-association of an IgG1, which exerts this association even at low protein concentrations, in detail to gain further understanding of this phenomenon by extensive characterization of the association as a function of multiple factors, namely pH, temperature, salt concentration, and protein concentration. The nature of the self-association of the full-length IgG1 as well as the corresponding Fab and Fc fragment was studied by viz. size exclusion chromatography combined with multiangle light scattering, batch dynamic and static light scattering, analytical ultracentrifugation, small angle X-ray scattering, asymmetric flow field flow fractionation coupled with multiangle light scattering, and intrinsic fluorescence. We rationalized the self-association as a combination of hydrophobic and electrostatic interactions driven by the Fab fragments. Finally, we investigated the long-term stability of the IgG1 molecule.

Exosomer är extracellulära vesiklar i nanostorlek som frigörs från cellerna till den extracellulära matrisen. Exosomer är laddade med nukleinsyror, proteiner, och lipider, och fungerar som kommunikatorer mellan celler. Det har gjort dem mycket attraktiva for forskning inom terapi, diagnostik och transport av läkemedel. För att använda exosomer i kliniska tillämpningar behöver standardiserade metoder för isolering, rening och analys av exosomer att utvecklas. Detta projekt syftar till att sätta upp en snabb metod som använder "multiangle light scattering" (MALS) i kombination med kromatografi for att bestämma storleken på exosomer. Olika storlekskromatografi (SEC)/jonbyte (IEX)-kolonner kommer att undersökas och användas for analys av storlekar på exosomer. Partikelstorleken som erhålls från MALS kommer sedan att verifieras med nanoparticle tracking analysis (NTA). För SEC-MALS-analyserna eluerades exosomerna i dödvolymen. IEX-MALS-metoden separerade exosomerna enligt resultatet. Exosomer i storlek mellan 60-110 nm eluerades ut med 800 mM NaCl. Större exosomer och eventuella aggregat i storlek 120-200 nm eluerades ut med 1200 mN NaCl. Resultatet visar att signalen från MALS indikerar var exosomer i kromatogrammen elueras ut och kan ge värdefull information om storleksfördelningen i en topp. SEC-MALS-resultatet är dock inte reproducerbart, eftersom det ibland sker en förändring i storleksfördelningen över toppen. Resultatet har också visat att arean for toppen i dödvolymen varierar mellan körningarna, vilket förmodligen orsakats av att exosomer interagerar med den stationära fasen eller filtret i kolonnen. Förskjutningen i storleksfördelning observerades också i IEX­MALS-metoden. Medelvärdet av partikelstorlekarna som beräknades från SEC-MALS överensstämmer med storlekarna beräknade med NTA. SEC-MALS-metoden behöver förbättras för att fa reproducerbara resultat. Den beräknade storleken från IEX-MALS stämde inte överens med storleken från NT A. Jonbytesanalysen skulle kunna upprepas och fraktioner skulle kunna analyseras med andra tekniker för att verifiera resultatet i framtida arbete. Effekten av den höga saltkoncentrationen på exosomerna behöver också undersökas ytterligare. Exosomes are nanosized extracellular vesicles released from the cells into the extracellular space. Exosomes are loaded with nucleic acids, proteins, and lipids, and work as communicators among cells. This has made them very attractive for research in therapeutics, diagnostics and drug delivery applications. Standardised exosome isolation, purification, and analysis methods need to be developed to use exosomes in clinical applications. This project aimed to set up a quick method using multiangle light scattering (MALS) combined with chromatography techniques to determine exosome sizes. Different size exclusion (SEC)/ion exchange (IEX) columns will be investigated and used to analyse exosome sizes. The particle size obtained from MALS will then be verified with nanoparticle tracking analysis (NTA). For the SEC-MALS analysis, the exosomes were eluted in the void volume. The IEX-MALS method separated the exosome sample. The exosome eluted with 800 mM NaCl ranged between 60-110 nm in diameter. The exosomes eluted with 1200 mM NaCl ranged between 120-200 nm in diameter. The result shows that the light scattering intensity from MALS indicates where the exosomes elute in the chromatograms and gives valuable information about the size distribution in a peak. However, the SEC-MALS result is not reproducible, as sometimes, a shift in the size distribution over the peak occurs. The result has also shown that the void peak area varies between the runs, caused mainly by the exosomes interacting with the resin or the column's filter. The shift in size distribution was also observed in the IEX-MALS method. The average sizes calculated from SEC-MALS agree with the sizes calculated with NTA. The SEC-MALS method needs to be improved to obtain reproducible results. The calculated size from IEX-MALS did not agree with the size from NTA. The ion exchange analysis could be repeated and further analysed with other techniques to verify the result in future work. The effect of the high salt concentration on the exosomes also needs to be further investigated.

International audience; Polymer-grafted chromatography media, especially ion exchangers, are high performance materials for protein purification. However, due to the pore size limitation, conventional chromatography beads are usually not considered for the downstream processing of large biomolecules such as virus-like particles (VLPs). Contrariwise, since the outer surface of the chromatography beads provides satisfactory binding capacity for VLPs and impurities of smaller size can bind inside of the beads, conventional porous beads should be considered for VLP capture and purification. We used HIV-1 gag VLPs with a diameter of 100-200 nm as a model to demonstrate that polymer-grafted anion exchangers are suitable for the purification of bionanoparticles. The equilibrium binding capacity was 1 × 1013 part/mL resin. Moderate salt concentration up to 100 mM NaCl did not affect binding, allowing direct loading of cell culture supernatant onto the column for purification. Dynamic binding capacity at 10% breakthrough, when loading cell culture supernatant, was approximately 6 × 1011 part/mL column; only 1-log lower than for monoliths. Endonuclease treatment of the cell culture supernatant did not increase the dynamic binding capacity, suggesting that dsDNA does not compete for the binding sites of VLPs. Nevertheless, due to simultaneous elution of particles and dsDNA, endonuclease treatment is required to reduce dsDNA contamination in the product. Proteomic analysis revealed that HIV-1 gag VLPs contain different host cell proteins in their cargo. This cargo is composed of conserved proteins and other proteins that vary from one particle population to another, as well as from batch to batch. This process allowed the separation of different particle populations. HIV-1 gag VLPs were directly captured and purified from cell culture supernatant with a total particle recovery in the elution of about 35%. Columns packed with beads can be scaled to practically any dimension and therefore a tailored design of the process is possible.

High-resolution structural analysis of flexible proteins is frequently challenging and requires the synergistic application of different experimental techniques. For these proteins, small-angle X-ray scattering (SAXS) allows for a quantitative assessment and modeling of potentially flexible and heterogeneous structural states. Here, we report SAXS characterization of the condensin HEAT-repeat subunit Ycg1(Cnd3) in solution, complementing currently available high-resolution crystallographic models. We show that the free Ycg1 subunit is flexible in solution but becomes considerably more rigid when bound to its kleisin-binding partner protein Brn1(Cnd2). The analysis of SAXS and dynamic and static multiangle light scattering data furthermore reveals that Ycg1 tends to oligomerize with increasing concentrations in the absence of Brn1. Based on these data, we present a model of the free Ycg1 protein constructed by normal mode analysis, as well as tentative models of Ycg1 dimers and tetramers. These models enable visualization of the conformational transitions that Ycg1 has to undergo to adopt a closed rigid shape and thereby create a DNA-binding surface in the condensin complex.

The fine structure and chain conformation of a heteropolysaccharide (PCIPS3) from mycelium of Paecilomyces cicadae were investigated via the analysis of HPLC, IR, methylation, NMR spectroscopy and multiangle light scattering. It was determined to be a 2.23 × 104 g/mol heteropolysaccharide primarily composed of glucose, galactose and mannose in a molar ratio of 23.8:2.1:1.0. The PCIPS3 backbone consisted of 1,4-linked α- d -Glcp and 1,4-linked 6-O-Me-α- d -Glcp residues, which were occasionally interrupted by branched β-Galf residues through 1,6-linkage. Moreover, the α (0.60) from Mark–Houwink–Sakurada (MHS) equation suggested that PCIPS3 adopted a flexible chain conformation in 0.1 mol/L NaNO3 at 25 °C. The worm-like chains model parameters for PCIPS3 were estimated as following: ML = 437 nm−1, q = 0.46 nm and 0.79 nm, which were further evidenced by AFM. Furthermore, PCIPS3 showed excellent scavenging capacities of 2,2-diphenyl-1-picrylhydrazyl radical, superoxide radical, hydroxyl radical, ORAC radical and moderate immunomodulatory activity.

An aqueous reversible‐deactivation radical polymerization (RDRP) approach is used to synthesize sodium polystyrene sulfonate directly from functionalized monomers to give uniformly and completely sulfonated materials. Reproducible gram scale syntheses are achieved under simple one pot reaction conditions at ambient temperatures, and full monomer conversions are achieved within approximately 3 h. Reaction variables such as pH, sodium chloride concentration, and methanol cosolvent have a significant effect on the molecular weights (Mₙ ≈ 20,000–400,000 g·mol⁻¹) obtained by gel permeation chromatography coupled multiangle light scattering. Observed dispersities were reasonably narrow: Ð ≈ 1.05–1.3. A parametric optimization, rather than direct variation of the monomer to initiator ratio, resulted in some of the highest molecular weight polymers by an RDRP approach. Linear progression between Mₙ and monomer conversion occurs at a neutral reaction pH, which results in narrow polymer molecular weight distributions, along with high end‐group fidelity as demonstrated with chain extension reactions. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019, 57, 1527–1537

With a constantly increasing complexity of macromolecular structures, advanced polymer analysis faces new challenges with regard to the comprehensive analysis of these structures. Today it goes without saying that comprehensive polymer analysis requires selective and robust fractionation methods in combination with a set of information-rich detectors. Thermal field-flow fractionation (ThFFF) has proven to be a powerful technique for the fractionation of complex polymers as well as polymer assemblies. In the present study, ThFFF is coupled to a set of five detectors to simultaneously provide quantitative information on a number of important molecular parameters, including molar mass, molecular size, chemical composition, molecular topology, intrinsic viscosity, and normal and thermal diffusion coefficients. The five-detector setup includes a triple detector device (multiangle light scattering (MALS), differential refractive index (dRI), and differential viscometer (dVis)) that is coupled to an ultraviolet (UV) detector for dual concentration detection and an online dynamic light scattering (DLS) detector. Triple detection consisting of MALS, dRI, and dVis provides information on molar mass, molecular size, and molecular topology. Dual concentration detection offers compositional analysis from a combination of UV and dRI detectors, whereas DLS provides information on diffusion coefficients and hydrodynamic radii. The power of this novel quintuple detector ThFFF (ThFFF-QD) is documented for three important fields of application, namely, the comprehensive analysis of (1) linear and star-shaped polymers, (2) hydrogenated and deuterated polymers, and (3) block copolymer self-assemblies. These applications highlight the novel approach of determining the most relevant molecular parameters, including Mark-Houwink and conformation plots, simultaneously in a single experiment.

Polymers functionally contribute to supersaturation and precipitation inhibition of the active pharmaceutical ingredient (API) in amorphous solid dispersions (ASD). Therefore, it is necessary to monitor physicochemical changes of the polymeric carrier caused by the manufacturing process. This is especially important when the material is exposed to heat and shear stress as in case of hot-melt extrusion (HME). This study evaluated the impact of HME process conditions on physical characteristics of poly(vinylpyrrolidone-co-vinyl-acetate) 60:40 (PVP-VA64) which is a widely used polymer for HME. Focus was set on molecular weight (Mw) and polydispersity index (PDI), by means of absolute molar mass detection via multi-angle light scattering. The generation of a high Mw fraction together with a decrease of the average Mw was detected. In a next step, the influence of these changes on the dissolution behavior of ASD was evaluated. Different stress conditions were applied onto PVP-VA64 in placebo extrusions. The obtained stressed polymer samples were subsequently used to prepare verum ASD with ketoconazole by spray drying (SD). SD dispersions (SDD) of thermally stressed PVP-VA64 were compared to SDD prepared with bulk powder. Although there were only slight changes in Mw and PDI, they significantly impacted supersaturation and precipitation of the formulation.

International audience; Particle size distribution and stability are key attributes for the evaluation of the safety and efficacy profile of medical nanoparticles (Med-NPs). Measuring particle average size and particle size distribution is a challenging task which requires the combination of orthogonal high-resolution sizing techniques, especially in complex biological media. Unfortunately, despite its limitations, due to its accessibility, low cost, and easy handling, batch mode dynamic light scattering (DLS) is still very often used as the only approach to measure particle size distribution in the nanomedicine field. In this work the use of asymmetric flow field flow fractionation coupled to multiangle light scattering and dynamic light scattering detectors (AF4-MALS-DLS) was evaluated as an alternative to batch mode DLS to measure the physical properties of lipid-based nanoparticles. A robust standard operating procedure (SOPs) developed by the Nanomedicine Characterization Laboratory (EUNCL) was presented and tested to assess size stability, batch to batch consistency, and the behavior of the lipid-based nanoparticles in plasma. Orthogonal sizing techniques, such as transmission electron microscopy (TEM) and particle tracking analysis (PTA) measurements, were performed to support the results. While batch mode DLS could be applied as a fast and simple method to provide a preliminary insight into the integrity and polydispersity of samples, it was unsuitable to resolve small modifications of the particle size distribution. The introduction of nanoparticle sorting by field-flow fractionation coupled to online DLS and MALS allowed assessment of batch to batch variability and changes in the size of the lipid nanoparticles induced by the interaction with serum proteins, which are critical for quality control and regulatory aspects. In conclusion, if a robust SOP is followed, AF4-MALS-DLS is a powerful method for the preclinical characterization of lipid-based nanoparticles.

The main principles of higher-order protein oligomerization are elucidated by many structural and biophysical studies. An astonishing number of proteins self-associate to form dimers or higher-order quaternary structures which further interact with other biomolecules to elicit complex cellular responses. In this study, we describe a simple and convenient approach to determine the oligomeric state of purified protein complexes that combines implementation of a novel form of clear-native gel electrophoresis and size exclusion chromatography in line with multi-angle light scattering. Here, we demonstrate the accuracy of this ensemble approach by characterizing the previously established pentameric state of the intracellular domain of serotonin type 3A (5-HT(3A)) receptors.

When the micellar aggregation number (Nagg) is sufficiently small (Nagg < 30), the micelle shows an abnormal aggregation behavior: monodispersity without any distribution in Nagg, whose values coincide with the vertex number of a regular polyhedral structure, i.e., they are termed Platonic solids. Micelles with these characteristics are named “Platonic micelles”. In this study, we investigated the aggregation behavior of calixarene-based micelles bearing primary amines—the first example of Platonic micelles—with increasing alkyl chain length by small-angle X-ray scattering, asymmetrical flow field flow fractionation coupled with multiangle light scattering, and analytical ultracentrifugation measurements. Morphological transition of the micelles from spherical to cylindrical was observed when the alkyl chain length was increased in this calixarene-based micellar system, which is similar to the case of conventional systems and is acceptable in terms of the packing parameter principle. However, although the micellar Nagg normally increases with an increase in the alkyl chain length, the structure of calixarene-based Platonic micelles bearing butyl (C4), heptyl (C5), and hexyl (C6) chains remains at 12-mer. This is presumably due to the relationship between the thermodynamic stability of the Platonic micelles and the coverage ratio defined by the Tammes problem.

Polymer nanocomposites containing nanometals became a subject of interest due to their bactericidal properties. Different polysaccharides have been used as matrices for nanosilver and nanogold synthesis. In this study, we present a novel, environmentally friendly method for the preparation of sodium alginate/nanosilver/graphene oxide (GOX) and sodium alginate/nanogold/graphene oxide GOX nanocomposites and their characteristics. The formation of approximately 10–20 nm ball-shaped Ag and Au nanoparticles was confirmed by UV–vis spectroscopy, scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectra. The incorporation of GOX sheets within the ALG matrix improved the thermal stability of the nanocomposites film, which was measured using the differential scanning calorimetry (DSC). We also estimated the molecular weights of polysaccharide chains of the matrix with the size exclusion chromatography coupled with multiangle laser light scattering and refractometric detectors (HPSEC-MALLS-RI). The composites were more prone to enzymatic hydrolysis. The strongest bacteriostatic activity was observed for the sample containing nanosilver.

Size exclusion chromatography equipped with a multi-angle, light-scattering online detector (SEC-MALS) measurements were carried out on a hydrophobically modified pullulan (PUL-OSA) with degrees of substitution (DS) of 0.14, 0.2, and 0.3 in 0.01 M aqueous NaCl to obtain the degree of polymerization (N0) dependence of the radius of gyration (⟨S2⟩1/2) for PUL-OSA in the aqueous NaCl. The result was consistent with the loose flower necklace model proposed in a previous study, and the increase in the chain size with introducing OSA groups was explained by the backbone stiffness of the loose flower necklace formed by PUL-OSA. For PUL-OSA samples with DS = 0.2 and 0.3, ⟨S2⟩1/2 obtained by SEC-MALS in a high N0 region deviated downward from ⟨S2⟩1/2 expected by the loose flower necklace model. This deviation came from a tiny amount of the aggregating component of PUL-OSA, taking a branched architecture composed of loose flower necklaces. Although the aggregating component of PUL-OSA was also detected by previous small angle X-ray scattering measurements, its conformation was revealed in this study by SEC-MALS.

The nucleocapsid phosphoprotein N plays critical roles in multiple processes of the severe acute respiratory syndrome coronavirus 2 infection cycle: it protects and packages viral RNA in N assembly, interacts with the inner domain of spike protein, binds to structural membrane (M) protein during virion packaging and maturation, and to proteases causing replication of infective virus particle. Even with its importance, very limited biophysical studies are available on the N protein because of its high level of disorder, high propensity for aggregation, and high susceptibility for autoproteolysis. Here, we successfully prepare the N protein and a 1000-nucleotide fragment of viral RNA in large quantities and purity suitable for biophysical studies. A combination of biophysical and biochemical techniques demonstrates that the N protein is partially disordered and consists of an independently folded RNA-binding domain and a dimerization domain, flanked by disordered linkers. The protein assembles as a tight dimer with a dimerization constant of sub-micromolar but can also form transient interactions with other N proteins, facilitating larger oligomers. NMR studies on the ∼100-kDa dimeric protein identify a specific domain that binds 1-1000-nt RNA and show that the N-RNA complex remains highly disordered. Analytical ultracentrifugation, isothermal titration calorimetry, multiangle light scattering, and cross-linking experiments identify a heterogeneous mixture of complexes with a core corresponding to at least 70 dimers of N bound to 1-1000 RNA. In contrast, very weak binding is detected with a smaller construct corresponding to the RNA-binding domain using similar experiments. A model that explains the importance of the bivalent structure of N to its binding on multivalent sites of the viral RNA is presented.

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.

The importance of polymeric nanocarriers in the field of drug delivery is ever-increasing, and the accurate characterization of their properties is paramount to understand and predict their behavior. Asymmetric flow field-flow fractionation (AF4) is a fractionation technique that has gained considerable attention for its gentle separation conditions, broad working range, and versatility. AF4 can be hyphenated to a plurality of concentration and size detectors, thus permitting the analysis of the multifunctionality of nanomaterials. Despite this potential, the practical information that can be retrieved by AF4 and its possible applications are still rather unfamiliar to the pharmaceutical scientist. This review was conceived as a primer that clearly states the “do’s and don’ts” about AF4 applied to the characterization of polymeric nanocarriers. Aside from size characterization, AF4 can be beneficial during formulation optimization, for drug loading and drug release determination and for the study of interactions among biomaterials. It will focus mainly on the advances made in the last 5 years, as well as indicating the problematics on the consensus, which have not been reached yet. Methodological recommendations for several case studies will be also included. Graphical abstract

Summary The aim of this study was focused on the investigation of supramolecular structure of humic acids (HA). This issue was studied on a number of HA samples which differed in source matrix (lignite, leonardite, biochar, soil, peat and compost). For these purposes, methods of size exclusion chromatography coupled with UV-VIS, MALS (multiangle light scattering) and dRi (differential refractometry), as well as dynamic and electrophoretic light scattering and UV-VIS spectrometry were used. In order to better understand the formation of the supramolecular structure of HA, the ways of its stabilization and how it was influenced by the change in pH, individual HA samples were studied in three pH-varying mediums (3,5; 7 and 12).