Size Exclusion Chromatography Method Research Articles

Characterisation of mass distributions of solvent-fractionated lignins using analytical ultracentrifugation and size exclusion chromatography methods

Lignins are valuable renewable resources for the potential production of a large array of biofuels, aromatic chemicals and biopolymers. Yet native and industrial lignins are complex, highly branched and heterogenous macromolecules, properties that have to date often undermined their use as starting materials in lignin valorisation strategies. Reliable knowledge of weight average molar mass, conformation and polydispersity of lignin starting materials can be proven to be crucial to and improve the prospects for the success of such strategies. Here we evaluated the use of commonly-used size exclusion chromatography (SEC)—calibrated with polystyrene sulphonate standards—and under-used analytical ultracentrifugation—which does not require calibration—to characterise a series of lignin fractions sequentially extracted from soda and Kraft alkaline lignins using ethyl acetate, methyl ethyl ketone (MEK), methanol and acetone:water (fractions F01–F04, respectively). Absolute values of weight average molar mass (Mw) determined using sedimentation equilibrium in the analytical ultracentrifuge of (3.0 ± 0.1) kDa and (4.2 ± 0.2) kDa for soda and Kraft lignins respectively, agreed closely with previous SEC-determined Mws and reasonably with the size exclusion chromatography measurements employed here, confirming the appropriateness of the standards (with the possible exceptions of fraction F05 for soda P1000 and F03 for Indulin). Both methods revealed the presence of low (~ 1 kDa) Mw material in F01 and F02 fractions followed by progressively higher Mw in subsequent fractions. Compositional analysis confirmed > 90% (by weight) total lignins successively extracted from both lignins using MEK, methanol and acetone:water (F02 to F04). Considerable heterogeneity of both unfractionated and fractionated lignins was revealed through determinations of both sedimentation coefficient distributions and polydispersity indices. The study also demonstrates the advantages of using analytical ultracentrifugation, both alongside SEC as well as in its own right, for determining absolute Mw, heterogeneity and conformation information for characterising industrial lignins.

An advanced method for the small-scale production of high-quality minicircle DNA

Minicircle DNA is a promising tool in the field of gene therapy, whose products are increasingly gaining market access. Greater transfection efficiency and longer expression time as well as lower immunogenicity contrast with cost-intensive production, which also stands in the way of a broader use of the advantages of this technology in research. Starting from a commercial minicircle production kit a simple protocol for the cost-effective small-scale production of high-quality minicircle DNA to be used at a research scale has been developed by combining and improving procedures of various publications. An optimized size-exclusion chromatography method led to almost pure minicircle DNA with a superior proportion of the desired supercoiled plasmid conformation. The pharmaceutical potential of the produced minicircle DNA was investigated in vitro by real-time impedance assays in a tumor cell model in case of coded suicide genes as well as by ELISA of the translation product in case of coded human coagulation factor IX.

Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method.

Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer microenvironment and immunology. We developed a combination method of size exclusion chromatography and concentration filters (SEC-CF) and aimed to characterize different EV types by their size, cargo types, and functions. A human monocytic leukemia cell line THP-1 was differentiated to CD14-positive macrophage-like cells by stimulation with PMA (phorbol 12-myristate 13-acetate) but not M1 or M2 types. Using the SEC-CF method, the following five EV types were fractionated from the culture supernatant of macrophage-like cells: (i) rare large EVs (500–3000 nm) reminiscent of apoptosomes, (ii) EVs (100–500 nm) reminiscent of microvesicles (or microparticles), (iii) EVs (80–300 nm) containing CD9-positive large exosomes (EXO-L), (iv) EVs (20–200 nm) containing unidentified vesicles/particles, and (v) EVs (10–70 nm) containing CD63/HSP90-positive small exosomes (EXO-S) and particles. For a molecular transfer assay, we developed a THP-1-based stable cell line producing a GFP-fused palmitoylation signal (palmGFP) associated with the membrane. The THP1/palmGFP cells were differentiated into macrophages producing palmGFP-contained EVs. The macrophage/palmGFP-secreted EXO-S and EXO-L efficiently transferred the palmGFP to receiver human oral carcinoma cells (HSC-3/palmTomato), as compared to other EV types. In addition, the macrophage-secreted EXO-S and EXO-L significantly reduced the cell viability (ATP content) in oral carcinoma cells. Taken together, the SEC-CF method is useful for the purification of large and small exosomes with higher molecular transfer activities, enabling efficient molecular delivery to target cells.

Distribution, speciation and composition of humic substances in a macro-tidal temperate estuary

In aquatic environments, the term humic substances (HS) encompasses terrestrial and autochthonous refractory organic matter. HS are one of the main fractions of natural organic matter and are important chelators of trace elements. In environmental studies, the determination of HS is often restricted to the dissolved fraction, and the content of HS in the suspended particles remains unknown. In this work, we present the composition and dynamics of HS along the mixing gradient of a macrotidal estuary in both the dissolved and particulate fraction. After the isolation of particulate HS using a solid-liquid alkali extraction method, HS were characterized by size exclusion chromatography (SEC) and electrochemical methods. The method, validated using a certified reference material, demonstrated a low detection limit (μg-C L−1), a good repeatability (7.7%) an excellent reproducibility (1.3%) and was poorly prone to contamination (filter blank < 1 μg-C). Analyses of environmental samples showed a particulate fraction contributing significantly to the total humic pool in the estuary (3–20%) and representing up to 35% of electroactive HS. Phase transfers from the dissolved to the particulate fraction were observed and the electroactive HS were strongly affected in the maximum turbidity zone. Multi-detection SEC analyses showed differences in the molecular composition between the dissolved and particulate fractions. Particulate HS were more nitrogen-rich and electroactive than dissolved HS. The non-conservative changes in the composition of HS along the land-sea continuum evidence the existence of abiotic and biotic processes that altered HS during their transit from river to marine waters.

Systematic Development of a Size Exclusion Chromatography Method for a Monoclonal Antibody with High Surface Aggregation Propensity (SAP) Index.

Size Exclusion Chromatography (SEC) has been widely used to assess aggregate content in bio-pharmaceutical drugs such as monoclonal antibodies (mAbs), and is routinely used during method development and release testing. Electrostatic interactions between protein analytes and SEC column resin are commonly observed besides the primary mode of size separation during SEC method development, which needs to be minimized. An effective method to minimize electrostatic interactions is through increasing mobile phase (MP) salt concentration. However; increasing salt concentration in MP will induce increased hydrophobicity of proteins and increased hydrophobic interactions between protein and stationary phase, as demonstrated for mAb-A in this paper, a protein with high surface aggregation propensity (SAP) score and an isoelectric point near mobile phase pH. In this work, a systematic, Design of Experimental approach was taken to identify optimal SEC method conditions including column type, buffer composition, ionic strength, pH and additives. The optimized method was demonstrated to be robust towards small changes in method operation conditions and was qualified for use in product release and stability studies. Additionally, biophysical and computational studies were performed to elucidate the role of MP additives, which supports the use of arginine as an essential additive to minimize undesirable hydrophobic interactions between proteins and stationary phase.

Characterization of Biological Material Adsorption to the Surface of Nanoparticles without a Prior Separation Step: a Case Study of Glioblastoma-Targeting Peptide and Lipid Nanocapsules.

PurposeCurrent preclinical therapeutic strategies involving nanomedicine require increasingly sophisticated nanosystems and the characterization of the complexity of such nanoassemblies is becoming a major issue. Accurate characterization is often the factor that can accelerate the translational approaches of nanomedicines and their pharmaceutical development to reach the clinic faster. We conducted a case study involving the adsorption of the NFL-TBS.40–63 (NFL) peptide (derived from neurofilaments) to the surface of lipid nanocapsules (LNCs) (a combined nanosystem used to target glioblastoma cells) to develop an analytical approach combining the separation and the quantification in a single step, leading to the characterization of the proportion of free peptide and thus the proportion of peptide adsorbed to the lipid nanocapsule surface.MethodsLNC suspensions, NFL peptide solution and LNC/NFL peptide mixtures were characterized using a Size-Exclusion Chromatography method (with a chromatographic apparatus). In addition, this method was compared to centrifugal-filtration devices, currently used in literature for this case study.ResultsCombining the steps for separation and characterization in one single sequence improved the accuracy and robustness of the data and led to reproducible results. Moreover the data deviation observed for the centrifugal-filtration devices demonstrated the limits for this increasingly used characterization approach, explained by the poor separation quality and highlighting the importance for the method optimization. The high potential of the technique was shown, proving that H-bond and/or electrostatic interactions mediate adsorption of the NFL peptide to the surface of LNCs.ConclusionsUsed only as a characterization tool, the process using chromatographic apparatus is less time and solvent consuming than classical Size-Exclusion Chromatography columns only used for separation. It could be a promising tool for the scientific community for characterizing the interactions of other combinations of nanosystems and active biological agents.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s11095-021-03034-8.

Process analytical technology for on-line monitoring of quality attributes during single-use ultrafiltration/diafiltration.

Process analytical technology (PAT) is a fast-growing field within bioprocessing that enables innovation in biological drug manufacturing. This study demonstrates novel PAT methods for monitoring multiple quality attributes simultaneously during the ultrafiltration and diafiltration (UF/DF) process operation, the final step of monoclonal antibody (mAb) purification. Size exclusion chromatography (SEC) methods were developed to measure excipients arginine, histidine, and high molecular weight (HMW) species using a liquid chromatography (LC) system with autosampler for both on-line and at-line PAT modes. The methods were applied in UF/DF studies for the comparison of single-use tangential flow filtration (TFF) cassettes to standard reusable cassettes to achieve very high concentration mAb drug substance (DS) in the order of 100-200 g/L. These case studies demonstrated that single-use TFF cassettes are a functionally equivalent, low-cost alternative to standard reusable cassettes, and that the on-line PAT measurement of purity and excipient concentration was comparable to orthogonal offline methods. These PAT applications using an on-line LC system equipped with onboard sample dilution can become a platform system for monitoring of multiple attributes over a wide dynamic range, a potentially valuable tool for biological drug development and manufacturing.

Small Extracellular Vesicles from adipose derived stromal cells significantly attenuate in vitro the NF-\u03baB dependent inflammatory/catabolic environment of osteoarthritis

The therapeutic ability of Mesenchymal Stem/Stromal Cells to address osteoarthritis (OA) is mainly related to the secretion of biologically active factors, which can be found within their secreted Extracellular Vesicles including small Extracellular Vesicles (sEV). Aim of this study was to investigate the effects of sEV from adipose derived stromal cells (ADSC) on both chondrocytes and synoviocytes, in order to gain insights into the mechanisms modulating the inflammatory/catabolic OA environment. sEV, obtained by a combined precipitation and size exclusion chromatography method, were quantified and characterized, and administered to chondrocytes and synoviocytes stimulated with IL-1β. Cellular uptake of sEV was evaluated from 1 to 12 h. Gene expression and protein release of cytokines/chemokines, catabolic and inflammatory molecules were analyzed at 4 and 15 h, when p65 nuclear translocation was investigated to study NF-κB pathway. This study underlined the potential of ADSC derived sEV to affect gene expression and protein release of both chondrocytes and synoviocytes, counteracting IL-1β induced inflammatory effects, and provided insights into their mechanisms of action. sEV uptake was faster in synoviocytes, where it also elicited stronger effects, especially in terms of cytokine and chemokine modulation. The inflammatory/catabolic environment mediated by NF-κB pathway was significantly attenuated by sEV, which hold promise as new therapeutic strategy to address OA.

Analytical method development and validation of mucopolysaccharide polysulfate in topical formulations by size exclusion chromatography.

In this study, a new size exclusion chromatographic method has been developed and validated for the analysis of mucopolysaccharide polysulfate used as an anti-inflammatory and antithrombotic agent in topical formulations. Mucopolysaccharide polysulfate was analyzed in Repromer OH-4000 (10 μm, 8.0 × 300 mm) and Repromer OH-5000 (10 μm, 8.0 × 300 mm) columns using a 0.05 M sodium sulfate isocratic elution mobile phase system at 40 °C with a flow rate of 1 mL min-1 and detected by using refractive index detection. The method was validated by means of the limit of quantification, limit of detection, linearity, robustness, recovery, precision and accuracy using the Bioanalytical Method Validation Guidance. The calibration curve showed linearity in the 0.090-1.575 mg mL-1 range. The limits of detection and quantification were found to be 45.000 and 90.000 μg mL-1, respectively. Assay recovery and precision of mucopolysaccharide polysulfate from topical formulations at 0.450, 0.900 and 1.350 mg mL-1 concentrations were evaluated. Intra-day and inter-day relative standard deviation values were calculated to be less than 2.46%. The mean recovery was calculated as 96.64%. The validated method was successfully applied to the determination of mucopolysaccharide polysulfate in cream and gel formulations.

Changes in poplar (Populus trichocarpa) wood porous structure after liquid hot water (LHW) pretreatment

Changes in poplar (Populus trichocarpa) wood porous structure after liquid hot water (LHW) pretreatment. The aim of this research was to investigate the effect of applying different hydrothermal pretreatment conditions on the porous structure of poplar wood. Porosity is recognised as an important factor considering efficiency of an enzymatic hydrolysis as a step of bioethanol production. Native poplar wood as well as solid fractions after pretreatment performed at different temperatures (160 °C, 175 °C and 190 °C) were analysed. Porous structure was examined with an inverse size-exclusion chromatography (ISEC) method. Results indicated a significant development of the porous structure of the biomass with increasing porosity along with the growing temperature of the LHW process. The temperature of 190 °C was chosen as the most promising condition of poplar wood LHW pretreatment in terms of the efficiency of the subsequent steps of bioethanol production. The obtained results were consistent with the previous experimental data procured during analysis of the LHW pretreated poplar wood and its subsequent enzymatic hydrolysis yield.

Metal-Polymer Complexes of Gallium/Gallium-68 with Copolymers of N-Vinylpyrrolidonewith N-Vinylformamideand N-Vinyliminodiacetic Acid: A Hint for Radiolabeling of Water-Soluble Synthetic Flexible Chain Macromolecules.

Copolymer of N-vinylpyrrolidone (VP) with vinylformamide (VFA) and N-vinyliminodiacetic acid (VIDA) was synthesized; its metal-polymer complexes (MPCs) with gallium were obtained. The complexes were characterized by size exclusion chromatography, hydrodynamic and optical methods, scanning electron microscopy, and spectral methods (UV, IR, 1Н NMR spectroscopy). It was demonstrated that in going from polymer to complex, hydrodynamic parameters of macromolecules change only slightly, although the polymer contains intramolecular Ga(VIDA)2 fragments in its structure. A new method for preparation of MPCs with gallium and gallium-68 radionuclide was suggested. The obtained metal-polymer complex is stable over a wide range of pH values as well as in the histidine challenge reaction. In vivo distribution experiments in intact animals showed high primary accumulation of thegallium-68 MPC in blood with subsequent excretion via urinary tract.

Characterization of Amphiphilic Cobaltocenium Copolymers via Size Exclusion Chromatography with Online Laser-Light Scattering and Viscometric Detectors

A sample of cobaltocenium copolymer (Copolym) obtained by a controlled polymerization method was characterized by a multi-detector size exclusion chromatography (MD-SEC) method eluted by various pure and mixed mobile phases. The value of number average molecular weight (), estimated by MD-SEC using the above elutes, was comparable with the ones obtained from the SEC standard calibration curve and estimation by the monomer conversion method. The solution properties of the Copolym were studied based on the Mark-Houwink-Kuhn-Sakurada (MH) relationship where the values of the MH exponent of the Copolym in the above solutions were within the values corresponding to a flexible random coil system. The unperturbed end-to-end dimensions of the Copolym were obtained based on the Stockmayer-Fixman extrapolation method. It was found that the degree of flexibility of the Copolym was higher than that of C-C backbone polymers such as atactic polypropylene, and in the range of poly(acrylic acid), which is suggested to be due to the short-range interference effect of the ionic side chains and solvent molecules on the degree of free rotation of the copolymer.

Purification, structural analysis, and stability of antioxidant peptides from purple wheat bran

Protein derived from purple wheat bran was hydrolyzed sequentially using alcalase proteases for the production of antioxidant peptides. Purple wheat bran protein (PWBP) hydrolysates were fractionated using size-exclusion (G-25) and ion-exchange chromatography methods to identify the structure of antioxidant peptides. The free radical scavenging activity of peptides purified from PWBP hydrolysates was evaluated using superoxide anion radical-scavenging activity and determination assays of Trolox equivalent antioxidant capacity (TEAC). Results demonstrated that purple wheat bran peptide F4-4 exhibited the highest antioxidant activity among other hydrolysates. F4-4 was further identified as Cys-Gly-Phe-Pro-Gly-His-Cys, Gln-Ala-Cys, Arg-Asn-Phe, Ser-Ser-Cys, and Trp-Phe by high performance liquid chromatography (HPLC) spectrometer coupled with Orbitrap Elite™ mass spectrometer (LC–MS/MS). Antioxidant peptides 2 and 4 showed improved stability when the temperature was lower than 80 °C. These peptides also demonstrated good digestive stability in vitro system by simulating gastrointestinal digestion.

Investigation on the interaction of brazilin with bovine serum albumin using multi-spectroscopic and computational methods: Exploring the binding mechanism and inhibitory effect on amyloid aggregation

Free and active concentration of drug in the blood plays a vital role in the disease treatment. Binding of drug to serum albumin, an important transport protein in plasma, usually improves its aqueous solubility, stability and bioactivity. Brazilin (Bra) is a natural compound extracted from Caesalpinia sappan with numerous pharmacological applications, while the behavior of Bra in circulatory system is still unknown. Therefore, in the present work, we firstly examined the interaction of Bra with bovine serum albumin (BSA) using various spectroscopic and computer simulation techniques. The static quenching mechanism and moderate binding affinity (2.18 × 104 M−1 at 298 K) were obtained through fluorescence spectroscopic measurement. Thermodynamic analysis results indicated that hydrophobic interactions and hydrogen bonds played a major role in the binding process. Site marker competitive experiments and molecular simulation suggested that the probable Bra-binding position was located in subdomain IIA of BSA. UV–vis absorption, circular dichroism (CD) and dynamic light scattering (DLS) experiments suggested the conformational changes of BSA induced by Bra. Additionally, the potential of Bra to inhibit the formation of amyloid fibrils, in vitro, has also been analyzed. Thioflavin T (ThT) binding, 8-anilino-1-naphthalenesulfonic acid (ANS) binding, Congo red binding, DLS, CD, fluorescence microscopic image, atomic force microscopy (AFM), size-exclusion chromatography (SEC) and native gel-electrophoresis methods were used, and all confirmed the anti-amyloidogenic behavior of Bra. In short, our findings have provided some important insights into the binding mechanism of Bra with BSA, and would be helpful in designing suitable agents against amyloid diseases.

An Immunosuppressive Effect of Melanoma-derived Exosomes on NY-ESO-1 Antigen-specific Human CD8+ T Cells is Dependent on IL-10 and Independent of BRAFV600E Mutation in Melanoma Cell Lines

ABSTRACT Exosomes, including human melanoma-derived exosomes (HMEX), are known to suppress the function of immune effector cells, which for HMEX has been associated with the surface presence of the immune checkpoint ligand PD-L1. This study investigated the relationship between the BRAF mutational status of melanoma cells and the inhibition of secreted HMEX exosomes on antigen-specific human T cells. Exosomes were isolated from two melanoma cell lines, 2183-Her4 and 888-mel, which are genetically wild-type BRAFWT and BRAFV600E, respectively. HMEX were isolated using a modified, size-exclusion chromatography (SEC) method shown to reduce co-isolation of non-exosome-associated cytokines compared to ultracentrifugation isolation. The immunoinhibitory effect of the exosomes was tested in vitro on patient-derived NY-ESO-1-specific CD8+ T cells challenged with NY-ESO-1 antigen. HMEX from both cell lines inhibited the immune response of antigen-specific T cells comparably, as evidenced by the reduction of IFN-γ and TNF-α in NY-ESO-1 tetramer-positive cells. This inhibition could be partially reversed by the presence of anti-PD-L1 and anti-IL-10 antibodies. IL-10 has been demonstrated to be a critical pathway for sustaining enhanced tumorigenesis in BRAFV600E mutant cells compared to BRAFWT melanoma cells. Thus, we demonstrate that HMEX inhibit antigen-specific T cell responses independent of the BRAF mutational status of the parent cells. In addition, PD-L1 and IL-10 contribute to the HMEX-mediated immunosuppression of antigen-specific human T cells. The inhibitory capacity of exosomes should be taken into consideration when developing therapies that are reliant upon the potency of customized, antigen-specific effector T cells.

Characterization of lysozyme PEGylation products using polarized excitation-emission matrix spectroscopy.

The growing use of therapeutic proteins requires accurate analytical techniques for measuring biophysical and structural changes during manufacturing. This is particularly true for the PEGylation of proteins, because characterization of PEGylation reactions and products can often be difficult due to the relatively small impact on protein structure, the lack of an accessible polyethylene glycol (PEG) chromophore, and the heterogeneous final product mixtures. Intrinsic fluorescence spectroscopy is one potential solution due to its relatively high sensitivity to small changes in protein structure and its suitability for online or atline measurements. In this study, we use the PEGylation of lysozyme as a model system to determine the efficacy of polarized excitation-emission matrix (pEEM) spectroscopy as a rapid tool for characterizing the structural variability of the lysozyme (LZ) starting materials and PEGylated products with varying PEG-to-protein ratios (PPR). Dynamic light scattering showed that as PPR increased from 0 to 2.8, the hydrodynamic radius increased from ∼2.2 to 4.8 nm. pEEM measurements provided several sources of information: Rayleigh scattering to identify size changes and aggregate/particle formation, and fluorescence emission to assess chemical and structural changes. PEGylation induced sufficient physicochemical changes in LZ, which produced changes in the pEEM spectra, largely due to variations in the hydrophobic environments of tryptophan residues close to a PEG attachment site. These significant spectral changes when modeled using conventional multivariate analysis methods were able to easily discriminate the raw product solutions according to the degree of PEGylation and were also able to predict PPR with reasonable accuracy (root mean square error for calibration ∼10%, relative error of prediction < 20%), considering the reference size exclusion chromatography method error of ∼7.2%. The variable selection of the pEEM data suggests that equivalent predictions could be obtained with faster and simpler two-dimensional spectra, making the method a more viable online measurement method.

Determination of glycosylation degree for glycoconjugate vaccines using a solid-phase extraction combined with liquid chromatography and tandem mass spectrometry method.

In this study, a solid-phase extraction with liquid chromatography and tandem mass spectrometry method was developed to determine the degree of glycosylation of glycosylation sites and the ratio of free carrier protein to total carrier protein for glycoconjugate vaccines. To remove and enrich the glycosylated peptides, a solid-phase extraction method was developed, optimized, and hyphenated to liquid chromatography-tandem mass spectrometry. The developed solid-phase extraction with liquid chromatography-tandem mass spectrometry method was shown to possess a wide linear dynamic range (0.03-100μg/mL), a high sensitivity (0.03μg/mL for CRM197), good interday and intra-day precision (relative standard deviation of peak area<3.3%), and good recoveries from vaccine matrix (90-105%). Finally, the method was utilized to determine the degree of glycosylation and free carrier protein to total carrier protein ratio for pneumococcal conjugate vaccines and meningococcal vaccines. For quality evaluation of glycoconjugate vaccines, the method could provide more information than the traditional size exclusion chromatography method. Fourteen and twelve reported glycosylation sites for CRM197- and tetanus toxin-based vaccines can be detected, respectively.

Purification Method of Silver Nanoparticles (AgNPs) and its Identification Using UV-Vis Spectrophotometer

The development of nanotechnology applications is rapidly growing in many sectors. One of them is silver nanoparticles (AgNPs) which are metal nanoparticles that play an important role, especially in nanomedicine. The most effective method of purifying to obtain stable AgNPs is very important to study. Experiments on the separation of AgNPs have been carried out using the size exclusion chromatography and centrifugation methods to see the effectiveness of refining the two methods. This experiment begins with the synthesis of AgNPs using the chemical reduction method. Then, the synthesized AgNPs were purified by Size Exclusion Chromatography (SEC) and centrifugation method then analyzed using UV-Vis spectrophotometer to determine the maximum peaks before and after purification. The experimental results were obtained that centrifugation methods and SEC having the same effectiveness in refining AgNPs. The centrifugation method at various speed (0, 3000, 6000, 9000, 12000 and 15000 rpm) gave wavelength results 403, 404, 404, 405, 404, and 404 nm. The SEC method using Sephadex-25 column showed the 4th to 8th fractions gave the maximum wavelength 404, 404, 404, 405, and 404 nm, respectively. The maximum wavelength of both methods showed the surface plasmon resonance characteristic of AgNPs. However, centrifugation at 3000 rpm has better homogeneity than SEC method.

Salivary Small Extracellular Vesicles Associated miRNAs in Periodontal Status-A Pilot Study.

This pilot study aims to investigate whether salivary small extracellular vesicle (sEV)-associated microRNAs could act as potential biomarkers for periodontal disease status. Twenty-nine participants (10 who were healthy, nine with gingivitis, 10 with stage III/IV periodontitis) were recruited and unstimulated whole saliva samples were collected. Salivary sEVs were isolated using the size-exclusion chromatography (SEC) method and characterised by morphology, EV-protein and size distribution using transmission electron microscopy (TEM), Western Blot and Nanoparticle Tracking Analysis (NTA), respectively. Ten mature microRNAs (miRNAs) in salivary sEVs and saliva were evaluated using RT-qPCR. The discriminatory power of miRNAs as biomarkers in gingivitis and periodontitis versus healthy controls was evaluated by Receiver Operating Characteristics (ROC) curves. Salivary sEVs were comparable to sEVs morphology, mode, size distribution and particle concentration in healthy, gingivitis and periodontitis patients. Compared to miRNAs in whole saliva, three significantly increased miRNAs (hsa-miR-140-5p, hsa-miR-146a-5p and hsa-miR-628-5p) were only detected in sEVs in periodontitis when compared to that of healthy controls, with a good discriminatory power (area under the curve (AUC) = 0.96) for periodontitis diagnosis. Our study demonstrated that salivary sEVs are a non-invasive source of miRNAs for periodontitis diagnosis. Three miRNAs that are selectively enriched in sEVs, but not whole saliva, could be potential biomarkers for periodontal disease status.

Characterization of Urinary Exosomes Purified with Size Exclusion Chromatography and Ultracentrifugation.

Exosomes, a subtype of extracellular vesicles secreted by mammalian cells with a typical size range of 30-150 nm, have been implicated in many biological processes as intercellular communication carriers. The isolation of exosomes is an essential and challenging step before subsequent analysis and functional studies, due to the complexity of body fluids, as well as the small size and low density of exosomes. Ultracentrifugation (UC) and size exclusion chromatography (SEC) are two methods that have been extensively used for exosomes isolation in biological studies in recent years. In this work, we compared the characteristics of urinary exosomes extracted with SEC and UC methods in detail. Results showed that the SEC isolation method was superior to UC in the recovery of exosomal particles and proteins. The results of proteomics analysis showed that more purified exosomes were extracted with the SEC method. We also observed that parts of exosomes were ruptured and precipitated insufficiently during UC isolations. It not only led to a low recovery of exosome proteins but also resulted in a considerable loss of exosomal particles. Moreover, the exosomal rupture and particle loss in UC could not be avoided by resuspension of the exosomal particles. Our results also showed that exosomes from SEC purifications possessed a high internalization capability from 4 to 6 h when incubated with EA.hy926 and HCV29 cell lines.