Efficient preparation of high-purity and intact mesenchymal stem cell-derived extracellular vesicles.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown great promise for regeneration and immunomodulation. However, efficient and scalable methods for their preparation are still lacking. In this study, we present the adoption of a label-free technique known as "EXODUS" to isolate and purify MSC-EVs from the conditioned medium. Our findings indicate that EXODUS can rapidly isolate EVs from 10 mL of conditioned medium with a 5-fold higher yield compared to conventional approaches, including ultracentrifugation (UC) and polyethylene glycol precipitation (PEG) methods. Additionally, pre-storing the conditioned medium at 4°C for 1 week resulted in a ~2-fold higher yield of MSC-EVs compared to the freshly prepared medium. However, storing the purified EV particles at 4°C for 1 month led to a 2-fold reduction in particle concentration. Furthermore, we found that MSC-EVs isolated using EXODUS exhibit higher expression levels of EV markers such as Alix, Flotillin1, CD81, and TSG101 in comparison to PEG and UC methods. We also discovered that MSC-EVs isolated using EXODUS are enriched in response to cytokine, collagen-containing extracellular matrix, and calcium ion binding compared to PEG method and enriched in extracellular structure organization, extracellular matrix, and extracellular matrix structure constituents compared to UC. Finally, we demonstrated that MSC-EVs isolated using EXODUS exhibit greater potential in animal organ development, tissue development, and anatomical structure morphogenesis compared to the UC. These findings suggest that EXODUS is a suitable method for the large-scale preparation of high-quality MSC-EVs for various clinical applications.

Preparation of zein-lecithin-EGCG complex nanoparticles stabilized peppermint oil emulsions: Physicochemical properties, stability and intelligent sensory analysis.

Peppermint oil emulsions were prepared by using zein-lecithin-EGCG (Z-L/E) complex nanoparticles as emulsifiers. The preparation conditions of emulsions were optimized via measuring the particle size, surface tension and stability of emulsions, and peppermint oil of 3% (particle size = 375 nm, polydispersity index (PDI) = 0.45), the zein:lecithin ratio of 4:1 (w/w) (particle size = 396 nm), and the zein:EGCG ratio of 10:1 (w/w) (surface tension = 47.32 N/m) was the optimal condition. The rapid stability analysis showed that the instability mechanism of emulsions was ascribed to creaming and stratification, and the stability mechanism of emulsions was explored, indicating that the complex nanoparticles adsorbed on the surface of oil droplets to give Pickering emulsions. Electronic tongue experiments showed that the Z-E/L4:1 stabilized emulsion was distinguished from the other three samples due to its good stability. The electronic nose experiment could distinguish the emulsions with different droplet sizes.

The fabrication of novel zein and resveratrol covalent conjugates: Enhanced thermal stability, emulsifying and antioxidant properties.

Novel zein and resveratrol conjugates were fabricated by alkaline and free radical grafting reactions. The grafting efficiency and total phenolic content of alkaline treated conjugates were slightly higher than those of free radical grafting. Compared to native and alkaline treated zein, the sulfhydryl contents of conjugates were obviously decreased, confirming that nucleophilic addition of resveratrol to sulfhydryl group of zein formed stable CS covalent bonds. The conformation changes of zein modified by resveratrol were revealed by fourier transform infrared spectroscopy and fluorescence spectroscopy. Moreover, covalent modification changed isoelectric point of zein from 6.5 to 5.4 (alkaline) or 5.6 (free radical grafting), and broadening the pH application range of zein. It was worth mentioning that the conjugates showed much higher thermal stability, antioxidant activity, and emulsify activity than those of native zein. This study provides an effective way for the design of novel delivery systems to encapsulate bioactive substances.

Characterization and stability of peppermint oil emulsions using polyglycerol esters of fatty acids and milk proteins as emulsifiers.

Three peppermint oil emulsions using polyglycerol esters of fatty acids-casein (PGFE-CN), polyglycerol esters of fatty acids-sodium caseinate (PGFE-NaCN), and polyglycerol esters of fatty acids-whey protein isolate (PGFE-WPI) as emulsifiers were fabricated, and the droplet size, zeta potential, viscosity, and stability of emulsions were determined. The experimental results showed that the emulsion containing PGFE-CN has relatively smaller droplet size of 231.77 ± 0.49 nm. No significant changes were observed on the average particle size, polydispersity index and zeta potential during 4-week of storage, indicating that the emulsions kept stable against pH, salt ion, freeze-thaw, and storage. Fourier transform infrared spectrometer (FTIR) results showed that the electrostatic interaction occurs between CN and PGFE in the emulsion. The confocal laser scanning microscope (CLSM) was used to observe the microstructure of the emulsion, proving that droplets were evenly distributed throughout the aqueous phase by PGFE-CN emulsifier. The protein-stabilized emulsions can be used as potential carriers for the delivery of the lipophilic nutrients such as peppermint oil. PRACTICAL APPLICATION: PGFE-CN emulsifier can be directly added to the beverage systems containing oil or protein, such as coconut milk, peanut milk, and walnut milk. It can enhance the stability of beverage, prevent the precipitation, stratification, and oil floating, improve the homogeneity of the system and therefore extend the shelf life.

Fabrication and characterization of oil-in-water pickering emulsions stabilized by ZEIN-HTCC nanoparticles as a composite layer.

In this work, the ZEIN-HTCC nanoparticles formed by zein and N-(2-hydroxy)propyl-3-trimethylammonium chitosan chloride (HTCC) were used as stabilizers to prepare oil-in-water (O/W) Pickering emulsions. The preparation conditions including shearing time, volume fraction of corn oil, mass ratio of ZEIN:HTCC and total concentration of ZEIN-HTCC of emulsions were optimized by measuring the droplet size, zeta potential, PDI and surface tension of emulsions. The ZEIN-HTCC emulsions are stable at the pH range of 4-9 and in the low salt ion concentrations up to 0.2 mol L-1, and can keep stable up to 21 d during low temperature storage. Fourier transform infrared spectroscopy (FTIR), the confocal laser scanning microscope (CLSM) and scanning electron microscopy (SEM) were used to analyze the interaction between emulsion components, revealing that zein and HTCC form a composite layer by flocculation to adsorb on the surface of oil droplets, thus preventing emulsion droplets from aggregation. This novel, long-term stable, surfactant-free, and edible zein-based Pickering emulsion could be used as potential carriers for lipophilic nutrients delivery.

Detection of Tear Components Using Matrix-Assisted Laser Desorption Ionization/Time-of-Flight Mass Spectrometry for Rapid Dry Eye Diagnosis.

The tear is a biological fluid that has the diagnostic potential for ocular diseases. Extracellular vesicles (EVs), widly detected in various biofluids including tears, are nanoparticles released by living cells and considered as promising detection sources for noninvasive liquid biopsy. Understanding the roles of tears and tear-EVs in ocular diseases such as dry eye can facilitate the studies of clinical diagnosis, which usually entails detecting such liquid objects with a rapid and effective method. In this study, we used a mass spectrometry-based strategy to analyze peptidome/proteome profiles of tears and EVs for rapid dry eye diagnosis. Nanosized EVs were isolated from tears of both healthy control (HC) individuals and dry eye syndrome (DES) patients, and the tear compositions were further analyzed by tracking their fingerprints with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. The fingerprints of tear-EVs could be observed in a dose-dependent manner and tears, allowing for comparison of the discriminant peaks between tears and EVs. By analyzing these peaks, the fingerprints of both tear and tear-EVs were showed to have the capability of distinguishing patients with DES from HC donors and providing an efficient way for screening potential DES biomarkers. The proposed tear and EV fingerprinting approach is expected to be a potential tool in the rapid diagnosis of ocular diseases and in-depth research on pathogenesis. Data are available via ProteomeXchange with identifier PXD020217.

Proliferation and differentiation of direct co­culture of bone marrow mesenchymal stem cells and pigmented cells from the ciliary margin.

Damage of retinal ganglion cells (RGCs) is the major consequence of glaucoma and regeneration of RGCs is extremely difficult once the damage has occurred. Retinal stem cells (RSCs) are considered an ideal choice for RGC regeneration. Pigmented cells from the ciliary margin (PCMs) have great retinal differentiation potential and may be an ideal RSC candidate. However, the ciliary margin is too small, so the number of cells that can be obtained is limited. Bone marrow­derived mesenchymal stem cells (BMMSCs) are another type of stem cell that have been previously investigated for RGC regeneration. BMMSCs expand sufficiently, whereas the retinal differentiation of BMMSCs is insufficient. The aim of the present study was to investigate whether the co­culture of PCMs and BMMSCs may combine the advantages of both cell types to establish a novel and effective stem cell source for RGC regeneration. Primary rat PCMs and BMMSCs were isolated and co­cultured. Cell growth was observed by an inverted microscope and proliferation was monitored by an MTT assay. Cell cycle analysis was performed by using a flow cytometer, while the expression of the photoreceptor­specific homeobox gene (cone­rod homeobox, Crx) was determined by reverse transcription­quantitative polymerase chain reaction and western blot analysis. In addition, retinal differentiation was confirmed by immunofluorescence staining of major markers of retinal differentiation, including rhodopsin, visual system homeobox 2 and heparin sulfate. The co­cultured cells expanded successfully, in a similar way to BMMSCs. In addition, the expression of Crx and retinal markers were significantly upregulated following BMMSC and PCM co­culture. The results of the present study demonstrated that the co­culture of BMMSCs and PCMs may be used as a source of RSCs.