AFM-TEM correlation microscopy and its application to lipid nanoparticles.

So far, only a few articles have demonstrated the possibility of correlated AFM-TEM imaging - sequential imaging of the same individual objects using atomic-force microscopy (AFM) and transmission electron microscopy (TEM). The current work contributes to the development of this approach by giving a step-by-step procedure, which yields pairs of correlated AFM-TEM images. We describe the application of correlation AFM-TEM microscopy to lipid nanoparticles (small extracellular vesicles and liposomes). The sizes of individual particles measured by the two methods were in good agreement, taking the tip broadening into account. The correlated AFM-TEM imaging can be valuable for single-particle analysis and nanometrology.

ScanEV - A neural network-based tool for the automated detection of extracellular vesicles in TEM images.

Transmission electron microscopy (TEM) is the most widely accepted method for visualization of extracellular vesicles (EVs), and particularly, exosomes. TEM images provide us with information about the size and morphology of the EVs. We have developed an online tool ScanEV (Scanner for the Extracellular Vesicles, available at https://bioeng.ru/scanev), for the rapid and automated processing of such images. ScanEV is based on a convolutional neural network; it detects the «cup-shaped¼ particles in the images and calculates their morphometric parameters. This tool will be useful for researchers who study EVs and use TEM for their characterization.

Stomatin is highly expressed in exosomes of different origin and is a promising candidate as an exosomal marker.

Proteins involved in the organizing of lipid rafts can be found in exosomes, as shown for caveolin-1, and they could contribute to exosomal cargo sorting, as shown for flotillins. Stomatin belongs to the same stomatin/prohibitin/flotillin/HflK/C family of lipid rafts proteins, but it has never been studied in exosomes except for extracellular vesicles (EVs) originating from blood cells. Here we first show the presence of stomatin in exosomes produced by epithelial cancer cells (non-small cell lung cancer, breast, and ovarian cancer cells) as well as in EVs from biological fluids, including blood plasma, ascitic fluids, and uterine flushings. A high abundance of stomatin in EVs of various origins and its enrichment in exosomes make stomatin a promising exosomal marker. Comparison with other lipid raft proteins and exosomal markers showed that the level of stomatin protein in exosomes from different sources corresponds well to that of CD9, while it differs essentially from flotillin-1 and flotillin-2 homologs, which in turn are present in exosomes in nearly equal proportions. In contrast, the level of vesicular caveolin-1 as well as its EV-to-cellular ratio vary drastically depending on cell type.

The miscibility and spatial distribution of the components in electrospun polymer-protein mats.

Biodegradable blended electrospun mats are promising for biomedical applications such as wound dressing, tissue engineering, and drug delivery. Electrospun mats based on polyesters can be modified by the addition of other polymers or proteins to accelerate the degradation, improve mechanical properties or biocompatibility. However, relatively little is known about the distribution of the components throughout the blended mats. In the present work, we prepared polylactide (PLA), bovine serum albumin (BSA), and the blended PLA-BSA electrospun mats. We demonstrated that PLA and BSA are miscible in a common solvent HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) at concentrations below 3%, but become immiscible as concentration increases. We used three methods (fluorescence microscopy, EDX, and Raman microspectroscopy) to validate that PLA and BSA can be blended in a single electrospun fiber despite the phase separation in the blend. The homogeneity of the blend influences on the homogeneity of the distribution of PLA and BSA components throughout the electrospun mat, as measured by Raman microspectroscopy. When the blended electrospun mats were incubated in water, they demonstrated the prolonged release of BSA. The presented results show a step-by-step approach for manufacturing blended electrospun mats made of immiscible components, which involves the analysis of component miscibility, the mat morphology, and composition. This approach can be used for the rational design of multicomponent electrospun mats.

Fabrication of hydrogel scaffolds via photocrosslinking of methacrylated silk fibroin.

Silk fibroin is a promising biomaterial for tissue engineering due to its valuable mechanical and biological properties. However, being a natural product and a protein, it lacks the processability and uniform quality of an advanced synthetic material. Here we propose a way to overcome this contradiction using novel fibroin photocrosslinkable derivative (FBMA). FBMA was synthesized by methacrylation of native fibroin nucleophilic side groups. It was dissolved in either formic acid (FA) or hexafluoroisopropanol (HFIP), and the obtained solutions were photocrosslinked into hydrogel scaffolds of various structural forms including films, micropatterns, pads and macroporous sponges. UV-exposition of dry FBMA films through a photomask created complex microscaled patterns of the polymer. The nature of the solvent affected the properties of resulting hydrogels. When HFIP was used as the solvent, the resulting hydrogels had a storage modulus ∼4 times higher than that of hydrogels fabricated using FA and ∼20 times higher compared to the reference hydrogel obtained from pristine fibroin. Both FBMA-based hydrogels were biocompatible and supported fibroblast adhesion and growth in vitro. Cells cultivated on FBMA scaffolds produced with HFIP exhibited more spread phenotype at 4 and 24 h of cultivation, consistent with increased stiffness of the hydrogel. Hence, FBMA is an attractive material for fabrication of micropatterned scaffolds of centimeter-scale size with minutely tunable physico-chemical properties via convenient and reproducible technological processes, applicable for rapid prototyping.

Wall Thickness of Industrial Multi-Walled Carbon Nanotubes Is Not a Crucial Factor for Their Degradation by Sodium Hypochlorite.

The propensity of multi-walled carbon nanotubes (MWCNTs) for biodegradation is important for their safe use in medical and technological applications. Here, we compared the oxidative degradation of two samples of industrial-grade MWCNTs-we called them MWCNT-d and MWCNT-t-upon their treatment with sodium hypochlorite (NaOCl). The MWCNTs had a similar inner diameter but they differed about 2-fold in the outer diameter. Electron microscopy combined with morphometric analysis revealed the different degradation of the two types of MWCNTs after their incubation with NaOCl-the thicker MWCNT-d were damaged more significantly than the thinner MWCNT-t. The both types of MWCNTs degraded at the inner side, but only MWCNT-d lost a significant number of the outer graphitic layers. Raman spectroscopy demonstrated that both MWCNTs had a similar high defectiveness. Using energy-dispersive X-ray spectroscopy, we have shown that the more degradable MWCNT-d contained the same level of oxygen as MWCNT-t, but more metal impurities. The obtained results suggest that the biodegradability of MWCNTs depends not only on the wall thickness but also on the defects and impurities. Thus, the biodegradability can be regulated by the synthesis conditions or the post-synthesis modifications. Such degradation flexibility may be important for both medical and industrial applications.