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1. Isolation of intact extracellular vesicles from cryopreserved samples

   https://doi.org/10.1371/journal.pone.0251290  

   Tessier, Shannon N. ; Bookstaver, Lauren D. ; Angpraseuth, Cindy ; Stannard, Cleo J. ; Marques, Beatriz ; Ho, Uyen K. ; Muzikansky, Alona ; Aldikacti, Berent ; Reátegui, Eduardo ; Rabe, Daniel C. ; et al ( May 2021 , PLOS ONE)

   Johnson, Colin (Ed.)

   Extracellular vesicles (EVs) have emerged as promising candidates in biomarker discovery and diagnostics. Protected by the lipid bilayer, the molecular content of EVs in diverse biofluids are protected from RNases and proteases in the surrounding environment that may rapidly degrade targets of interests. Nonetheless, cryopreservation of EV-containing samples to -80°C may expose the lipid bilayer to physical and biological stressors which may result in cryoinjury and contribute to changes in EV yield, function, or molecular cargo. In the present work, we systematically evaluate the effect of cryopreservation at -80°C for a relatively short duration of storage (up to 12 days) on plasma- and media-derived EV particle count and/or RNA yield/quality, as compared to paired fresh controls. On average, we found that the plasma-derived EV concentration of stored samples decreased to 23% of fresh samples. Further, this significant decrease in EV particle count was matched with a corresponding significant decrease in RNA yield whereby plasma-derived stored samples contained only 47–52% of the total RNA from fresh samples, depending on the extraction method used. Similarly, media-derived EVs showed a statistically significant decrease in RNA yield whereby stored samples were 58% of the total RNA from fresh samples. In contrast, we did not obtain clear evidence of decreased RNA quality through analysis of RNA traces. These results suggest that samples stored for up to 12 days can indeed produce high-quality RNA; however, we note that when directly comparing fresh versus cryopreserved samples without cryoprotective agents there are significant losses in total RNA. Finally, we demonstrate that the addition of the commonly used cryoprotectant agent, DMSO, alongside greater control of the rate of cooling/warming, can rescue EVs from damaging ice formation and improve RNA yield. 

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2. A novel method of high‐purity extracellular vesicle enrichment from microliter‐scale human serum for proteomic analysis

   https://doi.org/10.1002/elps.202000223  

   Ji, Xiaohui ; Huang, Sisi ; Zhang, Jie ; Bruce, Terri F. ; Tan, Zhijing ; Wang, Donglin ; Zhu, Jianhui ; Marcus, R. Kenneth ; Lubman, David M. ( February 2021 , ELECTROPHORESIS)

   We have developed a rapid, low‐cost, and simple separation strategy to separate extracellular vesicles (EVs) from a small amount of serum (i.e.,<100 μL) with minimal contamination by serum proteins and lipoprotein particles to meet the high purity requirement for EV proteome analysis. EVs were separated by a novel polyester capillary channel polymer (PET C‐CP) fiber phase/hydrophobic interaction chromatography (HIC) method which is rapid and can process small size samples. The collected EV fractions were subjected to a post‐column cleanup protocol using a centrifugal filter to perform buffer exchange and eliminate potential coeluting non‐EV proteins while minimizing EV sample loss. Downstream characterization demonstrated that our current strategy can separate EVs with the anticipated exosome‐like particle size distribution and high yield (∼1 × 10¹¹EV particles per mL of serum) in approximately 15 min. Proteome profiling of the EVs reveals that a group of genuine EV components were identified that have significantly less high‐abundance blood proteins and lipoprotein particle contamination in comparison to traditional separation methods. The use of this methodology appears to address the major challenges facing EV separation for proteomics analysis. In addition, the EV post‐column cleanup protocol proposed in the current work has the potential to be combined with other separation methods, such as ultracentrifugation (UC), to further purify the separated EV samples.

    

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3. Morphology and kinetics of asphalt binder microstructure at gas, liquid and solid interfaces

   https://doi.org/10.1111/jmi.12842  

   RAMM, A. ; DOWNER, M. C. ; SAKIB, N. ; BHASIN, A. ( November 2019 , Journal of Microscopy)

   We combined optical and atomic force microscopy to observe morphology and kinetics of microstructures (typically referred to as bees) that formed at free surfaces of unmodified Performance Graded (PG) 64‐22 asphalt binders upon cooling from 150°C to room temperature (RT) at 5°C min^–1, and changes in these microstructures when the surface was terminated with a transparent solid (glass) or liquid (glycerol) overlayer. The main findings are: (1) at free binder surfaces, wrinkled microstructures started to form near the crystallization temperature (∼45°C) of saturates such as wax observed by differential scanning calorimetry, then grew to ∼5 µm diameter, ∼25 nm wrinkle amplitude and 10–30% surface area coverage upon cooling to RT, where they persisted indefinitely without observable change in shape or density. (2) Glycerol coverage of the binder surface during cooling reduced wrinkled area and wrinkle amplitude three‐fold compared to free binder surfaces upon initial cooling to RT; continued glycerol coverage at RT eliminated most surface microstructures within ∼4 h. (3) No surface microstructures were observed to form at binder surfaces covered with glass. (4) Submicron bulk microstructures were observed by near‐infrared microscopy beneath the surfaces of all binder samples, with size, shape and density independent of surface coverage. No tendency of such structures to float to the top or sink to the bottom of mm‐thick samples was observed. (5) We attribute the dependence of surface wrinkling on surface coverage to variation in interface tension, based on a thin‐film continuum mechanics model.

   Asphalt binder, or bitumen, is the glue that holds aggregate particles together to form a road surface. It is derived from the heavy residue that remains after distilling gasoline, diesel and other lighter products out of crude oil. Nevertheless, bitumen varies widely in composition and mechanical properties. To avoid expensive road failures, bitumen must be processed after distillation so that its mechanical properties satisfy diverse climate and load requirements. International standards now guide these mechanical properties, but yield varying long‐term performance as local source composition and preparation methods vary.In situdiagnostic methods that can predict bitumen performance independently of processing history are therefore needed. The present work focuses on one promising diagnostic candidate: microscopic observation of internal bitumen structure. Past bitumen microscopy has revealed microstructures of widely varying composition, size, shape and density. A challenge is distinguishing bulk microstructures, which directly influence a binder's mechanical properties, from surface microstructures, which often dominate optical microscopy because of bitumen's opacity and scanning‐probe microscopy because of its inherent surface specificity. In previously published work, we used infrared microscopy to enhance visibility of bulk microstructure. Here, as a foil to this work, we use visible‐wavelength microscopy together with atomic‐force microscopy (AFM) specifically to isolatesurfacemicrostructure, to understand its distinct origin and morphology, and to demonstrate its unique sensitivity to surface alterations. To this end, optical microscopy complements AFM by enabling us to observe surface microstructures form at temperatures (50°C–70°C) at which bitumen's fluidity prevents AFM, and to observe surface microstructure beneath transparent, but chemically inert, liquid (glycerol) and solid (glass) overlayers, which alter surface tension compared to free surfaces. From this study, we learned, first, that, as bitumen cools, distinctly wrinkled surface microstructures form at the same temperature at which independent calorimetric studies showed crystallization in bitumen, causing it to release latent heat of crystallization. This shows that surface microstructures are likely precipitates of the crystallizable component(s). Second, a glycerol overlayer on the cooling bitumen results in smaller, less wrinkled, sparser microstructures, whereas a glass overlayer suppresses them altogether. In contrast, underlying smaller bulk microstructures are unaffected. This shows that surface tension is the driving force behind formation and wrinkling of surface precipitates. Taken together, the work advances our ability to diagnose bitumen samples noninvasively by clearly distinguishing surface from bulk microstructure.

    

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4. Preparation of Nanoparticle-Loaded Extracellular Vesicles Using Direct Flow Filtration

   https://doi.org/10.3390/pharmaceutics15051551  

   Mansur, Shomit ; Habib, Shahriar ; Hawkins, Mikayla ; Brown, Spenser R. ; Weinman, Steven T. ; Bao, Yuping ( May 2023 , Pharmaceutics)

   Extracellular vesicles (EVs) have shown great potential as cell-free therapeutics and biomimetic nanocarriers for drug delivery. However, the potential of EVs is limited by scalable, reproducible production and in vivo tracking after delivery. Here, we report the preparation of quercetin-iron complex nanoparticle-loaded EVs derived from a breast cancer cell line, MDA-MB-231br, using direct flow filtration. The morphology and size of the nanoparticle-loaded EVs were characterized using transmission electron microscopy and dynamic light scattering. The SDS-PAGE gel electrophoresis of those EVs showed several protein bands in the range of 20–100 kDa. The analysis of EV protein markers by a semi-quantitative antibody array confirmed the presence of several typical EV markers, such as ALIX, TSG101, CD63, and CD81. Our EV yield quantification suggested a significant yield increase in direct flow filtration compared with ultracentrifugation. Subsequently, we compared the cellular uptake behaviors of nanoparticle-loaded EVs with free nanoparticles using MDA-MB-231br cell line. Iron staining studies indicated that free nanoparticles were taken up by cells via endocytosis and localized at a certain area within the cells while uniform iron staining across cells was observed for cells treated with nanoparticle-loaded EVs. Our studies demonstrate the feasibility of using direct flow filtration for the production of nanoparticle-loaded EVs from cancer cells. The cellular uptake studies suggested the possibility of deeper penetration of the nanocarriers because the cancer cells readily took up the quercetin-iron complex nanoparticles, and then released nanoparticle-loaded EVs, which can be further delivered to regional cells. 

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5. Porous PDMS‐Based Microsystem (ExoSponge) for Rapid Cost‐Effective Tumor Extracellular Vesicle Isolation and Mass Spectrometry‐Based Metabolic Biomarker Screening

   https://doi.org/10.1002/admt.202201937  

   Marvar, Joseph ; Kumari, Abha ; Onukwugha, Nna‐Emeka ; Achreja, Abhinav ; Meurs, Noah ; Animasahun, Olamide ; Roy, Jyotirmoy ; Paserba, Miya ; Raju, Kruthi Srinivasa ; Fortna, Shawn ; et al ( January 2023 , Advanced Materials Technologies)

   Polydimethylsiloxane (PDMS) is an inexpensive robust polymer that is commonly used as the fundamental fabrication material for soft‐lithography‐based microfluidic devices. Owing to its versatile material properties, there are some attempts to use PDMS as a porous 3D structure for sensing. However, reliable and easy fabrication has been challenging along with the inherent hydrophobic nature of PDMS hindering its use in biomedical sensing applications. Herein, a cleanroom‐free inexpensive method to create 3D porous PDMS structures, “ExoSponge” and the effective surface modification to functionalize its 3D porous structure is reported. The ability of ExoSponge to recover cancer‐associated extracellular vesicles (EVs) from complex biological samples of up to 10 mL in volume is demonstrated. When compared to ultracentrifugation, the ExoSponge showes a significant increase in cancer EV isolation of more than 210%. Targeted ultra‐high pressure liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) is further employed to profile 70 metabolites in the EVs recovered from the lung cancer patient's plasma. The resulting profiles reveal the potential intraexosomal metabolite biomarker, phenylacetylglutamine (PAG), in non‐small cell lung cancer. The high sensitivity, simple usage, and cost‐effectiveness of the ExoSponge platform creates huge potential for rapid, economical and yet specific isolation of exosomes enabling future diagnostic applications of EVs in cancers.

    

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