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Extracellular vesicles (EVs) are membrane-bound nanoparticles (50–1000 nm) secreted by all cell types and play critical roles in various biological processes. Among these, exosomes, a smaller subset of EVs, have attracted considerable interest due to their potential applications in diagnostics and therapeutics. However, conventional EV isolation methods are often limited by inefficiencies in processing time, recovery, and scalability. Hydrophobic interaction chromatography utilizing capillary-channeled polymer (C–CP) fiber stationary phases offers a promising alternative, enabling rapid (<15 min), cost-effective (~$5 per column) EV isolation with high loading capacities (~1010–10¹² particles) and minimal sample pre-processing. Despite these advantages, achieving high-throughput EV isolation for larger-scale applications using the C–CP fiber platform is the present challenge. To this end, further optimization of stationary phase packing and adsorption conditions is necessary to maximize the available binding surface area in the current microbore column format. This study systematically investigates the influence of interstitial fraction (i.e. packing density) in polyester (PET) C–CP fiber columns on the dynamic binding capacity (DBC) of EVs isolated from human urine using a high-performance liquid chromatography platform. Microbore columns (0.76 mm i.d. × 300 mm) packed with PET C–CP fibers in both an eight-channel (PET-8) and a novel trilobal (PET-Y) configuration were evaluated using breakthrough curves and frontal analysis. The results reveal that lower packing densities correlate with higher mass- and surface areabased EV binding capacities, with a maximum DBCs of 2.86 × 10¹³ EVs g-1 fiber and 1.22 × 10¹⁴ EVs m⁻² fiber achieved in <2 min of sample loading. Under optimum conditions, surface utilization of >50 % is realized. These results establish a framework for optimizing C–CP fiber-based platforms to enhance EV capture efficiency, facilitating the development of scalable EV isolation techniques for biomedical research and therapeutic applications.
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Isolation of bovine milk–derived extracellular vesicles via a capillary-channeled polymer (C-CP) fiber stationary phase
Abstract Extracellular vesicles (EVs) are released by all cell types into the extracellular environment. A subset of EVs, known as exosomes, range in size from 30 to 200 nm and are of biochemical interest due to their function as vehicles of intercellular communication. Their ability to transport proteinaceous species and genetic material at the cellular level makes them prime candidates as vectors in gene therapies. Focusing on biotherapeutics, bovine milk–derived extracellular vesicles (MDEVs) hold particular promise as an alternative to other exosome sources for therapeutics delivery. Bovine milk poses unique challenges due to the complex colloidal matrix, composed predominantly of fats and proteins like casein, which form micelles that exhibit exosome-like characteristics, specifically size and density. When faced with complex matrices like milk, conventional size/density-based isolation methods including ultracentrifugation and size exclusion chromatography struggle to provide high purity yields on practical time and cost scales. When paired with a stepwise hydrophobic interaction chromatography (HIC) gradient, polyester (PET) capillary-channeled polymer (C-CP) fibers in column and spin-down tips formats have been used effectively to isolate exosomes from highly diverse sources. Here, PET C-CP fiber columns are demonstrated to isolate MDEVs from pre-treated raw milk, yielding concentrations of 1.5 × 1010particles mL⁻1with purities of ~2 × 1010EVs µg−1protein in less than 20 min. The efficacy of the isolation process is verified by a suite of characterization methods. Implementing PET C-CP fiber columns for MDEV isolation addresses the challenges associated with conventional isolation methods, holding promise for scale-up towards therapeutic applications.
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- Award ID(s):
- 2107882
- PAR ID:
- 10577320
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Analytical and Bioanalytical Chemistry
- Volume:
- 417
- Issue:
- 11
- ISSN:
- 1618-2642
- Format(s):
- Medium: X Size: p. 2345-2359
- Size(s):
- p. 2345-2359
- Sponsoring Org:
- National Science Foundation
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