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Aptamer loaded superparamagnetic beads for selective capturing and gentle release of activated protein C.

ABSTRACT: Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities which make it an attractive target for diagnostic and therapeutic applications. In this work, we present one-step activation of APC from a commercial source of protein C (PC, Ceprotin) followed by rapid and efficient purification using an APC-specific aptamer, HS02-52G, loaded on MyOne superparamagnetic beads. Due to the Ca²⁺-dependent binding of APC to HS02-52G, an efficient capturing of APC was applied in the presence of Ca²⁺ ions, while a gentle release of captured APC was achieved in the elution buffer containing low EDTA concentration (5 mM). The captured and eluted APC showed more than 95% purity according to SDS-PAGE gel analysis and an enzyme-linked fluorescent assay (VIDAS Protein C). The purification yield of 45% was calculated when 4.2 µg APC was used, however this yield reduced to 21% if the starting amount of APC increased to 28.5 µg. Altogether, this method is recommended for rapid and efficient PC activation and APC purification. The purified APC can be used directly for downstream processes where high concentration of pure and active APC is needed.

SUBMITTER: Hamedani NS

PROVIDER: S-EPMC9056527 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Aptamer loaded superparamagnetic beads for selective capturing and gentle release of activated protein C.

Hamedani Nasim Shahidi NS Happich Felix Lucian FL Klein Eva-Maria EM Rühl Heiko H Mayer Günter G Oldenburg Johannes J Müller Jens J Pötzsch Bernd B

Scientific reports 20220430 1

Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities which make it an attractive target for diagnostic and therapeutic applications. In this work, we present one-step activation of APC from a commercial source of protein C (PC, Ceprotin) followed by rapid and efficient purification using an APC-specific aptamer, HS02-52G, loaded on MyOne superparamagnetic beads. Due to the Ca<sup>2+</sup>-dependent binding of APC to HS02-52G, an efficient capturing of A ...[more]

PMID: 35490167

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Project description:The clinical benefit of therapies using Mesenchymal Stem Cells (MSCs) is attributable to their pleiotropic effect over cells and tissues, mainly through their secretome. This paracrine effect is mediated by secreted growth factors and extracellular vesicles (EV) including small EV (sEV). sEV are extra-cellular, membrane encompassed vesicles of 40 to 200 nm diameter that can trigger and signal many cellular responses depending on their cargo protein and nucleic acid repertoire. sEV are purified from cell culture conditioned media using several kits and protocols available that can be tedious and time-consuming, involving sequences of ultracentrifugations and density gradient separations, making their production a major challenge under Good Manufacturing Practices (GMP) conditions. We have developed a method to efficiently enrich cell culture media with high concentrations of sEV by encapsulating cells in semipermeable cellulose beads that allows selectively the release of small particles while offering a 3D culture condition. This method is based on the pore size of the capsules, allowing the release of particles of ≤ 200 nm including sEV. As a proof-of-principle, MSCs were encapsulated and their sEV release rate (sEV-Cap) was monitored throughout the culture and compared to sEV isolated from 2D seeded cells (sEV-2D) by repetitive ultracentrifugation cycles or a commercial kit. The isolated sEV expressed CD63, CD9, and CD81 as confirmed by flow cytometry analysis. Under transmission electron microscopy (TEM), they displayed the similar rounded morphology as sEV-2D. Their corresponding diameter size was validated by nanoparticle tracking analysis (NTA). Interestingly, sEV-Cap retained the expected biological activities of MSCs, including a pro-angiogenic effect over endothelial cells, neuritic outgrowth stimulation in hippocampal neurons and immunosuppression of T cells in vitro. Here, we successfully present a novel, cost, and time-saving method to generate sEV from encapsulated MSCs. Future applications include using encapsulated cells as a retrievable delivery device that can interact with the host niche by releasing active agents in vivo, including sEV, growth factors, hormones, and small molecules, while avoiding cell clearance, and the negative side-effect of releasing undesired components including apoptotic bodies. Finally, particles produced following the encapsulation protocol display beneficial features for their use as drug-loaded delivery vehicles.

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Aptamer loaded superparamagnetic beads for selective capturing and gentle release of activated protein C.

Publications

Aptamer loaded superparamagnetic beads for selective capturing and gentle release of activated protein C.

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