Extracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5–100 mL) can be significantly enriched (up to 1000 times), with up to 99% removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads to target EV subpopulations. Serum from a cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).
Apohemoglobin (apoHb) is a dimeric globular protein with two vacant heme‐binding pockets that can bind heme or other hydrophobic ligands. Purification of apoHb is based on partial hemoglobin (Hb) unfolding to facilitate heme extraction into an organic solvent. However, current production methods are time consuming, difficult to scale up, and use highly flammable and toxic solvents. In this study, a novel and scalable apoHb production method was developed using an acidified ethanol solution to extract the hydrophobic heme ligand into solution and tangential flow filtration to separate heme from the resultant apoprotein. Total protein and active protein yields were >95% and ~75%, respectively, with <1% residual heme in apoHb preparations and >99% purity from sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. Virtually no loss of apoHb activity was detected at 4°C, −80°C, and in lyophilized form during long term storage. Structurally, size exclusion chromatography (SEC) and circular dichroism indicated that apoHb was dimeric with a ~25% reduction of helical content compared to Hb. Furthermore, mass spectroscopy and reverse‐phase chromatography indicated that the mass of the α and β subunits were virtually identical to the theoretical mass of these subunits in Hb and had no detectable oxidative modifications upon heme removal from Hb. SEC confirmed that apoHb bound to haptoglobin at a similar ratio to that of native Hb. Finally, reconstituted Hb (rHb) was processed via a hemichrome removal method to isolate functional rHb for biophysical characterization in which the O2 equilibrium curve, O2 dissociation, and CO association kinetics of rHb were virtually identical to native Hb. Overall, this study describes a novel and improved method to produce apoHb, as well as presents a comprehensive biochemical analysis of apoHb and rHb.
Hemoglobin
(Hb)-based oxygen carriers (HBOCs) present an alternative
to red blood cells (RBCs) when blood is not available. However, the
most widely used synthesis techniques have fundamental flaws, which
may have contributed toward disappointing clinical application. Polymerized
Hb contains a heterogeneous distribution of particle size and shape,
while Hb encapsulation inside liposomes results in high lipid burden
and low Hb content. Meanwhile, there are a variety of other nanoparticle
synthetic techniques which, having found success as drug delivery
vehicles, may be well suited to function as an HBOC. We synthesized
desolvated Hb nanoparticles (Hb-dNPs) with diameters of approximately
250 nm by the controlled precipitation of Hb with ethanol. Oxidized
dextran was found to be an effective surface stabilizing agent that
maintained particle integrity. In vitro biophysical characterization
showed a high-affinity oxygen delivery profile (P
50 = 7.72 mm Hg), suggesting a potential for therapeutic
use and opening a new avenue for HBOC research.
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