Oxidative Stress and Extracellular Matrix Remodeling Are Signature Pathways of Extracellular Vesicles Released upon Morphine Exposure on Human Brain Microvascular Endothelial Cells.

Morphine, a commonly used antinociceptive drug in hospitals, is known to cross the blood-brain barrier (BBB) by first passing through brain endothelial cells. Despite its pain-relieving effect, morphine also has detrimental effects, such as the potential induction of redox imbalance in the brain. However, there is still insufficient evidence of these effects on the brain, particularly on the brain endothelial cells and the extracellular vesicles that they naturally release. Indeed, extracellular vesicles (EVs) are nanosized bioparticles produced by almost all cell types and are currently thought to reflect the physiological state of their parent cells. These vesicles have emerged as a promising source of biomarkers by indicating the functional or dysfunctional state of their parent cells and, thus, allowing a better understanding of the biological processes involved in an adverse state. However, there is very little information on the morphine effect on human brain microvascular endothelial cells (HBMECs), and even less on their released EVs. Therefore, the current study aimed at unraveling the detrimental mechanisms of morphine exposure (at 1, 10, 25, 50 and 100 µM) for 24 h on human brain microvascular endothelial cells as well as on their associated EVs. Isolation of EVs was carried out using an affinity-based method. Several orthogonal techniques (NTA, western blotting and proteomics analysis) were used to validate the EVs enrichment, quality and concentration. Data-independent mass spectrometry (DIA-MS)-based proteomics was applied in order to analyze the proteome modulations induced by morphine on HBMECs and EVs. We were able to quantify almost 5500 proteins in HBMECs and 1500 proteins in EVs, of which 256 and 148, respectively, were found to be differentially expressed in at least one condition. Pathway enrichment analysis revealed that the "cell adhesion and extracellular matrix remodeling" process and the "HIF1 pathway", a pathway related to oxidative stress responses, were significantly modulated upon morphine exposure in HBMECs and EVs. Altogether, the combination of proteomics and bioinformatics findings highlighted shared pathways between HBMECs exposed to morphine and their released EVs. These results put forward molecular signatures of morphine-induced toxicity in HBMECs that were also carried by EVs. Therefore, EVs could potentially be regarded as a useful tool to investigate brain endothelial cells dysfunction, and to a different extent, the BBB dysfunction in patient circulation using these "signature pathways".

Glucose-6-phosphate dehydrogenase immobilized onto magnetic beads (G6PDH-Mb) as a generator system for production of NADPH: Development and application in metabolism studies.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) participates in several anabolic and catabolic pathways, being essential in numerous biochemical reactions involving energy release. Most of these reactions require a high amount of NADPH, which can be expensive from an industry point of view. Thus, biotechnology industries developed a great interest in NADPH production. Currently, there are different ways to obtain NADPH in situ, however, the most common is by enzymatic reactions, known as generator systems. Although this approach can be beneficial in terms of cost, the major drawback is the impossibility of reusing the enzyme. To overcome this, enzyme immobilization is a proven alternative. Herein, we report the use of glucose-6-phosphate dehydrogenase immobilized onto magnetic beads (G6PDH-Mb) through glutaraldehyde coupling to produce high amounts of NADPH. The G6PDH-Mbs were kinetically characterized showing a sigmoidal curve. Besides, the stability was evaluated, and their reuse was demonstrated for a period superior to 40 days. The G6PDH-Mb was used to in situ production of the NADPH metabolism experiments, using human liver microsome solutions and either albendazole or fiscalin B as model targets. The production of in vitro metabolites from albendazole and fiscalin B was evaluated by comparing the use of NADPH generated in situ with those obtained by commercial NADPH. Moreover, the activity of the G6PDH-Mb was monitored after using it for five consecutive albendazole metabolism reactions, with only a minor decrease in the enzyme activity (3.58 ± 1.67%) after the fifth time of use. The higher concentration obtained when using the designed G6PDH-Mb generator system demonstrated proof of the concept and its applicability.

Micro- and nano-sized amine-terminated magnetic beads in a ligand fishing assay.

Functionalized micro- and nano-sized magnetic beads (MBs) have been widely used as versatile supports for proteins, enzymes, and drugs. Immobilized protein on MB surfaces has been successfully applied for ligand fishing assays allowing for direct identification of active ligands from complex mixtures, such as natural products and synthetic libraries. MBs with different properties such as different core compositions, sizes, coatings, and surface modifications are available commercially. Studies have been conducted to understand the role of these properties for ligand fishing assays. Here we evaluated, for the first time, the effect of MB size on the ligand fishing assay for acetylcholinesterase from Electrophorus electricus (AChE). For this purpose, four commercially available amine-terminated magnetic particles with diameters ranging from 4.5 nm to 106 µm were evaluated to fish out galantamine, a well-known AChE inhibitor, from an aqueous solution. All MBs were efficient at using glutaraldehyde to covalently immobilize AChE. The particles with diameters of about 1 µm (small microparticles) presented a higher protein mass capacity per milligram of particle than did those with diameters of about 4.5 nm (nanoparticles) and those with diameters of about 106 µm (large microparticles). The influence of these supports on the produced AChE-MBs with regards to hydrolysis turnover and ligand fishing was evaluated and is fully discussed.