The use of enzymes immobilized on magnetic nanoparticles to detect contaminants in aqueous samples has gained interest, since it allows the magnetic control, concentration and reuse of the enzymes. In this work, the detection of trace amounts of organophosphate pesticides (chlorpyrifos) and antibiotics (penicillin G) in water was attained by developing a nanoassembly formed by either inorganic or biomimetic magnetic nanoparticles used as substrates to immobilize acetylcholinesterase (AChE) and β-lactamase (BL). Other than the substrate, the optimization of the nanoassembly was done by testing enzyme immobilization both through electrostatic interaction (also reinforced with glutaraldehyde) and covalent bonds (by carbodiimide chemistry). Temperature (25 • C), ionic strength (150 mM NaCl) and pH (7) were set to ensure enzymatic stability and to allow both the nanoparticles and the enzymes to present ionic charges that would allow electrostatic interaction. Under these conditions, the enzyme load on the nanoparticles was ⁓0.1 mg enzyme per mg nanoparticles, and the preserved activity after immobilization was 50-60% of the specific activity of the free enzyme, being covalent bonding the one which yielded better results. Covalent nanoassemblies could detect trace concentrations of pollutants down to 1.43 nM chlorpyrifos and 0.28 nM penicillin G. They even permitted the quantification of 14.3 μM chlorpyrifos and 2.8 μM penicillin G. Also, immobilization conferred higher stability to AChE (⁓94% activity after 20 days storage at 4 • C) and allowed to reuse the BL up to 12 cycles.

Citation: Donini, M.; Pettinella, F.; Zanella, G.; Gaglio, S.C.; Laudanna, C.; Jimenez-Carretero, M.; Jimenez-Lopez, C.; Perduca, M.; Dusi, S. Effects of Magnetic Nanoparticles on the Functional Activity of Human Monocytes and Dendritic Cells. Int. J. Mol. Sci. 2023, 24, 1358. https:// (M.P.) † These authors contributed equally to this work. ‡ These authors equally contributed to conceptualization and supervision. Abstract: The use of nanoparticles in medicine is sometimes hampered by their potential to activate immune cells, eliciting inflammation or allergy. We investigated whether magnetic nanoparticles (MNPs) or biomimetic magnetic nanoparticles (BMNPs) affect relevant activities of human monocytes. We found that the nanoparticles neither elicited the production of pro-inflammatory mediators IL-6 and TNFα by resting monocytes (when BMNP dose < 300 µg/mL) nor enhanced their secretion induced by R848, a molecule engaging virus-recognizing receptors, or bacterial lipopolysaccharide (LPS). MNPs and BMNPs neither induced the generation of reactive oxygen species (ROS), nor affected the ROS production elicited by the NADPH oxidase activator phorbol myristate acetate (PMA) or the fungal derivative β-glucan. BMNPs, but not MNPs, caused an up-regulation of the maturation markers CD80, CD83, and CD86 in immature monocyte-derived dendritic cells (DCs), whereas both nanoparticles did not affect the LPS-induced expression of these markers. Moreover, the nanoparticles were greedily ingested by monocytes and DCs without altering their viability. Therefore, these nanoparticles are candidates for medical applications because they do not activate pro-inflammatory activities of monocytes. Furthermore, their ability to stimulate DC maturation could be used for the design of vaccines. Moreover, harmlessly engulfed nanoparticles could be vehicles to carry molecules inside the immune cells to regulate the immune response.