Instituto de Nanociencia y Nanotecnología - Nodo Bariloche, CNEA-CONICET, Centro Atómico Bariloche, Av. Bustillo 9500, 8400, S. C. Bariloche, Argentina.
Nanoscale. 2019 Oct 10;11(39):18393-18406. doi: 10.1039/c9nr05799d.
Iron oxide nanoparticles (IONPs) are frequently used in biomedical applications due to their magnetic properties and putative chemical stability. Nevertheless, their well-known ability to mimic some features of the peroxidase enzyme activity under specific conditions of pH and temperature could lead to the formation of potentially harmful free radical species. In addition to the intrinsic enzyme-like activity of IONPs, the buffer solution is an important external factor that can alter dramatically the IONP activity because the buffer species can interact with the surface of the particles. In our study, IONP activity was evaluated in different buffering solutions under different experimental conditions and predominant free radical species were measured by electron paramagnetic resonance using the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The formation kinetics of the reactive oxygen species were studied by UV-visible spectroscopy with TMB and DAB peroxidase substrates. We found that the highest catalytic oxidation of peroxidase substrates and free radical generation were achieved in acetate buffer, while phosphate buffer inhibited the peroxidase-like activity of IONPs in a concentration dependent manner. When emulating the physiological conditions, a lower catalytic activity was observed at pH 7.4 when compared to that at pH 5.0. Also, in phosphate buffered saline (PBS), we observed an enhancement in the peroxidase substrate oxidation rate that was not accompanied by an increase in DMPO/adduct formation which could be related to a non-specific oxidation catalyzed by the chloride ion. Similar observations were found after the addition of a bicarbonate to HEPES buffer. TMB oxidation did not occur when the reaction was conducted with free iron ions from metal salts with the same concentration of the IONPs (0.33 Fe2+ and 0.66 Fe3+). However, we observed even higher catalytic activities than those when doubling the IONP concentration when they are combined with the free iron salts. These results indicate that biological buffering solutions need to be carefully considered when evaluating IONP catalytic activity and their potential toxicological effects since under physiological conditions of pH, salinity and buffering species, the peroxidase-like activity of IONPs is dramatically reduced.
氧化铁纳米粒子（IONP）由于其磁性和潜在的化学稳定性，经常被用于生物医学应用。然而，它们在特定的 pH 和温度条件下模拟过氧化物酶活性的已知能力可能导致潜在有害自由基的形成。除了 IONP 的内在酶样活性外，缓冲溶液是一个重要的外部因素，可以极大地改变 IONP 的活性，因为缓冲物质可以与粒子表面相互作用。在我们的研究中，在不同的实验条件下，在不同的缓冲溶液中评估了 IONP 的活性，并通过使用自旋捕集剂 5,5-二甲基-1-吡咯啉 N-氧化物（DMPO）的电子顺磁共振测量了主要的自由基种类。通过使用 TMB 和 DAB 过氧化物酶底物的紫外可见光谱研究了活性氧物种的形成动力学。我们发现，在醋酸盐缓冲液中，过氧化物酶底物的最高催化氧化和自由基生成，而磷酸盐缓冲液则以浓度依赖的方式抑制 IONP 的过氧化物酶样活性。当模拟生理条件时，与 pH 5.0 相比，在 pH 7.4 时观察到较低的催化活性。此外，在磷酸盐缓冲盐水（PBS）中，我们观察到过氧化物酶底物氧化速率的增加，但没有伴随 DMPO/加合物形成的增加，这可能与氯离子催化的非特异性氧化有关。在向 HEPES 缓冲液中添加碳酸氢盐后，也观察到了类似的现象。当用具有与 IONP 相同浓度的（0.33 Fe2+和 0.66 Fe3+）金属盐中的游离铁离子进行反应时，TMB 氧化没有发生。然而，当它们与游离铁盐结合时，我们观察到甚至比 IONP 浓度加倍时更高的催化活性。这些结果表明，在评估 IONP 催化活性及其潜在的毒理学效应时，需要仔细考虑生物缓冲溶液，因为在 pH、盐度和缓冲物质的生理条件下，IONP 的过氧化物酶样活性会大大降低。
