关键词: 微/纳米塑料, 凝聚体, 超氧化物歧化酶, 结构变化, 酶活

Abstract:

The increasing presence of micro/nanoplastics (MNPs) in diverse ecosystems, coupled with their demonstrated capacity to infiltrate the human body via ingestion, inhalation, and dermal contact, has propelled intense scientific scrutiny of their environmental fate and potential health ramifications. Once they enter the human body, these small particles can aggregate or disperse in biological fluids, leading to significant changes in their effective size, further affecting the response of the antioxidant defense system to external substances. Within this system, key enzymes, such as superoxide dismutase (SOD), which catalyze the dismutation of the highly reactive superoxide radical into oxygen and hydrogen peroxide, serve as the first line of defense. Alterations in SOD activity or structure can lead to significant oxidative damage, which is implicated in inflammation. Therefore, understanding how MNPs influence SOD is crucial for assessing their true biological impact. This study systematically investigated the interactions between polyethylene terephthalate micro/nanoplastics (PET-MNPs) with distinct primary sizes (50 nm, denoted NP50; 100 nm, denoted NP100; and 1100 nm, denoted NP1100) on SOD, focusing on the aggregation behavior of the particles in artificial lysosomal fluid (ALF) and their impact on SOD enzyme activity and function, to explore the underlying mechanisms. The analysis of aggregation in ALF showed a pronounced effect on size. The small particles (NP50 and NP100) aggregated rapidly. NP50 aggregates grew beyond 1 μm within 1 h, whereas NP100 aggregates grew more gradually. In stark contrast, NP1100 remained stable. Remarkably, the presence of SOD significantly accelerated the aggregation rates of NP50 and NP100 at all temperatures. Zeta potential analysis provided mechanistic insights: SOD addition increased the zeta potential of NP50 from −15 to −7 mV and drove NP100 potential towards neutrality, reducing electrostatic repulsion. NP1100’s zeta potential remained unchanged at −3 mV. This size-dependent potential response difference may be related to the varying interaction capacity of SOD with particle surfaces of different sizes. Smaller NP50 particles have a higher specific surface area and stronger surface reactivity, facilitating easier binding/adsorption to SOD molecules. This partially neutralized the surface negative charges and significantly increased the zeta potential. Additionally, SOD binding may compress the thickness of the particle’s electrical double layer, further reducing electrostatic repulsion and promoting aggregation. In contrast, larger NP1100 particles have a lower specific surface area, reduced probability of binding with SOD, and less affected surface charge, resulting in minimal potential changes and higher colloidal stability. Notably, NP50’s size change rate significantly exceeded those of NP100 and NP1100, indicating that the particle size profoundly influences the aggregation behavior. Smaller NP50 particles exhibited greater surface energy and collision probabilities, resulting in pronounced aggregation. In contrast, although larger particles (NP1100) have a zeta potential with a lower absolute value (|−3 mV|), their relatively lower surface energy hinders the overcoming of the DLVO energy barrier, resulting in a higher colloidal stability. In the ALF system, SOD activity responses to PET-MNPs of different sizes were significantly different. The experimental results revealed that exposure to both NP50 and NP100 decreased SOD activity, indicating that smaller PET-MNPs disrupt enzyme function more readily. In contrast, NP1100 showed negligible inhibition of SOD activity, suggesting weaker interference from larger particles. Because small PET-MNPs (NP50 and NP100) rapidly aggregate in ALF, the impact of differently sized aggregates on enzyme activity was further examined. The results showed that during early aggregation (within 5 min), when NP50 and NP100 had not yet formed distinct aggregates, the SOD activity decreased. Subsequently, as aggregates formed (>500 nm), SOD activity began to recover, possibly reflecting an adaptive regulatory mechanism of SOD. When the aggregate size exceeded 1000 nm, the SOD activity continued to increase. As NP50 and NP100 form larger aggregates, partial surface sites become occluded, reducing the direct interactions between PET-MNPs and SOD and alleviating the structural interference. This demonstrates that the aggregation state of MNPs significantly modulates their impact on enzyme activity, providing experimental evidence for understanding the relationship between the physical properties of the particles and the functional interference of the enzyme. Spectroscopic analyses revealed the structural basis for the size-dependent inhibition of SOD activity. Circular Dichroism (CD) spectroscopy showed that SOD's native secondary structure comprised 38.1% α-helix, 15.2% β-sheet, 17.0% β-turn, and 29.8% random coil. Exposure to PET-MNPs, particularly NP50 and NP100, drastically reduced α-helix content to 12.94% and 13.05%, respectively, while increasing β-sheet structures, indicating significant protein unfolding, hydrogen bond rearrangement, and loss of conformational stability. NP1100 also reduced the α-helix content to 13.49%, but to a lesser extent than the smaller particles. The greater structural perturbation by small NPs is attributed to their higher surface reactivity, which facilitates stronger interactions with critical α-helical domains of proteins. Combined with the enzymatic activity results, we inferred that exposure to small NPs, especially before or during early aggregation, disrupts SOD’s secondary structure more readily. This triggers conformational disturbances near the active center of the enzyme, impairing its catalytic function. Thus, smaller Nps, such as NP50 and NP100, or those that have not yet formed large aggregates, induce stronger structural perturbations, causing significant α-helix loss and activity impairment. In contrast, NP1100 and larger aggregates exert weaker interference because of their smaller specific surface area, limited interaction sites, and potential spatial shielding effects after aggregation, which reduce directcontact with enzyme and provide buffering protection. Three-dimensional fluorescence spectroscopy revealed that the control SOD exhibited characteristic peaks at Ex=270/Em=280 nm for the peptide backbone and Ex=215/Em=280 nm for aromatic residues. Exposure to PET-MNPs, particularly NP50 and NP100, resulted in significant fluorescence quenching and peak shifts, indicating alterations in the microenvironment of tyrosine residues and overall structural changes in the protein. NP1100 induced comparatively weaker fluorescence quenching. NP50 and NP100, which readily aggregate in ALF, likely engage in stronger electrostatic interactions with superoxide dismutase (SOD). In contrast, NP1100 exhibited higher fluorescence intensity than the NP50/NP100 groups, suggesting that its interaction with SOD may involve more superficial adsorption, causing less structural damage and better retention of the fluorescence features. These findings highlight that the “effective size” of MNPs in biological compartments, determined by their intrinsic aggregation propensity and modulated by biomolecules such as enzymes, is a crucial parameter for accurately assessing their health risks, particularly in oxidative stress pathways in the respiratory system. Future research should focus on defining the stable size ranges of different MNPs under various physiological conditions and elucidating the in vivo consequences of MNP-induced antioxidant enzyme dysfunction using cellular and animal models.

Key words: micro/nanoplastics, aggregates, superoxide dismutase, structural changes, enzyme activity