(416a) Characterization and ROS Generation Study of Polyacrylic Acid-Coated Superparamagnetic Iron Oxide Nanoparticles for Spacecraft Water System Biofilm Control

Introduction: A primary challenge in space missions is providing vital resources like water for crew members without access to Earth resupply. Water Recovery Management Systems (WRMSs) are essential to sustain life on these travels by recovering nearly 98% of the initial water source [1]. However, due to their high surface area and the moist environment, WRMSs are susceptible to biofilm formation, potentially leading to crew infection and system malfunction. Biocides are being used in many space missions to inhibit bacterial growth [2], but biocide penetration into the biofilm structure is limited by the extracellular polymeric substance (EPS). High biocide dosages are therefore required for biofilm disruption, which can result in water toxicity. This project is focused on synthesizing water-soluble, superparamagnetic iron oxide nanoparticles (SPIONPs) designed to disrupt both established and newly forming biofilms in water recovery systems, reducing the need for high doses of biocides. The anti-biofilm action of SPIONPs is multifaceted; the particles are capable of catalytic production of reactive oxygen species (ROS)—recognized for their antibacterial effects—as well as the disruption of EPS via local hyperthermia and internal shear stresses induced by oscillatory particle motion, thereby enhancing biocide penetration. A challenge with iron oxide nanoparticles is their tendency to aggregate, which negatively impacts their magnetic characteristics. To address this, polymer coatings are used to prevent aggregation. Polyacrylic acid (PAA) was chosen because of its antibacterial and biocompatible properties. Currently, we have successfully synthesized PAA-coated SPIONPs (PAA@SPIONPs) and investigated the SPIONPs' role in modulating ROS production in presence of biocides. Presence of coating on the surface of SPIONPs can potentially enhance the stability and efficacy of ROS generation over time, especially at higher temperatures.

Methods: Water-soluble, stable, and magnetically responsive SPIONPs were synthesized with the co-precipitation method which was optimized in our previously published data [3]. For the coated nanoparticles, PAA (25% w/w with respect to the iron (II) salt) was added to the reaction just before addition of ammonium hydroxide. Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were employed to assess the hydrodynamic size and dry size of nanoparticles, respectively. Fourier transform infrared spectroscopy (FTIR) characterized the coating and its binding to the core SPIONPs. X-ray Photoelectron Spectroscopy (XPS) analyzed the oxidation state of the nanoparticles, crucial for determining their magnetic properties. The catalytic ability of coated and uncoated nanoparticles to generate ROS at various temperatures (21, 50, 60, 70 °C) was investigated by studying methylene blue degradation in the presence of two types of biocides: peracetic acid (PerAA), and hydrogen peroxide (H₂O₂). In this process, two types of biocides: 0.158 M PerAA and 32% w/w H₂O₂ were employed in combination with 0.6 ml of 0.5mM methylene blue and 0.464 ml of nanoparticles (1 mg/ml) for the PerAA biocide application. For the H₂O₂ biocide application, 2.32 ml of nanoparticles with the same concentration was used. The procedure was initiated by heating each test solution to a set temperature in a water bath. Upon reaching the desired temperature, all components were mixed to evaluate the reactive oxygen species (ROS) generation by the nanoparticles in the presence of each biocide. The degradation kinetics of methylene blue was evaluated for a period of 60 min. Prior to analysis of methylene blue degradation, the reaction in collected samples was halted by adding 7 ml of 1.58M sodium thiosulfate.

Results and Discussion: Our findings suggest that the addition of PAA to the synthesis step reduces the size of SPIONPs, with mean hydrodynamic sizes being 104.9±13.74 nm for uncoated SPIONPs and 84.88±3.52 nm for PAA-coated ones. Dry sizes, as measured via TEM, were 16.53±3.98 nm and 8.9±3.0 nm, respectively (Figure 1-(a,b)). TEM confirmed that our nanoparticles are within the size range (<30 nm) that is necessary for superparamagnetic behavior, which is crucial for hyperthermia applications [4], [5]. The XRD pattern (Figure 1-c) showed the six strong peaks that are correspond to crystalline planes of magnetite iron oxide nanoparticles, indicating the successful synthesis [6]. Figure 1-d shows the possible interactions between coating and iron oxide. FTIR analysis demonstrated a significant decrease in the intensity of the peak related to the COOH group on PAA, suggesting that the bonding between the coating and iron is of either type II or III. In XPS spectrum, the characteristic peaks of Fe 2p3/2 and 2p1/2 in Fe₃O₄ were observed, with the absence of a satellite line at 719 eV, indicating pure Fe₃O₄ without -Fe₂O₃ [7,8]. The temperature range of 21-70 °C is used to evaluate the effect of temperature on modulation of ROS generation by SPIONs in the presence and absence of biocides. The most effective degradation occurred at 60 °C in the presence of both biocides. Figure 1-e shows the methylene blue degradation rate using both coated and uncoated nanoparticles at 60 °C in presence of PerAA. For both coated and uncoated nanoparticles, the degradation rate was higher in the first 10 minutes, followed a decrease. The decreased degradation rate in the coated sample was less significant, resulting in a stable and prolonged degradation over time, whereas the uncoated samples exhibited almost no noticeable degradation after 10 minutes. Figure 1-f presents a similar study, but with H₂O₂ as the biocide. In this scenario, the degradation of methylene blue was more consistent for both nanoparticle types. However, the coated nanoparticles demonstrated significantly higher methylene blue degradation activity. Given our results, the presence of a coating on nanoparticles controls the release of ROS from biocides, leading to a more gradual and sustained generation of ROS, and higher methylene blue degradation rates. This indicates that the coating significantly enhances the particles' catalytic activity. The overall results demonstrated that the presence of both SPIONPs and PAA@SPIONPs could augment ROS generation in presence of biocides over time. As anticipated, the combination of SPIONPs with biocides at elevated temperature enhances their effectiveness through catalytic behavior of nanoparticles, leading to increased ROS production. Our preliminary hyperthermia studies using alternate magnetic field indicate that the temperature effects can be created to modulate ROS generation using biocides. However, we are still in the initial stages of this study, and further assessment is required before we can expose the nanoparticles to biofilms under an alternating magnetic field to study hyperthermia effects.

Conclusion: The synthesized iron oxide nanoparticles are appropriately sized and oxidized for superparamagnetic behavior. The coating has resulted in smaller, more stable nanoparticles and sustainable ROS generation at 60 °C, proving its potential for future hyperthermia studies. Future research will explore the impact of a magnetic field on ROS generation and expose these nanoparticles to bacterial cells to study the combined effects of hyperthermia and ROS generation on bacterial cells.

Acknowledgments: This work was supported by the National Aeronautics and Space Administration (NASA) under Grant No. 80NSSC21M0325.

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