Selenite-Incorporated Amorphous Calcium-Magnesium Carbonate Nanoparticles Reduce Bacterial Growth

Selenite-Incorporated Amorphous Calcium−Magnesium Carbonate Nanoparticles Reduce Bacterial Growth Yagmur Göctu, Cagatay M. Oral, and Batur Ercan* Cite This: ACS Appl. Nano Mater. 2023, 6, 16286−16296 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Amorphous calcium carbonate (ACC) is a nontoxic and degradable nanomaterial. ACC can be synthesized using the coprecipitation technique, which enables the incorporation of ions into its amorphous structure. Although ACC has been investigated for various applications, such as wastewater treatment, in vivo imaging, and drug delivery, its antibacterial properties have not been explored. Considering the extraordinary capability of bacteria to adapt antimicrobial strategies, as well as the extensive burden of bacteria-induced problems on healthcare systems and the world economy, the need for effective antibacterial agents is becoming a pressing issue. Herein, we introduced selenite-incorporated magnesium-stabilized amorphous calcium carbonate (ACMC) nanoparticles as a sustainable antibacterial material. For the first time, we demonstrated that selenite ions could be incorporated into ACMC nanoparticles while preserving the amorphous structure. Antibacterial activity analysis showed that selenite-incorporated ACMC (Se-ACMC) nanoparticles at 1 g/L concentration could significantly reduce the growth of Gram-positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria strains within 24 h of interaction. As an important observation, even the lowest selenite incorporation (4.38 ±0.19 mg selenium per g of nanoparticles) led to a more than 3-log reduction in the number of S. epidermidis colonies. Additionally, the antibacterial activity was enhanced with an increase in the amount of incorporated selenite. These results indicated that ion-incorporated ACMC nanoparticles can pave the way for applications as antibacterial agents. KEYWORDS: CaCO3, amorphous, ion incorporation, stabilization, antibacterial 1. INTRODUCTION Calcium carbonate (CaCO3) is a well-known mineral used in numerous industrial applications. 1 CaCO3has three anhydrous crystalline polymorphs (vaterite, aragonite, and calcite) and two hydrous forms (monohydrocalcite and ikaite). 2 Aside from its crystalline polymorphs, CaCO3is also present in a noncrystalline form as amorphous calcium carbonate (ACC). ACC is the transient precursor of the crystalline polymorphs, 3 and due to its metastable nature, it transforms into the more stable forms of CaCO3in aqueous environments via dissolution and recrystallization reactions. 4 Despite the metastable nature of ACC, several organisms in nature, such as sea urchins and mollusks, embody both the amorphous and crystalline CaCO3in their hierarchical structures along with various organic molecules. 5 Hence, inspired by nature, the stabilization of ACC using macromolecules and ions was investigated. 6,7 In the literature, various studies utilized Mg2+ ions to stabilize ACC and demonstrated the precipitation of amorphous calcium−magnesium carbonate (ACMC) nanoparticles. It is known that ACC has two forms: hydrous ACC and anhydrous ACC. Hydrous ACC transforms to anhydrous ACC through dehydration and then, following an energetically downhill path, crystallizes to calcite rapidly. 8 Although the effects of Mg2+ on the stabilization mechanism of ACC have not been clear, it was mainly attributed to the creation of a high dehydration-free energy barrier, which could decelerate the crystallization of calcite. 9 ACC is a nontoxic, biodegradable, and biocompatible nanomaterial, and, consequently, it is a potential candidate for numerous engineering applications. For instance, in the biomedical field, polymer-modified ACC nanoparticles were proposed for cancer treatment by utilizing their pH-dependent degradation and high drug-loading capacity. 10 Likewise, sodium alginate and phosphate-stabilized ACC nanocarriers loaded with curcumin were also investigated to fight against cancer. 7 Gadolinium-doped poly(acrylic acid)-stabilized ACC nanoparticles were investigated as a magnetic resonance Received: May 29, 2023 Accepted: August 29, 2023 Published: September 12, 2023 Articlewww.acsanm.org © 2023 The Authors. Published by American Chemical Society 16286 https://doi.org/10.1021/acsanm.3c02415 ACS Appl. Nano Mater. 2023, 6, 16286−16296 This article is licensed under CC-BY 4.0 Downloaded via BRNO UNIV OF TECHNOLOGY on February 19, 2024 at 14:11:43 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. imaging contrast agent due to the extensive water content of ACC. 11 Additionally, ACC nanoparticles were investigated to eliminate phosphate ions from wastewater due to their high adsorption capacity. 12 These nanoparticles were also proposed as a cement additive for the self-healing of microcracks, 13 and upon stabilization with magnesium, they could be used as an ink in 3D printing. 14 In spite of the potential use of ACC nanoparticles in various industries, their efficacy as antibacterial agents has not been explored. Bacterial colonization and bacteria-driven infection pose a growing threat to the health of individuals. Bacteria are usually apt to gather instead of existing as separate cells. Once bacteria colonize a surface, they synthesize an organic extracellular matrix layer. This structure is called biofilm and makes it much harder to fight against bacteria. 15,16 Furthermore, excessive antibiotic use along with the extraordinary genetic adaption capability of bacteria led to an uncontrollable increase in antibiotic-resistant strains. In fact, it is estimated that the cumulative loss of economic output due to antimicrobial resistance in the OECD countries will amount to approximately $20−35 trillion by 2050. 17 Although there are various strategies to limit bacterial colonization, generation of hazardous byproducts, cost, toxicity, and development of antibiotic resistance are among the problematic issues, which gradually led to decrease in efficacy for most of the utilized antibacterial strategies. 18,19 Consequently, there is a tremendous need to redesign the currently used disinfection methods and antibacterial materials. To address the aforementioned challenges, the utilization of several alternative antibacterial chemistries, such as carbon-based nanomaterials, silver ions, cationic polymers, and selenium (Se) nanoparticles, has captured growing interest in the past decade. 20 Se is an essential element for life with its involvement in the regulation of cellular metabolism for many organisms. 21 It is present in the amino acid selenocysteine (Sec), which eventually forms selenoproteins for various enzymes. 22 In addition, Se can neutralize the toxic effect of heavy metals, such as arsenic and lead. 23 It is also known to have antioxidant and anticancer properties. 24 Among the oxyanions of Se, selenite (SeO32−) has the potential to be incorporated into the ACC structure due to having a similar geometry and charge with carbonate. In the literature, the dose-dependent antibacterial effect of SeO32−ions against several bacteria strains, including Staphylococcus aureus,Escherichia coli, and Pseudomonas aeruginosa, was verified. 25,26 In this study, in an attempt to utilize the degradable nature of Mg2+-stabilized ACC (ACMC) nanoparticles, we introduced SeO32−ions, and thus provided antibacterial characteristics. This is the first study in the literature investigating SeO32−-incorporated ACMC (Se-ACMC) nanoparticles against several bacteria strains (Scheme 1). 2. RESULTS AND DISCUSSION 2.1. Synthesis and Characterization of Se-ACMC Nanoparticles. To synthesize ACMC nanoparticles, aqueous solutions of calcium acetate monohydrate (solution A), sodium bicarbonate, and magnesium chloride hexahydrate (solution B) were prepared separately. Having a high viscosity, ethylene glycol was added to each solution to decrease the diffusion rate of the ions and, consequently, decelerate the crystallization. 27 Afterward, solution A was poured into solution B to initiate ACMC precipitation. SeO32−-ionincorporated nanoparticles were synthesized using the same protocol except for the addition of sodium selenite (0.001, 0.005, or 0.01 M) into solution B prior to mixing with solution A (Figure 1a). According to inductively coupled plasma mass spectrometry (ICP-MS) analysis (Figure 1b), the quantity of Se incorporated into the nanoparticles increased when a higher amount of sodium selenite was used during their synthesis. For Se(I)-ACMC, 4.38 ±0.19 mg of Se was present for 1 g of the nanoparticles, whereas this value increases to 21.83 ±0.19 and 40.98 ±0.14 mg for Se(II)-ACMC and Se(III)-ACMC nanoparticles, respectively. Scanning electron microscopy (SEM) images showed that ACMC nanoparticles had an irregular particle morphology (Figure 1c,e,g,i). It is important to note that low-magnification SEM images did not reveal any sign of a secondary particle morphology independent of the amount of incorporated selenite (Supplementary Figure 1). The particle size distribution histograms were drawn for each nanoparticle specimen, and D50 values were found as 32.0 ±0.4, 31.1 ±0.4, 31.9 ±0.3, and 32.5 ±0.4 nm for ACMC, Se(I)-ACMC, Se(II)-ACMC, and Se(III)-ACMC nanoparticles, respectively (Figure 1d,f,h,j). Transmission electron microscopy (TEM) characterizations (Figures 1k,l) further confirmed that particles had an irregular morphology. The size measurement of the nanoparticles indicated that selenite ion incorporation did not lead to any changes in the particle size. The structures of the nanoparticles were investigated with X-ray diffraction (XRD; Figure 1m), which expressed the characteristic amorphous humps of the ACMC nanoparticles 2 without indicating any secondary crystalline phase (Supplementary Figure 2). Additionally, the selected-area electron diffraction (SAED) patterns of the nanoparticles further validated the amorphous nature of the particles prior to (ACMC) and after the highest selenite incorporation [Se(III)-ACMC]. These results confirmed that the incorporation of selenite into the ACMC nanoparticles did not alter the amorphous structure of the nanoparticles. Scheme 1. Se-ACMC Nanoparticles toward Antibacterial Applications ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.3c02415 ACS Appl. Nano Mater. 2023, 6, 16286−16296 16287 Fourier transform infrared spectroscopy (FTIR) was used to investigate the chemical bonding of the synthesized nanoparticles. In Figure 2a, the characteristic carbonate bands of ACMC are designated by gray sections. The symmetric stretching band at ∼1084 cm−1(ν1), the out-of-plane band at ∼859 cm−1(ν2), the asymmetric stretching bands at 1412 and 1477 cm−1(ν3), and in-plane deformation bands at around 688 and 728 cm−1(ν4) are associated with the carbonate ions in ACMC. 28 Furthermore, the lack of calcite peak at 718 cm−1, vaterite peak at 745 cm−1, and aragonite peaks at 700 and 712 cm−1indicated that crystalline polymorphs did not form. 29 Compared to the FTIR spectrum of ACMC, Se-ACMC nanoparticles expressed a band at ∼770 cm−1, which could be ascribed to the Se−O bending vibrations of selenite. 30 The intensity of this band increased with an increase in the selenite concentration. The wide band at 3400 cm−1and the small band at 1640 cm−1were attributed to O−H bonding: stretching and bending vibrations of water, respectively, which could stem from the physically and chemically bonded water and hydroxide ions in the molecule. 31 Wide-scan X-ray photoelectron spectroscopy (XPS) results show the elemental composition of the nanoparticles (Figure 2b). The presence of calcium, magnesium, and oxygen elements in all of the samples and Se in Se-ACMC Figure 1. (a) Sketch showing the synthesis protocol for the nanoparticles. (b) ICP-MS results of Se(I)-ACMC, Se(II)-ACMC, and Se(III)-ACMC nanoparticles showing milligrams of Se per gram of nanoparticles. SEM images of (c) ACMC, (e) Se(I)-ACMC, (g) Se(II)-ACMC, and (i) Se(III)-ACMC nanoparticles (the scale bars are 500 nm). Particle size histograms of (d) ACMC, (f) Se(I)-ACMC, (h) Se(II)-ACMC, and (j) Se(III)-ACMC nanoparticles. TEM images and SAED patterns of (k) ACMC and (l) Se(III)-ACMC nanoparticles (the scale bars are 100 nm). (m) XRD patterns of the nanoparticles. ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.3c02415 ACS Appl. Nano Mater. 2023, 6, 16286−16296 16288 nanoparticles was confirmed with the analysis. The presence of these elements was also in line with energy-dispersive X-ray spectroscopy (EDS) analysis of the nanoparticles (Supplementary Figure 1). Furthermore, a high-resolution scan of the 3d core level of Se was performed to determine its oxidation state (Figure 2c,d). Since Se(I)-ACMC nanoparticles had the lowest Se incorporation, they did not provide an intense signal sufficient enough to accurately perform the deconvolution process. On the other hand, the binding energy of Se 3d5/2 was found to be 58.97 eV for Se(II)-ACMC (Figure 2c) and 59.10 eV for Se(III)-ACMC (Figure 2d), which could indicate the oxidation state as Se4+, highlighting the presence of selenite. 32 The thermal properties and water fraction of the nanoparticles were determined by thermogravimetric analysis (TGA; Figure 3). The TGA curve of ACMC nanoparticles exhibited three major mass loss events upon heating to 800 °C (Figure 3a). These mass loss events could be attributed to the removal of physically and chemically bound water from the nanoparticles, decomposition to MgO, and decomposition to CaO, respectively. 33 XRD analysis of the heat-treated nanoparticles revealed that the amorphous structure of the ACMC nanoparticles could be maintained to a large extent until approximately 300 °C (Supplementary Figure 3). Once chemically bound water was removed from the system, ACMC nanoparticles crystallized as magnesian calcite. In addition to this, in the second event, magnesian calcite crystals decomposed into MgO, and the removal of CO2from the system led to the second mass loss. In the third event, complete decomposition to CaO and MgO occurred with the removal of the remaining CO2from the system. For the SeACMC nanoparticles (Figure 3b−d), similar to ACMC, the initial elimination of physically and chemically bound water and decomposition events were also observed. It is worth mentioning that, depending on the selenite concentration, the water content of the ACMC nanoparticles initially increased up to Se(II)-ACMC and then decreased for the Se(III)-ACMC nanoparticles. While ACMC nanoparticles had ∼17% water, Se(I)-ACMC, Se(II)-ACMC, and Se(III)-ACMC nanoparticles had approximately 28, 34, and 20% water, respectively. This indicated that selenite incorporation increased the hydration level of ACMC. For the second mass loss event, unlike ACMC, the derivative weight loss curves of Se-ACMC nanoparticles expressed double peaks. The expression of two peaks with overlapping temperatures indicated two separate decomposition events occurring for the Se-ACMC nanoparticles. We believe that a selenite phase formed during crystallization of the Se-ACMC nanoparticles, which further decomposed into a Se compound. We can speculate that the reason for the formation of a secondary selenite phase, rather than selenite being incorporated into the magnesian calcite crystal, could be the size of selenite. In the literature, it was shown that, during the crystallization of phosphate-incorporated ACC, calcite and hydroxyapatite phases formed separately since phosphate ions Figure 2. (a) FTIR and (b) wide-scan XPS spectra of the nanoparticles. High-resolution XPS spectra of (c) Se(II)-ACMC and (d) Se(III)-ACMC nanoparticles for Se 3d. ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.3c02415 ACS Appl. Nano Mater. 2023, 6, 16286−16296 16289 could not be incorporated into the calcite crystal. 34 Considering that selenite (0.239 nm) has a size similar to that of phosphate (0.238 nm), 35 selenite ions might not be incorporated into the crystal lattice and formed a secondary phase during crystallization. Hence, we deduced that amorphous CaCO3could be more favorable for selenite incorporation than crystalline CaCO3. Degradation tests were performed by measuring the pH values of the samples in 1x phosphate-buffered saline (PBS) at pH 7.4 (Supplementary Figure 4a). The dissociation of ACMC nanoparticles led to an increase in the pH values, and this could be associated with the release of selenite and carbonate to the aqueous solution. For the ACMC nanoparticles, there was a continuous increase in the pH value up to 24 h, and afterward, the pH value stabilized. However, for Se-ACMC nanoparticles, there was a swift increase in the pH, which indicated a sudden dissociation and release of ions within minutes. In fact, the pH value was stable within approximately the range of 9.0 ±0.05 at all time points. The earlier increase in the pH value for Se-ACMC nanoparticles indicated faster dissolution of the nanoparticles and faster release of the selenite ions into the aqueous solution. To demonstrate the time-dependent release profile of selenite from the nanoparticles, we conducted ICP-MS analysis using the extracts of Se(III)-ACMC nanoparticles (Supplementary Figure 4b). The amount of released Se in the solutions was measured to be 0.267 ±0.008, 0.251 ±0.002, and 0.155 ±0.002 mg at 5 min, 1 h, and 72 h time points, respectively. This analysis was in line with the pH change results and confirmed the sudden release of selenite ions into the solution. It is interesting to note that the amount of Se in the extracts decreased with time. One of the reasons for this behavior might be the crystallization of selenite compounds in the extracts. Particle size is one of the factors that affects the solubility of ACMC particles. In general, the smaller the particle size, the higher the solubility. 36 In this study, we synthesized highly small particles with sizes less than 40 nm, which might contribute to the solubility of the nanoparticles and the release of antibacterial selenite ions. In addition, the TGA curves exhibited that selenite ions enhanced the hydration level of ACMC. Specifically, water molecules heavily affect the ion mobility and structural alterations during crystallization, 37 consequently expediting the dissolution of ACC. 4 The degradation results showed that Se-ACMC nanoparticles degraded faster than ACMC nanoparticles. When the degradation result and TGA results are interpreted together, Figure 3. TGA showing the mass loss and derivative of the mass loss curves for (a) ACMC, (b) Se(I)-ACMC, (c) Se(II)-ACMC, and (d) Se(III)- ACMC nanoparticles. ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.3c02415 ACS Appl. Nano Mater. 2023, 6, 16286−16296 16290 it could be speculated that selenite ions in the ACMC structure could lead to an increase in the ion mobility by increasing the water content of the nanoparticles and, consequently, lead to higher solubility of the nanoparticles in an aqueous medium. These results were in line with the literature, where Mg2+ ions were shown to bring extensive amounts of water molecules into the ACC structure, which impaired the strength of the ionic structure in an aqueous medium. 37 In this study, Mg2+ contents were similar for all nanoparticles, and selenite ions further contributed to bringing more water to the nanoparticle structure, and thus increasing the solubility of the nanoparticles. 2.2. Antibacterial Activity of Se-ACMC Nanoparticles. To evaluate the antibacterial activity of the nanoparticles, Figure 4. Antibacterial activity of the nanoparticles against (a and c) S. aureus, (b and d) S. epidermidis, (e and g) E. coli, and (f and h) P. aeruginosa. (c, d, g, and h) Photographs of the agar plates showing the differences in the CFUs between the control and Se(II)-ACMC for different bacteria strains (***,p< 0.001; ns = not significant). ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.3c02415 ACS Appl. Nano Mater. 2023, 6, 16286−16296 16291 Gram-positive S. aureus and Staphylococcus epidermidis and Gram-negative E. coli and P. aeruginosa colonies were cultured with the nanoparticle extracts at 1 g/L concentration, and colony-forming units (CFUs) were counted (Figure 4a,b,e,f). To visually demonstrate the decrease in CFUs, S. aureus,S. epidermidis,E. coli, and P. aeruginosa colonies grown in the presence of Se(II)-ACMC nanoparticle extracts are displayed in Figure 4c,d,g,h, respectively. The results indicated that, after 24 h of culture, ACMC nanoparticles did not significantly reduce bacterial growth, except for S. epidermidis. On the other hand, the selenite-incorporated particles significantly decreased the growth of all four bacteria strains for all investigated compositions (p< 0.001). For S. aureus (Figure 4a,c), 81, 77, and 87% decreases in the CFUs were observed for Se(I)- ACMC, Se(II)-ACMC, and Se(III)-ACMC nanoparticles compared to the control group without nanoparticles (p< 0.001). For S. epidermidis, ACMC nanoparticles reduced the CFUs by 85%, and Se-ACMC nanoparticles reduced the CFUs by 99.9% compared to the control group (p< 0.001). For the case of E. coli, Se(I)-ACMC, Se(II)-ACMC, and Se(III)- ACMC nanoparticles reduced the CFUs by 30, 30, and 60%, respectively, compared to the control group (p< 0.001). Last, for P. aeruginosa, there were 27, 38, and 80% reductions in CFUs upon culturing with Se(I)-ACMC, Se(II)-ACMC, and Se(III)-ACMC nanoparticle extracts, respectively, compared to the control group (p< 0.001). Among the tested bacteria strains, Se-ACMC nanoparticles were the most effective toward S. epidermidis, decreasing the CFUs by more than 3-log. According to the CFU assay results, there was no statistically significant difference in the CFUs between the Se(I)-ACMC, Se(II)-ACMC, and Se(III)-ACMC nanoparticles for the Gram-positive bacteria strains. In contrast, Se(III)-ACMC nanoparticles were significantly more effective than either Se(I)-ACMC or Se(II)-ACMC nanoparticles toward Gramnegative bacteria strains. The half-maximal inhibitory concentration (IC50) values for Se(I)-ACMC nanoparticles were calculated to be 0.64 ±0.09, 0.35 ±0.14, 1.69 ±0.10, and 1.52 ±0.08 g/L against S. aureus,S. epidermidis,E. coli, and P. aeruginosa, respectively (Table 1). As the IC50 value of the nanoparticles, even at the lowest concentration of selenite incorporation [Se(I)-ACMC], was considerably higher than the concentration utilized in the CFU assay (1 g/L), it is plausible that the selected nanoparticle concentration did not result in a statistically significant difference in the CFUs for the Gram-positive strains. However, when the incorporated antibacterial selenite concentration increased 10-fold, transitioning from Se(I)-ACMC to Se(III)-ACMC nanoparticles, a nanoparticle concentration of 1 g/L could elucidate the differences in the antibacterial activity of the nanoparticles. CFU assay results clearly showed that Se-ACMC nanoparticles could significantly reduce both Gram-positive and Gram-negative bacteria growth. When the results were investigated in more detail, it was clear that the antibacterial activity of selenite ions toward Gram-positive bacteria (S. aureus and S. epidermidis) was more prominent than that toward Gram-negative bacteria (E. coli and P. aeruginosa). Although the precise antibacterial mechanism of action for Se and its compounds remains unclear, it is understood that certain bacterial strains, when capable of tolerating their concentration, have the ability to reduce selenite ions to elemental Se. 38 Wang et al. observed the presence of biogenic Se nanoparticles both within and outside cells along with cellular remnants as a result of the interaction between selenite-resistant bacteria and selenite. They hypothesized that the antibacterial effect might stem from the release of Se nanoparticles within the cells. 39 Based on this notion, it can be speculated that the bacterial cell wall could potentially hold significance in terms of providing protection against selenite. The difference between the antibacterial effects on Grampositive and Gram-negative bacteria could be explained by the distinct structural properties of the bacterial cells. The cell wall of Gram-positive bacteria consists of a thick peptidoglycan layer, while Gram-negative bacteria have a thinner peptidoglycan layer enclosed by an outer membrane 40 and possess an efflux pump system. 41 The lack of an outer membrane and efflux system can make Gram-positive bacteria more susceptible to environmental threats. As shown in Supplementary Figure 5, after a 24 h incubation of Se-ACMC nanoparticles with S. aureus,E. coli, and P. aeruginosa, the color of the Tryptic Soy Broth (TSB) medium was observed to change to red-orange. It was reported that this color change was an indicator of the involvement of bacteria in the reduction of selenite to Se, 42 and those elemental Se nanoparticles reduced by the bacterial enzymes have far less toxicity than selenite ions. 43 For instance, biogenic Se nanoparticles produced by S. aureus,E. coli, and P. aeruginosa strains at 25−175 g/L concentration did not exert any cytotoxic effect on the primary human dermal fibroblast cell line. 44 Considering the toxic and allergen nature of antibacterial agents, such as quaternary ammonium salts, the utilization of selenite anions to dope ACMC nanoparticles against bacteria could be a sustainable alternative. It is interesting to note that, upon 24 h culture of S. epidermidis with Se-ACMC nanoparticle extracts, an apparent color change was not detected (Supplementary Figure 5). The lack of or limited occurrence of selenite to Se reduction in S. epidermidis might explain the significant decline in the growth of S. epidermidis compared with the other strains. The high concentrations of selenite remaining inside the TSB solution, rather than being reduced to biogenic Se, might have led to higher antibacterial activity and led to a more than 3-log decrease in the S. epidermidis colony counts. It should be noted that, for S. epidermidis, Se-ACMC and ACMC nanoparticles at 1 g/L concentration significantly reduced bacteria growth. Although the reason behind this trend is not clear, it could be possible that the released Mg2+ ions might have contributed to the antibacterial activity against S. epidermidis. 45 Nonetheless, further research is required to explain why selenite-free ACMC nanoparticles were effective in the inhibition of S. epidermidis growth. Although the synthesized nanoparticles exhibited antibacterial properties, the concentration-dependent toxicity of Se and its compounds is a challenge. Se is advantageous when experienced in small amounts but can become harmful in larger quantities. In this regard, the spectrum between insufficiency and abundance is quite narrow. 38 To assess the Table 1. IC50 Values for Se(I)-ACMC Nanoparticles against S. aureus,S. epidermidis,E. coli, and P. aeruginosa Gram-positive Gram-negative S. aureus S. epidermidis E. coli P. aeruginosa Se(I)- ACMC (g/L) 0.64 ±0.09 0.35 ±0.14 1.69 ±0.10 1.52 ±0.08 ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.3c02415 ACS Appl. Nano Mater. 2023, 6, 16286−16296 16292 cytotoxicity of the nanoparticles, a cell viability assay was conducted (Supplementary Figure 6). While Se(II)-ACMC and Se(III)-ACMC nanoparticles did not support fibroblast proliferation at either 0.01 or 0.1 g/L concentration, Se(I)- ACMC nanoparticles promoted fibroblast viability and proliferation up to 5 days in vitro. However, Se-ACMC nanoparticles showed toxicity on fibroblasts at 1 g/L concentration independent of the amount of incorporated selenite. Since toxicity is a big concern for biomedical applications, we believe that the aqueous stability of the SeACMC nanoparticles requires further improvement for translation into commercial applications. 3. CONCLUSIONS In this study, we synthesized Se-ACMC nanoparticles for antibacterial applications. SEM, TEM, and XRD results confirmed that ACMC nanoparticles could maintain their particle size on the nanometer scale upon selenite incorporation, and XPS analysis verified the presence of selenite ions in the nanoparticles. Our results also revealed that the soluble nature of the nanoparticles led to the release of antibacterial selenite ions, which inhibited bacterial growth of both Grampositive (S. aureus and S. epidermidis) and Gram-negative (E. coli and P. aeruginosa) strains. The antibacterial activity of the ACMC nanoparticles increased with an increase in the concentration of the incorporated selenite ions. In summary, the results indicated that Se-ACMC nanoparticles have the potential to be used as an effective antibacterial agent. 4. MATERIALS AND METHODS 4.1. Materials. Calcium acetate monohydrate [Ca(CH3CO2)2· H2O], sodium bicarbonate (NaHCO3), magnesium chloride hexahydrate (MgCl2·6H2O), and sodium selenite (Na2SeO3) were purchased from Sigma-Aldrich. Ethylene glycol [CH2(OH)2] and ethanol (C2H5OH) were obtained from Isolab Chemicals. Ultrapure water obtained from a Millipore Milli-Q purification system was used in the experiments. All chemicals were used as received without further purification. 4.2. Nanoparticle Synthesis. ACMC nanoparticles were synthesized using the coprecipitation method. 5 mL of an aqueous calcium acetate monohydrate solution (0.5 M) and 5 mL of an aqueous sodium bicarbonate (0.5 M)/magnesium chloride hexahydrate solution (0.5 M) were prepared separately in two different beakers. Then, the total volume of each beaker was adjusted to 25 mL with the addition of ethylene glycol. The calcium acetate monohydrate solution was poured into the sodium bicarbonate/ magnesium chloride solution. The reaction took place for 15 min under magnetic stirring, and afterward, the solution was kept stationary for 1 h at room temperature. The precipitate was isolated by centrifugation, followed by washing with ethanol and ultrapure water, respectively. Last, the powder was lyophilized for storage. For the synthesis of Se-ACMC nanoparticles, the same protocol was followed, except the addition of Na2SeO3at 0.001, 0.005, and 0.01 M concentrations [referred to as Se(I)-ACMC, Se(II)-ACMC, and Se(III)-ACMC, respectively] into the solution of sodium bicarbonate. In Table 2, the molarities of the precursor solutions are shown. 4.3. Materials Characterization. The morphology of ACMC nanoparticles was observed using a FEI Verios 450L scanning electron microscope (Brno, Czechia) at 5 kV. The size of the nanoparticles was measured from 200 particles for each synthesized nanoparticle group using ImageJ software. Gaussian or log-normal distribution was used to curve-fit the nanoparticle size distribution histograms. TEM analysis was performed using an FEI Tecnai G2 F30 transmission electron microscope (Hillsboro, Oregon) to investigate the nanoparticle morphology and amorphous structure. Specimens were prepared for TEM characterization by dispersion in ethanol. 10 μL of the mixture was dropped onto a holey carbon-coated copper grid and dried for 10 min. To further demonstrate the amorphous nature of the synthesized nanoparticles, XRD analyses were performed using a Rigaku D/Max-2200 X-ray diffractometer (Tokyo, Japan) with monochromatic Cu Kαradiation (λ= 1.54 Å) at 2°/min scanning rate between 20 and 60°. For analysis of the chemical bonding, FTIR spectra were obtained in the 4000−400 cm−1range using a PerkinElmer Spectrum 100 (Waltham, MA) in attenuated-totalreflectance (ATR) mode. To analyze the chemical composition of the nanoparticles and the chemical state of Se, XPS analysis was performed using a Kratos Axis Supra (Manchester, U.K.), which has a monochromatic Al Kαexcitation source. The National Institute of Standards and Technology XPS online database was used to interpret the XPS data. All spectra were calibrated to the adventitious carbon peak at 284.8 eV. The binding energies of Se 3d5/2 and Se 3d3/2 were obtained by the deconvolution of peaks using CasaXPS software. Peak fitting was done via Shirley’s background using the combination of Gaussian and Lorentzian functions and putting constraints on the peak area ratio and peak positions. The peak separation between Se 3d5/2 and Se 3d3/2 was restricted to 0.86 eV. TGA was performed using a TA Instruments SDT650 analyzer (New Castle, DE) in a nitrogen atmosphere between 25 and 800 °C with a heating rate of 10 °C/min. ICP-MS analyses were conducted to reveal the amount of Se incorporated into the nanoparticles and to show the Se release profile of the nanoparticles using an X Series II ICP-MS (Thermo Fisher Scientific Inc., Waltham, MA). Prior to the ICP-MS analysis, the nanoparticles were microwave-digested in a HCl/HNO3solution to quantify the amount of incorporated Se. In another set of experiments, the nanoparticles were extracted in 1x PBS at 10 g/L concentration for 5 min, 1 h, and 72 h to analyze the Se release profile from the nanoparticles. Degradation tests were conducted in 1x PBS. For these tests, nanoparticles were added at a concentration of 1 g/L in 1x PBS and kept at 37 °C through the degradation experiments. The pH values of the solutions were measured at 1 min, 5 min, 15 min, 1 h, 24 h, 48 h, and 72 h time points. 4.4. Cell Viability Assay. L929 fibroblasts (ATCC CCL-1) were used to assess the cellular viability. Dulbecco’s modified Eagle medium (Sigma-Aldrich) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum was used as a growth medium. The fibroblasts were maintained in a humidified 5% CO2incubator at 37 °C and subcultured at 90% confluency. Prior to the viability assay, the nanoparticles were sterilized by UV irradiation for 1 h. Subsequently, the sterilized nanoparticles were extracted using growth media at 0.1 and 0.01 g/L concentrations at 37 °C for 72 h. The extracted media were used for the cell viability assay. For the viability experiments, fibroblasts were seeded into the wells of a 96-well plate at a concentration of 1 ×104cells/well. After a 24 h incubation period, the growth media were replaced with 200 μL of the nanoparticle extracts. On the first, third, and fifth days of incubation, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was conducted. Cells incubated with the nanoparticle extracts were rinsed with 1x PBS, and afterward, 125 μL of a sterilized MTT solution (1 g/L) was added to each well. Cells were incubated with MTT solutions for 4 h to form formazan crystals. Then, the crystals were dissolved with a 0.77% HCl solution prepared in isopropyl alcohol (1:1 volume ratio). The absorbance values were measured at 570 nm by using a Thermo Scientific Multiskan Go Table 2. Molarities of the Precursor Solutions Prepared for ACMC and Se-ACMC Nanoparticles solution A solution B sample designations Ca(CH3CO2)2· H2O (M) NaHCO3 (M) MgCl2·6H2O (M) Na2SeO3 (M) ACMC 0.5 0.5 0.5 - Se(I)-ACMC 0.5 0.5 0.5 0.001 Se(II)-ACMC 0.5 0.5 0.5 0.005 Se(III)- ACMC 0.5 0.5 0.5 0.01 ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.3c02415 ACS Appl. Nano Mater. 2023, 6, 16286−16296 16293 microplate spectrophotometer. Cells that were not treated with nanoparticle extracts were used as the control group. 4.5. Antibacterial Activity Tests. 4.5.1. Particle Sterilization and Preparation. ACMC nanoparticles were sterilized by UV-light exposure for 1 h. Sterilized nanoparticles were prepared at 1 g/L concentration using 0.3% TSB. After the nanoparticles were extracted in the TSB solution for 72 h at 37 °C, the supernatants were separated by centrifugation and the extracted media were used in the biological experiments. 4.5.2. CFU Assay. ACMC nanoparticles were tested against two different Gram-positive bacteria strains, S. aureus (ATCC 25923) and S. epidermidis (ATCC 35984), and two different Gram-negative bacteria strains, E. coli (ATCC 10536) and P. aeruginosa (ATCC 27853). Bacteria from the stock culture were inoculated onto Luria− Bertani agar plates and incubated overnight. Once the colonies grew, a single CFU was isolated and inoculated in 3% TSB. Afterward, the cells were transferred to a shaking incubator and cultured at 37 °C and 200 rpm for 18 h. At the end of 18 h, bacteria were diluted to an optical density of 0.01 at 625 nm (OD625), which was further diluted by 1:100 using 1x PBS. A total of 100 μL of the suspended bacteria was seeded into 96-well plates at a density of a×105/mL (a= 4, 9, 8, and 5 for S. aureus,S. epidermidis,E. coli, and P. aeruginosa, respectively). A total of 100 μL of the nanoparticle extracts were also added into each well for increasing the total volume to 200 μL/well. After 24 h of incubation, the bacteria in the wells were serially diluted with 1x PBS, and 20 μL of the dilutions were seeded onto agar plates to count the CFUs. 4.5.3. IC50 Determination. Nanoparticles were extracted at a 10 g/ L concentration in 0.3% TSB at 37 °C for 72 h. The extracts were serially diluted by half into the wells of a 96-well plate to a volume of 100 μL for each well. The concentration of each bacteria solution was adjusted to be 1 ×106CFU/mL. Then, 100 μL of a bacteria solution was added to the nanoparticle extracts to yield a bacteria concentration of 5 ×105CFU/mL. After 18 h of incubation, absorbance values were measured at 600 nm using a Thermo Scientific Multiskan Go microplate spectrophotometer. Dose-dependent inhibition data were analyzed using GraphPad Prism software. Extract concentrations that reduced the bacteria density to half were reported as IC50 values. 4.6. Statistical Analysis. All of the biological experiments were repeated three times, and three samples were used for each experiment. The results were reported as mean ±standard deviation. The one-way ANOVA method with Tukey’s posthoc test was used to determine whether the data showed a meaningful statistical difference. The statistical significance was fixed at p< 0.05. ■ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsanm.3c02415. Low-magnification SEM images and EDS spectra of nanoparticles, SEM image of sodium selenite salt, XRD spectrum of a mixture of ACMC and sodium selenite salt, XRD results of heat-treated nanoparticles, degradation test results and Se release analysis, images of the media color change, and the results of cell viability experiments (PDF) ■AUTHOR INFORMATION Corresponding Author Batur Ercan −Department of Metallurgical and Materials Engineering, Middle East Technical University, Cankaya, Ankara 06800, Turkey; BIOMATEN, Center of Excellence in Biomaterials and Tissue Engineering and Biomedical Engineering Program, Middle East Technical University, Cankaya, Ankara 06800, Turkey; orcid.org/0000-00031657-1142; Phone: +90 (312) 210-2513; Email: [email protected] Authors Yagmur Göctu−Department of Metallurgical and Materials Engineering, Middle East Technical University, Cankaya, Ankara 06800, Turkey; orcid.org/0000-0001-8312-3679 Cagatay M. Oral −Future Energy and Innovation Laboratory, Central European Institute of Technology, Brno University of Technology, Brno 61200, Czech Republic; orcid.org/ 0000-0001-5220-2104 Complete contact information is available at: https://pubs.acs.org/10.1021/acsanm.3c02415 Author Contributions Y.G. synthesized and characterized the nanoparticles and conducted in vitro experiments. C.M.O. obtained SEM images and XPS spectra of the nanoparticles. B.E. supervised the project. All authors contributed to the manuscript preparation. Notes The authors declare no competing financial interest. ■ACKNOWLEDGMENTS This research was funded by the Turkish Scientific Research Council under Grants 117M187, 118M652, and 219M480, and The Young Scientist Award Program of the Turkish Academy of Sciences. We acknowledge the Middle East Technical University (METU) Central Laboratory for TEM and TGA characterizations and the BIOMATEN-METU Center of Excellence in Biomaterials and Tissue Engineering (BIOMATEN) for FTIR characterization. CzechNanoLab Project LM2023051 funded by MEYS CR is gratefully acknowledged for financial support of the measurements at CEITEC Nano Research Infrastructure. ■REFERENCES (1) Morris, P. D.; Mcpherson, I. J.; Meloni, G. N.; Unwin, P. R. Nanoscale Kinetics of Amorphous Calcium Carbonate Precipitation in H2O and D2O. Phys. Chem. Chem. Phys. 2020,22, 22107. (2) Wang, Y. Y.; Yao, Q. Z.; Zhou, G. T.; Fu, S. Q. Transformation of Amorphous Calcium Carbonate into Monohydrocalcite in Aqueous Solution: A Biomimetic Mineralization Study. Eur. J. Mineral 2015,27 (6), 717−729. (3) Gower, L. B. Biomimetic Model Systems for Investigating the Amorphous Precursor Pathway and Its Role in Biomineralization. Chem. Rev. 2008,108 (11), 4551−4627. (4) Albéric, M.; Bertinetti, L.; Zou, Z.; Fratzl, P.; Habraken, W.; Politi, Y. The Crystallization of Amorphous Calcium Carbonate Is Kinetically Governed by Ion Impurities and Water. Adv. Sci. 2018,5 (5), 1701000. (5) Addadi, L.; Raz, S.; Weiner, S. Taking Advantage of Disorder: Amorphous Calcium Carbonate and Its Roles in Biomineralization. Adv. Mater. 2003,15 (12), 959−970. (6) Rani, R. S.; Saharay, M. Molecular Dynamics Simulation of Protein-Mediated Biomineralization of Amorphous Calcium Carbonate. RSC Adv. 2019,9, 1653−1663. (7) Nie, B.; Wang, H.; Rao, C.; Zhang, Y.; Wang, H.; Lian, X.; Gao, X.; Niu, B.; Li, W. Preparation and Characterization of Sodium Alginate/Phosphate-Stabilized Amorphous Calcium Carbonate Nanocarriers and Their Application in the Release of Curcumin. Nanotechnology 2021,32 (37), 375712. (8) Ihli, J.; Wong, W. C.; Noel, E. H.; Kim, Y. Y.; Kulak, A. N.; Christenson, H. K.; Duer, M. J.; Meldrum, F. C. Dehydration and Crystallization of Amorphous Calcium Carbonate in Solution and in Air. Nat. Commun. 2014,5, No. 3169. ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.3c02415 ACS Appl. Nano Mater. 2023, 6, 16286−16296 16294