Tannylated Calcium Carbonate Materials with Antacid, Anti-Inflammatory, and Antioxidant Effects

Calcium carbonate (CaCO3)-based materials have received notable attention for biomedical applications owing to their safety and beneficial characteristics, such as pH sensitivity, carbon dioxide (CO2) gas generation, and antacid properties. Herein, to additionally incorporate antioxidant and anti-inflammatory functions, we prepared tannylated CaCO3 (TA-CaCO3) materials using a simple reaction between tannic acid (TA), calcium (Ca2+), and carbonate (CO32−) ions. TA-CaCO3 synthesized at a molar ratio of 1:75 (TA:calcium chloride (CaCl2)/sodium carbonate (Na2CO3)) showed 3–6 μm particles, comprising small nanoparticles in a size range of 17–41 nm. The TA-CaCO3 materials could efficiently neutralize the acid solution and scavenge free radicals. In addition, these materials could significantly reduce the mRNA levels of pro-inflammatory factors and intracellular reactive oxygen species, and protect chondrocytes from toxic hydrogen peroxide conditions. Thus, in addition to their antacid property, the prepared TA-CaCO3 materials exert excellent antioxidant and anti-inflammatory effects through the introduction of TA molecules. Therefore, TA-CaCO3 materials can potentially be used to treat inflammatory cells or diseases.


Introduction
Inorganic minerals have been widely used in drug delivery systems for biomedical applications [1]. Calcium carbonate (CaCO 3 ), an inorganic biomineral, has been used as an antacid agent. It can be orally administered as a tablet, chewable tablet, capsule, or liquid. Furthermore, it has been used for the controlled and sustained delivery of chemical drugs [2][3][4], photosensitizers [5], and proteins [6,7] because of its biocompatibility and slow biodegradation [8]. CaCO 3 is stable at physiological pH, but can be dissociated under acidic conditions [9,10]. Owing to their pH sensitivity, CaCO 3 -based delivery systems concentrate drugs into targeted cancer tissues within the acidic tumor microenvironment (TME) [4,5,10]. In addition, these systems react with protons (H + ) to neutralize the acid [11,12]. CaCO 3 can generate carbon dioxide (CO 2 ) in the acidic TME, and this gas-generating property has extended its application as an ultrasound contrast agent for cancer imaging [5,10]. These previous studies have revealed the pH sensitivity, CO 2 gas generation, and antacid properties of CaCO 3 materials.
Polyphenol tannic acid (TA) is composed of five digalloyl ester groups that have been linked to a central glucose molecule, and exhibits various biological properties, such as antibacterial, anti-inflammatory, antioxidant, and anticancer activities [13,14]. Previous studies have revealed the TA-mediated scavenging of free radicals, leading to the inhibition of lipid oxidation and radical-mediated DNA damage [13,15,16]. Given their ability to undergo multiple interactions with various biomacromolecules (i.e., nucleic acids, peptides, proteins, and polysaccharides) through electrostatic and hydrogen bonding and/or hydrophobic interactions [17][18][19][20][21], TA-macromolecular complexes can be easily produced and used as surface modifiers on organic and inorganic substrates [22,23]. Thus, the TA-modified polymeric hydrogels and scaffolds greatly enhance anti-inflammatory effects in vitro or protect cells under reactive oxygen species (ROS) environments [23,24]. Moreover, as TA can coordinate with metal ions, it can be used to synthesize inorganic Ag and Au nanomaterials [25]. Our group recently prepared oxygen-generating calcium peroxide using TA through coordination between the catechol moieties of TA and calcium ions [26].
Based on these previous reports, we propose that the introduction of TA into CaCO 3 materials can endow them with anti-inflammatory and antioxidant functions. As CaCO 3 materials exert no anti-inflammatory or antioxidant functions, herein we fabricated tannylated CaCO 3 (termed as TA-CaCO 3 ) materials via the coordination of TA to calcium (Ca 2+ ) ions and further nucleation of CaCO 3 using carbonate ions (CO 3 2− ). We performed their physicochemical characterization and evaluated their antacid and antioxidant effects using colorimetric methods. In addition, we validated their in vitro antioxidant and anti-inflammatory properties in chondrocytes under inflammatory and ROS conditions.

Preparation of TA-CaCO 3 Materials
TA-CaCO 3 materials were synthesized by combining equimolar amounts of Ca 2+ and CO 3 2− at different concentrations with a fixed molar concentration of TA under constant stirring, as depicted in Figure 1. Given the tendency of TA to coordinate with metal ions [25,27,28], TA interacts with Ca 2+ ions and facilitates the nucleation of CaCO 3 to form TA-CaCO 3 nanoparticles, which then agglomerate into microparticles due to the interactions between TA-CaCO 3 nanoparticles.
studies have revealed the TA-mediated scavenging of free radicals, leading to the inhibition of lipid oxidation and radical-mediated DNA damage [13,15,16]. Given their ability to undergo multiple interactions with various biomacromolecules (i.e., nucleic acids, peptides, proteins, and polysaccharides) through electrostatic and hydrogen bonding and/or hydrophobic interactions [17][18][19][20][21], TA-macromolecular complexes can be easily produced and used as surface modifiers on organic and inorganic substrates [22,23]. Thus, the TA-modified polymeric hydrogels and scaffolds greatly enhance antiinflammatory effects in vitro or protect cells under reactive oxygen species (ROS) environments [23,24]. Moreover, as TA can coordinate with metal ions, it can be used to synthesize inorganic Ag and Au nanomaterials [25]. Our group recently prepared oxygengenerating calcium peroxide using TA through coordination between the catechol moieties of TA and calcium ions [26].
Based on these previous reports, we propose that the introduction of TA into CaCO3 materials can endow them with anti-inflammatory and antioxidant functions. As CaCO3 materials exert no anti-inflammatory or antioxidant functions, herein we fabricated tannylated CaCO3 (termed as TA-CaCO3) materials via the coordination of TA to calcium (Ca 2+ ) ions and further nucleation of CaCO3 using carbonate ions (CO3 2− ). We performed their physicochemical characterization and evaluated their antacid and antioxidant effects using colorimetric methods. In addition, we validated their in vitro antioxidant and antiinflammatory properties in chondrocytes under inflammatory and ROS conditions.

Preparation of TA-CaCO3 Materials
TA-CaCO3 materials were synthesized by combining equimolar amounts of Ca 2+ and CO3 2− at different concentrations with a fixed molar concentration of TA under constant stirring, as depicted in Figure 1. Given the tendency of TA to coordinate with metal ions [25,27,28], TA interacts with Ca 2+ ions and facilitates the nucleation of CaCO3 to form TA-CaCO3 nanoparticles, which then agglomerate into microparticles due to the interactions between TA-CaCO3 nanoparticles.

Characterization of TA-CaCO3 Materials
The morphologies of the synthesized TA-CaCO3 materials were examined using scanning electron microscopy (SEM). As shown in Figure S1, at 25 molar ratios of calcium chloride (CaCl2)/sodium carbonate (Na2CO3), aggregates comprising small TA-CaCO3 particles were predominantly observed, and very small amounts of micron-sized spherical TA-CaCO3 particles were formed. More spherical TA-CaCO3 microparticles were gradually observed as the molar ratio of CaCl2/Na2CO3 was increased to 75. Interestingly, although spherical TA-CaCO3 microparticles were still observed when the molar ratios of TA and CaCl2/Na2CO3 were 100 or 150, more irregular and broken TA-CaCO3 particles were detected. These results suggested that small TA-CaCO3 particles were formed at lower molar ratios of CaCl2/Na2CO3 and that spherical and broken TA-

Characterization of TA-CaCO 3 Materials
The morphologies of the synthesized TA-CaCO 3 materials were examined using scanning electron microscopy (SEM). As shown in Figure S1, at 25 molar ratios of calcium chloride (CaCl 2 )/sodium carbonate (Na 2 CO 3 ), aggregates comprising small TA-CaCO 3 particles were predominantly observed, and very small amounts of micron-sized spherical TA-CaCO 3 particles were formed. More spherical TA-CaCO 3 microparticles were gradually observed as the molar ratio of CaCl 2 /Na 2 CO 3 was increased to 75. Interestingly, although spherical TA-CaCO 3 microparticles were still observed when the molar ratios of TA and CaCl 2 /Na 2 CO 3 were 100 or 150, more irregular and broken TA-CaCO 3 particles were detected. These results suggested that small TA-CaCO 3 particles were formed at lower molar ratios of CaCl 2 /Na 2 CO 3 and that spherical and broken TA-CaCO 3 particles were more common at higher molar ratios of TA and CaCl 2 /Na 2 CO 3 . Based on SEM images, 1:75 TA-CaCO 3 particles were selected for further experiments because they produced more spherical TA-CaCO 3 microparticles than other TA-CaCO 3 particles.
Next, we investigated the particle size of the 1:75 TA-CaCO 3 materials and found it to range from approximately 3 to 6 µm (Figure 2a and Figure S1). Interestingly, the magnified SEM images revealed the presence of small particles on the surface of the 1:75 TA-CaCO 3 materials. These individual small particles ranged from 17 to 41 nm in size, and were approximately 26.18 ± 4.6 nm in diameter and spherical in shape (Figure 2b). SEM images revealed that the micron-sized TA-CaCO 3 materials consisted of small TA-CaCO 3 nanoparticles, probably owing to agglomeration following interactions between individual small nanoparticles.
CaCO3 particles were more common at higher molar ratios of TA and CaCl2/Na2CO3. Based on SEM images, 1:75 TA-CaCO3 particles were selected for further experiments because they produced more spherical TA-CaCO3 microparticles than other TA-CaCO3 particles.
Next, we investigated the particle size of the 1:75 TA-CaCO3 materials and found it to range from approximately 3 to 6 μm (Figure 2a and Figure S1). Interestingly, the magnified SEM images revealed the presence of small particles on the surface of the 1:75 TA-CaCO3 materials. These individual small particles ranged from 17 to 41 nm in size, and were approximately 26.18 ± 4.6 nm in diameter and spherical in shape (Figure 2b). SEM images revealed that the micron-sized TA-CaCO3 materials consisted of small TA-CaCO3 nanoparticles, probably owing to agglomeration following interactions between individual small nanoparticles. The preparation of TA-CaCO3 materials was confirmed using an SEM coupled with energy-dispersive (SEM-EDS) X-ray spectroscopy, inductively coupled plasma optical emission spectrometry (ICP-OES), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD) patterns, and X-ray photoelectron spectroscopy (XPS). The EDS mapping and spectrum data revealed the presence of carbon, oxygen, and calcium on the surface of TA-CaCO3 materials (Figure 2c,d). Consistent with a previous report [29], the EDS spectrum showed the peaks of Kα (0.277 eV) corresponding to carbon, Kα (0.523 eV) corresponding to oxygen, and Kα (3.691 eV) and Lα (0.341 eV) corresponding to calcium. ICP-OES analysis showed that 0.1 mg of 1:75 TA-CaCO3 contained 18.6 μg of TA and 81.4 μg of CaCO3. The FT-IR spectrum of commercial CaCO3 showed the characteristic vibrations of carbonate ions (at 1805, 1410, 1090, and 874 cm −1 ) (Figure 3), as previously reported [30,31]. The preparation of TA-CaCO3 materials was confirmed by the presence of the main asymmetric vibrations at 1460 and 1410 cm −1 . However, the symmetric vibration at 725 cm −1 disappeared, implying that TA-CaCO3 has an amorphous structure. Meanwhile, TA-CaCO3 showed characteristic peaks at 3300-3600 cm −1 (O-H stretching), The preparation of TA-CaCO 3 materials was confirmed using an SEM coupled with energy-dispersive (SEM-EDS) X-ray spectroscopy, inductively coupled plasma optical emission spectrometry (ICP-OES), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD) patterns, and X-ray photoelectron spectroscopy (XPS). The EDS mapping and spectrum data revealed the presence of carbon, oxygen, and calcium on the surface of TA-CaCO 3 materials (Figure 2c,d). Consistent with a previous report [29], the EDS spectrum showed the peaks of Kα (0.277 eV) corresponding to carbon, Kα (0.523 eV) corresponding to oxygen, and Kα (3.691 eV) and Lα (0.341 eV) corresponding to calcium. ICP-OES analysis showed that 0.1 mg of 1:75 TA-CaCO 3 contained 18.6 µg of TA and 81.4 µg of CaCO 3 . The FT-IR spectrum of commercial CaCO 3 showed the characteristic vibrations of carbonate ions (at 1805, 1410, 1090, and 874 cm −1 ) (Figure 3), as previously reported [30,31]. The preparation of TA-CaCO 3 materials was confirmed by the presence of the main asymmetric vibrations at 1460 and 1410 cm −1 . However, the symmetric vibration at 725 cm −1 disappeared, implying that TA-CaCO 3 has an amorphous structure. Meanwhile, TA-CaCO 3 showed characteristic peaks at 3300-3600 cm −1 (O-H stretching), 1445 cm −1 (C-C stretching of benzene ring and methylene; C-O stretching of phenolic), and 755 cm −1 (C-H torsion of benzene ring) [32], indicative of the presence of TA on the material. 1445 cm −1 (C-C stretching of benzene ring and methylene; C-O stretching of phenolic), and 755 cm −1 (C-H torsion of benzene ring) [32], indicative of the presence of TA on the material.  118), respectively, and these diffraction peaks were consistent with the vaterite crystal faces [33,34]. Based on XRD data, we think that the prepared TA-CaCO3 materials are the vaterite form of calcium carbonate.    118), respectively, and these diffraction peaks were consistent with the vaterite crystal faces [33,34]. Based on XRD data, we think that the prepared TA-CaCO3 materials are the vaterite form of calcium carbonate.  XPS data revealed that CaCO 3 and TA-CaCO 3 showed Ca, O, and C signals (Figure 4b). The binding energy peaks of these two materials appeared O1s at 531 eV, Ca2s at 441 eV, two Ca2p at 351 and 347.2 eV, and two C1s peaks at 289.3 eV (CO 3 in the CaCO 3 surface) and 284.6 eV (adventitious carbon peak), respectively. These data demonstrated the successful synthesis of TA-CaCO 3 , and the synthesized TA-CaCO 3 materials are vaterite calcium carbonate.

Antacid Effects of TA-CaCO 3
CaCO 3 exists as a stable crystalline solid at physiological pH, but can be dissociated into ionic species at or below weakly acidic pH [5,9]. Under acidic pH, CaCO 3 neutralizes acids by reacting with the proton (H + ) [11,12], and it has been used as an acid neutralizer [35].
To verify the antacid effects of TA-CaCO 3 materials, commercial CaCO 3 and TA-CaCO 3 were dispersed in phosphate-buffered saline (PBS; physiological pH = 7.4) and simulated gastric fluid (SGF, pH 1.5) containing bromothymol blue (BTB). The color and absorption changes of BTB were monitored before and after the reaction, because BTB is a useful acid/base indicator to distinguish the acidity, neutrality, and alkalinity of an aqueous solution [36,37]. As shown in Figure 5a and Figure S2, the aqueous BTB solution without commercial CaCO 3 and TA-CaCO 3 exhibited a blue color at pH = 7.4 and turned to a yellowish color in the presence of SGF (pH = 1.5). CaCO 3 and TA-CaCO 3 showed a deep blue color of BTB at pH = 7.4, indicating the slight pH increases of the solution following the degradation of CaCO 3 and TA-CaCO 3 , even at pH = 7.4. In contrast, the yellowish BTB solution at pH = 1.5 turned to a bluish green color after the reaction with CaCO 3 and TA-CaCO 3 , indicating that the pH of the solution increased to a nearly neutral pH (approximately 7). Consistent with these color changes, the λ max shift of BTB occurred from 615 nm at pH = 7.4 to 433 nm under SGF (pH = 1.5) (Figure 5b). However, the absorptions of both CaCO 3 and TA-CaCO 3 groups increased at 615 nm, while the λ max of BTB at pH = 1.5 was blue-shifted from 433 nm to 403 nm, implying the pH increases of the solutions after reacting with two kinds of CaCO 3 materials. Meanwhile, TA-CaCO 3 showed significantly higher absorbance below 400 nm at both pH = 1.5 and pH = 7.4, indicating the presence of TA in the solutions. Furthermore, TA-CaCO 3 had a lower absorbance than commercial CaCO 3 at 615 nm. This is attributed to the fact that TA-CaCO 3 contained a lower amount of CaCO 3 than commercial CaCO 3 .
XPS data revealed that CaCO3 and TA-CaCO3 showed Ca, O, and C signals ( Figure  4b). The binding energy peaks of these two materials appeared O1s at 531 eV, Ca2s at 441 eV, two Ca2p at 351 and 347.2 eV, and two C1s peaks at 289.3 eV (CO3 in the CaCO3 surface) and 284.6 eV (adventitious carbon peak), respectively. These data demonstrated the successful synthesis of TA-CaCO3, and the synthesized TA-CaCO3 materials are vaterite calcium carbonate.

Antacid Effects of TA-CaCO3
CaCO3 exists as a stable crystalline solid at physiological pH, but can be dissociated into ionic species at or below weakly acidic pH [5,9]. Under acidic pH, CaCO3 neutralizes acids by reacting with the proton (H + ) [11,12], and it has been used as an acid neutralizer [35].
To verify the antacid effects of TA-CaCO3 materials, commercial CaCO3 and TA-CaCO3 were dispersed in phosphate-buffered saline (PBS; physiological pH = 7.4) and simulated gastric fluid (SGF, pH 1.5) containing bromothymol blue (BTB). The color and absorption changes of BTB were monitored before and after the reaction, because BTB is a useful acid/base indicator to distinguish the acidity, neutrality, and alkalinity of an aqueous solution [36,37]. As shown in Figure 5a and Figure S2, the aqueous BTB solution without commercial CaCO3 and TA-CaCO3 exhibited a blue color at pH = 7.4 and turned to a yellowish color in the presence of SGF (pH = 1.5). CaCO3 and TA-CaCO3 showed a deep blue color of BTB at pH = 7.4, indicating the slight pH increases of the solution following the degradation of CaCO3 and TA-CaCO3, even at pH = 7.4. In contrast, the yellowish BTB solution at pH = 1.5 turned to a bluish green color after the reaction with CaCO3 and TA-CaCO3, indicating that the pH of the solution increased to a nearly neutral pH (approximately 7). Consistent with these color changes, the λmax shift of BTB occurred from 615 nm at pH = 7.4 to 433 nm under SGF (pH = 1.5) (Figure 5b). However, the absorptions of both CaCO3 and TA-CaCO3 groups increased at 615 nm, while the λmax of BTB at pH = 1.5 was blue-shifted from 433 nm to 403 nm, implying the pH increases of the solutions after reacting with two kinds of CaCO3 materials. Meanwhile, TA-CaCO3 showed significantly higher absorbance below 400 nm at both pH = 1.5 and pH = 7.4, indicating the presence of TA in the solutions. Furthermore, TA-CaCO3 had a lower absorbance than commercial CaCO3 at 615 nm. This is attributed to the fact that TA-CaCO3 contained a lower amount of CaCO3 than commercial CaCO3.

Antioxidant Effects of TA-CaCO 3
The antioxidant effects of TA-CaCO 3 were determined using the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) [38]. As shown in Figure 6, the DPPH solution showed a deep violet color, with an absorbance at approximately 520 nm, owing to the delocalization of the spare electron over the molecule [38]. CaCO 3 -treated DPPH solution also had a deep violet color and an absorption band at around 520 nm, indicating that CaCO 3 had no antioxidant effect. In contrast, TA-CaCO 3 -treated DPPH solution turned yellowish in color and lost its absorbance at 520 nm. This loss of violet color might be attributed to the presence of TA molecules within TA-CaCO 3 because TA molecules have an effective radical-scavenging activity, as its hydroxyl groups easily reduce the free radicals of DPPH [39,40]. These data imply that TA-CaCO 3 materials have antioxidant properties and effective ROS-scavenging activity.

Antioxidant Effects of TA-CaCO3
The antioxidant effects of TA-CaCO3 were determined using the stable free radica 2,2-diphenyl-1-picrylhydrazyl (DPPH) [38]. As shown in Figure 6, the DPPH solution showed a deep violet color, with an absorbance at approximately 520 nm, owing to the delocalization of the spare electron over the molecule [38]. CaCO3-treated DPPH solution also had a deep violet color and an absorption band at around 520 nm, indicating tha CaCO3 had no antioxidant effect. In contrast, TA-CaCO3-treated DPPH solution turned yellowish in color and lost its absorbance at 520 nm. This loss of violet color might be attributed to the presence of TA molecules within TA-CaCO3 because TA molecules have an effective radical-scavenging activity, as its hydroxyl groups easily reduce the free radicals of DPPH [39,40]. These data imply that TA-CaCO3 materials have antioxidan properties and effective ROS-scavenging activity.

In Vitro Anti-Inflammatory Effects of TA-CaCO3 in Inflamed Chondrocytes
The cytotoxicity study of TA-CaCO3 was examined against normal chondrocytes Figure S3 revealed that chondrocytes maintained their cell viabilities above 90% up to a 100 μg/mL concentration, indicating that TA-CaCO3 is non-toxic.
As shown in Figure 7, LPS significantly upregulated the mRNA expression of all pro inflammatory cytokines on day three compared to the control treatment. However, the mRNA levels of these pro-inflammatory cytokines were efficiently suppressed following treatment with TA-CaCO3 in a dose-dependent manner. These results indicate that TA CaCO3 materials are highly effective in suppressing pro-inflammatory factors in LPS

In Vitro Anti-Inflammatory Effects of TA-CaCO 3 in Inflamed Chondrocytes
The cytotoxicity study of TA-CaCO 3 was examined against normal chondrocytes. Figure S3 revealed that chondrocytes maintained their cell viabilities above 90% up to a 100 µg/mL concentration, indicating that TA-CaCO 3 is non-toxic.
As shown in Figure 7, LPS significantly upregulated the mRNA expression of all pro-inflammatory cytokines on day three compared to the control treatment. However, the mRNA levels of these pro-inflammatory cytokines were efficiently suppressed following treatment with TA-CaCO 3 in a dose-dependent manner. These results indicate that TA-CaCO 3 materials are highly effective in suppressing pro-inflammatory factors in LPS-stimulated chondrocytes. Consistent with previous studies, the present study also supports the anti-inflammatory effects of TA-based materials [24,44]. stimulated chondrocytes. Consistent with previous studies, the present study also supports the anti-inflammatory effects of TA-based materials [24,44].

In Vitro Antioxidant and Protective Effects of TA-CaCO3 in H2O2-Treated Chondrocytes
The exogenous treatment of cells with H2O2 results in the induction of oxidative stress and produces intracellular ROS [45,46]. To demonstrate the antioxidant activities of TA-CaCO3 at the cellular level, H2O2 (300 μM)-pretreated chondrocytes were incubated with different concentrations of TA-CaCO3 extracts, and the intracellular ROS level was detected from the fluorescence signal of 2′,7-dichlorodihydrofluorescein diacetate (DCFDA) using a confocal laser scanning microscope. As shown in Figure 8a, no

In Vitro Antioxidant and Protective Effects of TA-CaCO 3 in H 2 O 2 -Treated Chondrocytes
The exogenous treatment of cells with H 2 O 2 results in the induction of oxidative stress and produces intracellular ROS [45,46]. To demonstrate the antioxidant activities of TA-CaCO 3 at the cellular level, H 2 O 2 (300 µM)-pretreated chondrocytes were incubated with different concentrations of TA-CaCO 3 extracts, and the intracellular ROS level was detected from the fluorescence signal of 2 ,7-dichlorodihydrofluorescein diacetate (DCFDA) using a confocal laser scanning microscope. As shown in Figure 8a  Previous studies have reported that H2O2 can be toxic to cells because it produces hydroxyl radicals [45,46]. Substances with antioxidant properties prevent cell damage and protect the human body from free radicals or ROS by supplying electrons from antioxidants to the damaged cells [47,48]. Based on these facts, we investigated whether TA-CaCO3 with antioxidant properties is effective in mediating cellular protection by measuring the viability of chondrocytes treated with 300 μM of H2O2 (Figure 8b). In comparison with the control group, the chondrocytes cultured in 300 μM of H2O2 showed a significant decrease in viability owing to the oxidative damage to cellular components [49]. However, the viability of the cells treated with different concentrations of TA-CaCO3 significantly increased in a concentration-dependent manner, and was much higher than that of the chondrocytes treated with 300 μM of H2O2 alone. Consistent with our previous studies showing that substances with antioxidant properties could protect the cells against ROS environments [23,50], TA-CaCO3 effectively protected the cells and prevented cellular damage under ROS conditions. This protective effect might be associated with the effective radical-scavenging activity of TA molecules within TA-CaCO3 materials. Previous studies have reported that H 2 O 2 can be toxic to cells because it produces hydroxyl radicals [45,46]. Substances with antioxidant properties prevent cell damage and protect the human body from free radicals or ROS by supplying electrons from antioxidants to the damaged cells [47,48]. Based on these facts, we investigated whether TA-CaCO 3 with antioxidant properties is effective in mediating cellular protection by measuring the viability of chondrocytes treated with 300 µM of H 2 O 2 (Figure 8b). In comparison with the control group, the chondrocytes cultured in 300 µM of H 2 O 2 showed a significant decrease in viability owing to the oxidative damage to cellular components [49]. However, the viability of the cells treated with different concentrations of TA-CaCO 3 significantly increased in a concentration-dependent manner, and was much higher than that of the chondrocytes treated with 300 µM of H 2 O 2 alone. Consistent with our previous studies showing that substances with antioxidant properties could protect the cells against ROS environments [23,50], TA-CaCO 3 effectively protected the cells and prevented cellular damage under ROS conditions. This protective effect might be associated with the effective radical-scavenging activity of TA molecules within TA-CaCO 3 materials.

Characterization of TA-CaCO 3
For SEM analysis, the prepared TA-CaCO 3 materials were coated with platinum. Then, the morphologies and sizes of the as-prepared TA-CaCO 3 materials were examined using field-emission scanning electron microscopy (FE-SEM, S-4700, Hitachi, Japan). For size analysis of the prepared TA-CaCO 3 , randomly selected individual particles of each TA-CaCO 3 type were analyzed using ImageJ software (Version 1.47; US National Institutes of Health, Bethesda, MD, USA). The surface elemental composition of TA-CaCO 3 was examined using SEM-EDS.
The prepared TA-CaCO 3 materials were characterized using FT-IR (Shimadzu 8400S, Kyoto, Japan). The FT-IR spectrum was acquired using a KBr pellet at a resolution of 4 cm −1 between 4,000 and 400 cm −1 .
XRD and XPS analyses of 1:75 TA-CaCO 3 were done by using a K-alpha+ (Thermo Scientific, Waltham, MA, USA) with Cu Kα radiation and a D8 ADVANCE diffractometer (Bruker, Germany) with Cu Kα radiation, respectively.

Antacid Effects of TA-CaCO 3 Materials
To investigate the in vitro antacid effects, CaCO 3 (10 mg) or TA-CaCO 3 (1:75, 10 mg) was dispersed in 2 mL of two different solutions, PBS (pH = 7.4) and SGF (pH = 1.5). Each solution (1.4 mL) was mixed well with the as-prepared BTB solution (0.6 mL) and stirred. After 1 h, each solution was centrifuged at 13,500 rpm for 30 min. The color of the collected supernatant was observed by taking photos, and the absorbance was analyzed using a UV/vis spectrophotometer (NEO-S490, NEOGEN, Daejeon, Korea).

Antioxidant Effects of TA-CaCO 3 Materials
To examine and compare the in vitro antioxidant effects, CaCO 3 (1 mg) or TA-CaCO 3 (1 mg) was added to 1 mL of DPPH (0.1 mM) dissolved in MeOH and reacted at 600 rpm for 30 min. Then, each solution was centrifuged at 5,500 rpm for 10 min. The color of the supernatant was observed by taking photos, and the absorbance was recorded using a UV/vis spectrophotometer (NEO-S490).
The cytotoxicity of TA-CaCO 3 materials was determined with a cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Chondrocytes (1 × 10 4 cells/well) were seeded and incubated in a 96-well plate. After 24 h of incubation, TA-CaCO 3 materials at 0, 10, 50, and 100 µg/mL concentrations were treated into the cells. After 24 or 48 h of exposure, the CCK-8 reagent was additionally treated to the cells for 1 h, and the optical density was then recorded at 450 nm using a Multimode Reader. The cell viability of each group was represented as the percentage of viable cells versus the control group.
The in vitro anti-inflammatory effects of TA-CaCO 3 against LPS-stimulated chondrocytes were evaluated by determining the mRNA levels of pro-inflammatory factors using a real-time polymerase chain reaction (PCR). Cells (1 × 10 5 cells/well) were seeded into 24-well plates and cultured overnight. Cells were simultaneously treated with both LPS (100 ng/mL) and different concentrations of TA-CaCO 3 (0, 10, 50, and 100 µg/mL). After three-day treatment, the cells from each group were collected, and the total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's guidelines. Total RNA (1 µg) was reverse-transcribed into cDNA using AccuPower RT PreMix (Bioneer, Daejeon, Korea). The sequences of the target gene primers are provided in Supplementary Materials (Table S1). Real-time PCR analysis was conducted using an ABI7300 Real-Time Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The mRNA levels of the target genes were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and presented as relative levels.

Antioxidant Effects of TA-CaCO 3 at the Cellular Level
To investigate the antioxidant effects of TA-CaCO 3 at the cell level, we conducted DCFDA staining and a DCFDA assay. Cells (1 × 10 4 cells/well) were seeded and incubated in 24-well plates with microscope cover glasses for 24 h. Meanwhile, the extract solutions from the different concentrations of TA-CaCO 3 (0, 10, 50, and 100 µg/mL) were prepared by incubating them in DMEM medium at 37 • C for 24 h, followed by collecting the supernatants from each group. Next, cells were treated with 300 µM of H 2 O 2 at 37 • C for 30 min, followed by additional treatment of the collected extract solutions from each group for 24 h. After washing the cells with PBS, the cells were stained with DCFDA (25 µM) for 30 min under a dark condition, washed with PBS, and then fixed with 3.7% paraformaldehyde for 30 min. The fluorescence intensity within the cells was observed using a confocal laser scanning microscope (CLSM, LSM700, Zeiss, Germany).

Protection of Cell Viability in the ROS Environment
To investigate whether TA-CaCO 3 can protect cell viability in an ROS environment, chondrocytes (1 × 10 5 cells/well) were seeded in a 24-well culture plate. Then, the cells were treated with 300 µM of H 2 O 2 at 37 • C for 30 min, followed by additional treatment of TA-CaCO 3 at 10, 50, and 100 µg/mL concentrations. After 24 h of treatment, the CCK-8 (Dojindo) reagent was added to the cells for 1 h. The supernatant from each group was transferred into a 96-well plate, and its optical density was measured at 450 nm using a multimode reader.

Conclusions
In the present study, we prepared TA-CaCO 3 materials by reacting TA with CaCl 2 and Na 2 CO 3 , which led to the interaction between TA and Ca 2+ ions, followed by nucleation of CaCO 3 . Micron-sized 1:75 TA-CaCO 3 materials (ranging from 3 to 6 µm) comprised small nanoparticles in a size range of 17-41 nm. TA-CaCO 3 materials could effectively neutralize the SGF solution and scavenge free radicals. In addition, these particles significantly suppressed the mRNA expression of pro-inflammatory cytokines and mediators and scavenged intracellular ROS in cells. Their anti-inflammatory and antioxidant activities protected chondrocytes from ROS. These results suggest that TA-CaCO 3 materials have excellent antacid, antioxidant, and anti-inflammatory properties. Importantly, TA molecules can undergo multiple interactions with nucleic acids, peptides, proteins, and polysaccharides. Furthermore, due to the molecular adsorption of CaCO 3 materials, CaCO 3 -based materials can improve the incorporation efficacy of drugs. Thus, using TA-CaCO 3 materials, we will develop dual drug delivery systems that can ferry both a chemical drug and protein drug, and then apply them to treat inflammatory cells or diseases.