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

Abstract

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.

1. Introduction

Inorganic minerals have been widely used in drug delivery systems for biomedical applications [1]. Calcium carbonate (CaCO₃), 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]. CaCO₃ is stable at physiological pH, but can be dissociated under acidic conditions [9,10]. Owing to their pH sensitivity, CaCO₃-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]. CaCO₃ can generate carbon dioxide (CO₂) 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, CO₂ gas generation, and antacid properties of CaCO₃ 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 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 (Ca²⁺) ions and further nucleation of CaCO₃ using carbonate ions (CO₃²⁻). 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.

2. Results and Discussion

2.1. Preparation of TA-CaCO₃ Materials

TA-CaCO₃ materials were synthesized by combining equimolar amounts of Ca²⁺ and CO₃²⁻ 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 Ca²⁺ 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.

2.2. 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-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), 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.

Next, commercial CaCO₃ and synthesized TA-CaCO₃ crystal phases were identified via XRD analysis (Figure 4a). CaCO₃ exhibited the characteristic peaks, such as the plane of the calcite at 29.3° (104), and the calcite crystal faces at 23.02° (012), 35.9° (110), 39.4° (113), and 43.1° (202), respectively. Meanwhile, the diffraction peaks of TA-CaCO₃ were observed at 24.8° (110), 27.08° (112), 32.7° (114), 43.8° (300), 49.1° (304), and 50.08° (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 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.

2.3. 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 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 CaCO₃ and TA-CaCO₃ 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 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.

2.4. Antioxidant Effects of TA-CaCO₃

The antioxidant effects of TA-CaCO₃ 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]. CaCO₃-treated DPPH solution also had a deep violet color and an absorption band at around 520 nm, indicating that 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 antioxidant properties and effective ROS-scavenging activity.

2.5. 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.

In addition to antioxidant effects, TA molecules also exhibit anti-inflammatory properties [41]. We examined the in vitro anti-inflammatory effects of TA-CaCO₃ by analyzing the mRNA expression of pro-inflammatory factors, such as cyclooxygenase-2 (COX-2), interleukin (IL)-1β, IL-6, matrix metalloproteinase (MMP)-3, MMP-13, and tumor necrosis factor (TNF)-α in lipopolysaccharide (LPS)-stimulated chondrocytes, because LPS treatment leads to the overexpression of these pro-inflammatory factors [41,42,43].

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-stimulated chondrocytes. Consistent with previous studies, the present study also supports the anti-inflammatory effects of TA-based materials [24,44].

2.6. 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 fluorescence was observed in normal cells, whereas a strong signal was detected in cells treated with 300 μM of H₂O₂ alone. The treatment of the extracts containing different concentrations of TA-CaCO₃ led to a remarkable decrease in fluorescence intensities, indicative of the excellent scavenging of intracellular ROS by TA-CaCO₃.

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.

3. Materials and Methods

3.1. Materials

Calcium chloride dihydrate (CaCl₂·2H₂O, abbreviated as CaCl₂), sodium carbonate monohydrate (Na₂CO₃·H₂O, abbreviated as Na₂CO₃), TA, potassium bromide (KBr), BTB, DPPH, sodium hydroxide (NaOH), 26% sodium chloride (NaCl) solution, and hydrochloric acid (HCl) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol (MeOH, 99.5%) and absolute ethanol (EtOH, 99.9%) were provided by Samchun (Pyeongtaek, Korea) and DUKSAN (Ansan, Korea), respectively. Commercial CaCO₃ nanopowder (purity: 98%) was supplied by US Research Nanomaterials (Houston, TX, USA), and PBS by Lonza (Walkersville, MD, USA). SGF (pH = 1.5) was prepared using ultra-pure water (99.46% v/v), concentrated HCl (0.23% v/v), and 26% NaCl solution (0.21% v/v). To prepare the BTB solution, 10 mg of BTB was dissolved in 1 mL of 4% NaOH solution, and treated with 2 mL of 70% EtOH (2 mL) and 100 mL of ultra-pure water. Finally, a 10% NaOH solution was added dropwise to the BTB solution when the color of the BTB solution changed to blue.

3.2. Preparation of TA-CaCO₃

For the preparation of TA-CaCO₃ materials, TA (0.0294 mmol) was added to ultra-pure water (5 mL, pH = 7.0) and stirred to dissolve at 800 rpm for 1 h. Different moles of CaCl₂ (0.735, 1.47, 2.20, 2.94, and 4.41 mmol) were dissolved in ultra-pure water (3 mL, pH = 7.0). The prepared CaCl₂ solutions were added to the TA solution and stirred at 800 rpm for 2 h. In addition, different moles of Na₂CO₃ (0.735, 1.47, 2.20, 2.94, and 4.41 mmol) were dissolved in ultra-pure water (pH = 7.0) and added dropwise to the mixture solution of TA and CaCl₂. The mixture was stirred at 800 rpm for 24 h, and the resulting solution was centrifuged at 1200 rpm for 5 min and washed with ultrapure water. The collected TA-CaCO₃ materials were freeze-dried for two days. TA-CaCO₃ materials synthesized at different molar ratios of TA (molar ratio: 1), CaCl₂ (molar ratios: 25, 50, 75, 100, and 150), and Na₂CO₃ (molar ratios of 25, 50, 75, 100, and 150) were named as follows: 1:25, 1:50, 1:75, 1:100, and 1:150 TA-CaCO₃.

3.3. Characterization of TA-CaCO₃

For SEM analysis, the prepared TA-CaCO₃ materials were coated with platinum. Then, the morphologies and sizes of the as-prepared TA-CaCO₃ materials were examined using field-emission scanning electron microscopy (FE-SEM, S-4700, Hitachi, Japan). For size analysis of the prepared TA-CaCO₃, randomly selected individual particles of each TA-CaCO₃ type were analyzed using ImageJ software (Version 1.47; US National Institutes of Health, Bethesda, MD, USA). The surface elemental composition of TA-CaCO₃ was examined using SEM-EDS.

To determine the amount of Ca²⁺ in TA-CaCO₃, 1:75 TA-CaCO₃ materials (0.1 mg) were co-treated with nitric acid and H₂O₂, and the prepared samples were analyzed using ICP-OES (Optima 7300 DV, PerkinElmer, Waltham, MA, USA). Through ICP-OES analysis, the amount of TA and CaCO₃ within TA-CaCO₃ was determined using a standard curve (Y = 10,807x + 1340.9; R² = 0.999).

The prepared TA-CaCO₃ 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-CaCO₃ 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.

3.4. Antacid Effects of TA-CaCO₃ Materials

To investigate the in vitro antacid effects, CaCO₃ (10 mg) or TA-CaCO₃ (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).

3.5. Antioxidant Effects of TA-CaCO₃ Materials

To examine and compare the in vitro antioxidant effects, CaCO₃ (1 mg) or TA-CaCO₃ (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).

3.6. In Vitro Cytotoxicity and Anti-Inflammatory Effects

Human knee articular chondrocytes were obtained from Lonza (Basel, Switzerland) and cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Seoul, Korea), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C.

The cytotoxicity of TA-CaCO₃ materials was determined with a cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Chondrocytes (1 × 10⁴ cells/well) were seeded and incubated in a 96-well plate. After 24 h of incubation, TA-CaCO₃ 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-CaCO₃ 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 × 10⁵ 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-CaCO₃ (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.

3.7. Antioxidant Effects of TA-CaCO₃ at the Cellular Level

To investigate the antioxidant effects of TA-CaCO₃ at the cell level, we conducted DCFDA staining and a DCFDA assay. Cells (1 × 10⁴ 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-CaCO₃ (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 H₂O₂ 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).

3.8. Protection of Cell Viability in the ROS Environment

To investigate whether TA-CaCO₃ can protect cell viability in an ROS environment, chondrocytes (1 × 10⁵ cells/well) were seeded in a 24-well culture plate. Then, the cells were treated with 300 μM of H₂O₂ at 37 °C for 30 min, followed by additional treatment of TA-CaCO₃ 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.

4. Conclusions

In the present study, we prepared TA-CaCO₃ materials by reacting TA with CaCl₂ and Na₂CO₃, which led to the interaction between TA and Ca²⁺ ions, followed by nucleation of CaCO₃. Micron-sized 1:75 TA-CaCO₃ materials (ranging from 3 to 6 μm) comprised small nanoparticles in a size range of 17–41 nm. TA-CaCO₃ 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-CaCO₃ 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 CaCO₃ materials, CaCO₃-based materials can improve the incorporation efficacy of drugs. Thus, using TA-CaCO₃ 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.

Supplementary Materials

The supplementary materials are available online at https://www.mdpi.com/article/10.3390/ijms22094614/s1.

Author Contributions

Conceptualization, K.P. and S.-E.K.; methodology, S.-Y.J., H.H., H.-S.J. and S.C.; formal analysis, S.-Y.J., H.H., H.-S.J. and S.C.; writing—original draft preparation, S.-Y.J. and K.P.; writing—review and editing, H.-J.K., K.P. and S.-E.K.; funding acquisition, K.P. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the National Research Foundation of Korea (NRF-2019M3A9E2066883) and by the Chung-Ang University Research Grants in 2020.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Figure 1. Schematic illustration of the synthesis of TA-CaCO3 materials. TA was sequentially reacted with CaCl2 for 2 h and Na2CO3 for an additional 24 h in pure water (pH = 7.0).

Figure 2. (a) The representative SEM images and magnified surface of 1:75 TA-CaCO3 materials. The magnified SEM image revealed that the micron-sized TA-CaCO3 materials comprised small TA-CaCO3 nanoparticles. (b) Particle size distribution of the small TA-CaCO3 nanoparticles of 1:75 TA-CaCO3. (c) EDS mapping of elemental carbon (C; white), oxygen (O; green), and calcium (Ca; red). Scale bar: 1 μm. (d) EDS spectrum of 1:75 TA-CaCO3.

Figure 3. FT-IR spectra of commercial CaCO3 and TA-CaCO3.

Figure 4. XRD and XPS analyses of commercial CaCO3 and TA-CaCO3 materials. (a) XRD spectra of CaCO3 and TA-CaCO3. (b) Wide scan XPS spectra recorded from CaCO3 and TA-CaCO3.

Figure 5. Antacid effects of TA-CaCO3 using the colorimetric bromothymol blue (BTB) method. (a) Color changes and (b) UV/vis spectra of BTB under PBS (pH = 7.4) and simulated gastric fluid (SGF, pH = 1.5) after treatment with commercial CaCO3 and 1:75 TA-CaCO3. Green-colored arrows indicate a pH increase.

Figure 6. Antioxidant effects of TA-CaCO3 determined using the colorimetric DPPH method. Changes in the color and UV/vis spectra of DPPH after treatment with commercial CaCO3 and 1:75 TA-CaCO3. Inset: Photos of the DPPH solution treated with commercial CaCO3 and 1:75 TA-CaCO3.

Figure 7. In vitro anti-inflammatory effects of 1:75 TA-CaCO3 in inflamed chondrocytes. The mRNA levels of pro-inflammatory factors, including (a) cyclooxygenase-2 (COX-2), (b) interleukin-1β (IL-1β), (c) IL-6, (d) matrix metalloproteinase-3 (MMP-3), (e) MMP-13, and (f) tumor necrosis factor-α (TNF-α) in LPS-stimulated chondrocytes on day three. Data are represented as the mean ± SD (n = 5). ** p < 0.01 and * p < 0.05.

Figure 8. In vitro antioxidant effects of TA-CaCO3. (a) Representative fluorescence images of intracellular ROS in chondrocytes treated with extracts containing different concentrations of 1:75 TA-CaCO3 for 24 h after pretreatment with 300 μM of H2O2 for 30 min. Scale bar: 20 μm. (b) Viability of chondrocytes treated with different concentrations of 1:75 TA-CaCO3 for 24 h after pretreatment with 300 μM of H2O2 for 30 min. Results are expressed as the mean ± SD (n = 5). ** p < 0.01 and * p < 0.05.