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Reo Kimura, [Kota Shiba](https://orcid.org/0000-0001-7775-0318), Kanata Fujiwara, Yanni Zhou, Iori Yamada, Motohiro Tagaya

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[Precipitative Coating of Calcium Phosphate on Microporous Silica–Titania Hybrid Particles in Simulated Body Fluid](https://mdr.nims.go.jp/datasets/c44670b6-e400-47b1-bd7e-60ee589d91bc)

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Precipitative Coating of Calcium Phosphate on Microporous Silica–Titania Hybrid Particles in Simulated Body FluidCitation: Kimura, R.; Shiba, K.;Fujiwara, K.; Zhou, Y.; Yamada, I.;Tagaya, M. Precipitative Coating ofCalcium Phosphate on MicroporousSilica–Titania Hybrid Particles inSimulated Body Fluid. Inorganics2023, 11, 235. https://doi.org/10.3390/inorganics11060235Academic Editors: Roberto Nisticòand Silvia MostoniReceived: 11 April 2023Revised: 23 May 2023Accepted: 24 May 2023Published: 28 May 2023Copyright: © 2023 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).inorganicsArticlePrecipitative Coating of Calcium Phosphate on MicroporousSilica–Titania Hybrid Particles in Simulated Body FluidReo Kimura 1, Kota Shiba 2,* , Kanata Fujiwara 1, Yanni Zhou 1, Iori Yamada 1,3 and Motohiro Tagaya 1,*1 Department of Materials Science and Bioengineering Technology, Nagaoka University of Technology,Kamitomioka 1603-1, Nagaoka 940-2188, Japan2 Center for Functional Sensor & Actuator, Research Center for Functional Materials, National Institute forMaterials Science (NIMS), 1-1, Namiki, Tsukuba 305-0044, Japan3 Research Fellow of the Japan Society for the Promotion of Science (DC), 5-3-1 Koji-machi, Chiyoda-ku,Tokyo 102-0083, Japan* Correspondence: shiba.kota@nims.go.jp (K.S.); tagaya@mst.nagaokaut.ac.jp (M.T.)Abstract: Titania and silica have been recognized as potential drug delivery system (DDS) carriers.For this application, controllable biocompatibility and the suppression of the initial burst are required,which can be provided by a calcium phosphate (CP) coating. However, it is difficult to control themorphology of a CP coating on the surface of carrier particles owing to the homogeneous nucleation ofCP. In this study, we report the development of a CP-coating method that homogeneously correspondsto the shapes of silica–titania (SiTi) porous nanoparticles. We also demonstrate that controlled surfaceroughness of CP coatings could be achieved in SBF using SiTi nanoparticles with a well-definedspherical shape, a uniform size, and a tunable nanoporous structure. The precipitation of CP wasperformed on mono-dispersed porous SiTi nanoparticles with different Si/Ti molar ratios and poresizes. The pore size distribution was found to significantly affect the CP coating in SBF immersion;the surfaces of the nanoparticles with bimodal pore sizes of 0.7 and 1.1–1.2 nm became rough afterCP precipitation, while those with a unimodal pore size of 0.7 nm remained smooth, indicating thatthese two pore sizes serve as different nucleation sites that lead to different surface morphologies.Keywords: bioceramic nanoparticles; simulated body fluid; nanopore; CP precipitative coating;silica–titania nanohybrid1. IntroductionVarious nanomaterials composed of bioinert ceramics have been synthesized foruse as artificial joints, implants, and drug delivery system (DDS) carriers [1,2]. In theseapplications, a DDS carrier needs to fulfill many requisites, including not only the inherentbiocompatibility of bioceramic-based materials but also other properties including being ofa uniform shape, size, and size distribution and possessing high affinity for aqueous mediain order to form a stable suspension [3–5]. A sol–gel method based on the hydrolysis andcondensation of a metal alkoxide has been used to synthesize a variety of biocompatiblemetal oxide nanoparticles with controlled morphologies, for which titania and silica havebeen extensively studied [6–8]. For example, amorphous titania in nanotube form hasbeen investigated with respect to its use as a DDS carrier [9,10]. Since the surface of titaniaexhibits a Zeta potential of −18 mV [11] in water (at pH 7.4), serious aggregate formationcan occur depending on the experimental conditions. By contrast, amorphous silica showsa higher Zeta potential of −60 mV in water (at pH 7.4) [12], allowing for the formation ofa relatively stable suspension [13,14]. Although mixed oxide nanoparticles composed ofsilica and titania (SiTi nanoparticles) [15] have been explored as another potential option,their use also poses the problems such as biotoxicity due to the release of a silicate ionelusion into the biological solution [16] and difficulty in controlling drug release owing toInorganics 2023, 11, 235. https://doi.org/10.3390/inorganics11060235 https://www.mdpi.com/journal/inorganicshttps://doi.org/10.3390/inorganics11060235https://doi.org/10.3390/inorganics11060235https://creativecommons.org/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://www.mdpi.com/journal/inorganicshttps://www.mdpi.comhttps://orcid.org/0000-0001-7775-0318https://orcid.org/0000-0003-3695-7253https://doi.org/10.3390/inorganics11060235https://www.mdpi.com/journal/inorganicshttps://www.mdpi.com/article/10.3390/inorganics11060235?type=check_update&version=2Inorganics 2023, 11, 235 2 of 13the initial burst [17,18]. Therefore, the surfaces of SiTi nanoparticles need to be properlydesigned before being used for DDS applications.Calcium phosphate (CP) coating has been developed as a technique to improve theosteoconductivity of the surfaces of titanium implants [19–21]. The CP coating is thoughtto suppress the initial burst of drug molecules [22], allowing them to be released graduallyover several weeks. This is due to the fact that the CP coating itself can act as a reservoir fordrug molecules, which slowly dissolve and diffuse over time as the coating degrades. Thegeneral CP-coating methods are electrochemical deposition, sputtering, and plasma spray-ing, which are performed under unphysiological conditions such as at high temperaturesto provide different chemical and crystalline states with respect to the bone hydroxyap-atite [23–25], leading to lower bioactivity in vivo. The biomimetic method has attractedattention due to its potential benefits. In this approach, CP is precipitated on particles insimulated body fluid (SBF) under conditions that mimic the biological environment of aliving body [26,27]. Biomimetic CP synthesized under these conditions has been foundto be more bioactive than CP synthesized under higher-temperature conditions [28–30].However, biomimetic CP has generally only been coated on flat substrates [31–35]. In thecase of nanoparticles, uniform nucleation occurs at different positions from the particlesurfaces due to their high curvature and lower ability to induce heterogeneous nucleation.Therefore, a coating technique that adapts to the shapes of nanoparticles and provides auniform coating has not been developed.In this study, we demonstrate that controlled surface roughness of CP coatings can beachieved in SBF by using SiTi nanoparticles with a well-defined spherical shape, a uniformsize, and a tunable nanoporous structure (Scheme 1). We synthesized SiTi nanoparticlesusing a microfluidic approach [15], which allowed us to design their size and shape sothat they were suitable for DDS, and used them as a scaffold for the CP coating. The SiTinanoparticles serve two critical functions in the SBF: they (1) provide CP nucleation sitesthat promote the substitution of phosphate ions with silicate ions and (2) create nanoporesthat induce the selective adsorption of hydrated ions in SBF. As described, the silicateions elute readily into biological fluids [36,37] and can be replaced by phosphate ions [38],facilitating Ca2+ ion adsorption and subsequent CP nucleation. Moreover, the hydratedions in SBF, including Na+, K+, and Ca2+, can be adsorbed into the nanopores in theirhydrated states and their sizes differ from each other. We prove that SiTi nanoparticleswith tunable nanostructures can effectively function as an ion (molecular) sieve [39,40] thatenables the selective adsorption of Ca2+ from SBF, leading to the formation of CP coatings.We also discuss the effect of nanopore sizes on the surface roughness of CP coatings.Inorganics 2023, 11, x FOR PEER REVIEW 3 of 14     Scheme 1. Illustration of the CP precipitation process of the SiTi nanoparticles via immersion in SBF. 2. Results and Discussion 2.1. Synthesis Result of XSiTi Nanoparticles The Si/Ti molar ratios were measured via XRF and the values were X, and the sample was named as XSiTi (X = 0, 0.1, 0.7, and 1.2). The FE-SEM images and size distributions of the XSiTi nanoparticles are shown in Figure 1. All the SiTi nanoparticles exhibited spher-ical and mono-dispersed states. The diameter of the particles was around 150–200 nm, which is considered a size that does not induce cytotoxicity [41,42][41,42]. The XRD patterns (Figure S1) of the XSiTi nanoparticles indicated that all the nano-particles were amorphous. Comparing the properties of these particles (e.g., particle shape, particle size, and CV value) with those of previously reported particles [15], we confirmed that they were identical and that equivalent particles were synthesized. Figure 2 shows the N2 adsorption and desorption isotherms of the SiTi nanoparticles. In the results regarding the specific surface area calculated using the αs-plot (Figure 2a), it is evident that the surface area increased with the increase in the Si/Ti molar ratio. Ac-cording to the nanopore size distributions based on the MP (micropore) method in the results regarding the XSiTi nanoparticles (Figure 2b,d), 0SiTi and 0.1SiTi exhibited bi-modal nanopore sizes of 0.7 and 1.1~1.2 nm, and 0.7SiTi and 1.2SiTi exhibited only mon-omodal pores of 0.7 nm. The different nanopore sizes occurred due to the increase in the Si/Ti molar ratio. We propose that the hydrated ions of SBF were potentially diffused and adsorbed into the nanopores (Figure 2c,e). We suggest that nanopores 0SiTi and 0.1SiTi, with pore sizes of 1.1~1.2 nm, enable the diffusion and adsorption of the hydrated Ca2+, Na+, and K+ ions, while 0.7SiTi and 1.2SiTi, with a pore size of 0.7 nm, only allows for the diffusion and adsorption of the hydrated Na+ and K+ ions. According to the nanopore size distribution, 0SiTi and 0.1SiTi were defined as Group1.1, while 0.7SiTi and 1.2SiTi were defined as Group0.7. By comparing these nanopore diameters with those of previously reported particles [15], we confirmed that they are identical and that comparable particles had been synthesized. Formatted: Not HighlightFormatted: Not HighlightFormatted: Not HighlightScheme 1. Illustration of the CP precipitation process of the SiTi nanoparticles via immersion in SBF.Inorganics 2023, 11, 235 3 of 132. Results and Discussion2.1. Synthesis Result of XSiTi NanoparticlesThe Si/Ti molar ratios were measured via XRF and the values were X, and the samplewas named as XSiTi (X = 0, 0.1, 0.7, and 1.2). The FE-SEM images and size distributions ofthe XSiTi nanoparticles are shown in Figure 1. All the SiTi nanoparticles exhibited sphericaland mono-dispersed states. The diameter of the particles was around 150–200 nm, which isconsidered a size that does not induce cytotoxicity [41,42].Inorganics 2023, 11, x FOR PEER REVIEW 4 of 14    Figure 1. FE-SEM images and particle size distributions of the SiTi nanoparticles.   Figure 2. (a) N2 adsorption (●) and desorption (○) isotherms of the SiTi nanoparticles; (b,d) the MP pore size distributions; and (c,e) illustrations of the hydrated ion interactions with the micropores. The specific surface areas of 0SiTi, 0.1SiTi, 0.7SiTi, and 1.2SiTi were 382, 443, 466, and 570 m2・g −1, respectively. 2.2. Results Regarding the SBF-Immersed SiTi Nanoparticles The chemical element (Ca, Na, and K) amounts adsorbed by the SiTi nanoparticles through immersion in SBF were evaluated via XRF (Figure 3). The adsorbed elements in-creased with an increased immersion time. Referring to the results regarding the change in the amount of Ca on the nanoparticles (Figure 3a), the amount in Group1.1 was clearly larger than that in Group0.7 at the initial stage, indicating that Ca was preferentially ad-sorbed on the nanoparticles in Group1.1. By observing the amount changes of Na and K adsorbed on the nanoparticles (Figure 3b,c), it is evident that those of Group0.7 were sig-nificantly larger than those in Group1.1 at the initial stage, indicating that Na and K were Figure 1. FE-SEM images and particle size distributions of the SiTi nanoparticles.The XRD patterns (Figure S1) of the XSiTi nanoparticles indicated that all the nanopar-ticles were amorphous. Comparing the properties of these particles (e.g., particle shape,particle size, and CV value) with those of previously reported particles [15], we confirmedthat they were identical and that equivalent particles were synthesized.Figure 2 shows the N2 adsorption and desorption isotherms of the SiTi nanoparticles.In the results regarding the specific surface area calculated using the αs-plot (Figure 2a),it is evident that the surface area increased with the increase in the Si/Ti molar ratio.According to the nanopore size distributions based on the MP (micropore) method in theresults regarding the XSiTi nanoparticles (Figure 2b,d), 0SiTi and 0.1SiTi exhibited bimodalnanopore sizes of 0.7 and 1.1~1.2 nm, and 0.7SiTi and 1.2SiTi exhibited only monomodalpores of 0.7 nm. The different nanopore sizes occurred due to the increase in the Si/Timolar ratio. We propose that the hydrated ions of SBF were potentially diffused andadsorbed into the nanopores (Figure 2c,e). We suggest that nanopores 0SiTi and 0.1SiTi,with pore sizes of 1.1~1.2 nm, enable the diffusion and adsorption of the hydrated Ca2+,Na+, and K+ ions, while 0.7SiTi and 1.2SiTi, with a pore size of 0.7 nm, only allows for thediffusion and adsorption of the hydrated Na+ and K+ ions. According to the nanopore sizedistribution, 0SiTi and 0.1SiTi were defined as Group1.1, while 0.7SiTi and 1.2SiTi weredefined as Group0.7. By comparing these nanopore diameters with those of previouslyreported particles [15], we confirmed that they are identical and that comparable particleshad been synthesized.2.2. Results Regarding the SBF-Immersed SiTi NanoparticlesThe chemical element (Ca, Na, and K) amounts adsorbed by the SiTi nanoparticlesthrough immersion in SBF were evaluated via XRF (Figure 3). The adsorbed elementsincreased with an increased immersion time. Referring to the results regarding the changein the amount of Ca on the nanoparticles (Figure 3a), the amount in Group1.1 was clearlylarger than that in Group0.7 at the initial stage, indicating that Ca was preferentiallyInorganics 2023, 11, 235 4 of 13adsorbed on the nanoparticles in Group1.1. By observing the amount changes of Naand K adsorbed on the nanoparticles (Figure 3b,c), it is evident that those of Group0.7were significantly larger than those in Group1.1 at the initial stage, indicating that Naand K were preferentially adsorbed on the nanoparticles in Group0.7. These differencesare thought to be due to the difference in the nanopore sizes between Group1.1 andGroup0.7. The diameters of the hydrated ions that could diffuse and be adsorbed insidethe nanopores were determined as shown in Figure 2. The adsorption of Ca in Group1.1reached equilibrium within 1 day of immersion, while the other ions in Group0.7 did notreach equilibrium even after 3 days. In addition, most of the adsorption of Na and K forGroup0.7 reached equilibrium within 1 day of immersion, whereas that for Group1.1 didnot reach equilibrium until 3 days.Inorganics 2023, 11, x FOR PEER REVIEW 4 of 14    Figure 1. FE-SEM images and particle size distributions of the SiTi nanoparticles.   Figure 2. (a) N2 adsorption (●) and desorption (○) isotherms of the SiTi nanoparticles; (b,d) the MP pore size distributions; and (c,e) illustrations of the hydrated ion interactions with the micropores. The specific surface areas of 0SiTi, 0.1SiTi, 0.7SiTi, and 1.2SiTi were 382, 443, 466, and 570 m2・g −1, respectively. 2.2. Results Regarding the SBF-Immersed SiTi Nanoparticles The chemical element (Ca, Na, and K) amounts adsorbed by the SiTi nanoparticles through immersion in SBF were evaluated via XRF (Figure 3). The adsorbed elements in-creased with an increased immersion time. Referring to the results regarding the change in the amount of Ca on the nanoparticles (Figure 3a), the amount in Group1.1 was clearly larger than that in Group0.7 at the initial stage, indicating that Ca was preferentially ad-sorbed on the nanoparticles in Group1.1. By observing the amount changes of Na and K adsorbed on the nanoparticles (Figure 3b,c), it is evident that those of Group0.7 were sig-nificantly larger than those in Group1.1 at the initial stage, indicating that Na and K were Figure 2. (a) N2 adsorption (•) and desorption (#) isotherms of the SiTi nanoparticles; (b,d) theMP pore size distributions; and (c,e) illustrations of the hydrated ion interactions with the mi-cropores. The specific surface areas of 0SiTi, 0.1SiTi, 0.7SiTi, and 1.2SiTi were 382, 443, 466, and570 m2·g−1, respectively.Inorganics 2023, 11, x FOR PEER REVIEW 5 of 14   preferentially adsorbed on the nanoparticles in Group0.7. These differences are thought to be due to the difference in the nanopore sizes between Group1.1 and Group0.7. The diameters of the hydrated ions that could diffuse and be adsorbed inside the nanopores were determined as shown in Figure 2. The adsorption of Ca in Group1.1 reached equi-librium within 1 day of immersion, while the other ions in Group0.7 did not reach equi-librium even after 3 days. In addition, most of the adsorption of Na and K for Group0.7 reached equilibrium within 1 day of immersion, whereas that for Group1.1 did not reach equilibrium until 3 days.  Figure 3. Adsorbed amount changes of the chemical elements of (a) Ca, (b) Na, and (c) K on the SiTi nanoparticles from SBF with immersion time. The nucleation sites pertaining to the CP precipitation of SiTi nanoparticles im-mersed in SBF are discussed in Figure 4. Figure 4a shows the FT-IR spectra of the change in the absorbance band due to the OH group of the nanoparticles. The band intensity of Group1.1 did not change after immersion, whereas Group0.7 showed an increase in band intensity. The result of the change in the Si/Ti molar ratio of the nanoparticles after im-mersion is shown in Figure 4b. Group1.1 did not change in terms of its Si/Ti molar ratio, whereas Group0.7 showed a significant decrease, suggesting that the Si component was eluted from Group0.7 into SBF. Regarding the changes in the average particle sizes of the nanoparticles following immersion (Figure 4c), all the nanoparticles showed a decrease in the size, and a significant decrease was observed in Group0.7. Group1.1 containing lower Si-content did not show a change, while Group0.7 with higher Si-content showed a change, indicating that the Si component’s elution can induce CP precipitation. The mech-anism behind the CP nucleation in the precipitation on Group0.7 is suggested in Figure 4d. Group0.7 preferentially absorbed the hydrated Na+ and K+ ions inside the nanopores. The Si-components in Group0.7 were eluted as the silicate ions outside the nanopores, and the phosphate ions interacting with the H2O component in SBF were adsorbed into the eluted sites [38]. The intensity of the OH group of Group0.7 increased through immersion in SBF due to the subsequent adsorption of the hydrated Ca2+ ions and the consequent promotion of CP nucleation. Therefore, the outside nanopore surfaces are considered the CP nucleation sites for Group0.7. Formatted: Not HighlightFigure 3. Adsorbed amount changes of the chemical elements of (a) Ca, (b) Na, and (c) K on the SiTinanoparticles from SBF with immersion time.The nucleation sites pertaining to the CP precipitation of SiTi nanoparticles immersedin SBF are discussed in Figure 4. Figure 4a shows the FT-IR spectra of the change inthe absorbance band due to the OH group of the nanoparticles. The band intensity ofGroup1.1 did not change after immersion, whereas Group0.7 showed an increase in bandintensity. The result of the change in the Si/Ti molar ratio of the nanoparticles afterimmersion is shown in Figure 4b. Group1.1 did not change in terms of its Si/Ti molar ratio,whereas Group0.7 showed a significant decrease, suggesting that the Si component wasInorganics 2023, 11, 235 5 of 13eluted from Group0.7 into SBF. Regarding the changes in the average particle sizes of thenanoparticles following immersion (Figure 4c), all the nanoparticles showed a decreasein the size, and a significant decrease was observed in Group0.7. Group1.1 containinglower Si-content did not show a change, while Group0.7 with higher Si-content showeda change, indicating that the Si component’s elution can induce CP precipitation. Themechanism behind the CP nucleation in the precipitation on Group0.7 is suggested inFigure 4d. Group0.7 preferentially absorbed the hydrated Na+ and K+ ions inside thenanopores. The Si-components in Group0.7 were eluted as the silicate ions outside thenanopores, and the phosphate ions interacting with the H2O component in SBF wereadsorbed into the eluted sites [38]. The intensity of the OH group of Group0.7 increasedthrough immersion in SBF due to the subsequent adsorption of the hydrated Ca2+ ions andthe consequent promotion of CP nucleation. Therefore, the outside nanopore surfaces areconsidered the CP nucleation sites for Group0.7.Inorganics 2023, 11, x FOR PEER REVIEW 6 of 14     Figure 4. (a) FT-IR spectra, (b) Si/Ti molar ratio, and (c) average particle size changes of the SiTi nanoparticles with immersion time in SBF, and (d) illustrations of the possible interfacial reactions of Group0.7 with the ions in SBF. Xd represents the immersion time of X days (X = 0, 1, 3, and 7). In Figure 5, the characteristics of phosphate ion adsorption for the CP nucleation sites on Group1.1 and Group0.7 are evaluated and discussed. According to the changes of the absorption band generated by phosphate ions after immersion (Figure 5a), Group1.1 and Group0.7 showed increases in the absorbance bands of the stretching vibrations due to Ti–P–O [43], P–O, and P–OH bonds [44] at 1100, 1039–997, and 866–842 cm−1 following immersion. For Group0.7, the bands produced by Si–O–Si [45] and Si–OH [46] at 1039–997 and 866–842 cm−1 were also included in the spectra, and the shapes were different from those of Group1.1. The amount changes in the adsorbed phosphorous components of Group1.1 and Group0.7 showed an increase in the amount after immersion (Figure 5b). In particular, Group1.1 reached the adsorption equilibrium after approximately 1 day of im-mersion, whereas Group0.7 showed a slower adsorption rate, suggesting that the CP pre-cipitate emerged at a relatively earlier stage in Group1.1 compared to that of Group0.7. Figure 5c shows the possible illustrations of the nucleation sites of Group1.1 and Group0.7. In Group1.1, the hydrated Ca2+ ions in addition to the Na+ and K+ ions were preferentially diffused and absorbed inside the nanopores, which serve as sites for CP nucleation. In Group0.7, the hydrated Na+ and K+ ions were preferentially diffused and absorbed inside the nanopores, and the phosphate ions were replaced with the sites where the silicate ions were eluted, suggesting that the outside of the nanopores serve as CP nucleation sites. Formatted: Not HighlightFormatted: Not HighlightFormatted: Not HighlightFormatted: Not HighlightFigure 4. (a) FT-IR spectra, (b) Si/Ti molar ratio, and (c) average particle size changes of the SiTinanoparticles with immersion time in SBF, and (d) illustrations of the possible interfacial reactions ofGroup0.7 with the ions in SBF. Xd represents the immersion time of X days (X = 0, 1, 3, and 7).In Figure 5, the characteristics of phosphate ion adsorption for the CP nucleation siteson Group1.1 and Group0.7 are evaluated and discussed. According to the changes of theabsorption band generated by phosphate ions after immersion (Figure 5a), Group1.1 andGroup0.7 showed increases in the absorbance bands of the stretching vibrations due toTi–P–O [43], P–O, and P–OH bonds [44] at 1100, 1039–997, and 866–842 cm−1 followingimmersion. For Group0.7, the bands produced by Si–O–Si [45] and Si–OH [46] at 1039–997and 866–842 cm−1 were also included in the spectra, and the shapes were different fromthose of Group1.1. The amount changes in the adsorbed phosphorous components ofGroup1.1 and Group0.7 showed an increase in the amount after immersion (Figure 5b).In particular, Group1.1 reached the adsorption equilibrium after approximately 1 day ofimmersion, whereas Group0.7 showed a slower adsorption rate, suggesting that the CPprecipitate emerged at a relatively earlier stage in Group1.1 compared to that of Group0.7.Figure 5c shows the possible illustrations of the nucleation sites of Group1.1 and Group0.7.In Group1.1, the hydrated Ca2+ ions in addition to the Na+ and K+ ions were preferentiallydiffused and absorbed inside the nanopores, which serve as sites for CP nucleation. InGroup0.7, the hydrated Na+ and K+ ions were preferentially diffused and absorbed insidethe nanopores, and the phosphate ions were replaced with the sites where the silicate ionswere eluted, suggesting that the outside of the nanopores serve as CP nucleation sites.Inorganics 2023, 11, 235 6 of 13Inorganics 2023, 11, x FOR PEER REVIEW 7 of 14    Figure 5. (a) FT-IR spectral changes of the SiTi nanoparticles with immersion time in SBF and (b) the adsorbed amount changes of phosphorus from SBF. (c) Illustrations of the possible calcium phosphate nucleation processes of Group1.1 and Group0.7. According to the FE-SEM images and particle size distributions of the SiTi nanopar-ticles (Figure 6), even after immersion for 7 days, the particles still exhibited spherical shapes and mono-dispersed states, indicating a preserved particle size of approximately 150–200 nm. In particular, Group1.1 exhibited rough surfaces, whereas Group0.7 retained smooth surfaces. These results show that CP was roughly precipitated on Group1.1 but was smoothly precipitated on Group0.7, indicating that a smooth CP coating was achieved using Group0.7.  Figure 6. FE-SEM images and particle size distributions of the SiTi nanoparticles after immersion in SBF for 7 days. Figure 5. (a) FT-IR spectral changes of the SiTi nanoparticles with immersion time in SBF and (b) theadsorbed amount changes of phosphorus from SBF. (c) Illustrations of the possible calcium phosphatenucleation processes of Group1.1 and Group0.7.According to the FE-SEM images and particle size distributions of the SiTi nanopar-ticles (Figure 6), even after immersion for 7 days, the particles still exhibited sphericalshapes and mono-dispersed states, indicating a preserved particle size of approximately150–200 nm. In particular, Group1.1 exhibited rough surfaces, whereas Group0.7 retainedsmooth surfaces. These results show that CP was roughly precipitated on Group1.1 butwas smoothly precipitated on Group0.7, indicating that a smooth CP coating was achievedusing Group0.7.Inorganics 2023, 11, x FOR PEER REVIEW 7 of 14    Figure 5. (a) FT-IR spectral changes of the SiTi nanoparticles with immersion time in SBF and (b) the adsorbed amount changes of phosphorus from SBF. (c) Illustrations of the possible calcium phosphate nucleation processes of Group1.1 and Group0.7. According to the FE-SEM images and particle size distributions of the SiTi nanopar-ticles (Figure 6), even after immersion for 7 days, the particles still exhibited spherical shapes and mono-dispersed states, indicating a preserved particle size of approximately 150–200 nm. In particular, Group1.1 exhibited rough surfaces, whereas Group0.7 retained smooth surfaces. These results show that CP was roughly precipitated on Group1.1 but was smoothly precipitated on Group0.7, indicating that a smooth CP coating was achieved using Group0.7.  Figure 6. FE-SEM images and particle size distributions of the SiTi nanoparticles after immersion in SBF for 7 days. Figure 6. FE-SEM images and particle size distributions of the SiTi nanoparticles after immersion inSBF for 7 days.The elemental mapping results and TEM images of 0.1SiTi and 1.2SiTi after their immer-sion are shown in Figure 7. The particle images (i.e., BF: STEM HAADF images) and shapes(i.e., locations) of the chemical elements were similar between Group1.1 and Group0.7(Figure 7a,d), indicating that a homogeneous CP precipitation on the surfaces could beachieved by immersing the nanoparticles in SBF. The Ca signal for 0.1SiTi (Group1.1) wasweaker than that for 1.2SiTi (Group0.7), which is possibly due to the different CP nucleationInorganics 2023, 11, 235 7 of 13mechanisms between Group1.1 and Group0.7 (as shown in Figure 5c). In Group1.1, thehydrated Ca2+ ions in addition to the Na+ and K+ ions were preferentially diffused andadsorbed inside the nanopores, which served as CP nucleation sites. The results suggestedthat the number of nucleation sites in Group1.1 is smaller than that in Group0.7, indicatinga lower amount of the CP precipitation. The contrast of 0.1SiTi (Group1.1) was differentfrom that of 1.2SiTi (Group0.7), indicating the presence of rough surfaces due to the CPprecipitation of Group1.1 (Figure 7b,c,e,f).Inorganics 2023, 11, x FOR PEER REVIEW 8 of 14   The elemental mapping results and TEM images of 0.1SiTi and 1.2SiTi after their im-mersion are shown in Figure 7. The particle images (i.e., BF: STEM HAADF images) and shapes (i.e., locations) of the chemical elements were similar between Group1.1 and Group0.7 (Figure 7a,d), indicating that a homogeneous CP precipitation on the surfaces could be achieved by immersing the nanoparticles in SBF. The Ca signal for 0.1SiTi (Group1.1) was weaker than that for 1.2SiTi (Group0.7), which is possibly due to the dif-ferent CP nucleation mechanisms between Group1.1 and Group0.7 (as shown in Figure 5c). In Group1.1, the hydrated Ca2+ ions in addition to the Na+ and K+ ions were preferen-tially diffused and adsorbed inside the nanopores, which served as CP nucleation sites. The results suggested that the number of nucleation sites in Group1.1 is smaller than that in Group0.7, indicating a lower amount of the CP precipitation. The contrast of 0.1SiTi (Group1.1) was different from that of 1.2SiTi (Group0.7), indicating the presence of rough surfaces due to the CP precipitation of Group1.1 (Figure 7b,c,e,f).  Figure 7. (a,d) STEM and EDS elemental mapping (Ca, P, Si, and Ti) images of the SiTi nanoparticles after immersion in SBF for 7 days. The detected energies for Ca(K), P(K), Si(K), and Ti(K) were 3.69, 2.01, 1.74, and 4.52 keV, respectively. The dotted yellow circles indicate areas where chemical ele-ments are present. (b,c,e,f) TEM images of the SiTi nanoparticles after immersion in SBF for 7days. The αs plots of the SiTi nanoparticles after immersion in SBF are shown in Figure S2. The changes in the specific surface area of the nanoparticles, which were determined based on the aforementioned results, are shown in Figure 8. Group1.1 and Group0.7 showed a decrease in the specific surface area following immersion, indicating the adsorp-tion of the ions inside the nanopores. In particular, Group1.1 showed a faster rate of de-crease in surface area compared with Group0.7 since nitrogen (N2) molecules could not enter the nanopores where CP had effectively precipitated inside. Based on Figure 8, it can be observed that Group1.1 presents a higher rate of reduction in external surface area determined from the αs-plot, indicating that the effective precipitation of CP was due to pore blockage. As a result, Group1.1 exhibits a higher concentration of adsorbed phos-phorous (i.e., phosphate ions). The lower reduction in the specific surface area in Group0.7 suggests lesser pore blockage through calcium ion adsorption. Since the slight reduction in the surface area is attributed to Na+ and K+ ions, it can be inferred that this reduction in Figure 7. (a,d) STEM and EDS elemental mapping (Ca, P, Si, and Ti) images of the SiTi nanoparticlesafter immersion in SBF for 7 days. The detected energies for Ca(K), P(K), Si(K), and Ti(K) were 3.69,2.01, 1.74, and 4.52 keV, respectively. The dotted yellow circles indicate areas where chemical elementsare present. (b,c,e,f) TEM images of the SiTi nanoparticles after immersion in SBF for 7days.The αs plots of the SiTi nanoparticles after immersion in SBF are shown in Figure S2.The changes in the specific surface area of the nanoparticles, which were determined basedon the aforementioned results, are shown in Figure 8. Group1.1 and Group0.7 showed adecrease in the specific surface area following immersion, indicating the adsorption of theions inside the nanopores. In particular, Group1.1 showed a faster rate of decrease in surfacearea compared with Group0.7 since nitrogen (N2) molecules could not enter the nanoporeswhere CP had effectively precipitated inside. Based on Figure 8, it can be observed thatGroup1.1 presents a higher rate of reduction in external surface area determined from the αs-plot, indicating that the effective precipitation of CP was due to pore blockage. As a result,Group1.1 exhibits a higher concentration of adsorbed phosphorous (i.e., phosphate ions).The lower reduction in the specific surface area in Group0.7 suggests lesser pore blockagethrough calcium ion adsorption. Since the slight reduction in the surface area is attributedto Na+ and K+ ions, it can be inferred that this reduction in surface area is less significant inthe present paper. Therefore, the distribution of pores in Group 1.1 is considered a randomarray shape. Moreover, the peaks of Group1.1 with bimodal distributions decreased afterimmersion (Figure S3). The nanopores of 0SiTi at 1.2 nm decreased to 1.0 nm, while thenanopores of 0.1SiTi at 1.1 nm decreased to 0.9 nm. Figure S4 shows the N2 adsorptionand desorption isotherms during immersion. According to a previous report [47], theisotherm type of Group1.1 was type IV before immersion, which changed to type I afterimmersion. The isotherm of Group0.7 remained type I after SBF immersion, indicatingthat the nanopore structures in Group1.1 were preserved upon their immersion. Regardingpore size distribution, Group1.1 shows bimodal shapes in Figure S3. It was suggested thatpore blockage in the 1.1 nm sized particles of Group1.1 would occur, whereas the pores at0.7 nm remained unblocked, thereby maintaining microporous structures. After immersion,the adsorption isotherm of Group1.1 in SBF was changed such that is similar in shape tothat of Group0.7 (Figure S4).Inorganics 2023, 11, 235 8 of 13Inorganics 2023, 11, x FOR PEER REVIEW 9 of 14   surface area is less significant in the present paper. Therefore, the distribution of pores in Group 1.1 is considered a random array shape. Moreover, the peaks of Group1.1 with bimodal distributions decreased after immersion (Figure S3). The nanopores of 0SiTi at 1.2 nm decreased to 1.0 nm, while the nanopores of 0.1SiTi at 1.1 nm decreased to 0.9 nm. Figure S4 shows the N2 adsorption and desorption isotherms during immersion. Accord-ing to a previous report [47], the isotherm type of Group1.1 was type IV before immersion, which changed to type I after immersion. The isotherm of Group0.7 remained type I after SBF immersion, indicating that the nanopore structures in Group1.1 were preserved upon their immersion. Regarding pore size distribution, Group1.1 shows bimodal shapes in Figure S3. It was suggested that pore blockage in the 1.1 nm sized particles of Group1.1 would occur, whereas the pores at 0.7 nm remained unblocked, thereby maintaining mi-croporous structures. After immersion, the adsorption isotherm of Group1.1 in SBF was changed such that is similar in shape to that of Group0.7 (Figure S4).   Figure 8. Specific surface area changes of the SiTi nanoparticles with immersion time in SBF. The reduction percentages in the surface areas of 0SiTi, 0.1SiTi, 0.7SiTi, and 1.2SiTi were 50, 46, 26, and 38 %. Accoring to the XRD pattern results, after their immersion in SBF for 14 days, Group1.1 and Group0.7 remained amorphous (Figure S5). All of the nanoparticles showed an amorphous calcium phosphate (ACP) halo peak at 2θ = 30°, indicating the precipitation of ACP on their surfaces. 2.3. Mechanism of ACP Precipitation on XSiTi Nanoparticles After Immersion Based on the above results and discussion, the mechanisms of ACP precipitation in Group1.1 and Group0.7 are shown in Scheme 2. Regarding Group1.1, the Ca2+ ions were diffused and adsorbed inside the nanopores after immersion within one day. The Ca2+ ions inside the nanopores reacted with the phosphate ions in SBF, and the nanopores became the ACP nucleation sites, leading to rough ACP precipitation. For Group0.7, only the Na+ and K+ ions were diffused and adsorbed inside the nanopores after immersion for one day. The phosphate ions exchanged with the eluted silicate ions outside the nanopores and became the ACP nucleation sites. Therefore, it was determined that the ACP precipitation state was smooth without changing the surface morphology of Group0.7. Formatted: Not HighlightFigure 8. Specific surface area changes of the SiTi nanoparticles with immersion time in SBF. Thereduction percentages in the surface areas of 0SiTi, 0.1SiTi, 0.7SiTi, and 1.2SiTi were 50, 46, 26,and 38%.Accoring to the XRD pattern results, after their immersion in SBF for 14 days, Group1.1and Group0.7 remained amorphous (Figure S5). All of the nanoparticles showed anamorphous calcium phosphate (ACP) halo peak at 2θ = 30◦, indicating the precipitation ofACP on their surfaces.2.3. Mechanism of ACP Precipitation on XSiTi Nanoparticles after ImmersionBased on the above results and discussion, the mechanisms of ACP precipitation inGroup1.1 and Group0.7 are shown in Scheme 2. Regarding Group1.1, the Ca2+ ions werediffused and adsorbed inside the nanopores after immersion within one day. The Ca2+ ionsinside the nanopores reacted with the phosphate ions in SBF, and the nanopores becamethe ACP nucleation sites, leading to rough ACP precipitation. For Group0.7, only the Na+and K+ ions were diffused and adsorbed inside the nanopores after immersion for oneday. The phosphate ions exchanged with the eluted silicate ions outside the nanopores andbecame the ACP nucleation sites. Therefore, it was determined that the ACP precipitationstate was smooth without changing the surface morphology of Group0.7.Inorganics 2023, 11, x FOR PEER REVIEW 10 of 14    Scheme 2. Illustration of the precipitation processes of the SiTi nanoparticles in this study. 3. Materials and Methods 3.1. Chemicals TEOS (C8H20O4Si: CAS No. 78-10-4) and TTIP (C12H28O4Ti: CAS No. 546-68-9) were purchased from Tokyo Chemical Industry Co., Ltd. 2-Propanol (IPA, CAS No. 67-63-0), hydrochloric acid (HCl, 1 N, CAS No. 7647-01-0), ethanol (EtOH, 99.5 vol %, CAS No. 64-17-5), tris-hydroxymethylaminomethane (Tris, C4H11NO3, CAS No. 77-86-1), sodium chlo-ride (NaCl, CAS No. 7647-14-5), potassium chloride (KCl, CAS No. 7447-40-7), dipotas-sium hydrogenphosphate (K2HPO4, CAS No. 7758-11-4), magnesium chloride hexahy-drate (MgCl2･6H2O, CAS No. 7791-18-6), calcium chloride (CaCl2, CAS No. 10043-52-4), and sodium sulfate (Na2SO4, CAS No. 7757-82-6) were purchased from FUJIFILM Wako Pure Chemical Co., Ltd. Sodium hydrogen carbonate (NaHCO3, CAS No. 144-55-8) was purchased from Nacalai Tesque Co., Ltd. Octadecylamine (ODA, CH3(CH2)17NH2, CAS No. 124-30-1) was purchased from Sigma Aldrich Co., Ltd. All reagents are unpurified. 3.2. Synthesis 3.2.1. Synthesis of SiTi Nanoparticles In a previous report [15], SiTi nanoparticles were synthesized via microfluidic syn-thesis. Initially, three solutions (A–C) were prepared. Volumes of 1.63 mL of TTIP and 0, 0.187, 1.705, and 15.34 mL of TEOS were added to solution A to form Si/Ti molar ratios of 0, 0.15, 1.4, and 12, respectively, and 43.30, 43.11, 41.60, and 27.96 mL volumes of IPA were added according to the ratios. Solution B was prepared by mixing 44.60 mL of IPA and 0.277 mL of ultrapure water. Solution C was prepared by mixing 236.1 mL of IPA, 3.00 mL of ultrapure water, and 0.205 g of ODA. Solutions A and B were then mixed and reacted in a microreactor to generate nucleation via a sol–gel process, and the reaction solution was dropped into Solution C at a flow rate of 60 mL/min at 1000 rpm and left to grow the particles for 24 h under the room temperature. The liquid portion was removed via cen-trifugation, washed with ethanol and ultrapure water, and then dried at 60 °C for 24 h to obtain the SiTi nanoparticles with ODA (SiTi-ODA). Next, 10.2 mL of 1 N HCl and 150 mL of ethanol were added into 1 g of the dried SiTi-ODA, and the mixture was stirred at 700 rpm for 3 h at room temperature to remove ODA through solvent extraction. The solid phase was then removed via centrifugation and washed once with ethanol and once with Formatted: Not HighlightScheme 2. Illustration of the precipitation processes of the SiTi nanoparticles in this study.3. Materials and Methods3.1. ChemicalsTEOS (C8H20O4Si: CAS No. 78-10-4) and TTIP (C12H28O4Ti: CAS No. 546-68-9)were purchased from Tokyo Chemical Industry Co., Ltd. 2-Propanol (IPA, CAS No.Inorganics 2023, 11, 235 9 of 1367-63-0), hydrochloric acid (HCl, 1 N, CAS No. 7647-01-0), ethanol (EtOH, 99.5 vol%, CAS No. 64-17-5), tris-hydroxymethylaminomethane (Tris, C4H11NO3, CAS No. 77-86-1), sodium chloride (NaCl, CAS No. 7647-14-5), potassium chloride (KCl, CAS No.7447-40-7), dipotassium hydrogenphosphate (K2HPO4, CAS No. 7758-11-4), magnesiumchloride hexahydrate (MgCl2·6H2O, CAS No. 7791-18-6), calcium chloride (CaCl2, CASNo. 10043-52-4), and sodium sulfate (Na2SO4, CAS No. 7757-82-6) were purchased fromFUJIFILM Wako Pure Chemical Co., Ltd. Sodium hydrogen carbonate (NaHCO3, CASNo. 144-55-8) was purchased from Nacalai Tesque Co., Ltd. Octadecylamine (ODA,CH3(CH2)17NH2, CAS No. 124-30-1) was purchased from Sigma Aldrich Co., Ltd. Allreagents are unpurified.3.2. Synthesis3.2.1. Synthesis of SiTi NanoparticlesIn a previous report [15], SiTi nanoparticles were synthesized via microfluidic syn-thesis. Initially, three solutions (A–C) were prepared. Volumes of 1.63 mL of TTIP and 0,0.187, 1.705, and 15.34 mL of TEOS were added to solution A to form Si/Ti molar ratiosof 0, 0.15, 1.4, and 12, respectively, and 43.30, 43.11, 41.60, and 27.96 mL volumes of IPAwere added according to the ratios. Solution B was prepared by mixing 44.60 mL of IPAand 0.277 mL of ultrapure water. Solution C was prepared by mixing 236.1 mL of IPA,3.00 mL of ultrapure water, and 0.205 g of ODA. Solutions A and B were then mixed andreacted in a microreactor to generate nucleation via a sol–gel process, and the reactionsolution was dropped into Solution C at a flow rate of 60 mL/min at 1000 rpm and left togrow the particles for 24 h under the room temperature. The liquid portion was removedvia centrifugation, washed with ethanol and ultrapure water, and then dried at 60 ◦C for24 h to obtain the SiTi nanoparticles with ODA (SiTi-ODA). Next, 10.2 mL of 1 N HCl and150 mL of ethanol were added into 1 g of the dried SiTi-ODA, and the mixture was stirredat 700 rpm for 3 h at room temperature to remove ODA through solvent extraction. Thesolid phase was then removed via centrifugation and washed once with ethanol and oncewith ultrapure water. The particles were dried at 60 ◦C for 24 h to obtain nanoporousSiTi nanoparticles.3.2.2. Immersion of SiTi Nanoparticles into SBFThe 1.0 SBF (Na+, 142 mM; K+, 5.0 mM; Mg2+, 1.5 mM; Ca2+, 2.5 mM; Cl–, 148.8 mM;HCO3–, 4.2 mM; HPO42–, 1.0 mM; SO42–, 0.5 mM; and Tris, 50 mM) was prepared accordingto the method provided in a previous report [48], and the pH value was adjusted to 7.4 withHCl. Then, 0.5 SBF and 1.5 SBF were prepared at 0.5 and 1.5 times the inorganic ionconcentrations of 1.0 SBF. After the XSiTi nanoparticles were added to 0.5 SBF, the pH valuewas adjusted to 8.60 with Tris and kept at 37 ◦C for 1 day. The particles were then immersedin 1.5 SBF for 7 days. The solid phase was removed via centrifugation and dried at 37 ◦Cfor 24 h to obtain CP-coated SiTi nanoparticles.3.3. CharacterizationThe morphologies were observed on a carbonblack-coated Cu grid using a fieldemission scanning electron microscope (FE-SEM: HITACHI Co., Ltd., SU-8230) at an ac-celerating voltage of 200 kV; the vertical size, side size, and particle size distributions ofthe SiTi nanoparticles’ shapes were calculated by counting 150 particles, and their average(Ave.) and coefficient of variation (Cv.) values were also calculated. Size distributions of theSiTi nanoparticle images obtained through FE-SEM were calculated by randomly selecting150 particles.X-ray diffraction (XRD) patterns were obtained using a powder X-ray diffractometer(Rigaku Co., Ltd., Smart Lab) with CuKα radiation (λ = 0.15418 nm), a voltage of 40 kV,and a current of 200 mA.Specific surface area and pore size distribution determined via N2 adsorption anddesorption isotherms were measured at −196 ◦C with a BELSORP-Mini II instrumentInorganics 2023, 11, 235 10 of 13(Microtrac BEL Co., Ltd.) to estimate the total surface areas. Prior to measurement, 100 mgof each sample was degassed and pretreated at 80 ◦C under a vacuum. The followingmethods were used to analyze the nanopores. The specific surface area was evaluatedusing the αs-plots [49], and the pore size distribution was determined using microporeanalysis (MP). Furthermore, t-plots were used to calculate the specific surface area insidethe pores and the adsorbed layers’ thickness [50,51]; then, the pore volume was obtained.In this study, the Harkins−Jula equation representing the standard t-curve was used toinvestigate the standard isotherm. This curve is one of the most commonly used MPmethods. Pore size was defined as dp, which was plotted against dVp/dlog dp to showthe pore size distribution.Elemental composition was evaluated using an X-ray fluorescence analyzer (XRF: ZSXPrimus II, Rigaku Co., Ltd.). XRF analysis was performed on sample powders in thestate of pellets, which were pressurized and molded without dilution. The fundamentalparameter method was conducted using software for semi-quantitative analysis (EZ ScanProgram, Rigaku Co., Ltd.). Specifically, the amount of each element (Ca, Na, K, and P)adsorbed from SBF was detected and then evaluated in terms of mmol·(mg of sample)–1 ona semi-quantitative basis.Infrared absorption spectra were measured using a Fourier transform infrared spec-trometer (FT-IR: FT/IR-4600, Japan Spectroscopic Co., Ltd.) operating in the wavenumberrange 4000–500 cm–1 with a KBr background, 128 accumulation times, and a spectral reso-lution of 4 cm–1. FT-IR spectra were measured using KBr powder, and all weights weredetermined with 49 mg of KBr and 1 mg of sample. All the spectra were recorded aftersubtracting the background spectrum of KBr.Transmission electron microscopy (TEM) was performed using a JEOL JEM-2100Ftransmission electron microscope. Scanning TEM high-angle annular dark-field(STEM−HAADF) images and elemental mapping energy-dispersive X-ray (EDX) spectroscopyimages were recorded using a JEM-2100F and a JED-2300 instrument (EX-24200M1G2T, JEOLLtd.) at an accelerating voltage of 200 kV. The sample suspension was dropped onto a Cugrid (a high-resolution carbon substrate on STEM 100CuP grids, Okenshoji Co., Ltd.), andthe grids were dried under vacuum for a few days before each measurement. STEM andEDS elemental mapping (Ca, P, Si, and Ti) images of the SiTi nanoparticles were taken afterthe nanoparticles’ immersion in SBF for 7 days. The detected energies for Ca(K), P(K), Si(K),and Ti(K) were 3.69, 2.01, 1.74, and 4.52 keV, respectively.4. ConclusionsWe established a CP-coating method that homogeneously corresponds to the shapesof SiTi nanoparticles. CP precipitation was performed on mono-dispersed nanoporousSiTi nanoparticles with different Si/Ti molar ratios and pore sizes. The pore size distri-bution was found to significantly affect the CP coating in SBF immersion; the surfaces ofthe nanoparticles with bimodal pore sizes of 0.7 and 1.1~1.2 nm became rough after CPprecipitation, while those with unimodal pore sizes of 0.7 nm remained smooth, indicatingthat these two pore sizes work as different nucleation sites that lead to different surfacemorphologies. These CP-coated SiTi nanoparticles could improve osteoconductivity whileretaining the properties of SiTi nanoparticles, which we believe may be suitable for use inthe DDS carriers in the future.Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/inorganics11060235/s1, Scheme S1: XRD patterns of the SiTinanoparticles.; Figure S2: αs-plots of the SiTi nanoparticles with the immersion time Xd (X days,X=0, 1, 3, 7) in SBF; Figure S3: The MP pore size distribution of SiTi nanoparticles with the immersiontime in SBF; Figure S4: N2 adsorption (close marks) and desorption (open marks) isotherms of theSiTi nanoparticles with the immersion time in SBF; Figure S5: XRD patterns of the SiTi nanoparticlesat the immersion time in SBF for 14 days.https://www.mdpi.com/article/10.3390/inorganics11060235/s1https://www.mdpi.com/article/10.3390/inorganics11060235/s1Inorganics 2023, 11, 235 11 of 13Author Contributions: Conceptualization, R.K. and M.T.; methodology, R.K., K.F. and M.T.; software,R.K.; validation, K.F. and M.T.; formal analysis, I.Y. and K.S.; investigation, R.K.; resources, M.T.; datacuration, Y.Z.; writing—original draft preparation, R.K. and Y.Z.; writing—review and editing, K.S.,Y.Z., I.Y. and M.T.; supervision, K.S. and M.T.; project administration, K. S. and M.T. All authors haveread and agreed to the published version of the manuscript.Funding: This research was funded by the Japan Society for the Promotion of Science (JSPS) KAK-ENHI (Grant-in-Aid for Challenging Exploratory Research, Grant 22K18916).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: Not applicable.Acknowledgments: This study was partially supported by a grant from the Japan Society for thePromotion of Science (JSPS) KAKENHI (Grant-in-Aid for Challenging Exploratory Research, Grant22K18916). Additionally, a portion of this work was supported by NIMS Electron MicroscopyAnalysis Station, Nanostructural Characterization Group. The authors also thank the Analysis andInstrumentation Center at the Nagaoka University of Technology for providing their facilities.Conflicts of Interest: The authors declare no conflict of interest.References1. Lau, M.; Giri, K.; Garcia-Bennett, A.E. 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MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.https://doi.org/10.1016/S0022-3093(05)80557-5https://doi.org/10.1515/pac-2014-1117https://doi.org/10.1002/jbm.820240607https://www.ncbi.nlm.nih.gov/pubmed/2361964https://doi.org/10.1051/jcp/1984810791https://doi.org/10.1016/0021-9797(68)90270-1https://doi.org/10.7209/tanso.1997.159 Introduction  Results and Discussion  Synthesis Result of XSiTi Nanoparticles  Results Regarding the SBF-Immersed SiTi Nanoparticles  Mechanism of ACP Precipitation on XSiTi Nanoparticles after Immersion  Materials and Methods  Chemicals  Synthesis  Synthesis of SiTi Nanoparticles  Immersion of SiTi Nanoparticles into SBF  Characterization  Conclusions  References