A physicochemical process for fabricating submicrometer hollow fluorescent spheres of Tb³⁺-incorporated calcium phosphate

Abstract

We developed a laser fabrication process of submicrometer hollow fluorescent spheres of Tb³⁺-incorporated calcium phosphate. Pulsed laser irradiation was applied to a dispersion of carbon-integrated hydroxyapatite nanopowders in the presence of Tb³⁺. Thus, Tb³⁺ incorporation during calcium phosphate sphere formation through laser processing was achieved.

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Calcium phosphate (CaP) is the main mineral constituent of human bones and teeth. Certain types of CaPs, such as hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP), show excellent biocompatibility and osteoconductivity, and therefore, have been widely used as bone-substituting materials.^1,2 Since the 1990s, incorporation of metal ions into CaPs has been proposed to improve osteoconductivity of CaPs and/or provide CaPs with additional biomedical functions characteristic of the added ions.³ For example, Zn²⁺ was incorporated into β-TCP and β-TCP/HAp composite to improve osteoconductivity.⁴ Ag⁺, Cu²⁺, and Zn²⁺ were incorporated into HAp to provide antibacterial activity.⁵ Lanthanide ions, such as Tb³⁺ and Eu³⁺, were incorporated into HAp and amorphous CaP to provide fluorescence.^6–10

Such metal-ion-incorporated CaPs have potential for not only orthopedic applications but also drug delivery applications. For example, Eu³⁺-incorporated CaPs would be useful as a biocompatible, fluorescent delivery carrier that allows in vitro and in vivo live imaging.⁶ In such delivery applications, carriers in the form of submicrometer hollow spheres are of significant benefit. This is because the hollow structure enables loading of a large amount of drugs within the spheres. In addition, the spheres' submicrometer size is adequate for targeted delivery, since sphere permeability of the cell membrane and blood vessel wall can be controlled by appropriate design of sphere surface functionalities and structures on a submicrometer scale. Furthermore, a spherical shape has the practical advantages of reduced agglomeration, easy redispersion, and minimal physical irritation to living cells, tissues, and organs. Conventionally, the assembly approach and the template method have been used for fabrication of submicrometer hollow spheres of certain inorganic compounds^11–13 including CaP-based compounds.¹³ However, these processes usually need complex and time-consuming steps for fabrication, and often require synthetic surfactants.

Recently, we developed a simple, quick, and surfactant-free physicochemical process for fabricating submicrometer Fe-incorporated CaP-based spheres¹⁴ by combining a pulsed laser melting in liquid process^15,16 with a chemical precipitation process. In this physicochemical process, pulsed laser irradiation was performed without focusing to a labile CaP reaction mixture supplemented with Fe³⁺ as a light-absorbing agent. However, the resulting spheres were not hollow; they were homogeneous solid spheres.

Here, we aimed to develop a technique to fabricate submicrometer hollow spheres of metal-ion-incorporated CaPs using carbon nanopowders as the light-absorbing agent, as inspired by the technique developed by Li et al.^17,18 In their technique, optically transparent metal oxide nanopowders as raw materials are mixed and integrated with carbon nanopowders as the light-absorbing agent through mechanical milling. The resulting carbon-integrated metal oxide nanopowders are dispersed in a solvent and irradiated with pulsed laser light without focusing. Under laser irradiation, carbon nanopowders absorb laser light energy and transfer the energy to the raw material nanopowders, causing melting and spheroidization of the powder agglomerate in the solvent. During the irradiation process, most of the carbon nanopowders are sublimated or degraded into very fine carbon quantum dots and are not incorporated within the final spheres. The final spheres contain single or multiple internal pores because of gas trapping within the spheres. Using this carbon-assisted technique, submicrometer hollow spheres of Al₂O₃, MgO, and ZrO₂ have been fabricated.^17,18

Our first hypothesis was that submicrometer hollow CaP spheres could be fabricated by the carbon-assisted technique, i.e., pulsed laser irradiation of a dispersion of carbon-integrated CaP nanopowders. Our second hypothesis was that incorporation of metal ions into CaP spheres during carbon-assisted CaP sphere formation could be realized by adding metal ions to the dispersion of carbon-integrated CaP nanopowders. We employed Tb³⁺ as a functional lanthanide metal ion for incorporation with the intention of fabricating submicrometer hollow fluorescent spheres of Tb³⁺-incorporated CaP. Toxicity of Tb³⁺ is considered to be relatively low considering that oral LD₅₀ (lethal dose 50) value of terbium chloride is higher than 5000 mg kg⁻¹ in mice,¹⁹ which is almost the same as that of sodium chloride. Besides, it is reported that Tb³⁺-incorporated CaP nanoparticles show good cytocompatibility;¹⁰ hence they are the strong candidates of biocompatible cell imaging agents.

In this study, we obtained five different samples, CaP-1, CaP-2, CaP-3, CaP-4, and CaP-5, by varying the fabrication conditions (Table 1). To verify our first hypothesis, we fabricated Tb³⁺-free submicrometer CaP spheres using the carbon-assisted technique as follows (for Experimental details, see ESI†). Commercially available HAp nanopowders (Fig. 1a) were used as the CaP raw material. The HAp nanopowders and carbon nanopowders (Fig. 1b) with a mass ratio of 5 : 1 were mixed and integrated by mechanical milling. According to the scanning electron microscopy with energy dispersive X-ray spectrometry (SEM-EDX) results, the peak intensity of carbon of the raw HAp nanopowders (Fig. 2a) markedly increased after milling (Fig. 2b), suggesting integration with the carbon nanopowders. The carbon-integrated HAp nanopowders (CaP-1) were irregularly-shaped agglomerates, as shown in Fig. 1c. The nanopowders of CaP-1 were dispersed into ethanol. To this CaP-1 dispersion, pulsed laser irradiation (200 mJ per pulse per cm², 20 min, 355 nm) without focusing was performed under constant stirring. After irradiation, the powders in the dispersion were washed and purified by multistep centrifugation (CaP-2).

Table 1 Processing of samples obtained in this study and SEM results (for Experimental details, see ESI†)^a

Samples	Process			Submicrometer sphere formation
	Milling with carbon	Laser irradiation	Tb^3+ addition
a ○: Process applied. ×: process not applied.b Mixture of spheres and irregularly-shaped powders.
CaP-1	○	×	×	Not formed
CaP-2	○	○	×	Formed
CaP-3	×	○	×	Formed^b
CaP-4	○	○	○	Formed
CaP-5	○	×	○	Not formed

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Fig. 1 SEM images of raw nanopowders of (a) HAp and (b) carbon and obtained samples of (c) CaP-1 and (d) CaP-2.

Fig. 2 SEM-EDX spectra of (a) raw HAp nanopowders, and obtained samples of (b) CaP-1 and (c) CaP-2. The peaks of Si and Au were derived from the silicon substrate used for mounting the powders and gold coating on the powders, respectively.

With the above-mentioned carbon-assisted technique, submicrometer hollow spheres of CaP were successfully fabricated. As shown in the SEM image of CaP-2 (Fig. 1d), submicrometer spheres with a diameter of ca. 100–400 nm were obtained after laser irradiation of the dispersion of CaP-1 (Fig. 1c). This result indicates that the irregularly shaped agglomerates of the carbon-integrated HAp nanopowders (Fig. 1c) changed into submicrometer spheres (Fig. 1d) under laser irradiation. According to the SEM-EDX results, detected peaks derived from CaP (Ca, P, and O) had similar intensity ratios for both CaP-1 and CaP-2. On the other hand, the peak of C was greatly diminished (CaP-2, Fig. 2c) after laser irradiation of the dispersion of CaP-1 (Fig. 2b). This should be due to the sublimation or degradation of carbon nanopowders by laser irradiation, as reported previously.^17,18 Using transmission electron microscopy (TEM), single-hollow structures (type A) and multi-hollow structures (type B) were observed within the spheres in CaP-2 (Fig. 3). Solid spheres with no apparent phase separation (type C) were also observed, although their number was relatively small compared with that of hollow spheres (type A + type B). An outer diameter of hollow spheres (type A + type B) was relatively large (ca. 150–400 nm) compared with that of solid spheres (type C) (ca. 100–250 nm) in TEM observations. Internal pore size (hollow diameter) distribution in single-hollow spheres (type A) was relatively wide (20–240 nm) compared with that in multi-hollow spheres; over 90% of internal pores in multi-hollow spheres (type B) had diameters smaller than 60 nm (Fig. S1†). In the previous report on the fabrication of metal oxide spheres by the carbon-assisted technique^17,18 and some metal and semiconductor spheres by the conventional pulsed laser melting in liquid process,¹⁶ hollow spheres were also produced. The authors explained that due to the rapid heating and quenching during pulsed laser irradiation, the gases produced in the nanopowders (agglomerates of carbon-integrated metal oxide nanopowders^17,18 or raw metal and semiconductor nanopowders¹⁶) are sometimes not released before final solidification. In such cases, hollow spheres form; otherwise, solid spheres form. A similar phenomenon should occur in our experiment, judging from the TEM results (Fig. 3). Considering that the outer diameter of hollow spheres (type A + type B) were relatively large compared with that of solid spheres (type C), the gas release from the smaller agglomerates was easier than that from the larger agglomerates. The previously fabricated metal oxide (Al₂O₃ and ZrO₂) spheres possessed crystalline phases.^17,18 In contrast, according to transmission electron diffraction (TED) analysis, all spheres in CaP-2 possessed an amorphous phase irrespective of their structure type (Fig. 3). From X-ray diffractometry (XRD) analysis, even after milling (CaP-1), the HAp nanopowders retained their crystalline structure (Fig. S2b†), though crystallinity became lower compared with that of the raw HAp nanopowders (Fig. S2a†), probably because of the crystalline lattice distortion. Therefore, order–disorder transformation, i.e., change of the atomic arrangement from crystalline HAp (CaP-1) to amorphous CaP (CaP-2) occurred during sphere formation under laser irradiation. We believe that as a result of carbon-assisted laser melting in liquid, the agglomerates of HAp nanopowders experienced order–disorder transformation as well as spheroidizing (with the effect of surface tension), and the melted amorphous CaP droplets were quenched and retained their amorphous phase during the pulse intervals. This belief is supported by TEM and TED results showing that inadequately spheroidized agglomerates observed as residual dross in CaP-2 still possessed the crystalline structure of HAp (Fig. S3†). The free energy barrier for nucleation and quenching kinetics should be different for CaPs compared with that for the metal oxides (Al₂O₃, ZrO₂) due to their different thermodynamic and crystallographic properties (such as melting point, heat capacity, and step kinetic coefficient).

Fig. 3 TEM (a, b, d, and f) and TED (c, e, and g) images of CaP-2. Type A: single-hollow structure (b and c), type B: multi-hollow structure (d and e), and type C: solid structure (f and g).

As previously observed for metal oxide spheres,^17,18 the milling process with carbon nanopowders prior to laser irradiation was an important step in the CaP sphere formation in CaP-2. In fact, when the milling step was omitted from the fabrication process of CaP-2 (CaP-3), CaP sphere formation was obviously retarded. As confirmed by SEM observations, inadequately spheroidized irregular powders were observed to remain in large quantities in CaP-3 (Fig. S4†). In addition, the diameters of the spheres in CaP-3 (ca. 100–200 nm, Fig. S4†) were smaller than those in CaP-2 (ca. 100–400 nm, Fig. 1d). It appears that without the milling process, the integration of the HAp and carbon nanopowders was not sufficient to facilitate thermal energy transfer from the carbon to the HAp nanopowders.

Next, we fabricated Tb³⁺-incorporated submicrometer hollow CaP spheres (CaP-4) by adding Tb³⁺ to the dispersion of CaP-1 followed by laser irradiation. In the SEM images, the spheres in CaP-4 (Fig. 4a) were similar in shape and size to the submicrometer Tb³⁺-free CaP spheres in CaP-2 (Fig. 1d). In their SEM-EDX spectrum, the peak of Tb was clearly detected in addition to the peaks derived from CaP (Ca, P, and O) (Fig. 4b). As in the case of CaP-2 (Fig. 3), single-hollow structures (type A), multi-hollow structures (type B), and solid structures (type C) were found in the spheres in CaP-4 (Fig. 4c and S5a†), and all these spheres were amorphous (Fig. S5b†). Just as was collectively detected by SEM-EDX for the spheres (Fig. 4b), Tb was detected in one individual sphere by TEM-EDX (Fig. S5c†). No phase separation was observed in the solid phase of CaP-4 (Fig. 4c and S5a†), indicating that Tb³⁺ might be incorporated in the amorphous CaP matrix without forming any crystalline phases.

Fig. 4 CaP-4 (a) SEM image, (b) SEM-EDX spectrum, (c) TEM image, and (d) fluorescence spectrum with excitation at 228 nm and 488 nm (inset). FI stands for fluorescent intensity.

A putative mechanism for Tb³⁺ incorporation into the CaP spheres (CaP-4) is as follows. Even without laser irradiation, Tb³⁺ is incorporated in the outermost surfaces of the CaP-1 nanopowders through ionic adsorption and/or substitution for Ca sites. This was suggested by a control experiment in which the dispersion of CaP-1 in a 5 mM Tb³⁺ ethanol solution was just stirred for 20 min without laser irradiation. The obtained product (CaP-5) was irregularly shaped agglomerates of nanopowders similar to CaP-1 (Fig. S6a†) and surely contained Tb (Fig. S6b†), although the Tb content was relatively low compared with that in CaP-4 (Fig. 4b). Under laser irradiation in a Tb³⁺ solution, the CaP-1 nanopowders incorporate Tb³⁺ during the pulsed laser melting process, and this might be responsible for the relatively high Tb content in CaP-4. That is, Tb³⁺ on the outermost surfaces of the CaP-1 nanopowders could diffuse into the melted CaP droplets during the pulse duration (heating process). During the subsequent pulse interval (quenching process), Tb³⁺ in the solution is again incorporated in the outermost surfaces of the solidified CaP spheres. Because such heating and quenching are repeated throughout the pulsed laser irradiation, Tb³⁺ is likely to be incorporated deep within the CaP spheres of CaP-4 via the physicochemical ion-solid fusion process. Note that nitrate ion (NO₃⁻), a counter ion of Tb³⁺ in the Tb³⁺ source (terbium nitrate hexahydrate), was not incorporated in CaP-4. This was confirmed by the absence of an N peak in the SEM-EDX spectrum (Fig. 4b) and the X-ray photoelectron spectroscopy (XPS) spectrum (Fig. S7†) of CaP-4. This might be due to weaker affinity of NO₃⁻ to the powder surfaces of CaP-1 compared with Tb³⁺; the weaker affinity of NO₃⁻ is suggested by the XPS result that Tb was clearly detected in CaP-5, whereas N was not at all (Fig. S7†). Thermally unstable nature of NO₃⁻²⁰ might also be involved in the absence of NO₃⁻ in the final CaP spheres of CaP-4. Considering the above results and discussions, for ions to be incorporated in the final spheres, they might require high thermal durability in addition to sufficient affinity to the powder surfaces of CaP-1. Other metal ions besides Tb³⁺ that meet these requirements, e.g. rare-earth (Eu³⁺, Y³⁺, etc.) and heavy metal ions (Fe²⁺, Cu²⁺, etc.) with Ca-site substitution capability,²¹ might also be incorporated into CaP spheres with the present technique, although this is yet to be demonstrated.

The present carbon-assisted process had completely different source materials and light-absorbing agents than our previous process for fabricating Fe-incorporated CaP-based submicrometer spheres.¹⁴ In our previous process, pulsed laser irradiation was performed on a calcium phosphate reaction mixture supplemented with Fe³⁺ as a light-absorbing agent. In the reaction mixture, precipitates incorporating Fe were spontaneously formed via homogeneous nucleation and absorbed laser light, and were thereby heated and melted into a spherical shape. As a result, submicrometer Fe-incorporated CaP-based spheres were fabricated. In the present process, submicrometer Tb³⁺-incorporated CaP spheres were fabricated by laser irradiation of a Tb³⁺-supplemented dispersion of carbon-integrated HAp nanopowders. Under irradiation, Tb³⁺ was incorporated into CaP via a physicochemical ion–solid fusion process and did not act as a light-absorbing agent. Instead, carbon nanopowders integrated with HAp nanopowders acted as the light-absorbing agent. Unlike the Fe³⁺ light-absorbing agents in our previous experiment, carbon nanopowders in this experiment sublimated or degraded under laser irradiation; thus, they seldom remained in the final spheres.

The Tb³⁺-incorporated CaP spheres (CaP-4) exhibited fluorescence. The fluorescence spectrum of CaP-4 (dispersed in distilled water) with 228 nm excitation showed fluorescence peaks at wavelengths of 488, 543, 586, and 620 nm, which can be ascribed to ⁵D₄–⁷F₆, ⁵D₄–⁷F₅, ⁵D₄–⁷F₄, and ⁵D₄–⁷F₃ transitions of Tb³⁺, respectively (Fig. 4d).^7–10,22 Such fluorescence peaks that are characteristic of Tb³⁺ were not observed in CaP-2 due to the absence of Tb³⁺. The fluorescence peaks of 543 and 586 nm were also observed for CaP-4 with visible light excitation (488 nm) (Fig. 4d, inset). This result is significant for in vitro and in vivo imaging applications, since visible light is utilizable for the living cells and tissues.

Submicrometer hollow fluorescent spheres with low density, large specific area, and encapsulation ability have potential applications in catalysis, batteries, water treatment, drug delivery, and so on.^11,12 The submicrometer hollow fluorescent spheres of Tb³⁺-incorporated CaP fabricated by the present technique would be useful as multifunctional drug delivery carriers, as described in the second paragraph. Similar Tb³⁺-incorporated CaP powders were fabricated by other chemical processes; however, they were irregularly shaped or rod-shaped solid powders, not hollow spheres.^7–10 Hollow spheres of CaP with submicrometer sizes have been fabricated by the assembly approach and the template method using synthetic surfactants.¹³ In the present carbon-assisted technique, hollow spheres are spontaneously constructed from irregularly shaped nanopowders under laser irradiation without synthetic surfactants. Although further improvement in sphere size and structure control is required, the present surfactant-free technique would provide a new way to fabricate submicrometer hollow CaP-based spheres that could potentially be used as drug delivery carriers.

Conclusions

Submicrometer hollow fluorescent spheres of Tb³⁺-incorporated CaP were successfully fabricated by pulsed laser irradiation of a dispersion of carbon-integrated HAp nanopowders in the presence of Tb³⁺. A physicochemical ion–solid fusion process was proposed for the Tb³⁺ incorporation into the CaP spheres. The present carbon-assisted technique and the resulting Tb³⁺-incorporated CaPs with submicrometer spherical hollow structures would be useful in drug delivery applications.

Acknowledgements

This study was supported by the Cosmetology Research Foundation, Japan. A part of this study was conducted at Nano-Processing Facility supported by AIST. We thank Dr Yoshie Ishikawa from AIST for valuable discussions.

Notes and references

1. H. Zhou and J. Lee, Acta Biomater., 2011, 7, 2769.
2. K. Fox, P. A. Tran and N. Tran, ChemPhysChem, 2012, 13, 2495.
3. E. Boanini, M. Gazzano and A. Bigi, Acta Biomater., 2010, 6, 1882.
4. H. Kawamura, A. Ito, S. Miyakawa, P. Layrolle, K. Ojima, N. Ichinose and T. Tateishi, J. Biomed. Mater. Res., 2000, 50, 184.
5. J. Kolmas, E. Groszyk and D. Kwiatkowska-Różycka, BioMed Res. Int., 2014, 178123.
6. F. Chen, Y. Zhu, K. Zhang, J. Wu, K. Wang, Q. Tang and X. Mo, Nanoscale Res. Lett., 2011, 6, 67.
7. S. P. Mondéjar, A. Kovtun and M. Epple, J. Mater. Chem., 2007, 17, 4153.
8. L. Li, Y. Liu, J. Tao, M. Zhang, H. Pan, X. Xu and R. Tang, J. Phys. Chem. C, 2008, 112, 12219.
9. C. Yang, P. Yang, W. Wang, J. Wang, M. Zhang and J. Lin, J. Colloid Interface Sci., 2008, 328, 203.
10. X. Li, H. Zeng, L. Teng and H. Chen, Mater. Lett., 2014, 125, 78.
11. X. Yang, Y. Zhang, L. Xu, Z. Zhai, M. Li, M. Li, X. Liu and W. Hou, Dalton Trans., 2013, 42, 3986.
12. X. Yang, L. Xu, Z. Zhai, F. Cheng, Z. Yan, X. Feng, J. Zhu and W. Hou, Langmuir, 2013, 29, 15992.
13. K. Lin, C. Wu and J. Chang, Acta Biomater., 2014, 10, 4071.
14. M. Nakamura, A. Oyane, I. Sakamaki, Y. Ishikawa, Y. Shimizu, K. Koga, K. Kawaguchi and N. Koshizaki, RSC Adv., 2014, 4, 38442.
15. H. Wang, A. Pyatenko, K. Kawaguchi, X. Li, Z. Swiatkowska-Warkocka and N. Koshizaki, Angew. Chem., Int. Ed., 2010, 49, 6361.
16. H. Wang, M. Miyauchi, Y. Ishikawa, A. Pyatenko, N. Koshizaki, Y. Li, L. Li, X. Li, Y. Bando and D. Golberg, J. Am. Chem. Soc., 2011, 133, 19102.
17. X. Li, Y. Shimizu, A. Pyatenko, H. Wang and N. Koshizaki, J. Mater. Chem., 2011, 21, 14406.
18. X. Li, Y. Shimizu, A. Pyatenko, H. Wang and N. Koshizaki, Nanotechnology, 2012, 23, 115602.
19. T. J. Haley, N. Komesu, A. M. Flesher, L. Mavis, J. Cawthorne and H. C. Upham, Toxicol. Appl. Pharmacol., 1963, 5, 427.
20. N. P. Bansal, J. Mater. Sci., 1992, 27, 2922.
21. J. C. Elliott, Structure and chemistry of the apatites and other calcium orthophosphates, Elsevier, Amsterdam, 2nd edn, 1994, ch. 2.
22. X. Liu, W. Hou, X. Yang and J. Liang, CrystEngComm, 2014, 16, 1268.

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Footnote

† Electronic supplementary information (ESI) available: Experimental details and figures of pore size distribution, XRD patterns, TEM and TED images, SEM images, TEM-EDX spectrum, SEM-EDX spectrum, and XPS spectra. See DOI: 10.1039/c5ra01155h

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