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Preparation of calcium carbonate microparticles containing organic fluorescent molecules from vaterite
Advanced Powder Technology 25 (2014) 1147–1154
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Advanced Powder Technology journal homepage: www.elsevier.com/locate/apt
Original Research Paper
Preparation of calcium carbonate microparticles containing organic fluorescent molecules from vaterite Masahiro Fujiwara a,⇑, Kumi Shiokawa a, Takayuki Kubota b, Kenichi Morigaki c a
National Institute of Advanced Industrial Science and Technology (Kansai Center), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan National Institute of Animal Health, National Agriculture and Food Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan c Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada, Kobe 657-8501, Japan b
a r t i c l e
i n f o
Article history: Received 18 October 2013 Received in revised form 29 January 2014 Accepted 26 February 2014 Available online 13 March 2014 Keywords: Calcium carbonate Vaterite Fluorescent particles Encapsulation of organic molecules Drug delivery system
a b s t r a c t The encapsulation of fluorescent organic molecules into crystalline calcium carbonate was examined using calcium carbonate microcapsule, whose crystalline phase is vaterite as a metastable phase of calcium carbonate. A calcium carbonate microcapsule with impregnated pyrene that is a water insoluble fluorescent molecule was soaked into suitable aqueous solutions to promote the phase transition of vaterite toward calcite as the stable phase of calcium carbonate. When 0.2 M calcium chloride solution was used, the largest amount of pyrene (approximately 0.06 wt%) was encapsulated into the calcite particle. Pyrene thus included was not eliminated even after thorough washing with THF. The calcite particle thus prepared produced the excimer emission of pyrene by UV irradiation. Rhodamine B was also introduced into calcium carbonate by the immersion of the microcapsule into the aqueous solutions of Rhodamine B. The fluorescence of rhodamine B was observed from the calcium carbonate particles by visible light irradiation. Acetaminophen, a common drug poorly soluble in water, was also included in the calcium carbonate particle by the same procedures as the pyrene encapsulation. As acetaminophen thus encapsulated was released by the dissolution of the calcium carbonate particle in acidic solution, the particle is expected to be applied for a dissolution-triggered drug delivery. Ó 2014 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.
1. Introduction Fluorescent molecules and materials are useful probes for the extensive analyses of genes, proteins, drug targets in cells, tissues and other living systems [1–6]. While organic dyes and fluorescent proteins are still convenient materials [1–3], inorganic quantum dot microparticles are also applied to the biological sensing researches [4–6]. Since the direct use of these fluorophores and fluorescent materials often encounter some severe problems such as pH, pro-oxidant species and dissolved salts within organism environments, their functionalizations and decorations are usually required. However, the elimination and the egestion of these artificial decorations into living organisms must be carefully controlled. Calcium carbonate is one of the most useful and versatile materials for biomedical fields [7–10]. Its high environmentallyfriendly property and biocompatibility are undoubtedly appropriate for a broad range of utilizations for living objects and human being. As calcium carbonate is readily decomposed or metabolized ⇑ Corresponding author. Tel.: +81 72 751 9525; fax: +81 72 751 9628. E-mail address: [email protected] (M. Fujiwara).
within organisms to non-toxic calcium and carbonate ions (or carbon dioxide), calcium carbonate materials with properly designed functions must be powerful tools for biomedical related systems, including bio-sensing and drug delivery [11–17]. The preparation of fluorescent calcium carbonate materials have been examined by the doping of fluorescent metal cations such as Gd, Eu [18,19], Sn [20] and Ce [21], because the ionexchange of calcium to these metals effectively occurs due to their analogous ionic radii. Recently, the precipitation of calcium carbonate in the presence of mercaptopropyl acid-modified CdTe is reported to be an efficient method for incorporating quantum dot microparticles into crystalline calcium carbonate, which is beneficial for monitoring intracellular uptake [22]. On the other hand, as a number of fluorescent organic molecules and fluorescent pigments are known [23,24], the incorporation of these common phosphors in calcium carbonate must be versatile approaches to the preparation of fluorescent calcium carbonate materials. Although amorphous calcium carbonate [25–30] is used as a template for the encapsulation of fluorescent molecules into organic capsules like chitosan [31] and PMMA [32], the crystalline calcium carbonate materials containing fluorescent organic
http://dx.doi.org/10.1016/j.apt.2014.02.022 0921-8831/Ó 2014 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.
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molecules have not been examined. Therefore, new methods for the encapsulation of fluorescent organic molecules into crystalline calcium carbonate are required. We have studied the preparation of inorganic hollow microparticles (microcapsules) [33–35] such as calcium carbonate [36–39] by an interfacial reaction method. The crystalline phase of the calcium carbonate microcapsule is vaterite as a metastable phase of calcium carbonate, while the common calcium carbonate observed in nature is calcite as the most stable phase. We have recently found that the phase transition of the vaterite calcium carbonate microcapsule to calcite in the aqueous solutions of proteins such as bovine serum albumin (BSA), lysozyme and insulin achieves the encapsulation of those proteins into the resulting calcite calcium carbonate [40]. The application of this phase transition method to the incorporation of organic molecules is expected to develop a new procedure for creating functional crystalline calcium carbonate materials with organic molecules. In this paper, we report the encapsulations of pyrene and rhodamine B as fluorescent molecules into crystalline calcium carbonate particles by the phase transition method. Moreover, the incorporation of acetaminophen, a poorly water-soluble organic molecule, into calcium carbonate particle and its release behavior are also described.
2.3. Encapsulation of pyrene and acetaminophen into calcium carbonate particles This procedure consisted of two processes; the first step is the impregnation of pyrene onto the calcium carbonate microcapsule and the second one is the immersion of the microcapsule into an aqueous solution. The calcium carbonate microcapsule (1 g) was soaked in 50 mL of a THF solution with dissolved pyrene (0.2 g). After gentle mixing of this suspension for 30 min, the volatiles were removed under reduced pressure. This calcium carbonate microcapsule with impregnated pyrene was added to 20 mL of a 1 M Tris HCl buffer solution or a 0.2 M calcium chloride solution. The resulting suspension was stood at 22 °C for 24 days. The aqueous solution and white skim on the solution (identified as pyrene) were carefully separated from the calcium carbonate solid by decantation. The calcium carbonate solid was washed with deionized water more than three times, and finally dried at 60 °C for 12 h. This calcium carbonate was further washed with THF in order to remove pyrene until pyrene was not detected in the THF solutions by UV–vis spectrometry analysis (Jasco V-530 spectrophotometer). Double washing was enough to remove pyrene from the calcium carbonate particle completely. The encapsulation of acetaminophen was carried out by the same procedures as pyrene.
2. Materials and methods 2.4. Encapsulation of rhodamine B into calcium carbonate particles 2.1. Materials Ammonium carbonate [(NH4)2CO3], calcium chloride dihydrate (CaCl22H2O), pyrene, rhodamine B and other solvents (n-hexane, THF, methanol and ethanol) used in this work were purchased from Wako pure chemical industries. Tween 85 and a commercial calcite calcium carbonate were obtained from Kanto Chemicals and acetaminophen was from MP biomedicals. 1 M Tris HCl solution (pH = 7) was commercial available from Wako Pure Chemical Industries and 0.2 M calcium chloride solution was prepared with calcium chloride dihydrate in our laboratory. The structures of molecules encapsulated into calcium carbonate are summarized in Fig. 1. 2.2. Preparation of vaterite calcium carbonate microcapsule Calcium carbonate microcapsules were prepared by a described method in our recent papers [39,40]. A typical procedure is briefly describe as follows: 32 mL of a 3 M ammonium carbonate solution was mixed with 48 mL of a n-hexane solution dissolving 1.0 g of Tween 85, and the resulting solution was emulsified with a homogenizer at 8000 rpm for 1 min in order to obtain a water/oil (W/O) emulsion. This W/O emulsion was poured into 640 mL of a 0.3 M calcium chloride aqueous solution in one portion at 30 °C. After 5 min stirring, the precipitate was filtered, washed with deionized water twice and methanol once and finally dried at 80 °C for 12 h.
N
The calcium carbonate microcapsule (1 g) was immersed in 10 mL of a 1 M Tris HCl buffer solution or a 0.2 M calcium chloride solution with 0.2 g of rhodamine B. The resulting suspension was stood at 22 °C for 24 days. This solid was filtered, thoroughly washed with deionized water, and dried at 60 °C for 12 h. This solid was further washed with ethanol thoroughly until rhodamine B was not detected in the solutions by UV–vis spectrometry analysis. More than three times washing was necessary to remove the rhodamine B from the calcium carbonate particle perfectly. 2.5. Determination of the weight ratios of organic molecules encapsulated into calcium carbonate particles The amounts of organic molecules encapsulated into the calcium carbonate particles were estimated by the extraction of the molecules. The calcium carbonate particles were dissolved with an 8.5 M acetic acid solution, and the amounts of eliminated molecules were determined by UV–vis spectrometry (Jasco V-530 spectrophotometer). In the cases of acetaminophen and rhodamine B, after the complete dissolution of the calcium carbonate particles, the resulting acetic acid solutions were directly analyzed by the spectrometer. In the case of pyrene, after the complete dissolution of the calcium carbonate particles, pyrene was extracted from the solutions with toluene, and the pyrene contents in the toluene solutions were analyzed by the spectrometer. The extracted contents of pyrene, rhodamine B and acetaminophen were determined
O
N
H N
Cl COOH
Pyrene
Rhodamine B
HO
O
Acetaminophen
Fig. 1. Structures of molecules encapsulated into calcium carbonate.
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2.6. Characterization of materials The crystalline phases and the morphologies of the calcium carbonate samples were analyzed by XRD measurement using a Mac Science MXP3 V diffraction meter with Ni filtered Cu Ka radiation (k = 0.15406 nm) and scanning electron microscopy (SEM) images using a JEOL JSM-6390, respectively. The presences of the organic molecules in the calcium carbonate samples were ascertained by diffuse reflectance UV–vis spectra obtained by Jasco V-550 spectrophotometer equipped with integrating sphere (Jasco ISV-469). Fluorescence spectra were recorded by a Perkin Elmer luminescence spectrometer LS 50B with the excitation lights at 350 nm and 535 nm for pyrene and rhodamine B, respectively. Fluorescence microscopy observation was performed using an Olympus BX51WI upright microscope with a xenon lamp (AH2RX-T, Olympus). A 60 water-immersion objective (NA 0.90, Olympus) was used. Samples with included pyrene and rhodamine B fluorophores were observed using an Olympus WU filter set (excitation 330–385 nm, emission >420 nm) and the WIY filter set (excitation 545–580 nm, emission >610 nm), respectively. Fluorescence images were collected with a CCD camera (DP30BW, Olympus) and the brightness and contrast of the images were adjusted using the ImageJ program. Although the samples including pyrene and rhodamine B fluorophores emitted blue or red light, respectively, these color images were converted to black-and-white for showing the fluorescence clearly. 3. Results and discussion 3.1. Encapsulation of pyrene into calcium carbonate Pyrene (Fig. 1) was chosen as a fluorescent organic molecule that is poorly soluble in water, because pyrene and its derivatives are commonly used as fluorescent probes in biomedical and molecular analyses [41,42]. A calcium carbonate microcapsule with impregnated pyrene was soaked in a Tris HCl buffer solution (1 M, pH = 7.0) or a calcium chloride solution (0.2 M) for 24 days. These samples were named as CC–P–Tr and CC–P–Ca, respectively. After the careful separation of the aqueous solutions and white skims on the solutions, the calcium carbonate solids thus recovered were thoroughly washed with deionized water, and dried at 60 °C. Although the white skims on the solutions were identified as pyrene, the amount of the recovered pyrene was less than 10% of the impregnated one onto the microcapsule in both cases. The dried calcium carbonate particles were further washed with THF to eliminate removable pyrene. The same experiment was also carried out using a calcite calcium carbonate (commercial available) instead of the microcapsule (immersion in the Tris HCl buffer solution) and the sample obtained by this experiment was named as CL–P–Tr. Fig. 2 illustrates the XRD patterns of the original calcium carbonate microcapsule, two particles after the soaking (CC–P–Tr and CC–P–Ca), and the commercial available calcite. The XRD pattern of CL–P–Tr (not shown here) was similar to the commercial available calcite. According to these XRD patterns, the vaterite phase of the original microcapsule completely turned to the calcite phase. As shown in a SEM image of Fig. 3A, the original calcium carbonate microcapsule had a pseudospherical shape ranging in
Calcite
Intensity (au)
by the differences of absorptions between 337.5 nm and 400 nm, between 552–8 (peak) nm and 650 nm, and between 289 nm and 400 nm in wavelength, respectively. The weight ratios of the encapsulated organic molecules were estimated by the amounts of the calcium carbonate particles used and the extracted organic molecules.
CC-P-Tr CC-P-Ca Microcapsule 10
20
30
40
50
2 theta/ theta (CuKα )
60
70
Fig. 2. XRD patterns of the original calcium carbonate microcapsule (triplicate intensity), the calcium carbonate particles CC–P–Tr and CC–P–Ca and the commercial available calcite.
size from 2 to 10 lm. After the phase transition, the calcium carbonate particles CC–P–Tr and CC–P–Ca became larger particles (Fig. 3B and C). The shapes of these particles were changed to hexahedral ones ranging in size from 5 to 30 lm, which are the basic shape of calcite particles (Fig. 3D). The presence of pyrene in the calcium carbonate particles, CC– P–Tr and CC–P–Ca, was estimated by a diffuse reflectance UV–vis (DR UV–vis) spectrometry as illustrated in Fig. 4A. Those spectra clearly indicated the wide pyrene absorption around 250–380 nm in wavelength, which was absent in the original calcium carbonate microcapsule. As these samples were sufficiently washed with THF until no pyrene was detected in the washing solutions, the pyrene observed in the DR UV–vis spectra must be trapped in the crystalline calcium carbonate particles. On the other hand, no pyrene absorption was found in CL–P–Tr. In Table 1, the weight ratios of the encapsulated pyrene into calcium carbonate particles were summarized. The particle obtained by the immersion into a 0.2 M calcium chloride solution (CC–P–Ca) contained the higher pyrene (0.06 wt%) than the particle using a Tris HCl buffer solution (CC–P–Tr). On the other hand, no pyrene was certainly included in the sample obtained from calcite (CL–P–Tr). Therefore, the encapsulation of pyrene into calcium carbonate particle was achieved only when vaterite was used for the treatment. In Fig. 4B, the UV–vis spectrum of CC–P–Tr was compared with a solid pyrene and a dilute THF solution of pyrene. While the UV–vis spectrum of pyrene in the THF solution showed no absorption over 350 nm in wavelength, the solid pyrene had a broad absorption from 250 to 480 nm in wavelength. The DR UV–vis spectrum of pyrene in CC–P–Tr had broad peaks below 380 nm in wavelength, which is intermediate between the solution and the solid. It is reported that the microcrystalline pyrene had additional longer-wavelength absorptions around 360 nm [43,44]. The aggregated pyrene was encapsulated in the crystalline calcium carbonate particles. Fig. 5A shows the fluorescence spectra of pyrene encapsulated into the calcium carbonate particles, CC–P–Tr and CC–P–Ca, using an excitation light of 350 nm in wavelength. In these spectra, while the monomer emission of pyrene around 380 nm was slight, the excimer emission about 470 nm in wavelength [44,45] was strong. Since the pyrene molecules encapsulated into the calcium carbonate particles were agglutinated, the excimer emission was exclusively observed in these samples. Fig. 5B and C show the fluorescence and bright-field micrographs of the calcium carbonate particles encapsulating pyrene (CC–P–Ca) at the same position, respectively. Fluorescence emission was detected in some specific regions of the calcium carbonate particles. Thus, the fluorescent
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(A)
(B)
(C)
(D)
Fig. 3. SEM images of the original calcium carbonate microcapsule (A; magnified image in the inset), the calcium carbonate particles CC–P–Tr (B) and CC–P–Ca (C), and the commercial available calcite (D).
(B)
Kubelka Munk
CC-P-Ca
Kubelka-Munk (Abs.)
(A) CC-P-Tr
CL-P-Tr 200
300
400
500
Wavelength/nm
Pyrene solution Pyrene solid
CC-P-Tr
250
300
350
400
450
500
Wavelength/nm
Fig. 4. (A) Diffuse reflectance UV–vis spectra of the calcium carbonate particles, CC–P–Tr, CC–P–Ca and CL–P–Tr. (B) Comparison of UV–vis spectra among CC–P–Tr, solid pyrene as diffuse reflectance UV–vis spectra, and pyrene diluted solution in THF as an absorption spectrum.
Table 1 Calcium carbonate particles encapsulating organic molecules.
a
Sample
Organic molecule
Solution
Content / wt%
CC–P–Tr CC–P–Ca CL–P–Tra CC–R–Tr CC–R–Ca CC–A–Tr CC–A–Ca
Pyrene Pyrene Pyrene Rhodamine B Rhodamine B Acetaminophen Acetaminophen
1 M Tris HCl 0.2 M CaCl2 1 M Tris HCl 1 M Tris HCl 0.2 M CaCl2 1 M Tris HCl 0.2 M CaCl2
0.028 0.060 0 0.001 0.002 0.020 0.023
Commercial calcite calcium carbonate was used.
calcium carbonate microparticles were obtained by the pyrene impregnation to the vaterite microcapsule and the following immersion into aqueous solution.
The mechanism of the phase transition of vaterite to calcite has been intensively examined [46–48]. From these researches, it is concluded that the phase transition takes place not by the direct solid phase conversion but by the dissolution of vaterite and the recrystallization of calcite in aqueous solution. This crystallization to calcite occurs from the external surface of the microcapsule to grow toward the inside [49]. The high magnification SEM image of the microcapsule in the inset of Fig. 3A indicated that the microcapsule shell consisted of smaller nanoparticles (200 nm in diameter), which produced nano-voids among them. The impregnated pyrene was filled in the hollow space and the pore voids of the shell of the microcapsule. The calcite crystal formation might take place even on the exterior of the pore voids of the microcapsule with impregnated pyrene. Although the dissolution of vaterite eliminated pyrene from the microcapsule, the recovered pyrene
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(A)
(B)
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(C)
500
CC-P-Ca
Intensity
400 300 200
CC-P-Tr
100 0 400
450
500
550
600
Wavelength/nm Fig. 5. (A) Fluorescent spectra of pyrene encapsulated into the calcium carbonate particles, CC–P–Tr and CC–P–Ca, using an excitation light with a wavelength of 350 nm. Fluorescence (B) and bright-field (C) microscopy images of the calcium carbonate particle encapsulating pyrene (CC–P–Ca). The fluorescent microscopy color image with blue light emission was converted to black-and-white one for showing the fluorescence clearly. The scale bar corresponds to 40 lm.
3.2. Encapsulation of rhodamine B into calcium carbonate
Intensity (au)
Calcite
CC-R-Ca CC-R-Tr Microcapsule 10
20
30
40
50
60
70
2 theta/degree (CuKα) Fig. 6. XRD patterns of the original calcium carbonate microcapsule, the calcium carbonate particles, CC–R–Tr and CC–R–Ca, and the commercial available calcite (one third intensity).
solid skimming on the aqueous solution was less than 10% of pyrene used. A large part of the impregnated pyrene was still remained in the microcapsule during the immersion process. After the phase transition, some pyrene molecule especially located in the hollow space must be trapped into the calcite crystal formed. Finally, some portion of pyrene was encapsulated into the calcite crystal even after thorough washing of THF. On the other hand, as the phase transition of the commercial calcite did not progress, no encapsulation of pyrene into the calcite calcium carbonate occurred (CL–P–Tr).
(A)
Rhodamine B, a water-soluble fluorescent molecule (Fig. 1), can be encapsulated into crystalline calcium carbonate. In this case, the encapsulation procedure was simpler than that of pyrene. The calcium carbonate microcapsule was immersed into a 1 M Tris HCl buffer solution or a 0.2 M calcium chloride solution dissolving rhodamine B. After 24 days immersion, the calcium carbonate particles were filtered and washed with water and ethanol for the complete removal of rhodamine B. As shown in Fig. 6, the XRD patterns of vaterite were still strong in the immersed particles (CC–R–Tr and CC–R–Ca). As it is reported that the carboxylate groups of polymers prohibited the phase transition of vaterite to calcite [50], the carboxylate group of rhodamine B is likely to be responsible for the slow phase transition. From the SEM image of the calcium carbonate particle CC–R–Tr illustrated in Fig. 7A, the spherical particles of vaterite were still remained even after the immersion for 24 days. However, the crumbling shell of the microcapsule revealing the hollow structure indicated that the moderate dissolution of the microcapsule occurred (inset of Fig. 7A). On the other hand, a larger cubic-like particle that was considered as calcite was observed in the left-hand of the SEM image of CC–R–Ca (Fig. 7B). In the XRD pattern of CC–R–Ca (Fig. 6), the main peak of calcite detected at approximately 29 degrees in 2 theta was sharper than that of CC–R–Tr. The phase transition in the calcium chloride solution was faster than in the Tris HCl buffer solution, probably because the calcium ion promoted the crystallization of calcite. Fig. 8A represents the DR UV–vis spectra of calcium carbonate particles, CC–R–Tr and CC–R–Ca. The visible light absorption of
(B)
Fig. 7. SEM images of the calcium carbonate particles, CC–R–Tr (A; magnified image in the inset) and CC–R–Ca (B).
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(B) 1000
(A) CC-R-Ca
Intensity
Kubelka-Munk
800
CC-R-Tr
CC-R-Tr
600
CC-R-Ca
400 200 0
300
400
500
600
700
550
800
600
650
700
750
800
Wavelength/nm
Wavelength/nm
Fig. 8. (A) Diffuse reflectance UV–vis spectra of the calcium carbonate particles, CC–R–Tr and CC–R–Ca. (B) Fluorescent spectra of rhodamine B encapsulated into the calcium carbonate particles, CC–R–Tr and CC–R–Ca, using an excitation light with a wavelength of 535 nm.
(A)
(B)
(A)
(B)
Intensity (au)
Kubelka-Munk
Fig. 9. Fluorescence (A) and bright-field (B) microscopy images of the calcium carbonate particle encapsulating rhodamine B, CC–R–Ca. The fluorescent microscopy color image with red light emission was converted to black-and-white for showing the fluorescence clearly. The scale bar corresponds to 40 lm.
CC-A-Ca
CC-A-Ca CC-A-Tr
CC-A-Tr 10
20
30
40
50
60
70
2 theta/degree (CuKα)
200
250
300
350
400
Wavelength/nm
Fig. 10. (A) XRD patterns of the calcium carbonate particles, CC–A–Tr and CC–A–Ca. (B) Diffuse reflectance UV–vis spectra of the calcium carbonate particles, CC–A–Tr and CC–A–Ca.
rhodamine B around 550 nm in wavelength was clearly found in both spectra. Even though the phase transition was insufficient, rhodamine B was encapsulated into the calcium carbonate particles. The particle CC–R–Ca with faster phase transition had the stronger absorption than CC–R–Tr. As summarized in Table 1, the particle CC–R–Ca contained much more rhodamine B than CC–R– Tr. Fig. 8B shows the fluorescence spectra of rhodamine B encapsulated into the calcium carbonate particles using an excitation light of 535 nm in wavelength. The fluorescent emission was detected at about 570 nm to indicate that rhodamine B in these calcium carbonate particles maintained its fluorescence property. Fig. 9A illustrates the fluorescence microscope image of the calcium carbonate
particle encapsulating rhodamine B (CC–R–Ca) with 535 nm of excitation light. The bright-field micrograph of the same place is also shown in Fig. 9B. The fluorescent emission was clearly observed at the whole particles. As the main crystalline phase of these particles was still vaterite, the incorporation of rhodamine B occurred in the periphery of the calcium carbonate microparticles. 3.3. Encapsulation of acetaminophen into calcium carbonate by phase transition The encapsulation method of the fluorescent organic molecules described above was applicable to a drug molecule. N-(4-hydroxy-
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phenyl)acetamide (acetaminophen), a hardly water soluble drug widely used for analgesic and antipyretic (Fig. 1), was also encapsulated into the crystalline calcium carbonate particle by the phase transition method similar to pyrene. In this case, the phase transition from vaterite to calcite progressed completely as shown in Fig. 10A. The inclusions of acetaminophen in those particles, CC– A–Tr and CC–A–Ca, were confirmed from the corresponding DR UV–vis spectra (Fig. 10B). The UV absorption of acetaminophen around 250 nm in wavelength was detected in both spectra. The contents of acetaminophen in the calcium carbonate particles were in the same range as pyrene (Table 1). This drug molecule was released by the dissolution of the calcium carbonate particle in an aqueous solution, when the pH value of the solution was decreased to 5.5 by the addition of acetic acid. When 0.1 g of calcium carbonate particle was added to a 10 mL aqueous solution at pH 5.5 obtained from a 1 M Tris HCl buffer solution, the particle was completely dissolved within 10 min. In the UV spectrum of the resulting solution, the released acetaminophen was observed. This result indicated that the dissolution-triggered drug delivery induced by pH decrease will be possible using the calcium carbonate particles encapsulating organic drug molecules, where the residual products from the drug carrier (calcium carbonate in this case) are only non-toxic calcium and carbonate ions. 4. Conclusions This paper reports that organic molecules hardly-soluble in water such as pyrene and acetaminophen were encapsulated into the calcite calcium carbonate particles by simple two step treatments. In the first step, these molecules were impregnated to the vaterite microcapsule using a THF solution. In the second step, these calcium carbonate microcapsules with the impregnated molecules underwent the phase transition to calcite in suitable aqueous solutions. The utilization of vaterite was essential for this encapsulation method, while the encapsulation was unsuccessful using calcite. Rhodamine B, an organic molecule soluble in water, was also introduced by the simple immersion of the calcium carbonate microcapsule in the rhodamine B aqueous solutions. These organic molecules incorporated into the calcium carbonate particles were not eliminated unless the solid calcium carbonate was dissolved. The calcium carbonate particles encapsulating fluorescent pyrene and rhodamine B had the fluorescent emission abilities to be potentially applied to biological sensing and other microanalyses. The release of organic molecules through the dissolution of the solid calcium carbonate in low pH solution would be utilized to responsive drug delivery. The low content of the organic molecules less than 0.1 wt% in the calcium carbonate particles is still an unresolved issue. Since the higher porosity of calcium carbonate particles is beneficial to increase the contents, we will attempt the preparation of more porous calcium carbonate microparticles. Acknowledgement This study was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Research Project for ensuring food safety from farm to table: LP-5102 and LP-5103). References [1] M. Vendrell, D. Zhai, J.C. Er, Y.-T. Chang, Combinatorial strategies in fluorescent probe development, Chem. Rev. 112 (2012) 4391–4420. [2] M. Sameiro, T. Goncalves, Fluorescent labeling of biomolecules with organic probes, Chem. Rev. 109 (2009) 190–212. [3] M. Beija, C.A.M. Afonso, J.M.G. Martinho, Synthesis and applications of Rhodamine derivatives as fluorescent probes, Chem. Soc. Rev. 38 (2009) 2410–2433. [4] A.P. Alivisatos, W.W. Gu, C. Larabell, Quantum dots as cellular probes, Ann. Rev. Biomed. Eng. 7 (2005) 55–76.
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Advanced Powder Technology journal homepage: www.elsevier.com/locate/apt
Original Research Paper
Preparation of calcium carbonate microparticles containing organic fluorescent molecules from vaterite Masahiro Fujiwara a,⇑, Kumi Shiokawa a, Takayuki Kubota b, Kenichi Morigaki c a
National Institute of Advanced Industrial Science and Technology (Kansai Center), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan National Institute of Animal Health, National Agriculture and Food Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan c Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada, Kobe 657-8501, Japan b
a r t i c l e
i n f o
Article history: Received 18 October 2013 Received in revised form 29 January 2014 Accepted 26 February 2014 Available online 13 March 2014 Keywords: Calcium carbonate Vaterite Fluorescent particles Encapsulation of organic molecules Drug delivery system
a b s t r a c t The encapsulation of fluorescent organic molecules into crystalline calcium carbonate was examined using calcium carbonate microcapsule, whose crystalline phase is vaterite as a metastable phase of calcium carbonate. A calcium carbonate microcapsule with impregnated pyrene that is a water insoluble fluorescent molecule was soaked into suitable aqueous solutions to promote the phase transition of vaterite toward calcite as the stable phase of calcium carbonate. When 0.2 M calcium chloride solution was used, the largest amount of pyrene (approximately 0.06 wt%) was encapsulated into the calcite particle. Pyrene thus included was not eliminated even after thorough washing with THF. The calcite particle thus prepared produced the excimer emission of pyrene by UV irradiation. Rhodamine B was also introduced into calcium carbonate by the immersion of the microcapsule into the aqueous solutions of Rhodamine B. The fluorescence of rhodamine B was observed from the calcium carbonate particles by visible light irradiation. Acetaminophen, a common drug poorly soluble in water, was also included in the calcium carbonate particle by the same procedures as the pyrene encapsulation. As acetaminophen thus encapsulated was released by the dissolution of the calcium carbonate particle in acidic solution, the particle is expected to be applied for a dissolution-triggered drug delivery. Ó 2014 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.
1. Introduction Fluorescent molecules and materials are useful probes for the extensive analyses of genes, proteins, drug targets in cells, tissues and other living systems [1–6]. While organic dyes and fluorescent proteins are still convenient materials [1–3], inorganic quantum dot microparticles are also applied to the biological sensing researches [4–6]. Since the direct use of these fluorophores and fluorescent materials often encounter some severe problems such as pH, pro-oxidant species and dissolved salts within organism environments, their functionalizations and decorations are usually required. However, the elimination and the egestion of these artificial decorations into living organisms must be carefully controlled. Calcium carbonate is one of the most useful and versatile materials for biomedical fields [7–10]. Its high environmentallyfriendly property and biocompatibility are undoubtedly appropriate for a broad range of utilizations for living objects and human being. As calcium carbonate is readily decomposed or metabolized ⇑ Corresponding author. Tel.: +81 72 751 9525; fax: +81 72 751 9628. E-mail address: [email protected] (M. Fujiwara).
within organisms to non-toxic calcium and carbonate ions (or carbon dioxide), calcium carbonate materials with properly designed functions must be powerful tools for biomedical related systems, including bio-sensing and drug delivery [11–17]. The preparation of fluorescent calcium carbonate materials have been examined by the doping of fluorescent metal cations such as Gd, Eu [18,19], Sn [20] and Ce [21], because the ionexchange of calcium to these metals effectively occurs due to their analogous ionic radii. Recently, the precipitation of calcium carbonate in the presence of mercaptopropyl acid-modified CdTe is reported to be an efficient method for incorporating quantum dot microparticles into crystalline calcium carbonate, which is beneficial for monitoring intracellular uptake [22]. On the other hand, as a number of fluorescent organic molecules and fluorescent pigments are known [23,24], the incorporation of these common phosphors in calcium carbonate must be versatile approaches to the preparation of fluorescent calcium carbonate materials. Although amorphous calcium carbonate [25–30] is used as a template for the encapsulation of fluorescent molecules into organic capsules like chitosan [31] and PMMA [32], the crystalline calcium carbonate materials containing fluorescent organic
http://dx.doi.org/10.1016/j.apt.2014.02.022 0921-8831/Ó 2014 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.
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molecules have not been examined. Therefore, new methods for the encapsulation of fluorescent organic molecules into crystalline calcium carbonate are required. We have studied the preparation of inorganic hollow microparticles (microcapsules) [33–35] such as calcium carbonate [36–39] by an interfacial reaction method. The crystalline phase of the calcium carbonate microcapsule is vaterite as a metastable phase of calcium carbonate, while the common calcium carbonate observed in nature is calcite as the most stable phase. We have recently found that the phase transition of the vaterite calcium carbonate microcapsule to calcite in the aqueous solutions of proteins such as bovine serum albumin (BSA), lysozyme and insulin achieves the encapsulation of those proteins into the resulting calcite calcium carbonate [40]. The application of this phase transition method to the incorporation of organic molecules is expected to develop a new procedure for creating functional crystalline calcium carbonate materials with organic molecules. In this paper, we report the encapsulations of pyrene and rhodamine B as fluorescent molecules into crystalline calcium carbonate particles by the phase transition method. Moreover, the incorporation of acetaminophen, a poorly water-soluble organic molecule, into calcium carbonate particle and its release behavior are also described.
2.3. Encapsulation of pyrene and acetaminophen into calcium carbonate particles This procedure consisted of two processes; the first step is the impregnation of pyrene onto the calcium carbonate microcapsule and the second one is the immersion of the microcapsule into an aqueous solution. The calcium carbonate microcapsule (1 g) was soaked in 50 mL of a THF solution with dissolved pyrene (0.2 g). After gentle mixing of this suspension for 30 min, the volatiles were removed under reduced pressure. This calcium carbonate microcapsule with impregnated pyrene was added to 20 mL of a 1 M Tris HCl buffer solution or a 0.2 M calcium chloride solution. The resulting suspension was stood at 22 °C for 24 days. The aqueous solution and white skim on the solution (identified as pyrene) were carefully separated from the calcium carbonate solid by decantation. The calcium carbonate solid was washed with deionized water more than three times, and finally dried at 60 °C for 12 h. This calcium carbonate was further washed with THF in order to remove pyrene until pyrene was not detected in the THF solutions by UV–vis spectrometry analysis (Jasco V-530 spectrophotometer). Double washing was enough to remove pyrene from the calcium carbonate particle completely. The encapsulation of acetaminophen was carried out by the same procedures as pyrene.
2. Materials and methods 2.4. Encapsulation of rhodamine B into calcium carbonate particles 2.1. Materials Ammonium carbonate [(NH4)2CO3], calcium chloride dihydrate (CaCl22H2O), pyrene, rhodamine B and other solvents (n-hexane, THF, methanol and ethanol) used in this work were purchased from Wako pure chemical industries. Tween 85 and a commercial calcite calcium carbonate were obtained from Kanto Chemicals and acetaminophen was from MP biomedicals. 1 M Tris HCl solution (pH = 7) was commercial available from Wako Pure Chemical Industries and 0.2 M calcium chloride solution was prepared with calcium chloride dihydrate in our laboratory. The structures of molecules encapsulated into calcium carbonate are summarized in Fig. 1. 2.2. Preparation of vaterite calcium carbonate microcapsule Calcium carbonate microcapsules were prepared by a described method in our recent papers [39,40]. A typical procedure is briefly describe as follows: 32 mL of a 3 M ammonium carbonate solution was mixed with 48 mL of a n-hexane solution dissolving 1.0 g of Tween 85, and the resulting solution was emulsified with a homogenizer at 8000 rpm for 1 min in order to obtain a water/oil (W/O) emulsion. This W/O emulsion was poured into 640 mL of a 0.3 M calcium chloride aqueous solution in one portion at 30 °C. After 5 min stirring, the precipitate was filtered, washed with deionized water twice and methanol once and finally dried at 80 °C for 12 h.
N
The calcium carbonate microcapsule (1 g) was immersed in 10 mL of a 1 M Tris HCl buffer solution or a 0.2 M calcium chloride solution with 0.2 g of rhodamine B. The resulting suspension was stood at 22 °C for 24 days. This solid was filtered, thoroughly washed with deionized water, and dried at 60 °C for 12 h. This solid was further washed with ethanol thoroughly until rhodamine B was not detected in the solutions by UV–vis spectrometry analysis. More than three times washing was necessary to remove the rhodamine B from the calcium carbonate particle perfectly. 2.5. Determination of the weight ratios of organic molecules encapsulated into calcium carbonate particles The amounts of organic molecules encapsulated into the calcium carbonate particles were estimated by the extraction of the molecules. The calcium carbonate particles were dissolved with an 8.5 M acetic acid solution, and the amounts of eliminated molecules were determined by UV–vis spectrometry (Jasco V-530 spectrophotometer). In the cases of acetaminophen and rhodamine B, after the complete dissolution of the calcium carbonate particles, the resulting acetic acid solutions were directly analyzed by the spectrometer. In the case of pyrene, after the complete dissolution of the calcium carbonate particles, pyrene was extracted from the solutions with toluene, and the pyrene contents in the toluene solutions were analyzed by the spectrometer. The extracted contents of pyrene, rhodamine B and acetaminophen were determined
O
N
H N
Cl COOH
Pyrene
Rhodamine B
HO
O
Acetaminophen
Fig. 1. Structures of molecules encapsulated into calcium carbonate.
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2.6. Characterization of materials The crystalline phases and the morphologies of the calcium carbonate samples were analyzed by XRD measurement using a Mac Science MXP3 V diffraction meter with Ni filtered Cu Ka radiation (k = 0.15406 nm) and scanning electron microscopy (SEM) images using a JEOL JSM-6390, respectively. The presences of the organic molecules in the calcium carbonate samples were ascertained by diffuse reflectance UV–vis spectra obtained by Jasco V-550 spectrophotometer equipped with integrating sphere (Jasco ISV-469). Fluorescence spectra were recorded by a Perkin Elmer luminescence spectrometer LS 50B with the excitation lights at 350 nm and 535 nm for pyrene and rhodamine B, respectively. Fluorescence microscopy observation was performed using an Olympus BX51WI upright microscope with a xenon lamp (AH2RX-T, Olympus). A 60 water-immersion objective (NA 0.90, Olympus) was used. Samples with included pyrene and rhodamine B fluorophores were observed using an Olympus WU filter set (excitation 330–385 nm, emission >420 nm) and the WIY filter set (excitation 545–580 nm, emission >610 nm), respectively. Fluorescence images were collected with a CCD camera (DP30BW, Olympus) and the brightness and contrast of the images were adjusted using the ImageJ program. Although the samples including pyrene and rhodamine B fluorophores emitted blue or red light, respectively, these color images were converted to black-and-white for showing the fluorescence clearly. 3. Results and discussion 3.1. Encapsulation of pyrene into calcium carbonate Pyrene (Fig. 1) was chosen as a fluorescent organic molecule that is poorly soluble in water, because pyrene and its derivatives are commonly used as fluorescent probes in biomedical and molecular analyses [41,42]. A calcium carbonate microcapsule with impregnated pyrene was soaked in a Tris HCl buffer solution (1 M, pH = 7.0) or a calcium chloride solution (0.2 M) for 24 days. These samples were named as CC–P–Tr and CC–P–Ca, respectively. After the careful separation of the aqueous solutions and white skims on the solutions, the calcium carbonate solids thus recovered were thoroughly washed with deionized water, and dried at 60 °C. Although the white skims on the solutions were identified as pyrene, the amount of the recovered pyrene was less than 10% of the impregnated one onto the microcapsule in both cases. The dried calcium carbonate particles were further washed with THF to eliminate removable pyrene. The same experiment was also carried out using a calcite calcium carbonate (commercial available) instead of the microcapsule (immersion in the Tris HCl buffer solution) and the sample obtained by this experiment was named as CL–P–Tr. Fig. 2 illustrates the XRD patterns of the original calcium carbonate microcapsule, two particles after the soaking (CC–P–Tr and CC–P–Ca), and the commercial available calcite. The XRD pattern of CL–P–Tr (not shown here) was similar to the commercial available calcite. According to these XRD patterns, the vaterite phase of the original microcapsule completely turned to the calcite phase. As shown in a SEM image of Fig. 3A, the original calcium carbonate microcapsule had a pseudospherical shape ranging in
Calcite
Intensity (au)
by the differences of absorptions between 337.5 nm and 400 nm, between 552–8 (peak) nm and 650 nm, and between 289 nm and 400 nm in wavelength, respectively. The weight ratios of the encapsulated organic molecules were estimated by the amounts of the calcium carbonate particles used and the extracted organic molecules.
CC-P-Tr CC-P-Ca Microcapsule 10
20
30
40
50
2 theta/ theta (CuKα )
60
70
Fig. 2. XRD patterns of the original calcium carbonate microcapsule (triplicate intensity), the calcium carbonate particles CC–P–Tr and CC–P–Ca and the commercial available calcite.
size from 2 to 10 lm. After the phase transition, the calcium carbonate particles CC–P–Tr and CC–P–Ca became larger particles (Fig. 3B and C). The shapes of these particles were changed to hexahedral ones ranging in size from 5 to 30 lm, which are the basic shape of calcite particles (Fig. 3D). The presence of pyrene in the calcium carbonate particles, CC– P–Tr and CC–P–Ca, was estimated by a diffuse reflectance UV–vis (DR UV–vis) spectrometry as illustrated in Fig. 4A. Those spectra clearly indicated the wide pyrene absorption around 250–380 nm in wavelength, which was absent in the original calcium carbonate microcapsule. As these samples were sufficiently washed with THF until no pyrene was detected in the washing solutions, the pyrene observed in the DR UV–vis spectra must be trapped in the crystalline calcium carbonate particles. On the other hand, no pyrene absorption was found in CL–P–Tr. In Table 1, the weight ratios of the encapsulated pyrene into calcium carbonate particles were summarized. The particle obtained by the immersion into a 0.2 M calcium chloride solution (CC–P–Ca) contained the higher pyrene (0.06 wt%) than the particle using a Tris HCl buffer solution (CC–P–Tr). On the other hand, no pyrene was certainly included in the sample obtained from calcite (CL–P–Tr). Therefore, the encapsulation of pyrene into calcium carbonate particle was achieved only when vaterite was used for the treatment. In Fig. 4B, the UV–vis spectrum of CC–P–Tr was compared with a solid pyrene and a dilute THF solution of pyrene. While the UV–vis spectrum of pyrene in the THF solution showed no absorption over 350 nm in wavelength, the solid pyrene had a broad absorption from 250 to 480 nm in wavelength. The DR UV–vis spectrum of pyrene in CC–P–Tr had broad peaks below 380 nm in wavelength, which is intermediate between the solution and the solid. It is reported that the microcrystalline pyrene had additional longer-wavelength absorptions around 360 nm [43,44]. The aggregated pyrene was encapsulated in the crystalline calcium carbonate particles. Fig. 5A shows the fluorescence spectra of pyrene encapsulated into the calcium carbonate particles, CC–P–Tr and CC–P–Ca, using an excitation light of 350 nm in wavelength. In these spectra, while the monomer emission of pyrene around 380 nm was slight, the excimer emission about 470 nm in wavelength [44,45] was strong. Since the pyrene molecules encapsulated into the calcium carbonate particles were agglutinated, the excimer emission was exclusively observed in these samples. Fig. 5B and C show the fluorescence and bright-field micrographs of the calcium carbonate particles encapsulating pyrene (CC–P–Ca) at the same position, respectively. Fluorescence emission was detected in some specific regions of the calcium carbonate particles. Thus, the fluorescent
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(A)
(B)
(C)
(D)
Fig. 3. SEM images of the original calcium carbonate microcapsule (A; magnified image in the inset), the calcium carbonate particles CC–P–Tr (B) and CC–P–Ca (C), and the commercial available calcite (D).
(B)
Kubelka Munk
CC-P-Ca
Kubelka-Munk (Abs.)
(A) CC-P-Tr
CL-P-Tr 200
300
400
500
Wavelength/nm
Pyrene solution Pyrene solid
CC-P-Tr
250
300
350
400
450
500
Wavelength/nm
Fig. 4. (A) Diffuse reflectance UV–vis spectra of the calcium carbonate particles, CC–P–Tr, CC–P–Ca and CL–P–Tr. (B) Comparison of UV–vis spectra among CC–P–Tr, solid pyrene as diffuse reflectance UV–vis spectra, and pyrene diluted solution in THF as an absorption spectrum.
Table 1 Calcium carbonate particles encapsulating organic molecules.
a
Sample
Organic molecule
Solution
Content / wt%
CC–P–Tr CC–P–Ca CL–P–Tra CC–R–Tr CC–R–Ca CC–A–Tr CC–A–Ca
Pyrene Pyrene Pyrene Rhodamine B Rhodamine B Acetaminophen Acetaminophen
1 M Tris HCl 0.2 M CaCl2 1 M Tris HCl 1 M Tris HCl 0.2 M CaCl2 1 M Tris HCl 0.2 M CaCl2
0.028 0.060 0 0.001 0.002 0.020 0.023
Commercial calcite calcium carbonate was used.
calcium carbonate microparticles were obtained by the pyrene impregnation to the vaterite microcapsule and the following immersion into aqueous solution.
The mechanism of the phase transition of vaterite to calcite has been intensively examined [46–48]. From these researches, it is concluded that the phase transition takes place not by the direct solid phase conversion but by the dissolution of vaterite and the recrystallization of calcite in aqueous solution. This crystallization to calcite occurs from the external surface of the microcapsule to grow toward the inside [49]. The high magnification SEM image of the microcapsule in the inset of Fig. 3A indicated that the microcapsule shell consisted of smaller nanoparticles (200 nm in diameter), which produced nano-voids among them. The impregnated pyrene was filled in the hollow space and the pore voids of the shell of the microcapsule. The calcite crystal formation might take place even on the exterior of the pore voids of the microcapsule with impregnated pyrene. Although the dissolution of vaterite eliminated pyrene from the microcapsule, the recovered pyrene
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(A)
(B)
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(C)
500
CC-P-Ca
Intensity
400 300 200
CC-P-Tr
100 0 400
450
500
550
600
Wavelength/nm Fig. 5. (A) Fluorescent spectra of pyrene encapsulated into the calcium carbonate particles, CC–P–Tr and CC–P–Ca, using an excitation light with a wavelength of 350 nm. Fluorescence (B) and bright-field (C) microscopy images of the calcium carbonate particle encapsulating pyrene (CC–P–Ca). The fluorescent microscopy color image with blue light emission was converted to black-and-white one for showing the fluorescence clearly. The scale bar corresponds to 40 lm.
3.2. Encapsulation of rhodamine B into calcium carbonate
Intensity (au)
Calcite
CC-R-Ca CC-R-Tr Microcapsule 10
20
30
40
50
60
70
2 theta/degree (CuKα) Fig. 6. XRD patterns of the original calcium carbonate microcapsule, the calcium carbonate particles, CC–R–Tr and CC–R–Ca, and the commercial available calcite (one third intensity).
solid skimming on the aqueous solution was less than 10% of pyrene used. A large part of the impregnated pyrene was still remained in the microcapsule during the immersion process. After the phase transition, some pyrene molecule especially located in the hollow space must be trapped into the calcite crystal formed. Finally, some portion of pyrene was encapsulated into the calcite crystal even after thorough washing of THF. On the other hand, as the phase transition of the commercial calcite did not progress, no encapsulation of pyrene into the calcite calcium carbonate occurred (CL–P–Tr).
(A)
Rhodamine B, a water-soluble fluorescent molecule (Fig. 1), can be encapsulated into crystalline calcium carbonate. In this case, the encapsulation procedure was simpler than that of pyrene. The calcium carbonate microcapsule was immersed into a 1 M Tris HCl buffer solution or a 0.2 M calcium chloride solution dissolving rhodamine B. After 24 days immersion, the calcium carbonate particles were filtered and washed with water and ethanol for the complete removal of rhodamine B. As shown in Fig. 6, the XRD patterns of vaterite were still strong in the immersed particles (CC–R–Tr and CC–R–Ca). As it is reported that the carboxylate groups of polymers prohibited the phase transition of vaterite to calcite [50], the carboxylate group of rhodamine B is likely to be responsible for the slow phase transition. From the SEM image of the calcium carbonate particle CC–R–Tr illustrated in Fig. 7A, the spherical particles of vaterite were still remained even after the immersion for 24 days. However, the crumbling shell of the microcapsule revealing the hollow structure indicated that the moderate dissolution of the microcapsule occurred (inset of Fig. 7A). On the other hand, a larger cubic-like particle that was considered as calcite was observed in the left-hand of the SEM image of CC–R–Ca (Fig. 7B). In the XRD pattern of CC–R–Ca (Fig. 6), the main peak of calcite detected at approximately 29 degrees in 2 theta was sharper than that of CC–R–Tr. The phase transition in the calcium chloride solution was faster than in the Tris HCl buffer solution, probably because the calcium ion promoted the crystallization of calcite. Fig. 8A represents the DR UV–vis spectra of calcium carbonate particles, CC–R–Tr and CC–R–Ca. The visible light absorption of
(B)
Fig. 7. SEM images of the calcium carbonate particles, CC–R–Tr (A; magnified image in the inset) and CC–R–Ca (B).
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(B) 1000
(A) CC-R-Ca
Intensity
Kubelka-Munk
800
CC-R-Tr
CC-R-Tr
600
CC-R-Ca
400 200 0
300
400
500
600
700
550
800
600
650
700
750
800
Wavelength/nm
Wavelength/nm
Fig. 8. (A) Diffuse reflectance UV–vis spectra of the calcium carbonate particles, CC–R–Tr and CC–R–Ca. (B) Fluorescent spectra of rhodamine B encapsulated into the calcium carbonate particles, CC–R–Tr and CC–R–Ca, using an excitation light with a wavelength of 535 nm.
(A)
(B)
(A)
(B)
Intensity (au)
Kubelka-Munk
Fig. 9. Fluorescence (A) and bright-field (B) microscopy images of the calcium carbonate particle encapsulating rhodamine B, CC–R–Ca. The fluorescent microscopy color image with red light emission was converted to black-and-white for showing the fluorescence clearly. The scale bar corresponds to 40 lm.
CC-A-Ca
CC-A-Ca CC-A-Tr
CC-A-Tr 10
20
30
40
50
60
70
2 theta/degree (CuKα)
200
250
300
350
400
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Fig. 10. (A) XRD patterns of the calcium carbonate particles, CC–A–Tr and CC–A–Ca. (B) Diffuse reflectance UV–vis spectra of the calcium carbonate particles, CC–A–Tr and CC–A–Ca.
rhodamine B around 550 nm in wavelength was clearly found in both spectra. Even though the phase transition was insufficient, rhodamine B was encapsulated into the calcium carbonate particles. The particle CC–R–Ca with faster phase transition had the stronger absorption than CC–R–Tr. As summarized in Table 1, the particle CC–R–Ca contained much more rhodamine B than CC–R– Tr. Fig. 8B shows the fluorescence spectra of rhodamine B encapsulated into the calcium carbonate particles using an excitation light of 535 nm in wavelength. The fluorescent emission was detected at about 570 nm to indicate that rhodamine B in these calcium carbonate particles maintained its fluorescence property. Fig. 9A illustrates the fluorescence microscope image of the calcium carbonate
particle encapsulating rhodamine B (CC–R–Ca) with 535 nm of excitation light. The bright-field micrograph of the same place is also shown in Fig. 9B. The fluorescent emission was clearly observed at the whole particles. As the main crystalline phase of these particles was still vaterite, the incorporation of rhodamine B occurred in the periphery of the calcium carbonate microparticles. 3.3. Encapsulation of acetaminophen into calcium carbonate by phase transition The encapsulation method of the fluorescent organic molecules described above was applicable to a drug molecule. N-(4-hydroxy-
M. Fujiwara et al. / Advanced Powder Technology 25 (2014) 1147–1154
phenyl)acetamide (acetaminophen), a hardly water soluble drug widely used for analgesic and antipyretic (Fig. 1), was also encapsulated into the crystalline calcium carbonate particle by the phase transition method similar to pyrene. In this case, the phase transition from vaterite to calcite progressed completely as shown in Fig. 10A. The inclusions of acetaminophen in those particles, CC– A–Tr and CC–A–Ca, were confirmed from the corresponding DR UV–vis spectra (Fig. 10B). The UV absorption of acetaminophen around 250 nm in wavelength was detected in both spectra. The contents of acetaminophen in the calcium carbonate particles were in the same range as pyrene (Table 1). This drug molecule was released by the dissolution of the calcium carbonate particle in an aqueous solution, when the pH value of the solution was decreased to 5.5 by the addition of acetic acid. When 0.1 g of calcium carbonate particle was added to a 10 mL aqueous solution at pH 5.5 obtained from a 1 M Tris HCl buffer solution, the particle was completely dissolved within 10 min. In the UV spectrum of the resulting solution, the released acetaminophen was observed. This result indicated that the dissolution-triggered drug delivery induced by pH decrease will be possible using the calcium carbonate particles encapsulating organic drug molecules, where the residual products from the drug carrier (calcium carbonate in this case) are only non-toxic calcium and carbonate ions. 4. Conclusions This paper reports that organic molecules hardly-soluble in water such as pyrene and acetaminophen were encapsulated into the calcite calcium carbonate particles by simple two step treatments. In the first step, these molecules were impregnated to the vaterite microcapsule using a THF solution. In the second step, these calcium carbonate microcapsules with the impregnated molecules underwent the phase transition to calcite in suitable aqueous solutions. The utilization of vaterite was essential for this encapsulation method, while the encapsulation was unsuccessful using calcite. Rhodamine B, an organic molecule soluble in water, was also introduced by the simple immersion of the calcium carbonate microcapsule in the rhodamine B aqueous solutions. These organic molecules incorporated into the calcium carbonate particles were not eliminated unless the solid calcium carbonate was dissolved. The calcium carbonate particles encapsulating fluorescent pyrene and rhodamine B had the fluorescent emission abilities to be potentially applied to biological sensing and other microanalyses. The release of organic molecules through the dissolution of the solid calcium carbonate in low pH solution would be utilized to responsive drug delivery. The low content of the organic molecules less than 0.1 wt% in the calcium carbonate particles is still an unresolved issue. Since the higher porosity of calcium carbonate particles is beneficial to increase the contents, we will attempt the preparation of more porous calcium carbonate microparticles. Acknowledgement This study was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Research Project for ensuring food safety from farm to table: LP-5102 and LP-5103). References [1] M. Vendrell, D. Zhai, J.C. Er, Y.-T. Chang, Combinatorial strategies in fluorescent probe development, Chem. Rev. 112 (2012) 4391–4420. [2] M. Sameiro, T. Goncalves, Fluorescent labeling of biomolecules with organic probes, Chem. Rev. 109 (2009) 190–212. [3] M. Beija, C.A.M. Afonso, J.M.G. Martinho, Synthesis and applications of Rhodamine derivatives as fluorescent probes, Chem. Soc. Rev. 38 (2009) 2410–2433. [4] A.P. Alivisatos, W.W. Gu, C. Larabell, Quantum dots as cellular probes, Ann. Rev. Biomed. Eng. 7 (2005) 55–76.
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