Encapsulation of Proteins into CaCO3 by Phase Transition from

Jul 26, 2010 - (10-12) For example, the direct precipitation of calcium carbonate on yeast cell walls is reported to produce hybrid cellular-inorganic...
0 downloads 0 Views 2MB Size
Download PDF
DOI: 10.1021/cg100631v

Encapsulation of Proteins into CaCO3 by Phase Transition from Vaterite to Calcite

2010, Vol. 10 4030–4037

Masahiro Fujiwara,*,† Kumi Shiokawa,† Miyuki Araki,† Nobuyuki Ashitaka,†,# Kenichi Morigaki,‡ Takayuki Kubota,§ and Yoshiko Nakahara† †

National Institute of Advanced Industrial Science and Technology, Kansai Center, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan, ‡Research Center for Environmental Genomics, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501 Japan, and §National Institute of Animal Health, National Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki, 305-0856 Japan. # Main Address: Department of Applied Chemistry, Osaka Institute of Technology, 5 Ohmiya, Asahi-ku, Osaka 535-8585, Japan. Received May 10, 2010; Revised Manuscript Received June 21, 2010

ABSTRACT: Phase transition from vaterite to calcite is a general behavior of CaCO3 materials. The interfacial reaction method using water-in-oil-in-water (W/O/W) emulsion we reported before is an effective method to produce hollow CaCO3 particles (microcapsules) with vaterite crystalline structure. These vaterite CaCO3 microcapsules underwent the phase transition to calcite in various aqueous solutions. When some proteins were mixed in the solution used for the phase transition, their encapsulations were achieved satisfactorily at room temperature regardless of their molecular weight. Insulin, lysozyme, bovine serum albumin, and chicken IgY were successfully encapsulated into the phase transition CaCO3 particles, while the encapsulation of lysozyme was unsuccessful by the interfacial reaction method. Protein included in the phase transition CaCO3 particle was not discharged by simple water washing but by the dissolution of the CaCO3 matrix in an acid solution, being advantageous to a responsive protein delivery technology. The recycle uses of the insulin solution used for the encapsulation improved the utilization efficiency of insulin. It was ascertained that the phase transition of vaterite CaCO3 microcapsule to calcite is a simple, general, and convenient method to encapsulate proteins into CaCO3 small particles under very mild conditions.

Introduction The interactions between CaCO3 species and proteins (and related materials) capture the interests of materials chemists, biochemists, and biomedical scientists. One of the most attractive fields of these interactions are correlated to biomineralization of CaCO3 species,1 because the nanocomposites of CaCO3 with proteins such as seashells have a number of unique properties.2-9 On the other hand, CaCO3 small particles including functional molecules are also interesting research themes due to their potent applications to biological and biomedical fields.10-12 For example, the direct precipitation of calcium carbonate on yeast cell walls is reported to produce hybrid cellular-inorganic core-shell microparticles.13 This methodology provides an encapsulation technique of living cells into CaCO3 shells. Furthermore, various porous CaCO3 particles have been employed for biomedical technologies because of their superior biocompatibility. CaCO3 in calcite as a typical and stable crystalline phase is less porous than amorphous, aragonite, and vaterite CaCO3 as metastable phases. The higher porosity and specific surface area of these uncommon CaCO3 materials are favorable for the adsorbent and/or composites of functional molecules and biomacromolecules such as proteins.14-19 These materials donate various interesting techniques of the sustained releases of drugs from CaCO3 microparticles.12,20-25 The recent requests of drug delivery technology are not limited to the extended-release of drug molecules. The complete *To whom correspondence should be addressed. E-mail: m-fujiwara@ aist.go.jp. pubs.acs.org/crystal

Published on Web 07/26/2010

encapsulations and no-discharge of drug molecules without the destruction of the capsules materials are also important functions for the development of drug delivery. Therefore, special techniques to embed functional molecules into solid matrices are indispensable. Bioencapsulation using inorganic materials has been mainly studied in silica and its analogous compounds, because the sol-gel technique is quite advantageous to implant various molecules into silica matrices.26-29 CaCO3 is also a significant inorganic compound for the bioencapsulation or the embedding of functional molecules as mentioned above. We have already reported that our interfacial reaction method using water-in-oil-in-water (W/O/W) emulsion30,31 is a prominent procedure to encapsulate various solid materials and biomacromolecules such as proteins and duplex DNA into CaCO3 microcapsules32,33 and others.34,35 As the inorganic matrices are formed along the interface of W/O/W emulsion in the interfacial reaction method, the molecules added to the inner water phase are able to be directly incorporated into the solid matrices produced.30 However, the inclusion efficiencies of proteins strongly depend on their molecular weights, and no lysozyme as a small protein (Mw: 14 388 Da) is encapsulated.33 This is probably because the spreading of relatively small molecules such as lysozyme to the outer water phase is too rapid to be included in the CaCO3 matrix forming along the interface of W/O/W emulsion. In this paper, we report a novel approach for the encapsulation of a variety of proteins into CaCO3 solid matrix. The main new aspect of this finding is the combination of the crystalline phase transition of CaCO3 and the uptake of molecules. Vaterite as metastable phases of CaCO3 readily changes the crystalline phase to calcite in aqueous solution.36-39 r 2010 American Chemical Society

Article

Crystal Growth & Design, Vol. 10, No. 9, 2010

CaCO3 microcapsules prepared by our interfacial reaction method have the vaterite crystalline phase exclusively with hollow structure.40,41 These CaCO3 microcapsules in vaterite phase also undergo the phase transition to calcite when they are added into some suitable aqueous solutions. We found that this phase transition is an effective method for the encapsulation of various proteins including lysozyme and insulin into CaCO3 solid. The proteins thus encapsulated were not released without the destruction of the CaCO3 solid matrix, while the dissolution of CaCO3 solid readily discharged them. Although the interfacial reaction method is not the only way to prepare vaterite CaCO3,42-45 the phase transition of vaterite CaCO3 microcapsule reported here is able to combine the proteins encapsulated by the phase transition with other components included beforehand by the interfacial reaction method, providing new applications of CaCO3 particles with proteins. Therefore, this research aimed to examine the details of the encapsulation of proteins by the phase transition of CaCO3 microcapsules. Experimental Section Materials. The chemicals and proteins employed here are commercial available and listed as follows. (NH4)2CO3, CaCl2 3 2H2O, Tris-HCl buffer (0.05 mol/L Tris-HCl buffer; pH = 7.6), and 1 M NaCl solution are obtained from Wako Pure Chemical Industries. Tween 85 was purchased from Kanto Chemical Co. Inc. Normal saline solution was purchased from Otsuka Pharmaceutical Co., Ltd. Insulin was obtained from Sigma as 10 mg/mL solution from bovine pancreas. Lysozyme (lysozyme, from egg white) and BSA (albumin, from bovine serum, Cohn Fraction V, pH 7.0) were taken from Wako Pure Chemical Industries. Chicken IgY was obtained from Equitech-Bio Inc. as 0.01 M phosphate buffer in 0.15 M NaCl. Preparation of CaCO3 Microcapsules as Vaterite. CaCO3 microcapsules as vaterite were prepared by a modified method from a described one.32,33 A typical procedure is as follows: an aqueous solution (32 mL) of 9.23 g (96 mmol) of (NH4)2CO3 was mixed with a n-hexane solution (48 mL) of 1.0 g of Tween 85, and the resulting solution was emulsified with 8000 rpm for 1 min using a “Physcotron” homogenizer NS-51 with a shaft generator NS-20. This W/O emulsion was poured into another aqueous solution (640 mL) of 28.2 g (192 mmol) of CaCl2 3 H2O in one portion at 30 °C, which was found to be a more suitable temperature than room temperature for exclusive vaterite formation. The final solution was stirred for 300 rpm at the same temperature for 5 min, and the white precipitate yielded was filtered and washed with 1 L of deionized water twice and with 100 mL of methanol. After drying at 80 °C for 12 h, approximately 8.1 g of CaCO3 microcapsule was obtained. The spherical and hollow structure was confirmed by scanning electron microscopy (SEM) observation and the exclusive formation of vaterite was ensured by X-ray diffraction (XRD) measurement. Phase Transition of CaCO3 Microcapsule from Vaterite to Calcite. A given amount of CaCO3 microcapsule prepared by the above-mentioned procedure was added to some aqueous solutions such as Tris-HCl buffer solution and normal saline solution. After the solution was left for from 96 to 140 h, the resulting solids were filtered, washed with deionized water, and dried at 100 °C for 12 h. The weight of CaCO3 samples generally decreased to approximately 90%. The phase transition of CaCO3 microcapsule to calcite phase was confirmed by SEM observation and XRD measurement. Protein Encapsulation by Phase Transition of Vaterite CaCO3 Microcapsule to Calcite. A typical procedure of the encapsulation of proteins is mentioned as follows taking insulin as an example: 1 g of CaCO3 microcapsule was added to the mixed solution of 0.5 mL of Tris-HCl buffer solution and 1 mL of insulin solution (10 mg/mL; from bovine pancreas). The resulting solution with insoluble CaCO3 was permitted to stand for 160 h at room temperature. This solid was filtered and washed with sufficient amounts of fresh deionized water. The solid thus obtained was dried at room temperature for a few days. The crystal phase and the morphology of the solid were analyzed by XRD measurement and SEM observation, respectively.

4031

The presence of insulin in the CaCO3 solid was checked by diffuse reflection UV spectrum. The insulin included in CaCO3 solid was estimated by the recovered amounts of insulin in the filtrate and the washed aqueous solutions. In the encapsulation of lysozyme and BSA, 1.5 mL solutions of Tris-HCl buffer solution dissolving each protein were used. Fluorescence microscopy observation was performed using an Olympus BX51WI upright fluorescence microscope with a xenon lamp (AH2-RX-T, Olympus). A 60 water-immersion objective (NA 0.90, Olympus) was used. Fluorescence microscopic images were collected with a CCD camera (DP30BW, Olympus). [Uplan S Apo(X100); 1.40NA]. An aqueous solution of BSA with 1% of BSA bearing fluorescent substance [albumin, fluorescein isothiocyanate conjugate bovine (FITC-BSA): from Sigma] was used for the phase transition method. Samples were observed with excitation light by the OLYMPUS U-MNIBA2 filter set (excitation: 470-490 nm, emission: 510-550 nm). All fluorescent microscopy color images were converted to black-and-white ones for showing the fluorescence clearly. Multiple Encapsulations of Insulin by Recycling Insulin Solution. One gram of CaCO3 microcapsule was added to the mixed solution of 0.5 mL of Tris-HCl buffer solution and 1 mL of the insulin solution. After the solution was left to stand for 200 h, the resulting solution was recovered by decantation. The remaining CaCO3 sample was washed with 2 mL of Tris-HCl buffer solution, which was combined with the decanted solution and used for the second soaking with 2 g of another fresh CaCO3 microcapsule. After 180 h at ambient temperature, this solution was decanted and the residual solid was washed with 2 mL of Tris-HCl buffer solution. Three grams of fresh CaCO3 microcapsule was soaked in the gathered solution and was permitted to stand for 180 h at room temperature. Two CaCO3 samples obtained by the first and the second soaking and this CaCO3 sample (the third soaking one) were filtered and washed with sufficient amounts of deionized water separately. The amounts of insulin remained in these three sufficient washings were analyzed by the respective UV absorption spectra. The presence of insulin in the three samples of the phase transition CaCO3 was ascertained by diffuse reflectance UV spectra. Dissolution of Phase Transition CaCO3 Encapsulating Insulin by HCl. The dissolution of calcite CaCO3 encapsulating insulin was carried out by the dropwise addition of 1 M HCl to 5 mL of phosphate buffer solution (pH = 6.86) suspending 0.2 g of the CaCO3 sample with monitoring of the pH value using a common pH meter. The addition of approximately 3.5 mL of 1 M HCl decreased the pH value to about 3.0 and dissolved the CaCO3 sample completely. The amount of released insulin into the aqueous solution was estimated by UV absorption spectrum. Analyses. X-ray diffraction patterns were recorded using Mac Science MXP3 V diffraction meter with Ni filtered Cu KR radiation (λ = 0.15406 nm) using common glass plates. Scanning electron microscopy (SEM) images were measured using a JEOL JSM-6390 microscope apparatus. UV spectrum measurement of aqueous solutions was performed using a JASCO V-530 spectrometer by a common procedure with quartz cell. Diffuse reflectance UV spectra were obtained with a JASCO V-550 spectrometer equipped with an integrating sphere. Kubelka-Munk functions were plotted versus the wavelength. Nitrogen adsorption-desorption isotherms were obtained at -196 °C (in liquid N2) using a Bellsorp Mini instrument (BEL JAPAN, Inc.). The specific surface areas of CaCO3 samples were estimated by a BET calculation method. Thermogravimetric analysis (TGA) was carried out using a Shimadzu TGA-50 apparatus. All samples were held in a platinum sample holder and were heated under air at the rate of 5 °C/min.

Results and Discussion Phase Transition of Vaterite CaCO3 Microcapsule to Calcite. CaCO3 microcapsules are prepared by our interfacial reaction method as described in the Experimental Section. As shown in Figure 1, these CaCO3 microcapsules have a spherical shape, which is a typical morphology of vaterite. The hollow structure was also ascertained from SEM observation (Figure 1B). The image of this CaCO3 microcapsule in high

4032

Crystal Growth & Design, Vol. 10, No. 9, 2010

Fujiwara et al.

Figure 2. Typical XRD patterns of a CaCO3 microcapsule (A) and CaCO3 after phase transition (B).

Figure 1. SEM images of CaCO3 microcapsules prepared by the interfacial reaction method.33

magnification (Figure 1C) and our previous paper33 showed that the shell part consisted of nanoparticles approximately 50 nm in size. The exclusive formation of the crystalline phase of vaterite was evidenced by XRD patterns shown in Figure 2A. No peak was detected about 29.5 in 2 theta, which is a typical strong peak of calcite (Figure 2B). Although various procedures for the preparation of vaterite CaCO3 are reported,42-45 our interfacial reaction method is also a simple and effective method. Furthermore, the interfacial reaction method can involve other components in the vaterite CaCO3 microcapsule prior to the phase transition process and might have different functions from other procedures. This CaCO3 microcapsule in the vaterite phase was soaked in some aqueous solutions. It is well-known that the phase transition from vaterite to calcite is strongly influenced by ions dissolving in aqueous solutions.36,37 After the still standing of these solutions with CaCO3 microcapsule at room temperature for from 96 to 140 h, the spherical morphology of the CaCO3

microcapsules changed to cubic ones with the increases of their sizes (Figure 3). Tris-HCl buffer solution, CaCl2 solution (0.2 M), normal saline solution (0.9% solution), and 1 M NaCl solution were effective for this phase transition. According to the XRD pattern of the phase transition CaCO3 sample in Tris-HCl solution, the crystalline phase shifted from vaterite to calcite completely (Figure 2B). CaCO3 in vaterite phase is generally more porous than that in the calcite phase.36-39 The CaCO3 microcapsule in vaterite phase we used in this study had a BET specific surface area of approximately 2.02 m2/g, which decreased to 0.61 m2/g after the phase transition equivalent to commercially available CaCO3 (0.42 m2/g). Each nitrogen sorption isotherm is illustrated in Figure S1 of the Supporting Information. Therefore, it is experimentally demonstrated that the CaCO3 microcapsules obtained by the interfacial reaction method also undergo the phase transition to calcite in aqueous solutions. The porosities of CaCO3 samples varied from their crystalline phases as well. The time variation of this phase transition in Tris-HCl solution was monitored with SEM and XRD after 24, 60, 96, and 140 h. SEM images and XRD patterns at the indicated elapsed times are summarized in Figures 4 and 5, respectively. As shown in these SEM images of Figure 4, the spherical particles gradually changed to cubic ones, indicating that the phase transition from vaterite to calcite occurred. This phase transition was also supported by the shift of XRD

Article

Crystal Growth & Design, Vol. 10, No. 9, 2010

4033

Figure 3. SEM images of CaCO3 microcapsules after soaking in (A) Tris-HCl buffer solution, (B) CaCl2 solution (0.2 M), (C) normal saline solution (0.9% solution), and (D) 1 M NaCl solution for 96-140 h at room temperature.

patterns in Figure 5. The peaks of vaterite, for example, at approximately 25, 27, and 33 in 2 theta, diminished progressively with the steady increase of calcite peaks at about 29.5 in 2 theta. After 140 h, the peaks of vaterite completely disappeared, and only the peaks of calcite were observed. An interesting feature of this phase transition is the formation of semispherical depressions in the phase transition calcite CaCO3 (Figure 4E). The form and the size of the semispherical depressions are likely to be similar to spherical vaterite particles. Figure 4F shows that a spherical particle, presumably vaterite, was stuck in a larger cubic particle which is considered as calcite. Although the reverse phase transition from calcite to vaterite was reported recently,46 the calcite CaCO3 obtained by the phase transition of vaterite CaCO3 microcapsule did not return to vaterite CaCO3 by the soaking in the Tris-HCl solution at room temperature for over 7 days. The participation of the reverse phase transition could be excluded in our phase transition processes. Therefore, the phase transition of the vaterite CaCO3 microcapsule takes place in suitable aqueous solutions by the concurrent progresses of the dissolution of vaterite and the precipitation of calcite without backward phase transition. Encapsulation of Lysozyme by Phase Transition. As mentioned in the introduction part, the direct encapsulation of lysozyme into CaCO3 microcapsule was unsuccessful by the interfacial reaction method, probably because the diffusion of smaller proteins from internal water phase to outer one is sufficiently faster than larger proteins.33 Then, we attempted the inclusion of lysozyme into CaCO3 using the phase transition of vaterite to calcite. CaCO3 microcapsule in vaterite phase (1 g) was added to an aqueous solution (1.5 mL) of Tris-HCl buffer dissolving lysozyme (10 mg), and the resulting mixture was left out at room temperature for approximately 190 h. The solid material obtained was thoroughly washed with deionized water to remove adsorbed compounds, and dried. A SEM image (Figure 6A) and the XRD pattern (Figure 6B) of this CaCO3 solid clearly showed that the vaterite CaCO3 microcapsule perfectly changed to calcite in the Tris-HCl and lysozyme solution. For comparison,

Figure 4. Morphology of CaCO3 during the phase transition after (A) 24 h, (B) 60 h, (C) 96 h, and (D) 140 h; (E) semispherical depressions observed in phase transition calcite CaCO3; (F) a spherical particle stuck in a larger cubic particle.

Figure 5. Variation of XRD patterns of CaCO3 during the phase transition in Tris-HCl solution after 0, 24, 60, 96, and 140 h.

similar experiments were examined by using a commercial calcite and the phase transition calcite CaCO3 from the microcapsule prepared above instead of the CaCO3 microcapsule. SEM images of the commercial calcite CaCO3 before and after this soaking experiment shown in Figure S2 indicated no significant change of CaCO3 particles. Figure 7 is the diffuse reflectance UV (DR-UV) spectra of these CaCO3 samples after the lysozyme treatment and the thorough water washing. The UV spectrum of an aqueous solution of lysozyme is inserted in the inset, where the absorption band around 280 nm in wavelength mainly attributed from tryptophan and tyrosine is obviously detected. In the spectra of the commercial calcite

4034

Crystal Growth & Design, Vol. 10, No. 9, 2010

Fujiwara et al.

Figure 7. DR-UV spectra of CaCO3 samples soaked in lysozyme solution; CaCO3 microcapsule (vaterite), the phase transition calcite CaCO3 from CaCO3 microcapsule (in Tris-HCl solution in lysozyme), and the commercially available CaCO3. In the inset, a typical absorption UV spectrum of lysozyme solution is shown.

Figure 6. SEM image (A) and XRD pattern (B) of the phase transition CaCO3 in Tris-HCl solution with lysozyme.

and the phase transition calcite CaCO3 after the lysozyme treatment, no absorption or little absorption at 280 nm was found, respectively. Although various CaCO3 materials including the amorphous one adsorb proteins,21,22 proteins simply adsorbed are readily eliminated from CaCO3 by thorough water washing. These easily removable properties of proteins from CaCO3 materials have been applied to the sustained release of proteins.21,22 In our cases, the thorough washing of the CaCO3 samples completely eliminated the simply adsorbed lysozyme. Therefore, lysozyme adsorbed on the commercial calcite CaCO3 and the phase transition calcite CaCO3 was effectively removed by the washing treatment, and no or little UV absorption of lysozyme was found in the corresponding DR-UV spectra. On the other hand, the phase transition CaCO3 from the microcapsule still held lysozyme even after the thorough washing. The absorption around 280 nm and the strong absorption below 230 nm in wavelength from lysozyme were clearly found as shown in Figure 7. It is obvious that lysozyme found in the phase transition CaCO3 after washing was not simply adsorbed onto the surface of the CaCO3 crystalline but was incorporated into the CaCO3 crystalline matrix. The recovered amount of lysozyme from the TrisHCl solution used for the phase transition treatment was

estimated to be approximately 82.7% by the UV absorption spectrum of the solution. Therefore, about 17.3% (1.73 mg) of lysozyme employed was encapsulated into the phase transition CaCO3. The weight ratio of lysozyme in the phase transition CaCO3 was about 0.20 wt % calculated from the total weight of the obtained CaCO3 (0.865 g). Thus, the encapsulation of lysozyme into CaCO3 succeeded using this phase transition method, while the direct inclusion by the interfacial reaction method is unsuccessful,33 because no UV absorption around 280 nm in wavelength was detected in the CaCO3 microcapsule prepared by the interfacial reaction method with lysozyme. (DR-UV spectra of the CaCO3 microcapsules by the direct encapsulation of various proteins are illustrated in Figure S3.33) On the other hand, a certain level of the encapsulation of lysozyme was also found after about 60 h of the soaking of the CaCO3 microcapsule into the lysozyme solution, where CaCO3 material was still predominantly in the vaterite phase with slight calcite. While the weight ratio of encapsulated lysozyme in this step was less than 0.02 wt % in the obtained CaCO3, the encapsulated lysozyme was not removed after thorough washing as well. Thus, although no perfect phase transition is essential for the encapsulation of the proteins, the phase transition from vaterite to calcite was a significant factor for the high amount of inclusion of proteins. The size of the CaCO3 particles increased significantly after the phase transition of vaterite CaCO3 microcapsule as shown in Figure 6, while no change of particle size was found in the case of the commercial calcite CaCO3 (Figure S2). The invariance of the crystalline structure of the calcite CaCO3 during the soaking probably resulted from the poor solubility of calcite CaCO3 into the solution. On the other hand, the high crystalline growth during the phase transition from vaterite to calcite meant that CaCO3 is dissolved and recrystallized continuously in the solution of lysozyme. It is likely that lysozyme is adsorbed on the surface of CaCO3 materials during this phase transition of vaterite CaCO3 microcapsule, and the crystalline growth of CaCO3 takes place even on the surface with adsorbed lysozyme to incorporate the lysozyme into CaCO3 crystalline matrix. Finally, the considerable

Article

Crystal Growth & Design, Vol. 10, No. 9, 2010

4035

Figure 8. DR-UV spectra of phase transition CaCO3 particles encapsulating BSA (A) and chicken IgY (B).

amount of lysozyme in the solution was encapsulated tightly into the phase transition calcite CaCO3. These lysozyme molecules implanted in the calcite crystalline matrix were not removed from the solid even by the thorough water washing. The release of the encapsulated lysozyme into the phase transition CaCO3 was attempted by the dissolution of the CaCO3 particle in an acid solution. The phosphate buffer solution (pH = 6.86) with the undissolved CaCO3 including lysozyme was acidized by the dropwise addition of HCl. The CaCO3 particle dissolved slowly with the decrease of pH value. When pH value of the solution reached about 3, the complete dissolution of the CaCO3 particle was ensured. UV spectrum of this acid solution (Figure S4) showed a broad absorption around 260-270 nm in wavelength. This absorption is considered as that of lysozyme in acidic solution. This kind of a short wavelength shift of the absorption from 280 nm to 260-270 nm was also found when a simple lysozyme solution was acidified to pH = 2.97 (Figure S4). It is clear that the dissolution of the CaCO3 particle released the lysozyme encapsulated in the phase transition CaCO3. From the UV absorption, the amount of the released lysozyme was calculated to be around 0.15-0.25 wt %, which is roughly consistent with the estimated amount of lysozyme included (0.20 wt %). Thus, the proteins encapsulated in CaCO3 solid matrices by this phase transition method are potential for a controlled release system triggered by the dissolution of the CaCO3 solid. Encapsulation of BSA and Chicken IgY by Phase Transition. Other proteins were also encapsulated by the same technique as lysozyme. BSA (bovine serum albumin: 66 400 Da) and chicken IgY were successfully included in the phase transition CaCO3. The CaCO3 microcapsule perfectly changed to calcite in the Tris-HCl buffer solution dissolving BSA after 170 h. A SEM image and a XRD pattern of this phase transition CaCO3 are illustrated in Figure S5. All the particles were cubic, and the XRD measurement showed a typical calcite peak pattern exclusively. The efficiency of the encapsulation of BSA into CaCO3 was estimated at 38% by the recovered amount of BSA in the used Tris-HCl solution. The weight ratio of BSA in the resulting CaCO3 particle was approximately 0.40 wt %. This weight ratio was also ascertained by the TGA analysis. The weight loss of this phase transition CaCO3 encapsulating BSA from 200 to 500 °C was approximately 0.481 wt %, which was basically consistent with the above estimated value (This TGA profile is illustrated in Figure S6.). Using this phase transition technique, chicken IgY as an antibody protein was also encapsulated in the CaCO3 particle. Figure S5 clearly indicated the complete phase transition from vaterite CaCO3 microcapsule to calcite one. The efficiency of encapsulation and the

Figure 9. Fluorescence (A) and bright-field (B) microscopy images of the phase transition CaCO3 with encapsulated FITC-BSA. The color images with fluorescence green light were converted to be monochrome for the clear indication of the bright parts and dark parts. The scale bar corresponds to 10 μm.

weight ratio in the CaCO3 material were estimated at 17.3% and 0.19 wt %, respectively. BSA and chicken IgY were certainly encapsulated in each calcite CaCO3 particle, based on DR-UV spectra as shown in Figure 8, where the UV absorptions around 280 nm in wavelength were obviously found. The encapsulation manner of proteins was analyzed by fluorescent microscopy experiments. For this fluorescent microscopy experiment, we added fluorescein isothiocyanate conjugate BSA (FITC-BSA) to common BSA, and a similar encapsulation procedure was carried out. All color microscope images with fluorescence green light were converted to black-and-white ones for the sake of distinguishing the bright green parts and dark parts clearly. In Figure S7 of the Supporting Information, a SEM image and a XRD pattern of this phase transition CaCO3 with fluorescein are shown. The encapsulation method using the phase transition of CaCO3 generally divided the proteins equally into the each phase transition particles from the low magnification images shown in Figure S8. Figure 9 illustrated the fluorescence microscope image and the bright-field microscope image at approximately the same place in high magnification. Although the whole part of the cubic crystal of CaCO3 appeared to be luminous, the fluorescent proteins were located around the outer edge of the particle. In particular,

4036

Crystal Growth & Design, Vol. 10, No. 9, 2010

Figure 10. DR-UV spectra of the phase transition CaCO3 particles by the recycling uses of insulin solutions.

the corner parts of the particles had a stronger fluorescent emission than other places. According to the expected mechanism of the phase transition, proteins are preferentially incorporated into the domains where the calcite crystalline phase is growing. The crystal growth of the calcite phase occurred at these sharp corner edges. Therefore, the proteins are encapsulated much more into the regions where the crystal growth proceeded well. These observations might suggest that the preloaded compounds by the interfacial reaction method and the proteins encapsulated by the phase transition could be included in different parts, central domains, and edge domains of the particles, respectively. These divided inclusions of two components will also create new applications of proteins. Encapsulation of Insulin by Phase Transition. As mentioned above, lysozyme as a comparatively small protein was encapsulated in the phase transition CaCO3, which was not successful in the interfacial reaction method.33 Then, we also attempted to encapsulate insulin as a much smaller protein into CaCO3 particles, because this polypeptide hormone has important bioactivity and pharmacological activities as well as larger proteins. Insulin was also successfully included into the CaCO3 particle by the phase transition method. The complete phase transition from vaterite CaCO3 microcapsule to calcite one occurred in the insulin solution as well. The efficiency of the encapsulation and the weight ratio in the CaCO3 particle were estimated at 14.9% and 0.16 wt %, respectively. This insufficient efficiency is expected to be enhanced by the recycling uses of recovered insulin solutions. The decanted and washing solutions of the phase transition CaCO3 particles were reutilized for the encapsulation process. The combined solution with an additional Tris-HCl solution was added to a fresh CaCO3 microcapsule. After the phase transition of the CaCO3 microcapsule, both solutions decanted and used for washing CaCO3 particles were recovered and reused for the phase transition again. The complete phase transitions of these three CaCO3 microcapsules were confirmed by the corresponding SEM images and XRD patterns illustrated in Figure S9. DR-UV spectra of these three phase transition CaCO3 particles are shown in Figure 10. Before the DR-UV analyses, these CaCO3 particles were thoroughly washed with fresh deionized water to remove simply adsorbed insulin. All three samples contained insulin from the absorption around 280 nm in wavelength. The amounts of the each recovered insulin were estimated from

Fujiwara et al.

the UV analyses of these washed solutions to be 20.7% (first encapsulation), 19.0% (second one), and 25.7% (third one). Therefore, the total amount of the encapsulated insulin into these three CaCO3 particles was 34.6% more than twice of insulin included by the single encapsulation process (14.9% as noted above). Thus, the encapsulation method by the phase transition had access to the recycling utilization of recovered solutions, which is not generally available in the interfacial reaction method and other processes. The phase transition of CaCO3 microcapsule proceeded effectively in various proteins solutions irrespective of the molecular weight as described, while some other watersoluble polymers were not suitable for this encapsulation technique. For example, when the aqueous solutions of polyacrylamide or sodium polyacrylate were used for the phase transition, only the slight phase transitions were observed even after 720 h (30 days) soaking. According to the infrared spectra of the CaCO3 particles used for these treatments, no polymers were included in the resulting CaCO3. These results indicated that some properties of the proteins contributed the phase transition of CaCO3. Further examinations to reveal the effects of proteins on the phase transition are still under investigation in our group. Conclusions The phase transition of vaterite CaCO3 microcapsules to calcite occurred effectively in various aqueous solutions. When some proteins were mixed in the solution for the phase transition, they were encapsulated into the phase transition CaCO3 particles. The encapsulation of proteins into CaCO3 particles were achieved regardless of their molecular weight. A smaller protein, lysozyme, which was not included by the interfacial reaction method using W/O/W emulsion, was also successfully encapsulated into the CaCO3 particles. Insulin as a much smaller protein was also introduced in CaCO3 particle after the phase transition. The dissolution of the CaCO3 particle with encapsulated lysozyme in acid solution released the protein to indicate the possibility of a responsive protein delivery technology. The encapsulation efficiency of insulin into CaCO3 particles was enhanced by the recycle uses of the insulin solution used for the experiment. Thus, it is found that the phase transition of vaterite CaCO3 microcapsule to calcite is a simple, general, and convenient method to encapsulate proteins into CaCO3 small particles, which is expected to enhance the usefulness of proteins in various applications. Acknowledgment. This research 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-5201). Authors also acknowledge the assistance of Prof. Takayo K. Moriuchi in Osaka Institute of Technology. Supporting Information Available: The nitrogen sorption isotherms of some representative CaCO3 samples, SEM images, XRD patterns, UV spectra, fluorescence microscope image, and TGA profile of some CaCO3 samples not shown in the main text are available free of charge via the Internet at http://pubs.acs.org/.

References (1) (2) (3) (4)

Xu, A.-W.; Ma, Y.; C€ olfen, H. J. Mater. Chem. 2007, 17, 415–449. Naka, K.; Chujo, Y. Chem. Mater. 2001, 13, 3245–3259. Kato, T.; Sugawara, A.; Hosoda, N. Adv. Mater. 2002, 14, 869–877. Colfen, H. Curr. Opin. Colloid Interface Sci. 2003, 8, 23–31.

Article (5) Meldrum, F. C. Int. Mater. Rev. 2003, 48, 187–224. (6) Morse, J. W.; Arvidson, R. S.; Luttge, A. Chem. Rev. 2007, 107, 342–381. (7) Arias, J. L.; Fernandez, M. S. Chem. Rev. 2008, 108, 4475–4482. (8) Sommerdijk, N. A. J. M.; de With, G. Chem. Rev. 2008, 108, 4499–4550. (9) Xu, A.-W.; Dong, W.-F.; Antonietti, M.; C€ olfen, H. Adv. Funct. Mater. 2008, 18, 1307–1313. (10) Vallet-Regi, M.; Gonzalez-Calbet, J. M. Prog. Solid State Chem. 2004, 32, 1–31. (11) Cai, Y. R.; Tang, R. K. J. Mater. Chem. 2008, 18, 3775–3787. (12) Wang, Y. J.; Price, A. D.; Caruso, F. J. Mater. Chem. 2009, 19, 6451–6464. (13) Fakhrullin, R. F.; Minullina, R. T. Langmuir 2009, 25, 6617–6621. (14) Kim, I. W.; Robertson, R. E.; Zand, R. Cryst. Growth Des. 2005, 5, 513–522. (15) Ikoma, T.; Tonegawa, T.; Watanaba, H.; Chen, G. P.; Tanaka, J.; Mizushima, Y. J. Nanosci. Nanotechnol. 2007, 7, 822–827. (16) Liu, D. G.; Tian, H. F.; Jia, X. W.; Zhang, L. N. Macromol. Biosci. 2008, 8, 401–409. (17) Wang, X. Q.; Kong, R.; Pan, X. X.; Xu, H.; Xia, D. H.; Shan, H. H.; Lu, J. R. J. Phys. Chem. B 2009, 113, 8975–8982. (18) Njegic-Dzakula, B.; Brecevic, L.; Falini, G.; Kralj, D. Cryst. Growth Des. 2009, 9, 2425–2434. (19) Wang, X. Q.; Sun, H. L.; Xia, Y. Q.; Chen, C. X.; Xu, H.; Shan, H. H.; Lu, J. R. J. Colloid Interface Sci. 2009, 332, 96–103. (20) Seul, S. D.; Lee, S. R.; Kim, Y. H. J. Polym. Sci. A-Polym. Chem. 2004, 42, 4063–4073. (21) Volodkin, D. V.; Larionova, N. I.; Sukhorukov, G. B. Biomacromolecules 2004, 5, 1962–1972. (22) Sukhorukov, G. B.; Volodkin, D. V.; Gunther, A. M.; Petrov, A. I.; Shenoy, D. B.; Mohwald, H. J. Mater. Chem. 2004, 14, 2073–2081. (23) Ueno, Y.; Futagawa, H.; Takagi, Y. J. Controlled Release 2005, 103, 93–98. (24) Petrov, A. I.; Volodkin, D. V.; Sukhorukov, G. B. Biotechnol. Prog. 2005, 21, 918–925. (25) Wang, C. Y.; He, C. Y.; Tong, Z.; Liu, X. X.; Ren, B. Y.; Zeng, F. Int. J. Pharm. 2006, 308, 160–167. (26) Gill, I. Chem. Mater. 2001, 13, 3404–3421. (27) Avnir, D.; Coradin, T.; Lev, O.; Livage, J. J. Mater. Chem. 2006, 16, 1013–1030.

Crystal Growth & Design, Vol. 10, No. 9, 2010

4037

(28) Coradin, T.; Livage, J. Acc. Chem. Res. 2007, 40, 819–826. (29) de Vos, P.; Bucko, M.; Gemeiner, P.; Navratil, M.; Svitel, J.; Faas, M.; Strand, B. L.; Skjak-Braek, G.; Morch, Y. A.; Vikartovska, A.; Lacik, I.; Kollarikova, G.; Orive, G.; Poncelet, D.; Pedraz, J. L.; Ansorge-Schumacher, M. B. Biomaterials 2009, 30, 2559–2570. (30) Fujiwara, M.; Shiokawa, K.; Tanaka, Y.; Nakahara, Y. Chem. Mater. 2004, 16, 5420–5426. (31) Fujiwara, M.; Shiokawa, K.; Sakakura, I.; Nakahara, Y. Nano Lett. 2006, 6, 2925–2928. (32) Nakahara, Y.; Mizuguchi, M.; Miyata, K. J. Colloid Interface Sci. 1979, 68, 401–407. (33) Fujiwara, M.; Shiokawa, K.; Morigaki, K.; Zhu, Y.; Nakahara, Y. Chem. Eng. J. 2008, 137, 14–22. (34) Fujiwara, M.; Shiokawa, K.; Hayashi, K.; Morigaki, K.; Nakahara, Y. J. Biomed. Mater. Res. A 2007, 81, 103–112. (35) Fujiwara, M.; Shiokawa, K.; Morigaki, K.; Tatsu, Y.; Nakahara, Y. Mater. Sci. Eng., C 2008, 28, 280–288. (36) Wolf, G.; Konigsberger, E.; Schmidt, H. G.; Konigsberger, L. C.; Gamsjager, H. J. Therm. Anal. Calorim. 2000, 60, 463–472. (37) Berdonosov, S. S.; Znamenskaya, I. V.; Melikhov, I. V. Inorg. Mater. 2005, 41, 1308–1312. (38) Nan, Z. D.; Chen, X. N.; Yang, Q. Q.; Wang, X. Z.; Shi, Z. Y.; Hou, W. G. J. Colloid Interface Sci. 2008, 325, 331–336. (39) Nassrallah-Aboukaı¨ s, N.; Boughriet, A.; Laureyns, J.; A. Aboukaı¨ s, A.; Fischer, J. C.; Langelin, H. R.; Wartel, M. Chem. Mater. 1998, 10, 238–243. (40) Hadiko, G.; Han, Y.-S.; Fuji, M.; Takahashi, M. Mater. Lett. 2005, 59, 2519–2522. (41) Suzuki, M.; Nagasawa, H.; Kogure, T. Cryst. Growth Des. 2006, 6, 2004–2006. (42) Li, W.; Gao, C. Langmuir 2007, 23, 4575–4582. (43) Kasuga, T.; Maeda, H.; Kato, K.; Nogami, M.; Hata, K.; Ueda, M. Biomaterials 2003, 24, 3247–3253. (44) Han, Y.-S.; Hadiko, G.; Fuji, M.; Takahashi, M. J. Cryst. Growth 2006, 289, 269–274. (45) Mori, Y.; Enomae, T.; Isogai, A. Mater. Sci. Eng., C 2009, 29, 1409–1414. (46) Wu, Q.-S.; Sun, D.-M.; Liu, H.-J.; Ding, Y.-P. Cryst. Growth Des. 2004, 4, 717–720.