Synthesis of Mesoporous Calcium Phosphate Microspheres by

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Synthesis of Mesoporous Calcium Phosphate Microspheres by Chemical Transformation Process: Their Stability and Encapsulation of Carboxymethyl Chitosan Cong Sui, Yang Lu, Huai-Ling Gao, Liang Dong, Yang Zhao, Lahoussine Ouali, Daniel Benczédi, Huda Jerri, and Shu-Hong Yu Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/cg400595s • Publication Date (Web): 03 Jun 2013 Downloaded from http://pubs.acs.org on June 3, 2013

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Crystal Growth & Design

Synthesis of Mesoporous Calcium Phosphate Microspheres by Chemical Transformation Process: Their Stability and Encapsulation of Carboxymethyl Chitosan Cong Sui,† Yang Lu,† Huai-Ling Gao,† Liang Dong,† Yang Zhao,† Lahoussine Ouali,‡ Daniel Benczédi,‡ Huda Jerri,‡ Shu-Hong Yu†*

†

Division of Nanomaterials and Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, Department of Chemistry, University of Science and Technology of China, Hefei 230026, Anhui, P. R. China ‡

Corporate R&D Division, Firmenich SA, P.O. Box 239, CH-1211 Geneva 8, Switzerland

Correspondence should be addressed to S. H. Yu, Fax: +86 551 3603040, E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: We report on the transformation of calcium carbonate microparticles (CCMs) to calcium phosphate microparticles (CPMs) with modulated morphologies and phases. In this additive-free transformation process, the use of templates or surfactants was circumvented, thereby eliminating potential contamination of the final products. The hydroxycarbonate apatite (HCAP) microspheres in high yield were more stable than CaCO3 microspheres in aqueous solution and had higher specific surface areas, which suggested that they had higher loading capability than CaCO3 counterpart. In addition, the products had good biocompatibility because they were free from extraneous surfactants or stabilizers and hence, did not require further purification. In order to examine the loading efficiency of these microspheres, carboxymethyl chitosan (CMC) and doxorubicin which were both excellent biomedical materials, were taken as model high molecular weight and low molecular weight probes, respectively, to investigate the encapsulation capacity of CPMs. The CPMs showed high encapsulation efficiency for both molecules, with an impressive 40% loading efficiency of the adsorbed CMC biomacromolecules in the porous microparticles.

INTRODUCTION Encapsulation technologies have attracted much attention due to an increasing interest in diverse areas from biotechnology, tissue engineering to catalysis and synthetic chemistry. Calcium carbonate (CaCO3) is a fascinating material with excellent biocompatibility1, which has been used successfully for the encapsulation of molecules ranging from biomolecules (such as polysaccharides, proteins, enzymes) to small molecules (such as green tea extract) into calcium carbonate microparticles (CCMs).2 There are two different ways to load biomacromolecules into CCMs, namely physical adsorption and co-precipitation. As for CCMs, the loading efficiency of the co-precipitation has been found to be about five times higher than that of the physical adsorption, but the CCMs obtained by co-precipitation method did not have regular shapes.3 Since the CCMs 2


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are rather unstable in aqueous solution, they will transform into calcite entirely overnight.2a In this investigation, CaCO3 was used as a template to synthesize mesoporous structures with fixed crystalline phases.2-4 Calcium phosphate-based bioceramics have excellent biocompatibility and bioactivity due to their similarity to our human bone. It has been widely used as bone cavity filling materials and drug delivery systems.5 Hydroxyapatite (HAP) formed from natural inorganic skeletons by hydrothermal conversion has been studied since 1974.6 The CCMs can be transformed into calcium phosphate microparticles (CPMs) by dissolution-precipitation reaction at low temperature,7 and it can be converted to HAP completely by an ion-exchange reaction within a temperature range of 140-260 o

C.6,8 Guo et al converted calcium carbonate microparticles (CCMs) into hydroxycarbonate apatite

(HCAP) by treatment with phosphate buffer solution (PBS) at low temperatures.9 Since the synthesis of silica-based MCM-41 molecular sieves was first discovered in 1992, these mesoporous materials have received much attention.10 Mesoporous materials can be synthesized by using templates or surfactants like cetyltrimethylammonium bromide (CTAB),11 mono-n-dodecyl phosphate (MDP),12 and some block copolymers.13 The disadvantage of this approach remains the use of calcinations to get rid of the templates which could damage part of the mesoporous structure and decrease the BET surface area. Moreover, the remaining surfactants would contaminate the final products. However, the morphologies obtained so far with CPMs transformed from CCMs are irregular when no surfactant is used as a template.9 In this Article, we present a simple strategy to transform CCMs into CPMs via both a dissolution-precipitation reaction and an ion-exchange reaction without templates or surfactants. The CCMs were treated in a (NH4)2HPO4 solution at different temperatures, resulting in the formation of mesoporous CPMs with high BET surface areas. The resulting mesoporous CPMs are chemically very similar to biological apatite, they are more stable than CCMs and have much higher 3



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loading efficiency (~400 µg of captured protein per 1 mg of CPMs) than CCMs obtained by co-precipitation method (~100 µg of captured protein per 1 mg of CCMs).3 In addition, the obtained CPMs were regular spheres with mesoporous structures free from contamination due to the absence of any templates or surfactants. The loading efficiency of these microspheres for carboxymethyl chitosan (CMC) and doxorubicin has also been investigated.

EXPERIMENTAL SECTION Chemicals. Fluorescein isothiocyanate (FITC) was purchased from Sangon Biotech (Shanghai) Co., Ltd. Chitosan from crab shells was purchased from Bellancom Life Sciences Dep. Chloroacetic acid was bought from Tianjin Guangfu fine chemical research institute. Doxorubicin hydrochloride was obtained from Sangon Biotech (Shanghai) Co., Ltd. Calcium chloride (CaCl2), anhydrous sodium carbonate (Na2CO3), ammonium phosphate dibasic ((NH4)2HPO4), sodium hydroxide (NaOH) and 37% hydrochloric acid were obtained from Sinopharm Chemical Reagent Co. Ltd. (SCRC, China). All chemicals were of analytical grade and were used without further purification. Synthesis of Calcium Carbonate Microparticles (CCMs). 0.11 M, 0.33 M, 1 M, 3 M Na2CO3 solutions were rapidly poured into an equal volume of 0.11 M, 0.33 M, 1 M, 3 M CaCl2 solutions at room temperature respectively, and after intense agitation with a magnetic stirrer, the precipitate was filtered off, washed with deionized water (DIW) and ethanol, and then dried in the air. Conversion of Calcium Carbonate Microparticles (CCMs) into Calcium Phosphate Microparticles (CPMs). 2 g (NH4)2HPO4 were dissolved in 10 ml DIW. Then 2.5 g of calcium carbonate powder were added into this (NH4)2HPO4 solution. Subsequently, the calcium carbonate powder was treated for 48 h at different temperatures. The precipitate was filtered off, thoroughly washed with DIW, and dried in the air.

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Preparation of FITC-CMC. Carboxymethyl chitosan (CMC) was prepared according to the previously reported method.14 Typically, 10.0 g of chitosan and 13.5 g of sodium hydroxide, were added into 100 ml DIW, and reacted for 1 h. The temperature was maintained in a water bath. 15.0 g of monochloroacetic acid was dissolved in 20 ml of isopropanol, and added dropwise into the above described chitosan solution during 30 min and reacted for 4 h at the same temperature. The reaction was terminated by adding 70%(v/v) ethyl alcohol. The solid was filtered, rinsed and vacuum dried at room temperature. 1.0 g of the resulted product was dissolved in 100 ml 80%(v/v) ethyl alcohol, and then 10 ml of 37% hydrochloric acid were added to this solution and stirred for 30 min. The precipitate was filtered and rinsed in 70–90%(v/v) ethyl alcohol to neutral, and then vacuum dried. The products were CMC. 200 mg of CMC and 2 mg of FITC were dissolved in 20 ml of DIW, and then stirred in the dark at room temperature. The precipitate we obtained was FITC-CMC. Characterization. The morphologies of the as-prepared product were observed with SEM (JSM-6700F) and TEM (JEOL-2010). Powder X-ray diffraction (XRD) analysis was performed on a Philips X' Pert PRO SUPER X-ray diffractometer with Cu Kα (λ = 1.54056 Å). FTIR spectra were measured on a Bruker Vector-22 FTIR spectrometer from 4000 to 400 cm-1 at room temperature. Fluorescence microscopy images were measured on a fluorescence microscope (IX71, Olympus). The loading efficiency of FITC-CMC was measured with the FITC-CMC in the upper solution obtained by centrifugation. The fluorescence of FITC-CMC was investigated on a fluorescence spectrophotometer (F-7000, Hitachi), excited at 492 nm. The BET surface area was quantified by measuring N2 adsorption-desorption isotherms, using an accelerated surface area and a porosimetry instrument (ASAP 2000).

RESULTS AND DISCUSSION 5



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Synthesis of Calcium Carbonate Microparticles (CCMs) with Different Sizes. Porous CCMs were synthesized by the traditional method.15 As Ca2+ and CO32− ions were mixed together rapidly, they precipitated immediately. The XRD results demonstrate that the precipitate, CaCO3, is composed of a mixture of vaterite and calcite, in which the vaterite content increases with increasing the concentration of the Ca2+ and CO32− ions. However, at high concentration of Ca2+ and CO32− (3 M), all CCMs were transformed from spherical vaterite into rhombic calcites (Figure 1f). The SEM images show that all precipitates are CCMs with porous structures. 0.11 M and 0.33 M CaCl2 and Na2CO3 solutions were used as initial reactants, and homogeneous, spherical CCMs were obtained (Figure 1a, b). There was some rhombohedral calcite mixed in the vaterite microspheres in the CCMs obtained at lower concentration (0.11 M) (Figure 1a). However, the number of rhombohedral calcite particles obtained at a higher concentration of reactants (0.33 M) clearly decreased as shown in Figure 1b. In general, at low concentration of Ca2+ and CO32− (0.1 M-1 M), the CCMs are spherical. An increase in the concentration of initial reactants lead to CCMs with irregular morphology and the particle size became smaller (Figure 1c, d). An optimum was found at 0.33 M with CCMs of regular shape and a large output. The broken part of CaCO3 obtained by 0.33 M initial reactant shows that the microparticles have porous structures which are composed of a great number of small primary CaCO3 nanoparticles (Figure 1e).

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Figure 1. SEM images of obtained CaCO3 at different concentrations of Ca2+ and CO32-. (a) 0.11 M; (b) 0.33 M; (c) 1 M; (d) 3 M; (e) TEM images of broken part of CaCO3; (f) XRD pattern of CaCO3 obtained at different concentrations of Ca2+ and CO32-. (A) 0.11 M; (B) 0.33 M; (C) 1 M; (D) 3 M. Note: #, vaterite; ^, calcite. Transformation of CCMs into CPMs. When CaCO3 particles are treated in (NH4)2HPO4 solution, the Ca2+ ions are released from the surface of the particles. Ca2+ can then react with PO43and HPO42- to form HCAP by a dissolution-precipitation reaction7 which is influenced by immersion time and temperatures.9 CaCO3 can be completely converted into HAP by an ion-exchange reaction in the temperature range of 140-260 oC.6,8 The starting concentration of (NH4)2HPO4 solution ranged from 0.12 to 0.6 g ml-1 with an optimum value found at 0.2 g ml-1,16 and a Ca/P ratio of 1.67.17 In the present work, CaCO3 was transformed into calcium phosphate in (NH4)2HPO4 solutions at different temperatures. The temperature has a great influence on the phase and morphology of calcium phosphate converted from calcium carbonate. Here, calcium carbonate was converted to calcium phosphate after soaking in a (NH4)2HPO4 solution at 10 oC without any additives. The XRD pattern shows that only a small amount of CaCO3 was converted to HCAP, because the temperature was not 7



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sufficiently high. The content of calcium phosphate increased while the calcium carbonate content decreased with the increasing temperature. CaCO3 is more likely to transform into calcite at 37 oC, because the temperature does not provide sufficient energy for CaCO3 to form calcium phosphate, and the CaCO3 is less stable at higher temperatures. At 50 oC, HCAP is the main phase and the conversion of CaCO3 into HCAP is complete at 65 oC. The broad bands are due to a defective, low crystallinity of the formed apatite. As shown in Figure 2a, HCAP with better crystallinity was obtained at 80 oC and 160 oC. The XRD patterns shown in this study evidenced an increase of the average coherent domain size of HCAP from nano-scale to micro-scale with increasing temperature. The FTIR spectrum in Figure 2b shows the absorption bands at 564, 602 and 1040 cm-1 corresponding to PO43- ions, and the band at around 1090 cm-1 corresponding to HPO42- ions, indicating the product is calcium deficient apatite.18 OH- and PO43- in apatite lattice can be substituted by CO32- ions, namely A-type and B-type substitute respectively. Figure 2b shows the characteristic bands of B-type CO32- substitution at 1415 cm-1/1455 cm-1, and at 872 cm-1, and also the disappearance of band at 630 cm-1 demonstrates that it contain A-type substitute.19 The B-type CO32- substitution is covered by the bands of unreacted CaCO3 at 1437 cm-1 and 1491 cm-1 under low temperature. The bands of CO32- ion at around 1415 cm-1/1455 cm-1 can be clearly distinguished when the temperature reached 65 oC, and it decreased with the temperature increased. It illustrates that the CO32- ion can be partially reduced by treated with higher temperature.

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As the temperature increased, these microparticles are converted into villous microspheres. As reported elsewhere,9, 11 CPM with irregular shape can be obtained from CCM at 37 oC with regular shapes observed when adding CTAB as surfactant. Our SEM images show that all particles kept their spherical shapes when formed in the absence of any additives, and the XRD patterns show that the resulting CPMs are HCAP. The broken microparticle of HCAP (65 oC) shown in Figure 3f has a relatively dense texture and does not have large channels or cavities. Figure 4d shows that the microparticles obtained at 160 oC have needle shape structures on their surfaces. The XRD pattern shows no difference between 80 oC and 160 oC, but the SEM images of the broken particle show that the inner part of the HCAP microsphere is structured more loosely with more channels at 160 o

C than at 80 oC (Figure 4b, e). The TEM images illustrate that the HCAP (80 oC) is made of

slice-like platelets (Figure 4c), and the HCAP (160 oC) is made of small acicular, needle-like particles (Figure 4f). Thus, it indicates that HCAP obtained under 160 oC are structured more loosely, and also it can be concluded that the resulting microspheres at higher temperature tend to have looser structure. The high-resolution TEM image of the slice-like apatite platelets shows a characteristic lattice fringe of the well-crystallized apatite (Figure 4c). The selected area electron diffraction (SAED) pattern clearly indicates visible diffraction rings, whose interplanar spacings are in good agreement with the characteristic spacings of apatite (Figure 5). Our CCMs were converted into CPMs by dissolution-precipitation reaction below 80 oC, but by ion-exchange reaction at 160 oC. The CCM is made of plenty of small nanoparticles (Figure 1e), and the HCAP (160 oC) microspheres are composed of small nanoparticles (Figure 4f) whose shapes are similar with CCM nanoparticles. Therefore, the HCAP (160 oC) maintains its original shape, and the conversion mechanism should be through an ion-exchange process.6 However, the HCAP microspheres obtained below 80 oC consist of slice-like platelets, which means that the platelets of HCAP were not derived from the original material, but from the aggregation of nanoparticles. In 10


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addition, the obvious differences in morphologies between CCM and HCAP (80 oC) demonstrate that the formation mechanism of HCAP at temperatures below 80 oC is not by an ion exchange reaction, but the dissolution-precipitation reaction.7

Figure 3. SEM images of HCAP microparticles obtained from CCMs at different temperatures: (a) 10 oC; (b) room temperature; (c) 37 oC; (d) 50 oC; (e) 65 oC; (f) the broken part of a microparticle obtained at 65 oC.

Figure 4. SEM images of HCAP microparticles obtained from CCMs at different temperature: (a) 80 oC; (d) 160 oC; the broken part of a microparticle obtained at (b) 80 oC; (e) 160 oC; the TEM images of the broken part of HCAP microparticles at (c) 80 oC and (f) 160 oC. The inset in (c) shows a HR-TEM image of the broken part of HCAP microparticles obtained from CCMs under 80 oC 11



Figure 5. The SAED pattern taken on the outer layer of a HCAP (10 oC) microsphere shown in Figure 4c.

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Wavenumber (cm ) Figure 6. (a) XRD patterns and (b) FT-IR spectra of HAP-converted CaCO3 prepared at 80 oC with different reaction times. Note: #, vaterite; ^, calcite; *, apatite. Prolonging the immersion time will increase the conversion rate.9 Figure 6a shows the effect of different reaction times on the XRD pattern of HAP-converted CaCO3 prepared in (NH4)2HPO4 solution at 80 oC. The products were obviously transformed into HCAP after half an hour, with most of the vaterite converted to HCAP and still many calcite particles remaining. At longer reaction times, more CaCO3 was converted to HCAP, as shown by the main peak of calcite which is obviously smaller than the one observed at around 30°. It has been reported previously that calcite can be converted to HAP with additives such as CTAB11 and dopamine20. In the present research, all CaCO3 particles including calcite were converted to HCAP during 6 h without any additive. With reaction times above 36 h, the HCAP microparticle with better crystallinity was obtained. The FT-IR spectrum in Figure 6b shows that the absorption bands at 744 and 877 cm-1 correspond to vaterite and 712/877 cm-1 correspond to calcite.21 The bands of PO43- at 564, 602 and 1040 cm-1 indicate the presence of HCAP after soaking in (NH4)2HPO4 solution for 1 h.18 The bands 13



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of calcite disappeared by treatment with (NH4)2HPO4 solution for 6 h, meanwhile, the bands of CO32- can be clearly seen. The FT-IR result just meets the XRD pattern that HCAP was fully converted from CaCO3 at 6 h. The bands of CO32- were decreased with the soaking time prolonged. It can be clearly seen that the bands of OH- at 630 and 3440 cm-1 appeared when the soaking time up to 3 days. It means that the A-type CO32- substitution disappeared. Also, the bands of CO32- at 1415, 1455 and 872 cm-1 disappeared illustrates that the B-type CO32- substitution vanished.19 Therefore, HAP (hydroxyapatite) was fully converted from HCAP by treatment with (NH4)2HPO4 solution for more than 3 days under 80 oC. Stability of the CCMs and CPMs. The stability of CCM and CPM shows great differences in water. CaCO3 is very unstable in water. When the highly supersaturated solutions of Ca2+ and CO32− ions are rapidly mixed together, they precipitate quickly, and the resulted product is amorphous calcium carbonate, which is very unstable. The amorphous precipitates will transform into a more stable form composed of vaterite and calcite, and finally convert completely into the most stable calcite form.22 When CaCO3 was set in DIW for 3 h, only half of the amount of CaCO3 was converted to calcite whereas all of the CaCO3 was converted to calcite when setting in DIW for 2 days (Figure 7a). However, the HCAP (obtained at 160 oC) was very stable in DIW, and has not changed for a year (Figure 7b). Therefore, the obtained CPM was more stable than CCM, and may be a better candidate than CCM for application in aqueous solution.

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2 theta (degree) Figure 7. The stability of (a) CCMs and (b) HCAP microparticles (obtained at 160 oC) settled in DIW for different times. Note: #, vaterite; ^, calcite. BET Analysis. The BET analysis also shows a great difference between CCM and CPM. It reveals a higher surface area for CPMs than CCMs, as suggested by the SEMs where the CPMs are structured more loosely than the CCMs, with values decreasing at higher temperature (Table 1). Also, a pore size distribution from 10 to 30 nm illustrates that the obtained HCAP microspheres are 15



suitable for macromolecules adsorption (Figure 8). Thus, the CPMs converted from CCMs may have higher encapsulation ability because the higher BET value means higher surface area for the adsorption of macromolecules. The CPMs obtained in this study were more stable than CCMs and had a higher surface area, suggesting that they were better suited for an application as delivery systems or in some other fields of research. Table 1. BET Surface Area of CCM and CPM. BET Surface Area (m²·g-1)

Sample CaCO3

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0.0020 0.0015 0.0010 0.0005 0.0000 0

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Pore Diameter (nm) Figure 8. Pore size distribution of HCAP prepared at 80 oC. Adsorption of FITC-CMC by CPMs. The CPMs transformed from CCMs have a higher surface area, and they have many very large channels with negative charges.23 Thus, it may be a good carrier to encapsulate macromolecules with positive charge. In our work, carboxymethyl chitosan (CMC) was used as a model to investigate the encapsulation capability of CPM.

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In order to increase the water-solubility of chitosan, it was modified by carboxymethyl group, and CMC was labeled by FITC to detect the loading efficiency of CMC more conveniently. The rate of FITC connected onto CMC is not high, because some of the amino groups of the chitosan are occupied by carboxymethyl groups. Thus, there are fewer amino groups to bind with the isothiocyanate group of FITC (2.55 µg of FITC per 1 mg FITC-CMC). The CPM we obtained reveals a high ability to encapsulate macromolecules. As it is shown in the obtained fluorescent images (Figure 9), FITC-CMC is adsorbed into the CPM, and the loading capacity of CPM is high. The loading efficiencies achieved were of 40.23% in the case of HCAP (65 oC) with 402 µg of FITC-CMC per 1 mg of HCAP, and of 37.63% and 36.78% in the case of HCAP (80 oC) and HCAP (160 oC), respectively. This is much higher than the 20 µg of captured protein per 1 mg of CCMs,3 and is consistent with the results of the BET analysis of CPM (Table 1). This high loading capacity is probably due to electrostatic interaction. Meanwhile, the porous structure has a great influence on the encapsulation efficiency in the loading process. When CPMs were treated with phosphate buffer solution (PBS) at different pH conditions, an increase of the encapsulation efficiency was observed at lower pH (Figure 10).

Figure 9. Fluorescent images of (a) HCAP (65 oC); (b) HCAP (80 oC) and (c) HCAP (160 oC) microspheres absorbed FITC-CMC. Incubation time is 12 h.

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4 00 300 200 1 00

pH

c

7.4

0

FITC-CMC

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absorbed (µ g)


b pH

8

a

Figure 10. Adsorption of FITC-CMC per 1 mg CPMs (a) HCAP (65 oC); (b) HCAP (80 oC) and (c) HCAP (160 oC) at different pH values of PBS. Incubation time is 12 h. The amount of FITC-CMC adsorbed in CPMs at pH 7.4 (physiological condition) was higher than that at pH 8.0, due to the stronger interactions between FITC-CMC and CPMs. The CPMs were not treated with PBS at pH 6 because of the poor stability of CMC at lower pH values.24 At pH 7.4, a loading efficiency of 42.73% was achieved with HCAP (65 oC) with 427 µg of FITC-CMC per 1 mg of HCAP, while 42.68% and 42.29%, were achieved with HCAP (80 oC) and HCAP (160 oC), respectively. However, at pH 8, 37.0% loading was achieved with HCAP particles (65 oC) with 370 µg of FITC-CMC per mg of HCAP, while 36.69% and 36.31% were achieved with HCAP (80 oC) and HCAP (160 oC), respectively (Figure 10). Therefore, the CPM is a better and more stable carrier than CCM in many aspects. When loading CPMs with small molecules such as doxorubicin (DOX), a positively charged chemotherapeutic agent, a loading efficiency of 28% was achieved (WCPMs/WDOX = 1/2), demonstrating the effect of molecular dimensions beyond that of the charge. CPMs can adsorb both molecules and macromolecules with a positive charge, because they have many large tortuous channels or cavities. Small molecules can be easily adsorbed but they can escape with the same ease. 18


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CONCLUSIONS In summary, we have developed a simple and inexpensive approach to synthesize a large amount of mesoporous CPMs with regular shapes and a high ability to encapsulate positively charged biomacromolecules. The calcium carbonate microparticles (CCMs) can be converted into mesoporous calcium phosphate microparticles (CPMs) with different phases and morphologies without using any templates or surfactants. The FITC-CMC was used as a model to investigate the encapsulation capability of CPMs. The results demonstrate that the HCAP (65 oC) showed very high loading capacity of positively charged biomacromolecules with 402 µg of FITC-CMC per 1 mg of HCAP, which is much higher than the 20 µg of protein captured per 1 mg of CCMs. In addition, the obtained CPMs are chemically similar to biological apatite, which can be potentially used as delivery carriers, bone reconstruction materials and for other applications.

ACKNOWLEDGEMENTS. This work is supported by the National Basic Research Program of China (Grant 2010CB934700), the National Natural Science Foundation of China (Grants 91022032, 91227103, 21061160492), the International Science & Technology Cooperation Program of China (Grant 2010DFA41170), the Principal Investigator Award by the National Synchrotron Radiation Laboratory at the University of Science and Technology of China. Firmenich SA, Geneve, Switzerland is especially thanked for funding this joint research project.

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For Table of Contents Use Only A high yield additive-free transformation process has been reported for the transformation of calcium carbonate microparticles (CCMs) to calcium phosphate microparticles (CPMs) with modulated morphologies and phases. The loading efficiency of these microspheres for carboxymethyl chitosan (CMC) and doxorubicin has been investigated.

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2x1mm (600 x 600 DPI)


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Synthesis of Mesoporous Calcium Phosphate Microspheres by

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