Calcium CarbonateâMagnetiteâChondroitin Sulfate Composite

Article pubs.acs.org/crystal

Calcium Carbonate−Magnetite−Chondroitin Sulfate Composite Microparticles with Enhanced pH Stability and Superparamagnetic Properties Marcela Mihai,†,* Vlad Socoliuc,‡ Florica Doroftei,† Elena-Laura Ursu,† Magdalena Aflori,† Ladislau Vekas,‡ and Bogdan C. Simionescu†,§ †

“Petru Poni” Institute of Macromolecular Chemistry of Romanian Academy, 41A Grigore Ghica Voda Alley, 700487 Iasi, Romania Laboratory of Magnetic Fluids, Center for Fundamental and Advanced Technical Research, Romanian Academy - Timisoara Branch, 24 Bd. Mihai Viteazul, 300223 Timisoara, Romania § Department of Natural and Synthetic Polymers, “Gh. Asachi” Technical University, 73 Prof. Dimitrie Mangeron Street, 700050 Iasi, Romania ‡

S Supporting Information *

ABSTRACT: The CaCO3 microparticle growth from supersaturated aqueous solutions in the presence of oleic acid stabilized magnetite nanoparticles as a water-based magnetic fluid and a natural strong−weak polyanion, chondroitin sulfate A, has been investigated. The study follows the microparticle formation characteristics under different relative CaCO3−magnetite−polymer ratios. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to provide high resolution on particle morphology and distribution of magnetite in the composites and flow particle image analysis (FPIA) to evidence their mean size and circularity, whereas X-ray diffraction (XRD) and Raman spectroscopy were used to determine polymorph type and crystallite characteristics. Polymer and magnetite presence in the composite particles was evidenced by energy dispersive X-ray (EDX), TEM, particle charge density, and ζ potential. The magnetic properties of the obtained microparticles were also investigated. The pH stability of the new composites, given by the presence of acid oleic stabilized magnetite nanoparticles and polymer, has been followed by ζ potential variation.

1. INTRODUCTION Calcium carbonate is a common biomineral with a unique combination of properties, which include pH-sensitive decomposition, nontoxicity, biocompatibility, and low cost, and thus it is considered as a potential delivery vehicle for compounds such as drugs and proteins.1,2 Extensive attention has been paid to its mineralization mechanism and biomimetic synthesis,3,4 as a result of its wide application, such as filler in pigments, paper, rubber, and plastics industry.5,6 Development and optimization of such applications depend on the strategies controlling the structure of CaCO3 particles, in particular controlling the polymorph content, and on the pathways introducing functionality through the formation of nanocomposite structures. Coprecipitation with different excipients, such as divalent cations, organic solvents, and macromolecules (natural or synthetic), has a strong effect on the morphology of the formed CaCO3-based materials.7−12 The presence of polymeric additives can lead to the modification of the size and shape of the primary nanoparticles, as well as the number of the produced particles. Moreover, the selectivity in polymorph formation can be affected by the polymeric additives depending on the level of interactions of the additive © XXXX American Chemical Society

and calcium carbonate precursors. Generally speaking, the polymers containing carboxylate groups may act as inhibitors of CaCO3 crystal growth.13,14 A variety of macromolecular additives, including synthetic polymers,11−15 biomacromolecules,16−18 designed peptides,19 and dendrimers20 have been shown to exhibit a strong influence on CaCO3 crystallization. Currently, iron oxide superparamagnetic nanoparticles were introduced as a component in microspheres and hollow microcapsules,21 inorganic and organic nanotubes,22 microtubules,23 nanomagnetic sponges,24 hybrid organic−inorganic microgels,25 and anisotropic and Janus particles.26 The new materials provide a robust way of external manipulation using a permanent magnet, thus allowing the targeted delivery, separation, and extraction of magnetically modified materials. A similar approach was recently proposed for the separation of magnetically responsive calcite and aragonite microcrystals used as templates in the fabrication of cellosomes,27 where iron oxide nanoparticles were layerwise placed onto polyelectrolyte-coated Received: April 8, 2013 Revised: June 21, 2013

A

dx.doi.org/10.1021/cg400511d | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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M Na2CO3 was dissolved in the solutions. For the composite particles free of polymer, a specific amount of MF was added to a 50 mL 0.2 M Na2CO3 aqueous solution. The thus prepared solutions were rapidly mixed with an equal volume of 0.2 M CaCl2 aqueous solution; the pH of the mixtures was about 8.5. The mixtures were stirred for 1 min on a magnetic stirrer, at room temperature, and then the dispersions were kept under static conditions for 60 min. The obtained microparticles were separated by filtration, intensively washed with water, and finally washed with acetone and dried in an oven at 40 °C for 1.5 h. The dried microparticles were kept in hermetically closed tubes at room temperature. In the following, the composites samples were coded CaCO3−MFxCSAy, where x is the volume of MF (mL), and y is the polymer concentration in the initial mixture (wt %). The molar concentration of Na2CO3 and CaCl2 aqueous solutions were kept constant in all samples at 0.2 M. 2.3. Characterization of Composite Microparticles. 2.3.1. Scanning Electron Microscopy. The shape and surface of the new particles were examined with an environmental scanning electron microscope type Quanta 200, operating at 20 kV with secondary electrons. The SEM studies were performed on samples fixed on copper supports using carbon tape and covered with a thin layer of gold, by sputtering (EMITECH K550X). The Quanta 200 microscope was equipped with an energy dispersive X-ray (EDX) system for qualitative and quantitative analysis and elemental mapping. 2.3.2. Transmission Electron Microscopy. Morphological studies of oleic acid stabilized iron oxide magnetic nanoparticles and CaCO3− MFx(CSAy) composite microparticles were performed using a Hitachi HT7700 transmission electron microscope, operating at 100 kV. The samples were prepared by ultrasonication in water (for MF) or in acetone (for CaCO3/MF/CSA composite). To evidence the magnetite distribution into microparticles, some samples were ground in a mortar and then dispersed into acetone. One drop of the dilute dispersions was placed on a carbon-coated grid, and the solvent was evaporated in an oven at 40 °C for 72 h. After drying, the samples were examined in high resolution mode. The diameter statistics of the MF magnetite nanoparticles was determined from five TEM images (Figure 1S, Supporting Information) using ImageJ.31 The diameter of the magnetite nanoparticles was found to be 6.7 ± 1.7 nm, as determined on a population of 1200 sampled particles. Sampling of nanoparticle clusters was carefully avoided. The difference between the average diameter of the nanoparticles and the Z-average hydrodynamic diameter of surfactated particles (54 nm, Table 1) indicates particle clustering in the structure of the MF. 2.3.3. Raman Spectroscopy. The chemical composition of the CaCO3−MF−CSA microparticles was evaluated by Raman spectroscopy using a Renishaw inVia Reflex confocal microscope equipped with a He−Ne laser at 633 nm (17 mW) and a CCD detector coupled to a Leica DM 2500 M microscope. The range of vibrational frequencies was from 100 to 1500 cm−1. All measurements were performed in backscattering geometry using a 50× objective with a numerical aperture of 0.75. Three scans were accumulated for each spectrum, and the laser exposure for each scan was 10 s. The Raman measurements were performed at room temperature and atmospheric pressure. 2.3.4. Flow Particle Image Analyses. The particles size, size distribution, and the circularity of the carbonate particles has been evaluated using the Sysmex Dynamic Flow Particle Image Analyzer 2100. To obtain correct values, the Sysmex FPIA 2100 image analyzer was checked before starting the main experiment by using certified size standards. 2.3.5. X-ray Diffraction. X-ray diffraction characterization of the samples was carried out using a D8 Advance Bruker AXS device. The X-rays were generated using a Cu Kα source with an emission current of 36 mA and a voltage of 30 kV. Scans were collected over the 2θ = 20−60° range using a step size of 0.01° and a count time of 0.5 s/step. The semiquantitative analysis was performed with an EVA soft from DiffracPlus package and an ICDD-PDF2 database, based on the patterns’ relative heights. The criteria used to compare the simulated and the measured scan was the R/R0 ratio, where R is the weighted

microparticles. Hybrid magnetically responsive materials were also synthesized by coprecipitation of calcium carbonate rhombohedral microcrystals in the presence of citrate-stabilized iron oxide nanoparticles.28 In addition, the authors demonstrate the fabrication of hollow polyelectrolyte microcapsule templates on magnetically responsive calcite microcrystals, which retain their magnetic properties after decomposition of sacrificial cores and can be spatially manipulated using a permanent magnet. To produce magnetic calcium carbonate microcrystals via a single-step technique remains a challenge. This study describes a simple approach for the fabrication of magnetically responsive CaCO3 microcrystals by coprecipitation of CaCO3 in the presence of oleic acid stabilized iron oxide magnetic nanoparticles dispersed in water, as a magnetic fluid (MF), and of a natural strong/weak polyanion, chondroitin sulfate A (CSA). The study aims to highlight both the possibilities and the limitations of microparticle formation under different relative CaCO3−magnetite−polymer ratios. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to provide high-resolution images of particle morphology and flow particle image analysis (FPIA) to evidence their mean size and circularity, whereas X-ray diffraction and Raman spectroscopy were used to determine polymorph type and crystallite characteristics. The polymer and magnetite presence in the composite particles was investigated by energy dispersive X-ray (EDX), TEM, particle charge density, and ζ potential. The superparamagnetic properties of the obtained microparticles were also evidenced. The pH enhanced stability of the new composites, given by magnetite and polymer presence, was followed by ζ potential variation.

2. EXPERIMENTAL SECTION 2.1. Materials. CaCl2·2H2O and Na2CO3 from Sigma-Aldrich were used as received. Chondroitin sulfate A (CSA), with MV = 20 kDa, determined according to the method proposed by Wasteson,29 has been purchased from Sigma (C9819). The magnetic fluid (MF) was prepared by chemical coprecipitation followed by double layer steric and electrostatic (combined) stabilization of magnetite nanoparticles with oleic acid, M(OA)2, and dispersion in water.30 Some characteristics of the used MF are summarized in Table 1. 2.2. Preparation of CaCO 3−MF and CaCO 3−MF−CSA composite microparticles. The CaCO3−MF and CaCO3−MF− CSA composites were obtained in glass beakers at 25 °C. CSA aqueous solutions (50 mL) of different concentrations were first prepared, and then a specific amount of MF was added, and finally 0.2

Table 1. Some Characteristics of the Magnetic Fluid, M(OA)2/H2O saturation magnetization (Ms) surfactant contenta M(OA)2 contentb density at 21.6 °C (ρ) dispersion pH Z-average hydrodynamic diameter of M(OA)2 streaming potential (SP) ζ potential (ZP) electrophoretic mobility (μe) charge concentration (C) specific charge density (SC)

32 G 40 wt % 0.056 g/mL 1.0273 g/cm3 8.5 54 nm −1491 mV −40.93 mV −3.207 μm cm/(V s) −29.62 meq/l −28.80 μeq/g

a

Determined by thermogravimetry from the MF weight loss up to 500 °C. bDetermined gravimetrically (g of dry sample/mL of magnetic fluid). B



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Figure 1. SEM images of CaCO3−MFx particles. reliability and R0 represents the inevitable discrepancy due to the statistics of the X-ray diffraction (noise modeled by the Poisson’s law). For an ideal fit, the R/R0 value is 1. 2.3.6. Zeta Potential Measurements. The electrokinetic potential of carbonate samples was measured by a ZetaSizer Nano ZS (Malvern, UK) operating at 633 nm. The equipment measures the electrophoretic mobility of the particles and converts it into the ζ potential using the von Smoluchowski equation. The results were expressed as the average of at least three independent measurements performed on 0.5 mg/mL aqueous dispersions for each sample. 2.3.7. Particle and Solution Charge Density Determination. The ionic density on polymer chains and on particle surfaces was determined with a Mütek PCD 03 particle charge detector (BTG Instruments GmbH, Herrsching, Germany). The measured streaming potential is linearly correlated to the charge density of polyelectrolytes, and it becomes zero in case of charge neutrality. The concentration of the charged groups of each solution or particle aqueous dispersion was evaluated by titration with a standard solution of a strong oppositely charged polyelectrolyte, poly(sodium ethylenesulfonate) or poly(diallyldimethyl-ammonium chloride), with a concentration of 10−3 M. The concentration of the charged groups in the examined sample was calculated from the amount of standard solution needed to reach the zero value of the streaming potential. All measurements were performed at room temperature. 2.3.8. Magnetometry Measurements. The full magnetization curves of the samples (MF and CaCO3−MFxCSAy microparticles) were measured at room temperature using a vibrating sample magnetometer (VSM 880, ADE Technologies, USA) in the 0−800 kA/m field range.

3. RESULTS AND DISCUSSION The shape and surface morphology of CaCO3 composite microparticles, obtained by coprecipitation of calcium and carbonate ions in the presence of different amounts of oleic acid stabilized magnetic nanoparticles [M(OA)2], has been first followed by SEM (Figure 1). The carboxylic groups on the magnetite surface are ionized at the working pH (8.5) and therefore can be cross-linked by the Ca2+ ions in the solution. As the MF amount increases, a denser network is supposed to be formed. The synthesis approach used in this paper allows CaCO3 nucleation, at an outburst speed, forming a large number of small calcite and vaterite nanoparticles, which subsequently grow into calcium carbonate microcrystals in the electrostatically stabilized network, forming almost spherical (when vaterite is the main polymorph) and cubic (when calcite is the main polymorph) composite microparticles. Compared with the characteristic cauliflower shape of bare CaCO3 microparticles (Figure 1a) with a mean diameter of about 8 μm, the SEM images in Figure 1 show that smaller and smoother particles were obtained in the presence of MF, irrespective of MF content in the initial mixture. However, at higher MF content, some small aggregates seem to be attached on the surface of the composite microparticles, probably formed by M(OA)2 particles not included in the composite particles. C



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Some size characteristics, determined by FPIA, are given in Table 2. According to the values in Table 2, smaller particles Table 2. Particle Size Characteristics and Average Circularity Analysis Values of CaCO3−MF Particles, Obtained by FPIA Measurements 10% D,c μm

50% D,d μm

90% D,e μm

Cmeanf

sample

Dmean,a μm

Dmode,b μm

CaCO3 CaCO3− MF0.2 CaCO3− MF0.5 CaCO3− MF1.0

8.13 ± 2.55 5.06 ± 2.18

6.07 4.44

1.42 1.11

6.57 5.22

14.16 18.95

0.931 0.908

4.86 ± 2.29

4.34

1.85

5.37

19.56

0.905

6.13 ± 2.32

6.63

1.96

6.21

14.22

0.907

Figure 3. XRD paterns of (1) CaCO3−MF0.2, (2) CaCO3−MF0.5, and (3) CaCO3−MF1.0 microparticles.

a

Mean diameter as average size of particles. bMode diameter as the highest frequency size. cParticle diameter determined at the 10th percentile of undersized particles. dParticle diameter determined at the 50th percentile of undersized particles (the median diameter). e Particle diameter determined at the 90th percentile of undersized particles. fMean circularity, defined as perimeter of equivalent circle vs perimeter of particle.

Raman spectra of bare CaCO3 and composite particles as follows: at 155 cm−1 and 282 cm−1, due to lattice mode vibrations, at 712 cm−1 (ν4), attributed to the in-plane bending vibration of O−C−O, and at 1086 cm−1 (ν1) for symmetric stretching vibration of C−O bond.32 Vaterite crystal characteristic bands are located at 113 and 301 cm−1, corresponding to the translational and rotational lattice modes, at 750 cm−1 (ν4), and as a narrow double peak at 1074 and 1089 cm−1 (ν1). Moreover, the magnetite composite samples have a distinguishable peak around 670 cm−1 (A1g mode), accepted as the signature peak position of bulk magnetite.33,34 Three other theoretical phonon frequencies of magnetite exhibit peaks at 315 (Eg), 480 (T2g(3)), and 538 (T2g(2)) cm−1.35−37 The X-ray diffractograms confirm the crystal composition observed in Raman spectra. The XRD patterns demonstrate that the crystalline fractions of all investigated samples are mainly calcite (rhombohedral), vaterite (hexagonal), and magnetite (cubic) (Figure 3). As visible in Figure 3, X-ray diffractograms of the composite samples show some diffraction patterns characteristic of magnetite,28,38 in particular at 2θ equal to 21.15°, 30.1°, 35.4°, 37.1°, 43.1°, 47.2°, and 48.5°, calcite at 2θ equal to 29.45°, 35.9°, 39.4°, 43.2°, 47.1°, 47.6°, and 48.6°, and vaterite at 2θ equal to 24.9°, 27.1°, 32.7°, 38.9°, and 43.9°. The lattice constants for each CaCO3 polymorph were not influenced by the ratio between polymorph fraction or magnetite content: a = b = 4.99 Å and c = 17.002 Å for calcite; a = b = 4.12 Å and c = 8.556 Å for vaterite; a = 8.394 Å for magnetite. Table 3 lists the XRD semiquantitative analysis results, obtained with an EVA soft from DiffracPlus package and an ICDD-PDF2 database, based on the patterns’ relative heights. The criteria used to compare the simulated and the measured scan is the R/R0 ratio, where R is the weighted reliability and R0 represents the inevitable discrepancy due to the statistics of the X-ray diffraction (noise modeled by the Poisson’s law). For an ideal fit, the R/R0 value is 1. XRD patterns in Figure 3 show some peaks with very close positions (such as 43.1, 47.1, and 48.6 for calcite and 43.07, 47.2, and 48.5 for magnetite). However, for peak assignment, EVA software from DiffracPlus package and an ICDD-PDF2 database has been used. The database contains information about different kinds of CaCO3 polymorphs structure and verifies the reliability of the data fitting. The peak assignment for our samples using the database was close to the ideal fit, R/R0 values in Tables 3 ranging between 0.983 and 1.07. The CaCO3 polymorph ratio in the composite carbonate/magnetite particles is influenced by the magnetite content in the initial mixture. Thus, the X-ray

diameters were obtained when MF was used, compared with bare CaCO3 microparticles, irrespective of CaCO3/MF ratio. The relatively close values found for mean, mode, and median diameter suggest particles size homogeneity (low size dispersity) for all investigated samples. The circularity is a characteristic specific to particle shape; the more spherical particle, the closer its circularity to 1.0, whereas the cubic shape has a “circularity” value around 0.88. Thus, the values of mean circularity included in Table 2 suggest a combined population of spheres and cubes, a result corroborated by the SEM images (Figure 1). The CaCO3 polymorph content along with the magnetite crystals in the composite microparticles was followed by Raman spectroscopy (Figure 2) and X-ray diffraction (Figure 3). As shown in Figure 2, both calcite and vaterite characteristic peaks are evidenced in the Raman spectra, irrespective of CaCO3/MF ratio. Thus, calcite polymorphs are evidenced in

Figure 2. Raman spectra of (1) CaCO3, (2) CaCO3−MF0.2, (3) CaCO3−MF0.5, (4) CaCO3−MF1.0 microparticles. D



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Table 3. XRD Quantitative Analysis of CaCO3−MF Samples Performed with an ICDD-PDF2 Database calcite sample CaCO3−MF0.2 CaCO3−MF0.5 CaCO3−MF1.0

R/R0 1.070 0.983 0.984

% 13.5 33.1 35.6

vaterite

crystallite size,a Å

%

761.0 553.9 375.6

85.6 64.2 58.2

magnetite

crystallite size,a Å 141.4 157.3 189.9


% b

0.9 (1.94) 2.7 (4.71)b 6.2 (8.98)b

179.6 181.7 185.7

Determined by Scherrer equation. bMagnetite wt % percent in the initial mixtures = VMFρMF/ρMP, where VMF is the volume of magnetic fluid, ρMF the fluid density (1.0273 g/mL) and ρMP the density of pure magnetite (5.2 g/mL). a

Figure 4. SEM images of CaCO3−MFxCSAy composite particles.

particle size, shape, surface morphology, and polymorph ratio. However, as previously reported,11,12 a small amount of polyanion used as a template in the crystallization process would also control the crystallization pathway. The long macromolecular chain is expected to form a flexible network, due to the electrostatic cross-linking between the ionic sites on the polymer and the calcium divalent ions. Therefore, the next step in the present study was to introduce, beside the magnetic fluid, a strong/weak polyanion, CSA, during the formation of composite microparticles. The influence of CSA and MF content on the CaCO3−MF−CSA composite size, shape, and morphology has been followed by SEM (Figure 4). According to literature data,39−42 the growth mechanism of CaCO3/CSA/MF microparticles could take place as follows. The chains of CSA and the surfactated MF nanoparticles could electrostatically accumulate a large amount of Ca2+ and carbonate ions; Ca2+ ions form an ionically cross-linked network with the carboxylate groups on the CSA and oleic acid. As the ratio polymer/MF increases, the network flexibility increases due to the long chain flexibility, compared with that of oleic acid molecules on the MF nanoparticles. The nucleation of CaCO3 occurs in the preformed network, forming small calcite and vaterite nanoseeds, which further aggregate into

difractograms show that the intensity of calcite diffraction lines increases with the increase of magnetite amount up to 1.0, where a lower content of vaterite polymorph (∼58%) can be observed (Figure 3). The calcite crystallite size decreased significantly with increasing magnetite content in the composite particles (Table 3), the lower value of about 37 nm being obtained when the highest magnetite amount has been used for particle formation, whereas a small increase of vaterite crystallite size has been obtained. The decrease of crystallite size of calcite and vaterite polymorphs with increasing MF content can be ascribed to the formation of a dense network between the acidic groups on the magnetite surface and the Ca2+ ions in the solution, which restricts the space for CaCO3 crystals growth. The magnetite percent in the composite, determined by X-ray, is lower than that used in the initial mixtures, calculated from the volume of magnetic fluid used, fluid density, and density of pure magnetite, suggesting that just up to 70% of the initial magnetite amount is included in the composites. However, the values are just indicative and do not take into account the oleic acid presence in the composite. These results clearly showed that a small amount of magnetite used in the CaCO3 crystallization process strongly influences the composite particle characteristics in terms of E



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Figure 5. TEM images of some CaCO3−MFxCSAy composite particles.

higher crystals in the Ca2+−CSA/MF network. When the growth of CaCO3 particles reaches a critical size, the inorganic/ organic composite particles separate from solution and generally adopt a spherical morphology, which gives the minimum total surface energy for a given volume. As shown in Figure 4, a low amount of CSA in the initial mixture (0.04 wt %) induces the formation of almost spherical microparticles, the MF content having a small influence on particle size. However, upon increase of the MF content to 0.5 and 1.0 mL, respectively, part of the magnetite surfactated nanoparticles remain on composite microparticle surface, as also observed for particles prepared with the same MF amount and without

polymer (Figure 1). Increasing the CSA concentration to 0.1 and 0.2 wt % results in the formation of smoother particles, possessing a surface morphology very close to that of particles prepared without MF (sample CaCO3−CSA0.1). To further follow the embedment of MF into the composite particles, TEM images on particles were observed (Figure 5), and also on the particle edges (a2, b2, c2, and d2) and on particle sides (a3, b3, c3, and d3). The distribution of magnetite nanoparticles in composite CaCO3 microparticles was observed (some darkest dots of about 7 nm in diameter were marked with white arrows in TEM images on particle sides, Figure 5a3,b3), irrespective of magnetite amount used in the F



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composite material synthesis (samples CaCO3−MF0.2 and CaCO3−MF1.0). However, with increase of the MF content to 1.0 mL, a higher amount of magnetic nanoparticles on the composite microparticle surface is observed on the TEM images (Figure 5a2,b2). The presence of polymer in the CaCO3−MF0.2CSA0.04 samples yields a better embedment of MF into the polymeric network. Upon increase of the CSA content, almost no magnetite nanoparticles were observed at particles surface (Figure 5d2), even if inside the composites the magnetite presence was noticed, as observed in TEM images of CaCO3−MF0.2CSA0.1 sample (Figure 5d3). As evidence for magnetic nanoparticle distribution on the composite microparticles, EDX mapping was performed on the samples with the highest content in MF, that is, CaCO3−MF1.0 and CaCO3−MF1.0CSA0.04 (Figure 2S, Supporting Information). The EDX method allowed the quantitative determination of the atomic percent in all investigated samples. The EDX analysis demonstrates high carbon content, due to the contribution of C from the support material and to the spontaneously adsorbed hydrocarbon impurities, also known as adventitious carbon, and therefore the carbon content was not further considered. Table 4 summarizes the atomic ratios of the elements characteristic of MF (Fe and O) and CSA (N, S, and O) versus Ca content from CaCO3.

Table 5. Particle Size Characteristics and Circularity Average Values of CaCO3−MFxCSAy Particles Obtained from FPIA Measurements

Fe/Ca

N/Ca

S/Ca

O/Ca

0 0.0035 0.0105 0.0351 0.0019 0.0078 0.0168 0.0107 0.0062

0 0 0 0 0.460 0.368 0.287 0.680 1.016

0 0 0 0 0.0084 0.0062 0.0044 0.0215 0.0938

3.534 4.553 4.765 4.912 5.059 4.810 4.022 8.221 12.119

90% D,e μm

Cmeanf

sample

Dmean, μm

CaCO3− MF0.2CSA0.04 CaCO3− MF0.5CSA0.04 CaCO3− MF1.0CSA0.04 CaCO3− MF0.2CSA0.1 CaCO3− MF0.2CSA0.2

6.43 ± 8.64

6.15

1.05

5.75

9.64

0.945

6.28 ± 2.38

5.70

1.83

5.74

9.07

0.947

6.35 ± 2.06

5.77

1.69

5.25

9.14

0.942

4.79 ± 5.10

3.67

1.71

4.13

8.46

0.965

5.82 ± 2.87

5.74

1.82

5.41

7.32

0.964

Mean diameter as average size of particles. bMode diameter as the highest frequency size. cParticle diameter determined at the 10th percentile of undersized particles. dParticle diameter determined at the 50th percentile of undersized particles (the median diameter). e Particle diameter determined at the 90th percentile of undersized particles. fMean circularity, defined as perimeter of equivalent circle vs perimeter of particle.

content. The diameter values obtained at the 90th percentile of undersized particles suggest that the polymer presence prevents the aggregation of the particles, compared with the particles prepared only with MF, taking into account the values obtained for the particles prepared with the same MF content (Table 2). Moreover, the values of mean circularity are much closer to those characteristic to spheres, supporting the SEM images. At higher polymer concentration (0.1 and 0.2 wt %), the particles size decreases, probably due to a denser polymeric cross-linked network. Due to the polymer content and low magnetite content, it is difficult to clearly discriminate the magnetite beside vaterite and calcite polymorphs in XRD diffractograms (Figure 3S, Supporting Information). However, the XRD results were used for quantitative determination of CaCO3 polymorphs and their crystallite size (Table 6). The magnetite content is lower than that used in the initial mixtures but follows the same trend as in the samples prepared without polymer (Table 3). The CaCO3 polymorph ratios shown in Table 6 fully agree with the SEM images, where almost spherical particles were observed and therefore a high content in vaterite crystals was found. Thus, 0.04 wt % CSA chains seem to stabilize vaterite growth, even though increased MF content favors the calcite crystal growth (Table 3). Moreover, further increase of the CSA content (CaCO3− MF0.2CSA0.1 and CaCO3−MF0.2CSA0.2) determines higher vaterite content. Stable vaterite particles (although vaterite is metastable in pure water) were previously produced in the presence of organic additives that can stabilize the vaterite surface.43,44 The preferential vaterite growth could be explained by a kinetic inhibition of the calcite nuclei by the polymer presence. According to nucleation theory,45 subcritical nuclei of all potential polymorphs are stochastically formed and dissolved, and only those nuclei that pass a critical size (defined by crystallization enthalpy and interface energy) can continue to grow. If a polymer lowers the interface energy of a specific polymorph (and the interface energy is lowered already below the critical surface coverage), this polymorph will be nucleated. This concept is consistent with the previous findings that carboxylate copolymers are able to initiate vaterite nucleation

atomic ratio sample

50% D,d μm

a

Table 4. The Atomic Ratio of the Elements Characteristic of MF (Fe and O) and CSA (N, S and O) versus Ca Content from CaCO3

CaCO3 CaCO3−MF0.2 CaCO3−MF0.5 CaCO3−MF1.0 CaCO3−MF0.2CSA0.04 CaCO3−MF0.5CSA0.04 CaCO3−MF1.0CSA0.04 CaCO3−MF0.2CSA0.1 CaCO3−MF0.2CSA0.2

10% D,c μm

Dmode,b μm

a

As shown in Table 4, for the bare CaCO3 sample, the O/Ca ratio is higher (3.5) than the theoretical ratio (3.0). Similar results were reported in the literature11 and were ascribed to the vaterite crystallization form. Upon introduction of MF into the composite materials, the ratio O/Ca increases due to the contribution of oxygen from oleic acid and from magnetite (Fe3O4). Also, the Fe/Ca ratio increases with increasing the MF content, suggesting the increase of the magnetite content in the composite material. CSA-based composite materials showed, beside O and Fe, the presence of N and S. The relative atomic ratio of component elements supports the composite’s expected composition: increase of O/Ca ratio, due to organic content increase, and increase of Fe/Ca ratio with the increase of MF in the initial mixtures. For the same CSA content (0.04 wt %), the N/Ca and S/Ca ratios decrease, due to the increase of MF ratio into the composite. When MF content was kept constant (0.2 mL), the atomic ratio of the specific elements of CSA (N and S) increased with the increase of polymeric content. The particle size and circularity was determined by FPIA (Table 5). As shown in Table 5, the mean particle size of CaCO3−MFxCSA0.04 was almost constant, irrespective of MF G



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Table 6. XRD Quantitative Analysis of CaCO3−MFxCSAy Samples, Performed with an ICDD-PDF2 Database calcite sample CaCO3−MF0.2CSA0.04 CaCO3−MF0.5CSA0.04 CaCO3−MF1.0CSA0.04 CaCO3−MF0.2CSA0.1 CaCO3−MF0.2CSA0.2

R/R0 0.977 0.982 1.088 0.997 1.075

% 4.3 7.9 7.7 3.7 2.8

vaterite


%

184.8 257.4 254.5 182.2 184.1

94.0 89.7 86.9 95.1 96.1

magnetite

crystallite size,a Å 183.3 175.7 164.6 159.4 162.7


% 1.7 2.4 5.4 1.2 1.1

b

(1.90) (4.62)b (8.84)b (1.85)b (1.77)b

184.0 186.8 187.2 184.3 186.4

Determined by Scherrer equation. bMagnetite wt % in the initial mixture = VMFρMF/ρMP, where VMF is the volume of magnetic fluid, ρMF the fluid density (1.0273 g/mL), and ρMP the density of pure magnetite(5.2 g/mL).

a

for bare CaCO3 microparticles (ζ potential, −8.3 mV; specific charge density, −8.4 mequiv/g). The ζ potential and specific charge density values for the CaCO3-based composite microparticles are summarized in Table 7.

from stable supersaturated solutions through binding of the calcium ions at the ionized carboxylic groups.46 The polymer presence in the composite particles and the polymorph content were investigated through Raman spectroscopy (Figure 6). The Raman spectra in Figure 6 show the

Table 7. Zeta Potential (ZP) and Specific Charge Density (CD) of CaCO3−MFxCSAy Composite Microparticles sample

ZP, mV

CD, mequiv/g

CaCO3−MF0.2 CaCO3−MF0.5 CaCO3−MF1.0 CaCO3−MF0.2CSA0.04 CaCO3−MF0.5CSA0.04 CaCO3−MF1.0CSA0.04 CaCO3−MF0.2CSA0.1 CaCO3−MF0.2CSA0.2

−11.2 −18.1 −19.7 −22.8 −24.9 −26.5 −28.8 −38.3

−10.8 −12.8 −14.3 −15.3 −16.1 −17.1 −18.9 −25.4

The presence of the M(OA)2 nanoparticles in the CaCO3− MF composites increases the negative character of the composites, the ZP and CD values becoming more negative with increasing MF content compared with the bare CaCO3 particles. CSA is a strong (−SO3−)/weak (−COO−) polyanion, completely ionized at the working pH (8.5), and therefore even a very small amount of this polymer (0.04 wt %) significantly increases the negative values of ZP and CD (Table 7). A further increase in polymer content to 0.1 and 0.2 wt % yields a significant increase of the negative character of CaCO3−MF/ CSA composite microparticles. The release/uptake properties of CaCO3 composite particles containing weak polyelectrolytes can be easily tuned by pH changes of the environment.12 Therefore, to investigate the particle stability as a function of environmental pH, the variation of ζ potential vs pH has been followed for some CaCO3−MF/CSA particles (Figure 7). It is well-known that CaCO3 easily dissolves under acidic conditions, our previous study showing that bare CaCO3 particles, prepared with the same initial concentration in calcium and carbonate ions, are stable at pH higher than 5.4.12 As shown in Figure 7, 0.2 mL of MF in composite particles slightly moved the point of zero charge (pzc, considered as the numeric value of pH where ZP = 0) to about pH = 4.7; upon increase of the MF content, the pzc value is shifted to pH = 4.1, due to the increased ionic cross-linking between COO- groups from M(OA)2 surface and Ca2+ ions. The polymer presence in the composite particles also influenced the pH stability of the particles. Thus, comparison between the samples prepared with the same MF amount showed that the composites containing 0.04 wt % of CSA seem to be stable to more acidic pH values, that is, 3.5 and 2.9 for samples CaCO3−MF0.2CSA0.04 and CaCO3−MF1.0CSA0.04,

Figure 6. Raman spectra of composite microparticles: (1) CaCO3− MF0.2CSA0.04; (2) CaCO3−MF0.5CSA0.04; (3) CaCO3−MF1.0CSA0.04; (4) CaCO3−MF0.2CSA0.1; (5) CaCO3−MF0.2CSA0.2.

calcite, vaterite, and magnetite characteristic peaks at the similar wavelengths as in Figure 2. Moreover, the CSA characteristic peaks can also be observed,47 their intensity increasing with polymer content, as follows: the asymmetric −OSO 3 − stretching vibration at 1269 cm−1; the vibrations at 836 cm−1 as the asymmetric vibration of the C−O−S linkages; the 987 cm−1 band corresponding to the symmetric vibration of the C− O−(S) linkage, reflecting the effect of the sulfate group on the C−O link; the vibration at 939 cm−1 appearing to be a skeletal vibration of the C−O−C linkages; the amide III band at 1351 cm−1, characteristic of the cis arrangement of the C−O and N− H groups with respect to the C−N bond; the CH3 symmetric deformation frequency at 1373 cm−1 in all glycosaminoglycans. The strong band at 1421 cm−1 is due to the symmetrical vibration of the COO− group of the glucuronate residue. The magnetite nanoparticles are covered with a double layer of oleic acid, and thus the surface is negatively charged: at pH = 5.5, the ζ potential is −40.93 mV, and the specific charge density is −28.80 mequiv/g. Therefore, one should expect that the electronegative character of oleic acid stabilized magnetic nanoparticles further increases the negative character of CaCO3 composite microparticles, compared with the values obtained H



Article

The saturation magnetization values (Ms) of the samples are given in Table 8. The values of composite particle Ms are lower Table 8. The Saturation Magnetization Values (Ms) and Mass Percentage of the Coated Magnetite Nanoparticles in the Composite Microparticles sample

Ms, emu/g

magnetite, wt %

CaCO3−MF0.2 CaCO3−MF0.5 CaCO3−MF1.0 CaCO3−MF0.2CSA0.04 CaCO3−MF0.5CSA0.04 CaCO3−MF1.0CSA0.04 CaCO3−MF0.2CSA0.1 CaCO3−MF0.2CSA0.2

0.56 1.47 2.88 0.53 0.66 2.58 0.44 0.48

1.5 4.0 7.8 1.4 1.8 6.9 1.2 1.3

than those of the surfactant-coated magnetite nanoparticles (37.1 emu/g). This phenomenon has been observed and explained earlier by other authors48 and can be related to nanoparticle embedment in the composite materials. With this value, the mass percentage of the coated magnetite nanoparticles in the composite microparticles was calculated and is listed in Table 8. According to Table 8, the saturation magnetization values increase with increasing MF content and decrease with increasing polymer concentration. The magnetite percent in the composite, as determined by this method, is lower than that used in the initial mixtures, calculated from the volume of the used magnetic fluid but close to the values determined by X-ray (Tables 3 and 6). CaCO3 has a microporous nature and the ability to be dissolved in mild conditions of acidic pH and in the presence of complexing agents and has also been tested for pharmaceutical and biomedical applications owing to its biocompatible and biodegradable nature. Changing the environmental pH and thus tuning the polymer/MF/Ca2+ network flexibility and stability could control the partial CaCO3 dissolution and sorption of small molecules (such as drugs, chemotherapeutic agents, molecules and cells, etc) into microparticle pores. Magnetic force could carry the drug-loaded composite microparticles to the target sites and make them to stay there, whereas the pH at the target sites could allow the microparticle swelling, allowing thus pH controlled drug release. The synergetic action of the CaCO3 intrinsic characteristics and the superparamagnetism and pH sensitivity of as prepared composite particles, given by magnetite and weak polyelectrolyte presence, make them potentially a drug release systems.

Figure 7. Zeta potential vs pH variation for (1) CaCO3−MF0.2, (2) CaCO3 −MF 0.2 CSA 0.04 , (3) CaCO 3−MF1.0 , and (4) CaCO3 − MF1.0CSA0.04.

respectively. For these two samples, the ionic cross-links and the higher flexibility can be the reason of the increased composites stability. The magnetization curves of the dried composite microparticles are presented in Figure 8. None of the samples show a

Figure 8. Magnetization curves of the dried composite microparticles. Inset shows the magnetization of the dried surfactated magnetite nanoparticles.

4. CONCLUSIONS In this study, a simple and fast precipitation method was proposed to produce uniform calcium carbonate microspheres using MF and CSA as templates. The influence of the initial MF amount and polymer concentration on microparticle characteristics and particle pH stability was investigated by SEM, TEM, X-ray diffraction, Raman spectroscopy, FPIA, particle charge density, and electrokinetic measurements. The ratio between CaCO3, MF, and CSA has a remarkable effect on the morphology and polymorphism of CaCO3 particles. SEM images show that more or less spherical aggregates with a small size distribution were obtained, irrespective of the initial MF and CSA content. Compared with the typical cauliflower shape of bare CaCO3 particles, the presence of MF induces the

magnetic hysteresis, that is, the absence of magnetic memory of the sample once the applied field is removed, which indicates their superparamagnetic behavior. This observation strongly indicates that composite microparticle synthesis pathways do not compromise the magnetic properties of magnetite nanoparticles. The superparamagnetism of particles can be useful for the biomedical field with respect to magnetic localization and dispersion of actives. For example, particles that immobilize biospecific molecules and contain magnetic substances can be used as an easily collectable bioreactor, bioseparator, and diagnostic reagent. I



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formation of smoother particles of very small grain size. The presence of MF and polymer in the composite particles also influences the particle pH stability, the particle pzc values being shifted to more acidic pH values as a function of CaCO3/MF/ CSA ratio and particle morphology. Taking into account their biocompatibility, their enhanced pH stability, and superparamagnetic properties, these new composite materials could be of interest in different biorelated applications.

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ASSOCIATED CONTENT

* Supporting Information S

TEM image of MF, EDX mapping of samples with the highest content in MF, that is, CaCO 3 −MF 1.0 and CaCO 3 − MF1.0CSA0.04, and X-ray diffractograms of particles prepared at different the polymer concentration. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +40.2322217454. Fax: +40.232211299. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of Project ID_313/2011 is gratefully acknowledged. V. Socoliuc and L. Vekas acknowledge the financial support of AR-FT-CCTFA-LLM 2013-2015 research program. The authors are grateful to Dr. Ioana Moleavin from “Petru Poni” Institute of Macromolecular Chemistry of Romanian Academy, Centre of Advanced Research in Bionanoconjugates and Biopolymers, for ζ potential versus pH measurements, Mrs. Florica Balanean from Romanian Academy - Timisoara Branch, Center for Fundamental and Advanced Technical Research, Laboratory of Magnetic Fluids, for the synthesis of the magnetic fluid, and Ph.D. fellow Mrs. Oana Marinica from “Politehnica” University of Timisoara for the VSM measurements.

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REFERENCES

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