Improved suspension stability of calcium carbonate nanoparticles by ...

Biotechnology and Bioprocess Engineering 20: 794-799 (2015) DOI 10.1007/s12257-014-0898-3

RESEARCH PAPER

Improved Suspension Stability of Calcium Carbonate Nanoparticles by Surface Modification with Oleic Acid and Phospholipid Jin Kim, Seung Kang Bea, Yool Hee Kim, Dong-Woon Kim, Ki-Young Lee, and Chang-Moon Lee

Received: 30 December 2014 / Revised: 12 May 2015 / Accepted: 28 June 2015 © The Korean Society for Biotechnology and Bioengineering and Springer 2015

Abstract We prepared and characterized calcium carbonate nanoparticles (CC NPs) that were surface-modified with oleic acid (OA) and phosphatidylcholine (PC) in order to improve their suspension stability in an aqueous solution. The improvement in the suspension stability of CC NPs in an aqueous solution may be helpful to extend their applicability to a wider range of biological applications. The CC NPs were coated with OA by making use of their electrostatic potential and were then decorated with PC. The CC NPs surface-modified with OA and PC were successfully constructed, and the existence of the decorated OA and PC in the surface of the PC-OA-CC NPs was observed via transmission electron microscopy (TEM) and was confirmed by thermo gravimetric analysis (TGA), Xray diffraction (XRD), and Fourier transform infrared Jin Kim†, Yool Hee Kim, Ki-Young Lee Department of Advanced Chemicals and Engineering, Chonnam National University, Gwangju 500-757, Korea Seung Kang Bea†, Ki-Young Lee, Chang-Moon Lee Interdisciplinary Program of Perfume and Cosmetics, Chonnam National University, Gwangju 500-757, Korea Ki-Young Lee Faculty of Applied Chemical Engineering and Functional Food Research Center, Chonnam National University, Gwangju 500-757, Korea Dong-Woon Kim Department of Nursing, Gwangyang Health College, Gwangyang 545703, Korea Seung Kang Bea, Yool Hee Kim SION BSK Co., Ltd., Gwangju 501-081, Korea Chang-Moon Lee* Department of Biomedical Engineering, Chonnam National University, Yeosu 550-749, Korea Tel: +82-61-659-7361; Fax: +82-61-659-7369 E-mail: [email protected] †

These authors contributed equally to this work.

spectroscopy (FT-IR) analyses. The PC-OA-CC NPs floating in an aqueous solution exhibited better stability when compared to non-surface-modified CC NPs. The DLS and TEM results revealed that the degree of size agglomeration for the CC NPs was significantly reduced by the surface modification with OA and PC. The PC-OA-CC NPs showed a very low cytotoxicity at a high concentration in terms of the cell viability of the RAW264.7 cells. Consequentially, the stability in suspension of the CC NPs in an aqueous solution could be effectively improved through surface-modification with OA and PC. PC-OACC NPs could be useful in increasing the range biological applications for CC NPs. Keywords: calcium carbonate nanoparticles, surface modification, fatty acid, phospholipid, suspension stability

1. Introduction Calcium carbonate (CaCO3) is one of the most abundant minerals in nature and is the main component in the shells of marine organisms, including snails, clams, pearls, and eggs. This material has attracted an enormous amount of attention for research into its biomedical applications, including cosmetics, drug delivery, and tissue engineering because it has excellent biocompatibility. The technology used to produce CaCO3 nanoparticles (CC NPs) from eggshells, clamshells, or pearls has been previously developed [1], and in recent years, CC NPs have been used as a good drug delivery carrier [2-4]. For example, Wang et al. reported the amorphous ibuprofen-loaded CaCO3 microparticles which had a rapider release in the gastric fluid and a slower release in the intestinal fluid [3]. However, the use of CC NPs in biomedical applications require for several issues to

Improved Suspension Stability of Calcium Carbonate Nanoparticles by Surface Modification with Oleic Acid and Phospholipid

be resolved, including having the ability to control the size, increasing the drug entrapment efficiency, and improving the colloidal stability [5]. Above all, since CC NPs are very easy to agglomerate and therefore exhibit poor dispersion capacity in solution as a result to their large specific surface area and high surface energy, a major challenge is to find novel methods to improve their dispersion as well as their colloidal stability. This problem can be resolved by using some special preparation techniques, including surfacegrafting hydrophilic molecules and in-situ polymerization [6]. For example, a copolymer of poly(ethylene glycol)-bpoly(aspartic acid) and poly(vinylsulfonic acid) were used to control the crystallization of the calcium phosphate and to improve the aqueous solution stability of the CC NPs, respectively [7,8]. Recently, Zhao et al. reported that modifying CC NPs with alginate is useful in enhancing the delivery efficiency for genes and drugs [9]. An improvement in the colloidal stability of the CC NPs may be helpful to extend their range to more biomedical applications. Until now, the use of oleic acid (OA) and phospholipids (PL) for surface modification of the CC NPs to has yet been reported. In this study, we used OA and PL to improve the aqueous stability of CC NPs produced from ark shells for the first time. The use of OA and PL to modify the surfaces of the CC NPs may provide excellent biocompatibility, resulting in an increase in opportunities for use toward in vivo applications. The OA and PL-surface modified CC NPs were characterized via thermo gravimetric analysis (TGA), transmission electron microscopy (TEM), X-ray diffraction (XRD) analysis, Fourier transform infrared spectroscopy (FT-IR), and dynamic light scattering (DLS). In addition, we investigated the aqueous stability of the PC and OA-surface-modified CC NP solution by determining the changes in the turbidity.

2. Materials and Methods 2.1. Materials Calcium carbonate nanoparticles (CC NPs, from Ark shell, Ca 36%) were obtained from DreamLime Co., Ltd. (Yeosu, Korea). L-α-phosphatidylcholine (PC, 98%, from egg yolk), oleic acid (OA, 97%) and cholesterol (Chol, 99%) were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO). The other reagents for the experiments were analytically pure and were commercially available. 2.2. Preparation of the CC NPs surface-modified with OA The OA (10 mg) was dissolved in ethanol while stirring, and then the CC NPs (10 mg) were added into the solution.

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The mixture was heated to 70°C under vigorous stirring for 24 h, and the final solution was centrifuged and washed three times with ethanol. The CC NPs that were surfacemodified with OA (OA-CC NPs) were then freeze dried at -80°C. 2.3. Preparation of the PC-coated OA-CC NPs The PC-coated OA-CC NPs (PC-OA-CC NPs) were prepared by using a slightly modified thin film hydration method, as had been previously described [10]. Briefly, OA-CC NPs (10 mg/mL), PC (13 mg), and Chol (2 mg) were dissolved with a 10 mL mixture of chloroform/ methanol (2:1, v/v) in a round bottom flask. The solution was evaporated under vacuum at 50°C and was then treated with a sonicator (ultrasonic processor VCX-400/600, Sonics and Materials Inc., USA) equipped with a micro-tip over a few cycles of 2 sec of sonication and a l sec pause, for a total of 5 min. The aggregates were then removed via centrifugation at 10,000 rpm for 15 min. 2.4. Characterization of PC-OA-CC NPs The PC-OA-CC NPs were filtered with a 0.45 µm poresize syringe filter and were diluted to their appropriate scattering intensity. The size distribution of the PC-OA-CC NPs was determined via dynamic light scattering (DLS7000, Otsuka Electronics Co. Ltd., Osaka, Japan). Scanning electron microscopy (SEM) was performed in order to observe the morphology of the nanoparticles, and energy dispersive X-ray spectroscopy (EDX) was performed in order to investigate the composition of the nanoparticles by using a SEM analyzer (S-4700, HITACHI, Tokyo, Japan). Transmittance electron microscopy (TEM) for the PC-OACC NPs was performed with a JEOL JEM-2000 FX II at 80 kV (Tokyo, Japan). For the TEM analysis, the nanoparticle suspension was dropped onto a carbon film-coated copper grid. After negative staining with phosphotungstic acid solution, the observations were carried out. The chemical groups in the nanoparticles were determined via Fouriertransform-infrared spectroscopy (FT-IR) with an IR 8000 instrument (Shimadzu Co. Ltd., Kyoto, Japan) in order to confirm that the PC-OA-CC NPs had been prepared. The thermal behavior of the CC NPs, OA-CC NPs, and PCOA-CC NPs was investigated via thermogravimetric analysis (TGA) with a Perkin Elmer Diamond TG/DTA (Mettler-Toledo, Greifensee, Switzerland). Each sample was heated from 30 to 500°C at a scanning rate of 10°C/min under an argon atmosphere. The X-ray diffraction (XRD) patterns of the CC NPs, OA-CC NPs, and PC-OA-CC NPs were recorded using an X-ray diffractometer (Rigaku D/Max Ultima III) operating at 40 kV with 40 mA at a 2°/min scan rate. Cu Kα X-ray was filtered with nickel.

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Fig. 1. Schematic for the preparation of OA-CC NPs and PC-OA-CC NPs. The calcium carbonate powder was obtained from an ark shell and was surface-modified with oleic acid. Then phosphatidylcholine was introduced on the OA-CC NPs through a hydrophobic interaction.

2.5. Aqueous suspension stability The stability of PC-OA-CC NPs in an aqueous suspension was measured by determining the changes in transmittance for the solutions at 500 nm, as described in a previous paper [11]. The OA-CC NPs and the PC-OA- CC NPs (10 mg) was suspended in 7 ml of distilled water, and the transmittance of the solutions was then determined with a UV spectrophotometer (UV-1200, Shimadzu Co. Ltd., Kyoto, Japan) at 37°C (n = 3). 2.6. Cell viability test The cytotoxicities of the CC NPs, OA-CC NPs, and PCOA-CC NPs were assessed in vitro by using an MTT kit. The RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotics under a 5% CO2 atmosphere at 37°C. The cells were placed in a 96-well plate at a density of 1 × 104 cells/well. The medium was replaced with fresh medium containing various concentrations of nanoparticles incubated in PBS (0.1 M, pH 7.4) at 37°C. After 24 h, the cell cultures were incubated with the MTT assay reagent for 4 h and were then read on a multiwall microplate reader (BioTek Instruments Inc., VT, USA) at 570 nm. 2.7. Statistical Analysis The quantitative data are expressed as mean values (± standard deviation). The mean values were compared by using the independent samples t-test. P values of less than 0.05 were considered to be statistically significant.

3. Results and Discussion The technology used to produce calcium nanoparticles

from marine organism shells has rapidly developed, but their suspension stability in an aqueous solution is not yet adequate sufficient. Therefore, it is necessary to develop a new strategy to improve the aqueous stability of calcium particles. In this study, we report that CC NPs produced from ark shells can have an improved aqueous suspension through surface modification with OA and PC. 3.1. Characteristics of PC-OA-CC NPs As Fig. 1 shows, the CC NPs were surface modified into a core-shell structure with OA and PC. The carboxylic groups of the OA interact with the calcium ions through an electrostatic attraction, resulting in the dispersion of the CC NPs in water by reducing their surface tension [12]. After the surface modification, the PC-OA-CC NPs have a coreshell structure with a CC NP core and PC-OA layer (Fig. 2). The thickness of the layer formed by through the interaction of the PC-OA on the surface of the CC NPs was of about 15 ~ 20 nm. The size of the PC-OA-CC NPs was measured via DLS to be 98 ± 10 nm (Fig. 2D). The presence of PC and OA on the surface of the CC NPs was characterized via FT-IR spectroscopy. Fig. 3 shows the FT-IR spectra of the CC NPs and the PC-OACC NPs. The NPs exhibited characteristics peaks of calcium carbonate at 3404, 1419, and 871/cm. After introducing OA and PC on the surface of the CC-NPs, new peaks appeared at 1195, 1710, 2851, and 2910/cm (Fig. 3B). The peaks around 1195 and 1728/cm are attributed to P=O and carboxylic salt, and the strong absorption peaks at 2910 and 2880/cm are a result of the stretching vibration of the alkyl chains, indicating that OA or PC were attached to the surface of the CC NPs [13]. An EDX analysis was then performed to investigate whether the PC-OA-CC NPs were responsible for the peaks in the elemental level energy. Fig. 4 shows the EDX analysis spectrum for the PC-OA-


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Fig. 2. TEM images of (A) the CC NPs, (B) OA-CC NPs, and (C) PC-OA-CC NPs. The scale bars indicate a length of 100 nm. (D) Size distribution of the PC-OA-CC NPs.

Fig. 3. FT-IR spectra of (A) the CC NPs and (B) the PC-OA-CC NPs.

Fig. 4. Energy dispersive X-ray spectroscopic image of the PCOA-CC NPs and FE-SEM image.

Fig. 5. XRD patterns of (A) the CC NPs, (B) OA-CC NPs, and (C) PC-OA-CC NPs.

CC NPs in FE-SEM. The C, O, and Ca elements are present in the sample, indicating calcium carbonate was present in the PC-OA-CC NPs. The major elements observed in the EDX spectrum were CaO at 77% and carbon at 18%. The X-ray diffraction patterns of the CC NPs, OA-CC NPs, and PC-OA-CC NPs are shown in Fig. 5. The diffraction peaks of the CC NPs at the 2-θ positions correspond with calcite crystals (Fig. 5A). The surface modification of the CC NPs with OA and PC was performed sequentially, and the characteristic crystalline peaks at the 2-θ positions decreased dramatically (Figs. 5B and 5C). The thermal behavior of the CC NPs, OA-CC NPs, and PC-OA-CC NPs are shown in Fig. 6. The CC NPs lost about 42% of their weight between 620 and 780°C. After introducing the OA on the surface of the CC

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Fig. 6. TGA curves of the CC NPs (red line), OA-CC NPs (blue dotted line), and PC-OA-CC NPs (green dotted line).

NPs, the weight loss due to heating increased up to 15% of the weight from 200 to 600°C because the OA started to lose weight at 200°C and lost the entirety of its weight at 600°C. The thermogram of the PC-OA-CC NPs shows that the weight loss started at 180°C and the remaining weight was about 20% of total weight of the PC-OA-CC NPs. The thermogravimetric results indicate that OA and PC were successfully introduced on the surface of the CC NPs. 3.2. Suspension stability of PC-OA-CC NPs In order to investigate the suspension stability of the NPs in an aqueous solution, the changes in transmittance of the NPs in solution was determined during 120 h. As the photographs in Fig. 7 show, the CC NPs solution exhibited a small quantity of precipitate and the OA-CC NPs sank completely to the bottom (Figs. 7A and 7B). In contrast, the PC-OA-CC NPs were relatively well suspended in the aqueous solution (Fig. 7C). The measurements of the changes in transmittance revealed that the initial transmittance value of the CC NPs changed by 13%, whereas no transmittance changes were observed in the case of the PC-OA-CC NPs solution (Fig. 7D). The OA-CC NPs sank to the bottom in the aqueous solution because the hydrophobic part of the carbon chain in the OA comes to the front of the NPs, contacting with water [14]. PC can form a hydrophobic interaction with the carbon chain of OA, resulting in the hydrophilic part of the PC coming to the surface of the NPs. Consequentially, a surface modification using PC and OA enables CC NPs to improve their suspension stability in aqueous solution. 3.3. Cell cytotoxicity of PC-OA-CC NPs To evaluate the cytotoxicity of the CC NPs and the PCOA-CC NPs, the viabilities of the treated RAW264.7 cells were determine by using an MTT assay (Fig. 8). Even at a high concentration of 1 mg/mL, the cell viabilities of the

Fig. 7. Photographs of (A) the CC NPs, (B) OA-CC NPs, and (C) PC-OA-CC NPs in aqueous solution. (D) Transmittance changes of the CC NPs (blank circle) and PC-OA-CC NPs (blank triangle) suspended in an aqueous solution at 500 nm for 5 days (n = 3).

Fig. 8. Cell viabilities of the RAW 264.7 cells 48 h after exposure to CC NPs and PC-OA-CC NPs (n = 3, *P < 0.05).

RAW 264.7 cells were of 72.4 ± 8.3 and 89.7 ± 2.3%. When compared to CC NPs at the same concentration, the PC-OA-CC NPs showed a higher cell viability for the RAW264.7 cells. Calcium carbonate microparticles have been previously reported to be slightly cytotoxic [15]. For example, the cell viability of HeLa cells treated with calcium carbonate microparticles was 89.6% at a concentration of 0.4 mg/mL. The results indicate that CC NPs influence the cell viability with a significant improvement as a result of the surface modification with OA and PC.


4. Conclusion CC NPs were surface modified with OA and PC in order to improve their suspension stability in an aqueous solution. OA was attached to the surface of the CC NPs through an ionic interaction between the carboxyl groups of OA and the calcium cations. The PC was introduced to the OA-CC NPs through the hydrophobic interaction between PC and OA. In comparison with CC NPs, PC-OA-CC NPs showed an improvement in their suspension stability in an aqueous solution. Therefore, we expect for PC-OA-CC NPs to become more useful and to increase the range of applications for CC NPs in variety of biological applications.

Acknowledgement This study was financially supported by Chonnam National University in 2013.

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