Dual nutraceutical nanohybrids of folic acid and calcium containing layered double hydroxides

Journal of Solid State Chemistry 233 (2016) 125–132

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Dual nutraceutical nanohybrids of folic acid and calcium containing layered double hydroxides Tae-Hyun Kim, Jae-Min Oh n Department of Chemistry and Medical Chemistry, College of Science and Technology, Yonsei University, Wonju, Gangwondo 220-710, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 10 August 2015 Received in revised form 7 October 2015 Accepted 11 October 2015

Dual nutraceutical nanohybrids consisting of organic nutrient, folic acid (FA), and mineral nutrient, calcium, were prepared based on layered double hydroxide (LDH) structure. Among various hybridization methods such as coprecipitation, ion exchange, solid phase reaction and exfoliation-reassembly, it was found that exfoliation-reassembly was the most effective in terms of intercalation of FA moiety between Ca-containing LDH layers. X-ray diffraction patterns and infrared spectra indicated that FA molecules were well stabilized in the interlayer space of LDHs through electrostatic interaction. From the atomic force and scanning electron microscopic studies, particle thickness of LDH was determined to be varied with tens, a few and again tens of nanometers in pristine, exfoliated and reassembled state, respectively, while preserving particle diameter. The result confirmed layer-by-layer hybrid structure of FA and LDHs was obtained by exfoliation-reassembly. Solid UV–vis spectra showed 2-dimensional molecular arrangement of FA moiety in hybrid, exhibiting slight red shift in n-π* and π-π* transition. The chemical formulae of FA intercalated Ca-containing LDH were determined to Ca1.30Al(OH)4.6FA0.74
3.33H2O and Ca1.53Fe(OH)5.06FA2.24
9.94H2O by inductively coupled plasma-atomic emission spectroscopy, high performance liquid chromatography and thermogravimetry, showing high nutraceutical content of FA and Ca. & 2015 Elsevier Inc. All rights reserved.

Keywords: Calcium containing layered double hydroxide Folic acid Nutraceutical nanohybrids Intercalation Exfoliation-reassembly

1. Introduction Layered double hydroxide (LDH), a family of anionic clays, is one of the attractive materials in nanotechnology for bio-medical applications [1–5]. The structure of LDH is based on 2-dimensional nanolayers consisting of edge sharing polyhedrons of divalent and trivalent metal hydroxides. The nanolayers are known to possess positive layer charge with high density (2.5–4.2( þ)/nm2), which was compensated by interlayer anions [6]. LDHs have crystalline structure along crystallographic ab-plane through strong coordination bond of M–OH, whereas their c-axis direction has layerby-layer stacking through relatively weak electrostatic interaction. Thus LDHs can be hybridized with various kinds of anionic species, almost regardless of size, in their interlayer spaces through electrostatic interaction while preserving their 2-dimensional crystallinity. Due to their unique anionic capacity, during past couple of decades, there have been extensive researches on hybridizing biofunctionalized anionic molecules such as nucleotide phosphates [7], deoxyribonucleic acids [8], antisense oligonucleotides [9], n

Corresponding author. Fax: þ 82 33 760 2182. E-mail address: [email protected] (J.-M. Oh).

http://dx.doi.org/10.1016/j.jssc.2015.10.019 0022-4596/& 2015 Elsevier Inc. All rights reserved.

peptides [10], anti-inflammatory drugs [11,12], anticancer drugs [13], and vitamins [14,15] with nano-sized LDHs consisting of Mg (II), Zn(II) and Al(III) for safe and effective delivery of payload drugs. Recently, researchers became to be interested in developing nutrients delivery system, nutraceutical hybrids, with bio-LDH nanohybrids [16,17]. In terms of nutraceutical development, one of the best strategy is to incorporate nutrient moiety into LDHs which already consists of essential mineral components like calcium. However, due to unique hydrolysis behavior of Ca(II) [18,19], studies on Ca-containing LDHs for bio-LDH hybrids has been limited [20,21]. While Mg(II), Zn(II) and Cu(II) precipitates hydroxide at pH  10, 7 and 9, respectively, Ca(II) only forms hydroxide in highly basic condition (pH 411), which in other words stands for the possible dissolution of Ca-containing LDH in neutral pH. Thus, general hybridization methods for LDHs such as coprecipitation and ion-exchange could not be easily applied for Cacontaining LDHs. In our previous work, solid phase intercalation was applied to incorporate taurine molecules into CaAl- and CaFeLDHs to avoid dissolution of LDHs [20]. Leroux et al. reported alternative method like reconstruction to intercalate chloride ion into CaAl-LDHs [22]. In this paper, we are going to demonstrate the hybridization of folic acid (FA) and Ca-containing LDHs(CaM(III)-LDHs), in order to prepare nutraceutical nanomaterials having organic and mineral

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nutrient together. We chose FA as an organic nutrient, as it is an essential nutrient involved in DNA synthesis and cell proliferation, and its deficiency induces glossitis, depression or even serious diseases like fetal neural tube defect and brain defect during pregnancy [23–25]. Host LDHs were chosen to contain Ca(II), as it is an essential mineral nutrient in biological structure and signaling [26]. In order to introduce FA molecules into interlayer space of LDHs, we attempted various methods such as coprecipitation, ion-exchange, solid phase intercalation and exfoliation-reassembly. We investigated structure, stability and chemical composition of prepared FA/LDH nanohybrids with X-ray diffraction, thermal analysis, electron microscopies, high performance liquid chromatography, infrared and UV–vis spectroscopies.

2. Experimental 2.1. Materials Calcium nitrate tetrahydrate (Ca(NO3)2
4H2O) was purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan); aluminum nitrate nonahydrate (Al(NO3)3
9H2O), iron nitrate nonahydrate (Fe(NO3)3
9H2O) and folic acid (C19H19N7O6: FA) were obtained from Sigma Aldrich Co. LLC. (USA); sodium hydroxide (NaOH) pellet and formamide (CH3NO) were obtained from Daejung Chemicals & Metals Co., Ltd. (Gyeonggido, Korea) and used without further purification. Salt type FA, Na þ -folate, was prepared by dissolving it with equivalent amount of sodium hydroxide solution and utilized for hybridization.

2.4. Structural characterizations and quantification Powder X-ray diffraction patterns of all the prepared samples were obtained with Bruker AXS D2 Phaser with increments of degree and time step of 0.02° and 1 s/step, respectively. Fourier transform infrared (FT-IR; Perkin Elmer, spectrum one B.v5.0) spectroscopy was performed with conventional KBr method. The UV–vis absorbance spectra of all solid samples were obtained with UV spectrophotometer (Thermo EVOLUTION 220). The chemical formulae were obtained by inductively coupled plasma-atomic emission spectroscopy (ICP-AES: Perkin Elmer Optima-4300 DV), high performance liquid chromatography (HPLC) and thermogravimetric analysis (TG; TA Instruments SDT2960). The TG analysis was carried out under N2 atmosphere. FA quantification in each hybrid was evaluated with HPLC (Younglin, YL9100 HPLC with UV–vis detector) with C18 column (ZOBAX Eclipse, 4.6 mm × 150 mm, Agilent). Each hybrid (∼10 mg) was dissolved into 20 mL of phosphate buffer solution (pH ∼2) by sonicating for 15 min. The mobile phase of 0.1 M KH2PO4:methanol ¼90:10 was utilized and flow rate 1.0 mL/min was applied. The chromatogram was operated at 25 °C and collected at 282 nm wavelength. The size and morphology of pristine LDHs, exfoliated sheets and nanohybrids were obtained by scanning electron microscope (SEM; FEI Quanta 250 FEG) and transmission electron microscope (TEM; FEI TECNAI G2 F30 ST at Korea Basic Science Institute in Seoul). In order to verify the particle diameter and thickness of LDHs during exfoliation-reassembly, atomic force microscopy (AFM; Park systems NX10) was utilized.

2.2. Preparation of CaAl- and CaFe-LDHs

3. Result and discussions

For the preparation of CaAl and CaFe-LDHs, mixed metal nitrate solution (0.315 M of Ca(NO3)2
4H2O; 0.1575 M of Al(NO3)3
9H2O for CaAl- and Fe(NO3)3
9H2O for CaFe-LDHs) was titrated with NaOH solution (1.26 M) until pH reached coprecipitation region, 11.5 and  13.0 for CaAl- and CaFe-LDH, respectively, as previously reported [19]. The obtained precipitates were aged for 24 h with vigorous stirring under N2 atmosphere. Then the products were filtered and washed with decarbonated water and dried in vacuum at 40 °C.

In order to evaluate structure of FA/LDH nanohybrids prepared by various synthetic methods, powder X-ray analysis was carried out first. As shown in Fig. 1A(a) and 1B(a), both pristine CaAl- and CaFe-LDHs exhibited well-developed (00l) diffraction patterns and lattice peaks, which well corresponded to those of CaAl-LDH (hydrocalumite, ICSD ID: 54372) as previously reported [19]. The (00l) diffraction peaks of CaFe-LDH were slightly translocated to the lower angle region compared with hydrocalumite reference, which was due to the difference of ionic radius between Al3 þ (53.5 pm) and Fe3 þ (64.5 pm) [28]. Coprecipitation was not determined to be effective in hybridizing FA and Ca-containing LDHs, as the XRD patterns were amorphous (Fig. 1A(c), B(c)). Acidic property of FA might disturb effective formation of CaAl- or CaFe-hydroxide frameworks during coprecipitation process, giving rise to amorphous phase [29]. Nanohybrids prepared by ion exchange reaction exhibited (00l) peaks at low angle for FA/CaAl-I-LDH (Fig. 1A(d)), while amorphous phase was obtained for FA/CaFe-I-LDHs (Fig. 1B(d)). Ca-containing LDHs could be partially dissolved in neutral reaction media due to high basicity of Ca-LDHs. The basicity of CaFe-LDH was stronger than CaAl-LDH considering coprecipitation pH of 11.5 and 13.0 for CaAl- and CaFe-LDH. Thus, the CaFe-LDH could be more dissolved and lose structural integrity during ion exchange process. The peak position of (002) in FA/CaAl-I-LDH was 5.34 °, which corresponded to interlayer space of 11.9 Å, implying that FA molecules (18.9 Å) were arranged with tilting angle ∼51°. The XRD patterns of FA/ CaAl-S- and FA/CaFe-S-LDHs (Fig. 1A(e) and 1B(e), respectively) showed no change in peak positions compared with pristines, showing intercalation did not occur effectively. Although intercalation of small molecules through solid phase reaction was reported effective for CaM(III)-LDHs from previous report [20], it was not effective for large molecules like FA. Exfoliation-reassembly was the most successful in intercalating FA into both CaAl- and CaFe-LDH. The (002) peaks of FA/CaAl-E- and FA/CaFe-E-LDH at

2.3. Hybridization of folic acids with LDHs For coprecipitation method, metal nitrate and folate (Ca/Al/FA and Ca/Fe/FA molar ratio¼2/1/5) solutions were mixed and titrated with NaOH solution until pH reaches ∼11.5 and  13.0 for CaAl- and CaFeLDHs, respectively. Obtained slurry was aged for 24 h under N2 atmosphere. The resulting products were collected by centrifugation and washed several times with decarbonated water and then lyophilized. For ion-exchange reaction, powdered CaAl- and CaFe-LDH were dispersed into folate solution (FA/M(III)¼5/1 M ratio) and then stirred for 24 h under N2 atmosphere. The obtained product was washed several times with decarbonated water and lyophilized. In solid phase reaction, pristine LDH and Na þ -folate powder (FA/M (III)¼ 1/1 M ratio) was located in agate mortar and ground for 5 min adding 5 μL of decarbonated water according to previous report [20]. The mixture was dried for 12 h in vacuum at 40 °C. For exfoliationreassembly method, pristine LDH was exfoliated into nanosheets in formamide solution (1 mg/mL). After vigorous stirring for 24 h under N2 atmosphere [27], folate solution was added to the LDH nanosheet colloid and again stirred for 24 h. The product was washed with decarbonated water and dried in vacuum at 40 °C. Nanohybrids were denoted as FA/CaAl-x-LDH or FA/CaFe-x-LDH, where x stands for hybridization methods; C: coprecipitation, I: ion-exchange, S: solid phase reaction. E: exfoliation-reassembly.

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Fig. 1. Powder X-ray diffraction patterns of (A) (a) pristine CaAl-LDH, (b) FA and nanohybrids of (c) FA/CaAl-C-, (d) FA/CaAl-I-, (e) FA/CaAl-S-, and (f) FA/CaAl-E-LDH. (B) (a) pristine CaFe-LDH, (b) FA and nanohybrids of (c) FA/CaFe-C-, (d) FA/CaFe-I-, (e) FA/CaFe-S-, and (f) FA/CaFe-E-LDH.

(Fig. 1A(b) and B(b)), we could also suggest that FA molecules existed in the nanospace of LDH interlayers, not aggregated on the surface of LDHs. Quantitative analyses on prepared hybrids also suggested that exfoliation-reassembly was effective in FA intercalation into CaM (III)-LDH hybrids. Table 1 showed both metal ratio and FA content in each hybrid. Coprecipitation did not result in FA/CaFe-LDH nanohybrid, without showing significant amount of Ca and FA in the nanohybrid. FA/CaM(III)-I-LDH showed FA content higher than 50 wt/wt%, however, Ca/M(III) ratio significantly decrease, suggesting significant dissolution of LDH pristine during intercalation. Although FA/CaM(III)-S-LDH almost maintained metal ratio compared with pristine, their FA content was fairly low. Considering that XRD pattern of FA/CaM(III)-S-LDH was similar to that of pristine, the FA moiety in the nanohybrids exist at the surface of LDHs. Only exfoliation-reassembly showed meaningful quantification results in terms of metal ratio and FA content. As the exfoliation-reassembly was proven to be the most suitable method for FA intercalation into Ca-containing LDHs, we Table 1 Metal ratio and FA contents in FA/CaAl- and FA/CaFe-LDHs prepared with various synthetic methods.

Scheme 1. Molecular structure of folic acid (FA) and the schematic illustration of FA arrangement in the interlayer space of FA/CaM(III)-E-LDH.

6.11° and 6.06° (Fig. 1A(f) and 1B(f)) corresponded to interlayer spaces of 9.9 Å and 10.0 Å, respectively, standing for tilted orientation of interlayer FA with angle ∼58° along crystallographic c-axis (Scheme 1). As FA/LDH nanohybrids obtained with exfoliation-reassembly reaction did not show XRD pattern of FA itself

Sample

Ca/M(III) molar ratioa

FA content in hybrid (wt/wt%)b

CaAl-LDH CaFe-LDH FA/CaAl-C-LDH FA/CaFe-C-LDH FA/CaAl-I-LDH FA/CaFe-I-LDH FA/CaAl-S-LDH FA/CaFe-S-LDH FA/CaAl-E-LDH FA/CaFe-E-LDH

2.04 2.01 1.45 0.001 1.29 0.78 1.70 1.97 1.30 1.53

N.D. N.D. 42.76 N.D. 66.93 59.85 38.95 39.85 60.00 72.20

a

Metal ratios were measured on dissolved pristine or hybrid solution with ICP-

AES. b

FA content in hybrid was evaluated with HPLC on dissolved hybrid solution.

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Fig. 2. Fourier transform infrared spectra for (a) FA (b) Na þ -folate salt (c) CaAlLDH, (d) FA/CaAl-E-LDH nanohybrid, (e) CaFe-LDH and (f) FA/CaFe-E-LDH nanohybrids. The dotted lines are positioned at 1696, 1610, 1510, 1496, 1409, and 1390 cm  1.

further investigated chemical bonding of FA/CaAl-E- and FA/CaFeE-LDH nanohybrids with infrared spectroscopy. From the FT-IR spectra, we confirmed that the intact structure of FA was well preserved and they were chemically stabilized by LDHs through electrostatic interaction (Fig. 2). Molecular structure of folic acid is divided into three regions of pterin, p-aminobenzoic acid (PABA) and glutamic acid (Scheme 1). Prior to reassembly reaction, the glutamic acid residues were deprotonated, while preserving pterine and PABA moiety, for interaction with positively charged LDH layers. From the IR spectra (Fig. 2), we found the characteristic stretching vibrations of pterine and PABA (–NH2 (1610 cm  1), phenyl (1496 cm  1), and –NH– (1409 cm  1)) in the spectra of FA, Na þ -folate, and FA/LDH nanohybrids. The peaks corresponding to –COOH (in glutamic acid) were observed at 1696 cm  1 of which intensity decreased in Na þ -folate and FA/CaM(III)-E-LDH hybrids compared with FA alone. On the other hand, IR modes which were attributed to carboxylate such as νsym(COO  ) and νantisym(COO  ) appeared at around 1390 and 1510 cm  1 in both Na þ -folate and FA/CaM(III)-E-LDH hybrids. We calculated the percentage of ionic bonding (PIB) between carboxylate and LDHs utilizing the spectra of Na þ -folate as reference [30]. The PIB value for FA/CaAl-E-LDH and FA/CaFe-E-LDH were determined to be 0.94 and 0.95, respectively, verifying the formation of strong electrostatic interaction between LDH layers and glutamate residues. Figs. 3 and 4 showed microscopic analysis results for pristine LDHs and exfoliated ones. The exfoliation process was carried out in formamide condition according to the Sasaki et al.'s report [27]. The TEM images showed morphology of multi-layer stacking for pristine LDHs and a few layer stacking for exfoliated ones (Fig. 3). The inset images indicating selected area electron diffraction (SAED) patterns exhibited hexagonal spot patterns for exfoliated sheets. It has been reported that LDH with stacking structure showed SAED pattern of either several non-superimposable hexagonal spot patterns [31] or ring pattern [32]. On the other hand, that of exfoliated layer showed weak but hexagonal spot pattern

Fig. 3. Transmission electron microscopic images of (a) CaAl-LDH, (b) CaFe-LDH and exfoliated nanosheets of (c) CaAl-LDH, (d) CaFe-LDH (Inset: selected area electron diffraction pattern).

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129

Fig. 4. Atomic force microscopic images of (a) CaAl-LDH, (b) CaFe-LDH and exfoliated nanosheets of (c) CaAl-LDH, (d) CaFe-LDH (left: AFM images, right: height profiles).

coming from single layer diffraction [33]. The AFM results also supported that the LDHs were exfoliated into a few stacking of nanosheets. The height profile in Fig. 4 showed the particle diameter of ∼500 and 600 nm and thickness of ∼50 and ∼70 nm, for

pristine LDHs. After exfoliation, particle thickness significantly decreased to 6–8 nm and 2–8 nm for CaAl- and CaFe-LDH. Considering the single layer thickness of Ca-containing LDHs of ∼0.46 nm [20], the exfoliation of LDHs from hundred to a few

Fig. 5. Scanning electron microscopic images of (a) CaAl-LDH, (b) exfoliated CaAl-LDH, (c) FA/CaAl-E-LDH, (d) CaFe-LDH, (e) exfoliated CaFe-LDH and (f) FA/CaFe-E-LDH.

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Fig. 6. Solid UV–vis spectra for (a) FA (b) CaAl-LDH, (c) FA/CaAl-E-LDH, (d) CaFe-LDH and (e) FA/CaFe-E-LDH (solid line: obtained peak, dotted line: fitted peaks).

sheets was successfully preformed. Although, there seemed a slight destruction of layers in ab-plane directions, the delamination along c-axis was proven to be more significant. In order to examine the change in size and morphology of LDHs during exfoliation-reassembly reaction, we checked the SEM images at each step (Fig. 5). The average particle diameter of pristine CaAl- and CaFe-LDH were determined to be ∼500 and ∼400 nm and those of exfoliated ones were ∼200 nm, which was in good agreement with the results obtained from AFM. The morphology of pristine LDHs showed typical shape of layered

materials having large aspect ratio (diameter/thickness). After exfoliation, the aspect ratio became larger showing more platelike morphology. The final particle morphology of FA/CaM(III)-ELDH nanohybrid was relatively different from the pristine exhibiting sphere like shape, which might be attributed to the random stacking of LDH layers in the presence of FA molecules. Solid UV–vis spectra was utilized to evaluate intermolecular arrangement of FA moiety (Fig. 6). Pristine CaAl-LDH showed two absorption peaks at UV region (∼210 and 300 nm) which were attributed to the UV absorption of LDH lattice as previously

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reported [1]. In addition to UV absorption (280 nm), pristine CaFeLDH exhibited visible light absorption (∼500 nm) which was attributed to the allowed d–d transitions in Fe(OH)6 [34]. It has been reported that folic acid have several distinct UV–vis absorption at around ∼270 and ∼380 nm due to π-π* transition in carboxylate and n-π* transition in enone (Fig. 6(a)) [1]. All the three characteristic absorptions were found in FA/CaAl-E-LDH and FA/CaFeE-LDHs showing the preservation of intact FA structure. We could find a small red shift for the absorption band of π-π* transition in carboxylate and n-π* transition in enone. According to the previous studies, the red-shift of organic chromophore could be observed when molecules lost 3-dimensional crystallinity or they strongly interacted with counter-charged moiety through electrostatic attraction [1,35]. Thus, we could suggest that the FA molecules in FA/CaM(III)-E-LDH nanohybrids obtained by exfoliation-reassembly were well arranged in 2-dimensional arrays either between layers or at the surface of LDH. In order to determine the chemical formulae of nanohybrids and to evaluate the thermal stability of FA molecules in the LDH lattices, we carried out thermogravimetry analyses under N2 atmosphere. According to our previous study [19], CaAl- and CaFeLDH showed three main steps of thermal decomposition in the range of 25–100 °C, 100–250 °C and above 250 °C, attributed to dehydration of surface water, removal of interlayer water and dehydroxylation accompanied with decomposition of interlayer ion, respectively. The two nanohybrids showed similar temperature-dependent weight loss compared with its pristine; on the other hand, its weight loss above ∼250 °C was higher in amount and more complicated in pattern. The first two steps from 25 °C to ∼250 °C were attributed to the dehydration of surface absorbed and interlayer water, while the latter steps reflected the thermal decomposition of folic acids along with the dehydroxylation of LDH framework [36,37]. TG curve of FA itself showed three steps of decomposition in the range of 25–140 °C, 140–370 °C and above 370 °C. The first step was attributed to dehydration and the latter two steps were originated from sequential decomposition of glutamic acid residues, PABA and pterin groups (Fig. 7(c)) [37]. In the nanohybrids, decomposition of glutamate was retarded due to the electrostatic interaction with LDH layers, showing shifted temperature at ∼220 °C. Although it was not easy to clearly assign each step of thermal decomposition above 220 °C, this step includes thermal decomposition of FA moiety along with dehydroxylation. Taking into account metal ratio results obtained from ICP-AES and organic content information from TG and HPLC, we could establish the chemical formulae of Ca1.3Al(OH)4.6(FA)0.74
3.33H2O and Ca1.53Fe(OH)5.1(FA)2.24
9.94H2O for FA/CaAl-E-LDH and FA/ CaFe-E-LDH nanohybrids, respectively. It is worthy to note here that the content of folic acid in the nanohybrid was higher than expected. As an FA molecule has two carboxylate centers, the ideal molar ratio between trivalent metal and FA would be 2/1. In exfoliation-reassembly process, however, it might be possible that more anionic molecules than expected can be incorporated into the nanohybrid due to the random stacking of LDHs like in houseof-cards structure [38]. The Ca:M(III) ratios in the hybrids were slightly lower (1.30:1 and 1.53:1 for CaAl- and CaFe-hybrid) than those of pristine ones ∼2:1 (Ca2.04Al(OH)6(NO3)
5.25H2O and Ca2.01Fe(OH)6(NO3)
4.75H2O), which accounted the partial dissolution of LDH lattices, especially from the Ca(OH)7 moiety, during the exfoliation-reassembly reaction. However, this partial dissolution during exfoliation-reassembly was not serious, considering the previous report in which Ca-containing LDH lattices showed upto ∼50% of dissolution at pH 7 during 24 h [21]. According to the quantitative analyses, the contents of nutrient components, FA and Ca were determined to be 60 and 9.5 wt% for FA/CaAl-E-LDH and 72 and 4.5 wt% for FA/CaFe-E-LDH.

131

Fig. 7. Thermogravimetry and differential thermal analysis results for (a) FA/CaAlE-LDH, (b) FA/CaFe-E-LDH and (c) FA itself.

4. Conclusion Folic acid, which is an essential nutrient in biological structure and cellular signaling, was successfully intercalated into CaAl- and CaFe-LDHs for dual nutraceutical nanohybrids, as Ca(II) is an essential inorganic element in biological system. Among various intercalation routes, exfoliation-reassembly reaction was determined to be the most effective. The microscopic analyses including TEM, AFM and SEM showed that exfoliation of pristine LDHs into several sheets and random reassembly with FA moiety resulted in FA/LDH nanohybrids. XRD, IR and UV–vis spectra verified the stabilization of FA molecules in the LDH interlayer space

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without disintegration of FA moiety. Through thermogravimetry, it was also verified that the FA molecules acquired thermal stability compared to FA alone due to the electrostatic stabilization in LDH nanohybrids obtained by exfoliation-reassembly. Considering that FA/LDH nanohybrids showed high contents of essential nutrients in human health, i.e. FA and Ca, the FA/LDH nanohybrids prepared by exfoliation-reassembly could be considered as dual nutraceutical nanomaterials.

Acknowledgment This work was supported by a grant from the Postharvest Research Project (PJ01050201) of RDA, Republic of Korea.

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