Stabilization of antioxidant gallate in layered double hydroxide by exfoliation and reassembling reaction

Accepted Manuscript Stabilization of antioxidant gallate in layered double hydroxide by exfoliation and reassembling reaction Kanakappan Mickel Ansy, Ji-Hee Lee, Huiyan Piao, Goeun Choi, Jin-Ho Choy PII:

S1293-2558(18)30368-6

DOI:

10.1016/j.solidstatesciences.2018.04.001

Reference:

SSSCIE 5671

To appear in:

Solid State Sciences

Received Date: 31 March 2018 Accepted Date: 2 April 2018

Please cite this article as: K.M. Ansy, J.-H. Lee, H. Piao, G. Choi, J.-H. Choy, Stabilization of antioxidant gallate in layered double hydroxide by exfoliation and reassembling reaction, Solid State Sciences (2018), doi: 10.1016/j.solidstatesciences.2018.04.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Stabilization of antioxidant gallate in layered double hydroxide

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by exfoliation and reassembling reaction

Kanakappan Mickel Ansy, Ji-Hee Lee, Huiyan Piao, Goeun Choi,* and Jin-Ho Choy**

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Center for Intelligent Nano-Bio Materials (CINBM), Department of Chemistry and

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Nano Science, Ewha Womans University, Seoul 03760, Republic of Korea

** Corresponding author: Prof. Jin-Ho Choy (E-mail: [email protected]) * Corresponding author: Dr. Goeun Choi (E-mail: [email protected])

ACCEPTED MANUSCRIPT ABSTRACT As for the stabilization of chemically sensitive bioactive molecule in this study, gallic acid (GA) with antioxidant property was intercalated into interlayer space of layered double

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hydroxide (LDH), which was realized by exfoliation and reassembling reaction. At first, the pristine nitrate-type Zn2Al-LDH in solid state was synthesized via co-precipitation followed by the hydrothermal treatment at 80 °C for 6 h, and then exfoliated in formamide to form a

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colloidal solution of exfoliated LDH nanosheets, and finally reassembled in the presence of GA to prepare GA intercalated LDH (GA-LDH) desired, where the pH was adjusted to 8.0 in

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order to deprotonate GA to form gallate anion. According to the XRD analysis, GA-LDH showed well-developed (00l) diffraction peaks with a basal spacing of 1.15 nm, which was estimated to be larger than that of the pristine LDH (0.88 nm), indicating that gallate molecules were incorporated into LDH layers with perpendicular orientation. From the FT-IR

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spectra it was found that gallic acid was completely deprotonated into gallate, and stabilized in between LDH lattices via electrostatic interaction. The content of GA in GA-LDH was determined to be around 23 wt% by UV-vis spectroscopic study, which was also confirmed

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by HPLC analysis. According to the in-vitro release of GA out of GA-LDH in PBS solution (pH 7.4) at 4 ºC, GA was sustainably released from GA-LDH nanohybrid up to 86% within

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72h. The antioxidant property of GA-LDH was almost the same with that of intact GA which was examined by DPPH. The photostability of GA-LDH under UV light irradiation was immensely enhanced compared to intact GA. It is, therefore, concluded that the present GALDH nanohybrid can be considered as an excellent antioxidant material with high chemicaland photo- stabilities, and controlled release property.

Keyword: Layered double hydroxide; Gallic acid; Controlled release; Antioxidant activity; Photostability.

ACCEPTED MANUSCRIPT 1. Introduction Gallic acid (3,4,5-trihydroxybenzoic acid), a strong antioxidant and effectual apoptosis persuading agent, is a naturally occurring low molecular weight triphenolic

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compound which can be found in apple-peels, wines and etc. The byproducts of GA have also been used in a number of phytomedicines with wide-ranging pharmacological and biological actions, with scavenging, interfering with the cell signaling trails [1,2]. As well

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known, GA has a remarkable radical scavenging characteristics, which is a vital part in the aversion of tumor advancement [2-4]. According to the in-vitro studies, apoptosis actuated

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by GA is related to the oxidative procedures induced, and antioxidant activity of GA is linked to its antiviral capacity [5]. GA is, however, very sensitive to pH, and can easily be oxidized to form semi-quinone even under a mild condition, resulting in the decrease of biological functionality. And moreover, GA is not stable in plasma, and therefore, it is highly required to

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stabilize thermodynamically in order to protect it from degradation [6,7]. To overcome such problems, we attempted to encapsulate GA molecules in the interlayer space of LDH to explore single phasic GA-LDH nanohybrid.

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Ion exchangeable 2-demensional (2D) compound like LDH has been known to be not only good in biocompatiblity and solubility, but also high in cellular uptake and tissue

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targeting function, and therefore, proposed as a host reservoir to encapsulate fragile bioactive molecule like GA to form GA-LDH nanohybrid to improve its chemical and biological functions

[8-11].

The

chemical

formula

of

LDHs

is

generally

expressed

as

M(II)1−??M(III)??(OH)2(A??−)??/?? ⋅ ??H2O (M: divalent and trivalent metal ions, A: anionic molecules, ?? and ??: integer, 0 < ?? < 1). The individual metal hydroxide sheets are electrostatically stacked on the top of each other with interlayer anions along the out of plane direction to form a lamellar compound with 1 : 1 heterostructure. When a part of M(II) cations in M(II)(OH)2 sheet are isomorphously replaced with M(III) one, the positive layer

ACCEPTED MANUSCRIPT charge can be generated [12]. Such a layer charge is remunerated by interlayer anion to satisfy the charge neutrality condition. One thing to note here is that the interlayer anion is exchangeable, and therefore, various biomolecules such as anticancer drugs, antibiotics,

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vitamins and antioxidants could be intercalated into LDHs for pharmaceutical, cosmeceutical and neutraceutical applications [12-16]. As well documented [9-11], intercalated anions can be chemically stabilized by electrostatic interaction, and also physically and biologically

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protected by the metal hydroxide sheets from exterior harsh environments, and moreover controllably released out from the LDH lattice upon anion exchange reaction. Therefore,

biomedical applications [9-11].

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many attempts have been made to intercalate bio-functional molecules into LDHs for

To date, a number of synthetic routes to organic-LDH nanohybrids, such as coprecipitation, reconstruction, ion exchange and subsequent hydrothermal treatment

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[12,17,18], have been suggested, but some hydrophobic organic guests with stereo-chemical complexity could hardly been intercalated into the LDH lattice. According to the conventional ion exchange and co-precipitation routes, the GA anions could not be well

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intercalated into interlayer spaces of LDHs [3,19-21], whatever the synthetic routes were the co-precipitation or the ion-exchange [19,20] probably due to the kinetic hindrance in

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intercalation reaction.

An exfoliation and reassembling route [22-24] has been proposed as an alternative

way of preparing GA-LDH nanohybrid. In the present study, efforts have been made to prepare chemically and structurally well-defined GA-LDH nanohybrid, understand chemical stability and controlled release behavior of GA from host LDH, and eventually evaluate its antioxidant activity as well as photostabilities.

2. Experimental section

ACCEPTED MANUSCRIPT 2.1. Materials Gallic acid monohydrate [C6H2(OH)3CO2H·H2O, purity > 97%, molecular weight of 188.14 g/mol] and formamide (HCONH2) was purchased from Junsei, Japan, and used

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without further purification treatments. Chemicals including, zinc nitrate hexahydrate [Zn(NO3)2·6H2O, purity > 98.0%], aluminum nitrate nonahydrate [Al(NO3)3·9H2O, purity >

2.2. Preparation of the pristine Zn2Al-NO3-LDH

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98.0%], sodium hydroxide (NaOH) were purchased from Daejung Co. Ltd., Korea.

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By the direct co-precipitation and subsequent hydrothermal treatment, the pristine LDH was prepared. Under a nitrogen atmosphere, a mixed solution of ZnNO3.6H2O (0.012M) and AlNO3.9H2O (0.006M) was titrated with 0.5M NaOH solution. The solution pH was adjusted to 7.8 ± 0.2. The subsequent white precipitate was aged for 1 h, collected by

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centrifugation, washed with decarbonated water, and additionally treated under a hydrothermal condition at 80 °C for 6 h. And the products were finally freeze-dried after

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hydrothermal treatment.

2.3. Exfoliation-reassembly route to GA-LDH nanohybrid (GA-LDH-ER)

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For the exfoliation and reassembly reaction (Scheme 1), the hydrothermally treated white precipitate, the pristine LDH, was first dispersed in formamide (1 g/100 ml) and mechanically stirred for 48 h, and thus prepared colloidal suspension was then centrifuged with 2000 rpm for 5 min in order to separate homogeneously exfoliated and suspended nanosheets with Tyndall effect (Fig. S1) from unexfoliated precipitates, and finally used further for reassembling process. The colloidal suspension of exfoliated LDH nanosheets was added to an aqueous solution of GA with the given concentrations of 1.0, 1.5 and ~ 2.0 times excess of anionic exchange capacity (AEC) of pristine LDH, respectively. And all the

ACCEPTED MANUSCRIPT reassembling reactions took for 3 h at around 4 °C in order to avoid the decomposition of GA under an ambient condition [25]. To remove the nitrate ions externally attached and the GA ions unreacted, the resulting GA-LDH nanohybrid was carefully washed 5 times with a

dried.

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2.4. Ion Exchange route to GA-LDH nanohybrid (GA-LDH-I)

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mixed solution of decarbonated water and ethanol (1:1) solution, and then finally freeze-

An attempt was also made to prepare GA-LDH nanohybrid by ion-exchange reaction

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method (Scheme 1). At first, the GA solution was prepared by dissolving 1.06 g of GA in a mixed solution of 50 ml ethanol and water (1:1) at pH 7.8 ± 0.2, which was adjusted with 0.5 M NaOH, and then added to an aqueous suspension of 50 mL containing 1.0 g pristine LDH. In nitrogen atmosphere, the mixed solution was vigorously stirred for 24 h at room

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temperature. The subsequent products were carefully washed 5 times with a mixed solution of decarbonated water and ethanol (1:1), and then freeze-dried.

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2.5. Co-Precipitation route to GA-LDH nanohybrid (GA-LDH-C) The co-precipitation reaction (Scheme 1) for the formation of GA-LDH nanohybrid

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was performed by slowly adding an aqueous solution (50 : 50 v/v) of GA into a mixed solution of Zn(NO3)2·6H2O and Al(NO3)3·9H2O with a ratio of Zn/Al = 2 under a nitrogen flow condition for 24 h at room temperature. By adding an aqueous solution of 0.5 M NaOH simultaneously, the solution pH could be maintained in the range of 7.8 ± 0.2. The products were then washed 5 times with a mixed solution of decarbonated water and ethanol (1:1), and then freeze-dried.

2.6. Characterization of GA-LDH Nanohybrids

ACCEPTED MANUSCRIPT The structural analyses for GA-LDHs and the pristine LDH were made from the (00l) diffraction peaks obtained by Powder X-ray Diffractometer (Rigaku D/MAX RINT 2200Ultima+, Japan) with Ni-filtered Cu-Kα radiation (λ = 1.5418 Å). The diffractometer was

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worked at 40 kV and 30 mV. For Fourier transfer infrared (FT-IR) spectroscopic analysis, the samples were prepared on the basis of standard KBr disk method, and analyzed in the range of 400-4000 cm-1 with JASCO FT-IR 6100 spectrometer (JASCO, Japan). Atomic force

2.7. Determination of GA content

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microscope in tapping mode (Digital Instrument, USA).

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microscopy (AFM) image was obtained using a Veeco Dimension 3100 scanning force

To determine the content of GA, the GA-LDH-ER powder sample was dissolved in an acidic solution of 0.1 M HCl : EtOH, and then extracted with 0.45 µm PVDF filter. The

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content of GA was then determined by UV-visible spectroscopy, and also cross-confirmed by high performance liquid chromatography (HPLC, Agilent 1100 series Instrument, USA) with a column Zorbax SB RP-C18 (250 mm × 4.6 mm, USA). The flow rate and injection volume

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was 0.9 ml/min and 20 µl, respectively. The column temperature was adjusted to 25 °C and the UV absorbance was measured at 273 nm. The mobile phase was prepared with 0.1%

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acetic acid solution (pH 3.2) and acetonitrile (95:5, v/v).

2.8. In-vitro release test

The in-vitro release profiles of GA from GA-LDH and the solubility of intact GA

were obtained by the paddle stirring method with a dissolution tester DST-810 of LABFINE (Korea) at 4 ºC. The impeller was set at 100 rpm and the pH of phosphate-buffered saline (PBS) media was adjusted to 7.4. Each sample with an equivalent amount of 11.5 mg of GA was dispersed in 300 ml of dissolution media. An aliquot of each sample was collected at

ACCEPTED MANUSCRIPT scheduled intervals, extracted with a 0.45 µm pore PVDF membrane filter (Pall Corporation, USA), and then examined. The released amount of GA was decided cyclically with UVvisible spectrum at an absorption peak of 273 nm (λmax = 273 nm), and also confirmed by

2.9. Antioxidant activity test of GA-LDH

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HPLC measurements as described above.

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Antioxidant tests for intact GA and GA-LDH nanohybrid were determined on the basis of radical scavenging activity by DPPH. The solutions with lower concentration of

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intact GA and GA-LDH (0.5 µg/ml to 20 µg/ml) in ethanol were prepared by serially diluting the stock solution. The 0.1 mM solution of DPPH in ethanol (3.94 mg/100 ml Ethanol) was freshly prepared, and added to the samples, GA or GA-LDH, with different concentration in an equal volume (2 ml) to the ethanol solution of DPPH. All the experiments were performed

[19,26,27].

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in a dark box for 30 min at room temperature, the absorbance was recorded at 517 nm

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2.10. Photostability test of GA-LDH

The UV light stability for intact GA and GA-LDH nanohybrid was measured under

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UV lamp (UVB, 15 W: Black Light Lamp, Japan), and compared with that for intact GA. Each sample containing GA equivalent to 6 mg was dispersed in 10 ml ethanol, and irradiated with UVB for 12 h, and analyzed by HPLC as described in section 2.7.

3. Results and Discussion 3.1. Powder X-ray diffraction analysis As shown in the powder XRD patterns (Fig. 1) of the pristine LDH, GA-LDH-ER (exfoliation-reassembling), GA-LDH-C (co-precipitation), and GA-LDH-I (ion-exchange)

ACCEPTED MANUSCRIPT nanohybrids, the well-developed (00l) peaks of (003), (006), and (009) could be observed due to the 2D nature of pristine LDH, relating to progressive diffractions by the basal planes i.e. d003=2d006=3d009 [13,28]. For the co-precipitated sample, only the pristine LDH phase

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remained unchanged even in such a highly concentrated GA solution corresponding to ~ 2.0 anionic exchange capacity (AEC) of LDH, indicating that the reaction seemed to be kinetically inert, and thermodynamic stable due to the strong electrostatic interaction of LDH

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layers and nitrates. Therefore, the basal spacing for GA-LDH-C determined was around 0.89 nm, which was exactly the same as that (0.88 nm) for the pristine LDH with interlayer nitrate

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anions (Fig. 1 A(b) and Fig. S2). In the case of conventional intercalative ion-exchange reaction, no single phasic GA-LDH-I could be obtained as clearly demonstrated in Fig. 1 A(c) and Fig. S3, but two phases, the intercalated and the pristine LDH, are present due to the chemical stability of LDH lattice. It is, therefore, not that surprising to fail in preparing

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GA-LDHs single phasic, chemically and structurally well defined by simple ion exchange reaction. According to these experimental results and the previous [3,19-21], it is quite clear that neither ion exchange route nor co-precipitation one could be considered as an ideal route

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to GA-LDH nanohybrid due to the stereo-chemical complexity, size and charges of GA under pH condition of synthesis, as well as the difference in hydrophilic and hydrophobic nature

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between GA and LDH lattice.

In the present study, however, we were very successful in immobilizing GA molecules

in the interlayer space of the pristine LDH by exfoliation and reassembling reactions (Scheme 1). As shown in the XRD patterns for GA-LDH nanohybrid prepared from different concentrations of GA corresponding to 1.0, 1.5 and 2.0 AEC, the single phasic GA-LDH nanohybrid was successfully prepared, as well demonstrated in Fig. 1 A(d), with an increase of basal spacing from 0.88 nm to 1.15 nm (Fig. 1 B). But a negligible amount of carbonate impurity was monitored with the GA concentration of 1.0 and 1.5 AEC, without any

ACCEPTED MANUSCRIPT unreacted pristine nitrate LDH with a d value of 0.88 nm (Fig. S4). Upon intercalation of GA into LDH, the basal spacing was expanded to 1.15 nm with a gallery height of 0.67 nm [1.15– 0.48 nm (thickness of a single layer)]. If the gallate molecules were assembled such that they

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would have perpendicularly oriented with a tilting angle of ~ 68° and interdigitated structure with energetically favored π–π stacking of aromatic rings, as shown in Fig. 1 B(a), the length of gallate molecules along the c-axis would be around 0.67 nm (Fig. S5). However, its

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calculated gallery height with 90° orientation is equal to 0.72 nm, which is well-consistent

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with bonding interaction between positively charged LDH and di-anionic gallate molecules.

3.2. FT-IR analysis of GA-LDH nanohybrid

As shown in Fig. 2(a), the hydroxyl group of LDH and interlayer water molecules solvated could result in a broad absorption band around 3000-3500 cm-1 [29,30]. And the peaks at around 430 - 750 cm-1 found in LDH and GA-LDH can be ascribed to the M–O and

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O–M–O vibrations in the LDH lattice [10]. The presence of strong band at 1385 cm-1 can be assigned as stretching vibration of (NO3¯ ) in the pristine LDH [14,30]. The band at 1628 cm-1

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is δ(H2O) bending vibration due to the deformation mode of water molecules [29]. In Fig. 2(b), the (C=O) and (C=C) stretching bands at 1702 cm-1 and 1535 cm-1, respectively, are

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surely due to the presence of carboxylic acid group and aromatic ring in intact GA [19,31]. Different from intact GA, the characteristic bands at 1406 cm-1 and 1539 cm-1 from sodiumgallate salt (Fig. 2(c)), indicate the symmetric and asymmetric stretching vibrations of (COO¯ ) [19,20]. On the other hand, Fig. 2(d) shows all the bands from GA-LDH appeared are very similar with those from gallate salt as well evidenced by the presence of two bands at 1406 cm-1 and 1547 cm-1, corresponding to the (COO¯ ) symmetric and asymmetric ones, respectively. A slight blue shift of the asymmetric band could be due to the hydrogen bonding interaction between OH groups on the interlayer surface of LDH and carboxylate ones of

ACCEPTED MANUSCRIPT intercalated gallate anions. One thing to be underlined here is that a band placed at 1385 cm-1 is absent in the spectra of GA -LDH confirming the interlayer nitrate anions are replaced by gallate anions. From this FT-IR analyses, it is found that gallic acid is completely

3.3. Atomic force microscopy analysis of exfoliated LDH

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deprotonated into gallate, and stabilized in between LDH lattices via electrostatic interaction.

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In order to follow up the exfoliation and reassembling reaction, it is required to check whether the multilayered LDH sheets are well exfoliated into individual nanosheets in

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formamide solution. The colloidal LDH solution with high stability can be prepared in a way to maintain the exfoliated phase indirectly as shown in Fig. S1, but the measurement of the thickness of exfoliated LDH nanosheets dispersed in formamide can be a direct evidence of exfoliation. As shown in Fig. 3A, AFM images of exfoliated LDH nanosheets, which were

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deposited on a mica surface by spin-coating technique, were observed by typical tapping mode. The height profile was just the topographic one as determined to be around 0.53 nm (Fig. 3B), which was almost equal to the thickness of a single brucite layer (0.48 nm)

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indicating that 2D LDH lattice was really exfoliated into single sheets under the present

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preparation condition.

3.4. Determination of GA content The content of GA in GA-LDH-ER nanohybrid was determined by UV-visible

spectroscopy analysis and cross-confirmed by HPLC one. The content of GA in GA-LDH-ER was determined to be ~ 23.0% and ~ 22.6% by UV-vis spectroscopic analysis and HPLC one, respectively, which was rather different from the theoretical content of monovalent gallate (36.3 %) but similar with that of divalent one (22.1 %), indicating that the divalent anions

ACCEPTED MANUSCRIPT were preferentially stabilized in the interlayer space of LDH. Such a result could be understandable considering the pKa1 = 4.24 and pKa2 = 8.27, and the molar fractions of

3.5. In-vitro release study for GA-LDH nanohybrid

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anionic species of gallates in the synthetic pH domain of ~ 8.0 [7].

The controlled release of GA from GA-LDH-ER nanohybrid could be demonstrated

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as shown in Fig. 4. Since the Gallic acid was observed to be chemically unstable above room temperature [25], the release behavior was measured in the phosphate buffered solution

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(PBS) of pH 7.4 at 4 ºC equipped with an ice bath with respect to time from 0 to 72 h. The GA release was found to be rather fast, and reached to ~ 70% in 24 h, and ~86% at the end of 72 h. Based on this GA release study, it is concluded that gallate molecules in LDH could be better protected from a harsh environment, due to the strong electrostatic interaction between

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positively charged LDH and gallate anion.

In order to understand the release mechanism of GA from GA-LDH nanohybrid, the sample was fitted with the several kinetic models, as plotted in Fig. 5 [32]. The kinetic

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constants and the r2 values are given in Table 1. According to the present models, First order, Freundlich, and Elovich provided reasonable r2 values of more than 0.90. On the other hand,

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the parabolic model provided reasonable r2 value of 0.999, indicating that the GA release from GA-LDH is the controlled by a diffusion-controlled process.

3.6. Antioxidant assay

Strong antioxidant activity is one of the attractive features of GA itself. It is, however, also very interesting to estimate its antioxidant activity after intercalating into LDH whether the GA molecules could retain their chemical integrity and function without any degradation. And therefore, the antioxidant assay for GA-LDH nanohybrid was performed by measuring

ACCEPTED MANUSCRIPT the free-radical scavenging activity of DPPH at 4 °C, and compared with that for GA as a reference. In both cases, the solutions became changed from purple to light yellow representing the antioxidant activity of GA-LDH nanohybrid with the concentration-

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dependent manner as shown in Fig. 6. The intact GA showed ~ 95% of DPPH radical scavenging activity, while GA-LDH exhibited approximately the same activity of ~ 93% at

could be enhanced upon intercalation into LDH lattice.

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3.7. Photostability test of GA-LDH nanohybrid

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the concentration of 20 µg/ml. This is probably due to the fact that the photostability of GA

To determine the evolution of photostability, GA and GA-LDH-ER nanohybrid in ethanol solution were exposed to UV light with respect to irradiation time. As shown in Fig. 7, the GA concentration in both cases was gradually decayed upon irradiation, but a

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considerable enhancement of photostability could be observed for GA-LDH-ER nanohybrid compared to intact GA. In case of intact GA, its concentration was decayed down to ~ 50 % after 3h irradiation, which is approximately the same with the literature value at pH 7 [33],

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and further decomposed down to ~ 66 % after 12 h. On the other hand, the GA content of GA-LDH-ER was degraded only ~ 28 % under the same conditions after 12 h irradiation,

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which could be rationalized by the fact that gallate anions in positively charged LDH layers resulted in thermodynamic stabilization due to strong electrostatic interaction, compared to intact GA.

4. Conclusions GA-LDH nanohybrid was synthesized by intercalating gallic acid into Zn-Al-layered double hydroxide based on exfoliation and reassembling reactions. The powder X-ray diffraction patterns showed that the increase of interlayer arrangement to 0.88 nm and 1.15

ACCEPTED MANUSCRIPT nm is due to gallate anion into the interlayer. From the FT-IR spectra, it was found that gallic acid was completely deprotonated into gallate, and stabilized in between LDH lattices via electrostatic interaction. The content of gallic acid in LDH could be controlled in the range of

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~ 23 wt% with the layer charge density of LDH. UV-vis spectra of GA deintercalated from GA-LDH nanohybrid was the same as that of intact GA, indicating that GA was reserved in the interlayer space of LDH without any chemical degradation.

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From the release behavior of GA out of LDH lattice, GA molecules were slowly released from GA-LDH nanohybrid in a controlled manner under a condition of pH 7.4 at

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4 °C. It was also proven that GA-LDH nanphybrid could be an outstanding antioxidant to scavenge DPPH radicals with an activity of ~ 93% in ethanol for GA-LDH depending on the concentration level. Thus, LDH host can be applicable as an active inorganic host matrix to inhibit the oxidation of antioxidant drugs throughout the storage process prior to application,

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and to scavenge free radicals upon controlled release. The photostability assay of GA and GA-LDH nanohybrid were carried out under UV light. The content of GA in GA-LDH nanohybrid at 12 h after UV light irradiation retained more than ~ 72% of initial content,

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while that of intact GA was reduced down to ~ 34%, indicating that GA molecules could be stabilized by LDH inorganic host against the UV light. It is, therefore, concluded that the

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present GA-LDH nanohybrid can be considered as an excellent antioxidant material with high chemical- and photo- stabilities, and controlled release property.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) Grant funded by

the

Korean

Government

(MSIP)

(No.

2017R1A6A3A11034149,

2016R1D1A1A02937308, and No. 2017K2A9A2A10013104).

No.

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[15] S. J. Choi, J. M. Oh, H. E. Chung, S. H. Hong, I. H. Kim, J. H. Choy, In vivo anticancer activity of methotrexate-loaded layered double hydroxide nanoparticles, Curr. Pharm. Des. 19

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Table 1. Fitting parameters of GA release profiles to several kinetic models.

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kinetic equation

GA-LDH

First order : ln(Ct/C0) = -kd t

kd 2

r

0.0255 0.9053

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Parabolic diffusion : (1 - Ct/C0)/t = kd t -0.5 + a

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kd a

0.1876 -0.0076

2

0.9994

r

Freundlich : log(1 - Ct/C0) = log(kd) + a log t

kd

-0.7004

a

0.3777

2

0.9715

r

Elovich : 1 - Ct/C0 = a ln t + b a

0.0986

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0.1408

2

0.9860

r

Avrami-erofeev : ln[-ln(1 - Ct/C0)] = n ln(kd) + n ln t kd

0.0540

2

C0 = the amount of guest in the GA at 0 min.

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kd = the rate constant of release, a, b, n = constant

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Ct = the amount of guest in the GA at t min.

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0.9747

r

Scheme 1. Exfoliation and reassembling route to single phasic GA-LDH nanohybrid out of

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three representative methods.

Fig. 1. (A) Powder X-ray diffraction (PXRD) patterns of (a) pristine LDH, (b) GA-LDH-C (co-precipitation), (c) GA-LDH-I (ion-exchange), and (d) GA-LDH-ER (exfoliationreassembling), and (B) The most probable interlayer structures of (a) GA-LDH-ER, and (b) pristine LDH (For gallate and nitrate, carbon: gray, nitrogen: blue, oxygen: red, hydrogen:

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white.).

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Fig. 2. Fourier transform infrared (FT-IR) spectra of (a) pristine LDH, (b) intact gallic acid, (c) sodium gallate, and (d) GA-LDH-ER.

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Fig. 3. (A) Atomic force microscopy (AFM) image, and (B) height profile of exfoliated LDH

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nanosheets.

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Fig. 4. (a) Solubility of intact GA, and (b) release profiles of GA from GA-LDH-ER in PBS

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(pH 7.4) at 4 °C, respectively.

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Fig. 5. Plots of kinetic equation based on the models of (A) First order, (B) Parabolic

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GA-LDH-ER.

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diffusion, (C) Freundilich, (D) Elovich, and (E) Avrami-Erofeev for the release of GA from

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Fig. 6. DPPH radical scavenging effect of (a) GA-LDH-ER, and (b) intact GA in ethanol

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solution at 4 °C.

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Fig. 7. Photostability of (a) intact GA, and (b) GA-LDH-ER in ethanol solution with respect

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to UV irradiation time.

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Supporting Information

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Stabilization of antioxidant gallate in layered double hydroxide

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by exfoliation and reassembling reaction

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Kanakappan Mickel Ansy, Ji-Hee Lee, Huiyan Piao, Goeun Choi,* and Jin-Ho Choy**

Center for Intelligent Nano-Bio Materials (CINBM), Department of Chemistry and

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Nano Science, Ewha Womans University, Seoul 03760, Republic of Korea

** Corresponding author: Prof. Jin-Ho Choy (E-mail: [email protected]) * Corresponding author: Dr. Goeun Choi (E-mail: [email protected])

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Fig. S1. Tyndall effect of colloidal solution containing exfoliated LDH nanosheets.

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Fig. S2. Powder X-ray diffraction (PXRD) patterns of intact GA (a), pristine LDH (b), and

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GA-LDH-C (co-precipitation) for 6 h (c), 12 h (d), and 24 h (e) reaction, respectively.

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Fig. S3. Powder X-ray diffraction (PXRD) patterns of intact GA (a), pristine LDH (b), and

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GA-LDH-I (ion-exchange) for 6 h (c), 12 h (d), and 24 h (e) reaction, respectively.

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Fig. S4. Powder X-ray diffraction (PXRD) patterns of intact GA (a), pristine LDH (b), and

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GA-LDH-ER (exfoliation-reassembling) with given GA concentrations of 1.0 (c), 1.5 (d), and 2.0 (e) times excess of anionic exchange capacity (AEC) of pristine LDH, respectively.

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Fig. S5. Dimensions of gallate for (A) perpendicular orientation, and (B) tilted one with 68°

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by Chem3D Ultra 8.0 program (CambridgeSoft).