ar ticle

Copyright © 2014 by American Scientific Publishers All rights reserved. Printed in the United States of America

Science of Advanced Materials Vol. 6, pp. 1–11, 2014 (www.aspbs.com/sam)

Characterisations and Cytotoxicity Assessment of UV Absorbers-Intercalated Zinc/Aluminium-Layered Double Hydroxides on Dermal Fibroblast Cells Sumaiyah Megat Nabil Mohsin1 , Mohd Zobir Hussein1, ∗ , Siti Halimah Sarijo2 , Sharida Fakurazi3, 4 , Palanisamy Arulselvan4 , and Yun Hin Taufiq-Yap5 1

Material Synthesis and Characterization Laboratory (MSCL), Institute of Advanced Technology (ITMA), Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia 2 Faculty of Applied Science, Universiti Teknologi MARA, 40450 UiTM Shah Alam, Selangor, Malaysia 3 Faculty of Medicine and Health Sciences, Department of Human Anatomy, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 4 Laboratory of Vaccines and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 5 Faculty of Science, Catalysis Science and Technology Research Centre, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Zn/Al-layered double hydroxide (LDH) was used as a host to intercalate various organic ultraviolet (UV) radiation absorbers. The intercalation compounds were prepared via the co-precipitation method. Powder X-ray diffraction (PXRD) confirmed the successful intercalation of anions into the interlayer regions of the LDH nanocomposites. As a result of intercalation, the resulting nanocomposites loaded with UV-ray absorbers, cinnamic acid (CA), benzophenone 4 (B4) and Eusolex® 232 (EUS)—exhibited basal spacings of 17.9 Å, 21.3 Å and 21.0 Å, respectively. Photochemical analysis revealed an increase in the UV-ray absorption capability of UV absorber/LDH nanocomposites compared to pure UV-ray absorbers. The retention ability of the organic moieties in the LDH host was tested in a skin pH simulation and was found to demonstrate low release over an extended period of time. Cytotoxicity findings indicated that none of the nanocomposites exhibit significant cytotoxicity towards human dermal fibroblast (HDF) cells up to the test concentration of 25 g/mL. KEYWORDS: UV-Ray Absorber, Layered Double Hydroxide, Optical Properties, Intercalation, Nanocomposite.

1. INTRODUCTION The ultraviolet (UV) rays that reach the ground are mainly UVA (90∼99%) and UVB (1∼10%), as UVC is absorbed completely by the atmosphere. Prolonged exposure to UV radiation is linked to photo-aging, skin cancer (melanoma and non-melanoma), actinic keratoses and immunosuppression.1 2 Regular use of sunscreen is the leading preventive strategy recommended by physicians to curb excessive UV exposure. However, issues concerning the photo-stability of sunscreens have been raised, as several studies have demonstrated photo-degradation upon irradiation.3–5 The photochemical effects of UV radiation may be exacerbated by the chemical agents found in sunscreen itself. ∗

Author to whom correspondence should be addressed. Email: [email protected] Received: xx xxxx xx Revised/Accepted: xx xxxx xx

Sci. Adv. Mater. 2014, Vol. 6, No. 4

Several sunscreen carrier systems have been proposed to reduce the photo-degradation of organic sunscreen molecules, including the incorporation of organic UV absorbers in natural and synthetic polymers,6 7 complexation by cyclodextrins,8 encapsulation in liposome microspheres9 and intercalation in inorganic layered compounds.10 Amongst these, an inorganic layered material/sunscreen system is expected to have a high potential for scaled-up application because of its simplicity of synthesis, low cost of raw materials and low toxicity.11 Layered double hydroxide (LDH) is a type of layered compound in which the structure is built by stacking positively charged layers that contain two or more types of metallic cations. The positive charge is counterbalanced by anions located in the spaces between the layers. LDH is represented by the general formula z+ n− n− [MII1−x MIII is the interlayer x (OH)2 ] Az/n · yH2 O where A II III anion, and M and M are di- and trivalent metallic

1947-2935/2014/6/001/011

doi:10.1166/sam.2014.1752

1

ARTICLE

ABSTRACT

ARTICLE

Characterisations and Cytotoxicity Assessment of UV Absorbers-Intercalated Zinc/Aluminium-LDH on Dermal Fibroblast Cells

cations, respectively.12 Using LDH as a host for UV absorbing molecules, we hope to retain photo-protection in the UV range, reduce photo-contact allergic reactions and avoid possible photo-carcinogenesis from the photodegradation of sunscreen molecules. In this paper, organic UV-ray absorbers, namely, cinnamic acid (CA), 2-phenylbenzimidazole-5-sulfonic acid (EUS) and 2-hydroxy-4-methoxybenzophenone-5sulphonic acid (B4), were chosen as they have raised several health concerns when used as sunscreen materials. CA can cause skin irritations, such as contact urticaria, and has been identified as the primary allergen in Balsam of Peru, a resin that is used as flavouring and fragrance in many products.13 14 EUS can protect skin cells from the effects of sunlight. However, it exhibits photosensitising photochemical reactions upon irradiation and produces radical species that are capable of damaging DNA.15 16 Furthermore, B4 has been found to induce cosmetic dermatitis of the face and neck in test patients and also causes sensitisation at other locations.17 This paper presents a study of the physico-chemical and UV absorption properties as well as the anion retention in a skin pH simulation of the formed UV absorber/LDH nanocomposites. They were also tested on human dermal fibroblast (HDF) cells to assess whether the nanocomposites would induce toxicity in the cells. Dermal fibroblast cells were chosen as a model for our in vitro study because they are the most abundant cells in human skin, which is the primary organ that is exposed to sunscreen formulations.

2. EXPERIMENTAL DETAILS 2.1. Materials B4 (99.0%) was acquired from Norquay Technologies (USA) while CA (99.0%), EUS (99.0%) and sodium hydroxide (99.0%) were purchased from Merck (Darmstadt, Germany). Zinc nitrate hexahydrate (99.0%) and aluminium nitrate nanohydrate (99.0%) were obtained from PC Laboratory Chemicals. Phosphate-buffered solution and dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich (Missouri, USA) and sodium chloride (99.0%) was acquired from HmbG Chemicals (Hamburg, Germany). All reagents were used without further purifications. The water used for this study was purified and further deionised with a resistivity of 18 M. 2.2. Preparation Zn/Al-NO3 LDH was prepared by dissolving 0.1 mol/L Zn(NO3 )2 · 6H2 O and 0.05 mol/L Al(NO3 )3 · 9H2 O (Zn/Al molar ratio 2) in 250 mL of deionised water. The mixture was then precipitated with a 0.5 mol/L NaOH solution under nitrogen atmosphere until it reached its final pH of 7. The resulting white slurry was aged in an oil bath shaker at 70 C for 18 h. The pristine LDH was gathered via centrifugation, rinsed with deionized water several times 2

Mohsin et al.

and dried in an oven at 70 C for 3 days. The dried LDH was then powdered using a mortar and pestle and stored in a sample bottle for further use and characterisations. The formation of nanocomposites intercalated with anionic UV absorbents was conducted via the coprecipitation method in the presence of anion species. The organic UV absorbent/LDH nanocomposites were prepared by adding 100 mL of 0.1 mol/L guest-anion solution dropwise into 100 mL mixture of 0.1 mol/L Zn(NO3 )2 · 6H2 O and 0.05 mol/L Al(NO3 )3 · 9H2 O solution under nitrogen atmosphere and stirring vigorously. The pH of the solution was brought to 8 via the dropwise addition of 0.5 mol/L NaOH solution. The resulting slurry was placed in an oil bath shaker at 70 C for 18 h. It was then centrifuged and rinsed with deionised water. The final precipitate was dried in an oven at 70 C, for 3 days, finely ground and stored for further use and characterisations. 2.3. Characterisation Methods Powder X-ray diffraction (PXRD) patterns were recorded using an XRD-6000 (Shimadzu, Kyoto, Japan) with a CuK radiation ( = 15418 Å) between 2 and 60 . The carbon, hydrogen, nitrogen and sulphur contents in the samples were analysed using a CHNS-932 (LECO Instruments, Michigan, USA). Elemental analysis was carried out to determine the zinc and aluminium contents via inductively coupled plasma atomic emission spectrometry (ICP-AES) using a Perkin-Elmer spectrophotometer, model Optima 2000DV (Perkin-Elmer, Massachusetts, USA), under standard conditions. The morphologies of the products were observed via field-emission scanning electron microscopy (FESEM) using a Zeiss SUPRA 40VP (Oberkochen, German), and optical measurements were performed using a Shimadzu UV-3600 model (Kyoto, Japan) UV-VIS-NIR diffuse reflectance spectrometer with an integrating sphere attachment using ZnO as the reference. 2.4. Studies of Sunscreen Release from Nanocomposite Formulations The release of CA, B4 and EUS anions from the LDH host as a function of time was measured in situ at max = 272 nm (CA), 285 nm (B4) and 311 nm (EUS) using a Perkin-Elmer UV-VIS spectrometer Lambda 35 (PerkinElmer, Massachusetts, USA) by adding a 0.2 mg sample into 3.5 mL of release medium; deionised water, 0.5 mol/L NaCl and pH 5.5 phosphate buffer solution, at room temperature. The data were collected and fitted to zero-, first-, pseudo-second order and parabolic diffusion kinetic models. 2.5. Cell Culture and Determination of Cell Viability Healthy HDF cells obtained from ATCC (Virginia, USA) were cultured at 37 C and 5% CO2 in high glucose Dulbecco’s Modified Eagle Medium (DMEM) (ScienCell Sci. Adv. Mater., 6, 1–11, 2014

Mohsin et al.


Research Laboratories, California, USA) containing 2% fetal bovine serum, 0.5% penicillin–streptomycin, 1% glutamine and 1% non-essential amino acids. The cell viability of HDF cells was determined using an MTT assay, as described previously.18 Briefly, wells containing 1 × 104 cells/well were treated with the nanocomposites. After incubation for 24 h, the cells were treated with MTT in each well and incubated at 37 C for 3 to 4 h. The supernatant was then removed, and the formazan crystals that formed were dissolved by adding DMSO (100 L/well) and analysed at 570 nm using a Bio-Tek EL800 ELISA microplate reader (Vermont, USA). The percentage of cell viability was calculated as the ratio of the optical density in the medium (containing nanocomposites at each concentration) to that in the fresh control medium.

3. RESULTS AND DISCUSSION

Intensity (a.u.)

2θ (degree) Fig. 1. PXRD patterns of Zn/Al-NO3 , Zn/Al-CA, Zn/Al-B4 and Zn/Al-EUS. Sci. Adv. Mater., 6, 1–11, 2014

Sample Zn/Al-NO3 Zn/Al-CA Zn/Al-B4 Zn/Al-EUS

Empirical formulaea

d spacing (Å)

[Zn067 Al033 (OH)2 ][NO−3 ]033 · 056H2 O [Zn067 Al033 (OH)2 ][C3 H7 O−2 ]033 · 077H2 O [Zn069 Al031 (OH)2 ][C14 H11 O6 S− ]031 · 093H2 O [Zn069 Al031 (OH)2 ][C13 H9 N2 O3 S− ]031 · 106H2 O

88b 179c 213d 210d

Notes: a Estimated via ICP and TGA/DTG analysis; b Average basal spacing based on 3 harmonics; c Average basal spacing based on 5 harmonics; d Average basal spacing based on 6 harmonics.

are summarized in Table I. Pristine LDH exhibited diffraction peaks attributed to the (003), (006) and (009) reflections, while the UV absorber/LDH nanocomposites exhibited higher-order reflections up to (0012), (0015) and (0018), indicating a well-ordered layered structure. The average basal spacing recorded for Zn/Al-NO3 LDH is typical of Zn/Al LDH occupied by nitrates.19 The successful incorporation of the UV absorbers was demonstrated by the expansion of this interlayer as larger anions were intercalated. The degree of separation between the positively charged layers of the LDH depends on the intercalated anions’ size, charge, orientation and host-guest interaction. The estimated dimensions of energy-minimized CA, B4 and EUS molecular models were obtained using ChemDraw software, and they are presented in Figures 2(a)–(c), respectively. The expected gallery height that could be occupied was deducted by subtracting the layer thickness of 4.8 Å from the basal spacing obtained from the PXRD analysis. Figure 2(d) shows the most plausible spatial arrangement of the guests in the LDH. As the interlayer distance is much larger than the anion size, a bilayer arrangement is proposed in which the chargebalancing carbonate and sulphoxylate groups face towards the hydroxide layers. The functional groups are turned in the opposite direction, and the fields of the aromatic ring are opposed to maximise the – interaction between the benzene rings.10 3.2. Elemental Analysis The chemical compositions of CA, B4, EUS, Zn/Al-NO3 , Zn/Al-CA, Zn/Al-B4 and Zn/Al-EUS are listed in Table II. The UV absorber/LDH nanocomposites were specifically synthesized at a Zn/Al molar ratio of 2, as this ratio was found to yield the highest crystallinity. This high crystallinity has been observed previously, and it is attributed to the small difference in the ionic radii of Zn2+ and Al3+ provides a stronger bonding stability of the hydroxide layers networks of LDH crystal.20 21 However, ICP-AES analysis indicated the Zn/Al molar ratios for all Zn/Al LDH samples to be slightly higher than the calculated value, R = 2. This observed discrepancy of the experimental values may have been caused by the precipitation of nearly all 3

ARTICLE

3.1. X-Ray Diffraction and Spatial Orientation of the Guests Between LDH Interlayers The PXRD patterns of the nanocomposites Zn/Al-NO3 , Zn/Al-CA, Zn/Al-B4 and Zn/Al-EUS are shown in Figure 1. The average basal spacings of the LDH nanocomposites based on 3, 5 and 6 harmonics for Zn/AlNO3 , Zn/Al-CA, Zn/Al-B4 and Zn/Al-EUS, respectively,

Table I. Empirical formulae and d spacing of Zn/Al-NO3 , Zn/Al-CA, Zn/Al-B4 and Zn/Al-EUS nanocomposites.

Mohsin et al.

ARTICLE


Fig. 2. Molecular structure and three-dimensional molecular size of CA (a), B4 (b) and EUS (c) and the proposed spatial orientation of the molecules in the Zn/Al-LDH interlayer (d).

4

Sci. Adv. Mater., 6, 1–11, 2014

Mohsin et al.


Table II. Chemical compositions of CA, B4, EUS, Zn/Al-NO3 -LDH, Zn/Al-CA, Zn/Al-B4 and Zn/Al-EUS nanocomposites.

Sample

b b C H N S aAnion Zn Al Zn2+ / (%) (%) (%) (%) (%w/w) (%w/w) (%w/w) Al3+

CA B4 EUS Zn/Al-NO3 Zn/Al-CA Zn/Al-B4 Zn/Al-EUS

731 474 548 03 260 229 239

5.3 – – 4.5 – 105 3.1 170 97 2.3 139 04 4.0 – 30 4.1 – 43 4.0 32 52

– – – – 35.8 41.9 41.7

– – – 35.2 28.5 24.8 23.5

– – – 7.0 5.7 4.7 4.3

– – – 2.1 2.1 2.2 2.2

x – – – 0.33 0.33 0.31 0.31

Notes: a Estimated via CHNS analysis; b Estimated via ICP analysis.

3.3. Surface Characterisation Morphological analyses of the Zn/Al-NO3 , Zn/Al-CA, Zn/Al-B4, and Zn/Al-EUS nanocomposites were performed via FESEM and are shown in Figure 3. Pristine LDH consists of agglomerates of nanometre thin plate-like particles, whose diameters ranges from 0.5 m to 2 m. LDH intercalated with the various sunscreen molecules exhibit completely different surface morphologies from one another. In general, the nanocomposites exhibit agglomerates of nanometre sized thin platelike particles, a typical morphology for LDH. There is, however, a decrease in crystallite size from the pristine Zn/Al-NO3 to the intercalated products. Zn/Al-CA possesses coarse edges and surfaces. Zn/Al-B4, on the other hand, has a smooth surface and roundish shape. Zn/AlEUS presents blunt edges and smooth surfaces. Compared to Zn/Al-CA and Zn/Al-B4, the Zn/Al-EUS nanocomposite exhibits more shape and size irregularities.

(b)

(a)

1 µm

(c)

1 nm

(d)

100 nm

200 nm

Fig. 3. FESEM images of Zn/Al-NO3 at 10,000× magnification (a), Zn/Al-CA at 10,000× magnification (b), Zn/Al-B4 at 50,000× magnification (c) and Zn/Al-EUS at 50,000× magnification (d). Sci. Adv. Mater., 6, 1–11, 2014

5

ARTICLE

Zn and Al ions during the synthesis of the LDH inorganic layered host. A summary of the percentage loading of the intercalated anions is provided in Table II. The percentage loading of CA, B4 and EUS anions in the Zn/Al LDH host were 35.8 w/w%, 41.9 w/w% and 41.7 w/w%, respectively. In agreement with the PXRD patterns, elemental analyses confirmed the intercalation of all the guest anions into

the LDH interlayers. The empirical formulae of the Zn/Al LDH nanocomposites were determined via ICP-AES and TGA/DTG analyses, and they are listed in Table I.


(a)

ARTICLE

Absorbance

(b)

(c)

Wavelength (nm) Fig. 4. Solid-state absorbance spectra of Zn/Al-NO3 , organic sunscreen guests and the corresponding nanocomposites.

3.4. Analysis of UV-Ray Absorption Capability The UV-ray absorption capability of the resulting nanocomposites was monitored to inspect the effects of intercalating sunscreen molecules into LDH layers. The solid state absorbance spectra of CA, B4, EUS, Zn/AlNO3 , Zn/Al-CA, Zn/Al-B4 and Zn/Al-EUS are shown in Figure 4. Prior to the intercalation of anions with UV shielding properties, the UV absorption capability of pristine LDH was shown to be poor, with a maximum at 300 nm, which is ascribed to the presence of NO− 3 in the LDH interlayer.22 In general, the LDH nanocomposites inherited the broad and strong UV shielding capabilities of the UV-ray absorbing organic guest moieties. UV absorbers contain a chromophore group that influences the absorption characteristic of molecules. Thus, this attribute could be conferred by the retention of UV absorbent molecules in the 6

Mohsin et al.

inorganic lattice. Spatial confinement and host-guest interactions, namely, electrostatic attraction, hydrogen bonding and van der Waals forces, contributes to the broadened absorption range of the intercalated LDH. Figure 4(a) demonstrates that pure CA has an excellent UV absorption capability in the UVB region (280–320 nm) with a recorded maximum peak at 240 nm. The CAintercalated nanocomposite, Zn/Al-CA, exhibits a peak shift to 250 nm. This shift of the absorption maximum towards the longer-wavelength region, also known as a bathochromic shift, could be attributed to the stacking arrangement of chromophore molecules in an edge-to-edge manner.23 This arrangement is illustrated in Figure 2(d). The – interaction between aromatic groups of intercalated molecules results in a decrease in the excitation energy, which consequently shifts the absorbance band towards a higher-wavelength region. The intercalation of B4 molecules in LDH produced a more prominent bathochromic peak shift (273 nm to 337 nm) than that observed in the Zn/Al-CA nanocomposite. In addition to the edge-to-edge arrangement of intercalated B4 molecules (Fig. 2(d)), conjugation in the interlayer gallery is further induced by the electrondonating groups –OCH3 and –OH on the B4 molecules. In contrast, the Zn/Al-EUS nanocomposite (312 nm) demonstrated a shift of the absorption maximum towards the shorter-wavelength region despite the edge-to-edge association of the intercalated EUS molecules. To elucidate this effect, a closer look reveals that intermolecular N H hydrogen bonding exists between the intercalated EUS molecules. The electron pair of nitrogen causes conjugation to be removed, thus inducing a hypsochromic effect. The band gaps of the nanocomposites were investigated using the Kubelka-Munk equation;21 F · h 2 = Ah − Eg

(1)

where F is the Kubelka-Munk value, h is Planck‘s constant, A is a proportionality constant, h is the photon energy and Eg is the band gap energy. Using Eq. (1), band gap values can be extracted by extrapolating a straight line from the linear region to the h intercept. Figure 5 presents the Kubelka-Munk plot and band gap values of Zn/Al-NO3 , Zn/Al-CA, Zn/Al-B4, Zn/Al-EUS and their respective sunscreen compounds. A pure sunscreen compound exhibits more than one band gap because of the presence of multiple chromophores in its molecular structure. Therefore, it is expected that the intercalation of sunscreen molecules in LDH interlayers should permit the sunscreen to maintain the same band gap as an intercalated guest. It can be observed that the band gap values exhibit a shift towards narrower band gap values from pristine LDH to the intercalated products. As observed, intercalation increases the specific surface area of nanocomposites. As a result, visible-light photocatalytic availability is increased while the band gap value is narrowed.24 25 Sci. Adv. Mater., 6, 1–11, 2014

Mohsin et al.


3.5. Release Behaviour of Intercalated Anions from Nanocomposites It was observed that the UV absorber/LDH nanocomposites attained superior UV absorbing capability in comparison to the pristine LDH. However, it is important that the deintercalation of these sunscreen anions is suppressed under application conditions. Therefore, we investigated the release profiles of CA, EUS and B4 from their Zn/Al LDH matrices in pH 5.5 phosphate buffer solution. The release profiles of the anions are shown in Figure 6.

Fig. 6.

The release medium, pH 5.5 phosphate buffer solution was selected to simulate the pH conditions of skin. The objective of this simulation was to determine how much retention an LDH host could provide for each UV absorbing anions. By virtue of the anion exchange property, LDH hosts are able to exchange interlayer anions with those in release media. Therefore, a certain level of leaching into the release medium was observed. The nanocomposites exhibited an initial rapid release, which then slowed until the equilibrium state was achieved

Controlled release of CA (a), B4 (b) and EUS (c) from Zn/Al-CA, Zn/Al-B4 and Zn/Al-EUS, respectively, in pH 5.5 phosphate buffer solution.

Sci. Adv. Mater., 6, 1–11, 2014

7

ARTICLE

Fig. 5. Kubelka-Munk transformed reflectance spectra of Zn/Al-NO3 (a), Zn/Al-CA (b), Zn/Al-B4 (c) and Zn/Al-EUS (d). The inset provides the Kubelka-Munk transformed reflectance spectra of pure CA (b), B4 (c) and EUS (d).

Mohsin et al.

ARTICLE


Fig. 7. Fitting of the data regarding the release of CA (a)–(d), B4 (e)–(h) and EUS (i)–(l) from Zn/Al-CA, Zn/Al-B4 and Zn/Al-EUS, respectively, into pH 5.5 phosphate buffer solution for zeroth-, first-, and pseudo second order and parabolic diffusion kinetics.

8

Sci. Adv. Mater., 6, 1–11, 2014

Mohsin et al.


at a maximum accumulated release of approximately 59.83%, 54.68% and 57.69% for Zn/Al-CA, Zn/Al-B4 and Zn/Al-EUS, respectively. However, the Zn/Al-CA release profile exhibited an initial burst effect during the first 50 min of contact. This was caused by the slight dissolution of the metal layers upon contact with the slightly acidic medium.26 3.6. Release Kinetics of CA, EUS and B4 from Sunscreen Formulations The release of the UV absorbing anions was fitted into the following kinetic equations; zeroth-order (Eq. (2)),27 first-order (Eq. (3)),28 parabolic diffusion (Eq. (4))29 and pseudo-second-order kinetics (Eq. (5)).30 The equations are written below, where c is a constant, and Ceq and Ct are the anion concentrations at equilibrium and at time t, respectively. (2)

− log1 − Ct = kt

(3)

Ct /Ceq = c + kt 05

(4)

t/Ct =

2 1/k2 Ceq

+ 1/Ceq t

(5)

The plots are provided in Figure 7. The values of the correlation coefficient that were obtained (Table III) indicate that the release of the anions followed pseudo-second order kinetics. From the pseudo-second-order kinetic equation, we derived the time required for the guest release to reach half of the total accumulated release, the t1/2 value, to be 9 min, 20 min and 329 min for the CA, B4 and EUS anions, respectively. The ranking of the t1/2 values can be summarised as follows: CA < B4 < EUS The release rate constant, k, quantifies the speed of anion release, and its values can be ranked as follows: CA > B4 > EUS Table III shows that the rate constant for sunscreen anion release are 188 × 10−3 L mg−1 min−1 ,

3.7. Cytotoxicity Assessment of Nanocomposites Cosmeceuticals are often formulated with bioactive compounds and demonstrated to achieve multiple cellprotective properties for the reconstruction of normal skin on a cellular level. Nanobiotechnology applications have been realised in sunscreens, emollients, topical medications, and diagnostic imaging for the maintenance of skin health, and for the management of skin inflammation. The recent development of nanoscale “hybrids” is certain to considerably change the therapeutic manner in which we protect the skin from various factors. These hybrids are synthesised to facilitate the manipulation of single bioactive compounds while releasing them onto skin in a controlled release fashion, to achieve the most effective potential outcome of the treatment. In the present investigation, we synthesised nanocomposites with bioactive compounds for the effective treatment of skin related

Table III. Correlation coefficient, rate constant and half time obtained by fitting the data regarding the release of CA, B4 and EUS from their respective nanocomposites into pH 5.5 phosphate buffer solution using zeroth-order, first-order, parabolic diffusion and pseudo second-order kinetic models. Correlation coefficient, r 2

Zn/Al-CA Zn/Al-B4 Zn/Al-EUS

Saturated release (%)

Zeroth-order

First-order

59.83 54.68 57.69

0.5945 0.7219 0.7795

0.6482 0.7541 0.8224

Parabolic diffusion

Pseudo second-order

Rate constant of pseudo second order, k (L mg−1 min−1

t1/2 of pseudo second order (min)

1.0000 0.9999 0.9997

0.7042 0.8305 0.8840

188 × 10−3 904 × 10−4 497 × 10−5

9 20 329

2 Notes: Zeroth-order: Ct = kt + c; first-order: − log1 − C = kt + c; parabolic diffusion: Ct /Ceq = c + kt 05 ; pseudo second-order: t/Ct = 1/k2 Ceq + 1/Ceq t; c = a constant; Ceq = anion concentration at equilibrium; Ct = anion concentration at time t.

Sci. Adv. Mater., 6, 1–11, 2014

9

ARTICLE

Ct = kt + c

904 × 10−4 L mg−1 min−1 and 497 × 10−5 L mg−1 min−1 for CA, B4 and EUS anions, respectively. A lower t1/2 value and a higher k value indicate a slower release of anions from the LDH interlayers. CA exhibited significantly faster release than the other anions, reaching saturation after 9 h. A strong UVA and UVB absorber (Fig. 4), Zn/Al-B4, reached saturation after 13 h of release. Despite its relatively weak UV shielding ability, Zn/AlEUS showed promise in terms of its durability for longterm use. Indeed, deintercalation of sunscreen molecules from any such formulation is inevitable because of the ion-exchange property of LDHs. The results demonstrated that the direct contact of a sunscreen formulation with a skin pH medium could be reduced to an eventual maximum release of less than 60% of the intercalated sunscreen molecules. The inorganic metal layer extends the UV protection and slows the exposure of the sunscreen molecules to the photodegrading environment. As an alternative, further treatment such as an amorphous silica coating could also be used to suppress anion release and prevent the dissolution of the inorganic matrix without compromising the UV shielding capability of nanocomposites.31


Mohsin et al.

LIST OF ABBREVIATIONS CA B4 EUS Zn/Al-NO3 Zn/Al-CA Zn/Al-B4

ARTICLE

Fig. 8. Concentration–response curves obtained by plotting the percentage of viability of human dermal fibroblast cells exposed to Zn/Al-NO3 , CA, Zn/Al-CA, B4, Zn/Al-B4, EUS or Zn/Al-EUS for 24 h. The results are presented as the mean ± SD of triplicate values.

ailments. The synthesised nanocomposites exhibited a significant controlled release property; therefore, we suggest that these nanocomposites should prove to be a good Cosmeceutical formulation for effective skin care. To confirm the non-toxic nature of the LDH nanocomposites for further potential application in sunscreen formulation, the effect of the synthesised nanocomposites on the cell viability of HDF cells was evaluated by exposing the cells to various dose-dependent concentrations of nanocomposites. The cells were exposed to nanocomposites at various concentrations of 1.562, 3.125, 6.25, 12.5, 25 and 50 g/mL for 24 h, and the cell viability was analysed via cell viability assay (Fig. 8). Nanocomposites at concentrations between 1.562 and 25 g/mL did not cause any significant reduction in cell viability. However, cells exposed to 50 g/mL concentration of nanocomposite samples exhibited a reduction in cell viability of greater than 40%, with Zn/Al-EUS being the least toxic, followed by Zn/Al-B4 and Zn/Al-CA. Therefore, these findings indicated that overall, nanocomposites in concentration of up to 25 g/mL presents a non-toxic nature. In addition to this finding, further cellular and molecular preclinical investigations will be needed to confirm the non-toxic nature of these materials for further biomedical applications.

4. CONCLUSION The intercalation of organic UV absorbers CA, B4 and EUS into Zn/Al LDH was achieved via the co-precipitation method. The UV absorption capability of the intercalation products was enhanced by the incorporation of the UV absorbers into the LDH matrix. It is noteworthy that the sunscreen-nanocomposites exhibited good retention under skin pH condition, exhibiting a deintercalation of less than 60% of all guest anions. The UV absorber/LDH nanocomposites did not induce cytotoxicity at concentrations of up to 25 g/mL when tested on HDF cells. Overall, it could be concluded that LDH shows promise for use as a host for UV absorbing molecules. 10

Zn/Al-EUS UV LDH HDF

Cinnamic acid Benzophenone 4 Eusolex® 232 Zn/Al LDH intercalated with nitrates Zn/Al LDH intercalated with cinnamates Zn/Al LDH intercalated with benzophenone 4 Zn/Al LDH intercalated with eusolex® 232 Ultraviolet Layered double hydroxide Human dermal fibroblast.

Acknowledgments: The authors would like to thank the Ministry of Higher Education (MOHE) of Malaysia for the financial support provided under research grant FRGS/1/11/SG/UPM/01/2, Vot. No. 5524165. One of the authors (SMNM) would like to thank Universiti Putra Malaysia for providing a Graduate Research Fellowship (GRF).

References and Notes 1. F. P. Gasparro, M. Mitchnick, and J. F. Nash, Photochem. Photobiol. 68, 243 (1998). 2. J. H. Diaz and L. T. Nesbitt, J. Travel. Med. 19, 108 (2012). 3. N. Serpone, A. Salinaro, A. V. Emeline, S. Horikoshi, H. Hidaka, and J. Zhao, Photochem. Photobiol. Sci. 1, 970 (2002). 4. S. Zhang, J. Chen, X. Qiao, L. Ge, X. Cai, and G. Na, Environ. Sci. Technol. 44, 7484 (2010). 5. T. Hayashi, Y. Okamoto, K. Ueda, and N. Kojima, Toxicol. Lett. 167, 1 (2006). 6. Y. A. Gomaa, L. K. El-Khordagui, N. A. Boraei, and I. A. Darwish, Carbohydr. Polym. 81, 234 (2010). 7. D. Gogna, S. K. Jain, A. K. Yadav, and G. P. Agrawal, Curr. Drug Deliv. 4, 153 (2007). 8. S. Scalia, A. Molinari, A. Casolari, and A. Maldotti, Eur. J. Pharm. Sci. 22, 241 (2004). 9. H. Tønnesen, Int. J. Pharm. 225, 1 (2001). 10. Q. He, S. Yin, and T. Sato, J. Phys. Chem. Solids 65, 395 (2004). 11. P. Blasi, A. Schoubben, S. Giovagnoli, C. Rossi, and M. Ricci, Expert Rev. Dermatol. 6, 509 (2011). 12. G. G. C. Arizaga, K. Satyanarayana, and F. Wypych, Solid State Ionics 178, 1143 (2007). 13. D. Bickers, P. Calow, H. Greim, J. M. Hanifin, A. E. Rogers, J. H. Saurat, I. G. Sipes, R. L. Smith, and H. Tagami. Food Chem. Toxicol. 43, 799 (2005). 14. R. Brancaccio and M. S. Alvarez, Dermatol. Ther. 17, 302 (2004). 15. J. Inbaraj, P. Bilski, and C. F. Chignell, Photochem. Photobiol. 75, 107 (2002). 16. N. Bastien, J. Millau, M. Rouabhia, R. Jeremy, H. Davies, and R. Drouin, J. Invest. Dermatol. 130, 2463 (2010). 17. T. M. Hughes and N. M. Stone, Contact Dermatitis 56, 153 (2007). 18. S. M. N. Mohsin, M. Z. Hussein, S. H. Sarijo, S. Fakurazi, P. Arulselvan, and T. Y. Hin, Chem. Cent. J. 7, 1 (2013). 19. L. Perioli, M. Nocchetti, V. Ambrogi, L. Latterini, C. Rossi, and U. Costantino, Micropor. Mesopor. Mat. 107, 180 (2008). 20. T. Ishikawa, K. Matsumoto, K. Kandori, and T. Nakayama, J. Solid State Chem. 179, 1110 (2006). 21. A. A. A. Ahmed, Z. A. Talib, M. Z. Hussein, and A. Zakaria, J. Solid State Chem. 191, 271 (2012). Sci. Adv. Mater., 6, 1–11, 2014

Mohsin et al.


22. H. Chai, X. Xu, Y. Lin, D. G. Evans, and D. Li, Polymer Degrad. Stabil. 94, 744 (2009). 23. R. Marangoni, C. Taviot-Gueho, A. Illaik, F. Wypych, and F. Leroux, J. Colloid Interface Sci. 326, 366 (2008). 24. K. M. Parida and L. Mohapatra, Chem. Eng. J. 179, 131 (2012). 25. M. Shao, J. Han, M. Wei, D. G. Evans, and X. Duan, Chem. Eng. J. 168, 519 (2011). 26. M. Park, H. Kim, D. Park, J. Yang, and J. Choy, Bull. Korean Chem. Soc. 33, 1827 (2012).

27. P. Costa and J. M. S. Lobo, Eur. J. Pharm. Sci. 13, 123 (2001). 28. J. Singh, S. Gupta, and H. Kaur, Trends Applied Sci. Res. 6, 400 (2011). 29. T. Kodama, Y. Harada, M. Ueda, K. Shimizu, K. Shuto, and S. Komarneni, Langmuir 17, 4881 (2001). 30. L. Ling, J. He, M. Wei, D. G. Evans, and X. Duan, Wat. Res. 40, 735 (2006). 31. A. M. El-Toni, S. Yin, and T. Sato, Mater. Lett. 58, 3149 (2004).

ARTICLE

Sci. Adv. Mater., 6, 1–11, 2014

11