Synthesis of (cinnamate-zinc layered hydroxide) intercalation compound for sunscreen application
© Mohsin et al; licensee Chemistry Central Ltd. 2013
Received: 13 December 2012
Accepted: 25 January 2013
Published: 6 February 2013
Zinc layered hydroxide (ZLH) intercalated with cinnamate, an anionic form of cinnamic acid (CA), an efficient UVA and UVB absorber, have been synthesized by direct method using zinc oxide (ZnO) and cinnamic acid as the precursor.
The resulting obtained intercalation compound, ZCA, showed a basal spacing of 23.9 Å as a result of cinnamate intercalated in a bilayer arrangement between the interlayer spaces of ZLH with estimated percentage loading of cinnamate of about 40.4 % w/w. The UV–vis absorption spectrum of the intercalation compound showed excellent UVA and UVB absorption ability. Retention of cinnamate in ZLH interlayers was tested against media usually came across with sunscreen usage to show low release over an extended period of time. MTT assay of the intercalation compound on human dermal fibroblast (HDF) cells showed cytotoxicity of ZCA to be concentration dependent and is overall less toxic than its precursor, ZnO.
(Cinnamate-zinc layered hydroxide) intercalation compound is suitable to be used as a safe and effective sunscreen with long UV protection effect.
KeywordsSunscreen Zinc layered hydroxide Zinc oxide Optical properties Cytotoxicity
World Health Organization estimates that 60,000 deaths occur in 2000 from melanoma and other skin cancers due to solar ultraviolet (UV) radiation . Exposure to sunlight is unavoidable as we go about our daily errands. Sunscreen is our last defense against UV radiation. However, protection with sunscreen deemed inadequate due to photodegradation of organic UV absorber in sunscreen products that not only causes decrease in UV protection but also degrades with the occurrence of toxic degradation products. Therefore, development of a new sunscreen formulation is called for, to avoid previously mentioned effects.
Layered metal hydroxides can be categorized into layered double hydroxide (LDH) and layered hydroxide salt (LHS). Several studies have been done on the use of layered metal hydroxide-based sunscreen carrier system, in particular, LDH [2–4]. LDH can be represented by the general formula of [M3+1-xM3+x(OH)2]z+An-z/n.yH2O where An- is the interlayer anion, M2+ and M3+ are di- and trivalent metallic cation, respectively . Previous work has shown that cinnamate intercalated into Zn/Al LDH showed excellent UV ray shielding properties . However, to the best of our knowledge application of LHS as host has yet to be explored.
Zinc layered hydroxide (ZLH) is a type of LHS, having the general formula of; M2+(OH)2-x(A m- ) x/m ∙nH2O where M2+ in this case is the metal cation Zn2+ and A m- is the counter ion . They boast a structure consisting of positively charged layers that can expand or contract depending on the nature of interlayer anions. In recent years, there has been extensive research on the use of ZLH as drug carriers [6, 7], slow release herbicides , flame retardants  and anti-corrosion agents . In this study we further exploit its potential as an efficient host to organic molecules for possible application in sunscreen formulation.
In the present work, we investigated the intercalation of anion, cinnamate, into interlayer galleries of ZLH via direct method, as well as properties of the resulting cinnamate-ZLH (ZCA) intercalation compound. Direct method used involved a direct reaction between anion solution and ZnO precursor. Unlike other LHS synthesis methods like hydrolysis of salts and oxides , urea hydrolysis , precipitation with alkaline solution  and solid state reactions , this method is simple, environmentally friendly and economical as it involves fewer steps and fewer chemicals.
By intercalating organic UV absorbers into interlayer galleries of ZLH, the expected advantages are; UV absorber stabilization in interlayer region of a lamellar host to increase photo-stability and decrease degradation of UV absorber, absorption of ultraviolet light rays in UVA and UVB region and the absence of close contact between skin that subsequently eliminates allergy problems .
In the present investigation, we selected human dermal fibroblast (HDF) cells as a model to evaluate possible toxicity induction on the cells. Dermal fibroblasts are the most abundant cell in the human skin and represent the primary level of exposure to various environmental and other toxicants. Human skin is the primary anatomical barrier for various pathogens and damage, which acts as an important boundary marker between internal and external environment in the bodily defense system. Hence, the resulting intercalation compound of the present study was investigated for toxicity on human dermal fibroblast cells.
Materials and methods
Cinnamic acid (98%) was purchased from Acros (Geel, Belgium). Zinc oxide (99%) was obtained from PC Laboratory chemicals and was used without further purification. Sodium hydroxide (99%) from Merck (Darmstadt, Germany), dimethyl sulfoxide (DMSO) and phosphate-buffered solution from Sigma-Aldrich (Missouri, USA) and sodium chloride (99%) from HmbG Chemicals (Hamburg, Germany) were used without further purifications.
Synthesis of zinc layered hydroxide intercalated with cinnamate
About 0.2 g of ZnO was reacted with 100 mL of 0.1 mol/L CA solution. The intercalation compound was titrated with 2 mol/L NaOH to the final pH of 8 before it was magnetically stirred for 5 h at room temperature. Then it was aged in an oil bath at 70°C for 18 h, before being centrifuged and washed with deionized water. The final white solid (ZCA) was dried under vacuum at 70°C, overnight.
Powder x-ray diffraction (PXRD) patterns were recorded with a XRD-6000 (Shimazdu, Kyoto, Japan) using CuKα radiation (λ = 1.5418 Å) at 30 kV and 30 mA. The data was collected from 2 - 60º at a dwell time of 0.5º min-1. Fourier transform infrared (FTIR) spectra were recorded over the range of 280–4000 cm-1 on a Perkin-Elmer Spectrum 100 (Perkin-Elmer, Waltham, Massachusetts, USA) equipped with universal attenuated total reflectance (ATR) accessory. The carbon and hydrogen content in the intercalation compound were analyzed on a CHNS-932 (LECO Instruments, Michigan, USA). The chemical composition of the samples was analyzed for zinc by inductively coupled plasma atomic emission spectrometry (ICP-AES) using a Perkin-Elmer spectrophotometer model Optima 2000DV (Perkin-Elmer, Massachusetts, USA) under standard conditions. Thermogravimetric and differential thermogravimetric analyses (TGA/DTG) were performed on alumina crucibles with a Metter-Toledo instrument model TGA851e (Greifensee, Switzerland) at a heating rate of 10°C min-1 in the range of 25 – 1000°C and under nitrogen gas flow of about 50 mL·min−1. Surface characterization of the materials was carried out using a nitrogen gas adsorption-desorption technique at 77 K with a Micromeritics, ASAP2000 (Georgia, USA). The surface morphology of the samples was observed by a field emission scanning electron microscopy (FESEM) using a ZEISS supra 40VP (Oberkochen, German), and optical measurements were performed on a Shimadzu (Kyoto, Japan) UV–VIS-NIR diffuse reflectance spectrometer (UV-3600 model).
Release of cinnamate from ZCA
Release of cinnamate anion from ZLH host against time was measured in situ at λmax = 272 nm using a Perkin-Elmer UV–VIS spectrometer Lambda 35 (Perkin-Elmer, Massachusetts, USA) by adding 0.2 mg of sample into 3.5 mL of deionized water, 0.5 mol/L NaCl and pH 5.5 phosphate buffer solution, at room temperature. Data was collected and fitted to zero-, first-, pseudo-second order and parabolic diffusion kinetic models.
Human dermal fibroblasts were obtained from ATCC (Virginia, USA). Human dermal fibroblast cells were cultured at 37°C and 5% CO2 in high glucose Dulbecco’s Modified Eagle Medium (DMEM) (ScienCell Research Laboratories, California, USA) containing 2% fetal bovine serum, 0.5% penicillin–streptomycin, 1% glutamine, and 1% non-essential amino acids. Cultured cells were passaged using 0.25% trypsin. At 85% confluence, cells were harvested using 0.25% trypsin and were subcultured/seeded into 96-well plates.
Cell viability assay
Healthy human dermal fibroblast cells (at a density of 1 × 104 cells/well) were seeded in a 96-well culture plate. Cultured cells were incubated for 24 h prior to treatment. The cells were growing until 80% confluence, and the media were replaced with different gradient concentrations (from 0.781 μg/mL to 25 μg/mL) of sample in media for 24 h. After the exposure time, sample containing media were aspirated. The cells were then incubated with freshly prepared MTT solution in fresh medium for 4 h at 37°C until a purple colored formazan product developed. After the incubation time, 100 μL DMSO was added to each well to dissolve the formazan crystals. Absorbance of the solution in 96 well plates was measured at 570 and 690 nm using a Bio-Tek ELISA microplate reader EL800 (Vermont, USA). Cell viability was analyzed as a ratio of sample treated cells to untreated cells (control at 0 μg/mL).
Results and discussion
X-ray diffraction and spatial orientation of the guest between ZLH interlayers
Sun et al. reported intercalation of cinnamate anion into Zn/Al LDH as host. The reported basal spacing of intercalation compound is 18.0 Å . Subtracting layer thickness, the gallery available to be occupied is 13.2 Å which is relatively close to the value recorded for ZLH host. Hence we can deduce that cinnamate maintains similar orientation in both hosts. However, the observed basal spacing for ZLH host is significantly larger than what was reported for LDH host. We proposed that ZCA intercalation compound follows LHS type IIb structure which is formed by one quarter of the octahedral zinc cations displaced from main layer to tetrahedral sites located above and below each empty tetrahedron. The base of the tetrahedral share hydroxide groups with the octrahedral layer and the apex is occupied by water molecules. Thus, the tetrahedral sheet accounts for larger basal spacing recorded for cinnamate intercalation in ZLH host.
Physico-chemical properties of ZnO and ZCA intercalation compound
BET surface area (m2/g)
BJH pore diameter (Å)
BJH pore volume (m2/g)
ZnO has long been used in sunscreen products as it carries a strong capability to absorb UV rays. However, recent findings shows hair follicle allows penetration of nanoparticles up to 320 nm in size and excitation of ZnO due to sunlight and household items produces oxygen radical species that plays a role in skin aging and photocarcinogenesis [22–24]. In a way, conversion of ZnO precursor to particles of ZCA that are bigger in size will prevent penetration into hair follicles.
Release behavior of cinnamate anions
ZLH as host for sunscreen molecules will have some level of release as it has anion exchange capability but the release was shown to be very slow and achieved saturation state at low concentration. These results shows novelty of ZLH use in sunscreen formulation as it provides prolonged UV protection as well as prevention of UV ray absorbent molecules photodegradation into toxic degradation products.
Release kinetics of cinnamate from ZCA intercalation compound
Correlation coefficient, rate constant and half time obtained by fitting the release data of cinnamate from ZCA intercalation compound into various media using zeroth-, first-, parabolic diffusion and pseudo-second order kinetic models
Saturated release (%)
Correlation coefficient, r2
Rate constant of pseudo second order, k(L mg-1min-1)
t1/2of pseudo second order(min)
9.09 × 10-6
0.5 mol/L NaCl
1.07 × 10-5
Phosphate buffer pH 5.5
1.38 × 10-4
Release rate of the anion from the ZLH interlayer is influenced by the affinity of available anions in the media. The presence of carbonate, an anion known to have the strongest affinity towards ZLH interlayer, in deionized water was responsible for relatively fast and high accumulated release. Low accumulated release and slow release rate in 0.5 mol/L NaCl is attributed to low ion exchange affinity of chloride towards interlayer of ZLH. Phosphate buffer solution consists of phosphate, carbonate and chloride while 0.5 mol/L NaCl contains only chloride. Release rate and accumulated release will be elevated with the presence of phosphate combined with other anions due to multiple hydrolysis of phosphate [34, 35]. ZCA was found to have the highest accumulated release and fastest rate in skin pH simulation. Nonetheless, ZCA reached a saturated release in phosphate buffer after prolonged time (13 h) in comparison to actual use of sunscreen product. Furthermore, pH 5.5 phosphate buffer only reproduces skin pH value and not the real physiological conditions.
Effect of ZLH-sunscreen intercalation compound on cell viability
The biomedical applications of synthesized nanoparticles are currently attracting much research interest. The progress and application of nanotechnology enhances the quality of our human lives but also results in a health burden. Major issue in determining the efficacy of these materials is assessing their potential cellular toxicity either due to their inherent chemical composition/structure or as a consequence of their nanoscale biophysical properties . In order to evaluate directly if these synthesized nanoparticles were in a range to be physiologically harmful to human skin, we tested their effects on human dermal fibroblasts using a cell viability biological assays.
We found that synthesized intercalation compound exposure effectively reduced cell viability of human dermal fibroblasts at concentration 25 μg/mL and above. Based on this finding we suggest that intercalation compound dosage up to 12.5 μg/mL did not produce any cytotoxicity. Hence, further studies should focus at the range of the concentration not more than that 25 μg/mL, to develop the cosmetic product using the particular nanocarrier. At a concentration higher than 25 μg/mL, substantial study to focus on dermal toxicity with experimental animals for translational studies to provide systematic molecular mechanisms for biomedical application.
In the present work, organic UV-ray absorbing active agent, cinnamate anion has been successfully intercalated into ZLH interlayers spaces from zinc oxide precursor to generate ZCA intercalation compound with a basal spacing of 23.9 Å to accommodate cinnamates in a bilayer arrangement. ZCA intercalation compound retained excellent absorption capacity in the UV region of pure CA but with slight shift in absorption peaks and broadened absorption range due to arrangement in host and host-guest interactions. Retention of cinnamate in ZLH interlayers was tested with various media to show slow release and saturated release at a very low concentration. Hence it was demonstrated that the resulting material is suitable to be used as sunscreen with long term UV protection effect.
Prof. Dr. Mohd Zobir Hussein is a Professor of Chemistry in Institute of Advanced Technology (ITMA), Universiti Putra Malaysia. His major research areas include layered organic–inorganic nanohybrid for gene and drug delivery, nanoparticles and nanostructured materials, their design, synthesis and applications. He is a prolific author and has contributed to more than 200 technical papers. He is the assignor of 1 granted patent on the preparation method of nanomaterial for controlled release formulation and co-assignor of another 2 granted patents.
Zinc layered hydroxide
Human dermal fibroblast
Layered hydroxide salt
Cinnamate- zinc layered hydroxide intercalation compound
This work was financially supported by the Ministry of Higher Education (MOHE) of Malaysia under ERGS/1/11/STG/UPM/01/01/18. One of the authors (SMNM) would like to thank Universiti Putra Malaysia for Graduate Research Fellowship (GRF).
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