Effect of Ag and Ni-Doped Cerium Oxide Nanoparticles on the Formation of ROS and Evaluation as an Alternative Physical Sunscreen Material

Abstract

CeO₂ nanoparticles (nanoceria) were proposed as an alternative physical sunscreen agent with antioxidant properties and comparable UV absorption performance. Green synthesis of nanoceria with Ag and Ni dopants resulted in doped nanoceria with lower catalytic activity and biologically-safe characteristics. The doped nanoceria was characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), Rancimat Instrument, and UV-Vis Spectrophotometer for SPF (Sun Protection Factor) determination. XRD and TEM analysis showed that nanoceria had been successfully formed in nanoscale-sized with a change in crystallite size due to the crystal defect phenomenon caused by dopant addition. While the Rancimat test and band gap energy analysis were conducted to evaluate the oxidative stability and reactive oxygen species formation, it was confirmed that dopant addition could decrease catalytic activity of material, resulting in Ni-doped Ce with a longer incubation time (11.81 h) than Ag-doped Ce (10.58 h) and non-doped Ce (10.30 h). In-vitro SPF value was measured using the thin layer technique of sunscreen prototype with Virgin Coconut Oil (VCO)-based emulsion, which yielded 10.862 and 5.728 SPF values for 10% Ag-doped Ce and 10% Ni-doped Ce, respectively. The dopant addition of nanoceria could reduce catalytic activity and give a decent in vitro UV-shielding performance test; thus, Ag and Ni-doped nanoceria could be seen as promising candidates for alternative physical sunscreen agents.

Introduction

Indonesia is a tropical country crossed by the equator line, with high daily exposure to UV rays. Therefore, sunscreen is necessary to be applied since it can prevent various skin problems caused by overexposure to UV, such as erythema, hyperpigmentation, skin aging, and melanoma.¹⁾ Sunscreens are classified based on their mechanism of action into physical and chemical sunscreens. Physical sunscreens protect our skin by blocking and scattering UV radiation, meanwhile chemical sunscreens can absorb UV rays. However, chemical sunscreens have risk of unwanted UV agents absorption into the skin that can cause oxidative stress.²⁾ Until now, agents of physical sunscreens are limited to ZnO and TiO₂ in the form of bulk and nanoparticles. The nanoparticle of metal oxides could enhance anti-UV activity and be applied more evenly on the skin surface. However, ZnO and TiO₂ nanoparticles potentially produce reactive oxygen species (ROS) through their high photocatalytic activities. These could lead to DNA damage and enhance cell toxicity, which emerged as another health concern.³⁾

Cerium is a rare earth metal that exists in the form of Ce³⁺ and Ce⁴⁺. Shifting of the ion valence caused cerium could act as an antioxidant.⁴⁾ In addition, CeO₂ nanoparticles (nanoceria) have UV-shielding ability; hence they are proposed as alternative physical sunscreen agents.^3,5–7) However, the application of nanoceria in commercial sunscreen products has yet to exist. One of the concerns is its photocatalytic activity, which is still relatively high in oxidizing organic material contained in the sunscreen base. The addition of dopants is known to reduce the catalytic activity of nanoceria.⁶⁾ Therefore, in this study, nanoceria was synthesized using the green synthesis method, with the addition of Ag and Ni dopants. Ethanol extract from Moringa oleifera leaves was used as a reduction agent because of its antioxidant activity, making it safe for skin application. Then, the characteristics of Ag-doped Ce and Ni-doped Ce were analyzed, and these materials were dispersed into an emulsion base. In-vitro sun protection factor (SPF) value was measured using Mansur Equation.⁸⁾

Experimental

Materials

Materials used in this research are Cerium Nitrate Hexahydrate (Ce(NO₃)₃.6H₂O) and NaOH from Sigma-Aldrich, Germany; Nickel Nitrate Hexahydrate (Ni(NO₃)₃.6H₂O) from PT. Smart Lab, Indonesia; PEG-40 HCO from Emulsogen, Clariant, Switzerland. While Silver Nitrate (AgNO₃), Virgin Coconut Oil, Tween 20, Na-citrate, and demineralized water were purchased from chemical stores in Indonesia, and Moringa oleifera leaves powder was purchased from grocery stores in Indonesia.

Preparation and Pythochemical Screening Test of Leaf Extract

Simplisia powder of Moringa oleifera leaf was macerated for 3 × 24 h using ethanol and was concentrated using a rotary evaporator at 50 °C. Then Moringa oleifera leaf extract was dissolved in demineralized water with a composition of 2.79 g per 100 mL. The extract solution was then filtered using Whatman filter No. 1 and later would be used for nanoceria green synthesis. Pythochemical screening test was performed to detect components of the extract.

Green Synthesis of CeO₂ Nanoparticles (NPs)

0.1 M solution of cerium nitrate was mixed with 120 mL of Moringa leaves ethanol extract solution and stirred for 2 h at 1500 rpm, with a temperature of 85 °C. Silver nitrate and nickel nitrate were added to each mixture as nanoceria dopants with a concentration of 3% (w/w) calculated by cerium mass. The mixture was basified using NaOH until it had final pH of 9. Then, the brownish-green mixture was washed using demineralized water and was centrifuged at 20000 rpm. The supernatant was discarded, the pellet was collected, then dried in an oven at 90 °C for 3 h and calcined at 800 °C for 2 h to remove the remaining organic matter.

Characterization Technique

Characteristics of synthesized nanoceria were analyzed using Transmission Electron Microscope (TEM, Hitachi HT7700) at 120 kV to observe its morphology. Samples for TEM were dispersed using 1% PEG-40 HCO and 0.1% Na-Citrate as stabilizers. X-Ray Diffractometer (XRD) Bruker D8 Advance was used to analyze the crystal structure of the nanoparticles based on the Powder Diffraction File (PDF) database and the average crystallite size was estimated using the Rietveld refinement method on Profex graphical user interface (GUI) program. The lattice parameter of doped nanoceria can be calculated using Vegard’s law, assuming only substitution occurs in the matrix, as follow⁹⁾:

  

(1)

with a_Ce1−xMxO₂ is the lattice parameter of doped nanoceria, a_CeO2 is the lattice parameter of undoped nanoceria, R_O is the atomic radius of oxygen, R_M is the atomic radius of dopant, and n_x is dopant concentration. The atomic radius was retrieved from Shannon,¹⁰⁾ and dopant concentration was determined using Energy Dispersive X-ray Fluorescence (ED-XRF) analysis using Orbis micro-XRF from EDAX. UV-Visible Spectrophotometer (Thermo Fisher Scientific – Evolution 220 Series) at a wavelength range of 250–600 nm was used to calculate band-gap energy and determine in-vitro SPF value. First, band-gap energy analysis was carried out using nanoceria powder using UV-Vis reflectance measurement. Then, Rancimat Instrument (873 Biodiesel Rancimat, Metrohm) was used to analyze the catalytic activity of each nanoceria. Samples for the Rancimat test were dispersed into Virgin Coconut Oil (VCO) as organic material with a composition of 1 g nanoceria in 10 mL oil.

Preparation of Sunscreen Prototype

The emulsion base was prepared using VCO, Tween 20, and distilled water with a comparison of 32 : 32 : 36 (w/w). VCO was mixed with Tween 20 for 20 min at a speed of 600 rpm. Then, distilled water was added to the mixture slowly while stirring until a white-milky emulsion was formed. Nanoceria powder was dispersed into an emulsion base geometrically with a final concentration of 10% of nanoceria.

In-Vitro SPF Measurement

SPF value was determined using UV-Vis measurement of the sunscreen prototype. First, the UV-Vis sample was prepared by spreading 2 mg/cm² sunscreen prototype on quartz plates.¹¹⁾ Then, the absorbance values were taken in three repetitions for every 5 nm in the wavelength range of 290 to 320 nm. The UV-Vis data was then calculated using the Mansur equation to obtain the SPF value.⁸⁾

  

(2)

CF is the correction factor (10), EE(λ) is the erythemal efficiency spectrum, and I(λ) is the intensity spectrum of the solar simulator used in a previous study, meanwhile EE x I are constants.^8,12)

Results

Characterization of Doped-Cerium Nanoparticles

Nanoceria were synthesized using cerium nitrate hexahydrate and ethanol extract of Moringa oleifera leaf as the precursors. Phytochemical screening test showed that the ethanol extract of Moringa oleifera leaves contained flavonoids, hydrolyzed tannins, phenolic compounds, alkaloids, and steroids/triterpenoids. Nanoceria were synthesized with the addition of Ag and Ni dopants, resulting in nanoceria powder as shown in Fig. 1. Synthesizing non-doped Ce resulted in bright-yellow powder, while Ag-doped Ce appeared as grey powder and Ni-doped Ce as pale-yellow powder.

Fig. 1. Synthesized CeO₂ Nanoparticles: without Doping, Ag-Doping, and Ni-Doping

XRD Analysis

Peaks of Ag-doped Ce and Ni-doped Ceria nanoparticles from XRD analysis are shown in Fig. 2. XRD peaks correspond to crystal orientations of CeO₂ for (111), (200), (220), (311), (222), (400), (331), (420), and (422) based on the PDF database number 01-080-8533. The evaluation of XRD measurement results is presented in Table 1. Rietveld refinement results from XRD measurements give the lattice parameter, average crystallite size, and microstrain. The Rietveld lattice parameter for non-doped Ce is 5.413 Å, larger than the bulk CeO₂, which is 5.411 Å. Vegard’s law calculation gives lattice parameter values assuming only substitution occurs. Vegard’s law calculation shows that Ag-doped Ce leads to lattice expansion due to larger Ag⁺ ion (r = 1.75 Å), while for Ni-doped CeO₂, the lattice parameter tends to decrease due to smaller Ni²⁺ ion (r = 0.69 Å).

Fig. 2. XRD Result of CeO₂ Nanoparticles with Ag and Ni-Doping

Table 1. Evaluation of XRD Measurement Results

CeO2 samples	nx (at%)	avegard (Å)	arietveld (Å)	D (nm)	Microstrain
Non-doped	0	5.413099	5.413099	28.01	0.000002222
Ni-doped	3.38	5.380788	5.412502	34.98	0.000001451
Ag-doped	4.02	5.413662	5.412602	37.80	0.000001197

TEM Analysis

TEM analysis shows that cerium nanoparticles were successfully formed in nanoscale sizes. Figure 3 provides the size and morphology of Non-doped Ce, Ag-doped Ce and Ni-doped Ce.

Fig. 3. TEM Images of 0.1% CeO₂ Nanoparticles Dispersed in 1% PEG-40 HCO and 0.1% Na-Citrate as Stabilizers with the Addition of Dopant: (a) Non, (b) Ag, and (c) Ni

Rancimat Test

Rancimat test was established on VCO to assess the induction time of each nanoceria samples, thereby showing their catalytic activities. According to the Rancimat test showed in Fig. 4, the incubation time of test samples was as follows: 10.30 h for Non-doped Ce, 10.58 h for Ag-doped Ce, and 11.81 h for Ni-doped Ce. In the case of pure VCO, there was no incubation test shown in the result since there was no significant increase in the secondary product formation.

Fig. 4. Incubation Time of Rancimat Test for: (a) Pure VCO, and (b) Non-doped Ce, (c) Ag-Doped Ce, (d) Ni-Doped Ce Dispersed in VCO

Band Gap Energy and ROS Formation

Adding a particular dopant could modify the band gap energy of the materials. Figure 5 shows that non-doped Ce has a higher band gap energy of 2.370 eV. On the other hand, doping cerium nanoparticles resulted in lower band gap energy with Ag-doped and Ni-doped, resulting in band gap energy of 1.689 eV and 1.414 eV, respectively.

Fig. 5. Tauc Plot of Band Gap Energy from: (a) Non-doped Ce, (b) Ag-Doped Ce, and (c) Ni-Doped Ce for Direct Allowed Transition (γ = 1/2)

The value of band gap energy is crucial to determine the position of the conduction and valence band. The position of conduction band (CB) and valence band (VB) is related to the production of reactive oxygen species (ROS), as shown in Fig. 6 below.¹³⁾ Non-doped Ce would produce higher amount of ROS, consistent with Rancimat Test, due to the energy required to excite sample from the conduction band to the valence band surpassing the energy level for generating hydrogen peroxide.

Fig. 6. Position of Valence Band and Conduction Band Non-doped Ce, Ag-Doped Ce, and Ni-Doped Ce Related to ROS Formation

In-Vitro SPF Value Determination

Determination of SPF value in the form of thin film was conducted and the results were shown in Table 2. Nanoceria samples exhibited an increase in SPF value compared to the base emulsion. Furthermore, the addition of dopants resulted in even higher SPF values with Ag-doped Ce showing the highest SPF value compared to Ni-doped Ce and non-doped Ce.

Table 2. In-Vitro SPF Value of Doped-Ce

Material	SPF Value
Emulsion base (Blank)	2.80025
10% Non-doped CeO2	4.82227
10% Ag-doped Ce	10.8620
10% Ni-doped Ce	5.72825

Discussion

The green synthesis technique uses biologically safe and non-toxic materials compared to other nanoparticle synthesis methods that use chemical-reducing agents, with the risk of leaving toxic residue.¹⁴⁾ Nevertheless, this method is widely used because it is relatively harmless, particularly if these materials are applied directly to the skin. Nanoceria were evaluated using XRD, TEM, Rancimat Instrument, and UV-Vis Spectrometry. First, XRD and TEM were used to confirm the crystallite size and the morphology of CeO₂ nanoparticles from the green synthesis method. Then, the Rancimat test was conducted to evaluate the samples’ oxidative stability by detecting the formation of secondary reaction products. Finally, UV-Vis Spectrophotometry was performed to determine the UV-shielding activity of each nanoceria material by calculating the in-vitro SPF value and estimating the band-gap energy by the Tauc plot method.

The observed shift in the X-ray diffraction lattice parameters was assumed to be due to the formation of defects on the lattice, specifically oxygen vacancies.¹⁵⁾ The formation of oxygen vacancies introduces the reduction of Ce⁴⁺ to Ce³⁺, leading to the mixed valence of Ce ions in the unit cell. The size difference between Ce³⁺ (r = 1.034 Å) and Ce⁴⁺ (r = 0.92 Å) could induce strain in the lattice caused by local symmetry distortion, thus increasing in lattice parameter due to Ce³⁺ formation.^16,17) Meanwhile, the lattice parameter from Rietveld refinement shows lattice shrinking for both Ag-doped and Ni-doped Ce. Lattice parameter deviation of Rietveld refinement from Vegard’s law calculation could be due to oxygen vacancy and interstitial formation in the matrix. Interstitial formation in the case of Ni-doped Ce causes larger lattice shrinking compared to that of Ag-doped Ce since Ni²⁺ ion is smaller than Ce³⁺ (r = 1.034 Å) and Ce⁴⁺ (r = 0.92 Å).¹⁸⁾ Meanwhile, enhancing of microstrain value leads to smaller nanoparticle size.¹⁹⁾

Analysis with TEM was performed with the addition of PEG-40 HCO and Na-Citrate as stabilizers to reduce the agglomeration states of the nanoparticles.²⁰⁾ TEM analysis revealed morphological and size features which correspond to XRD analysis. Both non-doped and doped Ce have polygonal shapes, wherein Ag-doped Ce has a larger average size (28.046 ± 2.49 nm) than Ni-doped Ce (24.519 ± 3.16 nm) and non-doped Ce has the smallest size (15.705 ± 3.31 nm).

The Rancimat test is also known as the accelerated oxidation test to determine samples resistance against constant high temperatures during a certain period. The apparatus consists of reaction vessel and measuring vessel. Samples containing oil and/or fat will be heated in the reaction vessel and secondary reaction products will be formed and passed through the measuring vessel by the air stream. These secondary products will be absorbed in the measuring vessel consists of absorption solution and the electrical conductivity of the solution will be continuously recorded until the end of the reaction. Radical reactions would occur on unsaturated fatty acid, resulting in secondary products formation such as peroxides, alcohols, aldehydes, and carboxylic acids, that would affect the electrical conductivity as read in the Rancimat Instrument.²¹⁾

VCO would be used as an oil base in prototype sunscreen emulsions due to its antioxidant and UV-shielding properties.²²⁾ In addition, VCO could reduce skin damage caused by high UV exposure.²³⁾ Generally, lipid oxidation of VCO is assessed by detecting peroxide value. Lipid peroxidation causes rancidity in oils and fats due to the allylic positions of the fatty acid carbon chains being more reactive to oxygen, forming peroxides as the secondary products.²⁴⁾ Peroxides will be absorbed in the measuring solution, affecting the conductivity of the measuring solution. Alteration of the conductivity will be depicted on the graph of electrical conductivity against induction time of the sample. Significant increase in conductivity marks as the induction time. Hence, the longer induction time, the longer secondary product will be formed, which means that sample is more stable to catalytic activity. Therefore, it was proven that adding Ag and Ni dopants (induction time 10.58 h and 11.81 h, respectively) could prolong incubation time compared to non dopants (induction time 10.30 h), thereby reducing the catalytic activity.

The band gap energy of semiconductor material represents the energy needed to excite an electron from the valence band into the conduction band. An electron jump from the valence into the conduction band requires a specific minimum amount of energy for the transition. This parameter is crucial in predicting the photochemical properties of materials, particularly the photocatalytic activity of semiconductor materials.²⁵⁾ Diffuse reflection is commonly used as a standard method to determine the optical properties of nanoparticles in the form of a solid-state.²⁶⁾ Reflectance spectra can be measured corresponding to absorption spectra using the Tauc plot equation, as shown below.²⁷⁾

  

(3)

The exponent ‘γ’ depends on the type of transitions that occurs in the electron excitation phenomenon, such as direct allowed transition (γ = 1/2), direct forbidden transition (γ = 3/2), indirect allowed transition (γ = 2), and indirect forbidden transition (γ = 3). First, the type of transition is chosen based on the most abrupt slope changes around the tails obtained from the Tauc Plot. Then, band gap energy is determined using the point of intersection of the tangent line and the x-axis (hv).²⁷⁾ The decrease of band gap energy values is related to the changes in lattice parameters, which depends on the ionic radius, electronegativities, valence, and incorporation mechanisms. Therefore, modifying lattices and surface areas can affect band gap energy values and its photocatalytic properties synergistically, although no linear or monotonic relationship is found between lattice parameters, surface areas, and band gaps.²⁸⁾ Conduction band and valence band are calculated based on the absolute electronegativity of CeO₂ (5.56 eV) against the band gap value obtained from the Formula 3. Band gap value states the distance between absolute electronegativity of CeO₂ and between the valence band and the conduction band. Based on the energy level diagram in Fig. 6, the formation of hydrogen peroxide as ROS lies on energy level of 0.94 eV, which is passed by the energy required to excite Non-doped Ce from the valence band to the conduction band. However, this energy level is not exceeded by Ag-doped Ce and Ni-doped Ce, therefore Non-doped Ce tends to produce higher ROS. This is also in correspondence with the results of photocatalytic activity measured by Rancimat test.

Thin film measurement is more reliable than diluted form since it represents the actual shielding properties obtained from spreading the sample on the skin surface, based on previous study and sunscreen regulations.^29,30) Ag-doped and Ni-doped CeO₂ shows higher SPF value than non-doped Ce in the form of an emulsion. Ten percent of Ag-doped Ce and Ni-doped Ce showed low protection of UVB following the Recommendation 2006/647/EC published by European Commission.³¹⁾ However, this result is promising for further sunscreen development using nanoceria materials, with lower catalytic activity properties and reduction of ROS formation.

Acknowledgments

This research was completed during my study in School of Pharmacy, Institut Teknologi Bandung and was conducted at Research Center for Nanoscience and Nanotechnology, Institut Teknologi Bandung. I give thanks to Research Center for Nanoscience and Nanotechnology for the support of this study.

Funding

This project was financially supported by Bandung Institute of Technology under scheme of: Integrative Research Project 2022, granted to Heni Rachmawati.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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