An emulsion of pigmented nanoceria as a medicinal cosmetic

Abstract

Formulating nanoparticle based cosmetics with medicinal values to protect skin from environmental stress is a challenging task nowadays. In our work, we have prepared an emulsion of bluish pigmented cerium oxide nanoparticles (nanoceria) with potent free radical scavenging activity. The nanoceria were prepared by conventional thermal decomposition, and encapsulated by the amine-containing polyethylenimine. Characterizations such as Nuclear Magnetic Resonances (NMR), Dynamic Light Scattering (DLS), Atomic Force Microscope (AFM), Transmission Electron Micrographs (TEM) and X-ray Diffractograms (XRD) showed that the nanoceria were near spherical, monodispersed and sized 3–10 nm. UV-vis analysis confirmed that the particles were pigmented whereas circular dichroism revealed that the secondary structure of collagen, a fibrillar protein present in skin, was not affected by the presence of these pigmented nanoceria. In vitro anti-oxidant assays showed that the pigmented nanoceria were more effective than non-pigmented nanoceria against nitrile and peroxy radicals, with their activity depending on the concentration of CeO₂. Thus, the emulsion of pigmented nanoceria can be evaluated, in future, as a medicinal cosmetic for protecting the skin.

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1. Introduction

The skin is an important organ and the largest organ protecting our body from environmental stress and the invasion of microbes. As we are living in an environment which is being polluted every day and getting increasingly hot due to global warming, our skin needs to be protected. Cosmetics, being widely used by human beings, could play an important role. Developing a typical cosmetic formulation with good consumer acceptability is a challenge. According to the American Food Drug and Cosmetic (FDC) Act of 1938, the term ‘cosmetic’ means any article introduced for cleansing, beautifying and promoting attractiveness whereas a ‘drug’ means any article intended for use in diagnosis, cure, mitigation and prevention of disease.¹ A medicinal cosmetic would certainly meet the criteria of both terms, when used for external applications. Hence, we have formulated an emulsion, containing pigmented cerium oxide nanoparticles (nanoceria), which has medicinal properties.

The ingredients used for cosmetics can be either natural or synthetic. From ancient times, people have used naturally available pigments, such as anthocyanins and betalains as cosmetics.^2,3 The advent of talcum powder attracted customers after it was formulated with the desired particle size and shape to improve the perceived skin feel after application.⁴ Researchers nowadays are exploring whether nanoparticles can be used as the main ingredient of cosmetics. TiO₂ and ZnO have been reported to have a sun screening effect as they can reflect and scatter harmful UVA and UVB irradiation.⁵ Activated carbon adsorbs organic impurities and hence is widely used in commercial cosmetics for the purpose of cleansing skin.⁶ Silver based nanoparticles occupy the top of the list of the principal nanomaterials used in cosmetics. Fullerene and gold based nanoparticles are placed in second and third positions, respectively.⁷ There are no unanimous opinions amongst scientists about the toxicity of nanoparticle based cosmetics or their safety for dermal use, so far. Kokura et al. have shown that silver nanoparticles are not able to penetrate human skin. However, when the barrier function of human skin is disrupted, silver nanoparticles can penetrate the skin, but the level of 0.2–2% of Ag can be safe, not causing any toxicity to skin cells.⁸

Cerium oxide, investigated in our work, is a rare earth metal oxide belonging to the lanthanide group. Because of oxygen deficiencies in the crystal lattice, nanoceria entrap molecular oxygen (O₂) and are known as an ‘oxygen buffer’.⁹ They readily interact with and neutralize reactive oxygen species such as the superoxide anion (O₂˙⁻), peroxide radical (O₂˙²⁻), and hydroxyl radical (˙OH).¹⁰ Notably, there is an equilibrium existing between the states of reduced ceria, Ce(III) and unreduced ceria, Ce(IV), when CeO₂ undergoes redox reactions.¹¹ Nanoceria have been reported to have multi-enzyme mimetic activities imitating superoxide dismutase, catalase, oxidase, phosphatase and peroxidase.¹² Because of these unique properties, nanoceria have been investigated in our work in an emulsion formulation for their usage as a cosmetic. The other biological applications of cerium oxide include treating cancerous cells and various ailments including Alzheimer's disease.¹³ It has been reported that nanoceria enhance wound healing processes, effecting increased fibroblast growth and enhanced uptake of molecular oxygen.¹⁴

Polyethylenimine (PEI), used for stabilization of nanoparticles, has numerous reactive amine groups which can be involved in genipin based cross-linking reactions.¹⁵ Modified PEI nanoparticles have been reported to have surface antimicrobial activity and biocompatibility.¹⁶ In addition, the synthetic nature of PEI means that it can withstand the adverse effects of microbial attack and heat degradation during the storage of an emulsion. Genipin, extracted from the fruits of Gardenia jasminoides, has been used as a colouring agent, which has proven biocompatibility.¹⁷ According to the literature, genipin cross-links macromolecules containing primary amines in the pH range 7–10, and forms dimers which act as pigments and impart blue colour to the solution. Genipin is an oxygen based heterocyclic ring. When the cross-linking reaction takes place, genipin is modified into a nitrogen based heterocyclic ring and proceeds with the formation of a number of intermediates. The final process is free radical polymerization to form dimers in the presence of oxygen. As different structural intermediates are involved in dimer formation, the structure of the genipin dimers varies according to the source genipin.^18–20 Apart from pigmentation, genipin has some medicinal values including anti-inflammation, anti-oxidant and anti-tumour promoting factor effects.²¹ Our attempt, in this work, was to formulate an emulsion of pigmented nanoceria with anti-oxidant properties and to evaluate its fundamental physical, chemical and biological properties for its usage as a medicinal cosmetic.

2. Experimental section

2.1. Materials

Cerium(III) nitrate hexahydrate (CeNO₃)₃·6H₂O (99%), 1-octadecene (>95%, GC), oleylamine (70%), polyethylenimine (PEI, M_n 10 000, branched), hydrogen peroxide (H₂O₂, 30%), 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH), β-carotene, linoleic acid, genipin (>98%, 1R, 4aS, 7aS-methyl 1,4a,5,7a-tetrahydro-1-hydroxy-7-(hydroxymethyl)-4a,7a-dimethylcyclopenta[c]pyran-4-carboxylate), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and Tween 20 were purchased from Sigma-Aldrich. All other chemicals used throughout the work were of analytical grade. Type-I collagen was extracted from 6 month old albino rats' tail tendons. The procedures for acid based extraction, purification and determination of collagen concentration are described in our previously published work.^22,23 Mouse embryonic fibroblast cells (NIH-3T3) and human breast cancerous cells (MCF-7) were purchased from the National Centre of Cell Science (NCCS), Pune, India.

2.2. Synthesis of pigmented nanoceria

The preparation of nanoceria was adopted from the literature.²⁴ Briefly, a mixture of 1 mM of Ce(NO₃)₃·6H₂O, 3 mM of oleylamine and 5 g of octadecene was heated to 260 °C for 3 h under a nitrogen gas atmosphere to develop nanoceria. Purification of the nanocrystals was achieved by washing with methanol followed by acetone to eliminate any unreacted cerium precursor and additives. The oleylamine content that was physically adsorbed on to the nanoparticles was eliminated by raising the temperature to 800 °C for 1 h. A 2 mL sample of nanoceria was dispersed in chloroform and phase transferred to obtain water soluble nanoceria by mixing with 10 mL of water using probe sonication (3 min, 70% amplitude, full cycle). To encapsulate the nanoparticles, 1 mL of water soluble nanoceria was added to 2 μmol of PEI in chloroform and the mixture was sonicated with 10 mL of distilled water. The organic solvent was removed from the resulting phase transferred nanoceria by evaporation using a rotavapor at 40 °C. Dialysis was then carried out against distilled water for 24 h to remove free polymeric components from the pure encapsulated nanoceria (PEI ceria). 1 mL of genipin stock solution (2 mM) was added dropwise to the PEI ceria under stirring at 60 °C to cross-link the free amine groups of the polymeric components to produce pigmented nanoceria (PP ceria). Again the sample was subjected to dialysis for 12 h, to remove any non-reacted genipin. Finally, the sample was freeze dried to obtain a solid form. The stock solution of PP ceria was prepared by dissolving the solid in double distilled water in the concentration range 0.1–0.5% (w/v). The preparation, stabilization and pigmentation of nanoceria are shown in Scheme 1.

Scheme 1 Schematic diagram outlining the encapsulation followed by pigmentation of nanoceria. The reaction between genipin and PEI to form a blue colored adduct is shown in the rectangular box. The IUPAC nomenclature of the genipin dimer alone in the cross-linked polymer is (Z)-4-acetyl-7-((4-acetyl-2,7-dimethyl-2H-cyclopenta[c]pyridine-6-yl)methylene)-2,5-dimethyl-7H-cyclopenta[c]pyridine-2-ium named using ChemBioDraw Ultra 14.0 version.

2.3. Instrumentation

The samples PEI and PEI ceria were dissolved in D₂O and their ¹H NMR spectroscopic data were obtained using a Bruker 400 MHz spectrometer with 32 scans at 25 °C. UV-vis spectra were obtained using a Shimadzu UV-visible spectrophotometer UV-1800. To explore the H₂O₂ quenching efficiency of nanoceria, 20 μL of 10% H₂O₂ was added to 1 mL of 0.2% nanoceria samples and the spectra were recorded in 5 min. Thermogravimetric analyses (TGA) were carried out at a heating rate of 5 °C min⁻¹ for a temperature range of 50–800 °C purging with nitrogen gas (50 mL min⁻¹) using a TA instrument Q 50 V20.6 Build 31 thermogravimetric analyser. Fourier transform infrared (FTIR) transmittance spectra were recorded using a FTIR spectrophotometer ABB MB 3000 with the measured range of 600–4000 cm⁻¹ and an accumulation of 32 scans.

The photostability of the sample was assessed using a UVGL-25 compact UV lamp (4 W, 230 V to 50 Hz). The sample was irradiated for 180 min at the far UV wavelength of 365 nm and placed in a 1 cm quartz cuvette with a cover slip. The kinetic data was presented following the second order exponential decay function as given in eqn (1) and (2), and the data was plotted using origin software (version 9.0).

y(t) = y₀ + A₁e^−λ₁t₁ + A₂e^−λ₂t₂ (1)

where y(t) is the exponential decay function, t is the irradiation time and y₀ is the offset. A₁ and A₂ are the amplitude of the function and k represents the decay constants λ₁ and λ₂.

Dynamic light scattering (DLS) analyses of the samples were performed using a Malvern Nano ZS system equipped with a HeNe 633 nm laser (Malvern Zetasizer Nanoseries, Malvern, UK) operated at 25 °C. The pH values of the samples were adjusted to 7.0 using 0.1 N NaOH. The experiments were repeated in triplicate and the averages were taken. Transmission Electron Microscopy (TEM) images were captured using a JEOL 2100 field emission TEM gun at the operating voltage of 200 kV with a single tilt holder. The specimen was prepared by evaporating a drop of PP ceria onto ultrathin carbon type-A 400 mesh copper grids (Ted Pella Inc., CA, USA). X-ray powder diffraction (XRD) patterns of the nanoceria were recorded in the range of 10 to 80° (2θ) with Cu Kα radiation (1.54 Å) using a Philips X'Pert Super diffractometer with a zero background sample holder. Atomic Force Microscopy (AFM) images of liquid samples were prepared by spotting the liquid samples over glass cover slips and vacuum drying them in a desiccator for 12 h. The images were collected in air-dried conditions with T300R Vista probes (NanoScience, AZ, USA; force constant 40 N m⁻¹, resonance frequency 300 kHz) in tapping mode. Circular dichroic (CD) spectra of type-I collagen in the presence of PP ceria were performed in the far UV region (190–260 nm) using a Jasco 815 CD spectropolarimeter. The flow rate of nitrogen gas was 5 L min⁻¹ and the path length of the quartz cell used was 1 mm.

2.4. Evaluation of anti-oxidant activity

DPPH and β-carotene bleaching assays were carried out to evaluate the anti-oxidant properties of PP ceria against nitrile and peroxy radicals, respectively, from the procedures given in the literature.²⁵ 200 μL of DPPH solution (1 mM, EtOH) were mixed with 200 μL stock solutions (0.1–0.5%) of PP ceria and the mixtures were incubated in a water bath at 25 °C to measure the remaining DPPH radicals at 517 nm using UV-vis spectroscopy after 30 min. For the β-carotene bleaching assay, 0.02 mL of linoleic acid and 0.2 mL of Tween 20 were added to 1.0 mL of β-carotene solution (0.2 mg mL⁻¹, CHCl₃). The mixture was emulsified by agitating with 10 mL of distilled water. A 600 μL of emulsion was transferred to test tubes containing the samples (400 μL). The tubes were placed in a water bath at 45 °C for 60 min and the UV-vis absorbance was recorded at 456 nm. The emulsion without any sample was taken as the control and the emulsion without β-carotene was used as the blank. The inhibition percentages for PP ceria against nitrile and peroxy radicals were calculated by the following eqn (3):where A₀ is the absorbance of the control and A₁ is the absorbance of the sample. Each measurement was performed in triplicate and the values were expressed as mean ± S.D.

2.5. Determination of free amino acid content

To measure the degree of cross-linking during pigmentation, the numbers of free amino groups in PEI ceria and PP ceria were measured using a ninhydrin assay, as reported previously.²⁶ Ninhydrin reagents react strongly with amino groups in materials to produce the ninhydrin–amino complex, which has a purple color. A 0.5 mL of PEI ceria or PP ceria (0.1%, H₂O), was mixed with an equal volume of 20 mM ninhydrin solution. The solution was incubated at 80 °C for 20 min. Subsequently, the supernatant liquid was collected and its UV-vis absorbance was measured at 570 nm. The value corresponds to the number of amino groups in the nanoceria which are not involved in genipin dimer formation. The degree of cross-linking was calculated using eqn (4):where N₀ and N_p are the numbers of free amino groups present in PEI ceria and PP ceria, respectively.

2.6. MTT assay

An MTT assay was carried out to evaluate the biocompatibility of PP ceria in vitro. NIH-3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% CO₂, L-glutamine, and 100 units per mL of penicillin/streptomycin at 37 °C. The cells were seeded in 96-well plates (1 × 10⁴ cells per well) for 24 h at 37 °C. Subsequently, a series of concentrations of PP ceria (0.1, 0.3 and 0.5%) was added to the cell cultures. After 24 h incubation, MTT solution (0.5 mg mL⁻¹) was mixed into each well and this was followed by the addition of 100 μL dimethyl sulfoxide (DMSO) to each well. Finally, the cell viability was determined by measuring the optical density at 570 nm using a microplate reader. A control was prepared without the addition of PP ceria to the culture for the comparison of its results with those of treated cells. The morphology of the cell line was visualized using a Leica inverted microscope and staining with fluorescein diacetate (10 μg mL⁻¹).²⁷

2.7. Cellular ROS detection assay

MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% CO₂, L-glutamine, and 100 units per mL of penicillin/streptomycin at 37 °C. The cells were seeded into 96-well plates at 5000 cells per well (15 000 cells per cm²) and grown to sub-confluence. Subsequently, the cells were incubated with 25 μg mL⁻¹ of PP ceria for different time periods of 6, 12, 24 and 48 h. After the incubation, the cells were washed three time in PBS and then exposed to 800 μM H₂O₂ in culture medium for 8 h at pH 7.4. DCFH-DA dye was used to measure intracellular reactive oxygen species (ROS) production. Inside the cell, endogenous esterases deacetylate the DCFH-DA into non-fluorescent 2′,7′-dichlorofluorescein (DCFH), which is further converted into a green fluorescent dichlorofluorescein (DCF) compound in response to ROS production. The stock solution of DCFH-DA was prepared in DMSO. After the treatment with PP ceria, the cells were washed thoroughly in PBS and then incubated with 20 μM DCFH-DA at 37 °C for 30 min. At the end of the incubation period, the cells were washed thoroughly using PBS and subsequently the fluorescent intensities of the samples and control were measured using a Cary Eclipse fluorescence spectrophotometer at 489/529 nm. The relative fluorescence intensity (%) was calculated by the ratio of the mean intensity of the sample to the control, multiplied by 100, and the data was represented as mean ± SEM. The control sample comprised H₂O₂ treated cells without PP ceria pre-incubation.

3. Results and discussion

The encapsulation of nanoceria by PEI was confirmed through ¹H NMR spectra (Fig. 1). PEI has a peak for ethylene protons (–CH₂–CH₂–NH–) at 2.50–2.73 ppm, which agrees with the result in the literature.²⁸ In the case of PEI ceria, the spectral line of the peaks is broadened in the range of 2.48–2.85 ppm indicating the successful encapsulation of the nanoparticles by PEI.

Fig. 1 Representative ¹H NMR spectra of PEI and PEI ceria (solvent D₂O, 4.72 ppm).

UV-vis spectral analyses reveal that PP ceria are bluish in color with an absorption peak at 598 nm indicating the chemical cross-linkage of amine groups of PEI to generate genipin dimers (Fig. 2a). The chemical cross-linkage may occur between two encapsulated nanoparticles or within the same nanoparticle, as the branched polymer contains numerous reactive amine groups. It has been reported that cerium oxide nanoparticles quench H₂O₂, demonstrating the conversion of Ce³⁺ to Ce⁴⁺ under oxidising conditions²⁹ and resulting in a red shift in the UV-vis spectra, which is indicated with dashed arrows in Fig. 2a.

Fig. 2 (a) UV-vis spectra of PEI ceria and PP ceria (solid lines) and the shifts caused by the quenching behaviour of CeO₂ in the samples with H₂O₂ (dotted lines). The dashed arrows indicate the conversion of Ce³⁺ to Ce⁴⁺. (b) FTIR spectra of non-coated nanoceria (CeO₂), PEI, PEI ceria and PP ceria. (c) Thermograms (weight vs. temperature) of non-coated nanoceria (x), and PP ceria (y); the first derivatives of weight vs. temperature of the same are represented by x′ and y′, respectively.

FTIR spectra (Fig. 2b) of the nanoceria show the appearance of a characteristic peak at 486 cm⁻¹ corresponding to the Ce–O vibrational band. PEI shows –CH₃ and –CH₂ bands at 2922 and 2851 cm⁻¹, respectively. The amine functional group is found at 1572 cm⁻¹ and the peak at 1474 cm⁻¹ denotes the –CH bending vibrational mode. These results are in accordance with the results in the literature.^30,31 The encapsulation of the nanoparticles with ligands is apparent in the FTIR spectra from the broadness and shifts in the peaks. Co-ordinate bonding and hydrophobic interactions are the major forces involved in encapsulation.^32–34 The amine group of PEI undergoes broadening as well as a shift, as observed in the respective spectra of PEI ceria and PP ceria. Meanwhile, there is no significant difference between the spectra of PEI ceria and PP ceria, as the amide bond formed during cross-linking of the genipin dimer in PP ceria overlaps in the region around 1631 cm⁻¹. The reduction in intensity of the characteristic Ce–O vibrational band in the spectra of PEI ceria and PP ceria has been attributed to effective coating using PEI.

The degree of cross-linking, when PEI ceria was pigmented to PP ceria, was further investigated using the ninhydrin assay, which quantifies the number of amine groups present in the sample. The degree of cross-linking in PP ceria was found to be 81 ± 1.2%.

The TGA analysis revealed that the initial weight% of non-coated nanoceria was reduced from 100 to 97.8% at the final temperature of 800 °C (Fig. 2c). This non-significant weight loss (2.2%) may be due to removal of moisture content, and the lack of decomposition curves in the thermogram indicates that the compound has no ligands before encapsulation by PEI. The thermogram of PP ceria shows three segments in the temperature ranges of 30–154 °C, 155–255 °C and 256–485 °C. The first segment indicates loss of water and gaseous molecules such as CO₂ and NH₃, while the second and third stages of the curve yield organic decomposition products from the cross-linked PEI and genipin dimer. The saturation line observed after 485 °C indicates a complete decomposition of the encapsulated polymeric component leaving nanoceria alone; the amount of compound remaining was 33%.

The hydrodynamic diameter (D_H) and zeta potential (ζ) of each sample was estimated from dynamic light scattering (DLS) measurements. The values for the hydrodynamic diameter of the non-coated nanoceria, PEI ceria and PP ceria were 19 ± 1, 236 ± 3 and 263 ± 2 nm, respectively (Fig. 3a–c). The encapsulation of the nanoparticles by PEI resulted in an increase in diameter of the hydrated sphere of the nanoparticles. Meanwhile, the chemical cross-linkage among the nanoparticles through polymeric chains yielded a slight increase in the diameter of PP ceria when compared with that of the PEI ceria. The ζ value of non-coated nanoceria, which may vary depending on the size of the nanoparticles and pH of the solution, was 6 ± 0.8 mV at pH 7.0. PEI and PP ceria had ζ values of 28.4 ± 0.9 mV and 18.92 ± 1.1 mV, respectively. The larger positive charge is due to the presence of amine groups of PEI interacting with water molecules of the hydrated spheres. As the functional amine groups are involved in genipin dimer formation in the PP ceria, a reduction in ζ is observed. The long-term stability of PP ceria was also investigated by measuring ζ values at 7 and 14 days, which were found to be 17.7 ± 1.6 mV and 16.0 ± 1.8 mV, respectively. The slight changes observed in ζ values are due to agglomeration among the nanoparticles, which can be easily disrupted by agitation.

Fig. 3 DLS measurements demonstrating the hydrodynamic diameters of non-coated nanoceria (a), PEI ceria (b) and PP ceria (c); the characteristic crystal lattices of CeO₂ in PP ceria from XRD (d); TEM image of PEI ceria with a scale bar of 10 nm (e).

The XRD pattern in Fig. 3d demonstrates that the cerium oxide of PP ceria exhibits the typical diffraction peaks of a crystalline cubic fluorite structure consistent with the planes of (111), (200), (220), (311), (222), (400), (331) and (420), and reflecting literature results.³⁵ While DLS measurements provide details about the hydrodynamic diameters of nanoparticles in solution, TEM measurements show the actual size of the particles in their dried state. It was found that the PEI ceria nanoparticles are sized in the range of 3–10 nm, as shown in Fig. 3e.

The PP ceria sample was subjected to photo-bleaching to determine how it behaves upon exposure to light for the duration of 180 min. The UV-vis spectra showed neither new peaks nor a significant peak shift, but a decrease in intensity was observed (Fig. 4a). This indicates that the sample is bleached without being transformed into another product. The absorbance of PP ceria decays simultaneously via two different processes, as shown in Fig. 4b, and hence has two degradation constant values, λ₁ = 5.9 × 10⁻² min⁻¹ and λ₂ = 5.6 × 10⁻⁴ min⁻¹. Half-lives of the first and second processes were determined to be 11.71 ± 0.9 min and 1234 ± 8.2 min, respectively. It is known that the second process takes a longer time when compared to the first one.

Fig. 4 Photo-bleaching studies carried out using PP ceria during exposure to light (λ = 365 nm) for 180 min. (a) UV-vis spectra showing absorbance of PP ceria at times from t = 0 to t = 180 min. (b) Kinetic data showing time (X-axis) against absorbance (Y-axis).

The conformational stability of collagen, type I, was investigated using CD to establish the impact of PP ceria on its secondary structure. Being present in the skin matrix, collagen imparts tensile properties which allow the skin to serve as a protective organ against external trauma. Destruction of collagen generally causes deleterious effects, as observed in aged skin.^36,37 Hence, it is essential to know about any conformational changes of collagen induced by the presence of additives, in vitro, prior to animal studies for any kind of external applications. A typical CD spectrum of collagen exhibits a positive peak at 220 nm and a negative peak at 196 nm corresponding to the n–π* and π–π* transitions of amide bonds, respectively, and maintains a ratio of the positive to the negative peak (R_pn ratio) of around 0.115.³⁸ In our work, collagen does not show any significant changes caused by the presence of PP ceria (Fig. 5a). The calculated R_pn values also remained the same (0.115 ± 0.005) indicating that the native state of the triple helical structure of collagen was retained. The anti-oxidant activity of the PP ceria was demonstrated through the inhibition% against the radicals investigated. As given in Fig. 5b, PP ceria appear to be more effective against peroxy radicals than against nitrile radicals. In particular, 0.4% of PP ceria showed a scavenging efficiency of almost 100% against peroxy radicals. In the case of nitrile radicals, 0.1–0.5% of PP ceria gave inhibition values varying from 26 to 48%. CeO₂ particles adsorb oxy radicals, like oxygen molecules, and hence show more anti-oxidant activity against oxy radicals than nitrile radicals. In the DPPH assay, the odd electron of the nitrogen atom in DPPH is reduced by receiving a hydrogen atom from anti-oxidants to the corresponding hydrazine. DPPH is characterized as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole, so that the molecules do not dimerise, like most other free radicals. On mixing DPPH solution with an anti-oxidant substance, the reduced form is produced, which is accompanied by decolorization.³⁹

Fig. 5 (a) CD illustrating the conformational secondary structure of collagen, type-I, in the presence of PP ceria (0.1–0.5%); (b) anti-oxidant activity of PP ceria against nitrile and peroxy radicals, in terms of inhibition (%). The data represent the mean ± S.D. (n = 3).

Fig. 6a evidences that the morphology of NIH-3T3 cells is not affected by the presence of PP ceria. The viability of the cell line was found to be more than 66% for concentrations of PP ceria up to 25 μg mL⁻¹ (Fig. 6b). We further investigated the reduction in ROS generation in the MCF-7 cell line using a concentration of PP ceria of 25 μg mL⁻¹. After pre-incubating the cells with PP ceria for different time periods (6–48 h), a time dependent decrease in the level of ROS generation was observed upon exposure to H₂O₂ (Fig. 7). The significant decrease (from 100% to 67%) in the intracellular ROS level after 48 h pretreatment with PP ceria is due to the excessive internalization of the PP ceria into the cells. The results are concordant with a previous literature report which studied the scavenging potential of nanoceria-encapsulated albumin nanoparticles against reactive oxygen species in L-132 cells.⁴⁰ In addition, Alpaslan et al. reported that dextran-coated cerium oxide nanoparticles showed toxicity at 250–1000 μg mL⁻¹ in osteosarcoma cells at pH 6.0.⁴¹ The description of the toxicity of nanoceria needs more experimental studies in respect of various factors such as pH, concentration and time, and therefore we will investigate the toxicity of PP ceria with appropriate cell line models and report the results in future work along with in vivo animal model and human compatibility tests.

Fig. 6 (a) Morphology of NIH-3T3 cells after 24, 48 and 72 h at different concentrations of PP ceria (10, 25, 50, 75 and 100 μg mL⁻¹). The cell line without additives was taken as the control. The images are captured with a 20 μm scale bar. (b) Cell viability was measured by MTT assays. The data represent the mean ± S.D. of three independent experiments. *P < 0.05 by one way ANOVA, compared with the control.

Fig. 7 ROS generation, in terms of relative fluorescence intensity (%), in MCF-7 cells pretreated with PP ceria for time periods of 6, 12, 24 and 48 h at pH 7.4. The data represent mean ± SEM.

In brief, a nanoceria based cosmetic can work effectively against reactive oxygen and nitrogen species, thanks to the reversibility of the redox reactions and storage of molecular O₂. Unlike other nanoparticle based sun screens such as TiO₂ and ZnO, the photostability of our PP ceria emulsion has been preserved by the modified PEI-genipin dimer pigment, which, in addition, functions to endow the nanoparticles with long-term stability.

4. Conclusions

Pigmented PEI stabilized nanoceria displayed anti-oxidant properties and were capable of withstanding photo-bleaching for an extended duration. The viability of the NIH-3T3 cell line when cultured with PP ceria shows that the PP ceria have low toxicity at concentrations up to 25 μg mL⁻¹. CD data showed that the conformational stability of collagen was not altered when it was treated with PP ceria. The long-term colloidal stability of the collagen sample was good, with agreeable zeta potential values. The results of our fundamental work indicate that PP ceria can be further evaluated in in vivo animal studies and human compatibility tests including those for skin penetration and irritation.

Acknowledgements

The authors thank the STRAIT project (CSC 0201), sponsored by the Council of Scientific and Industrial Research (CSIR), India, for financial support.

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