Innovative Skin Product O/W Emulsions Containing Lignin, Multiwall Carbon Nanotubes and Graphene Oxide Nanoadditives with Enhanced Sun Protection Factor and UV Stability Properties

: In the present study, oil-in-water (O/W) sunscreen emulsions were prepared containing different portions of lignin (LGN), multiwall carbon nanotubes (MWCNTs) and graphene oxide (GO) nanoadditives. The stability in terms of pH and viscosity of emulsions was thoroughly studied for up to 90 days, exhibiting high stability for all produced O/W emulsions. The antioxidant activity of emulsions was also analyzed, presenting excellent antioxidant properties for the emulsion that contains LGN due to its phenolic compounds. Moreover, the emulsions were evaluated for their ultraviolet (UV) radiation protection ability in terms of sun protection factor (SPF) and UV stability. SPF values varied between 6.48 and 21.24 while the emulsion containing 2% w/v MWCNTs showed the highest SPF index and all samples demonstrated great UV stability. This work hopefully aims to contributing to the research of more organic additives for cosmetic application with various purposes.


Introduction
Skin cancer is the most common disease of human skin, with non-melanoma skin cancer (NMSC) alone consisting at least 40% of new cancer cases and is estimated to upsurge in the coming decades due to aging population [1]. Endogenous factors, such as UV radiation due to sun exposure is considered a complete carcinogen factor since it is both a mutagen and a non-specific damaging agent and has properties of both an initiator and a promoter of tumor. Specifically, UV is composed of UV-A (320-400 nm), UV-B (290-320 nm), and UV-C (100-290 nm). UV-C radiation is totally absorbed by the earth's ozone layer, however, the UV-A and UV-B radiations reach the earth surface and are accountable for several skin pathologies such as sunburns, photosensitivity, photoaging, phototoxicity, and most importantly skin cancer [2][3][4]. On the other hand, UV radiation offers some benefits to human health such as the natural synthesis of vitamin D and endorphins in the skin [3].
Sun Protection Factor (SPF) constitutes a significant feature for cosmetic oils, lotions, and emulsions. It is of great importance since the addition of UV protection agents could assistance the preservation of good skin health and the prevention of oxidative stress. SPF value implies the sun protection efficacy of a product, where usually, a higher SPF index corresponds to more effective sun protection. Nevertheless, various complications have been caused by currently commercial sunscreen products such as erythema, edema, and irritation, among others [2,5].
Cosmetic emulsions including sunscreen products can undergo a number of different instabilities such as gravitational separation, creaming, flocculation, coalescence and phase inversion [6]. The increased concern about these effects led to the addition of stabilizer Figure 1. Structures of (a) lignin [22], (b) GO [42] and (c) MWCNTs [43].
The current work is an attempt to fabricate three different model dispersed emulsions containing LGN, MWCNTs and GO nanoadditives, and to study their cochemical properties. More specifically, emphasis was given to study their pH an cosity stability, antioxidant activity, sun protection and UV stability of the final cr The results are exhibited in this article. Figure 1. Structures of (a) lignin [22], (b) GO [42] and (c) MWCNTs [43].
The current work is an attempt to fabricate three different model dispersed O/W emulsions containing LGN, MWCNTs and GO nanoadditives, and to study their physicochemical properties. More specifically, emphasis was given to study their pH and viscosity stability, antioxidant activity, sun protection and UV stability of the final creams. The results are exhibited in this article.

Materials
Kraft LGN was obtained from Aldrich chemicals (St. Louis, MO, USA). MWCNTs (purity > 95 wt%) were purchased from Chengdu Organic Chemicals Co., Ltd. Chinese Academy of Sciences (Chengdu, China). GO were purchased from Strem Chemicals Inc. (7 Mulliken Way, Newburyport, MA, USA). Glycerin, citric acid, xanthan gum, olive oil, cetyl alcohol, cetearyl alcohol, stearic acid, shea butter, beeswax, polysorbate 60, with appropriate purity for cosmetic emulsions, were kindly donated from Novita Group (Thessaloniki, Greece). All other materials used in the present study were of analytical grade.

Preparation of the Emulsions
The preparation of the emulsions was achieved according to O/W method as described in previous work [2]. O/W emulsions were prepared containing lignin, GO nanoadditives and MWCNTs in 0.5, 1 and 2% w/v ratios as exhibited in Figure 2. The list of the utilized ingredients for the preparation of 100 mL O/W emulsions oil are illustrated in the Table 1. The corresponding water and oil phase ingredients were weighed and added separately to glass beakers. The water phase constituted 75% while the oil phase constituted 25% of the final emulsion. The two mixtures were heated in parallel, in a water bath at 80 • C, under mechanical stirring until their homogenization. Finally, the oil phase was added drop-by-drop to the to the water phase, under continuous mechanical stirring, and the O/W emulsions occurred spontaneously. The produced O/W emulsions were left under mechanical stirring for 1 h at room temperature and after which phenoxyethanol and ethylhexylglycerin preservatives were added in each cream. A control emulsion (blank) without the presence of kraft LGN, MWCNTs and GO nanoadditives was also prepared according to the procedure mentioned above.
Appl. Nano 2022, 3, FOR PEER REVIEW 5 (blank) without the presence of kraft LGN, MWCNTs and GO nanoadditives was also prepared according to the procedure mentioned above.

Centrifugal Tests
Centrifugal tests of produced emulsions were determined after preparation as well as after 1, 3 and 20 days of storage. 3 g of each sample was placed in centrifugal tubes and then centrifugated at 4000 rpm and 25 • C for 15 min.

Antioxidant Activity
Tests measuring the antioxidant capability of the samples was determined using the 2,2-Diphenyil-1-picrylhydrazyl (DPPH) method, developed according to Blois in 1958 [44] 1 mL of each emulsion dispersed in EtOH (1% v/v) was added to 3 mL of a 5 × 10 −3 mg/mL ethanol DPPH solution. The reference sample consisted of 1 mL EtOH and 3 mL of the DPPH/EtOH solution. The samples were then sonicated, and their absorbance was recorded after 30 min with the aid of a UV-Vis spectrometer (UV Probe 1650, Shimadzu, Tokyo, Japan) at 517 nm. The Equation (1) proposed by Brand et al. was used to determine the free radical scavenging activity of the samples [45]: Free radical scavenging activity (%) = Absorbance of control − Absorbance of extracts Absorbance of control × 100 For all samples, experiments were performed in triplicate and results are presented in mean values.

SPF Determination
For the assessment of sun protection factor, the diluted solution transmittance technique was employed [46]. 1 g of each sample was transferred to a 100 mL volumetric flask, and then sonicated until complete homogenization, diluted to volume with ethanol, mixed for 5 min and filtered through Whatman filters. 2 mL of each sample was transferred to a 10 mL volumetric flask and diluted with ethanol. The absorption values of the samples were obtained in the range of 290-320 nm (every 5 nm) using a UV-Vis spectrophotometer (Shimadzu, Tokyo, Japan) while experiments for each sample were performed in trip-licate. Equation (2) proposed by Mansur was used to determine the SPF values of the formulations [47]: where CF = 10 (correction factor), EE (λ) = erythemogenic effect of radiation at wavelength λ, I(λ) = intensity of solar light at wavelength λ, and abs(λ) = absorbance of sample at wavelength λ. The values for the term "EE × I" are constants, which were determined by Sayre et al. [47]. For all samples, experiments were performed in triplicate and results are presented in mean values.

UV Irradiation
Emulsions (0.5 g of each sample), placed at an aluminum foil (Figure 3), were left under UV irradiation (chamber BS-09) at 280 nm at constant temperature (25 • C) and relative humidity (50%) for several hours. The irradiation intensity samples were exposed at a distance of 25 cm from the lamp, at an irradiated area of 35 cm × 45 cm. After exposure the creams were removed from the chamber (after 24 and 72 h of UV exposure) and the effect of UV exposure was evaluated by using spectrophotometer HUNTERLAB (Washington, VA, USA) MiniScan XE Plus. All measurements were performed in triplicates and an average value was selected.
Appl. Nano 2022, 3, FOR PEER REVIEW 7 Figure 3. Samples of prepared O/W emulsions on aluminum foil before UV irradiation.

Stability of the Prepared Emulsions
Emulsion stability is associated either with the decay or the persistence of the dispersed system under specific conditions. Viscosity constitutes an index of the inner stability and thus greatly affects the appealing of the final product to the consumers. Flaws in viscosity during storage may cause various failings, not only regarding aesthetic appearance (liquefaction) but also in the essential features of the final product, such as the sun protection capability of the emulsion and potentially provoke phase separation and liquefaction. The key mechanism describing the stabilization of the emulsion is the irreversible adsorption of solid particles at the oil-water interface, providing a steric barrier against droplet coalescence and Ostwald ripening. The above-described case mostly applies when solid particles have a good wettability for the oil phase, contributing to the formation of a dense layer of adsorbed particles around the droplets. Under this condition,

Stability of the Prepared Emulsions
Emulsion stability is associated either with the decay or the persistence of the dispersed system under specific conditions. Viscosity constitutes an index of the inner stability and thus greatly affects the appealing of the final product to the consumers. Flaws in viscosity during storage may cause various failings, not only regarding aesthetic appearance (liquefaction) but also in the essential features of the final product, such as the sun protection capability of the emulsion and potentially provoke phase separation and liquefaction. The key mechanism describing the stabilization of the emulsion is the irreversible adsorption of solid particles at the oil-water interface, providing a steric barrier against droplet coalescence and Ostwald ripening. The above-described case mostly applies when solid particles have a good wettability for the oil phase, contributing to the formation of a dense layer of adsorbed particles around the droplets. Under this condition, the energy barrier against the coalescence of oil droplets corresponds to the energy involved to expel the particles from the interface. Other factors benefiting the stabilization process include electrostatic repulsion induced by charged particles, or the setup of a three-dimensional network in the continuous phase induced by the solid particles that entrap the dispersed droplets and restrain their mobility [9].
The only complete method to determine viscosity stability is to count the number of drops in the system and to assess how it variates as time elapses. However, this is impracticable due to the fact that the majority of measurement techniques require dilution and thus modification of the emulsion [2,48]. Figure 4a shows the viscosity values in cP for all the prepared emulsions at 100 RPM, while Figure 4b shows the viscosity values at 50 RPM. Concerning the blank emulsion, an advanced still very low decrease of viscosities during storage, credited to the needed time of the emulsions to coagulate, is manifested. The adding of bioactives leads to a reduction of the emulsion viscosities during the first 20 days in all cases, as was expected, since these are solid units. After this period, all of the prepared emulsions manifested increased viscosity, attributed to the hydration of the added particles, resulting in the stability of the final emulsions. The above phenomenon is highly desirable for sunscreens since higher viscosity values can contribute to great photoprotection ability, since the consumer often tends to apply a thicker layer of the product, leading to a more effective film (higher SPF). At both rotation speeds, a correlation is observed between the ratio of added substance with the increase of viscosity values, which is due to a tighter packing and increased inter-drop interactions, while emulsion containing 2% w/v MWCNTs is stabilized in highest viscosity values. In addition, after 60 days we observed a decrease in the viscosity of emulsions containing lignin, possibly attributed to phase separation.
The pH values of a vigorous human skin vary between 4.5-6 range. Subsequently, the pH of the prepared O/W emulsions should vary between this region. Various skin disorders may occur from extreme pH values, thus the examination of the pH values of the prepared samples is of great significance [49]. pH values exhibited in Figure 5 for up to 90 days indicate the good pH stability of all prepared emulsions since their pH values are very close to the blank sample. In almost all cases, the addition of LGN, MWCNTs and GO at different ratios (0.5 up to 2% w/v), does not appear to have any effect on the pH values of the prepared emulsions. This was anticipated since the added portions were too low. A key factor contributing to the stability of the emulsion is the constant pH values observed over a period of time. Consequently, from the obtained measurements it can be established that all emulsions are likely to have steady pH values during storage, with no significant changes, thus constituting them appropriate for cosmetic dermal applications. Table 2 and Figure 6 present the SPF values of the prepared emulsions along with the blank one. The incorporation of lignin, graphene oxide and multi-walled carbon nanotubes into emulsions was able to produce a photoprotective formulation. All the emulsions exhibit improved SPF values compared to the blank sample, presenting increase of SPF index with increasing of the additives content. Specifically, the emulsion with 0.5% and 1%

Sun Protection
LGN presented SPF 6.48 and 7.4 respectively while the emulsion with 2% LGN exhibited a sharp increase with SPF 21.24 which is justified by the double lignin content. Emulsion with 0.5 and 1% graphene oxide with SPF 8.43 and 9.7 respectively showed similar behavior to the lignin emulsions. However, the emulsion with 2% graphene oxide presented low increase with SPF 11.29, which may be down to insufficient solubility of the additives in the emulsion [50]. All emulsions with multi-walled carbon nanotubes presented considerably high index SPF. Emulsions with 0.5 and 1% multi-walled carbon nanotubes exhibited 9.45 and 15.62 SPF values respectively while the emulsions with 2% multi-walled carbon nanotubes illustrated rapid increase with SPF 31.82.
between the ratio of added substance with the increase of viscosity values, which is due to a tighter packing and increased inter-drop interactions, while emulsion containing 2% w/v MWCNTs is stabilized in highest viscosity values. In addition, after 60 days we observed a decrease in the viscosity of emulsions containing lignin, possibly attributed to phase separation.     low. A key factor contributing to the stability of the emulsion is the constant pH values observed over a period of time. Consequently, from the obtained measurements it can be established that all emulsions are likely to have steady pH values during storage, with no significant changes, thus constituting them appropriate for cosmetic dermal applications.   Figure 5. pH values obtained from the prepared emulsions during storage stability tests. Table 2 and Figure 6 present the SPF values of the prepared emulsions along with the blank one. The incorporation of lignin, graphene oxide and multi-walled carbon nanotubes into emulsions was able to produce a photoprotective formulation. All the emulsions exhibit improved SPF values compared to the blank sample, presenting increase of SPF index with increasing of the additives content. Specifically, the emulsion with 0.5% and 1% LGN presented SPF 6.48 and 7.4 respectively while the emulsion with 2% LGN exhibited a sharp increase with SPF 21.24 which is justified by the double lignin content. Emulsion with 0.5 and 1% graphene oxide with SPF 8.43 and 9.7 respectively showed similar behavior to the lignin emulsions. However, the emulsion with 2% graphene oxide presented low increase with SPF 11.29, which may be down to insufficient solubility of the additives in the emulsion [50]. All emulsions with multi-walled carbon nanotubes presented considerably high index SPF. Emulsions with 0.5 and 1% multi-walled carbon nanotubes exhibited 9.45 and 15.62 SPF values respectively while the emulsions with 2% multi-walled carbon nanotubes illustrated rapid increase with SPF 31.82.    Figure 6. Sun protection ability of the emulsions. Figure 6. Sun protection ability of the emulsions.

Centrifugal Tests
Centrifugation tests confirmed the stability of the prepared O/W emulsions. A phenomenon commonly exhibited in commercially available products is the gravitational separation of emulsions. Images in Figure 7 confirm that no phase separation was observed at any sample during the 20 first days of centrifugation study. This finding is in total agreement with Smaoui et al. and is ascribed to accurate homogenization speed of the emulsions [51].

Antioxidant Properties
Emulsions with LGN were found to exhibit great antioxidant activities (Figure 8) unlike to emulsions with GO and MWCNTs, which did not show any antioxidant activity. The antioxidant ability of lignin emulsions is attributed to the presence of free phenolic hydroxyl groups in the lignin structure, which act as radical scavengers (Ar-OH, MeO-Ar-OH, (MeO)2-Ar-OH) [52,53]. The radical scavenging ability of phenolic groups depends not only on the ability to form but also on the stability of phenoxyl radical. Methoxyl groups in lignin structure at ortho position stabilize phenoxyl radicals by resonance as well as preventing them from propagation ( Figure 9). Conjugated double bonds increase the stabilization is through extended delocalization [54]. As was expected, results showed that the antioxidant ability was increased by increasing lignin amount due to higher content of phenolic hydroxyl groups. The relatively high antioxidant activity of lignin suggested that it could be used as potential antioxidant of emulsions.
Despite its phenolic nature, the high safety levels of different concentrations of lignin in emulsions make it ideal additive for cosmetic products [55]. Specifically, cell viability results for emulsions with 0.005-0.5 wt% concentration of lignin w greater than 80% [56]. Excellent non-cytotoxic activities have been also reported for materials containing 1 wt% lignin [22]. Similarly, emulsions with 3 wt% content of lignin did not show any evidence of toxic effects for fibroblast after 72 h [57]. These results demonstrate that despite the phenolic nature of lignin, it can be used as a material for applications in cosmetics.

Antioxidant Properties
Emulsions with LGN were found to exhibit great antioxidant activities ( Figure 8) unlike to emulsions with GO and MWCNTs, which did not show any antioxidant activity. The antioxidant ability of lignin emulsions is attributed to the presence of free phenolic hydroxyl groups in the lignin structure, which act as radical scavengers (Ar-OH, MeO-Ar-OH, (MeO) 2 -Ar-OH) [52,53]. The radical scavenging ability of phenolic groups depends not only on the ability to form but also on the stability of phenoxyl radical. Methoxyl groups in lignin structure at ortho position stabilize phenoxyl radicals by resonance as well as preventing them from propagation ( Figure 9). Conjugated double bonds increase the stabilization is through extended delocalization [54]. As was expected, results showed that the antioxidant ability was increased by increasing lignin amount due to higher content of phenolic hydroxyl groups. The relatively high antioxidant activity of lignin suggested that it could be used as potential antioxidant of emulsions.
Despite its phenolic nature, the high safety levels of different concentrations of lignin in emulsions make it ideal additive for cosmetic products [55]. Specifically, cell viability results for emulsions with 0.005-0.5 wt% concentration of lignin w greater than 80% [56]. Excellent non-cytotoxic activities have been also reported for materials containing 1 wt% lignin [22]. Similarly, emulsions with 3 wt% content of lignin did not show any evidence of toxic effects for fibroblast after 72 h [57]. These results demonstrate that despite the phenolic nature of lignin, it can be used as a material for applications in cosmetics. LGN 2% LGN 0.5% LGN 1%

UV Stability
UV irradiation effects on emulsions were determined in terms of color evaluation by means of the changes of Commission Internationale de l'Eclairage (CIE) Lab color components L*a*b*C*h. Five-dimensional color space measurement system in which the L* axis measures luminosity or lightness ranging from 0 (black) to 100 (white), the a* coordinate measures redness (a > 0) or greenness (a < 0), and the b* coordinate measures yellowness (b > 0) or blueness (b < 0), C*-chroma and H*-hue angle [58,59]. Creams prepared with lignin as active ingredient at application levels of 0.5, 1 and 2% w/v show progressively increasing brown coloration from pale brown (higher L value) to dark brown (lower L value), which can be attributed to the higher level of lignin employed in the preparation of the creams. Additionally, by increasing the lignin concentration, the creams are becoming reddish as this can be seen from the higher values recorded, which again can be LGN 2% LGN 0.5% LGN 1%

UV Stability
UV irradiation effects on emulsions were determined in terms of color evaluation by means of the changes of Commission Internationale de l'Eclairage (CIE) Lab color components L*a*b*C*h. Five-dimensional color space measurement system in which the L* axis measures luminosity or lightness ranging from 0 (black) to 100 (white), the a* coordinate measures redness (a > 0) or greenness (a < 0), and the b* coordinate measures yellowness (b > 0) or blueness (b < 0), C*-chroma and H*-hue angle [58,59]. Creams prepared with lignin as active ingredient at application levels of 0.5, 1 and 2% w/v show progressively increasing brown coloration from pale brown (higher L value) to dark brown (lower L value), which can be attributed to the higher level of lignin employed in the preparation of the creams. Additionally, by increasing the lignin concentration, the creams are becoming reddish as this can be seen from the higher values recorded, which again can be

UV Stability
UV irradiation effects on emulsions were determined in terms of color evaluation by means of the changes of Commission Internationale de l'Eclairage (CIE) Lab color components L*a*b*C*h. Five-dimensional color space measurement system in which the L* axis measures luminosity or lightness ranging from 0 (black) to 100 (white), the a* coordinate measures redness (a > 0) or greenness (a < 0), and the b* coordinate measures yellowness (b > 0) or blueness (b < 0), C*-chroma and H*-hue angle [58,59]. Creams prepared with lignin as active ingredient at application levels of 0.5, 1 and 2% w/v show progressively increasing brown coloration from pale brown (higher L value) to dark brown (lower L value), which can be attributed to the higher level of lignin employed in the preparation of the creams. Additionally, by increasing the lignin concentration, the creams are becoming reddish as this can be seen from the higher values recorded, which again can be attributed to the higher application level of the lignin employed ( Table 3). The colour stability of the lignin creams after 24 and 72 h of exposure to UV radiation is very stable as this can be seen from Table 3 with all the values of colour coordinates remaining relatively stable and very close to the same values of non-exposed samples. Creams prepared with MWCNTs as the active ingredient at application levels of 0.5, 1, and 2% w/v show grey black coloration with the cream prepared with 2% MWCNTs being the strongest colored (lower L value, Table 3). The colour stability of the creams prepared after UV exposure for 24 and 72 h remains excellent as it can be observed from Table 3 with all colour coordinates remaining very similar to the same values of non-exposed samples. Creams prepared with GO as active ingredient at application levels of 0.5, 1 and 2% w/v present dark grey coloration with the colour intensity remaining almost unaltered from the application level of the active ingredient (same L values, Table 3). This can be attributed to the fact that even the lower application level of 0.5% is enough to strongly colour the cream achieving a kind of colour saturation where further increase of the application level does not add to colour intensity. The colour stability of the creams prepared after UV exposure for 24 and 72 h is also excellent as this can be exemplified with the similar colour co-ordinates with the non-exposed samples.

Conclusions
In the present study O/W emulsions containing LGN, MWCNTs and GO in ratios 0.5, 1 and 2% w/v were prepared. Stability tests conducted in this study indicate great viscosity and pH stability for up to 90 days storage at room temperature for all fabricated samples. Regarding the sun-protective ability, obtained SPF values varied between 6.48 and 21.24. The highest SPF value was obtained by the emulsion containing 2% w/v MWCNTs, while the lowest by the 0.5% w/v LGN emulsion. The study of the antioxidant activity of the prepared emulsions, showed that only emulsions containing kraft lignin exhibited strong antioxidant activity associated with the existence of bioactive phenolic compounds in the structure of lignin. Finally, UV irradiation tests showed that all emulsions exhibit great UV stability after 24 and 72 h of exposure. The overall results exhibited in this study could further expand the usage of organic additives at cosmetic applications. Each additive offered excellent properties to the emulsions, so, in further research, it would be interesting to study the combination of LGN with GO and MWCNTs to synthesize emulsions with parallel enhanced antioxidant and UV protection properties. However, future cytotoxicity studies are required to fully evaluate the potential risk of produced emulsions to human health.