Eco-friendly sunscreen formulation based on starches and PEG-75 lanolin increases the antioxidant capacity and the light scattering activity in the visible light

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Journal of Photochemistry & Photobiology, B: Biology 222 (2021) 112264

Available online 22 July 2021

Eco-friendly sunscreen formulation based on starches and PEG-75 lanolin increases the antioxidant capacity and the light scattering activity in the visible light

Victor Hugo Pacagnelli Infante

^a^,^b

, Silke B. Lohan

^b

, Sabine Schanzer

^b

,

Patrícia Maria Berardo Gonçalves Maia Campos

^a

, Juergen Lademann

^b

, Martina C. Meinke

^b^,^*

aSchool of Pharmaceutical Sciences of Ribeir˜ao Preto, University of S˜ao Paulo, Av. do Caf´e, s/n - Vila Monte Alegre, Ribeir˜ao Preto, S˜ao Paulo 14040-900, Brazil

bCharit´e – Universit¨atsmedizin Berlin, Corporate Member of Freie Universit¨at Berlin, Humboldt-Universit¨at zu Berlin, Berlin Institute of Health, Department of Dermatology, Venereology and Allergology, Charit´eplatz 1, 10117 Berlin, Germany

A R T I C L E I N F O Keywords:

Sunscreen Optical properties

Radical protection factor (RPF)

Electron paramagnetic resonance spectroscopy Visible light

Starches

A B S T R A C T

Most modern sunscreens contain physical filters, which scatter the sunlight, increasing the photons’ pathway in the upper stratum corneum. This effect can lead to a better efficacy of the UV filters and improve the diffuse reflection. However, the addition of nanosized inorganic UV filters reduces the antioxidant capacity of sunscreen formulations. Two cream formulations (F1, F2) which differ in the ingredient PEG75 Lanolin (F2), have been characterized for their radical protection factor (RPF) and their optical properties in vitro using electron paramagnetic resonance (EPR) spectroscopy and UV/VIS spectrometry. The RPF for PEG-75 Lanolin was also determined. Furthermore, their radical protection properties were analyzed on porcine skin ex vivo after visible light irradiation by EPR. The structure of each formulation in the skin surface was determined by reflectance confocal microscopy in vivo. The addition of lanolin increased the reflectance and reduced the transmittance for visible light, improving the scattering drastically. Besides, the antioxidant capacity was also increased for F2, something unpublished until now. F1 presented a lower scattering provided by starches. The sunscreens showed high scattering properties and antioxidant capacity, especially for F2, which presented the lowest radical formation in the skin model. These results are consistent with the RPF measurements where F2 has a higher RPF value (193 ±3 ×10¹⁴radicals/mg) than F1 (155 ±4 ×10¹⁴radicals/mg) and for PEG-75 Lanolin (37 ±1 × 10¹⁴radicals/mg). The combination of starches and PEG-75 Lanolin is the first solution to provide both, light scattering and antioxidant capacity, in sunscreens.

1. Introduction

The damage induced by continuous exposure of the skin to ultraviolet (UV; 280–400 nm) radiation has already been reported in the literature [1,2]; however, the dermal effects of visible (VIS; 380–760 nm) and infrared (IR) radiation have not yet been fully elucidated, being more correlated with the free radical production, and even DNA damage was reported [3,4]. As more than 50% of the solar radiation reaching the earth’s surface is visible light, the need for studying the resulting dermal effects and their avoidance is growing [5].

In vivo studies on human skin have shown that UV light accounts for approximately 60% of the radicals formed in the skin by solar

irradiation. This illustrated that visible (VIS) and near infrared (NIR) irradiation is not negligible. VIS and NIR radiation penetrates deeper into the skin than UV light, promoting an increased ROS formation in deeper skin layers. Furthermore, after UV-, VIS-, and NIR-irradiation, EPR measurements in vivo show different radical concentrations depending on the skin type. Skin types I-III show a three times higher radical production after UV irradiation than skin types IV-V. On the other hand, darker skin is less protected against radical formation in the NIR region. Therefore, sun protection should be adapted to the skin type and the research on how to improve sunscreen for protection beyond the UV radiation needs to be intensified [6].

It is known that exposure to moderate doses of solar UV radiation are

* Corresponding author.

E-mail addresses: [email protected] (S.B. Lohan), [email protected] (S. Schanzer), [email protected] (P.M.B.G.M. Campos), juergen.lademann@

charite.de (J. Lademann), [email protected] (M.C. Meinke).

Contents lists available at ScienceDirect

Journal of Photochemistry & Photobiology, B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

https://doi.org/10.1016/j.jphotobiol.2021.112264

Received 11 March 2021; Received in revised form 11 June 2021; Accepted 17 July 2021

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vital for human vitamin D synthesis. Even with sunscreens this synthesis is not disturbed [7]. The therapeutic action of some wavelength of visible light radiation in a short time has also been reported, what in- cludes alleviation in inflammatory acne, for example [8]. However, photoprotectants are recommended, in particular sunscreens should regularly be applied, to counteract any detrimental effects. High UV filter protection leads to extended sun exposure accumulating radicals in the VIS and NIR spectral regions, and thus to an enhanced ROS formation in deeper skin layers.

Visible light appears to exert the same skin effects as UVA, mainly through the generation of reactive oxygen species (ROS) [9]. The ROS production due the blue light irradiation, for example, can correspond to 25% of UVA radiation in human keratinocyte mitochondria [10]. Hence, it is important to focus on the improvement of sunscreen formulations which reduce the action of free radicals in dermis or subcutaneous layers. This effect could be achieved using antioxidant substances capable of interacting with the free radicals and/or improving the scattering coefficient of sunscreen formulations [11]. Concerning visible light, the protection can be achieved by using pigmented sunscreen formulations with iron oxides. However, pigments with particles higher than the nanoscale can affect the sensorial properties and cause in- conveniences if constantly applied in sunscreen formulations [12].

The latest sunscreens contain nanosized physical filters that act as mirrors on the skin. These nanoparticles can reflect or scatter the sunlight radiation, thus enlarging the optical pathway of photons in the superficial layer of the stratum corneum. Theoretically, this increase in the photons’ pathways can improve both the efficacy of antioxidants and the diffuse reflection of photons [11,13]. However, practically, it is observed that the use of particulate UV filters such as nanosized titanium dioxide, which increase the scattering effect on sunscreen formulations, reduces the antioxidant capacity [2,4,11]. As this mechanism is highly dependent on the formulation’s structure, the complete system must be studied in depth to better understand the interaction between antioxidant and scavenging effect.

Another modern concern regarding the development of sunscreen formulations is the impacts on coral reefs. Oxybenzone (benzophenone- 3), 4-methylbenzylidene camphor (4-MBC), octocrylene and avo- benzone are organic UV filters which, according to literature, could contribute to the bleaching effect on coral reefs [14]. Recent studies show that nanosized inorganic UV filters can also present toxic effects on some coral reefs [15–17]. Even these findings are still preliminary. It could be an advantage to develop new ingredients which contribute to prevent environmental damage, finding more biodegradable solutions in the development of sunscreen formulations with a performance enhancement using less raw material.

Starches are well-known pharmaceutical biodegradable excipients.

Their strong affinity to the emulsion interface may result in stable emulsions. Due to its particulate behavior in micrometer size, starch is an excellent material to stabilize sunscreen formulations and improve the scattering effects, being a good alternative for the problem involving titanium dioxide, zinc oxide and coral reefs. Besides, starch can improve a lot of properties of pharmaceutical emulsions, such as stability, spreadability, oil absorption, film forming capacity, SPF improvement and heat tolerance [18]. Being more suitable for eco-friendly purposes, the utilization of starches is also an alternative to the use of acrylate polymers [19].

To improve the stability of sunscreen formulations based on particles, a small quantity of emulsifiers must be provided. PEG-75 Lanolin provides a good alternative for such improvement as it is an effective emollient, moisturizer, and fatting conditioner for skin care formulations, acting also as an emulsifier. Its utilization in combination with starches was already described in the literature, improving the film- forming capacity observed for these carbohydrates [19,20].

The objective of the present study was to investigate the optical properties of sunscreen formulations without inorganic UV filters, but with corn and tapioca starches, and the influence of a polyethylene-

glycolized lanolin. The correlation of the results with the radical protection factor, the sunscreen distribution on in vivo skin surface and the protection against VIS-induced free radicals using ex vivo skin model was studied.

2. Materials and Methods 2.1. Materials

For the sunscreen formulations: The UV filters bis- ethylhexyloxyphenol methoxyphenyl triazine (BASF, Germany); methylene bis-benzotriazolyl tetramethylbutylphenol (and) aqua (and) pro- pylene glycol (and) xanthan gum (and) decyl glucoside were provided by BASF, (Germany); octyl metoxicinamate was provided by Symrise (Galena, Brazil). PEG-75 lanolin (ChemyUnion, Brazil and Croda, Ger- many), corn starch (Maizena, Brazil), tapioca starch (DaTerrinha, Brazil), butyl hydroxy toluene, glycerin, phenoxyethanol and parabens, ethyelenediamine tetraacetic acid and butylenoglycol were provided by Mapric Pharmaceutical and Cosmetics Products (Sao Paulo, SP, Brazil), polyglyceryl-6-distearate (Gattefoss´e, France).

For EPR investigations, the nitroxide PCA (3-carboxy-2,2,5,5-tetra- methyl-1-pyrrolidinyloxy) and the radical DPPH (1,1-diphenyl-2-picrylhydrazyl) both were purchased from Sigma-Aldrich (Steinheim, Germany). Ethanol (UVASOL, Merck, Germany) was utilized for the RPF determination. All other chemical compounds used in the study at hand were of analytical grade.

2.2. Skin Samples

The porcine ears, which a local butcher had freshly delivered, were investigated within 48 h after slaughtering. Porcine ear skin has proven to be a suitable substitute for human skin [21]. For the experimental procedure, we cleaned the porcine ears with cold water and removed the hairs on the surface, carefully shaving them off. Subsequently, the skin was stored at 4 ^◦C until use.

2.3. Sunscreen Development

The only difference between F1 and F2 is the presence of 2% PEG-75 Lanolin (Table 1). No inorganic UV filters were selected in the development of these formulations to observe the optical properties of the starches in the formulations without any interferences from inorganic UV filters. We utilized a combination of starches well-described by Infante et al. (2021) [19] with medium particle size of 26.7 ±9 μm.

Besides, the native starches utilized in this work present more or less 29–35% of amylose, which are important due to their linear molecular morphology. Amylose polymer chains can easily entangle to form Table 1

Ingredients contained in the oil-in-water emulsion formulations. The UV filters utilized in this study and the PEG-75 Lanolin are printed in bold for better comparison. Content in % (w/w).

Ingredients (INCI Name) F1 F2

Tapioca starch 5% 5%

Corn Starch 5% 5%

Butyleneglycol 4% 4%

PEG-75 Lanolin – 2%

Polyglyceryl-6-distearate 3% 3%

Glycerin 6% 6%

Phenoxyethanol and parabens 0.8% 0.8%

Bis-Ethylhexyloxyphenol Methoxyphenyl Triazine 6% 6%

Octyl Metoxicinamate 6% 6%

Methylene bis-benzotriazolyl tetramethylbutylphenol, Aqua,

Decyl glucoside, Propyleneglycol, Xanthan gum 6% 6%

Ethyelene Diamine Tetra Acetic 0.1% 0.1%

Butyl Hydroxy Toluene 0.1% 0.1%

Distilled water 61% 59%

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continuous fibers.

2.4. Sunscreen Absorption Characterization

The absorption spectra in the UV and VIS region of sunscreen formulations were determined using a UV/VIS Spectrometer model Lambda 650 S (PerkinElmer, Waltham, USA) provided with a 150 mm integrating sphere. Solutions with 20 mg of each formulation in 10 mL of ethanol were prepared and 450 μL of each solution was placed in a spectrophotometer bucket.

2.5. Determination of RPF

The RPF technology is based on measuring the radical scavenging activity of a sample to determine its antioxidant capacity [11]. For this, EPR spectroscopy is utilized, using a marker radical which is reduced by the antioxidant system in the formulation. The number of reduced marker radicals represents the radical scavenging activity normalized to 1 mg of the antioxidant sample. In this case, we utilized 2,2-Diphenyl-1- picrylhydrazyl (DPPH, Sigma-Aldrich, Steinheim, Germany) as a test radical. 200 mg of the formulations were diluted in 10 mL of ethanol.

Subsequently, 400 μL of this solution were separated in a tube and 400 μL of DPPH (1 mM) were added. For PEG-75 Lanolin, 500 mg were diluted in 10 mL of ethanol.

The samples were kept dark at room temperature by constant panning until the end of the measurements. The measurements were performed directly after mixing with DPPH and stirring (0 h) until stabilization of the EPR DPPH signal which could take up to 30 h. An X- Band EPR spectrometer (MS5000, Magnettech GmbH by Freiberg In- struments GmbH, Freiberg, Germany) was used for the RPF analysis, whereby the reduction of the test radical DPPH by the antioxidative system was investigated. Based on the obtained data we can calculate the RPF, multiplying the concentration of radical (DPPH) [radicals * ml⁻¹] by the difference between the control test radical intensity and the decreased signal intensity after the treatment with the antioxidant in study. Subsequently, we divided the obtained value for the product input which represents the substance concentration we utilized in the study [mg.mL⁻¹]. The RPF will be expressed by a positive number N that presents the measuring unit 10¹⁴ radicals/mg, according to the following equation:

RPF = N ⋅ [

10¹⁴radicals⋅mg⁻¹]

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The skin samples were irradiated with a VIS-NIR optical fibre (LOT.

OrielGmbH&co.KG) +sun simulator filter +400FH90-50S filter (>400 nm). The intensity of each spectrum was measured before and after each measurement by means of a power meter 843-R (Newport Corporation, CA, USA) as described before [22,23].

An 8 mm (Ø) punch was taken from split skin (300 μm) and placed onto two filter discs (Ø 12 mm) (SmartPractice Europe GmbH, Germany) which were saturated with 150 μL of an 1 mM PCA solution ensuring a good EPR maker penetration into the whole split skin from the button.

The skin biopsy was incubated with the spin marker PCA for 5 min at 32 ^◦C. After incubation, a skin sample (Ø 4 mm) was punched out, placed into a tissue cell and measured in the EPR device (Bruker Elexsys E500, BioSpinGmbH, Karlsruhe, Germany). The measurements were performed 15 min without irradiation and 15 min with radiation, using the following magnetic parameters: center field [G]: 3479.65; sweep width [G]: 143.3; microwave [dB]: 22; delay [ms]: 100; number of scans: 1; power [mW]: 1.262; mod. frequency [kHz]:100; mod. ampl.

[G]: 1.5; receiver gain [dB]: 60; sweep time [s]: 45; conversion time [ms]: 43.95 and scan setting: points 1–44 (15 min).

The sunscreen formulations were tested according to the COLIPA standard procedure 389, whereby 2.0 mg/cm² of the relevant

formulation were distributed on the skin surface using a massage device (Rehaforum Medical GmbH, Elmshorn, Germany) for 2 min. The EPR investigations were performed 30 min after application [24,25].

2.7. Spectral Measurements and Evaluation of Optical Properties The double integrating sphere technique followed by iMCS (inverse Monte Carlo simulation) lends itself for determining the optical parameters of turbid media such as sunscreen formulations [1,11].

Utilizing an integrating sphere spectrometer (Lambda 650; Perki- nElmer, Rodgau-Jügesheim, Germany) as described by Meinke et al.

[11], we measured samples of approximately 100 μm in thickness for their Rt (total reflectance) and Tt (total transmittance) of sunscreens in a wavelength range from 400 to 800 nm.

The optical parameters absorption and effective scattering coefficients (μ_aand μ_s’) were calculated using inverse Monte Carlos simulation (iMCS) as described previously [11].

2.8. Distribution of the Sunscreen Formulations on the Skin Surface In Vivo

Subsequent to the approval of this study by the Ethics Committee of the School of Pharmaceutical Sciences of Ribeirao Preto/SP (CEP/

FCFRP 58368416.6.0000.5403), 6 subjects, aged between 18 and 28 years, were recruited for the in vivo investigations of the skin surface. All subjects gave their informed consent in writing before agreeing to participate in the study. This study was performed in accordance with the ethical standards of the Declaration of Helsinki of 1975, as revised in 2013.

The cellular characteristics of the different skin layers can be evaluated by Reflectance Confocal Microscopy (RCM) (Vivascope™ 1500) which uses a laser source with a wavelength of 830 nm and an immer- sion objective capable of detecting 20 images per second. Microscopic images were obtained using the Vivastack imaging system that provides multiple successive depth-confocal images at a certain site of the tissue, and 3-by-3-μm images were obtained up to a depth of 150 μm. The evaluation was performed in quintuplicate [26].

The distribution of the sunscreen formulations on the skin surface of the 6 volunteers was qualitatively evaluated for the filming effect 30 min subsequent to the application of the two formulations [26]. Using confocal laser reflectance microscopy, 3 regions of every subject’s forearm were analyzed: a control region, one with F1 formulation (in an area where 2 mg/cm²of sunscreen was deposited) and a third one with the F2 formulation at the same amount and in the same size as the second region. All regions were analyzed 30 min after sunscreen application. The forearm was utilized because of the low follicular density, which could interfere with the results for this evaluation, especially regarding the sunscreen formulation structure. Using this methodolog- ical step, the interaction of the formulations with the skin surface could be observed.

2.9. Statistical Analysis

For statistical analysis, we utilized the software Prism 8.4.3 (San Diego, USA) being p <0.05 fixed as the statistically significant. When the p value was next to 0.05 a tendency was considered. For the obser- vation of significant differences between the independent mean values obtained, Kruskal–Wallis and Mann–Whitney tests were utilized. For the cumulative radical production, a Two-way ANOVA was utilized (Tukey’s multiple comparison test), to detect the differences between the untreated and treated skin samples.

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3. Results

3.1. Radical Protection Factor of the Formulations

The RPFs for F1 and F2 were determined by EPR spectroscopy and resulted in 155 ±4 and 193 ±3 ×10¹⁴radicals/mg, respectively, which means that both creams present a good radical protection factor, but for F2 this value is higher. The RPF for PEG-75 Lanolin was 37 ±1 ×10¹⁴ radicals/mg, evidencing a low - but existent – antioxidant property.

3.2. Optical Properties of the Sunscreen Formulations

Not presenting any absorption in the visible light spectra, both formulations – F1 and F2 – showed absorption only in the UV region, with the absorption profiles being the same for both (Fig. 1). The formulation F1 presented higher transmittance and lower reflectance values for the visible light compared to F2 (Fig. 2).

The two sunscreen formulations were analyzed for their optical absorption properties and their effective scattering coefficients by measuring the total transmittance and reflectance in the visible light region followed by inverse Monte Carlo simulation. The absorption coefficients μ_aof both formulations were below 1 mm⁻¹for the visible light region, in agreement with the results observed in F1. The scattering effect for both formulations could be clearly determined, although the measured values were significantly higher for F2. The μ_s’ values decreased from 40 to 20 mm⁻¹in the visible light region for the formulation F2. The formulation F1 showed 4 times lower values in the high energy visible spectral region (400–500 nm) that decreased slightly with increasing wavelengths (Fig. 2).

3.3. Skin Protection Against Radical Formation in the VIS+NIR Spectral Region

The preventive action of the two formulations against radical formation during visible light irradiation was investigated using the ex vivo model, porcine ear skin. The radical formation due to VIS+NIR light irradiation is presented in Fig. 4. These results were compared to those obtained for untreated skin. A reduced radical formation in the VIS could be observed for both formulations, which became significant (p <

0.001) after 15 min. The protective effect of F2 turned out to be significantly higher than that of F1 (GEE, p <0.001) in the cumulative radical production over time (Fig. 3).

The radical production for both sunscreens and unprotected skin was analyzed at three different times after irradiation: 5, 10, and 15 min according to Tukey’s multiple comparison test. The results were

compared between the three samples for each time and for each sample according to the radical formation over time (Fig. 4).

It was possible to observe that after 5 min of irradiation only F2 presented a significant difference in the radical formation (p <0.001) when compared with untreated skin. After 10 and 15 min this difference between F2 and untreated skin remained highly significant, evidencing the good protective effect of this formulation. A significant difference to the untreated skin was found after 10 min, and the difference between F1 and F2 was already perceptible (p =0.016). After fifteen minutes, both formulations exhibited a significant and high reduction of radical formation compared to untreated skin, whereby the protective efficacy of F2 showing a reduction in radicals by 90% was superior to F1 (Fig. 4).

The punctual analyses for untreated skin at time points 5, 10 and 15

Fig. 1.Absorption spectra of the sunscreen formulations F1 (black) and F2 (light gray) dissolved in ethanol used in this study in the UV and visible light regions. The curves for F1 and F2 are almost overlapping.

Fig. 2. Effective scattering coefficient μs’ for F1 and F2 in the VIS/NIR region from 400 nm to 800 nm (mean ±SEM).

Fig. 3.Cumulative radical production for formulations F1, F2 and untreated skin after 15 min of visible light exposure (Mean ±SEM). *** means p <0.001 (GEE test) comparing the protective effect of both formulations and both formulations compared with untreated skin.

Fig. 4. Radical formation for F1, F2, and untreated skin at the time points 5, 10 and 15 min. Differences between the various samples within the respective time points, with * p <0.05; (**) p <0.01 and (***) p <0.001.

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min evidenced that the first 10 min are the most crucial ones in the radical production. The difference between 5 min and 10 min was significant, but not between 10 and 15. For the formulation F1 we noticed a tendency in the difference between 5 and 10 min (p =0.0677), while for F2 no differences were observed.

3.4. Distribution of the Sunscreen Formulations on the Skin Surface In Vivo

Using the RCM images, it was possible to observe a difference in the formulation structure for both sunscreens. The formulation F2 seems to disperse more homogeneously in the skin surface, presenting a “chain”

structure while F1 presents a dispersion more correlated with droplets (Fig. 5). Both sunscreen formulations, when applied at a quantity of 2 mg/cm², present a 12 μm thickness in the skin surface. Both formulations can deposit in the furrow areas. The blue arrows in Fig. 5 also show that F2 presents some adhesive interaction with the skin surface (stratum corneum region).

4. Discussion

The present study provides relevant data to better understand the action of modern sunscreen formulations according to their optical properties towards visible light. Besides, for the selected formulations it could be shown for the first time in the literature that antioxidant properties increased (higher RPF value for F2) with increasing optical scattering properties. This could be confirmed by a reduced radical formation in the skin during irradiation. The utilization of starches instead of synthetic polymers and inorganic UV filters is also an advantage presented in this study since it can reduce possible environmental impacts. The UV filters selected for the development were carefully chosen according to their safety and environmental impacts reported in the literature.

The protective action of sunscreen formulations action is based on different mechanisms, first, the absorption of UV radiation/photons by appropriate filters providing the SPF and UVA protection, second, the

reflection or diffuse scattering of photons is possible to be observed due the particle presence in the formulation. Finally, most modern sunscreen formulations are capable to neutralize free radicals by antioxidant substances [11].

The formulations developed in the present study did not contain inorganic UV filters, such as zinc oxide or titanium dioxide and/or substances able to absorb in the visible light range (400–800 nm).

However, two points in the sunscreen development need to be discussed:

some UV filters utilized and the formulation per se. The first one is the selection of the proper UV filters for the formulation. Three compatible UV filters were selected, and the concentrations were adjusted to achieve the SPF 30. The calculations were done using a BASF Sunscreen Simulator [27]. One of the UV filters is Methylene Bis-Benzotriazolyl Tetramethylbutylphenol (MBBT) based on micro-fine organic particles.

The photo-stable UV filter functions in the water phase, thus combining soluble components in the oil phase [28]. In micro-fine particles, UV filters may induce the effective scattering coefficient observed even in F1. The high RPF observed for both sunscreens can be correlated with the UV filters utilized in the study, since the presence of the organic UV filter, namely octyl-methoxycinnamate (OMC), can increase the antioxidant potential [29]. More than 90% of all types of commercial sunscreen formulations contain OMC, also known as ethylhexyl methoxycinnamate (EHMC) [30]. Kumar and collaborators observed that this UV filter is capable to present a high free radical scavenging activity using the DPPH assay [29]. Souza et al. (2017) [2] utilized in their study with sunscreen formulations, the UV filters MBBT and BEMT at similar concentrations as used in the presented study. However, the RPF was inferior (32×10¹⁴radicals/mg) when compared with the results of F1 and F2. Besides, the authors utilized the same preservatives/

antioxidants in the same concentration as we utilized. The difference to Souza et al. was the utilization of OMC/EHMC as UV filter in 6%. This compound could be responsible for the increased RPF, as the radical scavenging activity could be attributed to the group p-methoxy cin- namic in this molecule. In the present formulation, this is the only UV filter with a possible negative impact in the coral barrier environment

Fig. 5. RCM images of each sunscreen formulation at different skin depths after topical application on in vivo skin. The blue arrows represent the points with some adhesive interaction with the skin surface. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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[14], however, the utilization is reduced due the actions of other UV filters and the formulation structure.

On the other hand, the formulations present starches as polymeric phase. The utilization of corn and tapioca starch in synergy is well described elsewhere [19]. The tapioca starch is more disperse than the corn starch which presents a higher concentration of particles. The utilization of this technology is related to the Pickering emulsions, which can stabilize semisolid formulations with no or less emulsifiers [31]. Inorganic UV filters such as zinc oxide can be utilized for the stabilization of such emulsion [32], although an excessive use of particles may adversely affect the stability and sensorial properties of sunscreen formulations.

Other authors already described the utilization of starches as SPF enhancers, especially when combined with inorganic filters [18,33,34].

Another study, using the same sunscreen formulations as our study, revealed that the presence of starches in synergy can stabilize UV filters and achieve the theoretical SPF. However, the addition of 2% PEG-75 Lanolin provided F2 with a better film-forming property, improving its SPF efficacy [20]. We also could observe this when evaluating the distribution of the topically applied sunscreens in vivo (Fig. 5), where F2 showed a more homogeneous distribution on the skin surface.

In another study involving the same formulations, Infante et al. [20]

also demonstrate, the spatial distribution using two-photon tomography with fluorescence lifetime imaging (TPT/FLIM) on a microscopic slide.

The F1 formulation was found to appear in droplets, while F2 con- structed a better polymeric chain allowing a more homogeneous surface coverage. In the present work we could observe the same, however in vivo and using RCM.

The combination of ingredients presented at F2 offers more than one ecological alternative. The first important point is correlated with the reduction in the utilization of acrylate polymers since the polymers utilized in this study were starches (biodegradable) – and optimized by PEG-75 Lanolin. The second point is that, as presented in the work already published using the same formulations as in this study [20], the combination of starches and PEG-75 Lanolin could increase the SPF from 36 to 55 without increasing the amount of UV filters. This is an ecological advantage since the reduction in the utilization of UV filters with a better performance of the final formulation represents a reduction in the raw material and in the exposure of these UV chemical filters in the environment.

Despite the evidence for the stabilization of inorganic filters, the utilization of starches to stabilize – and even optimize – organic filters has not been well described in the literature, yet. Besides, this is the first evidence involving both, starches, and the visible light protection. This is important because it could be utilized in sunscreen formulations in face of inorganic filters, reducing the possible environmental impacts since starches are biodegradables. Thus the combination of starches and PEG lanolin can support the reduction of plastic polymers, organic UV filters – due to the SPF enhancement – [20] and nanoparticles (high scattering properties).

The better coverage leads to higher scattering processes and thus less transmittance. F2 presents a higher reflectance indicated by a higher effective scattering coefficient. The particles in sunscreen formulations can enlarge the optical pathways of the sun radiation in the superficial skin layers, an effect closely related to the solar hyperkeratosis, since the thicker stratum corneum may induce light scattering [35]. The enlarge- ment of optical pathways of the photons can increase the efficacy of the antioxidant molecules in sunscreens which are able to neutralize these photons or increase the diffuse reflectance. However, the enhanced radical scavenging effect has not been observed, so far, if physical filters in nanoscale size are used. In contrast, the addition of physical filters to formulations with high antioxidant properties reduce the antioxidant capacity clearly. Thus, high protection in the NIR or visible spectral region has been characterized, so far, by high scattering or high antioxidant properties [11,36].

The mechanism is not yet clear, but adsorption of the antioxidants to

the nanoparticles is one hypothesis. This is the first evidence that it is possible to increase the scattering and the antioxidant capacity of sunscreen formulations, in this case, using PEG-75 Lanolin.

The F2 formulation presents not just a higher RPF, but a large protection against visible light as observed in the ex vivo study. The RPF for F1 is high – probably because of the UV filters - but the presence of polyethylene glycol (C2nH4n +2On +1), a polyol, polymeric molecule, which can be utilized as rheologic modifier and emulsifier in cosmetic formulations, improved the results for F2 in both studies – in vitro and ex vivo [37]. In the study at hand incorporating this molecule into the sunscreen F2, some RPF results (37 ±1 ×10¹⁴radicals/mg) were obtained, evidencing a low - but existent – antioxidant property, being responsible for the increase in the RPF when F1 and F2 are compared.

Meinke et al. [11] showed that a scattering coefficient of 17 mm⁻¹at 1000 nm reduces the radical load in the skin by 60% compared to untreated skin. Alternatively, a cream with a RPF of 120 10¹⁴radicals/mg also reduces the radical formation strongly to 58%. Consequently, the combination of an RPF of 1,901,014 radicals/mg with a scattering coefficient of 45 to 20 mm-1 between 400 and 800 nm, as measured for F2, reduces the radical formation in the skin to less than 15%, which provides an excellent protection in the visible wavelength region. Good protection in the visible region by antioxidants and scattering could be demonstrated by Souza et al. [2].

A possible hypothesis for the improved scattering and antioxidant capacity could be the reaction of the polyol with the starches, increasing the plasticizer effect with the polymers. In the formulation F1 – without PEG-75 Lanolin – the formulation is composed of globules and stabilized with corn starches particles. On the other hand, the addition of PEG-75 lanolin to F2 results in a formulation which presents a more homogeneous distribution of the antioxidants and stabilization of the particles.

This more structured formulation allowed the increase in the scattering activity observed in the study. This is also a so far unpublished inno- vation, which may be fundamental for future research into sunscreen formulations.

Lanolin consists of several compounds, including esters, cholesterols and polyesters of long-chain alcohols and fatty acids, with a predomi- nance of unsaturated acids, represented by a high proportion of linoleic and docosahexaenoic acids [38,39]. PEG-75 Lanolin presents in its structure an ethoxylated lanolin condensate with an average chain length of 75 ethylene oxide units per mole, giving a better emulsifying property for formulation F2. This information is important for this study because it shows the presence of possible antioxidant properties for this ingredient. Juarez-Moreno and collaborators [40] showed in their study that PEG can be an interesting antioxidant counteracting reactive oxygen species (ROS), with their chain length directly being correlated with this antioxidant capacity.

Various commercial sunscreens provide protection in the visible and NIR region either by physical particles or by antioxidants [41]. PEG-75 Lanolin in combination with starches is the first solution to provide both, light scattering substances and antioxidant capacity in sunscreens. It may open good research prospects for visible light protection. Future research, addressing also formulations with starches and PEG-Lanolin combined with other UV filters and antioxidant mixtures, is important to support the findings presented in this study. However, the preservatives utilized in this study cannot be applied without restrictions.

Since starches are biodegradable, new studies are required to explore the potentials and limitations of these polymers in respect of their micro- biological stability.

In the face of all the results presented in this work, it is possible to observe that not only inorganic UV filters can affect the scattering activity of a sunscreen. The development of sunscreen formulations, which provide both an increase in the antioxidant capacity and a better light scattering activity, can be achieved with starches, and improved with PEG-75 Lanolin, reducing possible environmental impacts. This compound did not only enhance the optical scattering properties but also the antioxidant capacity of the formulation, which has not been observed, so

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far. Even F1, with results (always) inferior to F2, presented a significant protection against the radical formation from visible light. F2, on the other hand, presented better results because of the better formulation structures, entailing improved optical properties, skin coverage and protection consequently, being also correlated with the addition of PEG- 75 Lanolin to its composition. This demonstrated for the first time that high scattering properties can be combined with high antioxidant properties in the visible and NIR spectral region, evidencing the formulation composition in this process. Thus, a sunscreen with high protection in the UV can also provide a particularly good protection in the visible light region to prevent skin damage and premature skin aging.

Authors Contributions

Infante VHP performed the research, the analyses of the obtained data, developed the sunscreen formulations and wrote the manuscript;

Dr. Lohan SB. analyzed the obtained data, contributed to the design of the study and the elaboration of the manuscript; Mrs. Schanzer S.

contributed to the performance of RPF measurements; Prof. Dr. Maia Campos PMBG provided the laboratory, in which the sunscreen formulations were developed, and contributed to the design of the formulations; Prof. Dr. Meinke MC contributed with the analyses of the obtained data, the design of the study and the elaboration of the manuscript. Prof.

Dr. Lademann J developed the concept and design of the investigations.

All authors have read and approved the final manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by The S˜ao Paulo Research Foundation - FAPESP (grant numbers: 2016/13705-0 and 2019/12452-0) and Coor- denaçao de Aperfeiçoamento de Pessoal de Nível Superior Coordenaç˜ ao ˜ – CAPES – Code 001. The activity of access to Genetic Heritage/CTA, in the terms summarized below, was registered in the SisGen (Register number A93D2A1 for Tapioca Starch utilization), in compliance with the provisions of Brazilian Law 13.123/2015 and its regulations.

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