Prevention of UV-Induced Skin Cancer in Mice by Gamma Oryzanol-Loaded

20 Skin cancer is the most widespread cancer worldwide, mainly caused by exposure to ultraviolet 21 radiation (UV) in sunlight. Utilizing topical preventive agents in routinely daily used cosmetics 22 may prevent UV-related skin damages and skin cancers. γ-Oryzanol (GO) is a natural component 23 derived from rice bran oil, with potential antioxidant and skin anti-aging properties. We 24 biologically thorough studied the antioxidant and anticancer effects of GO in vitro to found the 25 effective signaling pathways, then evaluated the sun protection factor of prepared formulation, 26 and finally investigated the long-term preventive effects of GO-loaded nanoethosomes (GO27 NEs) against UVB-induced skin cancer in mice. Our results suggest that GO-NEs could 28 effectively prevent UVB-induced skin cancer and could be utilized as an innovative ingredient in 29


Introduction
Cancer is one of the serious human health problems globally, and its treatment is always associated with a variety of challenges 1 .Skin is the important and largest organ in the human body 2 which, undergoes the most exposure to the environment, including ultraviolet (UV) radiation from sunlight.Consequently, skin cancer is the most common cancer diagnosed in Caucasians in the United States and Australia 3 .Exposure to solar UVB radiation is a prominent cause of several skin disorders, particularly skin cancers caused by DNA damage and the generation of free radicals, i.e., reactive oxygen species (ROS) 4 .Cosmetic products are increasingly being used throughout the world and are now being developed using nanotechnology 5 .Nanotechnology-based cosmetics 6 , and new ingredients with anticancer 7 , antioxidant 8 , and anti-inflammatory properties 9 have been introduced into cosmetic products to prevent and/or treat skin diseases.Recently, a few studies have demonstrated the effect of specific compounds e.g., naproxen 10 , silver nanoparticles 11 , nicotinamide 12 , etc., on the prevention of UVB-induced skin cancers.The possible toxicity of chronic application of these compounds on the skin limits their routine administration as daily applied cosmeceuticals such as sunscreens.Natural products may overcome the problems mentioned above.In this regard, the anticancer activities of herbal extracts 13 like pterostilbene 14 , honokiol 15 , luteolin 16 , tea polyphenols 17 , sesame oil 18 , quercetin 19 , etc., have been shown against UVB-induced skin cancers.The main limitations of these natural products as far as the applications in the cosmeceutical industry is concerned, are dermal delivery obstacle due to considerable molecular weight, being expensive, having limited availability, and being obtained from difficult natural sources.Gamma-Oryzanol (GO) is a natural product derived from rice bran oil and consists of a mixture of at least ten phytosterol ferulates 20 .GO has been revealed to has good antioxidant activity 21 , anti-inflammatory 22 , skin anti-aging 23 , anticancer 24 properties, and its positive effect on increasing innate immune response in humans 25 has been reported.Despite the advantages of GO as mentioned above, its use as a novel agent for preventing solar UV-related skin cancer is hindered by the fact that its high molecular weight (>500 Da) and its hydrophobicity restrict dermal delivery across the stratum corneum (SC) 26 .It has been shown that nanotechnology can be efficiently used for the delivery of such materials across the SC down into the epidermis and the dermis.Ethosomes are a modified type of liposomes with high ethanol added (20-45%) (Fig. 1) that can function as a novel dermal drug delivery system 27 .The presence of a high amount of ethanol makes ethosomes more elastic and deformable than liposomes 28 , and consequently, it makes ethosomes an appropriate carrier for enhanced dermal drug delivery .
In this study, we prepared nanoethosomes (NEs) containing GO (GO-NEs) and carried out completely cellular studies on murine melanoma cell line (B16F10 cells) for the first time and found its apoptosis mediated signaling pathways.Besides, we evaluated possible sun protection factor (SPF) of GO-NEs.Moreover, long-termly (22 weeks) investigated the potentiality of GO-NEs to prevent UVB-induced skin cancer in vivo to introduce a novel cosmeceutical formulation.
The findings of the present study may open a new horizon on the formulation of novel sunscreens and other cosmeceuticals that could be used in populations faced with a high risk of skin cancers, such as Australians.

Characterization of nanoethosomes.
As illustrated in Fig. 2A, the characterization analysis reported the mean particle size (Fig. 2A-a) and zeta potential value (Fig. 2A-b) of NEs 80 ± 5 nm and -5 ± 0.9 mV, respectively.The SEM analysis displayed spherical morphology and confirmed particle size reports (Fig. 2A-c) and showed the narrow size distribution of particles reported by the PDI size study.TEM also revealed that NEs had ethosomal morphology (Fig. 2A-d).EE% and DLC% indexes of GO-NEs evaluated 96.36 ± 2.14% and 13.56 ± 1.46%.
Evaluation of sun protection factor.The SPF of gel-mediated GO-NEs and free gel-medium were recorded as 3.68 ± 0.3 and 0.75 ± 0.3, respectively.
Antioxidant stability assay and cellular ROS detection.The antioxidant activity for freshly prepared and stored for one year of GO, NEs, and GO-NEs, showed the concentration-dependent activity and the lack of significant scavenging activity of plain NEs (Fig. 2B-a).The IC50 for GO and GO-NEs according to DPPH assay on the first day of preparation were 90.8 and 103, respectively, and for GO-NEs was almost four times lower than free GO after one year (110 and 419.9 µM, respectively.).
Moreover, the ROS induction capacity of GO and GO-NEs was studied on B16F10 cells with LC and HC of GO and GO-NEs.According to Fig. 2B-b, the ROS levels significantly increased in H2O2 (100 µM) treated cells as compared to untreated cells (p < 0.01).Outcomes showed that the Vit.C, GO-NEs, and GO could reduce the oxidative stress in LC treatment around 29, 36, and 15 times compared with control positive, respectively.The statistical results between Vit C, GO-NEs, and GO showed a significant difference between GO and GO groups (p>0.01),demonstrate the high ROS scavenging ability of the GO-NEs as compared to GO.Also, GO, and GO-NEs in HC treatments substantially increase the ROS and approve the double-edged sword hypothesis 29 .GO, and GO-NEs could increase significantly substantially increase the ROS substantially in B16F10 cells, and these amounts were 14 and 34 times higher than control, respectively.Cellular cytotoxicity studies.Evaluation of cell viability with MTT assay showed a reduction in the cell viability by free GO and GO-NEs after 24 and 48 hours of treatment.As illustrated in Fig. 2C, it revealed that 1280 µM of free GO and GO-NEs were able to decrease the cell viability to 35% and 30% after 48 h of treatment, respectively.The IC50 values results showed a significant difference (P < 0.01) between free GO and GO-NEs with 838 µM and 255 µM after 48 h, respectively.GO-NEs could improve the IC50 value 3.27 times more than free GO after 48 h of incubation.NEs revealed no cytotoxic effects.
The consequences from cell cycle investigation showed a significant cell cycle arrest in the G2/M phase in GO (p < .01)and GO-NEs (p < .001)after 48 h of treatment.Encapsulation of the GO significantly decreased G0/G1 phase cells from 67.4% to 47.3%.On the other hand, the GO's encapsulation increased the number of Sub G1 and G2/M cells from 4.47 % to 7.81 % and 16.4 to 29.7%, respectively (Fig. 3a).B16F10 cells were stained by MDC, and cellular morphology changes were observed to investigate the effect of GO, GO-NEs, and blank NEs on autophagy induction in the cells.As shown in Fig. 3b, weak green fluorescence points are observed in blank NEs, GO, and control groups.While, cells treated with GO-NEs are entirely covered by fluorescence points, which indicated that GO-NEs affected a noticeable upsurge in the number of MDC-positive cells and a hallmark of autophagy.
Apoptosis and necrosis effects of GO and GO-NEs are shown in Fig. 4. Necrosis and apoptosis rate for GO were 15.7% and 14.23%, respectively, which were approximately equal, while for GO-NEs reported 2.18% and 29.23%, respectively.B16F10 cells treated with blank NEs showed 15% apoptosis and less than 1% necrosis.
From the results of DAPI staining, the blank NEs showed a low rate of DNA alteration.Also, GO-treated cells showed a low rate of DNA fragmentation, while GO-NEs increased the DNA fragmentation rate as compared to GO (Fig. 4b).
A quantitative cellular uptake study (Fig. 5a) showed that the NEs had considerable cell uptake, and its internalizations showed a dose-dependent response (Fig. 5b).These results were confirmed by fluorescent imaging of the cells as qualitative results (Fig. 5c).
The inhibitory effect of GO and GO-NEs on cell migration was measured using a scratch healing assay method.According to Fig. 6a and 6b, during 36 h of monitoring, both GO and GO-NEs could display an inhibitory effect on cell migration.The migration rates during 12, 24, and 36 h for GO were found to be 12.9, 26.4, and 36.8 %, respectively, additionally for GO-NEs were found to be 5.2, 6.5, and 14.31 %, respectively.According to Fig. 6c, GO-NEs could inhibit migration almost 1.4 times more than GO.
Moreover, utilizing formation assay, the effect of plain NEs, GO, and GO-NEs were evaluated.
As shown in Fig. 6d, the number of colonies decreased when treated by plain NEs and significantly reduced by GO.The number of colonies drastically diminished after treatment with GO-NEs.
Molecular cytotoxicity studies.Furthermore, we investigated the mRNA expression profile of apoptosis-related pathway genes to prove and reach support that GO-NEs have perceived the levels of apoptosis in B16F10 cells.Additionally, we completed the WB assay to quantify the targeted proteins to verify the consequences of the RT-PCR assay.As shown in Fig. 7, we remarked that both mRNA and protein expression levels of Bcl-2 and survivin were declined, and the levels of P53, BAX, caspase-3, caspase-8, and caspase-9 were increased under treatment B16F10 cells with GO and GO-NEs, while GO-NEs showed the largely effective in comparison GO.Cells under treatment with free NEs showed no significant change in selected mRNA and protein expression.

Weight changes monitoring.
Weekly body weight measurements for 22 weeks are reported in Fig. 8.After week 15, all groups showed weight loss.In week 15, the mice exposed with UV showed significant weight decreases compared with the Ctrl group.By continuation of the experiment, significant changes were observed between the groups.In the 18th week of the investigation, all groups except the group treated with GO-NEs showed substantial weight loss compared to the control group (Fig. 8 A).Finally, in the last week of the experiment, our results showed that mice treated with GO-NEs did not lose significant weight compared to the control group (Fig. 8

a).
A qualitative study of GO-NEs protective effect against UV radiation.Possible observational preventative effects of formulations are presented in Fig. 9A.By these results, the skin cancer prevention effect is seen in a group treated by GO-NEs, so that only a tiny lesion is visible (Fig. 9A-a).Skin lesions are observed in the groups treated by free GO (Fig. 9A-b) and NEs (Fig. 9Ac), but a bit lower than those in the non-treated group (negative control group).Skin injury with a large area and intensity was generated in the negative control group (Fig. 9A-d).
Histopathological studies.Histopathological results for skin samples from each group are presented in Fig. 9B.By these results, in the group treated by GO-NEs, the epithelial and connective tissues are entirely healthy.The hair follicle is reasonable, and there is no evidence for any hyperplasia (Fig. 9B-a).In the group treated by free GO, although the epidermal layer was completely healthy, there were still adnexal tumors indicated by red narrow in Fig. 9B-b.In a group where only free NEs were applied, there are a large number of adnexal tumors.But in this sample, the epidermis layer was less destroyed, and irregular connective tissue arrangement is shown (Fig. 9B-c).As shown in Fig. 9B-d, which belongs to the positive control group, the epidermal and connective tissues are destroyed, and a high number of adnexal tumors have been formed.Many tumors were generated in the lower layers of the dermis, indicating the lesion's depth in the positive control group.

Discussion
The increased incidence of skin cancer proposes the usage of sunscreens lonely is not adequate in preventing skin cancer.It needs additional strategies like adding novel ingredients to daily cosmetics with skin cancer-preventing effects.GO is a natural product derived from rice bran oil.
Previous studies verified that GO had well skin anti-aging effects 23 .Therapeutic, cosmetic agents must penetrate through SC to have beneficial effects.Studies have demonstrated that particles in the size range of sub 100 nm present the best skin penetration 30 .In this study, the dermal penetration problem was overwhelmed by nanotechnology to gain sub 100 nm and nearzero charged vesicular structure.The negatively close zero surface charge of particles is also promising for enhancing transdermal delivery 31 .Zero surface charge is necessary for colloidal stability; then the formulations were introduced in a gel-medium immediately after preparation to minimize dynamic changes in the particles and stabilize the NEs.DLC% of liposomes are usually not more than 10% 32 .In this case, more than 13% of DLC% was reported because of higher hydrophobicity of GO, and addition hydrogen/oxygen bonds of GO structure, which could form hydrogen bonds with the polar head group of lecithin 33 in the outer layer of the ethosomal system.These consequences could be the reason for the low negative zeta potential value and the higher ethosomal system DLC%.Free radical scavenging assessment displayed a bit reduced activity of the GO-NEs on the first day of preparation, which was possibly due to a temperature effect during the preparation procedure.However, after one year of storage showed a strong antioxidant protective effect.These results highlighted the carrier's effect 34 and displayed an advantage for the chemical stability of GO encapsulation into the ethosomal system.Studies demonstrated that the nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in acting as an emergent controller of cellular oxidants resistance to anti-inflammatory responses 35 .

Rungratanawanich et al. investigated the cellular antioxidant activity of GO and its related
signaling pathway.This study revealed that GO could successfully increase the upregulation of Nrf2, additionally enhanced glutathione peroxide and superoxide dismutase, which are antioxidant enzymes 36 .Antioxidant activity of our investigations at the cellular level showed that the GO-NEs and free GO could reduce the oxidative stress and the supremacy of oxidative stress reduction of GO-NEs in LC treatment, which highlighted the effect of the nanoethosomal system.Moreover, ROS have been identified as signaling molecules in various pathways regulating both cell survival and cell death 37 .Our results in HC treatment results shown that the GO and GO-NEs can significantly increase the ROS levels; this phenomenon can be justified by dubbing sword theory 38 .Recent research demonstrated that there is an effective relationship between increased oxidative stress and induction of autophagy 39 .Following further investigation, we found that GO and GO-NEs could activate the autophagy pathway in B16F10 cells.Our results can confirm that by increasing the ROS level, the autophagy pathway is activated in B16F10 cells.As mentioned, antioxidants can have a double effect on inhibiting or causing oxidative stress.To that end, due to GO effects on Nrf2 modulation and its role in antiinflammatory possesses, it is suggested to investigate the anti-inflammatory behavior of GO-NEs as well.Our results in most of the tests showed that the formulated GO performance was better than its free form.These results could be attributed to better cellular internalization of GO-NEs compared to GO, highlighting the impact of nanoparticles (especially sub 100 nm size) on cellular internalization 40 .Most of the antioxidants induce apoptosis in cancer cells 41 .Our apoptotic assessment results indicated that both GO and GO-NEs were able to induce apoptosis.
From the results, the blank NEs showed a low rate of DNA alteration and apoptosis rate due to ethanol in the nanoethosomal formulation.These outcomes indicated the effect of sub 100 nm GO-NEs that successfully increased B16F10 cells DNA alteration, apoptosis rate, and consequently effectively decreased necrosis rate.The progression of apoptosis is linked with higher endocytosis of the GO.Cell cycle plays a critical role in cell migration 42 ; cell migration occurred with a high velocity and directionality during the S-G2-phase of the cell cycle 43 .Our results showed that the GO increases the cell population in the G2 phase, and it can lead to the reduction of the cell migration rate.Scratch and colony formation assay tests were performed to confirming this claim.The following results showed that the GO could inhibit the colonyforming and cell migration.The obtained results proved its anticancer status and supremacy of GO-NEs.The most important issue in the introduction of an anticancer agent is the identification of the apoptosis pathways by alteration of the levels of apoptotic and anti-apoptotic genes.In our study, the protein level of caspase-8, as apoptosis promoter, effectively increased, and simultaneously, the levels of apoptotic inducers, including P53, BAX, and caspase-3, dramatically increased.Increasing the levels of cleaved caspase-3/caspase-8 and concomitantly describing the levels of pro-caspase-3/caspase-8 confirmed the activation of caspase-3 and caspase-8, respectively.Following, significantly describing in the protein level of survivin and Bcl-2 as anti-apoptotic inducers proved the anticancer behavior of GO and the superiority of GO-NEs by reducing anti-apoptotic proteins and increasing the levels of apoptotic proteins.In vitro, SFP measurements showed a bit of boost protection effects.It is important to note that the ingredient, which wants to be a novel cosmeceutical candidate, has sun protection effects, and these results highlighted the advantages of GO-NEs.Attained results proved in vitro cytotoxicity effects and showed a bit SPF benefit of GO-NEs that could be a justification to investigate the potential effect of GO-NEs on skin cancer prevention in animals.The safety of cosmetics that are taken routinely daily is more highlighted.Chaudhary et al. demonstrated the positive effect of naproxen on UVB-induced skin cancer 10 .This study did not investigate the safety of naproxen for daily usage.Although adding chemical products to daily cosmetics are controversial due to toxicity issues.Besides, rice derived components generally are well-thought-out to be safe and non-allergenic, as well as investigations had reported no irritation when rice components tested on the skin 44 .Nowadays, the safety of natural products is a fascinating topic in these issues 45  Preparation of nanoethosomes.NEs were prepared via a modified ethanol injection method 46 using soy lecithin as a surfactant.Briefly, 350 mg of soy lecithin, and 50 mg of GO, were dissolved entirely in 20 ml of ethanol as lipid/GO solution (Fig. 1a) and slowly injected with 1ml/min flow rate into the 50 ml of heated distilled water with 65 •C temperature with mixing by a homogenizer (Silent Crusher M, Heidolph, Germany) (20,000 rpm) (Fig. 1b).The homogenization step was continued for 15 min after finishing the lipid/GO solution to achieve GO-NEs in sub-100 nm size.The empty nanoethosomal formulation (NEs without GO) was also prepared using the same method.

Preparation of gel-mediated nanoethosomal formulation. Prepared hydroethanolic suspension
of GO-NEs was immediately transferred into a 250 ml beaker and 450 mg of xanthan gum was added slowly under stirring with a mixer (Ultraturrax, IKA, Germany) to prepare a gel-medium on a laboratory scale (Fig. 1d).For preparing GO gel-medium, GO (50 mg) was wholly dissolved at 20 ml of ethanol and added to 50 ml of distilled water.Next, xanthan gum was then added (1g of gel contains 0.085 % w/w GO).Gel-medium for plain NEs was also prepared in the same manner without the addition of GO.Encapsulation efficacy.The amount of loaded GO was estimated to evaluate the encapsulation efficacy (EE%) and drug loading capacity (DLC%) indexes.For this, due to the low density of GO (0.32 g/cm 3 ) 48 , the supernatant of nanoethosomal dispersion was precisely collected after gentle centrifugation at 500 rpm for 10 min.Then, 1 ml from the middle of the container was collected and mixed with 5 ml of chloroform, vortexed (LS100, Labtron, Iran) for 10 min for the destruction of the nanoethosomal structure and subsequently dissolving GO in chloroform.By measuring the absorbance of loaded GO spectrophotometrically (Ultrospec 2000, Pharmacia Biotech, UK) at 319 nm, the amount of entrapped GO was determined 49 .Following the EE% and DLC% were calculated using the following equations (1).

Radical scavenging activity.
The antioxidant activity and stability during one year storage time of the GO and GO-NEs were determined using DPPH radical scavenging activity test 50 .

Determination of sun protection factor.
The SPF of GO-NEs was determined using the classical in vitro spectrophotometric method 51 .To this purpose, 2 mg/cm 2 of gel-mediated GO-NEs was applied by a Transpore™ membrane, which mimics natural skin, and UV absorption of the sample was recorded from 290 to 400 nm.
Cell culture and seeding.Skin melanoma (B16F10) cells (Pasteur Institute, Iran) were maintained in 10% FBS contain RPMI-1640 and cultured at 37°C with 5% CO2.The cells were sub-cultured 24-48 h later with an initial concentration of 4 × 10 4 cells/ml and used in the logarithmic phase in all experiments.The cells seeded at the density of 1.2 × 10 4 , 1 × 10 5 , and 2 × 10 5 cell/well for 96, 12, and 6-well plates, respectively.The treatment was done when confluency reached 70% for MTT assay, flow cytometry, and fluorescent imaging experiments.
For the scratch migration assay, the required confluency was 100%, and the seeding concentration for colony formation assay was 1 × 10 3 cell/well.

Cell viability study. The MTT assay technique was used to evaluate the cytotoxicity effect of
GO and GO-NEs on cell viability 52 .After seeding the cells in a 96-well plate and incubation for 24 h, the cells were treated with 100 µl different concentrations of GO and GO-NEs (5-1280 µM).After 24 and 48 h of treatment, the media was discarded, and cells were washed with PBS twice.Next, 150 µl of fresh media containing 50 µl of MTT solution (2 mg/ml in PBS) was added to each well of the plate.After four h, the incubated medium was replaced with 180 µl of DMSO and 20 µl of Sorensen's phosphate buffer.Then, the plates' absorption was read using a wavelength of 570 nm and a reference wavelength of 630 nm by the Elisa Reader instrument (TCAN, Austria).
Cellular ROS detection.DCFH-DA was employed as a fluorescent dye to evaluate intracellular ROS accumulation through flow cytometry analysis 53 .Briefly, cells were cultured on a 6-well plate and treated for 48 h with Blank NEs, GO in low concentration (LC, based on DPPH assay IC50 concentration) and high concentration (HC, Based on MTT assay IC50 concentration), LC and HC of GO-NEs, Vit C as antioxidant control and H2O2 as a positive control.Concentrations were selected based on DPPH IC50 for LC treatments and MTT assay IC50 for HC treatments.
Then, cells were exposed to 500µl of 10 µM of DCFH-DA.Next, de-attached after two h incubation and washed twice with PBS.Cells were resuspended in 500 µl PBS and subjected to the flow cytometry (Becton Dickinson FACSCalibur, Franklin Lakes, New Jersey, United States) analysis using an FL2-H band-pass filter (FITC).
Cell uptake study.RhB as a fluorescent agent was used for labeling NEs to measure the cellular uptake intensity of the prepared formulations by cells 28 .Briefly, RhB was added to the NEs (0.05% w/w RhB ratio to lipid) during the same procedure carried out to prepare of GO-NEs, and unload RhB was separated with ultra-filtration method (Amicon® tube, 30 kDa Mw; Millipore, Germany).The cells were seeded in a 6-well plate, incubated overnight, and treated with 10, 100, 200, 400, 800, 1600, 3200, 6400, and 12800 µg/ml (based on the lipid weight) concentrations of RhB labeled NEs for four h.The quantitative cellular uptake of the prepared formulations was measured by a flow cytometry instrument (MACS Quant 10, Miltenyi Biotech GmbH).For qualitative evaluation of the cell uptake, the B16F10 cells were seeded into lamel coated six-well palate and incubated with the same concentrations of the quantitative cell uptake measurements method and incubated for four h; after the incubation, the upper media was discarded and replaced with 1 ml f formaldehyde 4% solution and fixed for four h.after fixing the cells were photographed with fluorescence imaging microscopy system to identify cell uptake intensity (BX50, Olympus, Japan).DAPI staining.The DAPI staining method was carried out for evaluating the DNA fragmentation in B16F10 cells 28 .For this purpose, cells were seeded onto 12 mm coverslips at the seeding density of 2 × 10 5 cells/well.After 48 h incubation, the cultured cells were treated with the IC50 concentration of blank NEs, GO, and GO-NEs.After 48 h of treatment, the cells were washed with PBS twice and fixed with formalin 4% solution for four hours.Next, the cells were washed with PBS once again and incubated for 15 min with Triton X100 solution (0.01% V/V) and DAPI solution (1 µg/ml) for 15 min.Finally, the stained cells were monitored by a fluorescence imaging microscopy system to identify apoptotic nuclei.
Detection of autophagic vacuoles by monodansylcadaverine. MDC is a fluorescent dye as the marker for visualization and identification of the autophagic process that was used for detection of the autophagy pathway activation level in the B16F10 cells.For this purpose, the cells were seeded onto 12 mm coverslips at the seeding density of 2 × 10 5 cells/well.After 48 h of incubation, the cultured cells were treated with the IC50 concentration of blank NEs, GO, and GO-NEs.After 48 h of treatment, the cells were washed with PBS twice and stained with 0.05 mmol/L of MDC at 37 °C for 10 min.Subsequently, the cells were washed three times to remove MDC in PBS and observed under a fluorescence imaging microscopy system 54 .
Cell cycle analysis.B16F10 cells were cultivated at a density of 2 × 10 5 cells/well into six-well plates in four groups, including 1) negative control cells, 2) blank NEs, 3) GO, and 4) GO-NEs.
The cells were harvested 48 h after the treatment, and following fixed with 75% of ice-cold ethanol overnight, treated with 500 l of 20 g/ml RNase A, stained with 500l of 0.1 mg/ml PI contain 0.01% of Triton X-100 solution.Then the cells were washed with PBS and finally analyzed by flow cytometry for DNA synthesis and cell cycle status 55 .
Apoptosis assay.The flow cytometry method was used to evaluate the cell death pathway treated with GO and GO-NEs.After seeding the cells into six-well plates and incubating overnight.The upper media of the plate wells were changed with 2 ml of fresh media containing the 48 h IC50 concentration of each treatment group.Then cells were incubated for 48 h.Following, cells were detached and stained via an annexin V/PI apoptosis detection kit (Exbio, Czech Republic) according to the manufacturer's protocol 56 .The rate of apoptosis was analyzed by flow cytometry (MACS Quant 10, Miltenyi Biotech GmbH).
In vitro scratch healing assay.The scratch healing assay was employed to evaluate the in vitro cell migration rate 57 .Briefly, cells were seeded into a 12-well plate and cultured until cells formed a confluent monolayer.Afterward, a linear wound was created in the monolayer with a 3 mm thickness scratch pen.Next, cells were treated with 48 h IC50 concentration of the free GO and GO-NEs and incubated.The migration percent of cells was measured according to the wound distance, which was recorded via an inverted microscope (LABOMED, TCM 400, USA) at 4X magnification during various times (0, 6, 12, 24, and 36 h) after incubation, and compared to the wound distance at the initial time.

Colony formation assay.
For evaluating the ability of the GO and GO-NEs in colony formation inhibition, the colony formation assay was performed.To perform this test, 2 × 10 3 cells per well of B16F10 cells were seeded into a 6-well plate.They were then incubated and followed for 15 days.The colonies were fixed and stained with crystal violet ten times diluted solution with PBS, and then photographed by iPhone 8 (Apple Inc.Cupertino, California, United States) and counted via ImageJSoftware. 58.
Real-Time PCR.Expression of subjected genes at the cells was assessed by real-time PCR (RT-PCR) 55 .TRIzol was used to extracting RNA from the cells.At the first step, 5 X 10 4 cells per well of B16F10 cells were seeded into six-well plates and incubated overnight; then the cells were treated with 48 h IC50 concentration of GO and GO-NEs.The cells were harvested after 48h and incubated with 1 ml of TRIzol solution (Thermo Fisher Scientific, Waltham, Massachusetts, U.S) for 20 min; at the next step, the cells 200 l of chloroform was added to the TRIzol cocktail and mixed gently, then the cocktail incubated in -20 refrigerator for 20 min.At the next step, the TRIzol/chloroform cocktail centrifuged at 12,000 RPM speed at 4-8 o C for 15 min and the colorless upper aqueous phase was harvested and mixed with 500 l of isopropyl alcohol and blended gently and incubated for 10 min then the mRNA cocktail for 10 minutes centrifuged at 12,000 RPM and the upper media was discarded and washed with isopropyl alcohol once again.At the last step, the mRNA sediment dried at room temperature and was solved with 20 l Rnase free water.The concentration of the isolated mRNA was measured by a Nano-Drop spectrophotometer (2000, Thermo Fisher Scientific Life Sciences, Waltham, Massachusetts).The complementary DNA was generated by the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany).RT-PCR was done by the Power SYBR GREEN mix (Applied Biosystems, Foster City, California).β-Actin was amplified as a reference gene.mRNA expression was measured with the 2−ΔΔCT method.Table 1 addressed the primer sequences used in RT-PCR reactions.
Western blot analysis.Total protein levels were extracted and quantified from the cells by RIPA protein extraction kit, and protein concentration was assessed by Nano-Drop spectroscopy.
Before loading proteins into the wells, they mixed with sample buffer and boiled at 100 °C for 5 min.Following, 50µg micrograms protein/lane whole cell lysates were electrophoresed in SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, Billerica, Massachusetts, U.S).
2% non-fat milk was used to block non-specific antigens, and then specific primary antibodies (1:1000, SCB) were added, then incubated 18 h at 4 °C.The membrane was washed three times with buffer TBS-T for 15 min, incubated with anti-rabbit secondary antibody (1:1000, SCB), shake for 75 min at room temperature, chemiluminescence reaction was performed 59 .
Animal care and treatment.A bank of two TL 40W/12 RS UVB lamps (Philips, Holland) with a peak emission of 313 nm was used to mimic sunlight UV and expose mice to UV irradiation for inducing skin damage.Thirty male BALB/C mice (6 weeks old with a mean weight around 36 g) were randomly divided into five groups and housed in a standard room (temperature 25-27 • C, 12h daylight cycle with air conditioning) one week before the start of animal studies.Animals in groups A, B, and C, were applied once daily GO-NEs gel, plain GO gel (1g of gel containing 0.085 % w/w GO), and blank nanoethosomal gel (gel-medium without GO), respectively.Group D and E did not receive any formulation as positive and negative control groups, respectively.Dorsal areas of animals were shaved weekly to diminish any UV covering and/or protective effects of animal hair and ease of applying the gels.For producing UVB induced skin lesions, groups A, B, C, and D were placed 25 cm under the UVB lamps and irradiated 200 J/m 2 /day, one h after topical application of formulations 15 .The 25 cm was the best distance to gain a calculated UV dose of 200 J/m 2 .While irradiating, to immobilize the animals and protect other areas from irradiation, mice were fixed in the mice restrainer was covered with aluminum foil and black tape.The limited upper part of the restrainer (the region in contact with the dorsal area of the animals) was only exposed area to UV light.Animal studies continued until skin lesions were observed, which were completed at 22 weeks.Weekly animal body weights were recorded during 22 weeks of study.
Histopathological study.After 22 weeks of study, the animals were sacrificed, and dorsal skin samples were removed and fixed in 10% formalin solution for hematoxylin and eosin (H&E) staining and subsequent examination under a light microscope in cooperation with a pathologist.
Statistical and data analysis.All experiments were repeated three times in succession.
Statistical analysis was done using the independent t-test and Two-way analysis of variance (Two-way ANOVA) with multiple assessments between confession data using a Tukey honest noteworthy difference test (Graphpad Prism, version 8, San Diego, CA).A P-value of <0.05 was considered significant.Data obtained from flow cytometry were analyzed using the FlowJo software package (V10.5.3,Treestar, Inc., San Carlos, CA).

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Our study, mean animal weight measurement during 22 weeks of the study, displayed no significant changes in GO-NEs treated group.Outcomes demonstrated a) the therapeutical effect of GO-NEs on preventing skin cancer and reducing its related stress, b) the safety of GO-NEs on long-term daily usage.Pathology results confirmed our hypothesis about the prevention of UV-Induced Skin cancer in mice by GO-NEs.Healthy epithelial tissue in the group treated by GO verified the potentiality therapeutical effect of GO.The generation of adnexal tumors in this group is due to GO could not penetrate through SC.Less destroyed epithelial tissue in the group treated only NEs could be related to the nature of lipid used in ethosomal structure, which could cover the wound and speed up recovery.ConclusionIn conclusion, GO-NE could effectively prevent UVB-induced skin cancer and suggest a new nanoethosomal formulation of GO.Our investigation opened up a new horizon, which may have novel clinical relevance as a pharmacologically active ingredient of cosmetics and sunscreens for the first time.Experimental Study design.This study has been approved by institutional review board of Faculty of Pharmacy and Drug Applied Research center, Tabriz University of Medical Sciences, Tabriz, Iran.All methods were carried out in accordance with relevant international regulations and guidelines.Materials.The Soy lecithin was purchased from Lipoid GmbH, Ludwigshafen, Germany.GO was a generously gifted gift from Tsuno Rice Fine Chemicals Co., Ltd, Japan.The xanthan gum, 4′,6-diamidino-2-phenylindole (DAPI), monodansylcadaverine (MDC), Rhodamine B (RhB), propidium iodide (PI), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), 2,2-diphenyl-1picrylhydrazyl (DPPH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and ethanol 96% were purchased from Sigma-Aldrich co., St Louis, MO.RNase A was obtained from CinnaGen co., Iran.RPMI 1640, fetal bovine serum (FBS), Trizol, trypsin EDTA (0.25%), and Penicillin-Streptomycin were purchased from Thermo Fisher Scientific, Waltham, Massachusetts.All of the WB regents were purchase from Santa Cruz Biotechnology, Inc. Dallas, Texas.

Figure 1 .
Figure 1.A brief illustration of the preparation of gamma oryzanol-loaded nanoethosomes (GO-

Figure 3 .
Figure 3. (a): Cell cycle progression and the percent of the cell population of B16F10 (murine

Figure 4 .
Figure 4. (a): Apoptotic rates of the control group as untreated (Ctrl) and treated B16F10

Figure 8 .
Figure 8. Mean mice body weight measurement of each group that applying (a) gamma oryzanol