In vitro and in vivo efficacy of a cosmetic product formulated with new lipid particles for the treatment of aged skin

The cumulative oxidative damage causes an acceleration in the skin aging.


| INTRODUC TI ON
The skin during its life undergoes an aging process, resulting in senescence.[3][4] Intrinsic and extrinsic components are involved in this process.For example, internal or also called chronological factors range from biological, biochemical, and molecular mechanisms.The intrinsic skin aging is a consequence of a combination of several events that occur into skin.In particular, a decreased proliferative capacity of cells, a reduced extracellular matrix synthesis in the dermis and an increase expression of enzyme degrading dermis matrix.4][5] The cumulative oxidative damage induced by the above-mentioned factors causes an acceleration in the skin aging.The main targets are mitochondria, proteins oxidation, and DNA damage at the level of telomeres.
The cell has several protective mechanisms that come into operation when one pathway is out of balance and not well regulated.
Under normal conditions, during aerobic metabolism, the cell produces reactive oxygen species (ROS), which are essential for maintaining homeostasis.However, when their concentration exceeds the cellular immune defenses, oxidative stress occurs, causing reversible or irreversible damage to macromolecules such as proteins, lipids, and nucleic acids.7][8] Vitamin E performs its protective function against the oxidation of polyunsaturated fatty acids, components of cell membranes. 9Vitamin C, on the contrary, performs its antioxidant activity, protecting the main macromolecules such as carbohydrates during cell metabolism and during exposure to risk factors. 10tamin A plays an important role in the preservation of cell membranes and fights free radicals, and for this reason it appears as an ingredient in the most effective anti-wrinkle creams, counteracts the harmful effects of smoke and pollution as well as providing valid help in fighting skin aging and protein synthesis. 11,12e literature shows that one of the causes of oxidative stress is the overproduction of ROS.4][15] The search for products or mechanisms capable of maintaining a well-groomed and youthful skin has always been of great interest, since the skin, being the most exposed organ of our body, shows its manifestations mainly on an aesthetic level.
The outputs will therefore be more wrinkled skin, characterized by pigmentary changes, loss of hydration, dryness, altered skin barrier function, and finally decreased elasticity and telangiectasias. 16 the last period, a winning system for topical treatment has proved to be the encapsulation of active substances with anti-aging and antioxidant functions in lipid matrices and/or nanoparticles. 17,18 this way, the final products containing these systems should have better efficacy and good stability.At the same time, the encapsulation of active ingredients can reduce skin irritations that could arise if these substances are left free. 191][22][23] Other intrinsic features known of the lipid nanoparticles are hydration, increased viscosity and skin occlusion.Depending on their composition and the active ingredient to be incorporated, [24][25][26] these characteristics will be higher or lower.
The choice of the lipid nanoparticles can be attributed to their chemical affinity to components of skin surface made of fatty acids, cholesterol and ceramides.In this work, new patented lipid particles (SIREN CAPSULE TECHNOLOGY™) are proposed for the topical treatment of a condition associated with the accumulation of ROS as an anti-aging solution. 27 this work, new patented lipid particles (SIREN CAPSULE TECH-NOLOGY™) are proposed for the topical treatment of a condition associated with the accumulation of ROS, as an anti-aging solution. 27is work is based on a scientific demonstration to establish the effect of a topical treatment using SIREN CAPSULE TECHNOLOGY™.To this end, both in vitro tests against ROS production and in vivo studies were carried out to assess the system's effects on skin hydration, skin barrier function, surface smoothness, and wrinkle depth.Vitamins are well-known in the literature as powerful agents against skin damage caused by oxidative stress. 28,29For these reasons, formulations containing free or encapsulated vitamins have been used as controls.

| SIREN CAPSULE TECHNOLOGY™ Preparation
The SIREN CAPSULE TECHNOLOGY™ system, hereinafter referred to as SIREN, is composed of lipid particles prepared by the emulsification method, properly modified. 30Briefly, (i) a blend of jojoba wax, mimosa wax, and sunflower seed wax was melted at a temperature of 55°C under magnetic stirring at 200 rpm for 10 min; (ii) a mixture of natural polyunsaturated fatty acids obtained from safflower oil and all other liposoluble active ingredients were added to the melted lipid mixture obtained in step (i), under magnetic stirring at 200 rpm while maintaining the temperature at 55°C until a homogeneous "fat" melted mixture is obtained and, just before emulsification, Retinyl palmitate as Vitamin A, is added under stirring; (iii) an aqueous dispersion was prepared by adding decyl glucoside and sodium ascorbyl phosphate as Vitamin C in water heated to 45-50°C subsequently under stirring (500 rpm for 10 min); (iv) the fat mixture prepared in point (ii) was added to the aqueous dispersion prepared in point (iii) by maintaining the temperature at 48-50°C under stirring at 3500 rpm for 10 min, obtaining an emulsion of lipid particles in an aqueous phase; then the emulsion obtained in step (iv) was sieved through a net with pores with a diameter of 25 μm, obtaining an emulsion of lipid particles with a diameter of up to 25 μm in an aqueous phase; (v) the emulsion obtained in the previous step was cooled to 10°C by means of a water bath to accelerate the solidification of the lipid part.Microparticles were stored at 4°C until use.
For the in vitro and in vivo evaluations, a gel base was prepared as described below and used as a placebo (gel code 6417): Table 1 summarizes all products used in the following experimental section.

| In vitro evaluation of protective effects against menadione-induced oxidative stress in cell cultures
The efficacy of SIREN CAPSULE TECHNOLOGY™ formulation to counteract the ROS production was tested by using menadione, a well characterized synthetic form of Vitamin K3.It has different aims, and one is related to the generation of mitochondrial ROS. 31 The cells selected for this study are human keratinocytes (Hacat).
Seeding took place in 96-well plates for 24 h at 37°C and 5% CO 2.
The exhausted medium was replaced with fresh one the next day by adding the samples at concentrations of 5 and 1.25 mg/mL diluted directly in the culture medium.The samples considered were the following: the gel placebo (gel code 6417), the gel containing free vitamins (gel code 6490), the gel containing encapsulated vitamins (gel code 6491), and the gel containing SIREN CAPSULE TECHNOL-OGY™ system (gel code 6418).
Four hours after the addition of the samples, 20 μM of menadione (2-methyl-1,4-naphthoquinone) was added to stimulate ROS production.
When ROS are formed, there is a one-electron reduction of quinone and the formation of the corresponding semiquinone radical.
In turn, these radicals will go on to produce the reactive species, recovering the initial quinone.
Cells treated neither with samples nor with menadione are used as a negative control, cells treated with menadione without sample as a positive control of ROS formation, finally, cells treated with samples alone are used as a further control.For each dilution, three replicates were performed.
After 2 h of exposure to the enhancer, the amount of hydrogen peroxide produced was studied.In particular, the hydrogen peroxide substrate is able to react directly with the luciferin precursor.This is followed by the addition of ROS-Glo™ Detection Reagent containing  The amount of ROS produced is determined as a percentage by the following formula: The acceptance criteria for the negative control was the following: ROS increase compared with cell not treated with menadione >3.

| In vivo enrollment
3][34] The studies were performed on 30 healthy female volunteers aged 36-58 hired on selected inclusion and exclusion criteria.Subjects were treated after their personal informed consent according to Italian law (GDPR 2016/679).
To participate in the study, people must be in good health, free from skin diseases and inflammatory lesions.Subjects who did not show allergies to the components introduced in the final formula and nonpregnant and breastfeeding women were selected.During the treatment period, the subjects should not undergo any type of other treatment in the investigated area during the trial period.
Subjects with chronic diseases, undergoing pharmacological therapy, allergic people and subjects who had participated in a similar study less than 60 days prior to the start of this study, have been also excluded.
The parameters were acquired at the beginning (T0) and after 4 weeks of applications (T1) in different body areas according to the aim of the specific test.
Specifically, to evaluate the moisturizing efficacy and the capability to maintain the skin barrier function of the products, the test was carried out on four different area selected on the forearms and volunteers were trained to apply each product to a specific area daily for a month.The choice of forearm side was randomized among subjects to reduce variability.
The anti-aging efficacy test was performed on the same 30 subjects selected according to the presence of signs of age on the face.Volunteers were trained to apply daily two products on face by the split-face method for 1 month.Then, after 1-month of interval subjects were trained to apply daily the third product for 1 month.
The choice of face side was randomized among subjects to reduce variability.
All measurements were made in a temperature and humidity controlled room (T = 22°C, relative humidity [RH] = 70 ± 5%).Before the assessments, each subject rest for at least 15 min to acclimate.
The probes used to evaluate the skin parameters were placed on the skin without causing alteration, pain and irritation.

| Parameter evaluated for moisturizing efficacy evaluation
The CM 825 corneometer (cutometer MPA580, Courage & Khazaka) was used to evaluate the amount of water present in the superficial layer of the epidermis, the stratum corneum (SCWC).This parameter will allow to evaluate the moisturizing power of the formula.The instrument is based on the technology of the corneometry, used to investigate the hydration of the outer layer of the epidermis, that is, the stratum corneum. 35,36The variations in hydration will be deter-

| Skin barrier function efficacy evaluation
The study of tolerability and the possible occlusive effect of the products under study were analyzed by examining the integrity of the skin barrier, by measuring transepidermal water loss (TEWL). 37For this purpose, the skin evaporimeter allowed to evaluate the TEWL (from 0 to 90 g/m 2 h).The probe consists of a small open cylindrical chamber (1 cm in diameter, 2 cm in height) with a pair of humidity sensors connected to a microprocessor inserted at the computer workstation, using Tewameter TM 300 (Cutometer MPA580, Courage & Khazaka).
The four products evaluated in this study were: the gel formulation containing SIREN CAPSULE TECHNOLOGY™; the gel containing free vitamins; the gel containing encapsulated vitamins and the gel base used as placebo.

| Anti-aging effect
The three products evaluated in this study were the gel formulation containing SIREN CAPSULE TECHNOLOGY™; the gel containing free vitamins and the gel containing encapsulated vitamins.
The study was carried out over 4 weeks of daily application on the right and left sides of each volunteer, treated with one of the products, respectively.Then, after 1-month of wash out, subjects were trained to apply daily the third product for 1 month.
The present study was aimed to evaluate the effectiveness of the product to improve skin texture and to reduce the appearance of wrinkles and fine lines.
The parameters taken into consideration, are designed to evaluate the micro-reliefs of the skin.The measurements were performed by means of a visual and topographical 3D analysis using the der-maTOP software, based on Breuckmann's OPTOCAT programme.
In particular, the average skin roughness (Ra) was analyzed and is expressed as the integral of the curve describing the skin roughness.This parameter is directly proportional to the roughness of the skin. 38,39e second parameter chosen is the mean depth of roughness (Rz).Rz represents the arithmetic average of the different segment roughness calculated from five succeeding measurement segments of the same length.

| Statistical analysis
The normal distribution of data was verified by the Shapiro-Wilk test.The distribution of the values obtained at the different experimental times was compared by means of an intra-group analysis (T28d vs. T0) by using Student's t-test.For each parameter analyzed in the clinical studies, a comparison was made between the values measured at time T 1 with respect to the initial value at time T 0 .A significance level of 5% was chosen, so changes were considered statistically significant for p < 0.05.

| Lipid particles preparation
The choice of the above-described mixture of waxes advantageously allows the particles to be prepared with a sustainable process that does not require high temperatures.
Furthermore, the process according to the invention does not include the use of organic solvents.To be sure of the oxidation stability of the prepared systems, the particles were previously tested by a validated test according to ASTM D1959-97 standard.The lipid particles demonstrated to be stable to oxidation, this means an iodine index <60 g Iodine/100 g. 27 During the production the emulsion is sieved through a net with holes obtaining a very fine emulsion with a cut-off of 25 μm of diameter.The formulation containing Siren capsules was subjected to accelerated stability test for 3 months at 40°C, RT and 4°C in its final packaging.During this period no significant changes have been observed (Color, pH, Viscosity, Odor, aspect and separation and aggregation) which make us think that Siren capsules Technology could be stable (data not reported).

| In vitro evaluation of protective effects against menadione-induced oxidative stress in cell cultures
The ROS-Glo™ H₂O₂ is a rapid and accurate assay in vitro, used to measure the levels of the reactive oxygen species with the longest and most stable half-life, the hydrogen peroxide (H₂O₂).This test is based on the phenomenon of bioluminescence.Briefly, first the derivatized luciferin substrate is incubated with the sample.
The reaction due to the presence of hydrogen peroxide, allows the production of the luciferin precursor.Subsequently, a detection solution, ROS-Glo™, is added, converting the precursor to luciferin by producing a light signal proportional to the level of H₂O₂ present in the sample.
The test chosen, had the aim to investigate one of the main ROS produced in our cells, in fact, H 2 O 2 is the main stable ROS and it could penetrate better through the membranes. 22Evidence of antioxidant activity of the sample is determined by a reduction in the amount of ROS produced in the cells treated with the sample compared to control cells.The less ROS will be produced by the cells, the more active the sample investigated.
In this way we are able to investigate the antioxidant capacity of the formulations.
First and foremost, it is important to verify the acceptance of the test by evaluating the ability to produce ROS in cells that have not been treated with the sample.
Table 2 shows the positive results concerning the acceptance criteria of the assay, for untreated cells used as control, expressed as the ROS increase in cells treated with menadione compared with cells not treated with menadione.
Table 3 reports results obtained from the in vitro study expressed as a mean of amount of ROS produced from all samples.The experiment was carried out on two plates and, for this reason, two controls were used.Based on these results, it is evident that the negative control (without menadione), the placebo gel, and the gel containing SIREN TECHNOLOGY exhibited similar levels of ROS production.The only exception is the sample 6490 at 5 mg/mL, containing free vitamins, that showed an amount of ROS production more than double respect to the control.These results could be related to the pro-oxidative activity of vitamin C, as recently reviewed by Baranska et al. 40 The sample 6418-5, formula with SIREN at the concentration of 5 mg/ mL, shows the highest protective activity against ROS production.As can be seen from Figure 2, the SIREN CAPSULE TECHNOL-OGY™ gel shows inhibitory activity against ROS production through menadione induction.In fact, at both tested concentrations, ROS production is lower than in the control samples.Specifically, this product results in 44.5% ROS production at concentration of 5 mg/mL, whereas at a concentration of 1.25 mg/mL, the percentage produced is 78.59%.By comparison of these data with the placebo gel, the gel containing free vitamin C and vitamin A, and the gel with the two encapsulated active ingredients, a dramatic increase in ROS production is observed.At concentrations of 5 mg/mL, the gel with free vitamin C and vitamin A, shows a percentage of 95.3%, while the gel with the encapsulated vitamins at the same concentration, a value of 88.51%.At lower concentration (1.25 mg/mL), the same samples show a 98.43% and 95.06% of ROS production, respectively.Finally, the placebo product without any actives, shows nearly 100% ROS production, in particular 97.83% and 94.98%, respectively at 5 and 1.25 mg/mL.
On the basis of the results obtained, the gel with SIREN CAP-SULE TECHNOLOGY™ exerts an antioxidant activity, showing a statistical inhibition of menadione-induced ROS release in human keratinocytes.These results can be related to the capability of lipid nanoparticles to successfully entrap actives.As already reported in literature lipid nanoparticles suitably modified can be able to entrap both lipophilic and hydrophilic ingredients. 41activity, of about +20.17% related to the placebo gel but it shows highest activity also respect to all other gels, +9.54% more than gel with free vitamins and + 7.51% more than gel containing encapsulated vitamins.

| Moisturizing effect of products
The net effect of SIREN CAPSULE TECHNOLOGY™ gel versus the others, as shown in Table 4, permitted to conclude that lipid particles exert a higher skin moisturizing effect after 4 weeks of treatment.

| Skin barrier function efficacy evaluation
The tolerability of the products was achieved by the assessment of skin barrier integrity by the measurement of transepidermal water loss (TEWL).
Variations in TEWL values over time, defined in Figure 4, revealed that all gels, by excluding the placebo one, produced a significant reduction in TEWL value, maintaining it in physiological value, thus all products are well respected and they do not provoke irritation to the skin barrier.
Table 5 reports the variations of the parameter over time and the comparison among the different formulations.
The net effect of SIREN CAPSULE TECHNOLOGY™ gel versus the others, as shown in Table 5, permitted to conclude that lipid particles exert a higher capability to reduce transepidermal water loss after 4 weeks of treatment.These results are strictly related to the hydrophobic character of lipid nanoparticles that can form a film on skin surface retarding the loss of moisture.The occlusion effect of particles depends on the applied concentration and lipid crystallinity. 41There is a relationship between the decrease of TEWL value and the increase of stratum corneum water content after product application highlighting the capability of lipid nanoparticles to restore the skin lipid barrier avoiding the evaporation of the water from the deeper layers of the skin.

| Anti-aging effect
One of the features that afflicts every individual on an aesthetic level, and which is widely of great interest in the field of medicine F I G U R E 2 Percent ROS production and statistical significance of results concerning inhibition of menadioneinduced ROS release in human keratinocytes.p < 0.05 is significant; **, p < 0.01 is strongly significant; ***, p < 0.001 is very strongly significant.

F I G U R E 3
Mean values and statistical analysis of the hydration parameter obtained from all volunteers.*, p < 0.05 is significant; **, p < 0.01 is strongly significant; ***, p < 0.001 is very strongly significant.
and dermatology, is the appearance of the skin with its relative laxity and roughness.Indeed, the appearance of the skin is indicative of its well-being and aging process.
The surface of the skin is characterized by a pattern of lines whose orientation depends on aging process.The polygonal structures of this microrelief become anisotropic with age and this process is strictly correlated with progressive atrophy of dermis matrix.
They become more pronounced when the dermis loses the quantity and quality of elastic fibers. 16erefore, analyzing these inhomogeneities can be helpful in understanding and investigating the state of aging of the skin.
Thanks to image analysis, the study of skin roughness can be resorted to in a quantitative manner, using methods that are scientifically validated and often used in clinical studies. 42 explained above, thanks to Ra and Rz values measured at time 0 and after treatment, we can investigate the functionality of the cosmetic product with an anti-aging claim.
The higher the values of Ra and Rz, the greater the skin roughness caused by texture, lines, and wrinkles.
The results obtained from the evaluation of roughness parameters of the skin are reported in Table 6; Figures 5 and 6.
Results highlighted that all serums are able to statistically reduce

1 .
Heat water to 30-35°C 2. Disperse carbomer under high-speed mixing until obtaining a homogeneous mass without agglomerations 3. Add preservative (commercial mixture of benzyl alcohol and dehydroacetic acid in water) under high-speed mixing until perfect solubilization 4. Neutralize with soda under high-speed mixing until obtaining a homogeneous gel without agglomerate The same gel containing vitamin A and vitamin C was used as Control 1 (gel code 6490).Briefly this gel was prepared as follow: 1. Add Retinyl palmitate to gel 6417 by high-speed mixing until a homogeneous mass is obtained 2. Solubilize in water sodium ascorbyl phosphate and add it to the previously prepared gel under high-speed mixing until a homogeneous mass is obtained 3. Dosing citric acid until the required pH The same gel containing encapsulated vitamin A and vitamin C was used as control 2 (gel code 6491) adding both encapsulated vitamins to gel 6417 under only stirring (No high-speed mixing).The same gel containing the patented SIREN CAPSULE TECH-NOLOGY™ system was prepared (gel code 6418) adding SIREN CAPSULE TECHNOLOGY™ to gel 6417 under only stirring (No highspeed mixing).

1 TA B L E 1
Composition of all batches produced.Ultra-Glo™, recombinant luciferase and D-cysteine.In this way the precursor is transformed into luciferin.this in turn reacts with the enzyme, luciferase, emitting a light signal proportional to the concentration of hydrogen peroxide present.The average Luminescence measured by a reader Promega-Glomax and the standard deviation of each set of cell cultures (controls and samples) are calculated, deducting the signals of the cells treated with the sample only to cells treated with the sample and menadione.Figure 1 shows a scheme of the experiment phases.

percentage of reduction = 100 − luminescence sample luminescence negative control × 100 F I G U R E 1
Scheme of assay detection (made by power point software).
mined by a different dielectric behavior of the skin.The device used in the present study is composed of a 49 mm 2 surface probe that allows precise measurement in 1 s within a depth range of 10-20 μm in the stratum corneum.Parameter was expressed using an arbitrary scoring scale (0-100 AU, Arbitrary Unit) and the treatment was carried out for 28 days for each volunteer by applying products once a day on the forearms.The four products evaluated in this study were: the gel formulation containing SIREN CAPSULE TECHNOLOGY™; the gel containing free vitamins; the gel containing encapsulated vitamins and the gel base used as placebo.

Figures 2
Figures 2 show results concerning the % of menadione-induced ROS release in human keratinocytes using all samples at two different concentrations, 1,25 mg/mL and 5 mg/mL.The results of ROS production are expressed as a percentage related to

Figure 3
Figure3shows the values of hydration of the stratum corneum obtained with the Corneometer.

4 TA B L E 5
both skin texture parameter and wrinkle depth, thus all serums exert anti-age activity.In particular, gel based on SIREN CAPSULE TECH-NOLOGY™ improves the skin texture (−8.19% of Ra after 4 weeks of daily application (T1)) as well as the gel containing encapsulated vitamins (−10.72% in 4 weeks); furthermore, also the anti-wrinkles effect of gel based on SIREN CAPSULE TECHNOLOGY™ (Rz variation of about −10.31% in 4 weeks), is as well as the efficacy obtained by gel containing encapsulated vitamins (Rz variation of about −11.6% in 4 weeks).Previously work demonstrated that solid lipid nanoparticle are able to react with stratum corneum components depending from the age of the subject and the time of contact between the TA B L E 4 Percentage variations of the hydration parameter over time and net effects of all formulations.Mean values and statistical analysis of the TEWL parameter obtained from all volunteers.*, p < 0.05 is significant; **, p < 0.01 is strongly significant; ***, p < 0.001 is very strongly significant.Percentage variations of the TEWL parameter over time and net effects of all formulations.the skin. 43In fact, in middle aged skin, lipid nanoparticles seem distributed in a homogeneous way in the relief and in the furrows, reducing the wrinkle appearance.Thus, in the present work, the efficacy of gel containing lipid nanoparticles is correlated both to the presence of lipidic matrix and the specific activity of encapsulated vitamins.

Figure 7
Figure 7 shows, as an example, 3D overlapped images of same area over time obtained from volunteers treated with one of the three products.The overlapping is a very powerful analysis tool in order to obtain reliable data.In fact the software is able, during the capture of T1 image, to match identical regions between the T0

Table 4
Amount of ROS produced in all samples.noM = cells non treated with menadione; M = cells treated with menadione; 6418 cells treated with formula with SIREN, 6417 cells treated with placebo, 6490 cells treated with gel containing free vitamins and 6491 cells treated with gel containing encapsulated vitamins.Five and 1.25 indicated the two different concentrations of sample tested in this study expressed in mg/ml.