The role of the ImmunatuRNA® complex in promoting skin immunity and its regenerative abilities: Implications for antiaging skincare

Recent advancements in cosmetic science have ushered in a new era of skincare strategies, with a focus on utilizing natural bioactive ingredients to enhance skin health and combat premature aging. The skin, as the largest organ of human body, provides as a vital protective barrier against external hazards such as environmental pollutions, toxins, and radiation. However, intrinsic and extrinsic factors, including various types of radiation, reduced air quality, and increased exposure to pollutants, lead to an imbalance in the skin's immune system, significantly reducing the skin's ability to regenerate and accelerating skin aging. Therefore, there is an emerging need to develop innovative skincare strategies that could support the skin's immune capacity by strengthening antioxidant protection, skin regeneration, and repair. Plant‐derived compounds, along with naturally sourced ingredients, show promise in accelerating wound healing, especially when incorporated into cosmetic formulation. ImmunatuRNA® stands as a prime example of a biologically active complex, uniquely comprising yeast‐derived RNA, marine exopolysaccharides, and natural hyaluronic acid, that exhibits high antioxidant activity and exerts beneficial modulatory effects on skin microbiota, thereby positively influencing skin immunity.


Introduction:
Recent advancements in cosmetic science have ushered in a new era of skincare strategies, with a focus on utilizing natural bioactive ingredients to enhance skin health and combat premature aging.The skin, as the largest organ of human body, provides as a vital protective barrier against external hazards such as environmental pollutions, toxins, and radiation.However, intrinsic and extrinsic factors, including various types of radiation, reduced air quality, and increased exposure to pollutants, lead to an imbalance in the skin's immune system, significantly reducing the skin's ability to regenerate and accelerating skin aging.Therefore, there is an emerging need to develop innovative skincare strategies that could support the skin's immune capacity by strengthening antioxidant protection, skin regeneration, and repair.
Plant-derived compounds, along with naturally sourced ingredients, show promise in accelerating wound healing, especially when incorporated into cosmetic formulation.
ImmunatuRNA® stands as a prime example of a biologically active complex, uniquely comprising yeast-derived RNA, marine exopolysaccharides, and natural hyaluronic acid, that exhibits high antioxidant activity and exerts beneficial modulatory effects on skin microbiota, thereby positively influencing skin immunity.

Methodology:
The main aim of this study was to investigate the potential of the ImmunatuRNA® complex in promoting skin regeneration and reducing signs of skin aging, both through the use of in vitro human skin cultures and the evaluation of clinical trials in healthy volunteers.

Results:
The results of conducted experimental studies have shown that the ImmunatuRNA® complex demonstrated significant positive effects on the immunity and repair capabilities of the skin, characterized by increased fibroblast proliferation, enhanced glycosaminoglycan synthesis, and reduced oxidative stress.Furthermore, use of the complex also significantly accelerated wound healing following mechanically-induced damage in the keratinocytes, demonstrated as reduction

| INTRODUC TI ON
The skin is the largest organ of the human body, which complex structure, composed of the key three layers including epidermis, dermis, and subcutaneous tissue, 1 allows skin to act as protective barrier against the environmental insults, that is necessary in maintaining body homeostasis.Therefore, as the first line of defense, skin must acquire an ability to regenerate and repair itself to protect the body. 2 Prolonged exposure to damaging factors over exceeding skin capability to mitigate their impact, can result in the skin damage, and additionally contribute to accelerated skin aging 3 To date, studies have shown that prolonged exposure to UV light from sun, as well as to air pollutants (e.g., particulate matter, nitrogen dioxide, sulfur dioxide, black carbon, carbon monoxide, and ground level ozone) and other chemicals, for example, tobacco and inappropriately chosen skincare products 4 or topical medications 5 might negatively impact on the skin appearance and functions.Prolonged exposure to environmental assaults, if not removed may significantly impact on the skin integrity, healing capacity, and appearance.
Skin regeneration and repair, as a complex and tightly regulated process, is driven by the interplay between various cell types, including skin-resident immune cells. 6Skin capacity to regenerate following injury, maintained by tightly regulated network of layered epithelial and immunocompetent cells, and extracellular matrix (ECM) enriched with collagen fibers, growth factors, and cytokines, allow skin regeneration and progressive wound healing.This process depending on the wound lesion may vary in time needed to completion and involves stages of hemostasis, inflammation, proliferation, and remodeling, that engage different components of the skin-immune network (Figure 1).
For example, at the initial phase of wound healing (Figure 1A), known as hemostasis, the blood vessels constrict to limit blood losses and enable platelet to aggregate and generate the clot, followed by fibrin matrix formation, acting as a scaffold for cell infiltration. 8Then, in the inflammation phase (Figure 1B), the immune cells, such as neutrophils and macrophages, being attracted by chemoattractants released from necrotic cells at the injury site, are attracted to remove debris and prevent infection. 9Once the site is clean, the proliferation (Figure 1C) occurs, in which macrophage-derived growth factor (MDGF) secreted by phagocytes promotes proliferation of endothelial cells. 10Following that, within 2-10 days after the lesion is confirmed within the dermis, the new tissue can form.A newly formed structure, also known as granulation tissue, being poorly differentiated and highly vascularized connective tissue, consists of cellular components, including fibroblasts, producing the fibrillar component; myofibroblasts, involved in the wound contraction and endothelial cells, that responsible for the neoangiogenesis. 11Finally, in the remodeling phase, starting around 3 weeks after the injury injurious advent and lasting in some cases to even for over a year, all processes activated in previous phases of wound healing are silenced allowing for resolving the immune response and returning to the homeostasis.In addition, the interactions between the epidermis and dermis, together with additional feedback, allow for continuous regulation of skin integrity and contribute to collagen replenishing in the ECM. 12 Glycosaminoglycans (GAGs) a family of long, linear, and negatively charged polysaccharides are a major component of the ECM, which is linked to their role in maintaining the structure, hydration, and elasticity of the skin.GAGs such as hyaluronic acid, dermatan in wound margins measurement, new cell production, and an increase in regeneration speed.In addition, conduced clinical study on healthy human volunteers with various skin types confirmed that use of cosmetic products that incorporate the ImmunatuRNA® complex within the formulation can visibly improve skin condition, appearance, and general health, achieved by increased skin hydration and elasticity, reduced wrinkles, and enhanced skin firmness.

Conclusions:
This study confirms the usefulness of the ImmunatuRNA® complex in the innovative antiaging cosmetic products that can be suitable for all skin types, including sensitive skin.The inclusion of naturally sourced bioactives, as those found in ImmunatuRNA® complex, represents a promising advancement in holistic natural skincare that consumers appreciate.The active ingredients of the complex support the skin's immunity, fostering its repair and protecting against oxidative damage, thus maintaining skin homeostasis and promoting its regenerative capacity.Further research is necessary to explore the long-term effects of ImmunatuRNA® complex on skin health and its potential applications in innovative skincare formulations.

K E Y W O R D S
antiaging, skin regeneration, skincare, wound healing, yeast RNA sulfate, and chondroitin sulfate are synthesized by fibroblasts in the dermis and are responsible for retaining water in the skin, which helps to maintain its plumpness and smoothness; nevertheless, during progressive aging, the production of GAGs decreases, leading to skin dehydration, loss of elasticity, and the formation of wrinkles. 13e efficiency of the skin ability to self-repair and regenerate has been shown to decline with age and that effect was linked to the development of age-related changes in the structure and function of the skin. 14During progressive aging, the number and function of skin-resident immune cells implicated in the skin repair (e.g., Langerhans cells and dendritic cells) gradually decrease, what may lead to impaired wound healing and decreased regenerative capacity. 15For example, studies have shown that the number and function of Langerhans cells decrease with age, leading to impaired immune surveillance and a decreased ability to clear pathogens or initiate an effective immune response of the skin 14 whereas dendritic cells, being essential for the immune activation in the skin, also decline with age, leading to a reduced capacity for antigen presentation and T-cell activation. 15Furthermore, the accumulation of senescent cells in the skin has been associated with increased production of proinflammatory mediators, leading chronic inflammation, and immune dysfunction in the skin, that can further compromise the skin's repair function and ability to regenerate. 14It is noteworthy that recent studies focused on the skin microbiome have demonstrated its ability to modulate skin aging and overall skin health, as skin microbiota dysbiosis has been linked to several skin diseases and age-related changes in skin immunity. 16nsequently, understanding the interplay between skin immunity, microbiome, and aging may aid the development of innovative strategies to enhance skin regeneration and combat age-related skin disorders.One of proposed strategy, which may promote skin healing and regenerative capabilities is appropriately selected skincare. 17utinely used cosmetics with moisturizing, antibacterial, and regenerative properties can significantly accelerate the healing process. 18r example, maintaining appropriate skin hydration is essential for tissue repair as it helps to preserve the skin barrier integrity thus prevents dehydration. 19Dry skin can delay wound healing and increase the risk of infection, 20 therefore regular moisturization with emollients should be encouraged.Second, the use of antibacterial preparations, along with cleansers, can help prevent infection, which is the most common complication of skin damage 21 that can significantly delay the healing process.Since the healing process of the skin may require certain time to form a new protective layer of the epidermis, the deeper skin layers are exposed to external environmental factors, of which air pollutants, oxidation, and UV radiation are the most critical.Therefore, it is extremely important to protect the skin against free radicals that limit the synthesis of collagen in the skin layers, which is the main component of the ECM, playing a key role in tissue repair and regeneration.In addition, the use of sunscreen and cosmetics with antipollutant properties may be necessary to effectively repair the skin. 22Similarly, topical application of hyaluronic acid, a major GAG found in the skin, has been shown to enhance skin immunity, by activating dendritic cells and inducing the expression of antimicrobial peptides, thereby protecting against infection and promoting healing 23 ; but also, through inhibiting the production of pro-inflammatory cytokines and promoting the production of anti-inflammatory cytokines. 24Therefore, GAGs could have therapeutic applications in chronic inflammatory skin conditions (e.g., eczema and psoriasis), and aged skin condition, by improving hydration, elasticity, and overall health and appearance of the skin.
Recent advancements in the cosmetics are leading to development of novel strategies that can support skin regeneration and promote wound healing.Some of the most emerging innovations including stem cells therapy, 3D printed biomimetic materials, as well as formulations based on platelet-rich plasma, 25 and more recently also nutraceuticals. 26A recently published a comprehensive literature review to evaluating the novel active ingredients added to the antipollution skincare products, highlighted the potential bioactives obtained both from plants and microorganisms, because of their ability to favorably modulate skin immunity and promote homeostasis of skin's microbiota.Interestingly, whole plants (e.g., fruits, seeds, leaves, and stalks) are considered as a source of various F I G U R E 1 Schematic outline of the progressive wound healing in the skin.Adopted from. 7ngredients with potent antioxidant and anti-inflammatory properties. 27Among them, saponins, polyphenols, flavonoids, anthraquinones, and polysaccharides seems to be most suitable for skincare products promoting skin regeneration and repair as they have shown to shorten wound healing time attributed to their antioxidant, antiinflammatory, and antimicrobial properties. 26To date, certain topical formulations added to the different skincare products have been shown to have potential in improving skin condition, through supportive effects on its regeneration capability.The most promising examples include moisturizers added with soy derivatives (e.g., soy proteins and phospholipids, essential fatty oils, isoflavones), green tea polyphenols, (e.g., epigallocatechin-3-gallate (EGCG), epicatechin and epicatechin-3-gallate (ECG)) that by enriching deeper layers of the skin, may enhance collagen synthesis within the ECM. 27ing implications of naturally sourced bioactives, use of novel biologically active complexes might holistically support skin regeneration and antiaging activity.A novel biologically active complex, ImmunatuRNA® (PCT/IB2022/000231 (in pending), VRFD SA/ VERDILAB Switzerland) is composed of the naturally obtained ingredients including yeast RNA, marine-derived exopolysaccharides, and hyaluronic acid (HA) with antioxidant activity and modulatory capabilities on skin microbiota experimentally proven in healthy subjects with various skin types. 28Therefore, the main purpose of this study is to experimentally, using human skin cultures, investigate the role of ImmunatuRNA® in promoting skin regeneration and repair as well as its role in antiaging processes tested in a clinical study on healthy subjects with different skin types using cosmetic preparations containing ImmunatuRNA®.It should be noted that the use of RNA by VRFD SA/VERDILAB Switzerland is based on number of studies by Dr. Neymann et al. on the biodynamic properties of yeast RNA as one of the most effective modulators of the immune system, activating all types of immunity and repairing its damage.According to the results of these studies, it was proven that RNA is a metabolic normalizer, preventing pathological disorders of the human microbiome, enhances tissue regeneration, and wound healing, including skin lesions, has antitoxic properties, eliminating the side effects of cytostatic, hormones, antibiotics, etc.In addition, yeast RNA molecules are able to enhance the cooperation of immunocompetent cells and their migration in the body and intercellular interactions by increasing the expression of adhesion molecules such as integrins.Most importantly, yeast RNA has no side effects and has been shown to be highly effective in treating dozens of different human infectious and somatic diseases.
The obtained ImmunatuRNA® complex preparation was in a form of fluid resembling the opaque, odorless, and gel-like with pH ranging between 4 and 7. Prior to the experiments, the INCI formulation of ImmunatuRNA® was determined.
For the series of the tissue culture studies, the various concentrations with ImmunatuRNA® complex were prepared based on their suitability for further incorporation into the cosmetic formulation and applicability in the scalable production of skincare products.These testing solutions were prepared from the stock solution of ImmunatuRNA® complex concentrate, as dilutions ImmunatuRNA® complex in deionized purified water with following concentrations 8.55%, 5.13%, and 0.855% used in the experiments.

| MTT assay
To evaluate cell viability, the MTT assay was performed.Prior to the assay, the human fibroblast cell culture was maintained in the 25 cm 2 flask with prepared cell medium including DMEM medium supplemented with fetal bovine serum 10%.Cells were incubated at 37°C and 5% CO 2 until full confluence.Then, cells were plated into 96-well plate and incubated at 37 and 5% CO 2 for 24 h.Cells in G0 phase were exposed to three concentrations of ImmunatuRNA® complex (8.55, 5.13, and 0.855%) for 24, 48, and 72 h.The testing MTT solution was prepared by dissolving 15 mg of MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent in 30 mL of cell medium to achieve a final concentration of 1 mg/ mL of MTT.Prior to addiction of MTT solution to the cell culture, cells were washed with 200 μL of PBS, and then 200 μL of MTT was added to the cell culture.Then, cell culture was incubated while being gently shaken on a rotatory plate for 30 min at the room temperature, to allow all the crystals to be dissolved from the cells and form a homogeneous solution.
The absorbance of cellular solution with dissolved MTT reagent was measured by using on a UV microplate reader at wavelength 540 nm.The results were determined as percentage of cell viability compared to the cell culture without treatment with ImmunatuRNA® complex.

| In vitro efficacy study
In vitro evaluation of cell proliferation after treatment with ImmunatuRNA® complex, at testing concentrations (e.g., 8.55%, 5.13%, and 0.855%) being dedicated for cosmetic formulations has been conducted by using human skin fibroblast culture.Briefly, the experiment has started from cell plating on the 96-well plate with incomplete culture medium, being without bovine fetal serum, what allowed for induction of phase of cellular quiescence.After 24 h from the plating, the testing concentrations (i.e., 8.55%, 5.13%, and 0.855%) of ImmunatuRNA® complex mixed with complete medium, was added in order to initiate progression of the cells through the cell cycle.The testing conditions were compared with the negative control (CTR-) being a complete medium only.
Culture medium containing tested concentrations of ImmunatuRNA® complex was added to the 96-well plate with human fibroblast cells in the G0 phase of the cell cycle.Cells were exposed to the product for 24, 48, and 72 h.At the end of incubation period, MTT assay was performed to evaluate cell viability and proliferation index compared to untreated control (CTR, 100% viability for definition).
Statistical analysis was conducted by T-test between the experimental samples and the negative control (CTR -).The results were considered significant for p < 0.05.

| Glycosaminoglycan synthesis
The in vitro effect of ImmunatuRNA® complex on the glycosaminoglycan synthesis in the skin was determined by using human fibroblasts cell culture.Briefly, the fibroblast cell culture (ATCC-CRL-2703) maintained in the culture media was exposed to series of testing concentrations of 8.55%, 5.13%, and 0.855% of ImmunatuRNA® complex for 24 h, 48 h, and 72 h.At the end of each time point of incubation period 50 μL of cell culture medium was collected to determine the concentration of glycosaminoglycans (GAGs) produced and released by the fibroblasts.GAGs synthesis was determined by a quantitative assay, a dye-binding method utilizing means of colorimetric assays (Blyscan assay).
Briefly, Blyscan assay involves use a 1,9-dimethylmethylene blue, which acts as specific dye label for the sulphated polysaccharide component of proteoglycans or the protein-free sulphated glycosaminoglycan chains.Obtained GAGs concentrations were presented as mean μg measured in 50 μL of cell culture medium by interpolation on a standard curve obtained with known and increasing glycosaminoglycan concentrations.The measurement was carried out in triplicates.

| Wound healing and regeneration efficacy
To determine the in vitro capability of ImmunatuRNA® complex to promote wound healing the experimental model represented by human skin keratinocytes with artificially induced damage was used according to the methodology described previously. 29iefly, human keratinocyte cell culture was used.The cells were maintained in the 25 cm 2 culture flask with prepared cell medium including Eagle medium (DMEM) supplemented with fetal bovine serum 10%.Cells were incubated at 37°C and 5% CO 2 until full confluence, then an artificial wound was mechanically created by sliding a tip of a pipette on the keratinocytes cell monolayer.After that cells were divided into two groups, the treatment group with added testing solutions at concentrations 8.55%, 5.13%, and 0.855% of ImmunatuRNA® complex; and control groups with added PBS (no treatment).Cells were exposed to each solution for 24 h and then MTT assay was performed in order to assess the percentage of cell viability.The repairing effect of the ImmunatuRNA® complex was determined by acquisition of photographic images of the cell cultures at T0 (baseline) after wound was created in 2 h (T 2 h), 8 h (T 8 h), and 24 h (T 24 h) after the treatment.At each experimental time points, cells were visually assessed by using optical microscope (OPTIKA, XDS-2) with a 10× magnifications and pictures taken by optical camera (TiEsseLab PrimoCam HD 5.0 with sensor Micron MT9P001) were collected.These images were then analyzed with Image J Software.The distances between the wound margins for each image were measured in μm and were recorded in 3 points of the wound length.The variation and significance between wound margins change was statistically determined by using t-test.Experiment was conducted in duplicates.

| Participant recruitment
The main aim of this study was to objectively assess the application properties of in-use skincare products containing ImmunatuRNA® The volunteers were chosen to the study were recruited by the study specialist according to the following criteria.Briefly, female participants aged 35+ with all skin types, including sensitive skin and/or with visible wrinkles.The participants were excluded in they were pregnant or breastfeeding; and/or were on the pharmacological treatment.

| Study intervention
Recruited participants who voluntary agreed to participate in the study were provided with one package of each of the skincare products (Table 1) and were provided with detailed instructions of use for whole duration of the study being in total 28 days (4 weeks) (Table 2).
In addition, the study volunteers selected for instrumental test were obligated to appear for the baseline measurements (before starting the use of the tested skincare product) and following testing days indicated in the plan (28 days).During testing, participants were asked not to use other products of identical or analogous use during the study duration.

| Antiradical testing
The antiradical activity of the cream sample containing ImmunatuRNA® complex in the formulation was determined by the ABTS method.Briefly, ABTS (2,2′-azino-bis(3-ethylbenzothiazo line-6-sulfonic acid)) method is a common assay for measuring the antioxidant capacity of various compounds, including those incorporated into the formulations of skincare products. 30,31Following TA B L E 1 Detailed description and characteristics of the skincare products containing ImmunatuRNA® complex used in the intervention.

Product name Instructions for application Duration of use
LIFT & GLOW ROSE SERUM Apply the serum on the cleansed skin of the face, neck, and décolleté and massage it in gently.
Use in the morning and evening during 28 days.

ANTI-WRINKLE ROSE SUPREME CREAM
Apply the cream after the serum on the skin of the face, neck and décolleté and massage it in gently.The obtained results were presented as g Trolox/ g ImmunatuRNA® complex sample.The antioxidant properties of the testing solution with ImmunatuRNA® complex were also presented as percentage (%) inhibition of the ABTS radical that was used during radical neutralization.This experiment was conducted in standardized environmental conditions, including room temperature of 20-25°C and relative humidity of 40%-60%.

| Assessment of antiaging properties
Participants were asked to assess the perceived effectiveness of the product and its cosmetic properties based on the parameters that they observed or felt over intervention duration.In this study, recruited volunteers were not given any special requirements based on the assumption that the effects of cosmetic preparation and its functional properties should be testes in the natural conditions in which the product will be used in practice.Therefore, the results might be affected by subject-related factors, including type and condition of the skin, genetics, individual preferences, lifestyle, and environmental conditions.
In order to confirm the claimed properties of the skin products

| Skin assessment
The impact of declared effect of the skincare products containing ImmunatuRNA® complex in the formulation on skin parameters reflecting skin condition such as change of skin elasticity and skin firmness levels, level of moisture, changes in the overall appearance including a reduction in the visibility of wrinkles, were assessed at the prior the intervention (baseline) and after 28 days of continuous use of the given cosmetic products.The skin assessment was carried out under the supervision of a qualified person conducted the test as well as a dermatologist on volunteers.
Briefly, the skin parameters were assessed before (baseline) and after (28 days of regular cream use) the application of the tested cosmetic products with ImmunatuRNA® complex in formulation.
The selected tested areas of skin included the face skin area.
Prior testing, the selected face skin areas were cleaned, without the makeup and not treated with the test preparation.Moisture content was performed with a device Corneometer CM825; whereas the measurement of wrinkles depth and length was conducted with Visioscan VC20 plus, and skin elasticity and skin firmness levels were measured with Cutometer dual (MPA580).
The fullface images were obtained from the Visioface RD device.
To confirm the declaration of the product benefits, over 50% of the positive answers from the responders were required to be reported during the study.

| Statistical analysis
The

| ImmunatuRNA® complex preparation
ImmunatuRNA® complex which formulation was described in the detail in patent (PCT/IB2022/000231 (in pending), VRFD SA/ VERDILAB Switzerland) was composed of naturally derived ingredients including sodium yeast RNA, sodium hyaluronate obtained from lactic bacteria by biosynthesis, saccharide isomerate from microplankton, sea water from Atlantic cost of France, and organic apple fruit extract with addition of lecithin, glycerin, tocopherol, aqua, phentylene glycol, sodium hydroxide, and benzyl alcohol.For the purpose of the in vitro studies, water dilutions of ImmunatuRNA® complex concentrate to the following testing concentrations 8.55%, 5.13%, and 0.855% were used.
The assessment of qualitative formulation according to INCI nomenclature confirmed the presence of active ingredients in the ImmunatuRNA® complex concentrate, which indicated that key bioactive components, yeast RNA sodium salt, exopolysaccharides (saccharide isomerate), and medium weight hyaluronic acid (sodium hyaluronate) at concentrations ranging between 5.57% and 9.70%.The formulation of active ingredients was supplemented with apple fruit extract, seawater, and a biomimetic liposome delivery system composed of lecithin derived from organic soybean, to achieve optimal penetration capabilities of active ingredients in the ImmunatuRNA® complex, especially reaching deeper layers of epidermis.

| In vitro efficacy study
The results of in vitro study investigating the efficacy of cell proliferation after treatment with ImmunatuRNA® complex at following concentrations, 8.55%, 5.13%, and 0.855%, in human skin fibroblast culture (Table 3; Figure 2) have shown a significant increase in cell proliferation rate at 24 h (p < 0.05) with all the tested concentrations.Similar effects were observed also at 48 h and 72 h time points (p < 0.05), however, exclusively for treatment with 0.855% concentration of ImmunatuRNA® complex.

| Glycosaminoglycan (GaGs) synthesis
The results of the effect of ImmunatuRNA® complex on GAGs production in human fibroblast in vitro are presented in Table 4.
Treatment with ImmunatuRNA® complex led to increase of the glycosaminoglycan synthesis starting from 24 h-treatment in the human fibroblast culture.This effect was in particular noticeable for the treatment with ImmunatuRNA® complex at concentration 0.855%, which significantly increased GAGs production after 24 h, 48 h, and 72 h when compared to control culture without the treatment.
Similar effect was reported for treatment with ImmunatuRNA® complex at concentration 5.13% and 8.55% accordingly (Figure 3.).

| Wound healing and regeneration efficacy
The evaluation of the ImmunatuRNA® complex capability to promote wound healing and skin regeneration was assessed by using an experimental model represented by human skin keratinocytes based on the methodology according to Lauder et al. 29 The findings obtained from that study were presented in Tables 5 and 6.Briefly, the results have shown that treatment with ImmunatuRNA® complex enhanced human keratinocyte ability to heal and promoted regeneration of artificially induced mechanical damage.This was observed as significantly reduced mean distance between the wound margins (μm) when compared with the baseline and control cell culture not treated with ImmunatuRNA® complex (Figure 4).Among all tested concentrations of ImmunatuRNA® complex, concentrations 0.855% and 5.13% resulted in the statistically significant reduction of the mean distance between the wound margins (μm) in the keratinocyte cell culture compared to the control without the treatment in 8 h and 24 h after treatment (Table 5).
Addition of the ImmunatuRNA® complex to the mechanically injured human keratinocytes led to the statistically significant reduction in the percentage of the distance between wound margins when compared to the wound size at the baseline (T0) subsequently after 8 h and after 24 h time points (Table 5).Among all ImmunatuRNA® complex TA B L E 3 Results from the human fibroblast proliferation study at 24 h, 48 h, and 72 h following treatment with experimental concentrations of ImmunatuRNA® complex.

TA B L E 5
The mean distance (SD ±) between the wound margins (μm), recorded at the different experimental times.TA B L E 6 Regeneration activity of ImmunatuRNA® complex represented as percentage reduction of the distance between the wound margins in cell culture treated with ImmunatuRNA® complex and control without treatment compared to the baseline (T0).concentrations, use of 0.855% treatment was the most effective in the reducing wound size compared to the baseline size of the artificial wound after 2, 8, and 24 h (Figure 4).Assessment of the repairing activity, calculated as ability of the ImmunatuRNA® complex to reduce the distance (%) between the wound margins in a model of injured human skin keratinocytes, indicated that treatment with all tested concentrations of the complex, 0.855%, 5.13%, and 8.55%, led to significantly reduced that space at two experimental time points, T8 and T24, when compared to T0 and the untreated control (Table 6.).

| Antiradical testing ex vivo
Cosmetic products involved in the study intervention were independently analyzed for their antiradical activity.Each response was carefully reviewed by qualified researcher, and findings were separately presented for each skincare product.
The details are outlined in the Table 8.

| Clinical study: an in vivo skin assessment
Skin assessment was conducted in the 10 randomly selected individuals out of the total group of 29 human volunteers completing the intervention with the skincare products.
The results from assessment of skin moisture level after 28 days of using skincare products containing ImmunatuRNA® complex, such a Moisture is presented in the units of measurement.
Note: A change in the moisture content of 5% was considered an error of measurement.W, woman; P0, initial measurement before starting the intervention with skincare products (baseline); P1, measurement after 28 days of using the skincare products in the end of intervention.

TA B L E 1 0
The wrinkles measurement obtained from the Visioscan (VC20plus).

Subject Age Sex
SEw parameter a P0 P1 a SEw parameter is presented in the units of measurement.

Note:
The wrinkles measure was presented as the value of the SEw parameter.A decreasing value of SEw parameter indicated an improvement (i.e., skin smoothing of wrinkles).A change of 5% was considered an error of measurement.W, woman; P0, initial measurement before starting the intervention with skincare products (baseline); P1, measurement after 28 days of using the skincare products in the end of intervention.
value of 19% on average when compared to baseline measures (P0 average 96.00 vs. P1 average 77.33) with the best result of −33%.
Results obtained from the assessment of the skin elasticity parameter following application of the skincare products containing Furthermore, before and after 28 day long intervention with tested skincare products, the skin appearance was compared by using a photo images taken with Visioface®RD (Figure 5).Briefly, the presented pictures have shown positive visible changes in the appearance of the face skin surface following intervention.In particularly, participants who were taking part in the intervention after 28 days of product use, displayed a reduction in wrinkles, the nasolabial fold, followed by improved skin tone demonstrated as reduced redness and visibility of broken blood vessels.Interestingly, favorable effect on the skin tone by reduced of skin face discoloration visualized by the polarized UV light.

| DISCUSS ION
The scientific evidence to date supports the use of naturally sourced bioactive substances in holistic skincare products, helping to maintain skin homeostasis and promoting its regenerative capacity, thus protecting against damage.Therefore, the main objective of this study was to experimentally evaluate the potential of novel biologically active complex ImmunatuRNA®, composed of naturally derived ingredients including sodium RNA obtain from yeast, sodium hyaluronate obtained from lactic bacteria, saccharide isomerate obtained from microplankton to promote the immunological, regenerative, and antiaging capabilities of the skin in both human skin cultures and healthy adult volunteers.
The overall results of this study confirmed the ability of the a Skin elasticity is presented as dimensionless value.
Note: An increasing value of skin elasticity indicated an improvement.A change of 5% was considered an error of measurement.W, woman; P0, initial measurement before starting the intervention with skincare products (baseline); P1, measurement after 28 days of using the skincare products in the end of intervention.
TA B L E 1 2 Skin face firmness measurement obtained from the Cutometer dual (MPA580).Previous studies, similarly, to the results of this study, supported use of skincare preparations containing natural bioactives with antioxidant properties, such as vitamin C, vitamin E, and ferulic acid, as well as those from plant extracts, including green tea, grape seeds, rosemary, and ginseng, which can effectively protect the skin against oxidative damage caused by environmental factors and improve its hydration, reduce the appearance of fine lines, increase skin elasticity. 33The ImmunatuRNA® complex, which is a combination of natural bioactives when added to cosmetic preparations as an active ingredient, has shown the potential to strengthen skin immunity, promoting its regenerative abilities resulting from increased fibroblast proliferation and GAGs production in in vitro studies.In our previous study, 28 we have demonstrated that this novel biologically active complex, ImmunatuRNA® (PCT/ IB2022/000231 (in pending), VRFD SA/VERDILAB Switzerland) has proven antioxidant properties and improves skin microbiota in healthy individuals with various skin type. 28ile the exact mechanism of action of the ImmunatuRNA® complex role in skin repair remains to be further explored, reported observations indicate that the ImmunatuRNA® complex has potential to promote tissue regeneration.These beneficial effects can be explained by the combination of bioactive ingredients that have previously shown beneficial effects on the physiology of the skin.For example, the sodium salt of yeast-derived RNA, present in formulation of the ImmunatuRNA® complex, probably plays a major role in facilitating wound healing.Experimental studies in rodents showed that treatment with RNA salts increased GAG synthesis by increasing ECM gene expression in vivo and significantly improved skin barrier function and hydration levels. 34These observations are in line with findings from human fibroblast cultures where RNA salts upregulated expression of genes involved in GAG synthesis and transport and increased cellular hyaluronic acid content, 13 explained by the ability of RNA salts to facilitate transition from inflammatory to proliferative wound healing as well as modulating cytokine production and controlling keratinocyte proliferation. 35Similarly, sodium hyaluronate, another component of ImmunatuRNA® complex, as well as essential component of the ECM of the skin, have been proven to exhibit stimulatory effects on GAGs synthesis of GAGs in various cell types, including human fibroblasts viability following the exposure to pro-oxidative agent, when compared to control after 72 h in the dose-dependent manner. 28In addition, a beneficial effect was also observed in fibroblast culture in the context of wound healing, indicating that treatment with sodium hyaluronate significantly increased the GAG synthesis in human fibroblasts in vitro up to 96 h after treatment.Moreover, the addition of sodium hyaluronate also increased the expression of genes involved in ECM synthesis and skin tissue remodeling, further supporting tissue repair and regeneration 36 -thanks to its ability to activate molecular pathways involved in GAG synthesis 37 and interaction with cell surface receptors such as CD44, regulating cellular responses responsible for tissue repair and regeneration. 38GAGs as important components of the ECM in skin, play a critical role in skin regeneration and repair.During the healing process, GAGs are synthesized and deposited in the newly formed ECM where they interact with growth factors and cytokines to regulate newly established cellular processes such as cell proliferation, migration, and differentiation; but also, by interacting with various components of the ECM, such as fibronectin, they form a scaffold for collagen deposition, cell adhesion, and migration during regeneration. 39Hyaluronic acid, the main component of GAGs in the ECM, is a large polysaccharide with hydrophilic properties, which allows it to absorb large amounts of water and maintain tissue hydration, thus increasing cell migration and proliferation by activating signaling pathways involved in wound healing. 40Consequently, the increased GAGs synthesis observed in recent study might play a critical role in promoting skin healing, perhaps promoting cell migration, proliferation, and differentiation, as well as modulating the inflammatory response, which may be particularly important in improving the condition of mature or damaged skin, in which age-related changes may limit its regenerative abilities.Furthermore, ImmunatuRNA® complex contains exopolysaccharides of marine origin, 41 which are a rich source of N-acetyl-glucosamine of planktonic origin, which was credited with a beneficial effects on the condition of the skin, such as maintaining skin cohesiveness and smoothness, increasing its hydration, and enhancing skin-immune capabilities as well as all important healing process. 7teworthy, formulation of ImmunatuRNA® complex has been enriched with various phytochemicals derived from organic soybeans, and apple fruit extract, which are a rich source of flavonoids that can sooth inflammation and reduce oxidative stress in inflamed tissues. 42is effect was confirmed in previous experiments, the results of which showed that treatment with the ImmunatuRNA® complex at concentrations of 0.855%, 5.13%, and 8.55% can reduce synthetic radicals production in a dose-dependent manner in a free radicals scavenging assay using DPPH, as well as protects human fibroblasts from exposure to oxidative stress in vitro, which is observed as a significantly increased viability of human fibroblasts after exposure to oxidative stress, and subsequently increases the expression of SIRT-1 protein, 28 that serves as an important modulator of DNA repair, gene expression, and cellular apoptosis. 43The confirmed in vitro ability of the ImmunatuRNA® complex can be especially important for explaining the beneficial effects observed in clinical experiment in this study, as significant reduction in oxidative damage upon free radical exposure, 26 may provide explanation of noticeable improvement in the skin condition such as reduced redness due to potential inflammation, 44 along with increased smoothness and hydration, observed in the volunteers following use of the cosmetics with ImmunatuRNA® complex.In addition, components of with ImmunatuRNA® complex, by influencing on SIRT-1 expression and regulating vital physiological functions of the skin, can protect the skin against further oxidative stress and cell death, thereby preventing against aging and effects of exposure to air pollutants. 45rthermore, ImmunatuRNA® complex may promote skin healing and reduce risk of inflammation by modulating the skin microbiota in vivo, which has been shown to reduce the number of potentially pathogenic microorganisms associated with wound infections, [46][47][48] such as gram-negative bacilli, Citrobacter koseri, and Enterococcus spp.(fecal streptococci); and aerobic gram-positive rod-shaped bacteria including genus Bacillus, Alcaligenes, and Pantoea. 28is study has some limitations that should be considered when interpreting the results.Undoubtedly, individual factors, such as skin type and condition, genetic makeup, lifestyle habits, and environmental influences may affect the results of human study.
Therefore, further research on wider group are planned, which will allow to investigate the mechanisms of the effect of treatment with ImmunatuRNA® complex on the skin physiology in vivo.

| CON CLUS IONS
The results of this study demonstrated both in vitro and in vivo evi-

R E FE R E N C E S
complex, such as LIFT & GLOW ROSE SERUM, ANTI-WRINKLE ROSE SUPREME CREAM, and EXTREMELY NOURISHING ROSE RADIANCE CREAM MASK; and verify the declared benefits for skin condition in a group of 29 healthy human adult female volunteers.Participant recruitment and intervention methodology with the skincare products were conducted in accordance with the Declaration of Helsinki 1964, Cosmetics Europe directives including regulation of the European Parliament and Council Regulation (EC) No 1223/2009 of 30 November 2009 and No 655/2013 of 10 July 2013 on cosmetics.Furthermore, the recommendations provided by Cosmetics Europe-The Personal Care Association Guidelines, including Product test guidelines for the Assessment of Human Skin Compatibility 1997 and Guidelines for the Evaluation of the Efficacy of Cosmetic Products 2008 were also applied.

Use a week 7
times in the morning and 3 times in the evening during 28 days EXTREMELY NOURISHING ROSE RADIANCE CREAM MASK Apply generous layer on the cleansed skin of the face, neck, and décolleté leave for 20 min or overnight use.Clean the excess amount with the face cloth.Use 4 times per week, instead of cream during 28 days.assay, by involving the oxidation of ABTS reagent by a radical initiator (e.g., potassium persulfate) to generate a blue/green chromophore compound, which then can be neutralized by the tested bioactive compound with expected antiradical properties, leading to a decrease in the absorbance measured spectrophotometrically.In this study, the antiradical activity of ImmunatuRNA® complex was measured in the methanol solution and the methanolic solution of radical with a final absorbance of ~0.7.Changes in absorbance of the colored solution were monitored for 60 min at wavelength of 744 nm using UV-VIS spectrophotometer.The obtained results from ImmunatuRNA® complex sample were compared to the standardized sample by using Trolox (an artificial equivalent of vitamin E).
results obtained from cell culture studies are presented as mean and standard deviation (SD) of measured concentration.The percentage of change in the mean value was calculated by comparing post-treatment sample with control.The statistical significance of the results was determined through the student t-test.All the comparisons between the experimental samples with the negative and positive control were considered statistically significant for p value of <0.05.
Firstly, assessment of antiradical properties of ANTI-WRINKLE ROSE SUPREME CREAM, indicated that 1 g of its formulation corresponds to the antiradical activity of standardized antioxidant compound, Trolox (Vitamin E) of about the 1163.82g mass.Consequently, the results of ABTS radical assay have confirmed high antiradical activity of ANTI-WRINKLE ROSE SUPREME CREAM as its prepared solution was able to neutralize it in 82.12% of free radical species, analogously prepared solutions of butylhydroxytoluene (E321 BHT) and Trolox.Second testing conducted on the samples of LIFT & GLOW ROSE SERUM, which 1 g had antiradical activity equivalent to the antiradical activity of 1328.10 g Trolox; after 60 min of reaction with the ABTS radical, the LIFT & GLOW ROSE SERUM solution was able to neutralize 93.28% of the created radicals, thus confirming a very high value of the antiradical activity of the preparation, compared to the activity of E321 BHT and Trolox.Last assay, conducted on the samples of EXTREMELY NOURISHING ROSE RADIANCE CREAM-MASK, have shown that 1 g of that cosmetic corresponded to the antiradical activity of 913.28 g of Trolox.Following 60 min of reaction with the ABTS radical, the preparation made of CREAM-MASK solution neutralized 65.1% of created radicals, also indicating the high value of the antiradical activity when, compared to the activity of the analogously prepared solutions of E321 BHT and Trolox.Nevertheless, among all tested skincare products, ANTI-WRINKLE ROSE SUPREME CREAM and LIFT & GLOW ROSE SERUM demonstrated the highest antioxidative activity value.

3. 6 |F I G U R E 4 3 . 6 . 2 |
Clinical study 3.6.1 | Participant characteristics Participant characteristics are presented in Table 7.In this study, 29 females were involved in the study, with average age of 47.5 years (range from 35 to 70 years old), having three different skin types, including combination (18 subjects; 60%), normal (2 subjects; 7%), Graphical representation of the observed progressive reduction of the distance between artificially induced wound size over 24 h time point.and dry (10 subjects; 33%).In this group, 24 females (80%) claimed to have a sensitive skin phenotype.Results of participant questionnaire assessing tested antiaging product claims After the intervention with skincare products, that included 28 days of regular use of LIFT & GLOW ROSE SERUM, ANTI-WRINKLE ROSE SUPREME CREAM, and EXTREMELY NOURISHING ROSE RADIANCE CREAM MASK, 29 study participants filled a designed questionnaire to assess the perceived antiaging products claims.
ImmunatuRNA® complex, such as LIFT & GLOW ROSE SERUM, ANTI-WRINKLE ROSE SUPREME CREAM, and EXTREMELY NOURISHING ROSE RADIANCE CREAM MASK (Table 11.) during 28 days of use indicated a change in the average skin elasticity from the baseline, translating to 10% improvement (P0 average 0.605 vs. P1 average 0.666) with the best result +23% on improvement on skin elasticity.The final assessment, including the evaluation of the face skin firmness in just 28 days of using skincare products containing ImmunatuRNA® complex, such as LIFT & GLOW ROSE SERUM, ANTI-WRINKLE ROSE SUPREME CREAM, and EXTREMELY NOURISHING ROSE RADIANCE CREAM MASK (Table 12) showed for overall improvement in the skin firmness, demonstrated by a change in the skin firmness by +16% change from the baseline, indicating for an average decrease upon use the skincare regime (P0 average 0.241 mm vs. P1 average 0.203 mm) with the best result of 64% improvement.
keratinocytes consistently demonstrated a beneficial effect of the ImmunatuRNA® complex at concentrations of 0.855% and 5.13% on wound healing at the 8 h and 24 h time points, noticeable as a reduction in the wound margins compared to control.Finally, results obtained from experiments with human skin tissue cultures have been confirmed in vivo, thus proving that the regular use of skincare products, including face serum, cream, and cream-mask containing ImmunatuRNA® complex, regularly for 4 weeks, can increase hydration, firmness, and elasticity of the skin, while reducing visibility of wrinkles, which contributes to improving the appearance and condition of the skin.Further studies of regenerative properties of ImmunatuRNA® complex added to formulas of skincare products such as face cream, serum, and face cream-mask demonstrated in vitro antiradical properties of these products, equal to standardized derivative of vitamin E, 32 thus suggesting that synergistic action of the components of ImmunatuRNA® complex can effectively neutralize free radicals, protecting skin from oxidative stress, one of the main causes of premature skin aging associated with skin damage.32Furthermore, the antiaging properties of skincare products with ImmunatuRNA® complex were noticed by participants of the clinical trial where healthy adult volunteers with various skin types routinelyF I G U R E 5The images taken with Visioface®RD to demonstrate visible effect following intervention with skincare products.used a face serum, cream, and cream-mask with ImmunatuRNA® complex for a period of 4 weeks, reported a subjective improvement in the skin conditions, including increased skin hydration and refreshing, increased elasticity, youthful appearance of the skin with the additional effect of smoothing wrinkles.The participants' opinion was consistent with the results of the clinical skin examination, which demonstrated a significant improvement in the skin condition, including increased skin hydration (+15%), smoothing of wrinkles (decrease in SEw parameter by 19%), greater skin elasticity (+10%), and firmness (+16%).In addition, these qualitative benefits were reflected in an improvement of the overall appearance of the skin as an improvement in skin tone, reduction in redness, and the visibility of broken blood vessels when visualized in Visioface® and with polarized UV light.
dence of the ability of the ImmunatuRNA® complex to visibly improve the overall condition of the skin; thus confirming the usefulness of the ImmunatuRNA® complex in cosmetic products with antiaging properties dedicated to all skin types.A series of in vitro studies have shown that the ImmunatuRNA® complex can effectively accelerate wound healing and skin repair, increase fibroblast proliferation and glycosaminoglycan synthesis, which are important contributors to the regenerative capacity of the skin, also involved in maintaining skin health.Noteworthy, glycosaminoglycans as a vital component of the ECM may additionally contribute to hydration level and moisture retention in the skin structures, which is manifested by the youthfullooking complexion.The ImmunatuRNA® complex used in cosmetic products also has potent antioxidant properties, protecting against free radicals, which largely contribute to premature aging of the skin and the formation of wrinkles.Finally, further clinical study in healthy volunteers with different skin types indicated that the use of skincare products with the addition of the ImmunatuRNA® complex in the formulation can visibly improve the appearance and condition of the skin, noticeable as an increase in skin hydration, reduction of wrinkles, increased elasticity and firmness of the skin.In conclusion, the results of this study indicate that incorporation of naturally sourced bioactives, such as those found in the ImmunatuRNA® complex formulation can offer promising advancement for skincare with antiaging properties.The findings of this study demonstrate that active ingredients from ImmunatuRNA® complex can promote skin repair by mechanisms involved in the skin immunity and protection against oxidative damage.Further studies are needed to explore the potential use of the ImmunatuRNA® complex in the development of new, innovative skincare products.ACK N OWLED G M ENTSThe contributions of Dr Andrea Poggi and Dr Silvana Giardina (Comp life Italia) are extremely acknowledged.FU N D I N G I N FO R M ATI O NThe research was founded and covered by VRFD SA, via Serafino Balestra 12, 6900 Lugano, Switzerland.CO N FLI C T O F I NTER E S T S TATEM ENTDr.Joanna Michalina Jurek was a main person involved in the manuscript preparation and submission to the Journal.This work was carried out as own initiative independent of the employment and conducted in the spare time based on the research collaborative agreement with VRFD SA/VERDILAB Switzerland.Victoria Neymann, MD is a founder of VERDILAB Switzerland/VRFD SA, as well as the main owner of the intellectual property and chief lead on conducted research including conceptualization, methodology, formal analysis, investigation, and data curation.The research activities described in the manuscript was conducted in collaboration with Complife Group and Dr. Koziej Laboratory.The authors bear primary responsibility for the accuracy of made statements and for the content and writing of the paper.DATA AVA I L A B I L I T Y S TAT E M E N T Data sharing not applicable to this article as no datasets were generated or analysed during the current study.E TH I C S S TATEM ENT Participant recruitment and testing procedures with use of the skincare products were conducted in accordance with the Declaration of Helsinki 1964 Cosmetics Europe directives including regulation of the European Parliament and Council Regulation (EC) No 1223/2009 of November 30, 2009, and No 655/2013 of July 10, 2013, on cosmetics.In addition, the recommendations provided by Cosmetics Europe-The Personal Care Association Guidelines, including product test guidelines for the Assessment of Human Skin Compatibility 1997 and Guidelines for the Evaluation of the Efficacy of Cosmetic Products 2008, were also applied.
Outlined characteristics of participants involved in the study.Summary of the questionnaire results assessing the claims behind use of skincare products.Note: Noteworthy, none of 29 females participated in the study, did not report any unexpected effects from using all tested skincare products.Moisture assessment obtained from the Corneometer (CM825) measurement.
TA B L E 7 MASK; (Table 9.) indicated an average + 15% increase in the moisture level, being equal to 8.31 units increase of moisture content when compared to average baseline (P0 average 55.70 vs P1 average 64.01) with best results of +28% increase.Findings of the assessment of wrinkles on the face skin following use of the skincare products containing ImmunatuRNA® complex, such as LIFT & GLOW ROSE SERUM, ANTI-WRINKLE ROSE SUPREME CREAM, and EXTREMELY NOURISHING ROSE RADIANCE CREAM MASK; (Table 10) have shown that regular use of these skincare products during 28 days resulted in decrease of Skin microrelief-SEw parameter (number and width of wrinkles) in TA B L E 8 TA B L E 9 The face skin elasticity measurement obtained from the Cutometer dual (MPA580).
Skin firmness is presented in mm. a