1. Introduction
As the body’s first line defence against environmental damage, the skin is constantly exposed to multiple stressors which negatively impact its health and beauty. It is now widely accepted that solar damage accounts for approximately 80% of facial aging [1] , and increasingly urban air pollution is implicated as another major threat to human skin health worldwide.
Distinct from intrinsic aging primarily caused by genetic factors and hormonal status changes over time, extrinsic aging is essentially photoaging caused by the combined effects. The combined effects of sunlight (including ultraviolet (UV), visible light (VL) and near-infrared (NIR) radiation) are universally acknowledged by dermatologists [2] [3] , although our understanding of the biochemical mechanisms involved remains incomplete.
Despite this knowledge, however, formulations and regimens designed to protect the skin from solar and environmental damage by day and repair cellular injury at night are not well defined or researched.
The authors previously reported that most globally available sunscreen formulations, whether they are SPF 50+, PA+++ or ++++ are incapable of providing complete skin protection from the entire solar spectrum, which includes not only UV but also VL and NIR radiation [4] - [13] . Most sunscreens only offer protection in the UV range, when in reality skin damage also results from VL and NIR exposure [4] - [13] . Increasingly, dermatologists are recommending comprehensive skin regime that protects from UV, VL and NIR.
We also reported that a comprehensive solar protection and repair regime (including skin immune boosters, antioxidants, zinc oxide sunscreens, barrier lipids, hydroxy acids and retinoids) elicits significant improvements skin texture, appearance, clarity, and firmness as demonstrated in multidimensional 2D and 3D assessments [4] - [13] .
To investigate the efficacy of a solar repair Formulation following UV and environmental exposure in order to improve overall skin health and appearance, we assessed in vitro gene expression changes and in-vivo efficacy in improving overall skin appearance through reducing signs of inflammation, elevating hydration, reinforcing skin firmness and amplifying radiance.
2. Materials and Methods
2.1. Topical Formulation
The Formulation is a complex composition of functional ingredients forming the delivery system as well as performant active ingredients such as Vitamin A in two different forms, 15 pure amino acids and one modified amino acid and more (Table 1). This complex blend, #6NIC1.V1.1-1, is the studied Test Material (TM).
Test Materials
Four TMs were used to complete the studies described below. The Untreated Control consists of tissue samples not treated with any substances. This TM was used to assess cytotoxicity. Triton X-100, a nonionic surfactant with cell lysing properties that disrupt cell walls leading to cellular death was used as a positive control for cytotoxicity assessment. 100% cell death is expected when cells are exposed to Triton X-100. In order to assess the gene expression changes in the 3-dimensional reconstucuted skin tissue model as well as to confirm the cytotoxicity rate of the Formulation, a 0.9% Saline solution was used as a negative control. This solution is considered neutral and inert to the skin cells. Therefore it should induce very low to zero cytotoxicity and changes in gene expression. The results obtained with this solution serve as a comparison with the tested Formulation results. Finally, #6NIC1.V1.1-1 also referred to as the Full Formulation is described in Table 1. This is the main material to be assessed in order to determine how much change will be induced after using the Full Formulation in the cytotoxicity, gene expression changes and In-Vivo studies. Table 2 expands further on the Test Materials design.
2.2. Skin Model and Gene Expression
The gene expression and cytotoxicity studies were performed on a commercially available 3-dimensional in vitro skin model (Mattek EFT-400) composed of epidermal keratinocytes and dermal fibroblasts. Tissues were equilibrated prior to inoculation with 15 μL of any of the above test materials (TM). For each treatment
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Table 1. List of ingredients functions composing the formulation.
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Table 2. Test groups and result expectations.
group, four tissues were included. Post distribution of treatment materials, the tissues were placed in an incubator at 37˚C with 5% CO2 and ~95% relative humidity for 24 hours. At the issue of the 24 hour incubation, each tissue’s surface was washed to remove TM. Depending on the treatment group, tissues were exposed to UVB at 200 mJ/cm. Following full exposure to UVB, 15 uL of TM was re-applied to the tissues which were then re-incubated at 37˚C with 5% CO2 and ~95% relative humidity for 24 hours. Gene expression was assessed after a total of 48 hours exposure to the TM utilising Genemarkers’ qPCR-based Environmental Stress gene Panel (ESP) containing 107 target genes (Table A1). After full incubation, tissue surfaces were washed, and each culture was placed in contact of RNA later solution ready for RNA isolation (Figure 1).
Following a qPCR process, statistical data analysis was performed using relative quantitation (RQ) method and converting any linear RQ values into linear fold-change values.
2.3. LDH Cytotoxicity Assessment
Controls:
- High Control: 100 μL of 15 Triton X-100 was inoculated on the surface of two tissues as per process above.
- Low Control: Four untreated (UNT) tissues
To assess the LDH activity (cytotoxicity) of the TM, the culture medium of each treated tissue was used and diluted 1:10 with Phosphate Buffered Saline (PBS). Every dilution was then combined with the LDH reaction mix at a ratio 1:1 and followed by a 20 minute incubation at room temperature in the dark. A 1.0 N HCl solution was used to stop the reaction of each dilution and absorbance was measured at 492 nm with a reference filter at 620 nm.
The LDH activity or cytotoxicity was calculated relative to the absorbance of the low control (0% cytotoxicity) and high control (100% cytotoxicity) following the below formula:
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Figure 1. Gene expression process from inoculated tissues to raw data generation.
% Cytotoxicity = [(Test Media Value − Low Control)/(High Control − Low Control)] * 100
2.4. In-Vivo Clinical Evaluation
Patients
Patients were selected based on inclusion and exclusion criteria defined as Tables 3-5. Patients were directed not to use any other skincare products or undergo any form of esthetic procedure during the study. Additionally, they were instructed to continue with their normal diet throughout the study. As this study involved a retrospective review of patients previously treated, the approval of an ethics committee was not required. All patients signed an informed consent document of participation following an explanation of the study design and execution, and agreed to publication of results and images.
Kinetics
Parameters tested and the time points are described below.
3. Results
LDH Cytotoxicity Assessment
The cytotoxicity and gene expression studies were performed on the below Test Groups incorporating some of the test materials referenced in Material and Methods:
Increased LDH activity is interpreted as an indication of cells being damaged or dead. The positive control, Triton X-100 is the reference for full cytotoxic LDH activity. Levels of cytotoxicity were assessed after 24 and 48 hours and were compared to the untreated, negative control.
After 24 hours, the cytotoxicity levels of the test groups were minimal (Figure 2). After 48 hours, the Full Formulation exposed to UV showed slightly higher cytotoxicity although still very low and lower than the tissues inoculated with 9% Saline Solution and exposed to UV radiation.
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Table 3. Patients inclusion criteria for selection.
Each patient is his/her own control.
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Table 4. Formulation application instruction.
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Table 5. Kinetics known as In-Vivo tests parameters and check points.
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Figure 2. Relative % LDH activity (cytotoxicity) as 24 and 48 hours. Note. The * (p ≤ 0.05) symbol and. ** (p ≤ 0.001) designates statistical significance after performing unpaired t-test.
Measured Changes in Gene Expression Following Treatment with Formulation (#6 NC1. V1.1)
Of the 107 genes tested, 60 - 80 presented significant changes in genetic expression with and without UV exposure. Each gene change is reported to influence structural and functional activity within skin cells (anti-inflammation, DNA repair, survival, apoptosis, antioxidation etc). A selection of 5 genetic markers was curated and reported above as their functions and mechanism of action are the most relevant to the study (Table 6).
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Table 6. Gene expression linear fold change before and after UV exposure.
In-Vivo Clinical Evaluation
Each participant’s skin was assessed at Day 0, 28 and 56 under dermatological supervision, thus representing its own control.
Each subject was provided with the full Formulation to apply every night, a self-assessment questionnaire and a daily log to report any unusual or adverse occurrences.
The population recruited is detailed in Table 7.
Cutaneous Compatibility
This assessment was performed by a dermatologist. None of the patients experienced nor reported any clinical, functional nor physical signs of skin irritation during the trial period (Table 8). The full results obtained are described in Table A2.
Cutaneous Microcirculation, Soothing Effect
Cutaneous microcirculation, assessed via Tivi700 revealed a reduction of redness (Figure 3), which may be regarded as a visible symptom of inflammation (Figure 4) [24] . A 9% mean reduction in redness across 70% of patients was recorded after 28 days of usage and a further 7.3% across 60% of patients after 56 days (Table A3).
Cutaneous Hydration
Cutaneous hydration was measured with a Corneometer [25] , and results were compared before and after using the Formulation (Figure 5). 100% of the patients experienced an increase in hydration after applying the Formulation nightly for 28 days and continuing using it for 56 days. An average 39% elevation in epidermal moisture levels was recorded after 56 days of continual use (Table A4).
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Table 7. In-Vivo test demographics (total number of patients n = 22).
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Table 8. Clinical and reported signs of non-acceptability.
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Figure 3. Variations of cutaneous microcirculation measured with TIVI700 after using the Formulation for 28 and 56 days compared to the initial state. A significant decrease of cutaneous microcirculation characterizes a soothing effect of the Formulation.
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Figure 4. An amelioration of inflammatory products is supported by a reduction in porphyrin count (yellow dots) after 28 days of using the solar repair Formulation. Further investigation is required to determine the effect of this change on the skin microbiome.
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Figure 5. Variations of cutaneous hydration rate measured with a corneometer (in arbitrary units) after using the Formulation for up to 56 days compared to the initial rate. A significant increase of hydration rate is a sign of moisturizing effect, no change of the hydration rate shows a non-drying effect.
Clinical Grading of overall radiance through measurements of evenness of skin tone, texture and brightness
Upon Dermatologist assessment, each patient’s skin was graded on three different parameters to determine the radiance-enhancing effect of the Formulation (Figure 6 and Figure 7).
- Evenness of the skin tone
- Brightness and complexion radiance
- Skin texture refinement
Overall, all of the patients experienced an average of 20% increase in radiance (Table A5).
Biomechanical properties: firmness, tonicity, and suppleness
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Figure 6. Variations in clinical grading of evenness of skin tone, brightness, skin texture and overall radiance by a dermatologist after using the Formulation for 28 days, continuing on to 56 days compared to initial assessments.
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Figure 7. Visible improvements in skin tone, texture and radiance after 28 days of using the solar repair Formulation nightly
Using a Cutometer, the firmness of each patient’s skin was measured (see Table 9) [26] . After 28 days and after 56 days, 95% of the patients experienced, respectively, an average 13% and 21% improvement in skin firmness. Results for tonicity and suppleness improvement were not significant after 28 or 56 days as this was deemed to be too short a period to affect changes in skin connective tissue integrity (Table A6).
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Table 9. Variations of the skin biomechanical properties using a cutometer after using the Formulation for 28 days compared to initial assessments. Statistical significance was examined by T-test.
*Not clinically relevant.
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Figure 8. Patients self-assessment after using the formulation for 28 days.
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Figure 9. Patients self-assessment after using the formulation for 28 days.
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Figure 10. Patients self-assessment after using the formulation for 56 days.
Self-Assessment Questionnaire
Each participant was requested to fill a questionnaire about perceived efficacy of the Formulation based on various criteria. Affirmations were proposed and patients were asked to rate their degree of agreement with the statement on a four-point scale from agree to disagree (Figure 8, Figure 9 and Figure 10). Where patients agreed or strongly agreed, the results were considered positive. Only 21 patients were considered for this assessment.
Overall, the vast majority of patients observed and reported a global improvement in the appearance and feel of their skin.
4. Discussion
Dermatologist assessment protocols on the safety of the Formulation for even the most sensitive skin revealed that all of subjects were able to use the Formulation nightly without skin irritation. On the contrary, in response to TIVI700 microcirculation testing, 70% of patients reported a reduction in redness (mean 9%) and sensitivity. Subjects reported that their skin felt firmer, stronger and more resilient. All of the patients experienced an increase of skin hydration measured with the corneometer and could feel a positive change in skin moisturisation and firmness after 28 days of use. Improvements in skin radiance were reported by all of patients, with enhancement of skin tone evenness, brightness and texture also observed in majority of patients after 28 and 56 days. This could potentially be explained by the improved hydration and a complex of active ingredients purposefully curated for this Formulation.
Subjective self-assessment revealed that skin firmness, plumpness, appearance of wrinkles and fine lines were also significantly improved as measured via the cutometer and self-assessment. These results could be attributed to the elevated complex of Vitamin A and peptides incorporated into the Formulation to enhance the skin tone and texture via reparative retinoid and cellular signalling pathways.
With an overwhelmingly positive in-vivo response to the Formulation, it can be concluded that the Formulation enhances skin appearance and health based on objective and subjective assessments.
It should be noted that this was an initial clinical study based on 40 patients, 20 of whom were assessed for physiological changes (hydration, skin firmness, radiance etc.) as reported by subjects and confirmed by dermatologist assessment, while the remaining 20 subjects were assessed solely for visual improvements (captured via Visia imaging). These documented outcomes serve as foundational data that could form the basis of further investigation.
In-vitro genetic studies on changes in gene expression in 3-D reconstructed human skin models revealed that four highly significant genes responsible for solar skin repair processes following UV assault were significantly upregulated.
Dehydrogenase/reductase SDR family member 9 (DHRS9) is described as a moonlighting protein that functions simultaneously as a metabolite enzyme and as a transcriptional repressor [14] . DHRS9 is a specific, identifiable marker of human regulatory macrophages. In keratinocytes, DHRS9 is involved in the conversion of retinol and its metabolites into retinoic acid, and its activation is evidenced by production of retinoic acid [15] . Its expression is influenced by UVB exposure and existing skin retinoic acid reserves. When inoculated with the solar repair Formulation and exposed to UVB, a 1738% increase in the expression of DHRS9 was recorded.
Lecithin Retinol Acyltransferase (LRAT) responsible for conversion of all-trans-retinol into retinyl esters, LRAT has been characterized at the molecular level. Because of the physiological relevance of LRAT, particularly in the metabolism and storage of vitamin A, its molecular characterization is of vital importance [16] . Acute UVB impairs retinoid storage in the epidermis, and inhibiting retinoic acid synthesis disrupts epidermal differentiation and structure [17] . In this study, we observed a 574% increase in LRAT expression in the order of LRAT following inoculation and UVB exposure.
Annexin A1 (ANXA1) belongs to the annexin family of Ca2+-dependent phospholipid-binding proteins, preferentially located on the cytosolic face of the plasma membrane, and is a reliable marker of collagen synthesis activation [18] [19] . ANXA1 also acts an anti-inflammatory agent because of the biosynthesis of potent mediators of inflammation, leukotrienes and prostaglandins [19] . In our study investigating the potential of a solar repair Formulation to reduce inflammation and increase collagen reduction, we documented a 398% increase in the expression of ANXA1 in a 3-D reconstructed human skin model.
Colony Stimulating Factor 2 (CSF2) is a monomeric glycoprotein that functions as a cytokine, and involved in multiple biological activities, including regulating extracellular matrix proteins expression, inducing keratinocyte proliferation and differentiation in order to facilitate early wound healing and modulating epidermal melanocytes proliferation and differentiation in response to UVB exposure [20] [21] . Our research demonstrated an increase in CSF2 expression of 1064%.
Interleukin-1 alpha (IL-1A) is an epidermal resident cytokine acting as a regulator of skin structure and function [22] [23] . IL-1A orchestrates dermal collagen turnover when upregulated, as well as stimulating the production of hyaluronic acid and Natural Moisturising Factors. It posses the ability to inhibit tyrosinase activity leading to a reduction of melanogenesis.
Each of these results is supported by the in-vitro genetic study using a 3-dimensional skin model of epidermal keratinocytes and dermal fibroblasts. 107 genes were assessed but only 5 were selected for this paper, not only for their outstanding significant improvement but also for their link to visual results observed through the in-vivo study. Effectively, an improvement in the collagen production will have physical impacts such as a firmer and smoother skin.
Some of these results were, in fact, expected in relation to the active ingredients used in the Formulation. For example, upon incorporating retinoids in the form of retinol and retinaldehyde as well as vitamin A functional boosters, it is expected that the skin will be better equipped to convert, store and utilise retinoid metabolites including retinoic Acid, a known promoter of connective tissue synthesis and repairer of extrinsic photoaging.
5. Summary
In-Vivo testing has demonstrated that when applied to human skin for 28 and 56 days, significant improvement in skin appearance and skin health were observed:
- Skin radiance improved
- Skin firmness increased
- Skin redness reduced
- Skin hydration elevated.
To confirm these clinical improvements in skin tone and texture, in vitro genetic testing validated that not only do skin cells remain healthy upon exposure to the Formulation even when exposed to UV light but also that significant positive changes can be observed in gene expression after use of the Formulation.
- DHRS9, instrumental in modulating the conversion of retinol into retinoic acid was upregulated by up to 1738% after usage of the Formulation.
- LRAT, an enzyme encoding genes responsible for regulating the absorption and storage of retinoids was upregulated up to 574% after usage of the Formulation.
- ANXA1, encoding for collagen synthesis was upregulated by up to 398% after use of the Formulation.
- CFS2, responsible for regulating extracellular matrix proteins expression and inducing keratinocyte proliferation and differentiation was upregulated by 1064% following use of the Formulation
6. Conclusion
Each of the gene expression changes recorded in this study corresponds to functional proteins responsible for skin hydration, calmness, firmness (collagen and elastin production) and overall radiance, confirming each of the three proposed hypotheses.
1) DNA repair mechanisms markers are enhanced following daily use of the Formulation.
2) By reducing signs of inflammation, elevating hydration, reinforcing skin firmness and amplifying radiance, the Formulation elicits a visual and tactile improvement in skin appearance and tactility.
3) In-vitro changes in gene expression confirm clinical in-vivo results.
Limitations
The absence of a clinical control group and a comparison dosage may limit the significance of these findings.
Disclosure
The authors disclose that this study was entirely funded by RATIONALE Skincare Pty Ltd., Victoria, Australia. Amaryllis Aganahi and Richard Parker are paid employees of RATIONALE. Yohei Tanaka is a paid consultant plastic surgeon for RATIONALE.
Appendix
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Table A1. Gene markers environmental stress gene panel list.
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Table A2. Cutaneous acceptability, detailed patients assessment results after 28 and 56 days of usage.
Legend: NC: not come, ()*: not included in data analysis, UN: untraceable, ()*: not included in data analysis.
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Table A3. Patients TIVI 700 results and variations of cutaneous microcirculation after 28 and 56 days.
Legend: AV: aberrant value. MV: missing value, NC: not come, UN: untraceable, ()*: not included in data analysis.
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Table A4. Patients Corneometer results and variations of cutaneous hydration rate after 28 and 56 days.
Legend: NC: not come, UN: untraceable subject, ()*: not included in data analysis.
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Table A5. Patients radiance clinical score assessment by Dermatologist results and variations after 28 and 56 days.
Legend: UN: untraceable, NC: not come.
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Table A6. Patients Cutometer biomechanical properties results and variations after 28 and 56 days.
Legend: AV: aberrant value, NC: not come, UN: untraceable, ()*: not included in data analysis.