Prolonged sun exposure disrupts the skin’s structural, mechanical, and functional properties, accelerating aging and contributing to skin disorders. To counteract these effects, we developed a formulation containing potent antioxidant, anti-inflammatory, moisturizing, and reparative ingredients to protect and repair sun-damaged skin. The efficacy of the formulation was evaluated through in vitro, ex vivo, and in vivo studies. Results demonstrated that the formulation reduced oxidative stress and suppressed the production of pro-inflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), as well as lactate dehydrogenase (LDH) secretion, in a photodamaged skin model, nearly reaching levels observed in undamaged skin. It also restored collagen levels, improving structural integrity. In vivo, no adverse reactions were observed when used for 28 or 56 days. The formulation improved skin hydration by up to 46%, reduced transepidermal water loss by 20%, increased luminosity by 70%, and reduced hyperpigmented spots by 14%. It also enhanced skin firmness and elasticity by 30% and reduced wrinkle volume and density by up to 53% and 19%, respectively. These findings demonstrate that the formulation’s active ingredients effectively target the pathways altered by UV exposure, offering considerable potential for preventing and reversing sun-induced skin damage while improving both the appearance and functionality of the skin.
The skin serves as a vital barrier that shields the body from external influences, including physical, chemical, and microbiological challenges, while maintaining hydration by preventing water loss. However, being the body’s outermost layer, it is constantly exposed to environmental factors that accelerate its natural aging process. Among these, prolonged exposure to ultraviolet (UV) radiation from sunlight is the most significant contributor to extrinsic aging, a process referred to as photoaging. Clinically, photoaged skin manifests as dryness, roughness, deep wrinkles, sagging, loss of elasticity, and hyperpigmentation. This appearance results from tissue-level structural changes involving both cellular components and the extracellular matrix (ECM). Key alterations include reduced collagen levels, driven by increased degradation and mediated by matrix metalloproteinases (MMPs), and decreased biosynthesis of new collagen fibers. Additionally, there is an accumulation of abnormal elastin fibers, known as elastosis, and a significant reduction in glycosaminoglycans (GAGs) such as hyaluronic acid, which are critical for maintaining skin hydration and elasticity. At the cellular level, UV radiation impairs the function of keratinocytes, fibroblasts, and melanocytes, leading to compromised barrier integrity, diminished regenerative capacity, hyperpigmentation, and an overall decline in skin resilience. These changes contribute to the structural and functional decline observed in photoaged skin, increasing its susceptibility to skin disorders, including non-melanoma and melanoma skin cancers.
The detrimental effects of photoaging result from complex biological mechanisms triggered by UV radiation, visible light, and infrared radiation, all of which penetrate the skin at varying depths. A primary mechanism is the generation of reactive oxygen species (ROS), which directly damage cellular and extracellular biomolecules, including DNA, proteins, lipids, and polysaccharides. These oxidative events destabilize cellular components, impair repair processes, and activate signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade. This activation leads to the nuclear translocation of transcription factors such as nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1), which promote the expression of inflammatory mediators, such as interleukin-6 (IL-6) and interleukin-8 (IL-8), and matrix metalloproteinases (MMPs). MMPs are responsible for ECM degradation, particularly of collagen and elastin, which are vital for maintaining the skin’s structural integrity and elasticity. In addition to ECM remodeling, UV radiation induces premature cellular senescence, limiting the replicative capacity of fibroblasts, keratinocytes, and melanocytes, and promoting a senescence-associated secretory phenotype (SASP) characterized by the release of inflammatory cytokines and proteases. Furthermore, chronic low-grade inflammation, termed “inflammaging,” perpetuates oxidative damage and ECM degradation, creating a self-sustaining cycle of tissue damage. Chronic UV exposure also disrupts the skin microbiota. The microbiota play a vital role in maintaining skin health, including protection against harmful pathogens, modulation of immune responses, and enhancement of skin’s barrier function. UV radiation alters this microbial balance, reducing the skin’s natural defenses and exacerbating inflammation.
In response to this need, we have designed a reparative product that incorporates our novel OxIR PhotoRescue Technology® to target and counteract the primary mechanisms of photodamage. This advanced formulation combines antioxidants such as encapsulated beta-carotene, a corticosteroid-like anti-inflammatory agent that mimics hydrocortisone’s effects without associated adverse reactions, and a unique blend of plant-derived extracts with potent regenerative effects. When synergized with specially selected hyaluronic acid, an advanced combination of natural sugars comprising natural moisturizing factors (NMF) and physiomoisturizers, and a product that enhances skin microbiota protection, this formula is intended to improve signs of aging while providing robust protection against sun exposure-associated skin disorders. In order to validate the combined effects of the formulation in mitigating skin photodamage, a series of in vitro, ex vivo, and in vivo studies have been conducted in which antioxidant and anti-inflammatory capacity, its ability to counteract cellular damage and the effects of sun exposure on the extracellular matrix, as well as its impact on visible signs of skin aging have been evaluated.
The product to be evaluated is a multi-active formula with antioxidant, anti-inflammatory, and regenerative ingredients combined in the novel technology OxIR PhotoRescue Technology®, manufactured by Unikare Bioscience SL (Vitoria-Gasteiz, Spain), part of i+Med Cooperative of Scientists group, for Sibari Republic® brand and commercialized under the trademark name of Cellular Rescue.
The specific ingredients of the product, listed according to the INCI (International Nomenclature of Cosmetic Ingredients), are as follows: AQUA, GLYCERIN, ISOTRIDECYL ISONONANOATE, ETHYL OLEATE, BATYL ALCOHOL, STEARIC ACID, GOSSYPIUM HERBACEUM SEED EXTRACT, PUNICA GRANATUM SEED EXTRACT, HYLOCEREUS UNDATUS FRUIT EXTRACT, KRAMERIA TRIANDRA ROOT EXTRACT, CAPRYLIC/CAPRIC TRIGLYCERIDE, SODIUM ACRYLATES COPOLYMER, PENTYLENE GLYCOL, PHYSALIS ANGULATA EXTRACT, HYDROXYACETOPHENONE, GLYCERYL BEHENATE, HYDROGENATED POLYDECENE, TREHALOSE, 1,2-HEXANEDIOL, CAPRYLYL GLYCOL, FRUCTOSE, UREA, CITRIC ACID, SODIUM HYDROXIDE, LECITHIN, PHOSPHOLIPIDS, POLYGLYCERYL-10 STEARATE, HELIANTHUS ANNUUS SEED OIL, SODIUM HYALURONATE, HYDROGENATED PHOSPHATIDYLCHOLINE, LYSOLECITHIN, MALTOSE, SODIUM PCA, SODIUM CHLORIDE, SODIUM LACTATE, TREHALOSE, ALLANTOIN, POTASSIUM SORBATE, FRUCTOOLIGOSACCHARIDES, TOCOPHEROL, PHYTIC ACID, GLUCOSE, DAUCUS CAROTA SATIVA ROOT EXTRACT, BETA-CAROTENE.
Ascorbic acid (A4544), hydrogen peroxide solution (H 2 O 2, H1009), and Hank’s Balanced Salts Solution (H-2387) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). 5 (and-6)-carboxy-2′,7′-difluorodihydrofluorescein diacetate (carboxy-H2DFFDA) (C-13293) was sourced from Invitrogen (Waltham, MA, USA), while PBS (10×) was obtained from Roche (11 666 789001) (Basel, Switzerland). The IL-8 Human ELISA Kit (DY208) and IL-6 Human ELISA Kit (DY206) were provided by R&D Systems (Minneapolis, MN, USA). The Collagen Assay Kit (S1000) was acquired from Biocolor (Belfast, UK), and the CytoTox 96 kit (G1780) was supplied by Promega (Madison, WI, USA). Skin culture medium without animal components with Pen/Strep was supplied by Biopredic International MIL215 (Saint-Grégoire, France).
The antioxidant activity of the testing product was determined by using the in-tube DFFDA assay. This assay measures the capacity of the testing product to block the oxidation of DFFDA by H 2 O 2. Different concentrations of the product (10%, 1%, and 0.1% v/v, diluted in assay buffer) were incubated in a solution with 5 µM DFFDA and 4 mM H 2 O 2. After 1 h, fluorescence was measured at Ex/Em = 485/535 nm. Ascorbic acid (50 mM) was used as reference material (positive control). The percentage of antioxidant activity (AA%) was calculated by comparing the fluorescence values of the control (negative control) and test samples by applying the following formula:
AA%=100−(F 485/535 CX−F 485/535 B−CX)×100 F 485/535 Control
F 485/535 CX: fluorescence at Ex/Em = 485/535 nm of the product at a given concentration in DFFDA and H 2 O 2 presence.
F 485/535 B − CX: fluorescence at Ex/Em = 485/535 nm of the product at a given concentration (Blank).
F 485/535 Control: fluorescence Ex/Em = 485/535 nm of the assay buffer in DFFDA and H 2 O 2 presence (C−).
Human organotypic skin explant cultures (hOSECs) were obtained from healthy donors undergoing plastic surgery (authorization granted by the French government’s ethical committee according to French law L.1245 CSP). Up to 2 h from the surgery, the skin was cut into 0.8 cm 2 pieces and shipped in transport medium. Upon receipt, samples were placed with dermis facing down and epidermis facing up in culture plates containing skin culture medium without animal components supplemented with Pen/Strep (1%). Tissue cultures were incubated for at least 48 h at 37 °C under 5% CO 2 for recovery prior to study initiation.
In order to mimic skin photoaging, UV-Vis (ultraviolet-visible) light irradiation (5 J/cm 2) was applied daily to the hOSEC by using the SOL 500 Solar simulator (Dr. Hönle, Gräfelfing, Germany), based on previously published studies using similar doses and exposure times. At the same time, the product was administered topically at 2 mg/cm 2. The product was in contact with the hOSEC throughout this study, for a total of 9 days. Four replicates of each experimental group (control, photoaged skin, and photoaged skin + product) were carried out.
The IL-6 and IL-8 cytokine quantification assays were performed in the supernatants of the skin culture. Culture supernatants were collected, clarified by centrifugation, aliquoted, and stored at −70 °C until analysis. Once thawed, cytokine levels were determined by typical sandwich ELISA assay kits according to the manufacturer’s recommendations.
The LDH Cytotoxicity test is a colorimetric assay that quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released into the culture medium supernatant upon damage to the cytoplasmic membrane. The released LDH in culture medium supernatants can be measured by a 30 min coupled enzymatic reaction; LDH oxidizes lactate to pyruvate, which then reacts with the tetrazolium salt WST-1 to form formazan. The increase in the amount of formazan measured in the culture supernatant directly correlates to the increase in the number of damage cells in the skin explant. 50 µL of supernatant were removed from each sample and transferred into a 96-well microplate. After that, 50 µL of formazan dye was added to each sample, and, following a 30-min incubation period, absorbance was read using a standard ELISA plate reader at 490 nm.
The soluble collagen content was determined using the collagen kit from Biocolor Ltd. (Carrickfergus, UK). The collagen assay is a dye-binding method for the analysis of acid and pepsin-soluble collagens. hOSECs were incubated with a solution of pepsin concentration at 0.1 mg/mL and acetic acid at 0.5 mM at 4 °C overnight. Collagen Dye Reagent (Sircol Dye Reagent) (1 mL) was added to each supernatant and shake
