Advantages Of Hyaluronic Acid And Its Combination With Other Bioactive Ingredients in Cosmeceuticals

Keywords: hyaluronic acid; cosmeceuticals; biological activity; skin health; moisturising effect;anti-aging effect; bioactive compounds; molecular weight; hyaluronan derivates

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1. Introduction

Hyaluronic acid (HA) is a polysaccharide belonging to glycosaminoglycans, made up of disaccharide units constituted of N-acetylglucosamine and D-glucuronic acid (Figure 1). It is a component of the connective, epithelial, and neural tissues and it represents a substantial constituent of the extracellular matrix (ECM) [1–6]. HA was discovered for the first time in the vitreous humor of the eye in 1934, and in 1964 it was synthesized in vitro [7–9]. HA has a wide range of molecular weights ranging from 2 × 105 to 107 Da [10–13]. The HA average molecular weight can influence its physicochemical properties [3,14]. Among the many biological effects, HA is involved in cell differentiation, embryological development, inflammation, wound healing, viscoelasticity, etc. [15]. As it has been observed, the molecular mass and the mode of its synthesis or degradation define the HA biological effects [3,16,17]. By a passive mechanism, high molecular weight HA (HMW-HA) permits tissue hydration, contributes to osmotic balance, and stabilizes the ECM structure. On the other hand, HA interacts with different receptor-binding proteins, and its molecular weight can influence the receptor affinity or its uptake by the cells, leading to opposite effects.

For example, HMW-HA inhibits cell growth (angiogenetic activity) and protects the articular cartilage due to its lubrication properties. Low molecular weight HA (LMW-HA) has angiogenetic activity and can induce tumor progression or present pro-inflammatory activity [15,16]. Thus, the biological activity of HA is due to its binding to different receptors. For example, the binding HA-CD44 transmembrane receptor mediates cell adhesion and migration in many physiological or pathophysiological processes: (a) angiogenesis; (b) ECM structure (linking the HA with cytoskeleton); (c) inflammation (upregulation of the receptors overexpresses the interleukin-1); (d) wound healing; (e) malignant tumors (e.g., pancreatic, breast, lung, etc.). The CD-168 receptor (Receptor for Hyaluronan-Mediated Motility, RHAMM) localized on the cell surface has important relevance in cell migration. When the receptor is situated intracellularly, it affects the activity of the mitotic spindle. As a result, the HA-RHAMM links can influence the inflammation and tissue repair processes. The HARE (Hyaluronan Receptor for Endocytosis) receptors modulate glycosaminoglycan clearance. The lymph absorption of HA, implicitly the HA turnover, is controlled by LYVE1 (Lymphatic Vessel Endothelial Hyaluronan receptor-1). As a result, HA-LYVE1 interaction influences the tissue's biomechanical properties, including its hydration. Referring to the HA interaction with TLRs (Toll-Like Receptor), it is noticed that LMW-HA has an inflammatory effect, because of its agonist activity on TLR-2 and TLR-4. On the other hand, a high mass of HA decreases the binding capacity to the receptors, forming a dense coat around the cell and covering the receptor surface [15,16,18,19]. 

Figure 1.Chemical structure of hyaluronic acid (HA). 

Nowadays, there are a lot of studies conducted to elucidate the mechanism of action and the biosynthetic pathways of HA, or to optimize its biotechnological production, to synthesize derivatives with superior properties, and to improve its therapeutic utilization [16]. The list of substances that are restricted or prohibited in the EU for use in cosmetic products does not include hyaluronic acid and sodium hyaluronate (NaHA). As an example, using hyaluronic acid, sodium hyaluronate, or potassium hyaluronate (KHA) in cosmetics is not restricted in Japan [20]. Some studies realized by the Cosmetic Ingredient Review (CIR) experts panel, based on the application of cosmetic HA in various concentrations, showed acute, short-term, or chronic toxicity [20]. 

Additionally, some side tolerable effects (scaling, erythema, and pruritus) were observed, after the use of a topical product with hyaluronic acid (0.01%), hydroquinone (4%), and glycolic acid (10%) for melasma treatment [4]. Subsequently, HA and NaHA can be nebulized and used in cosmetic products which can be applied as aerosols (e.g, hair spray) (20]. The nebulized particles can be stored at different levels of the respiratory system, depending on their size and concentration. Because of this fact, the safety assessment of cosmetic aerosols is an important issue. The protective effect of HA on the respiratory system was noticed in some studies 21], however the propellant gas, vapors, and other soluble compounds (e.g., alkanes, alcohols, stabilization polymersoentonite, aluminum chlorhydrate, perfume oils, cosmetic colorings, complexation agentsanolin derivates, plant extracts, etc.) associated with hyaluronan in cosmetic aerosols could induce respiratory sensitization effects such as: rhinitis, conjunctivitis, wheeze, dyspnea, or asthma. Moreover, the insoluble particles from aerosols could be responsible for pulmonary overload, leading to chronic toxicity (e.g., chronic inflammation, fibrosis, including lung tumors). These effects are related to their concentration, exposure duration, or particle size for example, the assessment of the inhalation toxicity of products with insoluble particles with a size below 10 um is recommended. Regarding these aerosols, an exposure duration of 5 min is indicated, and also it is necessary to avoid the exposure to fine droplets of lipophilic substances, which could produce acute respiratory syndrome” (22). Due to various biological activities, HA products are increasingly in demand. Thus, in 2016, the total market of HA (pharmaceuticals, beauty, and personal care) exceeded 141tones and it is expected to grow more than 30% in 2021 (Figure 2). The most significant increase of HA market is estimated to be in Europe and Asia (23).

Figure 2. Hyaluronic acid market-regional comparison. 

Some formulations containing HA are already available on the market, with a large experience in their use. At the same time, for other products, it is necessary to perform subsequent investigations to confirm their efficacy. HA is a special moisturizing active ingredient, used in cosmetics, particularly formulated as emulsions or serums, claiming hydration and skin elasticity effect. These skin biophysical parameters are closely related to the anti-wrinkle effect, but no rigorous scientific evidence does justify this statement completely. Additionally, it should be taken into consideration that the efficacy of hyaluronic acid depends largely on molecular weight [10]. Hyaluronic acid is one of the most efficient and safe ingredients used frequently in cosmetics. HA properties can be improved by other bioactive ingredients (e.g., plant Figure 2. Hyaluronic acid market-regional comparison. Some formulations containing HA are already available on the market, with a large experience in their use. At the same time, for other products, it is necessary to perform subsequent investigations to confirm their efficiency. HA is a special moisturizing active ingredient, used in cosmetics, particularly formulated as emulsions or serums, claiming hydration and skin elasticity effect. These skin biophysical parameters are closely related to the anti-wrinkle effect, but no rigorous scientific evidence does justify this statement completely.

Additionally, it should be taken into consideration that the efficacy of hyaluronic acid depends largely on molecular weight [10]. Hyaluronic acid is one of the most efficient and safe ingredients used frequently in cosmetics. HA properties can be improved by other bioactive ingredients (e.g., plant extracts, vitamins, amino acids, peptides, proteins, minerals, saccharides, probiotics, etc.). Nowadays, there is a multitude of cosmetics containing HA, marketed by different manufacturers. The previously published papers present separately these advantages of HA or bioactive ingredients. In our paper, we present first the biological effect of HA on skin level, after which the portfolio of some popular manufacturers was analyzed, commercial cosmetic brands and products containing HA were identified, and their declared qualitative composition was evaluated. Subsequently, the additional biologic effects and the toxicological potential of the other active ingredients were presented. 

2. Applications of Hyaluronic Acid

Taking into account its biological actions, physicochemical properties, its biocompatibility or safety profile, HA has multiple applications. Figure 3 depicts the utilization of HA and its derivates in: medical (arthrology, cancer therapy, pneumology, odontology, ophthalmology, otolaryngology, rhinology, soft tissue regeneration, urology, wound treatment, etc.), pharmaceutical (e.g., drug delivery systems), nutritional (nutraceuticals, nutricosmeceuticals), or cosmetic field [3,8,9]. 

Figure 3. Cosmetic, pharmaceutical, and medical applications of HA and its derivates

Being an essential component of the ECM and due to its available derivatization scenarios, HA is widely used in drug delivery through several routes: cutaneous, ocular (intravitreal, periocular, subretinal), topical, nasal, oral, etc. HA can be conjugated with drug molecules (in the form of prodrugs) or can be incorporated in several molecular ararchitectures (nanoparticles, microparticles, microspheres, gels, polyplexes, polymersomes, liposomes, micelles, implants, etc.). The resulting HA structures possess superior physicalchemical properties and higher therapeutic efficacy. A brief list of HA applications in drug delivery includes: targeting for skin diseases, cancer therapy, and controlled release of proteins, antiseptics, and antibiotics [16,24–30]. Normal body cells have a poor expression of HA receptors while many tumor cells generate overexpressed receptors that bind HA. This fact can lead to several approaches in cancer therapy, involving HA. Firstly, the conjugation of paclitaxel (PXT) and docetaxel (DOX), should be mentioned. PXT alone is not suitable for intravenous injection due to its hydrophobicity and adverse events. The PXT-HA conjugate is hydrophilic enough Figure 3. Cosmetic, pharmaceutical, and medical applications of HA and its derivates. Being an essential component of the ECM and due to its available derivatization scenarios, HA is widely used in drug delivery through several routes: cutaneous, ocular (intravitreal, periocular, subretinal), topical, nasal, oral, etc. HA can be conjugated with drug molecules (in the form of prodrugs) or can be incorporated in several molecular architectures (nanoparticles, microparticles, microspheres, gels, polyplexes, polymersomes, liposomes, micelles, implants, etc.). The resulting HA structures possess superior physicochemical properties and higher therapeutic efficiency. A brief list of HA applications in drug delivery includes: targeting for skin diseases, cancer therapy, and controlled release of proteins, antiseptics, and antibiotics [16,24–30]. Normal body cells have a poor expression of HA receptors while many tumor cells generate overexpressed receptors that bind HA. This fact can lead to several approaches in cancer therapy, involving HA. Firstly, the conjugation of paclitaxel (PXT) and docetaxel (DOX), should be mentioned. PXT alone is not suitable for intravenous injection due to its hydrophobicity and adverse events. The PXT-HA conjugate is hydrophilic enough and seems to overcome limitations. Hydrophobic drug molecules can be loaded in HA micelles in order to achieve target delivery to cancer cells. Both lipophilic and hydrophilic drugs can be loaded in polymersomes. The main advantages of the previously mentioned structure modulations are solubility increase and targeting CD44 receptors on tumor cells.

Modifying mesoporous silica nanoparticles with HA leads to an increased uptake in the case of CD44 over-expressing cells. Other nanomaterials with potential efficiency in cancer therapy include dendrimers and liposomes. Additionally, HA-coated nanoparticles (NP) are of considerable interest in cancer therapy. Several HA-based nanomaterials are used in hyperthermia (an increase in the temperature of the cancer cells at about 42–46◦C): NIR-loaded nanoparticles, gold nanoparticles, functionalized graphene, oxide nanoparticles, Prussian Blue nanoparticles, and other particles (related to magnetic hyperthermia treatment). In addition, HA-based nanoparticles were used in photodynamic therapy, immunotherapy, and son dynamic therapy [31–37]. 

Two major steps in wound healing (in which HA is involved) are inflammation and angiogenesis. Several biomaterials (wound dressings based on HA or combinations of HA with other biopolymers) were synthesized and tested: sponges, films, hydrogels, and electrospun membranes. The main advantages of incorporating HA in these biomaterials are porosity and swelling improvement, together with exudate absorption. Tissue engineering uses HA for the regeneration and reconstruction of several tissues: cartilage, ocular tissues, skin, vascular tissue, adipose tissue, and peripheral nerve [38–45]. The intended use of HA in dentistry is mainly the regeneration of soft tissues, but also wound healing and the regeneration of hard tissues. HA may be used as a co-material (together with other biopolymers) in several procedures related to dentistry: papilla reconstruction, osseointegration of implants, sinus lifting, periodontitis, and stomatitis therapy [46–49]. Orthokeratology is a treatment for correcting patients’ refractive error by means of wearing a special lens overnight. 

Viscous artificial tears (based on HA) proved to be superior (in terms of patients’ comfort) in comparison to the saline solution when used for the fitting of the orthokeratology lenses. Nisin was grafted on HA by means of amide bonds. The biocidal capacity of this modified polysaccharide (incorporated in solutions or gels) was tested on Gram-positive organisms with promising results. Additionally, HA conjugated with ciprofloxacin and vancomycin was used for the prevention of infections in ophthalmic surgery. Ophthalmic viscoelastic devices (OVDs) are used during cataract surgery due to their multiple advantages. However, long retention times of OVDs can lead to an increase in intraocular pressure (IOP). The use of two OVDs (Healon GV—1.8% sodium hyaluronate and Healon 5—2.3% sodium hyaluronate) showed a non-significant increase of IOP. The use of artificial tears is the most common therapeutic solution for dry eye syndrome. Tears based on HA and carmellose (carboxymethylcellulose) proved to be superior in terms of stability of the tear film and quality of vision in comparison with normal saline solution [26,50–54]. The physical and physicochemical properties of HA are highly dependent on its molecular weight (MW). The main benefits of HA use in arthrology are related to the treatment of osteoarthritis, rheumatoid arthritis, and bone cancers. In the case of advanced osteoarthritis, knee joint distraction is a promising procedure for spontaneous cartilage repair, in about 8 weeks. The key factors are HA (in synovial fluid) and mesenchymal stromal cells (MSCs). MSCs are able to adhere to the cartilage under the influence of HA (especially the ones with MW > 9 MDa) [55–58]. Interstitial Cystitis/Bladder Pain Syndrome is a chronic inflammatory syndrome and seems to be related to the destruction of the bladder mucosa, especially glycosaminoglycan coating, both Chondroitin Sulphate (CS) and non-sulfated–HA [59,60]. A therapeutic approach is the intravesical instillation of CS or a combination of CS and HA while evaluating: Female Sexual Function Index (FSFI), Visual Analog Pain Scale, Interstitial Cystitis Syndrome, and Interstitial Cystitis Problem Index. FSFI was higher for the control group (CS group). The last three parameters were improved in a higher manner when comparing CS/HA and CS groups [60]. No adverse reactions were reported during CS or CS/HA installations [61,62]. Vesicoureteral reflux (VUR) affects children and is linked to the patient’s Urinary Tract Infection (UTI) history [63]. Surgery is the main therapeutic approach for VUR, but in this case complications may occur. Another approach is endoscopic injection therapy with teflflon, polydimethylsiloxane, dextranomer/hyaluronic acid copolymer (Dx/HA), and polyacrylate polyalcohol copolymer. In the case of Dx/HA combination, the short time success rate was high, but more studies are needed regarding long-term success rate [64]. 

Cystic fibrosis is an inflammatory lung disease linked with high airway levels of neutrophil elastase. Polysulfated GAGs (including polysulfated HA) are currently used for neutrophil elastase inhibition because of their anti-inflammatory properties [65]. HA is also used in other airway-related diseases: chronic sinusitis, asthma, bronchiectasis, and chronic obstructive pulmonary disease [66]. HA was successfully used in the postoperative recovery of nasal mucosa after sinus surgery when administered topically or by means of nebulization [67,68].

Due to its anti-inflammatory and tissue regeneration properties, HA has been used for gene delivery in otology and there are some promising results in tympanic membrane perforation treatment [68,69]. 

The activity of fibroblasts (in the epidermis) and keratinocytes (in the dermis) seems to slow down together with age and also became less responsive to growth factors [70]. The aging process consists of both intrinsic and extrinsic aging which leads to a reduction of HA in the skin [71]. Because of the great number of polar groups present in its molecule, hyaluronic acid is a hydrophilic macromolecule with anti-aging and hydrating claims. In aqueous solutions, it can form viscoelastic gels, and when it is applied to the skin it ensures moisturizing, firming, and rejuvenation and has improved wound healing effects [10,12]. In contact with water, HA has the capacity to augment its volume, having the effect of softening wrinkles by filling the spaces between the cells of the skin forming a viscid gel matrix. The half-life of HA in the tissues, in its natural form, is of just 12–24 h. As a result, crosslinked forms of HA are used in topical and cosmetic preparations [72–74]. Nevertheless, the high molecular weight of HA does not allow it to penetrate the deeper layers of the skin which restricts its benefits to topical effects [75]. Many studies showed the exogenous HA significant role in the epidermis and especially in the dermis, and its involvement in remodeling, tissue repair, and healing [2,76–80]. In a randomized, placebo-controlled, single-blind trial (daily oral intake, for 60 days, of 200 mg of hyaluronic acid, 500 mg of L-carnosine, and 400 mg of methylsulfonylmethane) it was proven that skin hydration and elasticity were improved and glabellar sebaceous secretion decreased [81]. Ingestion of HA/hyaluronans can improve skin moisture content and reduce aging symptoms and signs [82,83]. 

3. Use of Hyaluronic Acid in Cosmetology

Nowadays, HA is one of the most widely used active ingredients in cosmetic formulations. The general perception of skin regeneration is of constant interest to both industry professionals and consumers. It is evident that the skin is an indicator of an individual’s health and HA is one of the main factors for healthy skin [84]. As shown above, hyaluronic acid is a biopolymer considered of primary interest from a scientific point of view, due to its multitude of applications in cosmetic and biomedical fields. Such being the case, exploration of this ingredient is increasing in many interdisciplinary domains targeting, on the one hand, the improvement of production processes in terms of biotechnology and on the other hand the development of new formulations incorporating hyaluronan or HA-based innovative ingredients. Scientific efforts are moving nowadays toward the production of appropriate molecular-weight biopolymers. This specific aspect relies precisely on the biological function, as indicated by bibliographic studies. Although HA was synthesized a very long time ago, it is still needed to investigate this active ingredient in terms of physicochemical and biological properties [16]. HA has a multitude of applications based on specific properties such as: (1) high hygroscopicity; (2) viscoelastic nature; (3) biocompatibility; (4) non-immunogenicity. Nevertheless, the HA skin penetration mechanism is still barely understood. A multitude of factors is studied, including the existence of HA receptors for active transport and a particular structure of the hydrated HA. The general hydration effect of the skin may also optimize dermal absorption of active ingredients and can assist their retention within the moisturized epidermal layers. HA is appropriate for biomacromolecules because it ensures protein-stabilizing properties. However, the precise mechanism for the transdermal transport of HA remains to be elucidated. 

In wound regeneration, HA has mainly cosmetic applications. In skin care formulations, it can be used as a moisturizing component, because of its hydrophilic nature. Using cosmetic products such as creams or lotions that contain HA helps to moisturize the skin and to improve elasticity, thereby decreasing the depth of wrinkles. It is assumed that, when applied onto the surface of the skin, HA solutions form an occlusive layer, absorb moisture, thereby hydrating the skin, and default wrinkles filling occurs. HA is assumed to stimulate the migration of epidermal cells. Additionally, the occlusive properties given by HA may allow the biologically active substances incorporated in cosmetics to persist in the skin layers and possibly make it easier for them to penetrate the epidermis. According to previous studies, some cosmetic HA products have been proven efficient in protecting the skin from UV irradiation. At the same time, sunscreen products containing hyaluronic acid help to maintain firmer skin, protecting it from the injurious impact of UV radiation, due to the potential antioxidant effect of HA [87]. 

In cosmetic formulations, hyaluronic acid has the function of a viscosity modifier and/or a skin conditioning agent. HA is mainly used in anti-aging cosmetic products. LMW-HA has the ability to enhance the level of moisture in the skin and expedite regeneration. HMW-HA forms a viscoelastic film when applied to the skin and has a moisturizing effect. The main action of the HMW-HA polymer is film forming and it reduces the evaporation of water from the skin and thus possesses an occlusive effect. Additionally, HMW-HA, Medium molecular weight (MMW-HA), and LMW-HA hygroscopic properties justify the ability to maintain skin hydration [87,88]. HA is also of particular importance as a delivery system of active ingredients. Currently, there are some commercially available formulations incorporating actives in different concentrations. These products are designated for the topical treatment of actinic keratosis and skin inflammatory diseases. In fact, it has been proven that HA enhances the penetration of the active ingredient through the stratum corneum (SC), which behaves as a barrier to the entry of the molecule into the deeper layers of the skin, and the holding and locating the active ingredient in the epidermis. Topical preparations containing HA in the formulation are used for their healing properties, decreasing the skin irritation. A topical preparation that contains HA (0.2% w/w sodium hyaluronate (NaHA)) as a main component is currently available for the amelioration of acute and chronic wounds (areas of grafted skin, post-surgical incisions, etc.) [13,77,88]. A significant number of in vitro and in vivo studies have shown the effectiveness of HA treatment as: anti-inflammatory, skin regeneration and chondroprotective effect, anti-aging and immunosuppressive effects, etc. Although hyaluronan has various applications, subsequent research and technological development are needed, because there are currently certain issues to be elucidated. Firstly, further consideration of aspects regarding HA metabolism and receptor clustering analysis is necessary in order to explain the various biological actions and to foresee the effects that can vary with the molecular weight of HA. Some pharmaceuticals and/or cosmetics can incorporate HA with different molecular weight. Thus, studies are necessary for assessing the implications of molecular weight in the HA effects. Next-generation products with derivatives of crosslinked HA-conjugated polymer-delivery systems and drug substances should be developed, granting a high level of biocompatibility, prolonged half-life, and permanent in situ performance. Therefore, clinical exploration is imperative to fully characterize the safety and efficacy profile of these substances. So far, recent in vitro studies have shown promising results regarding the safety and efficacy of these promising and novel compounds: for example, HA-CL (urea-crosslinked hyaluronic acid) showed a signifificant biocompatibility with human corneal epithelial cell, having antioxidant, antiinflflammatory, and skin regeneration properties.

HA is used in cosmetic formulations in concentrations ranging from 0.2 to 1%. The maximum concentration of NaHA in a body lotion is 2%. When a rate of 1 mg/cm2 of a product is applied, the contribution of hyaluronic acid is 0.02 mg/cm2 of skin [20]. Interest in using hyaluronic acid as a cosmetic ingredient in skin care products occurred with the discovery that the amount of HA found in natural skin diminishes with age, and when reintroduced into the skin care products, it keeps skin hydrated, attenuates the appearance of wrinkles, and smooths the skin. 

HA has many qualities that make it superior to other substances used in skin regeneration, with pronounced moisturizing and anti-ageing effects [87,88]. Biological activity and HA penetration into the skin depends on the molecular weight of this substance showing different effects on the skin, as presented in Figure 4.

Figure 4. Hyaluronic acid activity, molecular weight dependence and claimed effect of HA. (TEWL—Transepidermal Water Loss; DEJ—Dermoepidermal junction).

It has been demonstrated by some researchers, that HA has extraordinary cosmetic and nutricosmetic effificacy in improving diverse skin imperfections such as wrinkles, periorbital and nasolabial folds, and skin ageing. These types of effects of HA have been correlated with their capacity to induce the augmentation of soft tissue, to hydrate the skin, stimulate collagen, and rejuvenate the faceas summarized in Figure 5 [89]

3.1. Hydratation Effect of HA in Cosmetic Formulations

The amount of hyaluronic acid synthesized is more substantial in the epidermis than in the dermis. Since the dermis is much thicker than the epidermis, it comprises four to nine times more HA, but it was demonstrated that for equivalent tissue quantities, the epidermis synthesizes four times more hyaluronic acid than the dermis. In the epidermis, HA is located in the intercellular matrix of the basal and spinous layers. 

Similarly, as in the dermis, the hygroscopic properties of hyaluronic acid are of substantial relevance in hydrating the deep layers of the epidermis, but its contribution goes further than conventional hydration [90–92]. HA, which has the ability to bind water up to 1000 times its volume, has a relevant contribution to cellular growth, adhesion, and membrane receptor function. The major biologic role of HA in the intercellular matrix is to reinforce the intercellular structures and to produce the elastoviscous fluid matrix that firmly envelops collagen and elastin fibers. HA holds moisture and provides firmness and radiance to the skin as well [93,94]. HA can be used topically to regenerate the skin and support hydration, although it's very high molecular weight prevents its penetration through the SC [95,96]

3.2. Anti-Ageing Effect of HA in Cosmetic Formulations HA also has an important role in skin aging. Cells lose their ability to produce HA with aging. The skin becomes drier, thinner, and looser, leading to wrinkling, among other signifificant changes [97]. Skin aging is also associated with a decrease of skin moisture. Hyaluronic acid (hyaluronan) has a unique capacity to link and retain water molecules [98]. As it was shown, hyaluronic acid is a natural component that is present in the whole body. In a 70 kg individual, there are 15 g of hyaluronic acid, 5 g of which are replaced daily. HA is naturally and constantly renewed because of its rapid degradation, but its renewal tends to slow with age and external aggressions. Therefore it is necessary to act very early, sustaining an optimal hyaluronic acid turnover, similar to that of young skin, in order to prevent the signs of aging [76,99–101]. In relation to its biological effects at skin level, it is known that hyaluronic acid is actively involved in skin cell signaling (by binding the CD44 and LYVE-1 receptors) and thus influences ECM stability. It has been noticed that HA has an impact on the growth of keratinocytes which protect the epidermis from aging [10,16,79,93,102]. Hyaluronic acid is used in cosmetic preparations for its elasticity effect and for giving shape to the periorbital area after HA cosmetic treatment (103. Additionally, the chemical double binding structure of the D-glucuronic acid unit confers antioxidant properties to hyaluronic acid. Furthermore, HA restrains the proliferation of the skin cells via the CD44 receptor and HA also has anti-inflammatory properties on the skin [76,104]. Hyaluronic acid is applied in a multitude of anti-aging products. For example figure 6 presents the effect of an anti-aging cream incorporating 0.5% (w/w) LMW-HA(20-50 KDa) and 3% (w /w) encapsulated HMW-HA (1-1.4 MDa) on periorbital wrinkles before treatment and after 28 days of treatment (Protocol Report No. 300924/19 /ISHRAgreement No. 331/30 August 2019 JS. Hamilton Romania S.R.L) [1051.