Current Topics: Review
Hyaluronan Tetrasaccharides Penetrate into the Skin by Passive Diffusion and Contribute to Skin Health
2025 Volume 73 Issue 4 Pages 284-290
Details
2025 Volume 73 Issue 4 Pages 284-290
Hyaluronan (HA) is a commonly used material in cosmetics and pharmaceuticals because of its various pharmacological activities. However, because of its large molecular weight, HA penetrates the skin very poorly and most of it remains on the skin surface. Thus, topically applied HA could not be expected to function biologically in the skin. However, we have confirmed that HA tetrasaccharides (HA4), which is the smallest unit of HA, penetrate into the skin by passive diffusion and affect epidermal metabolism. Topical treatment of HA4 rescues the epidermal damage caused by long-term UVA irradiation. Furthermore, various biological functions of HA4 to maintain healthy skin was observed in cell culture studies. This review describes the skin permeability of HA4 and how it contributes to healthy skin.
Hyaluronan (HA) is a major extracellular matrix (ECM) component that exists throughout the whole body and is abundant in the skin, especially in the dermis.1–3) It has a very simple structure consisting of repeats of disaccharide structures of glucuronic acid and N-acetylglucosamine with an unbranched form. It was first discovered and isolated from the vitreous body of a cow in 1934,4) and a few years later HA was shown to be effective in treating racehorses suffering from arthritis because of enormous water holding capacity and maintains organ viscoelasticity, which resulted in initiating research into medical fields. Because of the specific properties of HA, such as its non-immunogenicity,5) high biocompatibility and excellent biodegradability, its applications have expanded to include medical care6–12) and beauty care.13–20)
Adding to these properties, HA works in regulating cellular functions and intercellular communications21) and plays a very important role in the inflammatory response in the skin’s innate immunity, particularly in regulating the functions of neutrophils, macrophages and dendritic cells, and in the production and secretion of cytokines and chemokines.22–26) Interestingly, these physiological effects of HA vary greatly depending on its molecular weight (glycan length). HA is synthesized in the cell membrane by hyaluronan synthase (HAS)27–32) and is subsequently degraded into fragments of various sizes by hyaluronidase.33–35) High molecular weight of HA (H-MW-HA) has anti-inflammatory effects36–40) and anti-angiogenic effects.36) Additionally, H-MW-HA accelerates cutaneous wound healing41) and reduces adhesion formation after intra-abdominal surgery.42) In contrast, low molecular weight HA (L-MW-HA) promotes the production and secretion of cytokines and chemokines43–45) and promotes angiogenesis.46) In addition, HA oligosaccharides, which have even shorter sugar chains than L-MW-HA, has more unique biological and biochemical functions.47–51) Hascall and Heinegard reported in 1974 that the smallest HA fragment that binds to aggrecan is a decasaccharide.52) In the skin, HA oligosaccharides produced by hyaluronidase 1 (Hyal1) trigger adaptive immunity by activating toll-like receptor 4 (TLR4) and CD44, the HA receptor, that leads to increased allergic inflammation via dendritic cell trafficking and the improved clearance of bacterial skin infections.53–56) These effects are not observed with H-MW-HA, and it has been shown that when HA is broken down into smaller fragments, it acquires new functions and activities. L-MW-HA and HA oligosaccharides may recognize specific proteins that cannot be recognized by H-MW-HA. Thus, the degradation of HA by hyaluronidase might control cellular metabolisms. Furthermore, abnormalities in HA metabolism are associated with the severity of inflammatory diseases and the aggressiveness of cancer.57–64) It is reported that HA tetrasaccharides (HA4), which is the smallest unit of HA, has antitumor activity.65,66) Currently, many HA-binding proteins and receptors have been discovered.67–74) In addition, HA oligosaccharides also inhibit the binding of endogenous HA to HA-binding proteins.75,76) For these reasons, the molecular weight (glycan length) of HA and the role of HA-binding proteins and receptors will be important tools in research.
Although HAs are multifunctional material, the administrating approach is limited, and direct injection are mainly used because of its molecular size. There are many foods and supplements containing HA as oral ingestion,77–79) however, it is hard to deliver enough amount of HA to certain organs because of digestion and absorption process. Since HA does not penetrate by simple topical application, it has been administered using various approaches, including direct transdermal administration.80–83) Physical promotion methods such as electroporation (EP)84,85) and iontophoresis (IP)86,87) are often used for cutaneous penetration. However, EP and IP were unable to deliver H-MW-HA (>1200 kDa) into the skin using physical enhancement methods.88) In recent years, various nano-formulations containing HA-based drug delivery systems have been developed, such as, niosomes, ethosomes, transferosomes, polymeric micelles and microneedles.89–103)
Because of its physical properties and biochemical functions, HAs are quite useful materials for dermatological care and cosmetics. Increase in HA improves skin wrinkling and sagging100–108) and HA with suitable molecular weight may control cellular functions and cutaneous inflammations. There are also many reports regarding HA production109–116) and there are materials to increase cutaneous HA contents. However, it is hard to control the size of HA by de novo synthesis. H-MW-HA may penetrate under conditions where the skin barrier is compromised. In addition, it can be made to penetrate into the skin through special formulation83) or techniques.117) A quick method would be to deliver HA of a specific molecular weight into the skin in hopes of achieving an effect, but in general, it is generally difficult to penetrate the skin when used externally.118)
We found that HA4 permeates through passive diffusion119) and control the cellular metabolism in vivo. Therefore, HA4 can be expected to be physiologically active in the skin just by applying it. Furthermore, decomposed HA may indicate different biochemical functions from original HA. However, topically applied HA4 may function directly in the skin. Thus, HA4 is a useful compound for medical and cosmetical treatments. This article provides an overview of HA4, focusing on its function in the skin in vivo and in vitro, which was mainly obtained from our research during the past decade.
The stratum corneum (SC), located at the outermost layer of the skin, has a strong permeability barrier function that prevents water evaporation from the body and the invasion of foreign substances and viruses into the body. Thus, compounds applied to the skin need to penetrate by passive diffusion through the SC barrier. Generally, hydrophilicity and molecular weight are important factors for skin permeation. Hydrophilic compounds have lower skin permeability than lipophilic compounds, and skin permeation can be controlled if the molecular weight is less than 500 Da.120)
The permeability of HA4 and H-MW-HA were evaluated in human and murine skin tissue using a Franz type diffusion cell. HA is highly hydrophilic with a high molecular weight, thus, most HA does not penetrate the SC barrier of human skin118) (Figs. 1A, B). On the other hand, HA has a large molecular weight distribution and HA oligosaccharide MIX penetrates more efficiently into skin118) (Figs. 1A, C). Furthermore, we confirmed that HA4 permeates through the skin by passive diffusion using murine skin119) (Fig. 2). The cumulative amounts of 0.5% HA4 through SC-stripped skin and full-thickness skin after 8 h were 2000 and 0.8 µg/cm2, respectively, indicating that HA4 can penetrate even the SC with high barrier function. Thus, it can be expected that the physiological activity within the skin may be induced by the topical application of HA4.
Fluorescein-labeled H-MW-HA and HA oligosaccharide mix were applied to human skin. A. Hematoxylin–eosin stain; B. Application of H-MW-HA; C. Application of HA oligosaccharide mix. The scale bars indicate 100 µm length. H-MW-HA was observed on the surface of the SC, but the HA oligosaccharide mix penetrated into the skin.
Cumulative amount of HA4 that permeated through HR-1 hairless mice skin over time. HA4 penetrated the skin by passive diffusion. The amounts are expressed as mean values ± standard deviation (S.D.) (n = 3).
From the data of penetration of HA4, the efficacy of topically applied HA4 was evaluated in vivo. Chronic UVA irradiation induces epidermal hyperproliferation121) and disturbs SC functions.122,123) However the daily topical application of 0.1% HA4 significantly prevented those skin disturbances.119) The dorsal skin of hairless mice was irradiated with UVA 5 times a week for 3 weeks. The SC water content, indicated by cutaneous capacitance, significantly decreased on day 5 and that decrease continued for 3 weeks. Accompanied by the decrease in SC water content, the cutaneous barrier function was perturbed and the transepidermal water loss (TEWL) was increased significantly. However, the daily topical application of HA4 prevented that decrease in SC water content for 3 weeks119) (Fig. 3). The increase in TEWL was also prevented by the topical application of HA4 from day 9. This prevention was also kept by the end of week 3. Histological analysis at the end of week 3 indicated that the murine epidermis was thickened more than 7 times by chronic UVA irradiation, but epidermal skin treated with HA4 was thickened only less than 3 times, which was not significantly different from the normal control119) (Fig. 4). These results show that HA4 can permeate the skin and can affect the cellular functions in murine epidermis, resulting in improving the skin after UVA irradiation. Moreover, these data suggest that HA4 is involved in the healing process of the skin after UVA irradiation. Interestingly, topical application of HA4 without UVA also significantly increased SC water content.119) It is presumed that this increase may be caused by the moisture holding property of HA4 itself. However, it is also suspected that the HA4 that penetrated may increase water holding properties such as natural moisturizing factors in the SC in murine epidermis.
The scale bars indicate the relative value as the capacitance value of normal mice was 1.0. Values represent the means ± S.D. (n = 3). ** p < 0.01, Tukey’s post-hoc multiple comparison test. The amounts are expressed as mean values ± S.D. ** indicates p < 0.01 significant difference. Although the water content of the SC decreased following UVA irradiation, that decrease was significantly suppressed by treatment with HA4. Furthermore, the application of HA4 to untreated skin increased the water content of the SC from day 7 onwards.
Hematoxylin–eosin stained mouse skin tissue 28 d after HA4 treatment. A. Normal mouse skin without UVA; B. UVA-irradiated mouse skin with vehicle treatment (Control); C. UVA-irradiated mouse skin with HA4 treatment; D. Epidermal thickness on day 28. The scale bars indicate 100 µm length. Values are the means ± S.D. (n = 3). ** p < 0.01, Tukey’s post-hoc multiple comparison test. UVA irradiation increased the epidermal thickness, but the topical treatment with HA4 significantly suppressed the epidermal thickening.
From the result that topically applied HA4 prevents the murine epidermal thickness induced by chronic UVA irradiation may indicate that HA4 controls keratinocyte proliferation and differentiation. It has been reported that H-MW-HA induces epidermal cell differentiation mediated by activation of the predominant HA receptor CD44.124) Interactions between HA and CD44 induce intracellular signaling cascades that suppress cell adhesion, cell migration, cell proliferation, morphogenetic repair and wound healing.125–131) It has been reported that HA–CD44 interactions are involved in regulating keratinocyte differentiation, cholesterol synthesis and lamella formation, which are essential for normal SC structure and epidermal barrier function.126,129) In the skin of CD44-knockout mice, endogenous HA is almost undetectable and the expression levels of several differentiation markers such as involucrin and filaggrin are reduced.124) This means that both HA and CD44 have important roles in normal epidermal physiology and functions such as keratinocyte differentiation.
When cultured human keratinocytes were treated with HA4, the CD44-phosphorylated protein levels were significantly increased after 8 h.132) Furthermore, treatment of keratinocytes with HA4 increased protein levels of keratin 10 (K10, an early differentiation marker) on day 3, and those of involucrin and filaggrin (late differentiation markers) on day 9. H-MW-HA also increased protein levels of involucrin and filaggrin on day 9, but the protein level of K10 did not change on day 3. Thus, it is presumed that HA4 also induces keratinocyte differentiation through CD44, and this efficacy of HA4 might be higher than that of H-MW-HA.
As described above, HA4 can rescue the disruption of SC function caused by chronic UVA irradiation. It is well known that intercellular lipids are crucial components for barrier function and for SC water content. Ceramides account for approximately 50% of the intercellular lipids in the SC, and are involved in the skin’s moisture retention function133–135) and epidermal permeability barrier function.136–139) The decrease in the ceramides in the SC causes water loss and barrier dysfunction in the epidermis, including the loss of protection against antigens, including bacteria,139) and it can cause other skin problems such as atopic dermatitis.140–143)
Therefore, we examined the effects of HA4 on ceramide production. The mRNA expression levels of ceramide synthesis-associated enzymes were enhanced by the application of HA4 on cultured human keratinocytes.144) Similar results were obtained for H-MW-HA on this evaluation. One of the mechanisms by which skin function was restored by treatment with HA4 to skin damaged by UVA irradiation may be that HA4 enhances the production of ceramides.
The collagen, elastin, and HA exist in the dermis as ECM. The collagen and elastin give the skin elasticity, and the HA present in the gaps between those fibers retains water. By aging and exposed to UV, these ECM decrease, making the skin less elastic and less resistant to external stimuli and more susceptible to skin tears and bedsores.3,145–154) Thus, various treatments are applied to increase ECM. In cosmetic surgery, HA injections are often performed as a minimally invasive, non-surgical treatment that as an anti-aging medical treatment that seeks to rejuvenate a person’s appearance. The injection of cross-linked HA stimulates collagen synthesis, which partially restores the dermal matrix components that are lost in photo-damaged skin.155) Also, there are many components have been reported to inhibit the degradation or enhance the synthesis of ECM.156,157) Type I Collagen is a major type of fiber in the dermis. In our research, we have reported that the addition of HA4 increases gene expression levels of HAS1 and collagen type I alpha 1 chain (COL1A1) within 24 h in fibroblasts that produce collagen and HA.30) Treatment of the skin with HA4 stimulates dermal fibroblasts and may have anti-aging effects. Although HAS1 activity is low compared to other HAS,158,159) HAS1 synthesize H-MW-HA160) which has anti-inflammatory functions. Thus, increase in H-MW-HA caused by higher HAS1 activity prevents the inflammation in the dermis.36–40) It is presumed that the topically applied HA4 may suppress the inflammation caused by UVA irradiation by increase in H-MW-HA as one of the anti-inflammatory pathway in our study.119)
Although HA may be an excellent material for healthcare including dermatological treatments because its properties are involved in many biological processes, the decreased transparency of topically applied HA is a limitation of its treatment. However, HA4 has a better permeability through the SC and affects epidermal function. HA works on inflammation of the skin and our results that topically applied HA4 prevents the hyperproliferation and barrier perturbation caused by chronic UVA irradiation suggest that HA4 might be effective for treating skin diseases such as atopic dermatitis and psoriasis. Therefore, we are now investigating whether HA4 is effective against those skin diseases. Recently, the effectiveness of topical application of HA4 on atopic dermatitis and psoriasis has been confirmed using model mice. The increase in SC water content by topically applied HA4 was also observed in mice that had not been UVA irradiated, thus the efficacy of HA4 as a skin moisturizer not only retains water but also increases water holding materials such as natural moisturizing factors and/or intercellular lipids in the SC. Furthermore, in vitro research using keratinocytes and fibroblasts indicates additional biological functions of HA4 such as enhancing ceramide synthesis. In addition, EP (100 V, 10 ms, 50 times) treatment increased the amount of HA4 permeation by approximately 8-fold, and IP (cathodal IP, current density: 0.1 mA/cm2) treatment increased it by more than 7000-fold compared to passive diffusion.160) We reported that topically applied 0.1% HA4 improve cutaneous damage caused by chronical UVA irradiation.119) And it has been reported that treatment of higher intradermal dose of HA4 which was applied by intradermally or intraperitoneally injection (100 µg/mouse) improved skin symptoms of rosacea model mice induced by LL-37.161) Therefore, with the usage of skin penetration enhancing methods such as IP, the required efficacy of topically applied HA4 may be expected. The topically applied HA4 might improve cutaneous conditions by the normalizing and/or enhancing the keratinocyte metabolisms. We have found that 1 µg/mL HA4 induced keratinocyte differentiation in vitro.124) Furthermore, we have detected that the cumulative amounts of 0.1% HA4 through full-thickness skin after 8 h were 22 ng/cm2 on Franz cell analysis.119) Thus, approximately this amount of HA4 might affect keratinocytes during passing through the epidermal layers. Keeping HA4 much longer in the epidermal layer might be effective for higher efficacy on improving epidermal functions.
Although HA is quite an attractive material for healthcare including dermatological treatment, its decreased penetration limits the use of topical treatment with HA. However, the high transparency and multiple functions of HA4 may reduce that limitation and expand the usage of HA.
The authors declare no conflict of interest.
