Hair is a specialized derivative of the skin that is ideally organized to protect the human scalp. The well-known structure of hair consists of an external cuticle composed of overlapping scales that acts as a barrier to protect the inner structure. The central medulla is surrounded by the cortex, which is mainly composed of the protein keratin [1]. This protein plays an important role in the physical and mechanical properties of hair. At the molecular level, keratin is a helical protein composed of two types of fibers: type I, with acidic amino acid residues, and type II, with basic amino acid residues. One strand of each type combines to form a coiled-coil dimer. These dimers coil together in antiparallel tetramers known as protofilaments. Finally, the protofilaments interact to form a single intermediate filament organized into micro-fibrils and larger macro-fibrils [2]. The conformation of the keratin chain is controlled by hydrogen bonds, ionic forces, Van Der Waals interactions, and disulfide bonds. The disulfide bonds, originating from sulfur-containing cysteine residues, create strong crosslinks between adjacent chains, giving each individual’s hair its unique shape. Indeed, the more α-helical conformation the keratin has, the greater the curve in the protein chain, and the curlier the hair fiber. Conversely, a β-sheet conformation in the keratin decreases the formation of disulfide bonds and produces smoother hair fibers [3]. In the haircare industry, many products have been developed to modify the disulfide bonds, with the aim of shaping or straightening the hair. These include chemical agents such as alkaline reducing agents, thioglycolates, or guanidine, which are used to break and rearrange bonds. Unfortunately, all these products damage the hair and reduce its strength [4]. As a consequence, alternative, safe, active ingredients that smooth without inducing damage to the hair, the scalp, or the environment, are actively sought.

Hyaluronic acid (HA) is a key active ingredient in skincare products, but its benefits for hair have been little described. To date, interactions between HA and keratin have been described in the stratum corneum of the skin, where it is known to enhance skin hydration [5]. In previous work, we first posed the hypothesis that HA can also interact with keratin in hair fiber, promoting keratin interconversion from α-helix to β-sheet conformation. Our data showed an increase of β-sheet conformation and a significant reduction in disulfide bonds after applying HA, thereby delivering an anti-frizz property [3]. This interconversion was already observed during mechanical or chemical hair straightening, which leads to hair smoothing and ultimately produces an anti-frizz effect [4]. Due to the cuticle on hair fibers, only low molecular weight molecules (˂ 10 kDa) have been found to penetrate the cortex [6]. For molecules > 500 kDa, the penetration may only occur when the cuticle is damaged, e.g., after bleaching [6]. In addition, no evidence of interaction of HA deep inside the hair has yet been established; exploring the penetration of such molecules into hair fibers presents several challenges. A range of techniques, including confocal fluorescence microscopy, have been used in attempts to examine the penetration of tagged molecules, but autofluorescence consistently compromises results [7]. Raman spectroscopy is a promising method for this type of evaluation. This non-invasive technique detects the characteristic vibrational energy levels of molecules, and provides structural information. In confocal mode, it can gather information at various depths inside a tissue, and quantification is possible because inelastic Raman scattering correlates linearly with molecule concentration [8]. In a previous study, we were able to track HA penetration ex vivo through the skin using this technique [9]. Other studies performed on natural and intact human hair fibers demonstrated that confocal Raman spectroscopy can be used to investigate the influence of cosmetic ingredients on keratin structure. An example is the evaluation of a hair-smoothing or heat-protecting effect by analyzing α-helix and β-sheet keratin conformation and the S-S disulfide bridges [10].

In the present study, we investigated whether Raman spectroscopy could be used to measure HA penetration in hair, and to assess the benefits of a blended and optimized HA formulation for use in haircare products.

The aim of this study was to develop a method to measure HA penetration in hair, and to assess the benefits of a blended and optimized HA formulation for use in haircare products.

We established an innovative method to demonstrate the penetration of hyaluronic acid into the hair fiber in rinse-off conditions. UV irradiation damage is a leading cause of hair protein loss and color change [11]. Using UVA and UVB irradiation, we damaged the hair to open the cuticle and enhance product penetration. The semi-quantification of the total Raman signal showed an increase in the quantity of LMW, HMW, and placebo (shampoo without the active substance) detected in damaged hair (over undamaged hair) (Data not shown). It is interesting to note that the total quantity of HA-Blend detected in irradiated hair fibers is the same but the penetration profile is different, with a lower quantity of HA-Blend in the cuticle and a higher amount in the cortex. That could suggest that the penetration of HA-Blend is already optimal in undamaged condition. We have thus compared the penetration of HA in normal or damaged hair locks to gain a greater understanding of how the product works. When using non-irradiated hair locks, the three active shampoos (LMW, HMW, and HA-Blend) had a similar ability to penetrate the cuticle; they were consistently better than the placebo shampoo. In contrast, when examining the cortex, the HA-Blend outperformed all the other formulations. With UV-damaged hair, penetration of the HA-Blend into the cuticle was significantly reduced, but penetration into the cortex was still significantly higher than that of the placebo or the two other active shampoos. This apparent discrepancy is due to the fact that when hair has undergone UV-induced damage, the placebo more readily penetrates the cuticle, whereas hydrophilic compounds such as HA bind more tightly to proteins such as keratin, which is in the cortex [12]. In other words, once the HA-Blend penetrates the cuticle in damaged hair, this optimal formulation may be attracted to the keratin in the cortex, leaving only small amounts on the cuticle to repair the cortex. Even in the non-irradiated condition, only the HA-Blend significantly penetrated the cortex, due to its optimized ratio of low- to high-molecular weight HA formulation, and its capacity to bind to keratin. Besides, the optimal process behind the HA-Blend involved the addition of lactic acid to reduce the pH of the formula. It has been found that pH plays a critical role in molecule absorption and can have opposite effects depending on the initial ionization of the molecule (a cationic or anionic compound). Indeed, change in pH could lead to structural changes in keratin including changes in the binding affinity and the number of available binding sites [12]. At pH > 6.0, hair keratin is negatively charged and attracts positively charged molecules. Inversely, at pH 2.0, cationic species showed a low level of binding to keratin due to the electrostatic repulsion [12]. HA is an anionic compound and we hypothesize that acidification of the pH induced by lactic acid improved binding of HA-Blend to keratin.

By binding to the keratin, this optimal HA-Blend modified the keratin conformation, decreasing α-helices and promoting the formation of β-sheets, leading to smoothing of the hair. These data corroborated results from our previous study, in which analyses showed that this HA-Blend decreases the number of disulfide bonds in keratin and induces a change in the α-helix/β-sheet ratio [3]. The results presented here establish that the change in keratin conformation is correlated to the effective penetration of the HA-Blend, allowing a direct interaction between HA and keratin. This anti-frizz potential was confirmed in ex vivo experiments, with the HA-Blend producing a significant smoothing effect 11% better than the placebo, thereby resulting in a visible benefit.

Current straightening methods, involving the thermal method (with an iron) or chemical agents, have been found to damage hair by reducing its physico-mechanical properties [13]. Those treatments break and rearrange disulfide bonds, change the structure of keratin in favor of a β-sheet conformation, and disrupted cuticle [14,15]. We have shown that HA can straighten the hair and change the keratin conformation without damaging the fiber, as shown by the tensile-strength analysis. Indeed, we have demonstrated that HA treatment smoothed the hair while improving the elastic modulus and the maximum strength needed to break a fiber by filling damaged hair from the inside. We have demonstrated that the HA-Blend is also able to repair keratin integrity after UV irradiation.

Moreover, thermal straightening has been found to lead to a reduction of water content into the hair fiber [16]. The authors of that paper explained that the changes in protein conformation and a reduction of the α-helix conformation may change the water accessibility. We have shown that even if HA interacts with keratin and changes its conformation, it is able to significantly increase water content in the fiber. This interaction between HA and keratin has already been described in the skin to provide hydration and maintain skin-barrier integrity [5]. We hypothesize that the damage caused at the cuticle level by thermal straightening is mainly responsible for water loss, but using high molecular weight HA in the HA-Blend coats the fiber and improves hair hydration while smoothing the fiber.

Our results demonstrated that Confocal Raman Spectroscopy is a powerful, non-invasive technique to investigate the penetration of cosmetic ingredients into human hair fibers. To the best of our knowledge, this study is the first evidence of HA penetration into hair fibers, and the first comparison of the penetration of different molecular weights of HA. Our results show that an optimized formulation containing different molecular weight HA plus lactic acid penetrates deeply into the hair cortex to interact with keratin and exert a smoothing, hydrating, and reparative effect. This insight opens new doors for the haircare industry to understand the mechanisms of action of active molecules for applications in haircare.

This optimal blend is protected by the international patent application WO 2018/162604.