in the industry include carboxylic acids [1–3], divalent metal-car- boxylic acid salts [4, 5], specially designed hydrogen bond builders [6] and cross-linkers [7, 8]. However, one of the most widely used classes of hair protein repair agents in the industry is hydrolysed proteins (peptides). Wool-derived keratin peptides are particularly popular. The use of proteins to repair damage in hair protein has substantial consumer appeal. Several studies have investigated the hair benefits provided by peptides. Gamez-Garcia (1993) showed that treatment with hydrolysed wheat proteins and wheat oligosaccharides modified the stress relaxation of virgin (undamaged) hair at high and low humidities [9]. Barba et al. [10–12] showed that wool keratin peptides can restore the sur- face properties, moisture content, tensile properties and water adsorption of bleach-damaged hair. Benaiges et al. [13] claimed that protein-rich plant extracts can improve the mechanical prop- erties of bleach-treated hair. Cruz et al. [14] have performed a very wide-ranging screening of 10–13 amino acid synthetic pep- tides and determined those peptides that bind well to hair ker- atins. Subsequent work showed hair benefits such as straightening for curly hair and volume for straight hair [15, 16]. Although the effect of proteins on hair fibres has been well stud- ied over recent decades, the majority of studies have looked at pro- tein effects on straight or wavy hair and on repair from bleaching or semi-permanent colorant-related damage. Little research has been performed with proteins on textured hair, or textured hair that has been chemically straightened. Textured hair fibres have a unique, highly curled and coiled struc- ture that causes increased tangling and grooming damage. The structure of these fibres also makes them more prone to fracture when stretched [17, 18]. Many consumers with textured hair use chemical treatments, including relaxers and perms, to straighten their hair. These treatments damage hair proteins, making the hair even more fragile [19]. Clearly, there is a consumer need for hair care products that can, somehow, restore these damaged hairs. This study investigated the effects of a series of wool-derived hydrolysed keratins on very curly hair that had been straightened with a sodium hydroxide-based relaxer treatment. There are relax- ers available that do not contain sodium hydroxide (no-lye relaxers which use lithium hydroxide and guanidine carbonate) however, we selected the relaxer that is popular in the region where the hair came from. The hydrolysed keratins varied in average MW and comprised of the following: (i) a low-MW hydrolysed keratin (221 Da), (ii) a mid-range MW hydrolysed keratin (2577 Da) and (iii) a high-MW hydrolysed keratin (75440 Da). Leucine (131 Da) was used as an amino acid reference treatment. Leucine and the respective hydrolysed keratins’ effects on the dimensional, mechani- cal and thermal properties of the hair were investigated using hair diameter measurements, single-fibre tensile and differential scan- ning calorimetry (DSC), respectively. Additionally, the penetration of the mid-range and high-MW peptides into the relaxed textured hair was investigated using fluorescence microscopy. Materials and methods Hair samples Single-source hair was collected from South African volunteers who had not previously used any chemical treatments. The L’Oreal classification [20] was used to assess hair curl type. The hair was assessed to have type VII curl. Relaxing procedure Hair was gathered into 0.5 g bundles. Each bundle was washed first with 15% w/v sodium lauryl ether sulphate solution in deionized (DI) water (pH 5.2) and dried at ambient conditions. Then, 5 g of relaxer (Sofn’ Free Creme Relaxer, M & M, South Africa) was applied to 0.5 g hair, combed through and left for 15 min, while massaging every 5 min. Hair was then rinsed for 2 min under a tap with a fixed water temperature and flow rate (IntellifaucetTM tap by Hass Manufacturing Company, Averill Park, NY 40°C, flow rate 3.8 L minÀ1). A total of 0.2 mL of pH 6 neutralizing shampoo (Softn’ Free, M & M, South Africa) was applied per 1 g hair, and massaged for 30 s, and rinsed for 30 s under a tap (40°C, flow rate 3.8 L minÀ1). The washing process was repeated one additional time, and the hair was left to dry under ambient conditions. Amino acid and keratin peptide ingredients L-Leucine (≥98% purity) was obtained from Sigma-Aldrich, (MO, USA). Aqueous solutions of low-MW (221 Da) hydrolysed keratin (INCI: hydrolysed keratin), mid-range MW (2577 Da) hydrolysed keratin (INCI: Aqua (and) hydrolysed keratin) and high-MW (75 440 Da) hydrolysed keratin (INCI: Aqua (and) keratin (and) hydrolysed keratin), respectively, were supplied by Croda (UK). Preparation of fluorescently labelled keratin peptides An amine reactive label, 5(6)-carboxytetramethylrhodamine N-suc- cinimidyl ester (TMR-SE, Sigma-Aldrich, USA), was used for label- ling the peptides, henceforth referred to as the label. Before labelling, hydrolysed keratin solutions were dialysed against phos- phate-buffered saline (PBS) using a dialysis cassette (Slide-a-lyzer, Thermo Scientific, USA) with a MW cut-off of 2000 Da to remove any amine-containing substances from the products. Hydrolysed keratin concentrations in dialysed solutions were determined using BCA protein assay (PierceTM BCA Protein Assay Kit, Thermo Scien- tific, USA) per manufacturer instructions. A total of 1 mg of the label was used per 10 mg peptide. For label-only control, the same amount of label was added to a sodium bicarbonate buffer without any peptides in it. The reaction was incubated for 1 h at room temperature with continuous stirring. The labelled peptides were further purified by dialysis or centrifugal filter unit (Amicon Ultra-15 Centrifugal Filter Units, Millipore Sigma, USA) to remove any unbound free label. Labelled peptides were used for hair soaking treatments to determine penetration. Penetration of labelled keratin peptides into hair fibres Penetration studies were performed on relaxed textured hair. Peo- ple with this hair type tend to use long-duration treatments, including those designed for overnight use (while wearing a cap). To understand penetration behaviour, three-hour soaking was selected for this study. A total of 10 mg of hair (~20–30 9 1-inch hair fibres) was soaked in 1.5 mL labelled keratin peptide solution such that hair fibres were fully immersed in the solution. Hair sam- ples were incubated for 3 h with gentle rocking. After incubation, hair was rinsed in DI water for 1 min and air-dried on a paper towel. Hair fibres were embedded as a bundle in OCT compound (Optimal cutting temperature, Sakura Finetek USA, Inc, Torrance, 2 © 2020 The Authors. International Journal of Cosmetic Science published by John Wiley & Sons Ltd on behalf of Society of Cosmetic Scientists and Societe Francaise de Cosmetologie International Journal of Cosmetic Science, 1–12 Larger hydrolyzed keratins reduce hair breakage E. Malinauskyte et al.