OBTAINING HUMAN HAIR KERATIN-BASED FILMS AND THEIR CHARACTERISTICS

Conclusions. The polymer keratin films with the addition of glycerol demonstrated better mechanical properties and can be used in biomedicine.


INTRODUCTION
The number of studies of protein-containing compounds such as gelatin, collagen, albumin, silk fibroin and keratin for their use in biomedicine and engineering is constantly growing. This is due to the significant demand for natural and renewable materials, the production of which would be environmentally friendly and cost-effective [1,3,5,18]. Keratins are a potential raw material for the creation of biomaterials due to their biodegradability, self-assembly ability and low cytotoxicity [8].
One of the promising ways of the creation of biomaterials is the production of keratin films used in biomedicine [4,7,11], in particular as a substrate for cell cultivation. In this regard, keratins have successfully demonstrated the possibi lity of replacing polystyrene previously used for the manufacture of materials of this type [12]. According to literature, pre-treatment of substrates with keratins stimulates cell adhesion and proliferation. It has been shown that keratin films are used in particular for culturing mouse L929 fibroblasts [12].
Yamauchi et al. studied the physicochemical properties and biodegradation ability of keratin films [21]. According to their research, the combination of extracted keratin with glycerol promotes their biodegradability, makes the films transparent, strong and more flexible [21], and the combination with chitosan also gives them antibacterial properties [17,20]. Tonin et al. [19] demonstrated the prospects for combining keratin films with a synthetic polymer such as polyethylene oxide. The films of this type can be used not only as a framework for cell growth, but also as a membrane for drug delivery. For this purpose, keratins were combined with hydrotalcite nanoparticles containing diclofenac [14]. Such hybrid films, compared to those containing unbound diclofenac, were characterized by a lesser degree of porosity and swelling, but were more heat-resistant and released diclofenac better under physiological conditions than non-hybrid ones.
Based on literature analysis, one can conclude that natural polymers, in particular keratins, are a promising biomaterial with ta possibility of application in biomedicine and bioengineering.

MATERIALS AND METHODS
Only samples of pigmented hair, which had not previously been dyed with either natural or synthetic dyes were used. All hair samples were provided by healthy females aged 20 to 25 years with their written consent. The research was conducted in accordance with the decision of the Commission of bioethical examination of the Institute of Animal Biology of NAAS of 16.06.2020, protocol No. 83, which meets the requirements of bioethical examination according to the Order of the Ministry of Health of Ukraine No. 281 of 01.11.2000, European Convention for the protection of vertebrate animals used for experimental and other scientific purposes of 18.03.1986 andthe EEC Directive No. 609 of 24.11.1986. The experiments were conducted in the laboratory of metabolism named after Stepan Gzhytskyi.
The keratin extract obtained from human hair was used for the production of films. Hair samples weighing 0.5 g each were ground, washed in 1% sodium dodecyl sulfate (SDS) solution, rinsed with distilled water and dried at room temperature. For keratin extraction, we modified the method proposed by Nakamura [13] using dithiothreitol of the same concentration instead of 2-mercaptoethanol. Protein solubilization was performed in an extraction mixture consisting of 25 mM Tris-HCl, 5 M urea, 2.6 M thiourea and 250 mM dithiothreitol. Each hair sample was placed in 25 mL of the extraction mixture. Protein extraction was performed at a temperature of 60 °C at pH 8.5 for 72 hours. During this time, the samples were stirred several times with a glass rod for better contact of hair with the extraction mixture. The obtained extraction mixture was filtered and dialyzed against distilled water. The protein concentration in the solution was determined by colorimetric method using Bradford's reagent.
To determine the composition of the extracted proteins, we used PAGE in the Lemmly buffer system with the addition of SDS.
After electrophoresis, the gels were stained with a 0.2% solution of Coomassie R-250, and then washed with a 7% solution of acetic acid in distilled water.
Two types of films were prepared from the obtained proteins. The first type was a keratin-glycerol film prepared from a 4% solution of keratin in distilled water with the addition of 1% glycerol. The film of the second type consisted of only 4% aqueous solution of keratin. Suitable solutions were poured into Petri dishes in a thin layer that did not exceed 3.0 mm, and incubated in a thermostat for 24 h at 37 °C. After that, the films were fixed for 24 h in room temperature water vapor in a desiccator.
The features of the films surface were examined using a scanning electron microscope REMMA-102. The elemental composition of the films was determined using an X-ray microanalyzer.

RESULTS AND DISCUSSION
Keratins extracted from wool and human hair in aqueous solutions have the ability to self-assemble, forming the films [16], which has been used to study the structure and functions of self-organized keratins. Keratin-based films are successfully used in tissue engineering because they have the ability to support and improve cell growth, adhesion, migration and proliferation [3,4,12,16], as well as for controlled drug delivery [13,16,18].
Keratin-only films are very brittle and fragile [23]. Literature describes many ways of solving this problem [6,9,22]. In our experiments, we used glycerol to provide the films with elasticity. In addition, glycerol can be washed out of the film with aqueous solutions, if necessary [16]. Glycerol reduces intramolecular and intermolecular forces, thus improving the flexibility of the polymer. According to the studies of the effect of different concentrations of glycerol on the physical properties of keratin films, it was found that the use of this plasticizer in the amount of 0.09 g/g increases the tensile strength of the film by about 15 times [12], and has the greatest impact on moisture retention [10].
As a result of extraction of keratins using DTT as a reducing agent, we obtained a protein solution at a concentration of 3.75 mg/mL. DTT is used due to its ability to effectively restore disulfide bonds. As a result of such extraction keratins that retain the properties of native keratin are formed. It is very important for the production of biomaterials.
In Fig. 1, the electrophoretic profile of human hair keratins is cha racterized by the presence of two polypeptide chains with a molecular weight in the range of 40-50 kDa, which correspond to type I and type II proteins of intermediate filaments (IF) with low Sulfur content.
In the low molecular weight region, bands of proteins with a molecular mass of 10-30 kDa were detected, which are sulfur-rich keratin-associated proteins (KAP). After the composition of the extracted keratins had been studied, the obtained protein solutions were used for the production of polymer films. Depending on the purpose of production of polymeric films they can be combined with both synthetic and natural materials. In the research, we used glycerol as a safe and cheap plasticizer. We also made a polymer film without the addition of plasticizer for comparison. Fig. 2 shows keratin films after incubation in a thermostat at the temperature of 37 °C. The method of stabilizing keratins extracted from human hair with water vapor was tested for the first time. As can be seen from Fig. 2, the film based on keratin and glyce rol (A) acquired a gel-like consistency after stabilization in water vapor, which is explained by the property of glycerol to accumulate moisture, as mentioned above. At that time, the film made only from an aqueous solution of keratin (B) was represented by a thin polymer layer, which was easily torn when trying to separate it from the Petri dish, which was not typical of keratin-glycerol film. This confirms that glycerol improves mechanical properties of the film.
In Fig. 3, scanning electron microscopy shows the surface of the film made by combining a 4% aqueous solution of keratin with the addition of 1% glycerol (A) and the surface of the film made without the addition of glycerol (B). As can be seen from Fig. 3A, the surface of the film is mostly homogeneous, without cavities and holes, with a small number of built-in granules of different shapes. The presence of single cracks may be due to a large number of intermolecular bonds [2]. The surface the film without glycerol is not homogeneous and has a large number of recesses and protrusions (Fig. 3B). Fig. 4 shows the chemical composition of the film made of 4% reduced keratin solution with the addition of 1% glycerol (A) and without glycerol (B). Qualitative analysis revealed that differences between both types of film are nearly indistinguishable. We detected such elements as Sodium, Silicon, Sulfur, Potassium. Among the studied elements, Sulfur has the largest share, while the other elements have much lower shares. High Sulfur content is caused by the presence of a large number of disulfide bonds in the keratin molecule, mainly due to cysteine residues.
Apart from Sulfur, another important element is Silicon that provides hardness to keratinized tissues and promotes their high chemical resistance.
The Table shows the content of elements of both types of films. This elemental composition is characteristic of the regenerated keratin. CONCLUSIONS 1. Protein extraction was performed using a modified method of Nakamura using DTT as a reductant in the extraction mixture. The chosen method of solubilization of keratins allows for obtaining IF proteins of type I and II with a molecular weight of 60-70 kDa and low molecular weight KAP with a molecular weight of 10-30 kDa. The fibrillar structure of these proteins makes them a potential raw material for the production of biomaterials. 2. The polymer film made on the basis of protein extract with the addition of glycerol demonstrated better mechanical properties and homogeneous structure, in contrast to the film made without the addition of glycerol. Improved mechanical properties of the film facilitate their application. In addition, the safety of glycerol, which we used as a plasticizer, allows for the use of keratin films in biomedicine. 3. The method of stabilization of films made on the basis of keratins of human hair with the help of water vapor was tested. 4. The results of X-ray microanalysis of films indicate a high presence of Sulfur. Apart from Sulfur, such elements as Potassium, Sodium and Silicon were detected, which is characteristic of the elemental composition of the regenerated keratin.

COMPLIANCE WITH ETHICAL STANDARDS
Human Rights: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards