Characteristics of regenerated keratin and keratin-based film

Aim. Determination of the characteristics of regenerated keratin obtained from human hair, development of keratin-based films, and studing their surface morphology and adsorption capacity. Methods. Keratins were extracted by sulfitolysis. The structure of regenerated kera­ tin was studied by IR spectroscopy, thermogravimetry, and electrophoresis. Keratin-based films were made by casting. Their biocompatibility was analyzed in the adsorption test by incubation in human serum. Scanning electron microscopy was used to evaluate the surface morphology of the films. Results. The regenerated keratin is represented mainly by proteins of intermedi­ ate filaments and is characterized by better thermal properties than the native one. The keratin-based films had a pronounced relief structure. The adsorption test shows the ability of the films to adsorb albumin from human serum on their surface. Conclusions. The mild extraction of keratins by sulfitolysis ensures the preservation of their native properties and the ability of keratin-based films to form bonds with blood proteins.


Introduction
One of the most important innovative ap proaches in various fields for the development of sensors, the creation of optoelectronic ma terials, robotics, and biomedicine is the design of new materials. Special attention is now paid to the development of materials based on na tu ral biopolymers. Fibroin, elastin, chitosan, keratin are widely used in cell engineering and regenerative medicine due to their wide avai la bility, low toxicity, biocompatibility, and bioactivity [1][2].
One of the most promising biopolymers is keratin, which is characterized by a complex hierarchical structure of subunits -from α-chains through microfibrils to fiber (Fig. 1). At the molecular level, keratins differ from other structural proteins by a high level of disulfide bonds, which ensure the formation of V. V. Havryliak a compact threedimensional structure that is resistant to biological and chemical degrada tion [3]. It is known that the structure of keratin is similar to the extracellular matrix of biological tissues, due to which the biomaterials based on it are widely used as matrices for cell adhe sion and as a basis for a cellular support of native tissues [5].
A unique characteristic of extracted kera tins is their ability to selfassembly and self aggregation [6]. There is enough information in the scientific literature on the use of keratin for the development of nanofibers [7], hydro gels [8], films [9], 3D scaffolds for tissue engineering [10], nanocontainers for con trolled drug deli ve ry [11], biomaterials for wound healing [12], regeneration of nerve fibers [13], heavy metal binding [14]. There are many different methods for extracting keratin, but all of them are mainly based on the oxidation or reduction of disulfide bonds in its molecules [15]. The materials based on regenerated keratins can differ significantly in composition, structure, and properties, de pending on the specific source and methods of their extraction [16].
The purpose of our work was to investigate the biochemical characteristics of solubilized keratins obtained from human hair by sulfi tolysis, to develop films on their basis, and to determine their main characteristics.

Obtaining regenerated keratin
The hair samples were obtained from volun teers. This study was conducted according to the international bioethical norms, legislative documents of Ukraine, and protocol approved by the Committee for Bioethics of the Institute of Animal Biology of the National Academy Fig. 1. The ahelical structure of ke ratin [4] Characteristics of regenerated keratin and keratin-based film of Agrarian Sciences of Ukraine (№91, 05.08.2020).
For the extraction of keratin, the method of sulfitolysis was used. Sulfitolysis is the reversib le process of keratin extraction where sodium metabisulfite (Na 2 S 2 O 5 ) is used as a reducing agent. As a result of sulfitolysis, S-sulfonate residue (RSSO 3 ), and cysteine thiol (RS ) are produced. The sulfitolysis reaction is shown schematically in this formula: R -S -S -R + Na 2 SO 3 ↔ R -S + R -S -SO 3 - [17].
This method involves the use of 8 M urea, 0.1 M sodium dodecyl sulphate (SDS), and 0.5 M sodium metabisulfite (m-BS) in the composition of the extraction mixture [18].
In order to separate the keratin solution from the extraction mixture, the solution re sulting from extraction was filtered and dia lyzed for 3 days against deionized water using dialysis cassettes for the low molecular weight proteins and centrifuged at 12000 g for 20 min.
The protein content in the supernatant was determined by the colorimetric method, using the Bradford reagent [19]. 1 ml of Bradford reagent and 1 ml of the obtained protein extract were mixed, the mixture was then incubated for 30 minutes at room temperature and the absorbance was measured at a wavelength of 595 nm. To determine the concentration of keratin, a calibration curve was built, using the standard solutions of serum albumin (Sigma).
Protein electrophoresis was performed in 12.5 % polyacrylamide gel under denaturation conditions with SDS in the Laemmli buffer system [20]. After the electrophoretic separa tion, the gels were stained with the solution of 0.2 % Coomassie R-250 for 1 hour. The dye was washed with the solution of 7 % acetic acid in distilled water.
The protein solution was lyophilized and used as a regenerated keratin for the research.

Thermal analysis of the regenerated keratin
The complex thermal analysis of keratin was carried out on a derivatograph Q-1500 of the PaulikPaulikErdey system connected to a personal computer. The studies were performed in the temperature range of 20-400 °C, in the air atmosphere. The heating rate of the samples was 3 °C per minute. The thermal behavior of the native keratin was compared with that of the regenerated one. Aluminium oxide served as the reference substance.
In the given thermograms, the thermogravi metric curves (TG) reflect the weight loss of the samples during heating. The differential thermogravimetric curves (DTG) correspond to the rate of the weight loss of the samples and are the result of the differentiation of the TG curves. The differential thermal analysis (DTA) curves illustrate the thermal effects of the processes.

IR spectroscopy of regenerated keratin
Lyophilized keratin was compressed into tab lets with potassium bromide. IR spectra were recorded using a SPECORD 80 M spectro scope, which automatically detects IR trans mission spectra in the wavelength range of 4000 to 400 cm 1 .
Preparing the films 5 ml of 4 % solution of keratin were prepared in distilled water from the regenerated keratin adding 1 ml of 1 % aqueous solution of glyce rol as a plasticizer. The resulting protein glycerol mixture was poured in a thin layer with a thickness of no more than 3.0 mm into Petri dishes for cultivating cells, which were then placed in a thermostat for 24 h at a tem perature of 37 °C. After that, they were fixed with water vapour in a desiccator with a ground lid for 24 h. Water vapour fixation is one of the safest methods for stabilizing and shaping polymer biofilms. It is particularly important in producing biofilms for implantation in a living organism. Besides, such a method of fixation is easy to perform. The water vapour formed in the desiccator is then accumulated by hydrophilic glycerol, which leads to the film's swelling, thus making it more flexible [21,22].

Scanning microscopy of the film surface
The surface features of the films were studied using a REMMA102 scanning electron mic ro scope. Scanning the sample surface was carried out using an electron beam with a dia meter of a few nanometers and electron ener gy of 0.2-40 kV.

The study of the biocompatibility of the films based on regenerated keratin
The adsorption properties of the films were studied by electrophoresis in 12.5 % PAGE. The film fragments of approximately 0.6x0.6 cm 2 were cut out, placed in a special plate, and incubated with human blood serum for 5 min and 15 min in a thermostat at 37 ºС. After the incubation, the serum was collected in signed test tubes and used for electrophore sis as a contact serum.
After serum collection, all film fragments were washed in distilled water and placed on a new plate. These fragments were incubated in SDS for 5 min and 15 min in a thermostat at 37 ºC. After incubation, the SDS solution was collected in specially signed test tubes and used for electrophoresis as a desorbed protein solution. The contact serum was used to assess the binding of serum proteins to the keratin films, while the desorbed protein solution made it possible to estimate residual proteins on the keratin film from their initial amount in the contact serum [23].

Results and Discussion
The first stage of this work was to obtain regenerated keratin and to study its charac teristics. Figure 2 shows an electrophoregram of keratin obtained from human hair by sul fitolysis. As a result of electrophoretic anal ysis, two polypeptide chains with the mo lecular weights in the range of 40-65 kDa were identified. According to the available literature data, the proteins with such mo lecular weights belong to the proteins of intermediate filaments (IF) of types I -acid ic and ІІ -basic [5]. Intermediate filaments proteins are composed of 8 subunits of poly peptides that differ from each other in their molecular weight and electric charge. These polypeptides are found in all keratins, but the ratio of their components may vary. They are characterized by low sulphur content, have an alphahelix conformation, are localized in the fiber cortex, and are responsible for its strength [24].
In this case, the low molecular weight ke ra tinassociated proteins were not found. It is obvious that the method of sulfitolysis pro vides the predominant extraction of fibrillar proteins, which are often used in various areas of biomedicine due to their ability to the self organisation in complex structures. For the analysis of protein structure, the most interesting are the three bands in the in frared range of wavelengths, which correspond to the vibrations of the appropriate bonds pre sent in the peptide chain. These include the bands produced by valence vibrations of the NH (3300 cm 1 ) and C = O (1640-1660 cm 1 ) bonds present in IF of type I, and the band as sociated with the deformation vibrations of NH bonds (1520-1550 cm 1 ) present in IF of type II [25].
During the formation of the secondary structure of the protein, the energies of these three vibrations change. As a result, the shifts The first two bands, corresponding to the valence vibrations of NH and C = O bonds, are shifted to the region of lower frequencies. This is due to the fact that the formation of a hydrogen bond leads to the weakening of bonds in the amide and carbonyl groups be cause of the displacement of the nitrogen atom of the amide group and the oxygen atom of the carbonyl group towards the acceptor or proton donor, respectively.
The band of deformation vibrations of the NH bond, which is present in IF of type II, is shifted towards high frequencies. This is due to the fact that the hydrogen bond prevents the deformation of the NH bond in the amide group.
This indicates that the applied soft extrac tion methods allowed the obtaining of reduced keratin, which for its characteristics corre sponds to the native keratin of human hair.
The thermal stability of native and rege ne ra ted keratin was investigated using a complex thermal analysis, the results of which are pre sented in Fig. 4.
The weight loss (10.96 %) of native keratin in the temperature range of 20-180 °C corre sponds to the processes of keratin dehydration [26]. This process is accompanied by the ap pearance of an endothermic effect on the DTA curve and a clear extremum on the DTG curve. A slight weight loss (0.66 %) of native keratin in the temperature range of 180-206 °C can be explained by initial destructive processes in the keratin molecule. A shallow endothermic effect accompanies the weight loss of keratin on the DTA curve.
The intense weight loss of the native kera tin sample in the temperature range of 206-276 °C (19.48 %) corresponds to the simulta neous processes of thermal denaturation of a-helices of intermediate filaments of native keratin and pyrolysis of the matrix [27]. This process is accompanied by a clear endothermic effect on the DTA curve and a deep extremum on the DTG curve.
In the temperature range of 276-400 °С, deep destructive and thermooxidative proces ses take place in native keratin, which end with the combustion of destruction residues. An en dothermic effect appears on the DTA curve in A B Fig. 4. Thermogram of native (A) and regenerated (B) keratin this temperature range, which turns into a clear exothermic effect at higher temperatures. Fig.4 (B) shows the thermogram of the regenerated keratin sample. The appearance of shallow endothermic effects on the DTA curve in the temperature range of 20-170 °C, ac companied by a slight loss of mass (0.88 %), correspond[s] to the release of water residues and plasticization of the matrix. The slight weight loss (2.23 %) in the temperature range of 170-215 °C is accompanied by the appearan ce of a shallow endothermic effect, which corresponds to the initial destructive proces ses of regenerated keratin.
In the temperature range of 215-282 °C, the regenerated keratin undergoes profound de structive changes caused by denaturation of ahelices of IF and pyrolysis of matrix com ponents. The significant weight loss (14.13 %) and the appearance of a clear endothermic effect on the DTA curve correspond to these processes. In the temperature range of 282-400 °С, the deep destructive processes in the keratin molecule have been recorded, accom panied by thermal oxidation of [the] degrada tion residues. This is evidenced by a significant loss of sample mass (41.49 %) and the appearan ce of first endo-and then exothermic ef fects on the DTA curve.
Noteworthy, in comparison with native keratin, regenerated keratin is characterized by a higher thermal stability. The beginning of intense weight loss of regenerated keratin (215 °C), as compared with the native keratin sample (206 °C), is shifted to the region of higher temperatures. In the temperature range of 206-282 °C, regenerated keratin loses weight less intensively (14.13 %), as compared with native keratin (19.48 %).
An increase in the thermal stability of the regenerated keratin can be explained by a higher relative content of molecules of internal filaments, which, in comparison with the ma trix components, are noted by a higher thermal stability. The change in the composition of regenerated keratin predetermines the forma tion of a more homogeneous structure in it, accompanied by an increase in the amount of the crystalline phase [26].

The study of the films based on regenerated keratin
Keratins extracted from wool and human hair in aqueous solutions have the ability to self assembling, forming films. This type of kera tinbased biomaterials is successfully used in tissue engineering because they may support and improve cell growth, adhesion, migration, and proliferation, and can be applied for con trolled drug deliveries [28].
As can be seen from Fig. 5, the keratinbased film after stabilization in water vapour acquired a gellike consistency due to the abi li ty of keratin to accumulate moisture.  6 shows the surface of the film based on an aqueous solution of keratin, obtained using scanning electron microscopy. As it can be seen from the micrographs, the surface of the film is inhomogeneous, structured, and has a characteristic pattern.

The study of biocompatibility of keratin film
The protein adsorption analysis is an integral part of material biocompatibility studies [29]. Considering that the implanted material in the body is primarily in contact with blood, our task was to test the ability of the films based on the extracted keratins to form bonds with blood serum proteins.
It is well known that serum albumin quan titatively exceeds all other human blood pro teins. It has been established that the adsorp tion of albumin on the surface inhibits throm bus formation, whereas fibrinogen, in contrast, participates in blood clotting, promotes plate let adhesion and aggregation, and plays an important role in the processes of hemostasis and thrombosis [30]. Therefore, the enhanced adhesion of albumin against fibrinogen is ex tremely desirable for the successful functio na li zation of biocompatible coatings.
On the other hand, it is known that the ad sorption coefficients of albumin and fibrinogen are significantly related to the nature of the coating, because fibrinogen, being more hy drophobic, is mainly adsorbed on hydrophobic surfaces, whereas albumin being hydrophil ic -on hydrophilic surfaces during competi tive bonding [31]. Fig. 7 shows the results of electrophoretic separation of proteins adsorbed by the keratin glycerol film. As can be seen from the electro phoregram, the keratin-based film is suffi ciently hydrophilic and capable of absorbing a significant amount of albumin, as evidenced by a decrease in the band intensity in the mo lecular weight range of 60 kDa, after 5 and 15 minutes of the incubation in the blood se rum. Our results have shown predominant desorption of serum albumin after the incuba tion of the keratin film in the solution with sodium dodecyl sulfate, which is confirmed by a clear band with a molecular weight of 60 kDa. The detected bands in the range of 60-70 kDa may correspond to the molecular weight of hemoglobin. Additionally, the inten sity of protein bands with a molecular weight below 40 kDa was almost the same in all sam ples. These results indicate that keratinbased films do exhibit a selective ability to adsorb serum albumin.
Therefore, the obtained results indicate that the sulfitolysis method provides a predominant extraction of the keratins with the molecular weight in the range of 45-65 kDa. The obtained regenerated keratin is characterized by better thermal stability compared with the native one. The films, based on regenerated keratin with adding of glycerol, have an inhomogeneous relief surface and are characterized by the abi li ty to selectively adsorb albumin from serum. This indicates that our soft method for the extraction of keratins ensures the preservation of their native properties and the ability to form bonds with proteins of living organisms.

Funding
This research was not funded by any organiza tion.