Development of keratin based hydrogels for biomedical applications

Synthesis of hydrogel was developed by using keratin protein extracted from a chicken feather. Further, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and starch have been commonly using for biomedical applications hydrogels due to their non-toxicity and, water solubility. The first sample, (KS-50) was mixed keratin, PVA/PVP and starch, the second sample, (K-50) was mixed the total weight of the (KS-50) without starch. KS-50 and K-50 were synthesized through cyclic freezing/thawing technique. Moreover, characteristics of the hydrogels, scanning electron microscopy (SEM) examined clearly exhibited porous structures, and the chemical structure of the compound was investigated by Fourier transform infrared spectroscopy (FTIR). The swelling degree of KS-50 exhibited slightly more pore size compared with K-50 hydrogels.


Introduction
The hydrogels are generally polymeric networks seen in cross-linked structures. They can be distinguished by their indistinct viscoelastic performance, hydrophilic nature. Elevation-water content facilitates the transfer the metabolic waste, nutrients, and oxygen [1]. Due to the presence of physical or chemical crosslinks like crystallites networks are collected from copolymers and homopolymers insoluble [2]. Modulating the polymer concentration and methods of synthesis allows tuning of specific hydrogel properties like biodegradability, swelling behavior, and porosity [3]. The hydrogels have been applied as scaffold materials the controlled release of drugs, proteins, and other applications [4][5][6]. Also, hydrogels are attractive for biomedical applications because of their diffusion properties/good water sorption biocompatibility [7].
Keratin turns into one of the most significant biomaterials through commercially available biomacromolecules due to its relatively good biocompatibility, excellent mechanical properties, high physicochemical, and good formatting-film ability [8][9]. Feather keratins from chicken feathers, that are considered to be the essential waste in the industry of poultry, because of coloring-dyeing and perming the control of the quality of the feather keratin is easier than hair keratin [10-11]. The featherweight of pure keratin is 90% feathers waste are a source of amino acids, protein, and nitrogen [12- [3]. Due to a great number of disulfide bonds, the keratin is a group of cysteine-rich structural proteins established in the cells of vertebrates that display high-rise mechanical strength [14][15][16]. Keratin has been utilized in several biomedical applications because of its biodegradability and biocompatibility [17][18].
Starch is one of the cheapest and most abundant polysaccharides. Also, the starch is a very cheap polysaccharide due to every starch derivatives are biodegradable and biocompatible polymers that will be utilized biomedical applications [19][20][21]. PVA based hydrogel is one of the famous polymer gel because of its good biocompatibility and utilized in several numbers of biomedical applications, such as, implants, dressings in wounds management, contact lenses, and drug delivery devices [22]. It has raised attention due to that it's watersoluble, non-toxic, and biocompatible properties [23][24][25]. PVP is one of the most common watersoluble, biodegradable, biocompatible, and low toxicity synthetic polymers [19,24]. It is utilized in many biomedical applications and separation processes to raise the hydrophilic character of the mixed polymeric materials [26].
This paper presents a methodology for the development of hydrogels, composed of keratin protein, PVA, PVP, and Starch by using physical crosslinking by the freeze-thawing method for potential biomedical applications.

The keratin protein Extraction from chicken feather
Keratin protein solution was prepared in the chemical engineering lab at University Malaysia Pahang by cleaning feathers according to previously studied methods. The 100g of cleaned, dried and blended chicken feathers were added in 1L of (1N) sodium hydroxide solution in a 2L of the conical flask. The temperature of the mixture of the solution was 50°C, pH was 12 and the mixture was continuously stirred for 4h and cool to room temperature. After that, the solution was filtered through filter stainless steel then centrifuged at 10,000rpm for 10min [27], [28]. The keratin protein was collected carefully and then was filtered through filter paper and of the solution was adjusted to pH 7 with HCl (2N) and stored in the laboratory bottle for the synthesis of hydrogels.

Preparation of Polyvinyl Alcohol (PVA) Solution
A mass of 10g PVA powder was weighed by using the analytical balance. Then, the powder was added into 100ml distilled water with stirring until PVA dissolved completely. In the same time, the temperature was kept constant in the range of 70-80°C [29]. The stirring process was done on a hot plate stirrer and continuously stirred until all the solutes dissolved thoroughly [30].

Preparation of Polyvinylpolypyrrolidone (PVP) Solution
A mass of 10g PVP powder was weighed by using the analytical balance. Then, the powder was added into 100ml distilled water with stirring until PVP dissolved completely. The stirring process was done manually by using a stirring rod for about 15 minutes [31].

Preparation of hydrogel film
The first sample, the keratin hydrogel (KS-50) was mixed with 50mL of keratin, 30mL of PVA 10mL of PVP, and 10g of starch with continuous stirring at 60 °C for 30min. The second sample, keratin hydrogel (K-50) was the total weight of the (KS-50) without starch and under stirred at 60°C for 30min. The proper amount of this mixture (15mL) were poured in plastic Petri dishes, followed by freezing at −20°C for 8h and thawing for 6h at 25°C for 3 continuous cycles to form hydrogel for further analysis [32], [33].

Scanning Electron Microscope (SEM)
Hitachi's Tabletop Electron Microscope (TM3030) was selected for carrying out the analysis related to the molecular structure of the study of the hydrogel samples [25].

Fourier Transform Infrared Spectroscopy (FTIR)
The Perkin-Elmer Model 1000 Series FTIR device was utilized to select spectra of the hydrogel. The frequency scope of the spectra was between 4000 cm −1 to 500 cm −1 . The all data obtained for hydrogel was through the utilizing of the software of FTIR [25].

Degree of the Swelling ratio
The hydrogel ability for fluid uptake was measured utilizing an electronic balance by immersion in phosphate-buffered saline (PBS). The hydrogels pieces were weighed ( ) and put in PBS at 37 °C, and pH 7.4. The swollen hydrogel discs were measured at 10, 20, 30, 40, 50, 60, 1440 minutes, the surface was wiped gently with blotting paper to eliminate surface-adsorbed fluid, and the hydrogels weighed again ( ). The degree of swelling defines the ability for fluid uptake by the following equation (1) [34]: Where is the initial weight of the disc at time 0, and is the weight of the disc at time t.

SEM analysis
SEM micrographs of the top view of the KS-50 (A) and K-50 (B) smooth with no particular structure. Figure 1, freeze-dried KS-50 and K-50 hydrogels clearly exhibited porous structures. Pore sizes can affect the physicochemical characteristics of the outcoming KS-50 and K-50 hydrogels, like the degree of swelling [35,26].

FTIR analysis
FTIR measurement was utilized to recognize the chemical structure of the hydrogel and the results are shown in figure 2. The attained results validate the presence of keratin chemical structure when compare to the previous study in which keratin extracted from the chicken feathers [28]. The prepared hydrogels demonstrated transmission bands of the peptide bonds (-CONH) and are known as Amide A (3298cm -1 ) ascribed to stretching vibration of -N-H and-O-H, Amide I (1651cm -1 ) which are related to C=O stretching bonds, Amide II (1427cm -1 ) is for N-H bending and C-H stretching, Amide III (1252cm -1 ) derived from C-N stretching and N-H bending [36], [37]. The closeness of hydrophilic and hydrophobic moieties which was confirmed by FTIR makes them soluble in both aqueous and organic medium. This made it essential for medication framework [38]. The vibration bands from 950cm -1 recognized to alcohol. The presence of more -OH gatherings contributed to hydrogen bonding in the hydrogel. Thus, FTIR spectra the similar peaks demonstrated the presence of chemical groups in hydrogels that they can improve their physical and chemical properties for various for potential biomedical applications.

Equilibrium degree of swelling
The swelling percentage of the KS-50 has increased only slightly with the time compared with the K-50 and the equilibrium swelling was reached at 50minutes for both KS-50 and K-50 as shown in figure  3. The size of pores is the major factor that controls the swelling degree of the hydrogel [39]. The sample could rapidly restore to the original state after being pressed, suggesting a sponge-like characteristic [40]. For most of the keratin hydrogels, the swelling properties are effective for biomedical applications.

Conclusion
In summary, the synthesis of keratin-based hydrogel was successfully developed using PVA/PVP/starch. Although the analysis of the results described good properties of hydrogel (K-50), the desired development was seen in the presence of starch (KS-50). FTIR spectra showed that peaks appeared in the hydrogel designating that they provide the macromolecular composition of the keratin, and SEM exhibited a large of pore sizes in the smooth surface. Moreover, the swelling percentage of the KS-50 has increased only slightly compared with the K-50 and the samples have developed like a