Pure Appl. Chem. 2017; 89(12): 1829 1839 Conference paper Alina Sionkowska*, Beata Kaczmarek, Marta Michalska, Katarzyna Lewandowska and Sylwia Grabska Preparation and characterization of collagen/ chitosan/hyaluronic acid thin films for application in hair care cosmetics https://doi.org/10.1515/pac-2017-0314 Abstract: In this study thin films based on a blend of collagen, chitosan and hyaluronic acids were prepared and their surface and mechanical properties were studied. The structure of the films was studied using FTIR spectroscopy, contact angle measurement and AFM images. Swelling and mechanical analyses were also performed. The hair protection possibility of collagen/chitosan/hyaluronic was studied using SEM microscopy and the mechanical testing of hair coated by the blends. It was found that the addition of hyaluronic acid to a collagen/chitosan blend improves the mechanical resistance of biopolymeric films. Samples with the addition of hyaluronic acid were more stable in aqueous conditions and provided higher roughness of surface. Keywords: chitosan; collagen; cosmetic; hair; hyaluronic acid; NICE-2016. Introduction Biopolymers are widely used in the biomedical and cosmetics field. Biopolymers which are components of the skin or hair in living organisms are very important for cosmetic application. Single biopolymers, such as collagen, keratin, chitosan and hyaluronic acid are widely used in cosmetic formulations. A new approach in cosmetic science could be the preparation of new materials based on the blends of two or more biopolymers based on the molecular interaction between them. Blending two or more polymers together may improve the properties of the material in comparison with one component [1]. In fact, biomaterials based on the blends of two polymers and/or biopolymers are already used in the medical field [1 4]. The aim of this work was to create biopolymer films based on the blends of collagen, chitosan and hyaluronic acid. It is worthy of study in order to investigate whether such a blend can offer any added value to the conditioning of hair. Collagen is the most abundant protein in a mammalian s body, where it constitutes one-third by weight of all body protein tissue [5 7]. For cosmetic application collagen from mainly fish skin is used [8, 9]. Chitosan is a cationic polyelectrolyte copolymer derived from chitin. Chitin is a homopolymer comprised of 2-acetamido- 2-deoxy-β-D-glucopyranose units. The majority of units in chitosan chains exist in a deacetylated form as Article note: A collection of invited papers based on presentations at the 3 rd International Conference on Bioinspired and Biobased Chemistry and Materials: Nature Inspires Creativity Engineers (NICE-2016), Nice, France, 16 19 October 2016. *Corresponding author: Alina Sionkowska, Nicolaus Copernicus University in Torun, Faculty of Chemistry, Department of Chemistry of Biomaterials and Cosmetics, Gagarin 7, 87-100 Torun, Poland, e-mail: as@chem.umk.pl Beata Kaczmarek, Katarzyna Lewandowska and Sylwia Grabska: Nicolaus Copernicus University in Torun, Faculty of Chemistry, Department of Chemistry of Biomaterials and Cosmetics, Gagarin 7, 87-100 Torun, Poland Marta Michalska: Nicolaus Copernicus University in Torun, Faculty of Chemistry, Department of Chemistry of Biomaterials and Cosmetics, Gagarin 7, 87-100 Torun, Poland; and Nicolaus Copernicus University in Torun, Faculty of Biology and Environmental Protection, Department of Environmental Microbiology and Biotechnology, Lwowska 1, 87-100 Torun, Poland 2017 IUPAC & De Gruyter. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. For more information, please visit: http://creativecommons.org/licenses/by-nc-nd/4.0/
1830 A. Sionkowska et al.: Preparation and characterization of collagen/chitosan/hyaluronic acid 2-amino-2-deoxy-β-D-glucopyranose. When chitin is deacetylated to at least 50 %, it becomes soluble in dilute acids and is referred to as chitosan [10 12]. Hyaluronic acid (HA) is a type of non-sulfated glycosaminoglycan composed of N-acetyl glucosamine and D-glucuronic acid [13]. HA is widely distributed in an extracellular matrix (ECM). Hyaluronic acid provides suitable viscoelastic properties, has good biocompability and biodegrability and it is used as a biomaterial [14]. Collagen is miscible with chitosan due to several interactions and such a blend exhibits film-forming properties [15]. Research on collagen and chitosan blends has been done with interesting results having already been found thus far [16, 17]. It was also found that the mixture of collagen and hyaluronic acid with the addition of chitosan exhibit film-forming properties. Collagen/chitosan/ hyaluronic acid films were made as a two layer film, however the homogenous mixtures were not studied [18]. The film-forming properties of blends with a wide range of composition have not been studied yet for hair treatment. So far we have studied the miscibility of collagen, chitosan and hyaluronic acid in solution by viscometry technique and in the case of such ternary blends, the polymeric components showed some miscibility [19]. The study of the blends by AFM showed different morphology and wettability when comparing with that of pure components [19]. In this work the properties of hair covered by thin films made of the blend of collagen/chitosan/hyaluronic acid were studied. Materials and methods Films preparation Collagen (Col) type I was obtained in our laboratory from rat tail tendons. Tendons were washed in distilled water and dissolved in 0.1 M acetic acid for 3 days at 4 C, the non-dissolved parts were removed by centrifugation for 10 min at 10 000 rpm. Completely frozen mixtures were lyophilized at 55 C and 5 Pa for 48 h (ALPHA 1 2 LD plus, CHRIST, Germany). A 1 % solution was prepared by dissolving collagen in 0.1 M acetic acid. Chitosan (CS) (low molecular weight, Sigma) and hyaluronic acid (HA) (sodium salt from Streptococcus equi, Sigma) were dissolved in 0.1 M acetic acid to obtain a 1 % solution of each. Chitosan and collagen were mixed together in the volume ratios, and. To each solution 1, 2 and 3 % of hyaluronic acid was added. Mixtures without HA were left as a control samples. Samples were dried at room temperature until solvent evaporated. FTIR spectroscopy The interactions between functional groups of polymers were evaluated by attenuated total reflection infrared spectroscopy using Nicolet is10 equipment. All spectra were recorded by absorption mode at 4 cm 1 intervals and 64-times scanning. The absorption values were obtained in the range of 400 4000 cm 1. Mechanical properties Mechanical properties of films were tested by using Zwick and Roell testing machine. Parameters of testing program: 200 mm/min speed starting position, 0.1 MPa initial force, 5 mm/min the speed of the initial force. Samples were cut with a scalpel and the tensile strength as well as Young Modulus of each film were measured. Surface properties The surface properties of the polymeric films were observed using an atomic force microscope. Topographic was performed in air using a commercial AFM a MultiMode Scanning Probe Microscope Nano-
A. Sionkowska et al.: Preparation and characterization of collagen/chitosan/hyaluronic acid 1831 scope IIIa (Digital Instruments Veeco Metrology Group, SantaBarbara, CA) operating in the tapping mode in air with the resolution 1.7 μm. Roughness calculations were made using the NanoScope Analysis program (v. 1.40). Contact angle measurements The contact angle of two liquids: diiodomethane and glycerol on polymeric films was measured. All analyses were made at room temperature. The surface free energy was calculated using the Owens-Wendt method, as this method is commonly used for the calculation of polar and dispersive components of surface free energy [20]. Swelling test The test was performed according to the instruction proposed previously [21] with slight modifications. Pieces of each of the films were weighed (W d ) on the analytical balance and then immersed in 5 ml of phosphate buffer saline (PBS, ph = 7.4) for 1, 2, 3 and 24 h at room temperature. After removal from PBS, samples were placed on filter paper to remove excess fluid and were weighed again (W w ). The materials ability to absorb water was assessed according to the following equation: Swelling ratio = [(W W)/W ] 100 (1) w d d Treatment of hair The film forming properties of the blends on hair surface were measured by immersing the curly brown human hair of a 26 year old volunteer in polymer solution for 1 h and drying for 24 h at room temperature and humidity. Mechanical tests for hair were made for hair covered with polymeric films and without such treatment. The mechanical properties of films were tested by using a Zwick and Roell testing machine with 5 mm/min speed starting position and 0.1 N initial force. Analysis was repeated for 10 samples and the standard deviation was calculated. Scanning electron microscopy The surface of human hairs was studied using scanning electron microscope (SEM) (LEO Electron Microscopy Ltd, England). Samples were covered by gold and images were made with the resolution 50 μm. The thickness of hair shafts was calculated for native hair and for hair covered with the polymer film. Results and discussion FTIR spectroscopy FTIR spectra in the range of 1000 1650 cm 1 contain absorption bands related to the chemical structure of biopolymers studied and structure features of the obtained blends based on collagen/chitosan/hyaluronic acid. FTIR analysis of chitosan/collagen composites allows us to distinguish characteristic groups for chitosan and collagen. Peaks at 3300 cm 1 (Amide A), 1630 cm 1 (Amide I), 1550 cm 1 (Amide II) and 1085 cm 1
1832 A. Sionkowska et al.: Preparation and characterization of collagen/chitosan/hyaluronic acid (C O C) are usually observed. The IR spectra of collagen/chitosan blends are presented in Fig. 1. After 3 % addition of hyaluronic acid to the collagen/chitosan blend one can observe changes at 1401 cm 1 (C=O) and 1054 cm 1 (C O). Such alterations in infrared spectrum are due to the presence of characteristic groups of hyaluronic acid. Small differences in the position of the main peaks of chitosan and collagen can be observed after mixing with hyaluronic acid which is possibly a consequence of interactions between polymers. Usually, a small shift is observed when functional groups of biopolymers are involved in hydrogen bonding between them. Mechanical properties Mechanical properties such as Young s Modulus and tensile strength were measured for each kind of sample. Results with standard deviations are presented in Figs. 2 and 3. The addition of hyaluronic acid has different influence on the mechanical parameters depends on the collagen/chitosan ratio. For the Col/CS mixture in ratio the 1 % hyaluronic acid addition improves the Young s Modulus as well as tensile strength. However, 2 and 3 % addition decreases them. The 1 % addition of hyaluronic acid to the Col/CS mixture in ratio improves the Young s Modulus, but for the 2 or 3 % hyaluronic acid addition the decrease of the mechanical parameter value is observed. For the third combination of Col/CS (the mixture in ratio) 0.4 Absorbance 0.3 0.2 Col/CS/3HA Col/CS 0.1 1000 1100 1200 1300 Wavenumber (cm 1 ) 1400 1500 1600 Fig. 1: FTIR spectra of collagen/chitosan (Col/CS) composites in ratio 50/50 with 3 % addition of hyaluronic acid (Col/CS/3HA) and without it. 3 2.5 Young modulus (GPa) 2 1.5 1 0.5 0 Col/CS Col/CS/1HA Col/CS/2HA Col/CS/3HA Fig. 2: Young modulus (E mod ) [GPa] of collagen and chitosan composites (Col/CS) in different ratios (, and ) with 1 % (Col/CS/1HA), 2 % (Col/CS/2HA) and 3 % (Col/CS/3HA) addition of hyaluronic acid.
A. Sionkowska et al.: Preparation and characterization of collagen/chitosan/hyaluronic acid 1833 80 70 Tensile strength (MPa) 60 50 40 30 20 10 0 Col/CS Col/CS/1HA Col/CS/2HA Col/CS/3HA Fig. 3: Tensile strength (F max ) [MPa] of collagen and chitosan composites (Col/CS) in different ratios (, and ) with 1 % (Col/CS/1HA), 2 % (Col/CS/2HA) and 3 % (Col/CS/3HA) addition of hyaluronic acid. the improvement of mechanical parameters values were noticed after the 2 % addition of hyaluronic acid compared to the blend without hyaluronic acid. The results may suggest that the influence of hyaluronic acid addition on the collagen/chitosan films stiffness is different. Surface properties The hydrophilicity of the surface of polymeric films made of collagen/chitosan with and without the addition of hyaluronic acid was measured by contact angle measurements. Two different liquids were used in this experiment: glycerin and diiodomethane. On the basis of the contact angle measurements, surface free energy and its polar and dispersive components were calculated by Owens-Wendt method. The results are presented in Table 1. An increasing amount of chitosan content in the blend results in the increase of surface p d Table 1: Surface free energy (γ) and its polar ( γ ) and dispersive component ( γ ) of chitosan/collagen composites (Col/CS) in s s the weight ratio 72:25,, and with 1 % (Col/CS/1HA), 2 % (Col/CS/2HA), and 3 % (Col/CS/3HA) addition of hyaluronic acid. Specimen Surface free energy (mj/m 2 ) (γ) Dispersive component (mj/m 2 d ) γ s Polar component (mj/m 2 p ) γ s Col/CS 26.03 ± 0.43 25.88 ± 0.11 0.15 ± 0.07 Col/CS/1HA 23.89 ± 0.21 23.79 ± 0.13 0.10 ± 0.11 Col/CS/2HA 24.40 ± 0.17 24.28 ± 0.08 0.12 ± 0.14 Col/CS/3HA 25.07 ± 0.19 25.00 ± 0.21 0.07 ± 0.15 Col/CS 28.41 ± 0.25 24.52 ± 0.07 3.88 ± 0.09 Col/CS/1HA 27.55 ± 0.19 25.75 ± 0.13 1.97 ± 0.05 Col/CS/2HA 27.33 ± 0.11 25.05 ± 0.10 2.31 ± 0.10 Col/CS/3HA 28.13 ± 0.15 26.76 ± 0.17 1.37 ± 0.06 Col/CS 28.60 ± 0.17 26.71 ± 0.09 1.89 ± 0.03 Col/CS/1HA 28.42 ± 0.22 26.68 ± 0.12 1.74 ± 0.06 Col/CS/2HA 29.13 ± 0.34 28.07 ± 0.14 1.06 ± 0.04 Col/CS/3HA 28.71 ± 0.19 26.87 ± 0.20 1.84 ± 0.08
1834 A. Sionkowska et al.: Preparation and characterization of collagen/chitosan/hyaluronic acid Table 2: Swelling test for composites of collagen/chitosan (Col/CS) in the weight ratio 72:25,, and with 1 % (Col/CS/1HA), 2 % (Col/CS/2HA) and 3 % (Col/CS/3HA) addition of hyaluronic acid. Specimen Swelling (%) 0.5 1 2 3 24 Col/CS 84.61 84.65 169.01 430.07 220.00 Col/CS/1HA 184.43 130.43 105.88 130.09 242.50 Col/CS/2HA 133.33 121.04 154.70 179.22 229.85 Col/CS/3HA 203.72 164.12 187.14 198.16 225.00 Col/CS 65.86 74.06 160.34 415.71 185.71 Col/CS/1HA 115.76 131.70 117.88 338.23 155.55 Col/CS/2HA 87.22 267.64 149.23 221.43 145.71 Col/CS/3HA 179.43 179.68 210.89 181.45 105.12 Col/CS 114.21 189.95 180.03 552.51 240.63 Col/CS/1HA 113.22 134.04 188.43 164.29 101.25 Col/CS/2HA 109.75 181.91 187.05 147.22 92.86 Col/CS/3HA 154.09 172.01 184.52 212.05 124.42 free energy; however, for the mixture of Col/CS in ratio the change is not statistically significant. After addition of hyaluronic acid the surface free energy decreases. The polar and dispersive components values depend on the blend composition. The values of the polar component of surface free energy decrease with an increasing amount of HA in the blend. Hyaluronic acid contains a number of carboxylic and hydroxyl groups which can form hydrogen bonds with water as well as with collagen and chitosan [22]. As a result of such interactions the hydrophilic character decreases due to the lower number of free carboxylic and hydroxyl groups present on the surface. Swelling test The swelling behavior of collagen/chitosan films with and without addition of hyaluronic acid is shown in Table 2. Films were immersed in PBS solution for 0.5; 1; 2; 3; 24 h and they were taken out of the solution and put between two absorbing papers. After gently drying, the samples were weighed. Swelling tests allow us to observe changes in the blends behavior in aqueous conditions. Addition of hyaluronic acid improves the swelling in time initially, nevertheless after 24 h the films swelling decreases compared to the samples without it. Atomic force microscopy The topography of the film surface was detected by atomic force microscopy. The images made for composites of collagen/chitosan composites in ratio 50/50 with 1, 2 and 3 % addition of hyaluronic acid in 2 and 3D scales are shown in Fig. 4. Moreover, the roughness was calculated and listed in Table 3. As the results show, the addition of hyaluronic acid increases the roughness of the film surface. It may cause the improvement of the surface contact area with any agent to be applied to the top of the film.
A. Sionkowska et al.: Preparation and characterization of collagen/chitosan/hyaluronic acid 1835 Fig. 4: AFM images of composites (a) chitosan/collagen in ratio 50/50, (b) chitosan/collagen in ratio 50/50 with 1 % addition of hyaluronic acid, (c) chitosan/collagen in ratio 50/50 with 2 % addition of hyaluronic acid, (d) chitosan/collagen in ratio 50/50 with 3 % addition of hyaluronic acid. Table 3: Roughness of chitosan/collagen composites in ratio 50/50 (Col/CS) with 1 % (Col/CS/1HA), 2 % (Col/CS/2HA) and 3 % (Col/CS/3HA) addition of hyaluronic acid and without it. Blend R q (nm) Col/CS 18.1 Col/CS/1HA 42.4 Col/CS/2HA 59.5 Col/CS/3HA 80.8
1836 A. Sionkowska et al.: Preparation and characterization of collagen/chitosan/hyaluronic acid Table 4: Mechanical parameters of human hair with and without treatment by the blends of biopolymers. Specimen/hair E mod (GPa) F max (GPa) Without polymer 4.70 ± 1.10 0.244 ± 0.067 Col/CS 5.78 ± 1.21 0.321 ± 0.052 Col/CS/1HA 6.72 ± 1.17 0.342 ± 0.065 Col/CS/2HA 5.76 ± 1.90 0.287 ± 0.077 Col/CS/3HA 5.84 ± 1.85 0.309 ± 0.107 Col/CS 6.70 ± 1.63 0.333 ± 0.072 Col/CS/1HA 6.40 ± 1.61 0.331 ± 0.083 Col/CS/2HA 5.06 ± 0.52 0.303 ± 0.053 Col/CS/3HA 5.50 ± 1.29 0.342 ± 0.069 Col/CS 6.78 ± 1.97 0.327 ± 0.112 Col/CS/1HA 4.75 ± 2.66 0.357 ± 0.550 Col/CS/2HA 5.54 ± 1.44 0.308 ± 0.092 Col/CS/3HA 4.72 ± 1.04 0.267 ± 0.052 Film forming properties on the surface of hair The possibility protecting hair using collagen/chitosan/hyaluronic acid thin films was studied using SEM microscopy and the mechanical properties of hair after the topical application of the blend were studied. Samples of human hair were cut from the hair shaft without any chemical treatment. Hair were immersed in polymeric mixture for 1 h and then dried at room temperature and humidity for 24 h. The mechanical tests were made and the results are shown in Table 4. Mechanical parameters such as Young s modulus and the breaking force increase after the treatment of hair by polymer mixture are as follows. The highest increase of Young Modulus was observed after the immersion of hair in Col/CS/1HA (Col/CS in ratio 75 % of chitosan and 25 % of collagen) and the highest maximum tensile strength for the sample Col/CS/1HA (Col/CS in ratio 25 % of chitosan and 75 % of collagen). Addition of hyaluronic acid improves the mechanical parameters of hair, which is a result of film formed on the hair surface. During the formation of biopolymeric film on the hair s surface several interactions are possible. Hydrogen bonds can be formed between side groups of keratin present in hair and functional groups of collagen, chitosan and hyaluronic acid. Moreover electrostatic interactions are possible between hair components and biopolymers. Scanning electron microscope (SEM) The thickness of hair shafts with and without polymer covering was calculated from the SEM images in three places and it is shown in Table 5. SEM images of hair shafts are shown in Figs. 5 and 6. The thickness of a hair shaft with the polymer covering is bigger than without it. SEM images showed that scales of hair are more detached in a shaft without the polymer covering. The presence of 3 % hyaluronic acid additive improves the hair smoothness. It can be noticed that the increasing hyaluronic acid content decreases the scales detachment. The highest thickness was observed for hair covered with Col/CS in ratio with 2 % addition of hyaluronic acid. It may suggest that such a composition of the blends leads to the formation of film with very good adhesion to the hair surface. Nevertheless, for each studied composition of the mixtures, an increase of hair shaft thickness was observed. This suggests that the polymer mixture is adsorbed on the hair surface and a smoothing of hair can be observed. When the hair surface is smooth, the general appearance of the hair is better. In general, as conditioning agents for hair, biopolymers have no effect on growth and cannot affect cellular repair, however, they can temporarily improve the cosmetic appearance of damaged hair. There are
A. Sionkowska et al.: Preparation and characterization of collagen/chitosan/hyaluronic acid 1837 Table 5: The thickness of hair shafts without and with covering by biopolymers mixture, where CS is chitosan, Col is collagen, HA is hyaluronic acid. Hair covering Thickness (μm) Without polymer 62.5 CS 69.6 Col 100.3 Col/CS 94.5 Col/CS/1HA 82.8 Col/CS/2HA 81.0 Col/CS/3HA 100.3 Col/CS 85.7 Col/CS/1HA 91.6 Col/CS/2HA 82.8 Col/CS/3HA 72.7 Col/CS 85.8 Col/CS/1HA 82.8 Col/CS/2HA 125.0 Col/CS/3HA 95.9 Fig. 5: SEM images of hair shafts with and without polymer covering on the surface (a) without polymer (b) covered by chitosan (c) 50CS/50Col/2HA (d) 75CS/25Col (e) 75CS/25Col/2HA (f) 75CS/25Col/3HA.
1838 A. Sionkowska et al.: Preparation and characterization of collagen/chitosan/hyaluronic acid Fig. 6: The SEM image of hair cross-section. several mechanisms by which conditioners can improve the cosmetic appearance of a weathered hair shaft. They can increase shine, decrease static electricity, improve hair strength and protect against ultraviolet radiation. Biopolymeric films formed on the surface of the hair shaft help hair to look and feel better by improving the physical condition of these surfaces. Hair covered by biopolymeric film has a greater volume than those which are non-treated which leads to the reduction of the force required when combing and flyaway hair which, in turn, leads finally to an improved manageability of hair. Conclusion Triple component blends of chitosan, collagen and hyaluronic acid can be obtained in the thin film form through solvent evaporation. Mechanical properties, for example, in addition to, swelling, surface free energy, and the roughness of blends depend on their composition. The increasing chitosan content improves mechanical parameters, surface free energy and the stability in aqueous conditions. The addition of hyaluronic acid improves the mechanical resistance to the applied force. Moreover, the blends are more stable in aqueous conditions which means that such materials will not immediately dissolve after wetting with water. The presence of hyaluronic acid in the sample leads to a decrease of surface free energy and to an increase of roughness. Triple component blends of chitosan, collagen and hyaluronic acid can cover the hair surface well. Such material is less hydrophilic than a collagen/chitosan blend but the contact area, due to bigger surface roughness, is more suitable for additional treatment. The covering of hair with triple component blends of chitosan, collagen and hyaluronic acid leads to an increase in hair thickness and to the improvement of its mechanical properties. Overall, this leads to an improvement in the general appearance and conditioning of the hair. Acknowledgements: Financial support from the National Science Centre (NCN, Poland) Grant no UMO-2013/11/B/ST8/04444 is gratefully acknowledged. References [1] A. Sionkowska. Prog. Polym. Sci. 36, 1254 (2011). [2] C. Yu, J. Chang, Y. Lee, Y. Lin, M. Wub, M. Yang, C. Chien. Mater. Lett. 93, 133 (2013). [3] A. Sionkowska, B. Kaczmarek, K. Lewandowska. J. Mol. Liq. 199, 318 (2014). [4] A. Sionkowska, B. Kaczmarek, J. Stalinska, A. M. Osyczka. Key Eng. Mater. 587, 205 (2014).
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