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Journal of the Chilean Chemical Society

On-line version ISSN 0717-9707

J. Chil. Chem. Soc. vol.59 no.1 Concepción Mar. 2014

http://dx.doi.org/10.4067/S0717-97072014000100008 

 

PHYSICOCHEMICAL CHARACTERIZATION OF GELATIN-CMC COMPOSITE EDIBLES FILMS FROM POLYION-COMPLEX HYDROGELS

 

CHETOUANI ASMA*, ELKOLLI MERIEM, BOUNEKHEL MAHMOUD AND BENACHOUR DJAAFER

Laboratory multiphase polymeric materials (LMPMP), Faculty of technology, Setif -1 University, Setif 19000, Algeria
* e-mail. achetouani@hotmail.fr


ABSTRACT

This study is concerned to elucidate the interaction behavior of films consisted of gelatin and carboxymethylcellulose, which are polyelectrolytes and have applications in tissues engineering. These films were chemically cross-linked using glutaraldehyde.

The decrease of framework in triple helix of gelatin in the presence of polysaccharide and/or the cross-linking agent increases until a total disappearance was showed by XRD, the disappearance of macroporous gelatin structure in the presence of additives has been confirmed by SEM and AFM.

According to DSC analysis, glass transition temperature (Tg) increases and decreases as GTA and CMC were added, respectively.

It was shown that swelling of macroporous structure in pseudophysiological mediums was more absorbent and used electrolytes exert an osmotic pressure or ionic strength inducing higher swelling. Also, it was found that diffusion mechanism is directly related to gelatin structure. On the other hand, the incorporation of CMC improves the flexibility of matrice.

Key words: Biocompatible polymers, electrostatic interaction, crosslinking, mechanical properties, AFM.


 

1. INTRODUCTION1

Natural polymers have large applications notably in packaging materials or coating agents 1. Gelatin is largely used in the pharmaceutical industry as wound dressing, absorbent film and adhesive, and particularly as excipient in controlled drug release 2-4. However, it has a very hydrophilic nature and relatively poor in mechanical properties which limited their potential applications 5. Several studies have been widely used to improve gelatin properties by blending gelatin with other proteins or polysaccharides such as alginate 6, chitosan7 and hyaluronic acid 8.

These, are known for their self adhesiveness and permeability as well as their ability to form physical and chemical hydrogels, self-adhesiveness and permeability properties. Also, they are the support matrices for drug release and tissue regeneration9.

Carboxymethylcellulose is one of the endogenous polysaccharides which play an important role in the cellular growth, differentiation and as a buffer in pressure changes. Due to its enormous biological properties, it is used in the medical field 10,11.

Polysaccharides/proteins mixtures were greatly thermally influenced. Above the isoelectric point of the protein thermodynamic incompatibility between the protein and polysaccharide occurs because of the repulsive electrostatic interactions and different affinities towards the solvent. Therefore, the protein and polysaccharide may co-exist in a single phase but in domains in which they mutually exclude one another or segregate into different phases 12. Many studies of the interactions between proteins and polysaccharides in solution have been reported 13-16, that the formation or dissociated of protein-polysaccharide complexes and their solubility depends on parameters such as the type of biopolymers involved and their concentration, the pH, ionic strength, calcium ion concentration, the temperature 17.

The purpose of this work is to study the influence of sodium carboxymethyl cellulose on the physical and mechanical properties of gelatin.

The formation of integrated materials (permanent films polyionic-complex hydrogels) have been demonstrated in two stages: the first, CMC-gelatin mixture produces a new reversible polyionic biomaterial, whereas, in the latter, the addition of glutaraldehyde as cross-linking agent ensure the formation of a permanent chemical network and its mechanical resistance has been proved to be promising for the development of new renewable biomaterials.

2. MATERIALS AND METHODS

Pharmaceutical and food grade pig skin gelatin (Type A, 500 Bloom), powder (isoelectric point, IEP~9.0) and Carboxymethylcellulose sodium (CMC), pKa =3.5, viscosity: 300-600 cps at T=25°C were supplied from Sigma. Glutaraldehyde (GTA) aqueous solution (50wt.%) : Sodium azide (NaN3) were purchased from Aldrich and Chemopharma, respectively. These reagents were used as received.

2.1. Preparation of films

Biopolymer films were prepared from a mixture of gelatin and CMC at 5 wt. % total solids by casting using the following procedure. 5 grams of gelatin/CMC (80/20) were dissolved in 100 ml distilled water along with 0.02% sodium azide to prevent bacterial contamination for 30 min. Then, the mixture was placed in a water bath at 60°C under a gentle shaking for 30 min to obtain a homogeneous solution. 10 ml of this solution was poured into 9.5 cm diameter polystyrene Petri dish and air dried at ambient temperature for 3 to 4 days. The obtained film was inserted in 20 ml of aqueous solution of GTA (2%) for 24 h. The cross-linked film was washed with distilled water and dried.

The same procedure has been applied for gelatin film.

2.2. Fourier transformed infrared spectroscopy analysis

FT-IR spectra of dried films (1%) were obtained using a SHIMADZU 84005 spectrometer. Scanning was carried out in the range 4000-400 cm-1.

2.3. X-ray diffraction

X-ray diffraction patterns (XRD) were collected on a D8-ADVANCE-BRUKER-AXS diffractometer equipped with a Cu Kα radiation source (λ=1.5418 Å) operating at 40KV and 40 mA. Data were recorded in the range of 2Ө =5-600 at scanning rate (0.020/min).

2.4. Differential scanning calorimetry

Calorimetric measurements were performed using a DSC (DSC 200PC). Thermograms were obtained from (2-4 mg) of preheated samples up to 100°C for 30 mn using sealed aluminum pans at a heating rate of 5 0C/min up to 1500C, under O2/N2 atmosphere (50 ml/min and 20 ml/min), respectively.

2.5. Tensile testing

Stress-strain curves of strip-shaped (50 × 20 mm, thickness around 0.26 mm) films equilibrated at 75% relative humidity conditions 18, 19 were recorded using a Zwick /Roell Z. 0.1 testing machine, with a crosshead speed of 5 mm min-1. The elastic modulus E (or Young's modulus, MPa), the stress at break σb (MPa), and the strain at break εb (%) of the strips were measured in a static mode.

2.6. Atomic force microscopy analysis (AFM)

Samples surface was scanned in a liquid medium using a microscope with commercial atomic force (PSIA Xe-100) equipped with a scanner and a liquid quartz cell. The topographic images (512 X 512 pixels) are acquired in semi-contact mode comparable with the tapping mode at ambient temperature using points "silicon nitride V" Si3N4. In each case, the excitation frequency of the tip is fixed around 300 KHz and the scanning rate on the surface of the sample is 0.5 HZ.

2.7. Scanning electron microscopy analysis (SEM)

Film samples were attached by double sided electrically conductive carbon type and their surface morphologies were observed by scanning electron microscopy (SEM) using a JEOL JSM-840 instrument at 5.01V accelerating voltage.

2.8. Swelling

Mediums

Solution1: pseudo extracellular fluid (PECF) which is simulated wound fluid, was prepared by dissolving (0.68g, 11,6 mmol) NaCl, 0.22g KCl, 2.5g of NaHCO3, and 0.35g of NaH2PO4 in 100 ml of distilled water to obtain a solution with pH 8 ± 0.2 (Balgit, 2008).

Solution2: buffer solution (pH 7.4) was prepared by taking (1.36g, 0.2M) KH2PO4 and (0.31g, 0.2M) NaOH in volumetric flask to make volume 200ml with distilled water.

Solution3 and 4: [NaCl]=1.5N and 0.5N with pH=6.95 and 5.78, respectively.

Solution5: distilled water, used as reference pH=7.

Solution6: Buffer solution (pH=2.2) was prepared from (0.74g, 0.2M) KCl and (7.8ml, 0.2N) HCl in 200 ml of distilled water.

Swelling test:

80 mg of each dried square piece of prepared films (20/20/0.26)(l/w/t) was immersed in six different mediums (PECF, PBS, [NaCl]=1.5N, [NaCl]=0.5N, distilled water and pH=2.2) at 25 and 37°C for 24h. The swelling ratio (S) was calculated using the following equation:

Where Wt and Wi are weights of the wet and dry samples, respectively.

The swelling type of samples was determined using the following equation:

Where t is the time, K is the swelling constant, and n is the swelling exponent.

The short time approximation is applied only for S≤ 60%.

3. RESULTS AND DISCUSSION

3.1. FTIR spectroscopy

The FTIR spectra of gelatin, CMC and gelatin/CMC films are presented in fig. 1 which shows major changes in the functional groups of gelatin due to the interaction between gelatin and CMC. Gelatin film revealed absorption bonds at 3395 and 3317 cm-1 corresponding to OH and NH stretching vibration, respectively.

 

Fig 1. FT-IR Spectra of G (a), CMC (b), G/CMC (c), G/GTA(d) and G/CMC/GTA (e)

 

Fig 2. Crosslinking reactions with glutaraldehyde occurring in CMC-Gel mixture.

The absorption at 1651 cm-1 and 1554 cm-1 are attributed to (□ (C=O)) and (□ (C-N)) vibrations of amide I and amide II, which is representative of the collagen secondary structure. According to Yakimet & al., the bond at 1651 cm-1 gives information on the helicity of the protein.

The CMC spectrum (fig 1.b) exhibits bands at 2920, 1602 and 1424 cm-1 attributing to stretching vibrations of C-Haliphatic, asymmetric and symmetric of the carboxylate group (-COO-), respectively. The absorptions at 1154 and 1064 cm-1 are due to the stretching vibrations of C-O-C the glucosidic units and ββ β-(1-4) glucosidic linkage, respectively 21.

A broad absorption band at 3390 cm-1 of OH groups corresponding to absorbed water, secondary alcohols (CMC) and (intramolecular/ intermolecular) hydrogen bonding 22 23.

The IR spectrum of gelatin /CMC shown in fig. 1.c. Peaks have been shifted from 3317, 1452 to 3326, 1455. This modification can be explained by coupling of the anionic groups in CMC with the cationic ones in gelatin. In our case, the absorption of the carboxylate group has been shifted to 1650 and 1455 cm-1, which indicates the formation of monodentate I or II:

??(COO- ) complexe - ??(COO - ) Na sel = [(1650 -1455) - (1602-1424)] cm-1 = +17 cm-1.

Cross-linked gelatin has been widely studied by different authors 24 27. Visually, the change of color to yellow is due to the condensation of primary amine of lysine with GTA to produce imine "schiff-bases".

The IR spectrum of G/GTA (fig. 1.d) exhibits an intensification absorption at 1650 cm-1 which becomes stronger and broader ranging from 1656 to 1633 cm-1. Also, the absorption at 1456 is corresponding to γC-N "vibration of C-N deformation" intensifies with crosslinked reaction.

The IR spectrum of G/CMC/GTA (fig. 1.e) shows that the absorption of CH aliphatic group has been increased to 2952 cm-1. In the other hand, the addition of glutaraldehyde shows a drastic change to the absorption below 1200 cm-1, which confirm the reaction of the GTA with CMC forming hemiacetal structure.

3.2. XRD

X-ray diffraction analysis was performed to quantify the crystalline structure of gelatin in gelatin, gelatin/CMC and crosslinked by GTA. The diffractogram acquired on the gelatin films were typical of a partially crystalline gelatin with peaks at 2θ=7.8° (d101=11.08 Å) and at 22.12° (d101=4.01 Å), shown in fig 3. These characteristic peaks are usually assigned to the triple-helical crystalline structure in collagen and gelatin 20 28. On the other hand, Bigi & al., 29 have demonstrated that the first diffraction peak at 8° (sharp and intense) is directly related to the diameter and the rate of the triple helix, respectively. It was also found that the addition of polyols such as tannin 28, glycerol 30 most often decreases the intensity of the first peak (2θ=8°). It has been reported 31, that CMC has a crystallinity of about 8%. The diffraction peak at 2θ = 19° decreases with the degree of substitution (DS). Thus is due to the rate of alkylation of cellulose by chloroacetic acid. Also, when DS is equal to 1, Lin et al., 32 have revealed the complete disappearance of the peak of cristallinity.

 

Fig. 3. X-ray diffractograms of G (a), G/CMC (b) G/CMC/GTA (c).

According to data present in Table 1, it has been observed that the addition of CMC is shifting the diffraction angle from 8.06° to 7.86° and increasing the diameter of the inter reticular triple helix. On the other hand, the intensity of peaks decreases as the amount of CMC increases. These results are in agreement with Langmaier et al., 33 and Rivero 30.

Table 1. X-ray data for gelatin and modified gelatin films.

 

The disappearance of X-ray diffraction peaks corresponding to CMC confirms the interaction between two biopolymers. These results are supported by the work of Yang et al., 34. This phenomenon illustrates the reduction hydrogen bonds between hydroxyls of the anhydroglucose of cellulose group.

The crosslinked films by GTA exhibits a negligible triple helix approaching ~ 0.6%.

3.3. DSC characterization

The differential scanning calorimetry (DSC) thermograms present in fig. 4 and table 2. All traces display the classical thermal behavior of gelatin samples, with the first drop of the thermogram related to the glass transition, followed by an endothermic peak associated with the helix-to-coil transition.

 

Fig. 4 DSC Thermograms of Gelatin (a), (G/CMC) (b) and (G/CMC/GTA) (c).

Table 2. The DSC data for films based on gelatin: G, (G/CMC) and (G/CMC/GTA).

 

Each thermogram exhibits a single endothermic peak (Tm) at 61,78 , 58,24 and 84,79 for gelatin, G/CMC, and G/CMC/GTA, respectively. More specifically, the addition of CMC to gelatin decreases Tg and Tm, and the increases of enthalpy (ΔH) is due to the evolution of residual water. So, the interaction between two macromolecules increases and CMC tends to plasticize the gelatin. Finally, crosslinked film shows higher stability. Exhibits a higher Tg and Tm, as well as ΔH than gelatin and gelatin/CMC.

In all cases, the appearance of the endothermic peak in DSC thermograms is normally due to the breakage of hydrogen bonds 29.

3.4. Tensile properties of films

The mean stress-strain curves for the three types of films differ from each other as shown in fig. 5 and confirmed by the results reported in table 3. The elastic modulus values were calculated for all specimens from the slope of the linear climbing tract of the stress-strain plot within the fixed strain region 0.5-1.0%.

 

Fig. 5 Tensile stress-strain curves of G, G/CMC, and G/CMC/GTA at RH =75%.

Table 3. Mechanical properties of films based on gelatin.

 

According to curves of gelatin, G /CMC, G /CMC/ GTA, it has been observed that the addition of CMC leads to a decrease in tensile strength and εmax while the Young's modulus decreases after addition of CMC and increases after cross-linking.

3.5. AFM

According to Farris et al. 35, gelatin molecules assemble into aggregates containing shorter segments than those expected for collagen triple helices. In our case, this protein presents, uniaxial fibers whose size varies from 0.57 to 1.21μm length and of approximately 23-40 nm height (fig. 6).

 

Fig. 6 AFM photographs of G (a), (G/CMC) (b), (G/CMC/GTA) (c).

After addition of CMC, the fibrilary homogeneous structure was disappeared and took a relief form (surface in projections and hollows). Whereas, film of (G/CMC/GTA) became thinner and smoother.

3.6. SEM

The porosity of biomaterials is a very required field in tissue engineering and drugs release. In our case gelatin film has a macroporous droplet form structure as indicated by Petterson, (2009) 2.

In notorious way, the presence of CMC and GTA particularly affects the porosity of gelatin as shown in fig. 7.

 

Fig. 7 SEM of G (a), (G/CMC) (b) and (G/CMC/GTA) (c), at two different magnifications (100 X and 2000 X).

3.7. Swelling

To improve the swelling at pH = 7 and 6.95, it is necessary to use an electrolyte as shown in table 4. On the other hand, it has been observed that the maximum swelling (Smax) has been increased in the presence of CMC as well with higher pH and lower degree of crosslinking.

Table 4. Maximum swelling data for G, G/CMC and G/ CMC/GTA at different pH (2.2-8.2).

 

Attempts to determine the diffusion mechanisms systems, swelling results have been shown that, the value of n is always lower than 0.5 for non-crosslinked films and agrees with Fickian diffusion "Less Fickian behavior"" and the rate of water penetration is lower than that of chains relaxation.

The exponent n for crosslinked films is greater than non-crosslinked films, reaching maximum n at 0.7 (G / CMC / GTA) at room temperature and decreases at higher temperatures (table 5).

Table 5. Swelling kinetics data (n, K, R and D) for G, (G/CMC) and (G/CMC/GTA) at pH=7 and 8.2.

 

CONCLUSION

FT-IR spectra of G/CMC have been confirmed an electrostatic interaction between gelatin and carboxymethyl cellulose. The addition of crosslinking agent (GTA) to gelatin/CMC has been (physically and / or chemically) established leading to three-dimensional networks.

X-ray diffractograms show that the framework in triple helix of gelatin decreases as polysaccharide and/or the crosslinking agent increases until a total disappearance.

According to DSC thermograms, the glass transition temperature is directly related to the denaturation of gelatin (transition helix/coil). Also, it has been observed that Tg increases and decreases as GTA and CMC were added, respectively.

Atomic force microscopy analysis has been presented the change of morphology of gelatin which was initially in the form of regrouped uniaxial fibers, then, it is transformed into relief, by the addition of CMC. On the other hand, micrograph of G/CMC/GTA film reveals a complete irregular junction.

SEM data are in agreement with AFM. Furthermore, macroporous gelatin structure has been disappeared in the presence of additives.

Swelling in pseudophysiological mediums shows that macroporous structure is more absorbent and used electrolytes exert an osmotic pressure or ionic strength inducing higher swelling.

Also, it was found that diffusion mechanism is directly related to gelatin structure, particularly porosity and Tg. In general, linear films are Fickien diffusion, which is one of characteristic porous materials. Whereas, crosslinked films (no Fickien) became less porous and Tg tends to increase, so, the rate of relaxation (toward the glassy state) decreases and becomes much lower than that of water diffusion. At high moisture content (63-75%), tensile tests have been showed that CMC and GTA tend to plasticize and make gelatin more rigid, respectively.

 

REFERENCES

1.- S. Patil, D. R. Jadge, Latest Reviews 6, 246, (2008).         [ Links ]

2.- S. Pettersson, J. Wetterö, P. Tengvall and G. Kratz, Journal of Tissue Engineering and Regenerative Medicine 3, 450, (2009).         [ Links ]

3.- W. Lee & S. Lee, J Mater Sci: Mater Med. 18, 1089, (2007)         [ Links ]

4.- A.G. Ward, A. Courts The Science and technology of gelatin. Academic press, London, 1977.         [ Links ]

5.- F. Wiwatwongwana and S. Pattana, The Second TSME International Conference on Mechanical Engineering, Krabi, 2011.         [ Links ]

6.- Y.S. Choi, S.R. Hong, Y.M. Lee, K.W. Song, M.H. Park and Y.S. Nam, Biomaterials 20, 409, (1999).         [ Links ]

7.- J.S. Mao , L.G. Zhao, Y.J. Yin and K.D. Yao, Biomaterials 24, 1067, (2003).         [ Links ]

8.- F. Wiwatwongwana and S. Pattana, The First TSME International Conference on Mechanical Engineering, Ubon Ratchathani, 2010.         [ Links ]

9.- W. S. Anthony, Advanced Drug Delivery Reviews 57, 1539, (2005).         [ Links ]

10.- S. Kamel, N. Ali, K. Jahangir, S. M. Shah, A. A. El-Gendy, eXPRESS Polymer Letters 2, 758, (2008).         [ Links ]

11.- S. Sungur, E. Emregül, Macromolecular reports 33, 319, (1996).         [ Links ]

12.- I. R. Baeza and A. M. R. Pilosof, Lebensm.-Wiss. u.-Technol. 35, 393, (2002)        [ Links ]

13.- C.G. De Kruif, R. Tuinier, Food Hydrocolloids 15, 555, (2001).         [ Links ]

14.- D. J. McClements, Biotechnology Advances 24, 621, (2006).         [ Links ]

15.- J. L. Doubliera, C. Garniera, D. Renarda, C. Sanchezl, Current opinion in Colloid & interface Science 5, 202, (2000).         [ Links ]

16.- C. Schmitt, C. Sanchez, S. Desobry-Banon & J. Hardy, Critical Reviews in Food Science and Nutrition 38, 689, (1998)         [ Links ]

17.- L. Jourdain, M. E. Leser, C. Schmitt, M. Michel, E. Dickinson, Food Hydrocolloids 22, 647, (2008)        [ Links ]

18.- G. Champetier, L. Monnerie Introduction à la chimie macromoléculaire, Masson, Paris, 1969.         [ Links ]

19.- A. Bigi, B. Bracci, G. Cojazzi, S. Panzavolta, N. Roveri, Materials 19, 2335, (1998).         [ Links ]

20.- I. Yakimets, N. Wellner, A. Smith, R. Wilson, I. Farhat, J. Mitchell, Polymer 46, 12577, (2005).         [ Links ]

21.- A. Ibrahim Atef, M. Adel Abeer, H. Abd El-Wahab Zeinab, T. Al-Shemy Mona, Carbohydrate Polymers 83, 94, (2011).         [ Links ]

22.- D.R. Biswal, R.P. Singh, Carbohydrate Polymer 57, 379, (2004).         [ Links ]

23.- L. I. Kutsenko, A. M. Bochek, E. N. Vlasova, B. Z. Volchek, Russian Journal of Applied Chemistry 78, 2014, (2005).         [ Links ]

24.- J. F. Martucci, R. A. Ruseckaite, A. Vazquez, Materials science and engineering. A, Structural materials: properties, microstructure and processing 435, 681, (2006).         [ Links ]

25.- J. Sharma, H. B. Bohidar, European polymer journal 36, 1409, (2000).         [ Links ]

26.- P. M. A. Alves, R. A. Carvalho, I. C. F. Moraes, C. G. Luciano, A. M. Q. B. Bittante, P. J. A. Sobral, Food hydrocolloids 25, 1751, (2011).         [ Links ]

27.- S. S. Kim, H. W. Kim, S. H. Yuk, S. Y. Oh, P. K. Pak., H. B. Lee, Biomaterials 17, 813, (1996).         [ Links ]

28.- C. Peña, K. de la Caba, A. Eceiza, R. Ruseckaite, I. Mondragon, Bioresour Technol. 101, 6836, (2010).         [ Links ]

29.- A. Bigi, S. Panzavolta, K. Rubini, Biomaterials 25, 5675, (2004).         [ Links ]

30.- S. Rivero, M. A. García, A. Pinotti, Journal of Food Engineering 90, 531, (2009).         [ Links ]

31.- M. P. Adinugraha, D. W. Marseno, M. Haryadi, Carbohydrate Polymers 62, 164, (2005).         [ Links ]

32.- X. Lin, T. Qu, S. Qi, ActaPolymerica 41, 220, (1990).         [ Links ]

33.- F. Langmaier, P. Mokrejs, K. Kolomaznik, M. Mladek, Thermochimica Acta 469, 52, (2008).         [ Links ]

34.- F. Yang , G. Li, Y. He, F. Ren, G. Wang, Carbohydrate Polymers 78, 95, (2009).         [ Links ]

35.- S. Farris, J. Song, Q. Huang, J. Agric. Food Chem. 58, 998, (2010).         [ Links ]

 


(Received: January 28, 2013 - Accepted: October 10, 2013)