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Characterizations of Thai silk fibroin/gelatin blend films: effect of silk variety.

Introduction

There are many reports about advantages of the biomaterials. Recently, they have been used in various applications [1]. This was due to their relation on health and friendly environment. Among them, natural polymers were interested in last decade due to their excellent properties such as non-toxicity, biodegradability and biocompatibility.

Silk is a kind of natural protein produced by silkworm [2]. In general, silk divided into 2 catagories: family Bombycidae (domestic or mulberry silk) and Saturniidae (wild or non-mulberry silk) [3]. Silk is well known fiber from the past until now. It has been used for various applications [2,3]. With previously reported, silk was used in various applications such as cosmetics, food additives and medical materials [4-7]. At present, silk is gradually interested for biomedical applications [8]. In addition, it can be modified into many forms depending on applications [9,10]. Each silk fiber composed of at least 2 main proteins; sericin and fibroin. Silk fibroin (SF), a structural protein has been studied and applied. Even natural polymers are attractive to used, some desirable properties do not consist in only one type of polymer. The efforts of study and development the biomaterial by blending or co-forming with bioactive substances are the aims in the last decade. SF was blended with other natural materials such as cellulose [11], chitosan [12], calcium alginate [13], gelatin [14] to adjust their properties and shapes.

Gelatin (G) is a derivative biopolymer of collagen. It is mostly found in many organs of animal tissues such as skin, muscle and bone [14]. The structure of gelatin composed of high hydrophilic amino acids such as arginine and aspartic acid with peptide linked [15]. This point implied that it is polar molecule. Gelatin composed of excellent properties such as biocompatibility, high strength and solubility [16,17], which was suitable for medical and pharmacological applications [13,18]. Moreover, gelatin is a cheap and environmental friendly material. Gelatin can be used by its only or blend with other materials [13,19].

Basic and advance information of SF/G blend films have been proposed. However, the effect of silk variety on the SF/G blend film, especially about Thai silk was rarely reported. Therefore, the aim of this study is to prepare SF solution from different domestic Thai silks for blending with G solution to give the blend films. Some properties of the blend films were characterized and compared between silk varieties. Furthermore, in vitro study about releasing of methylene blue (MB) and film dissolution of each blend films were also described.

Materials and Methods

Materials

Thai domestic silk locally called Nanglai, Kaki and Mo varieties cocoons were kindly supplied by Silk Innovation Center (SIC), Mahasarakham University, Thailand. The cocoons were degummed twice using 0.5% [Na.sub.2]C[O.sub.3] (w/v) and thoroughly rinsed 2 times in warm distilled water. They were then air-dried at room temperature. Gelatin powder was purchased from Sigma (Singapore). All of chemical used were analytical grade.

Preparation of SF solution

The SF was dissolved with tertiary system of Ca[Cl.sub.2]: Ethanol: [H.sub.2]O [1:2:8 by mol]. Briefly, 1 g SF and 10 mL of dissolving solution was mixed and magnetic stirred at 90-95[degrees]C on the hot plate until SF completely dissolved. The SF solution was filtrated, and then dialyzed in cellulose tube (MC = 10 kDa) against distilled water for 3 days at room temperature. The obtained SF solution was adjusted to 1% weight by distilled water.

Preparation of G solution

The G solution was prepared at 1% wt. by weighing of 1 g gelatin powder and then added distilled water until total volume reach to 100 mL. The mixture was placed on the stirring hotplate with stirred for 30 min at room temperature until gelatin powder completely dissolved.

SF/G blend films preparation

The SF/G blend films were prepared by mixing 10 mL of SF and 10 mL of G solutions, and then poured on 9cm polystyrene plates. The plates were dehydrated in an oven at 40[degrees]C for 3 days. Both native SF of each silk varieties and G films were also prepared as control. In addition, SF/G blend films loaded 1% methylene blue (MB) was prepared using the same method. Firstly, 20 mL of SF/G mixing solution was prepared before adding 1% (V/V) of MB.

Characterization of films

All of films were observed under SEM (JEOL, JSM 6460LV, Japan) for their morphology. The films were cut into small pieces, coated with gold for enhancing the surface conductivity before observation.

The secondary structures of the films were analyzed with FTIR (Perkin Elmer-Spectrum Gx, USA) in the spectral range of about 2000-400 [cm.sup.-1] at 4 [cm.sup.-1] spectral resolution and 32 scans.

Thermal properties were measured using TA instruments, SDT Q600 (Luken's drive, New Castle, DE). The SF weight of 8-10 mg were prepared and loaded in a platinum crucible. The samples were non-isothermal heated from 50[degrees]C to 600[degrees]C at a heating rate of 20[degrees]C/min. The TGA was carried out in nitrogen with the flow rate of 100 mL/min. The DTG and heat flow values were recorded with TA Instrument's Q series explorer software. The analyses of the data were done using TA Instrument's Universal Analysis 2000 software (version 3.3B).

In vitro releasing study

The SF/G blend films loaded MB were cut and weighed for 0.15 g. They were then immersed in 20 mL of phosphate buffer saline (PBS), pH 7.4. The mixture was left and collected for 10 mL every 2 h interval times until 72 h of immersion. The solution was then measured at 640 nm using UV-Vis spectrophotometer.

In vitro dissolution study

The SF/G blend films loaded MB were cut and weighed for 0.15 g. They were then immersed in 20 mL of phosphate buffer saline (PBS), pH 7.4. After 24 h of study, the buffer solution was discarded and collected the film residues. They were washed twice with distilled water before drying in an oven until completely dehydrated. Finally, they were weighed to know the remaining weight. The collected films were immersed again in 20 mL fresh PBS until 12 days. Besides duration study, the films were gathered at every 24h designation times. The dissolution of the films was calculated as follow equation;

% Dissolution = W0-W1/W0 x 100 (1)

W0 and W1 are the weight of films before and after immersion in PBS, respectively.

Results and Discussion

Morphology

Morphology of all films was indicated by SEM micrographs as shown in Figure 1. The SF/G blend films have homogeneous texture without phase separation. Among the native SF films, Nanglai shows bead-like particles cover its surface but did not occur in other native films. With cross-section, G blend with SF from Mo variety was rougher than other blend films [Figure 1-e]. The results may be caused from different amino acids composition in each SF which was depending on silk variety. Moreover, it may affect from the interaction and chemical structure in each silks. However, all of blend film has homogeneous texture illustrated that SF and G can miscible forming.

[FIGURE 1 OMITTED]

Secondary structures

The structures of all film were analyzed from FTIR spectra as shown in Figure 2-4. The native SF of each silk varieties slightly differed of absorption bands. Generally all of dominate peak was changed except amide III region when blended SF with G, as listed in Table 1. This was due to the amide III region was unique band of domestic SF. Comparison with both native materials, G blended SF from Nanglai (Figure 3) and Mo (Figure 4) varieties have similar spectrum pattern. The wave number of amide bands, especially I and II are be found between the wave number of native film. In case of Kaki, amide I band has shift to lower wave number but amide II was the middle value between SF and G (Figure 3). The results suggested that interactions between amino acids of SF and G were formed via hydrogen bonds [20]. FTIR is a powerful method for determination protein structures [21,22]. FTIR spectra of the blend films indicated that G enhanced the flexibility of the SF films since most of films were co-existed of [alpha]-helix and [beta]-sheet structures [13,23].

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Thermal properties

Thermal property is another factor which was wildly tested for film study. The detail of those decomposition regions were clearly described by differential thermogravimetric (DTG) curves (Figure 5). All of film did not completely decompose even the final temperature. The blend films started to decompose at temperature under 100 [degrees]C according to water evaporation [24]. Decomposition of films was maximum rate at the temperature of 300-400[degrees]C. G native film rapidly degraded at the initial temperature than that of native SF. However, the decomposition of SF was higher than G after 300[degrees]C. Among silk variety, Mo decomposed rapidly while Nanglai was the lowest. Interesting, the degradation of films gradually decreased when added G. Heat flow thermograms of each silk varieties indicated in Figure 6. The endo/exo-thermic peaks were slightly differed depending on the variety of silk. SF/G blend films have endo/exo-thermic peaks higher than native SF but lower than native G films as shown in Table 2.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Releasing of MB and dissolution of films in vitro

Releasing of drugs or substances was affected by various factors. The interactions between materials and type of molecules loaded on the film might be main effect on the releasing profile. In this work used methylene blue (MB), a cationic dye as model study. The MB was rapidly released at the first 30 min of study and then gradually increased up to 10h. The released content from Mo SF variety blend film has the highest value of MB releasing with about 2 times of Nanglai and Kaki [Figure 7]. However, the dissolution of SF from Mo variety blend film was lower than other film types at 2 days, but was higher after 3 days and gradually increased until 12 days of study [Figure 8]. The releasing of MB should be affected by the dissolution of the films as well as the thermal properties [14, 25]. In addition, each silk varieties might be contained low content of bulky amino acids which were decreased the interaction with the MB. However, each silk fiber may differ in that bulky amino acids contents.

[FIGURE 7 OMITTED]

[FIGURE 8 OMITTED]

Conclusion

SEM micrographs showed native Nanglai, Kaki and Mo SF, G and SF/G blend films have homogeneous texture. FTIR spectra indicated that SF and G were interacted via hydrogen bond and helped to enhance the flexibility of the SF. Adding G result to improve thermal properties of films. The G/SF from Mo variety blend films showed rapidly released of MB and highest percentage of dissolution. However, more details of all result did not clear which was significantly differed in each silk varieties.

Acknowledgements

Firstly, I like to thank my B.Sc. student Miss Prapatsorn Thummapitak and Miss Achara Kumprai, for their help to do my work. I gratefully thank Division of Research Facilitation and Dissemination, Department of Chemistry, Faculty of Science, Mahasarakham University, and the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education, Thailand for financial support this work.

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Srihanam Prasong

Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahasarakham

University, 44150 Thailand

E-mail: prasong.s@msu.ac.th
Table 1: Amide regions of different films.

 Absorption peaks ([cm.sup.-1])
Film Types
 Amide I Amide II Amide III Amide IV Amide V

Nanglai 1671 1635 1558 1245 1061 670
Nanglai/G 1684 1666 1540 1245 1072 670
Mo 1684 1600 1523 1240 1065 665
Mo/G 1653 1537 1245 1081 668
Kaki 1639 1551 1245 1061 668
Kaki/G 1685 1631 1597 1248 1061 630

Table 2: Exo/Endo-thermic peaks of different films.

Film Types Exothermic Endothermic
 ([degrees]C) ([degrees]C)

G 175 258 415 210 350
SF Mo 200 375 297
SF Nanglai 219 375 330
SF Kaki 325 375 220
SF Mo/G 210 375 330
SF Nanglai/G 210 375 322
SF Kaki/G 220 375 330
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Author:Prasong, Srihanam
Publication:International Journal of Applied Chemistry
Date:Sep 1, 2011
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