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Biodegradable silk fibroin/chitosan blend microparticles prepared by emulsification-diffusion method

  • Onanong Cheerarot EMAIL logo and Yodthong Baimark
Published/Copyright: January 21, 2015
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e-Polymers
From the journal e-Polymers Volume 15 Issue 2

Abstract

In this study, biodegradable microparticles of silk fibroin (SF)/chitosan (CS) blends with SF/CS blend ratios of 1:0, 2:1, 1:1, 1:2 and 0:1 (w/w) were prepared using the water-in-oil emulsification-diffusion method without any surfactants. An aqueous SF/CS blend solution and ethyl acetate were used as the water and oil phases, respectively. The plain SF had an irregular shape, while the CS microparticles were spherical. The blend microparticles were spherical in shape with a rough surface. Average microparticle sizes were in the range of 73–80 μm. The microparticle matrices had a sponge-like morphology. Conformational changes in the SF component, from random coil to the β-sheet form, were observed in the FTIR data. The dissolution of the blend microparticles was lower than those of the plain SF and CS microparticles. Microparticle density steadily increased and porosity slightly decreased as the amount of CS in the blend increased.

Keywords: blend microparticles; chitosan; conformational transition; morphology; silk fibroin

1 Introduction

Silk fibroin (SF) is a biodegradable and biocompatible natural protein polymer that is produced by the Bombyx mori silkworm (1) and has recently been extensively investigated as a biomaterial for use in biomedical applications such as tissue engineering (2) and drug delivery applications (3–5). Minimal inflammatory reactions for in vitro and in vivo tests of SF have been reported by Meinel et al. (6). Many forms of SF including fibers, films, foams, tubes, gels and particles have been investigated for these purposes (7–9). SF has been blended with chitosan (10–14), cellulose (15), poly(vinyl alcohol) (16) and poly(ethylene oxide) (17, 18) in order to enhance its physicochemical, mechanical and biodegradation properties for specific applications. The SF blends have been fabricated in film, fiber and sponge forms but not in the form of microparticles.

Many researchers have reported the formation of plain SF microparticles by diverse methods including spray-drying (19, 20), lipid vesicle templates (21), water-in-oil (W/O) emulsion solvent evaporation (22), W/O emulsion solvent diffusion (23, 24), phase separation of SF/polyvinyl alcohol blend films (25) and salting out (4). The dissolution of SF matrix has been modified and controlled via the use of a cross-linker, alcohol, heat treatments and by blending with hydrophilic polymers for drug delivery applications.

Chitosan (CS) is a natural hydrophilic polysaccharide that, thanks to its biocompatibility, biodegradability and desirable physical properties, has been prolifically investigated in a variety of applications including wound dressings, drug delivery and food packaging (26, 27). Chitosan microparticles have been widely investigated for use as drug delivery systems (28–33). Several studies on CS/SF blends (in a variety of structural formats) have shown that blending of CS with SF induced conformational changes in the SF component from the random coiled (water-soluble) to the water-insoluble β-sheet form (11–14). Many methods for the preparation of CS microparticles have been reported (34); however, investigations focusing on the preparation of SF/CS blend microparticles have yet to be published.

In our previously published studies, SF and CS microparticles were prepared using the W/O emulsion solvent diffusion method (23, 35, 36). The microparticles were formed via the diffusion of water from dispersed droplets of aqueous polymer solution in an organic continuous phase. In this study, SF/CS blend microparticles, prepared using the W/O emulsion solvent diffusion technique, will be examined. The effect of blend ratios on particle morphology and physicochemical properties will be investigated.

2 Materials and methods

2.1 Materials

Aqueous SF solution was prepared using a chemical de-gumming method prior to dissolution and dialysis. For the de-gumming process, silk cocoons from B. mori were boiled for 30 min in an aqueous solution of 0.5% (w/v) Na2CO3 twice and then rinsed thoroughly with distilled water to remove the glue-like sericin proteins. The de-gummed SF fibers were then dissolved in a CaCl2-ethanol-water mixture (mole ratio 1:2:8), by stirring at 80°C for 2 h. The resulting SF solution was then dialyzed in distilled water using a cellulose tube (molecular weight cut-off 7000 Da) for 3 days. The distilled water was replaced every day. The final SF concentration was adjusted to 0.5% w/v with distilled water. Chitosan with a 90% degree of de-acetylation and molecular weight of 100 kDa was purchased from Seafresh Chitosan Lab Co., Ltd. (Bangkok, Thailand) and used without further purification. An aqueous solution of chitosan (0.5% w/v) was prepared by dissolving chitosan flakes in an aqueous acetic acid (analytical grade) solution (1% v/v).

2.2 Preparation of SF/CS blend microparticles

SF/CS blend microparticles were prepared using a surfactant-free W/O emulsion solvent diffusion method. The aqueous solutions of SF and CS were combined and magnetically stirred for 30 min before the fabrication of the microparticles. Blend microparticles with SF/CS blend ratios of 1:0, 2:1, 1:1, 1:2 and 0:1 (w/w) were produced. For a given ratio, the solution of SF/CS blend (0.5 ml) was added drop-wise to analytical grade ethyl acetate (100 ml), while the latter was magnetically stirred (IKA Yellowline RST) at 900 rpm for 60 min. To prevent the evaporation of the ethyl acetate, the beaker was tightly covered with aluminum foil after the addition of the blend solution. The resulting blend microparticles were isolated by centrifugation before drying in a vacuum oven at room temperature for 1 week.

2.3 Characterization of blend microparticles

Morphological observations were performed using a JEOL JSM-6460LV scanning electron microscope (JEOL, Japan). The samples were sputter-coated with gold to produce a conductive coating and to prevent charging. The average size (diameter) and standard deviation of the blend microparticles were determined from several SEM images by measuring a minimum of 100 particles using the Smile View software (version 1.02).

The chemical structure and SF conformation within the microparticles were investigated using transmission Fourier transform infrared (FTIR) spectroscopy (Perkin-Elmer Spectrum GX FTIR, USA) The FTIR spectra were taken over the wave number range 4000 to 400 cm-1 with a spectral resolution of 4 cm-1, using 32 scans. The microparticles were diluted in KBr, and the mixture was then compressed into a disc for transmission FTIR using established methods.

The dissolution assessment of the blend microparticles was performed by taking microparticles and adding them to a 0.1 mmol/l of phosphate buffer solution (pH 7.4 at 37°C). The flask was then shaken at 150 rpm for 24 h. The determination was repeated three times for each sample. The percentage of dissolution was calculated from Equation 1. Each dissolution value was averaged from the three measurements.

[1]Dissolution (%)=initial weight of microparticles (g)-remaining weight of microparticles (g)initial weight of microparticles (g)×100 [1]

The density of the microparticles was determined by the gas displacement technique using a Quantachrom 1000 ultrapycnometer (Quantachrome Corporation, USA) with helium gas at 23°C. Each average density value was measured from a mean of five determinations. The porosity (P) of the porous material was calculated based on a comparison of densities between porous and non-porous forms (37) according to Equation 2. The SF, CS and their blend films designed as non-porous matrices were prepared by the film casting method at 40°C. The film density was also measured by the gas displacement method.

[2]P=[1-(ρ/ρo]×100 [2]

where ρ and ρo are the densities of the porous matrix (microparticle) and non-porous matrix (film), respectively.

3 Results and discussion

In this paper, surfactant-free SF/CS blend microparticles were prepared by a W/O emulsion solvent diffusion method. This method has been successfully used to prepare both SF (23, 24, 35) and CS (36) microparticles. For this method, the aqueous SF/CS blend solution was emulsified as dispersed droplets in the ethyl acetate continuous phase before solidification during the diffusion of water and ethyl acetate, as previously described in our work (38). All the SF/CS blend microparticles without aggregates were obtained with more than 90% yields. This suggests that the W/O emulsification-diffusion method is appropriate for the preparation of SF/CS blend microparticles.

3.1 Morphology and size of blend microparticles

The microparticle morphology was investigated using SEM imaging (Figure 1). The plain SF microparticles had an irregular shape (Figure 1A). Protein-based microparticles of SF (20) and insulin (39) with irregular or erythrocyte-like shapes were prepared via the spray-drying technique. The rough surface texture of the last two microparticles was formed because the surface regions of the droplets were the first to dry and solidify, forming a skin that shriveled when the interior regions dried out. For the W/O emulsion solvent diffusion method used in our study, the irregular shape of the SF microparticles may form due to the rapid solidification of the SF droplet surface regions during the solvent diffusion process (23, 24, 35). However, the CS microparticles and SF/CS blend microparticles were nearly spherical in shape (Figure 1B–E). This may be due to the slow solidification (36, 38). The microparticle surfaces are illustrated in Figure 2, where it can be seen that the surface of the SF microparticles (Figure 2A) was smoother than those of the CS and the blend microparticles. The rougher surfaces of the CS and blend microparticles may be explained by the diffusion of water from the solution droplets to the continuous organic (ethyl acetate) phase, during the solidification of the microparticles.

Figure 1 SEM images of microparticles prepared with SF/CS blend ratios of (A) 1:0, (B) 2:1, (C) 1:1, (D) 1:2 and (E) 0:1 (w/w). All scale bars represent 100 μm.
Figure 1

SEM images of microparticles prepared with SF/CS blend ratios of (A) 1:0, (B) 2:1, (C) 1:1, (D) 1:2 and (E) 0:1 (w/w). All scale bars represent 100 μm.

Figure 2 SEM images of microparticle surfaces prepared with SF/CS blend ratios of (A) 1:0, (B) 2:1, (C) 1:1, (D) 1:2 and (E) 0:1 (w/w). All scale bars represent 5 μm.
Figure 2

SEM images of microparticle surfaces prepared with SF/CS blend ratios of (A) 1:0, (B) 2:1, (C) 1:1, (D) 1:2 and (E) 0:1 (w/w). All scale bars represent 5 μm.

The SEM images of the fractured microparticles revealed their internal morphology (Figure 3). The SF, CS and blend microparticles exhibited a sponge-like or porous structure. This may be due to phase separation taking place within the emulsion droplets before solidification. The porous structure could form when a small amount of non-solvent (ethyl acetate) diffused into each emulsion droplet and reduced the solubility of the SF and CS, which then solidified and precipitated. The porous microparticles may have been formed due to the phase separation within the water1-in-oil-in-water2 double emulsion droplets before solidification (24, 36, 40). However, the internal porous structures were completely covered with continuous particle surfaces. It should be noted that the internal pores of the CS and blend microparticles were larger in size than those of the SF microparticles.

Figure 3 SEM images of fractured microparticles prepared with SF/CS blend ratios of (A) 1:0, (B) 1:1 and (C) 0:1 (w/w). All scale bars represent 5 μm.
Figure 3

SEM images of fractured microparticles prepared with SF/CS blend ratios of (A) 1:0, (B) 1:1 and (C) 0:1 (w/w). All scale bars represent 5 μm.

The average particle size of the blend microparticles was determined from at least 100 particle diameter measurements over several SEM images. The direct method was used instead of laser scattering of a particle suspension in water, to avoid partial swelling and dissolution of the microparticles. Average particle sizes and standard deviations are summarized in Table 1, with the exception of the SF microparticles due to their irregular shape. However, SF particles that are <100 μm in size can be observed from the SEM images. The average sizes of the CS and all the blend microparticles varied between 73 and 80 μm, thereby indicating that particle size was not measurably affected by blend ratio.

Table 1

Average particle size, porosity and dissolution of SF, CS and SF/CS blend microparticles.

SF/CS blend ratio (w/w)Average particle sizea (μm)Dissolutionb (%)Porosityc (%)
1:0–d63±340
2:180±1149±541
1:175±1346±440
1:273±940±335
0:178±1260±628

aDetermined from several SEM images.

bCalculated from Equation 1.

cCalculated from Equation 2.

dNot measured due to its irregular shape (see Figure 1A).

3.2 FTIR Spectra of blend microparticles

FTIR spectroscopy has been widely used to investigate functional groups of SF and CS, and their intermolecular interactions. Figure 4 shows the FTIR spectra of the SF, CS and blend microparticles. The amide I, II and III absorption bands of SF were used to monitor the formation of the random coil and β-sheet forms of SF. The FTIR spectrum of plain SF microparticles (Figure 4A) shows absorption bands at 1655 cm-1 (amide I), 1541 cm-1 (amide II) and 1249 cm-1 (amide III), which can be assigned to the random coil conformation (10). The FTIR spectrum of plain CS microparticles (Figure 4E) shows absorption bands at 1654 cm-1 (amide groups of residue chitin units) and 1586 cm-1 (free amino groups of chitosan units). The absorption bands at 1103 cm-1 are attributed to the saccharide structure of CS.

Figure 4 FTIR spectra of microparticles prepared with SF/CS blend ratios of (A) 1:0, (B) 2:1, (C) 1:1, (D) 1:2 and (E) 0:1 (w/w).
Figure 4

FTIR spectra of microparticles prepared with SF/CS blend ratios of (A) 1:0, (B) 2:1, (C) 1:1, (D) 1:2 and (E) 0:1 (w/w).

The FTIR spectra of the blend microparticles in Figure 4B–D showed both SF and CS characteristics. Predictably, the intensity of the saccharide bands significantly increased as the amount of CS in the blend increased. Interestingly, on blending with CS, the SF amide I and II bands were shifted to a lower wave number, while the SF amide III bands were shifted to a higher wave number. It is significant that the magnitude of the shift increased as the amount of CS in the blend increased. The FTIR data suggest that the conformation of the SF in the blend microparticles changed from the random coil to the β-sheet form. This effect may be related to the intermolecular interactions between the SF and the CS components that induced a change in the SF conformation. The rigid CS molecules may act as templates that encourage the SF molecules to assemble into the more ordered β-sheet form (11, 14).

3.3 Dissolution of blend microparticles

Table 1 shows the percentage dissolution of the blend microparticles in phosphate buffer solution (pH 7.4 at 37°C) for 24 h compared to the pure SF and CS microparticles. The dissolution values of these last two microparticles were 63% and 60%, respectively. Blending of the CS and SF caused the percentage dissolution to fall below these values: the dissolutions of the 2:1, 1:1 and 1:2 (w/w) SF/CS blend microparticles were 49%, 46% and 40%, respectively. Increasing the CS content in the blend led to a further decrease in the percentage dissolution. The conformational transition of SF from a random coil (water soluble) to a β-sheet (water-insoluble) structure was induced by blending with CS (11, 14), according to previous FTIR results. The control of the dissolution behavior of the microparticles is important for drug delivery applications as control over dissolution can result in control of the drug release rate.

3.4 Density and porosity of blend microparticles

The porous structure within the SF, CS and their blend microparticles was confirmed by the density data (Figure 5). The densities of the SF and CS microparticles were 1.28 and 1.68 g/ml, respectively. Meanwhile, the densities of the non-porous SF and CS films prepared by the solvent evaporation method were 2.14 and 2.34 g/ml, respectively. This may be explained by the SF and CS microparticles containing a porous structure, as previously described in the SEM results.

Figure 5 Densities of (□) microparticles and (■) films of SF, CS and SF/CS blends.
Figure 5

Densities of (□) microparticles and (■) films of SF, CS and SF/CS blends.

The density of the blend microparticles steadily increased as the amount of CS in the blend increased. The densities of the 2:1, 1:1 and 1:2 (w/w) SF/CS blend microparticles were 1.30, 1.35 and 1.48 g/ml, respectively. This was due to the density of the CS component being higher than that of the SF component. The density results showed that the blend microparticles with various SF/CS blend ratios and densities can be prepared by the emulsification-diffusion method. Figure 5 also shows that the density of the non-porous SF/CS blend films was slightly increased as the amount of CS in the blend increased, similar to the density change of the SF/CS blend microparticles. The densities of the SF/CS blend films were higher than those of the SF/CS blend films for the same blend ratio.

The porosities of the microparticles calculated from the ratio of microparticle and film densities using Equation 2 are summarized in Table 1. The porosities for the SF and CS microparticles were 40% and 28%, respectively, indicating that the SF microparticles have a more porous structure than the CS microparticles. This may be explained by the viscosity of the SF aqueous solution being lower than the CS aqueous solution from our observations. The external continuous phase, ethyl acetate, can more easily diffuse into the dispersed droplets of the SF solution than into those of the CS solution. The diffused ethyl acetate induced a porous structure within the microparticles after solidification and drying (24).

The porosity of the blend microparticles increased from 28% to 35% and 40% when the SF/CS blend ratio was changed from 0:1 to 1:2 and 1:1 (w/w). This may be due to the viscosity of the blend solution decreasing as the SF content increased. However, the porosities of the 1:1 (40%) and 2:1 (41%) (w/w) SF/CS blend microparticles were similar to those of the SF microparticles (40%). This may be due to the viscosities of these solutions being similar. The porosity is an important functional property for drug-loaded microparticles that can affect drug release from microparticles. Decreasing the porosity of the microparticles led to decreases in the drug mobility and drug release rate (41).

4 Conclusions

SF/CS blend microparticles were successfully prepared by the W/O emulsification-diffusion method in ethyl acetate with no surfactants added. The SF microparticles were irregular in shape, while the CS and SF/CS blend microparticles were spherical with a rough surface texture. The interior regions of the microparticles had a sponge-like (porous) structure. The average microparticle size was found to vary between 73 and 80 μm. The addition of CS to SF induced conformational changes in the latter from the random coil to β-sheet form. The dissolution, density and porosity of the blend microparticles can be controlled by adjusting the SF/CS blend ratio. These blend microparticles should be considered as promising biodegradable and biocompatible drug carriers for sustained release of water-soluble drugs.

Corresponding author: Onanong Cheerarot, Biodegradable Polymers Research Unit, Faculty of Science, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Mahasarakham University, Mahasarakham 44150, Thailand, e-mail:

Acknowledgments

This work was supported by the National Metal and Materials Technology Center (MTEC), National Science and Technology Development Agency (NSTDA), Ministry of Science and Technology, Thailand (MT-B-52-BMD-68-180-G) and by the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education, Thailand. The authors would like to thank Dr. Christopher M. Liauw, Manchester Metropolitan University, England, UK, for proofreading of the manuscript.

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Received: 2014-7-7
Accepted: 2014-12-17
Published Online: 2015-1-21
Published in Print: 2015-3-1

©2015 by De Gruyter

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  10. UV-Cured polypropylene mesh-reinforced composite polymer electrolyte membranes
  11. Silica aerogel/epoxy composites with preserved aerogel pores and low thermal conductivity
  12. Copper-amine complex solution as a low-emission catalyst for flexible polyurethane foam preparation
  13. Using an artificial neural network for the evaluation of the parameters controlling PVA/chitosan electrospun nanofibers diameter
https://doi.org/10.1515/epoly-2014-0134
Keywords for this article
blend microparticles; chitosan; conformational transition; morphology; silk fibroin

Articles in the same Issue

  1. Frontmatter
  2. In this Issue
  3. Editorial
  4. Editorial March 2015
  5. Full length articles
  6. Biodegradable silk fibroin/chitosan blend microparticles prepared by emulsification-diffusion method
  7. Living radical polymerization of polyacrylamide with submicrometer size by dispersion polymerization
  8. Investigation of pH-dependent swelling behavior and kinetic parameters of novel poly(acrylamide-co-acrylic acid) hydrogels with spirulina
  9. Significantly improving the performance and dispersion morphology of porous g-C3N4/PANI composites by an interfacial polymerization method
  10. UV-Cured polypropylene mesh-reinforced composite polymer electrolyte membranes
  11. Silica aerogel/epoxy composites with preserved aerogel pores and low thermal conductivity
  12. Copper-amine complex solution as a low-emission catalyst for flexible polyurethane foam preparation
  13. Using an artificial neural network for the evaluation of the parameters controlling PVA/chitosan electrospun nanofibers diameter
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