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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effect of shear viscosity on the preparation of sphere-like silk fibroin microparticles by electrospraying
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Moo Kon Kim a , Jeong Yun Lee a , Hanjin Oh b , Dae Woong Song a , Hyo Won Kwak a , Haesung Yun a , In Chul Um c , Young Hwan Park a , Ki Hoon Lee a,d,e,∗ a
Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea National Instrumentation Center for Environmental Management, Seoul National University, Seoul 151-921, Republic of Korea c Department of Bio-fibers and Materials, Kyungpook National University, Daegu 702-701, Republic of Korea d Center for Food and Bioconvergence, Seoul National University, Seoul 151-921, Republic of Korea e Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea
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Article history: Received 26 December 2014 Received in revised form 20 May 2015 Accepted 22 May 2015 Available online xxx
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Keywords: Silk fibroin Electrospraying Microparticle Cell carrier
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1. Introduction
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Silk fibroin (SF) is known to be a biocompatible material, and different forms of SF are used for various applications. However, the application of SF in particle form is rarely reported, compared to other forms. In this study, SF microparticles with a diameter of approximately 250 m were prepared by the electrospray method, using 1 M LiCl/DMSO as a solvent. The dissolution time of SF in the CaCl2 /CH3 CH2 OH/H2 O solution and the concentration of the SF dope solution affected the final morphology of the microparticles. A long dissolution time and a low SF concentration led to the formation of irregular microparticles, but a short dissolution time and a high concentration produced sphere-like microparticles. The shear viscosity of the SF dope solution was the main parameter that affected the morphology of the SF microparticles. Regardless of the dissolution time in the CaCl2 /CH3 CH2 OH/H2 O solution and the concentration of the SF dope solution, the shear viscosity of the dope solution must be higher than 0.33 Pa s to produce spherelike microparticles. Finally, cell adhesion experiments demonstrated that these SF microparticles show potential for use as cell carriers. © 2015 Published by Elsevier B.V.
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Polymeric materials have been widely adopted in biomedical research, for use as scaffolds or as drug carriers. One of the main advantages of polymeric materials is that they can easily be formed into various shapes, such as films, fibers, gels, particles, and sponges, with sizes ranging from nanometers to centimeters. Both synthetic and natural polymers are currently used, but natural polymers show better bioactivity than synthetic polymers [1]. Silk fibroin (SF) is a well-known biomaterial, and it has been applied in both tissue engineering and drug delivery [2]. Several studies have reported that SF could prove to be an excellent scaffold for various cell types, including stem cells [3,4]. In addition, some studies have also reported on the use of SF as a drug carrier [5]. As a biomaterial, SF is not only biocompatible and slowly
∗ Corresponding author at: Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea. Tel.: +82 2 880 4625; fax: +82 2 873 2285. E-mail address: [email protected] (K.H. Lee).
biodegradable, but it also has a relatively good processing ability. Both mulberry and non-mulberry SF are now used as biomaterials [6,7]. SF can be shaped into various forms, such as films, hydrogels, sub-micron fibers, and nano- and microparticles. Many studies are currently underway on the application of electrospun SF nanofibers (or sub-micron fibers) as scaffolds in tissue engineering [8,9]. However, relatively few applications of the particulate forms of SF have been reported, compared to SF nanofibers. SF nano- and microparticles can be prepared using several methods. Although emulsion techniques are frequently used to prepare polymeric microparticles, this technique has only been used to prepare SF microparticles in a few cases. Baimark et al. prepared porous SF microparticles using a water-in-oil emulsion technique [10]. The particles were spherical and ranged in size from 50 to 150 m. Self-assembly is the most widely used method for SF nanoand microparticle preparation. Jin and Kaplan reported that SF forms sphere-like micelles via a self-assembly mechanism [11]. This mechanism can be induced either by adding a hydrophilic polymer to the mixture [12] or by adding water-miscible organic solvents, such as acetone [13], ethanol [14], or dimethylsulfoxide [15]. When a hydrophilic polymer is mixed with the SF solution,
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phase separation of the two polymers occurs, and the SF assembles into nano- or microparticles [12]. In addition, some water-miscible organic solvents can serve as non-solvents for SF; therefore, the addition of SF solution to an organic solvent or vice versa can induce SF precipitation. Gradual addition of water-miscible organic solvents induces a transition to a secondary SF structure, followed by precipitation. Precipitation will not progress to large aggregates due to electrostatic repulsion, and nano- or microparticles will be formed [13–15]. Another technique that has been used for SF microparticle preparation is spray drying [16]. Although it is difficult to prepare nanometer-scale particles using this process, it is relatively simple compared to other methods. Various other methods for SF nano- or microparticle preparation have been reported, including techniques such as the use of supercritical CO2 [17], a template technique using liposomes [18], a salting out system [19], a capillary microdot technique [20], a milling process [21], and a laminar jet break-up technique [22]. Electrospraying, sometimes referred to as electrohydrodynamic spraying, employs strong electrostatic forces to cause emission of polymer particles from the meniscus of a polymer solution. Particles that range in size from nano- to micrometers can be prepared; if the optimal conditions are identified, a narrow size distribution can be produced. In addition, two-needle coaxial electrospraying can be used to prepare capsules and bubbles [23]. The basic principles of electrospraying are similar to those of electrospinning, except that fibers are formed in the latter case. Therefore, using the same polymer and equipment, either particles or fibers can be formed, depending on the specified parameters. For example, particles will be formed if the polymer solution has a low viscosity or low concentration, but fibers will be formed if a higher viscosity or higher concentration solution is used [24]. Several other parameters can affect particle size, including the viscosity and conductivity of the polymer solution, the voltage applied, and the feeding rate of the dope solution. The electrospraying technique requires only simple instrumentation and allows for generation of monodisperse particles with high efficiency. In addition, the electrospraying technique applies less shear stress than the emulsion technique, allows easier control of microparticle size than the self-assembly method, and requires no heat, unlike spray drying [25,26]. Recent studies have suggested that various kinds of polymer particles can be formed by electrospraying and then used for drug delivery, because the electrosprayed particles are so small [27,28]. SF has also been formed by electrospraying, and SF nanopowder was prepared using formic acid as a solvent. SF nanopowder particles of approximately 80 nm in diameter were produced when a 0.25% (w/w) solution was applied [29]. In this study, SF microparticles were prepared using the electrospray method by dissolving SF in a 1 M LiCl/DMSO solvent, which has previously been used for sericin electrospraying [30]. First, we investigated the dissolution of SF in the LiCl/DMSO solvent, because this solvent had not previously been used for SF dissolution. We also investigated the effect of the molecular weight distribution (MWD) of the SF raw material and the concentration of the dope solution on the electrospray technique, to determine the optimum conditions for the preparation of SF microparticles. Finally, we examined the potential of these microparticles as cell carriers.
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2. Materials and methods
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2.1. Materials
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Bombyx mori cocoons were purchased from Heungjin (Korea). Dimethylsulfoxide (DMSO) was purchased from Amresco (USA), and methanol was purchased from Samchun (Korea). Lithium
Table 1 Types of SF used in this study. Sample
Solvent
Dissolution temperature
Dissolution time
SFL SFC5 SFC30 SFC60 SFC180
LiBr CaCl2 /CH3 CH2 OH/H2 O
r.t.a 80 ◦ C
15 h 5 min 30 min 60 min 180 min
a
Room temperature.
chloride, calcium chloride, and other reagents were purchased from Sigma–Aldrich (USA). 2.2. Preparation of regenerated SF The cocoons were degummed twice with a 0.2% (w/v) sodium carbonate and 0.3% (w/v) Marseille soup solution at 100 ◦ C for 30 min, and rinsed with distilled water to remove the sericin. The SF fibers were dried in an oven overnight at 50 ◦ C. Then, the SF fibers were dissolved in one of two different solutions: CaCl2 /CH3 CH2 OH/H2 O (SFC) or LiBr (SFL). For the first solution, the SF fibers (20 g) were dissolved in 500 mL of a CaCl2 /CH3 CH2 OH/H2 O (1/2/8 in molar ratio) solution at 80 ◦ C, with dissolution times that ranged from 5 min to 180 min. The second solution was prepared in a similar manner; the same weight of SF was dissolved in 500 mL of a 9.3 M LiBr solution at room temperature for 15 h. Each of the SF solutions was dialyzed in a dialysis tube (Spectra/Por® , MWCO 6-8000, USA) against distilled water for 72 h. The dialysis water was changed every 2 h during the day. Finally, after dialysis, the SF solutions were lyophilized to obtain an SF powder. Table 1 shows the sample codes for each of the SF powders used in this study. 2.3. Solubility of SF in LiCl/DMSO solvent SFL and SFC5 powders were dissolved in LiCl/DMSO solvents with various concentrations of LiCl. For each, 100 mg of SF powder was added to 1 mL of LiCl/DMSO solvent, and stirred at 40 ◦ C for 60 min to allow the SF to dissolve. Then, any undissolved SF was precipitated by centrifugation at 10,000 × g, and the absorbance of the supernatant was measured using a UV spectrometer (Optizen UV2120, Duksan Mecasys, Korea) at 280 nm. A calibration curve was used to calculate the total amount of dissolved SF, using a standard SF solution that contained no observable precipitate following centrifugation. The standard solution was prepared by dissolving SFC5 in 1 M LiCl/DMSO. The maximum solubility of SFC samples was measured in a similar manner, and a predetermined amount of SFC was added to 1 mL of 1 M LiCl/DMSO solvent. Finally, any undissolved portion of the solution was removed by centrifugation, and the absorbance of the supernatant was measured at 280 nm. 2.4. Preparation of SF microparticles by electrospraying SF microparticles were prepared by electrospraying the dope solution to a methanol coagulation bath (Scheme 1). The dope solution was prepared by dissolving a predetermined amount of SFC powder in 1 M LiCl/DMSO solvent to attain the desired concentration; the solution was then filtered through a nonwoven filter prior to electrospraying. One millimeter of dope solution was delivered to the electrospray apparatus via a syringe fitted with a 22 G needle. The dope solution was transferred to the needle tip using a syringe pump (KD Scientific, KDS100, Korea). The applied voltage and the flow rate were fixed at 22 kV and 0.4 mL/h, respectively; these values were determined based on experimental reproducibility. The distance from the needle tip to the methanol coagulation bath was
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(pH 7.2), at 4 ◦ C for 2 h. The cells were further washed with a sodium cacodylate buffer, and then post-fixated with 1% osmium tetroxide in a 0.05 M cacodylate buffer (pH 7.2) at 4 ◦ C for 2 h and then washed with distilled water at room temperature. Following this, the cells were dehydrated for 10 min using a graded ethanol series, with concentrations of 30%, 50%, 70%, 80%, 90%, and 100%. Finally, cell adhesion onto the SF microparticles was observed by FE-SEM. To observe the proliferation of fibroblast cells on carriers, an MTT assay was performed. The proliferation of fibroblasts on SF microparticles was compared with that on SFC5 cast film and commercial cell carrier, Cyclodex1 (GE Healthcare, USA). SFC5 cast film was prepared by evaporating the SFC5 solution on a Petri dish. Samples were sterilized in 70% EtOH for 1 h and washed thrice in PBS, for 1 h each time. Ten milligrams each of the microcarriers and the SF film (6 mm in diameter) were placed on the 96-well-plate and cultured fibroblast cells were seeded on the sample at 5000 cells/well in 200 L of DMEM. Cells were incubated at 37 ◦ C and 5% CO2 for 1, 4, and 7 days. After each incubation period, 20 L of the MTT solution (5 mg/mL in PBS) was added to each well and incubated at 37 ◦ C and 5% CO2 for 2 h. The culture medium was removed and 200 L of DMSO was added to each well for solubilization of formazan crystals and incubated for another 30 min with shaking. Finally, 150 L of the solubilized MTT solution was transferred to another 96-well plate and the absorbance at 570 nm was measured. 2.7. Statistical analysis
Scheme 1. Electrospraying of SF solutions.
10 cm. The collected SF microparticles were washed three times with methanol to remove the solvent and the salt. The scheme for 171 Q3 the electrospray apparatus is shown in the Supporting information. 172
Data were expressed as the mean ± SD from at least three separate experiments. Data were analyzed using Student’s t-tests for multiple comparisons using Origin software (Origin 8.0, OriginLab Cooperation, USA).
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3. Results and discussion
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2.5. Instrumental analyses
3.1. SF solubility in LiCl/DMSO
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The MWD of the SF was measured by gel filtration chromatography (GFC) (ÄKTA purifier, GE Healthcare, USA) using a Superdex column (Superdex 200 10/300GL, GE Healthcare, Sweden) at a flow rate of 0.5 mL/min; a 4 M urea solution was used for elution. To calculate the number average molecular weight (MN ) and the weight average molecular weight (MW ) of the SFCs, the following standard molecular weight markers were used for GFC: apoferritin (443 kDa), -amylase (200 kDa), alcohol dehydrogenase (150 kDa), and carbonic anhydrase (29 kDa). The shapes and sizes of the SF microparticles were measured after drying using a field emission scanning electron microscope (FE-SEM, JSM-7600F, JEOL, Japan). A rheometer (Advanced Rheometric Expansion System, ARES, Rheometric Scientific, UK) was used to measure the shear viscosity, using a cone and plate geometry, with a shear rate of 1.0–700 s−1 at 25 ◦ C. The diameter and angle of the cone were 50 mm and 0.04 rad, respectively. The shear viscosity of each sample was determined at a shear rate of 16 s−1 .
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2.6. Cell adhesion
The preparation of the polymer dope solution is critical, because the solution affects the entire downstream process. Generally, the SF dope solution can be prepared in three steps. First, the sericin should be removed via a degumming process, to obtain pure SF fibers. Second, the pure SF fibers can be dissolved using various methods to produce an SF aqueous solution. Currently, a highly concentrated LiBr solution or a tertiary solvent system of a CaCl2 /CH3 CH2 OH/H2 O solution is commonly used at this stage, and the salts are then removed by dialysis. Several studies have used the aqueous SF solution as the dope solution; however, the poor stability of SF in aqueous solutions can induce irreversible aggregation, which can be problematic at later stages of the procedure [31]. Therefore, in most studies, a third step is required to produce a stable SF dope solution. Typically, the SF aqueous solution is lyophilized into a powder and then dissolved in another solvent such as hexafluoroisopropanol (HFIP) or formic acid. In this study, LiCl/DMSO, instead of HFIP or formic acid, was used to prepare the SF dope solution. We had previously used this solvent, which is less expensive and less toxic than HFIP, to prepare sericin microparticles [27]. In addition, using this LiCl/DMSO solution, no indications of degradation during dissolution were observed, unlike when using formic acid. Fig. 1 shows the solubility of SF powder in a LiCl/DMSO solvent system. Two types of SF powder were prepared for the experiment. SF fiber was dissolved either in a 9.3 M LiBr (SFL) solution or in a CaCl2 /CH3 CH2 OH/H2 O (SFC) solution. The result indicates that addition of LiCl is necessary for dissolving SF in DMSO, regardless of the method of preparation. When 1 M LiCl was added to DMSO, the SFC5 completely dissolved within 60 min; however, in the 2 M LiCl/DMSO solvent, the solubility of the SFC5
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SF microparticles prepared with 10 wt% SFC5 were used for the cell adhesion experiment. SFC5 microparticles (10 mg) were sterilized with 70% EtOH and washed three times with a PBS buffer. Fibroblasts (1.5 × 105 per cm2 ) were seeded to the microparticles in the culture medium (DMEM) and cultivated in a CO2 incubator at 37 ◦ C. Cell adhesion on the microparticle was observed for a total of 4 days. At 24-h intervals, the cells on the microparticles were fixed with a modified Karnovsky’s fixative, containing 2% paraformaldehyde and 2% glutaraldehyde in a 0.05 M sodium cacodylate buffer
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Fig. 1. Effect of concentration of LiCl in DMSO solvent on the solubility of SFL and SFC5. The data represent the mean ± SD of 10 samples. * Significant at P < 0.05, ** significant at P < 0.01 (n.d., not dissolved).
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decreased to 75%, due to salting out. Compared to SFC, SFL had limited solubility in LiCl/DMSO. When a 0.5 M LiCl solution was added to DMSO, nearly 40% of the SFC5 dissolved; the SFL did not dissolve at all in this solvent. Maximum SFL solubility within the given time of 1 h was approximately 80% in 1 M LiCl/DMSO. Previous reports have indicated that LiCl can cleave hydrogen bonds in cellulose and protein [32,33], thereby increasing the solubility of these polymers in the organic solvent. The observed differences in solubility can be attributed to the difference in the MWD of SFL and SFC. We previously reported on the differences between SFL and SFC, based on MWD and hydrodynamic radius [31]. Although SFL retains the same molecular weight as SF, a significant decrease in molecular weight occurs during the preparation of SFC. As a result, the hydrodynamic radius of SFL is smaller than that of SFC, because non-degraded SFL maintains its original compact structure. Therefore, for a specified dissolution period, the solubility of SFL will be limited by its compact structure, which hinders LiCl penetration. Therefore, we used SFC as the raw material and 1 M LiCl/DMSO as the solvent for further experiments. The molecular weight of SFC, as discussed above, is significantly smaller than that of SF. Therefore, we hypothesized that the solubility of SFC would differ accordingly. In this study, four different SFC samples were prepared according to their dissolution time in a CaCl2 /CH3 CH2 OH/H2 O solution; Table 2 shows the MN , MW , and polydispersity index of each sample. The GFC graphs of each sample are provided in the Supporting Information (Fig. S1). The molecular weight of SFC decreased as the dissolution time increased. In addition, the polydispersity index of SFC also increased as the dissolution time increased, which indicates a broader MWD in those samples dissolved for a longer time in CaCl2 /CH3 CH2 OH/H2 O solution. These differences in molecular weight and its distribution could affect the solubility of SFC in 1 M LiCl/DMSO. To determine the
Table 2 Number average molecular weight (MN ), weight average molecular weight (MW ), and polydispersity index of SFCs. Sample
MN (kDa)
MW (kDa)
Polydispersity index
SFC5 SFC30 SFC60 SFC180
324 286 257 114
398 378 359 219
1.23 1.32 1.40 1.92
Fig. 2. Absorbance of the supernatant after dissolving a predetermined amount of various SFC samples in 1 mL of 1 M LiCl/DMSO solvent. The insoluble portion was precipitated by centrifugation (mean ± SD, n = 3).
highest dope concentration achievable, a predetermined amount of each SFC sample was dissolved in 1 M LiCl/DMSO. Fig. 2 shows the absorbance of the supernatant of each solution, according to the amount of SFC initially added to the solvent. The absence of a further increase in the absorbance indicates that the highest dope concentration has been reached. The highest achievable dope concentration for SFC5, SFC30, SFC60, and SFC180 was 10, 11, 13, and 14 wt%, respectively. Thus, more highly degraded SFC can reach a higher dope concentration in 1 M LiCl/DMSO, perhaps because of its lower molecular weight. 3.2. SF electrospraying A standard technique for electrospraying uses a volatile solvent that is volatized before it reaches the collecting plate. However, this study used DMSO, which has a boiling point that is much higher than room temperature. Therefore, the SF dope solution was electrosprayed toward a coagulant bath instead of the collecting plate, which solidified the SF droplets emitted from the needle tip. Methanol was used as a coagulant because it is a known standard and the most common solvent that can make SF insoluble. During electrospraying of the polymer, the shape and size of the nano- or microparticles greatly depends on several parameters, including the concentration of the polymer solution, the molecular weight of the polymer, the voltage applied, the flow rate, and the tip-tocollector distance. In this study, we focused on the concentration of the dope solution and the MWD of the SF; the applied voltage, flow rate, and tip-to-coagulant bath distance were fixed. 3.2.1. Effect of concentration Fig. 3 shows the effect of SFC concentration on microparticle shape. In the analysis of SFC5, the shape of the microparticles changed from irregular (6 wt%) to oval (9 wt%) and finally became sphere-like (10 wt%) as the SFC concentration increased. Previous studies have indicated that the concentration of the polymer has a high impact on the shape of the particles produced during electrospraying. Recently, Bock et al. [25] reviewed the process for electrospraying of polymers and reported the effect of polymer concentration on the shape of the resulting polymer particles. Polymer concentration is important because it is correlated with polymer chain entanglement. In a dilute polymer solution, there will be no chain entanglement between individual polymer chains. However,
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Fig. 4. Shear viscosity of SFCs in 1 M LiCl/DMSO solvent. The concentration of each sample was 10 wt%.
Fig. 3. SEM images of SFC microparticles according to dissolution time and concentration (scale bar = 200 m).
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when the concentration increases, the polymer chains will start to overlap; the concentration at which this occurs is referred to as the critical chain overlap concentration (Cov ). Therefore, if the concentration of the polymer in the solution is lower than the Cov , no chain entanglement will occur; this is known as the dilute regime. In this condition, electrospraying is impossible or debris is formed. If the concentration is higher than the Cov , three different regimes are possible. The first is the semi-dilute unentangled regime, in which the concentration of the polymer is sufficient for polymer chain overlap, but entanglement is limited. In this regime, particles have an irregular, non-reproducible morphology. The second is designated as the semi-dilute moderately entangled regime, which occurs at higher polymer concentrations, when polymer chains have moderate entanglement, creating dense particles. The semidilute moderately entangled regime is the only regime in which the formation of sphere-like particles can be achieved by electrospraying. Finally, a further increase in the concentration produces fibers or beaded fibers, particularly if the polymer concentration greatly exceeds the Cov . This third regime is known as the semi-dilute highly entangled regime; the process that occurs is referred to as electrospinning. Similar results were also observed in our study. For each SFC sample, irregular shapes were formed at relatively low SFC concentrations, as follows: SFC5 ≤ 8 wt%, SFC30 ≤ 9 wt%, and SFC60 ≤ 11 wt%. No concentration of SFC180 produced regularly shaped particles. These irregular shapes indicate that stable SFC droplets cannot be formed during flight, due to limited
entanglement between the SFC chains; this could be referred to as the semi-dilute unentangled regime. In some samples, however, hemispherical particles were observed (SFC5 at 7 and 8 wt% and SFC30 at 9 wt%), which indicates that the SFC droplets initially had a spherical shape during flight, but were not sufficiently dense to hold that shape and deformed when they collided with the surface of the coagulant bath. Uniform particles cannot be prepared in these conditions. However, when the concentration increases further, more sphere-like particles could be obtained. Spherelike microparticles were observed, along with some bean-like or oval microparticles. When using SFC5 at 10 wt%, SFC30 at 11 wt%, and SFC60 at 12 wt%, the occurrence of sphere-like microparticles increased significantly. At these concentrations, chain entanglement was sufficient to create dense droplets, which could overcome the surface tension of the coagulant. These concentrations represent the semi-dilute moderately entangled regime of SFC. The semi-dilute highly entangled regime was not observed in this study, because a sufficiently concentrated SFC solution could not be prepared (Fig. 2). The diameter of the sphere-like SFC microparticles was approximately 200 m. Interestingly, during the production of the SFC180 microparticles, sphere-like microparticles did not form, even at the maximum concentration. This result indicates that the MWD of SF is also important; we therefore investigated the effects of the MWD of SF. 3.2.2. Effect of dissolution time in calcium chloride/ethyl alcohol/water solvents Examination of the 10 wt% concentration of all SFCs indicated that a similar change, from an irregular to a sphere-like shape, can be observed between SFC180 and SFC60 samples, to SFC30 and SFC5 samples. The MWD of each SFC would cause this result, since the MWD of SFC is greatly affected by the dissolution time in a CaCl2 /CH3 CH2 OH/H2 O solution, as shown in Table 2. A previous study reported that the shear viscosity of SFC in water and formic acid is also greatly affected by the dissolution time [31]. Because the shear viscosity is an important factor in the electrospraying process, we investigated how the shear viscosity of SFC in 1 M LiCl/DMSO affects the final shape of the microparticles. Fig. 4 shows the rheological behavior of 10 wt% SFCs in 1 M LiCl/DMSO solvent. Original SF in the silkworm gland exhibits non-Newtonian behavior, typically shear thinning [34]. In this study, SF in 1 M LiCl/DMSO solvent also exhibited shear thinning, although the extent was very small. Similar shear thinning of SF has been observed in other solvents, such as formic acid [31]. SFC subjected to a longer dissolution time had a lower shear viscosity. The increase in the low-molecular weight SFC would allow for less entanglement
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Fig. 5. Shear viscosity of SFC solutions according to concentration and dissolution time (mean ± SD, n = 3).
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between SFC chains, which would result in a lower shear viscosity. Based on the knowledge that entanglement is the main factor that determines microparticle shape, a decrease in entanglement would have the same effect as a decrease in concentration. Therefore, it can be concluded that both concentration and dissolution time in a CaCl2 /CH3 CH2 OH/H2 O solution can affect the final shape of SFC microparticles. 3.3. Shear viscosity of SF solutions for preparation of sphere-like microparticles This study has demonstrated that both the concentration and the dissolution time in a CaCl2 /CH3 CH2 OH/H2 O solution affect the shape of SF microparticles. Because polymer concentration and molecular weight are the primary factors that determine the viscosity of a polymer solution, their effect on the shear viscosity of SF solutions was investigated. Here, the molecular weight of the SFC was controlled by the dissolution time in a CaCl2 /CH3 CH2 OH/H2 O solution, as shown in Table 2. Fig. 5 presents a summary of the
previously discussed results in the form of a graph that shows concentration versus shear viscosity. Based on the flow rate of the SF solution in the needle, the shear rate during electrospraying was calculated to be 16 s−1 . We compared the shear viscosity at this shear rate with the concentration and the final shape of the SF microparticles in Fig. 5. The shear viscosity of each sample is provided in the Supporting information (Figs. S2–S5). Based on the shape of the SF microparticles, the graph can be divided into three regions. First, where the shear viscosity was low, only irregular particles were formed due to the low degree of entanglement. As the shear viscosity increased, bean-like or oval microparticles were formed, but the degree of entanglement was not yet sufficient to create sphere-like particles. Finally, when the shear viscosity exceeded approximately 0.33 Pa s, solid sphere-like microparticles were formed due to the relatively high degree of entanglement, which resulted in formation of dense droplets. This figure illustrates that the shear viscosity is a critical factor in determining the shape of SFC microparticles. Furthermore, any combination of concentration and dissolution time that produces a particular shear viscosity range would create SF microparticles of similar shape. 3.4. Application of SF microparticles The potential applications of these sphere-like SF microparticles include targeting of biomedical therapies; we therefore investigated their application as cell carriers for cell therapy. In cell therapy, cells are delivered to a target site, where they will proliferate and differentiate, ultimately regenerating the tissue. Initially, bone and cartilage were the main target tissues, but recent work with stem cells has allowed this therapy to be applied to all kinds of tissues. Various materials have been considered as cell carriers such as poly(␣-hydroxyesters), collagen, gelatin, fibrin, chitosan, alginate, dextran, and others [35]. For cell culture experiments, SFC microparticles were prepared using SFC5, which has a non-porous structure and an average diameter of 250 ± 35 m (Fig. 6). Fig. 7 shows the images of growing fibroblasts on SFC microparticles during 4 days of culture. Cell adhesion was observed beginning on day 2, and some cells formed focal adhesions. Usually, cell adhesion on SF substrates is rapid; however, cells were observed to initially adhere to the tissue culture plate on
Fig. 6. SEM images of SFC5 microparticle in (a) low (500×, scale bar = 100 m) and (b) high (30,000×, scale bar = 1 m) magnification.
Fig. 7. SEM images of fibroblast cell culture on SFC5 microparticles (scale bar = 100 m).
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We prepared sphere-like SF microparticles via electrospraying, using a 1 M LiCl/DMSO solvent. We also demonstrated that shear viscosity is the primary parameter that affects the final morphology of SF microparticles under the described conditions; this is also true using other solvent systems such as HFIP and formic acid. The use of a chaotropic solvent and a coagulant bath would reduce the effectiveness of the SF microparticles for the delivery of therapeutic agents. However, the preliminary results of this study showed that SF microparticles have potential for applications in cell delivery. Additional studies are needed to further evaluate the technique and to optimize the application of these microparticles in tissue engineering. Fig. 8. SEM image of fibroblast morphology attached onto SFC5 microparticle (scale bar = 20 m). Inserted image is magnified image of boxed area (scale bar = 1 m).
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This work was supported by a Marine Biomaterials Research Center grant from the Marine Biotechnology Program funded by the Ministry of Land, Transport and Maritime Affairs, Korea, Q4 and by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (NRF-2010-0025378 and NRF2013R1A1A2059239). References
Fig. 9. MTT assay of fibroblast cultured on SFC5 film, Cyclodex1 and SFC5 microparticles for 7 days (mean ± SD, n = 5, * significant at P < 0.01).
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day 1, before migrating to the SFC microparticles. Therefore, no cells were observed on SFC particles on day 1. Cell adhesion proceeded through day 3; the entire surface of the SF microparticles was covered by cells on day 4. Fig. 8 shows the morphology of fibroblast cells on SFC5 microparticle after 4 days of incubation. The smooth surface of SFC5 microparticle is covered with dense and compact cell layer. Cells were also covered with fibrillar network indicating successful production of extracellular matrix (inserted image). To quantify fibroblast adhesion and proliferation, a standard MTT test was performed. SFC5 film and the commercial dextranbased non-porous microcarrier, Cyclodex1 were chosen for comparison. As shown in Fig. 9, SFC5 microparticles showed the highest rate of proliferation among the samples tested. Compared to SFC5 film, the high surface area of SFC5 microparticles allowed much greater cell adhesion and proliferation. SFC5 microparticles showed better cell attachment than the commercial Cyclodex1 microcarrier, due to the higher affinity of fibroblasts to SF than to dextran. After 4 days of culture, further increase of fibroblast on the SFC5 microparticles could not be observed because almost all surface area were already occupied at day 4 (Figs. 7 and 8). Fibroblast cells were used here because they are easy to culture, but SF shows great potential for use with various cell types, such as osteoblasts [6,36], chondrocytes [37,38] and even stem cells [4,39,40]. Thus, SF microparticles show the potential to contribute to the development of cell therapy.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effect of shear viscosity on the preparation of sphere-like silk fibroin microparticles by electrospraying
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Moo Kon Kim a , Jeong Yun Lee a , Hanjin Oh b , Dae Woong Song a , Hyo Won Kwak a , Haesung Yun a , In Chul Um c , Young Hwan Park a , Ki Hoon Lee a,d,e,∗ a
Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea National Instrumentation Center for Environmental Management, Seoul National University, Seoul 151-921, Republic of Korea c Department of Bio-fibers and Materials, Kyungpook National University, Daegu 702-701, Republic of Korea d Center for Food and Bioconvergence, Seoul National University, Seoul 151-921, Republic of Korea e Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea
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Article history: Received 26 December 2014 Received in revised form 20 May 2015 Accepted 22 May 2015 Available online xxx
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Keywords: Silk fibroin Electrospraying Microparticle Cell carrier
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1. Introduction
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Silk fibroin (SF) is known to be a biocompatible material, and different forms of SF are used for various applications. However, the application of SF in particle form is rarely reported, compared to other forms. In this study, SF microparticles with a diameter of approximately 250 m were prepared by the electrospray method, using 1 M LiCl/DMSO as a solvent. The dissolution time of SF in the CaCl2 /CH3 CH2 OH/H2 O solution and the concentration of the SF dope solution affected the final morphology of the microparticles. A long dissolution time and a low SF concentration led to the formation of irregular microparticles, but a short dissolution time and a high concentration produced sphere-like microparticles. The shear viscosity of the SF dope solution was the main parameter that affected the morphology of the SF microparticles. Regardless of the dissolution time in the CaCl2 /CH3 CH2 OH/H2 O solution and the concentration of the SF dope solution, the shear viscosity of the dope solution must be higher than 0.33 Pa s to produce spherelike microparticles. Finally, cell adhesion experiments demonstrated that these SF microparticles show potential for use as cell carriers. © 2015 Published by Elsevier B.V.
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Polymeric materials have been widely adopted in biomedical research, for use as scaffolds or as drug carriers. One of the main advantages of polymeric materials is that they can easily be formed into various shapes, such as films, fibers, gels, particles, and sponges, with sizes ranging from nanometers to centimeters. Both synthetic and natural polymers are currently used, but natural polymers show better bioactivity than synthetic polymers [1]. Silk fibroin (SF) is a well-known biomaterial, and it has been applied in both tissue engineering and drug delivery [2]. Several studies have reported that SF could prove to be an excellent scaffold for various cell types, including stem cells [3,4]. In addition, some studies have also reported on the use of SF as a drug carrier [5]. As a biomaterial, SF is not only biocompatible and slowly
∗ Corresponding author at: Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea. Tel.: +82 2 880 4625; fax: +82 2 873 2285. E-mail address: [email protected] (K.H. Lee).
biodegradable, but it also has a relatively good processing ability. Both mulberry and non-mulberry SF are now used as biomaterials [6,7]. SF can be shaped into various forms, such as films, hydrogels, sub-micron fibers, and nano- and microparticles. Many studies are currently underway on the application of electrospun SF nanofibers (or sub-micron fibers) as scaffolds in tissue engineering [8,9]. However, relatively few applications of the particulate forms of SF have been reported, compared to SF nanofibers. SF nano- and microparticles can be prepared using several methods. Although emulsion techniques are frequently used to prepare polymeric microparticles, this technique has only been used to prepare SF microparticles in a few cases. Baimark et al. prepared porous SF microparticles using a water-in-oil emulsion technique [10]. The particles were spherical and ranged in size from 50 to 150 m. Self-assembly is the most widely used method for SF nanoand microparticle preparation. Jin and Kaplan reported that SF forms sphere-like micelles via a self-assembly mechanism [11]. This mechanism can be induced either by adding a hydrophilic polymer to the mixture [12] or by adding water-miscible organic solvents, such as acetone [13], ethanol [14], or dimethylsulfoxide [15]. When a hydrophilic polymer is mixed with the SF solution,
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phase separation of the two polymers occurs, and the SF assembles into nano- or microparticles [12]. In addition, some water-miscible organic solvents can serve as non-solvents for SF; therefore, the addition of SF solution to an organic solvent or vice versa can induce SF precipitation. Gradual addition of water-miscible organic solvents induces a transition to a secondary SF structure, followed by precipitation. Precipitation will not progress to large aggregates due to electrostatic repulsion, and nano- or microparticles will be formed [13–15]. Another technique that has been used for SF microparticle preparation is spray drying [16]. Although it is difficult to prepare nanometer-scale particles using this process, it is relatively simple compared to other methods. Various other methods for SF nano- or microparticle preparation have been reported, including techniques such as the use of supercritical CO2 [17], a template technique using liposomes [18], a salting out system [19], a capillary microdot technique [20], a milling process [21], and a laminar jet break-up technique [22]. Electrospraying, sometimes referred to as electrohydrodynamic spraying, employs strong electrostatic forces to cause emission of polymer particles from the meniscus of a polymer solution. Particles that range in size from nano- to micrometers can be prepared; if the optimal conditions are identified, a narrow size distribution can be produced. In addition, two-needle coaxial electrospraying can be used to prepare capsules and bubbles [23]. The basic principles of electrospraying are similar to those of electrospinning, except that fibers are formed in the latter case. Therefore, using the same polymer and equipment, either particles or fibers can be formed, depending on the specified parameters. For example, particles will be formed if the polymer solution has a low viscosity or low concentration, but fibers will be formed if a higher viscosity or higher concentration solution is used [24]. Several other parameters can affect particle size, including the viscosity and conductivity of the polymer solution, the voltage applied, and the feeding rate of the dope solution. The electrospraying technique requires only simple instrumentation and allows for generation of monodisperse particles with high efficiency. In addition, the electrospraying technique applies less shear stress than the emulsion technique, allows easier control of microparticle size than the self-assembly method, and requires no heat, unlike spray drying [25,26]. Recent studies have suggested that various kinds of polymer particles can be formed by electrospraying and then used for drug delivery, because the electrosprayed particles are so small [27,28]. SF has also been formed by electrospraying, and SF nanopowder was prepared using formic acid as a solvent. SF nanopowder particles of approximately 80 nm in diameter were produced when a 0.25% (w/w) solution was applied [29]. In this study, SF microparticles were prepared using the electrospray method by dissolving SF in a 1 M LiCl/DMSO solvent, which has previously been used for sericin electrospraying [30]. First, we investigated the dissolution of SF in the LiCl/DMSO solvent, because this solvent had not previously been used for SF dissolution. We also investigated the effect of the molecular weight distribution (MWD) of the SF raw material and the concentration of the dope solution on the electrospray technique, to determine the optimum conditions for the preparation of SF microparticles. Finally, we examined the potential of these microparticles as cell carriers.
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Bombyx mori cocoons were purchased from Heungjin (Korea). Dimethylsulfoxide (DMSO) was purchased from Amresco (USA), and methanol was purchased from Samchun (Korea). Lithium
Table 1 Types of SF used in this study. Sample
Solvent
Dissolution temperature
Dissolution time
SFL SFC5 SFC30 SFC60 SFC180
LiBr CaCl2 /CH3 CH2 OH/H2 O
r.t.a 80 ◦ C
15 h 5 min 30 min 60 min 180 min
a
Room temperature.
chloride, calcium chloride, and other reagents were purchased from Sigma–Aldrich (USA). 2.2. Preparation of regenerated SF The cocoons were degummed twice with a 0.2% (w/v) sodium carbonate and 0.3% (w/v) Marseille soup solution at 100 ◦ C for 30 min, and rinsed with distilled water to remove the sericin. The SF fibers were dried in an oven overnight at 50 ◦ C. Then, the SF fibers were dissolved in one of two different solutions: CaCl2 /CH3 CH2 OH/H2 O (SFC) or LiBr (SFL). For the first solution, the SF fibers (20 g) were dissolved in 500 mL of a CaCl2 /CH3 CH2 OH/H2 O (1/2/8 in molar ratio) solution at 80 ◦ C, with dissolution times that ranged from 5 min to 180 min. The second solution was prepared in a similar manner; the same weight of SF was dissolved in 500 mL of a 9.3 M LiBr solution at room temperature for 15 h. Each of the SF solutions was dialyzed in a dialysis tube (Spectra/Por® , MWCO 6-8000, USA) against distilled water for 72 h. The dialysis water was changed every 2 h during the day. Finally, after dialysis, the SF solutions were lyophilized to obtain an SF powder. Table 1 shows the sample codes for each of the SF powders used in this study. 2.3. Solubility of SF in LiCl/DMSO solvent SFL and SFC5 powders were dissolved in LiCl/DMSO solvents with various concentrations of LiCl. For each, 100 mg of SF powder was added to 1 mL of LiCl/DMSO solvent, and stirred at 40 ◦ C for 60 min to allow the SF to dissolve. Then, any undissolved SF was precipitated by centrifugation at 10,000 × g, and the absorbance of the supernatant was measured using a UV spectrometer (Optizen UV2120, Duksan Mecasys, Korea) at 280 nm. A calibration curve was used to calculate the total amount of dissolved SF, using a standard SF solution that contained no observable precipitate following centrifugation. The standard solution was prepared by dissolving SFC5 in 1 M LiCl/DMSO. The maximum solubility of SFC samples was measured in a similar manner, and a predetermined amount of SFC was added to 1 mL of 1 M LiCl/DMSO solvent. Finally, any undissolved portion of the solution was removed by centrifugation, and the absorbance of the supernatant was measured at 280 nm. 2.4. Preparation of SF microparticles by electrospraying SF microparticles were prepared by electrospraying the dope solution to a methanol coagulation bath (Scheme 1). The dope solution was prepared by dissolving a predetermined amount of SFC powder in 1 M LiCl/DMSO solvent to attain the desired concentration; the solution was then filtered through a nonwoven filter prior to electrospraying. One millimeter of dope solution was delivered to the electrospray apparatus via a syringe fitted with a 22 G needle. The dope solution was transferred to the needle tip using a syringe pump (KD Scientific, KDS100, Korea). The applied voltage and the flow rate were fixed at 22 kV and 0.4 mL/h, respectively; these values were determined based on experimental reproducibility. The distance from the needle tip to the methanol coagulation bath was
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(pH 7.2), at 4 ◦ C for 2 h. The cells were further washed with a sodium cacodylate buffer, and then post-fixated with 1% osmium tetroxide in a 0.05 M cacodylate buffer (pH 7.2) at 4 ◦ C for 2 h and then washed with distilled water at room temperature. Following this, the cells were dehydrated for 10 min using a graded ethanol series, with concentrations of 30%, 50%, 70%, 80%, 90%, and 100%. Finally, cell adhesion onto the SF microparticles was observed by FE-SEM. To observe the proliferation of fibroblast cells on carriers, an MTT assay was performed. The proliferation of fibroblasts on SF microparticles was compared with that on SFC5 cast film and commercial cell carrier, Cyclodex1 (GE Healthcare, USA). SFC5 cast film was prepared by evaporating the SFC5 solution on a Petri dish. Samples were sterilized in 70% EtOH for 1 h and washed thrice in PBS, for 1 h each time. Ten milligrams each of the microcarriers and the SF film (6 mm in diameter) were placed on the 96-well-plate and cultured fibroblast cells were seeded on the sample at 5000 cells/well in 200 L of DMEM. Cells were incubated at 37 ◦ C and 5% CO2 for 1, 4, and 7 days. After each incubation period, 20 L of the MTT solution (5 mg/mL in PBS) was added to each well and incubated at 37 ◦ C and 5% CO2 for 2 h. The culture medium was removed and 200 L of DMSO was added to each well for solubilization of formazan crystals and incubated for another 30 min with shaking. Finally, 150 L of the solubilized MTT solution was transferred to another 96-well plate and the absorbance at 570 nm was measured. 2.7. Statistical analysis
Scheme 1. Electrospraying of SF solutions.
10 cm. The collected SF microparticles were washed three times with methanol to remove the solvent and the salt. The scheme for 171 Q3 the electrospray apparatus is shown in the Supporting information. 172
Data were expressed as the mean ± SD from at least three separate experiments. Data were analyzed using Student’s t-tests for multiple comparisons using Origin software (Origin 8.0, OriginLab Cooperation, USA).
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2.5. Instrumental analyses
3.1. SF solubility in LiCl/DMSO
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The MWD of the SF was measured by gel filtration chromatography (GFC) (ÄKTA purifier, GE Healthcare, USA) using a Superdex column (Superdex 200 10/300GL, GE Healthcare, Sweden) at a flow rate of 0.5 mL/min; a 4 M urea solution was used for elution. To calculate the number average molecular weight (MN ) and the weight average molecular weight (MW ) of the SFCs, the following standard molecular weight markers were used for GFC: apoferritin (443 kDa), -amylase (200 kDa), alcohol dehydrogenase (150 kDa), and carbonic anhydrase (29 kDa). The shapes and sizes of the SF microparticles were measured after drying using a field emission scanning electron microscope (FE-SEM, JSM-7600F, JEOL, Japan). A rheometer (Advanced Rheometric Expansion System, ARES, Rheometric Scientific, UK) was used to measure the shear viscosity, using a cone and plate geometry, with a shear rate of 1.0–700 s−1 at 25 ◦ C. The diameter and angle of the cone were 50 mm and 0.04 rad, respectively. The shear viscosity of each sample was determined at a shear rate of 16 s−1 .
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2.6. Cell adhesion
The preparation of the polymer dope solution is critical, because the solution affects the entire downstream process. Generally, the SF dope solution can be prepared in three steps. First, the sericin should be removed via a degumming process, to obtain pure SF fibers. Second, the pure SF fibers can be dissolved using various methods to produce an SF aqueous solution. Currently, a highly concentrated LiBr solution or a tertiary solvent system of a CaCl2 /CH3 CH2 OH/H2 O solution is commonly used at this stage, and the salts are then removed by dialysis. Several studies have used the aqueous SF solution as the dope solution; however, the poor stability of SF in aqueous solutions can induce irreversible aggregation, which can be problematic at later stages of the procedure [31]. Therefore, in most studies, a third step is required to produce a stable SF dope solution. Typically, the SF aqueous solution is lyophilized into a powder and then dissolved in another solvent such as hexafluoroisopropanol (HFIP) or formic acid. In this study, LiCl/DMSO, instead of HFIP or formic acid, was used to prepare the SF dope solution. We had previously used this solvent, which is less expensive and less toxic than HFIP, to prepare sericin microparticles [27]. In addition, using this LiCl/DMSO solution, no indications of degradation during dissolution were observed, unlike when using formic acid. Fig. 1 shows the solubility of SF powder in a LiCl/DMSO solvent system. Two types of SF powder were prepared for the experiment. SF fiber was dissolved either in a 9.3 M LiBr (SFL) solution or in a CaCl2 /CH3 CH2 OH/H2 O (SFC) solution. The result indicates that addition of LiCl is necessary for dissolving SF in DMSO, regardless of the method of preparation. When 1 M LiCl was added to DMSO, the SFC5 completely dissolved within 60 min; however, in the 2 M LiCl/DMSO solvent, the solubility of the SFC5
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SF microparticles prepared with 10 wt% SFC5 were used for the cell adhesion experiment. SFC5 microparticles (10 mg) were sterilized with 70% EtOH and washed three times with a PBS buffer. Fibroblasts (1.5 × 105 per cm2 ) were seeded to the microparticles in the culture medium (DMEM) and cultivated in a CO2 incubator at 37 ◦ C. Cell adhesion on the microparticle was observed for a total of 4 days. At 24-h intervals, the cells on the microparticles were fixed with a modified Karnovsky’s fixative, containing 2% paraformaldehyde and 2% glutaraldehyde in a 0.05 M sodium cacodylate buffer
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Fig. 1. Effect of concentration of LiCl in DMSO solvent on the solubility of SFL and SFC5. The data represent the mean ± SD of 10 samples. * Significant at P < 0.05, ** significant at P < 0.01 (n.d., not dissolved).
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decreased to 75%, due to salting out. Compared to SFC, SFL had limited solubility in LiCl/DMSO. When a 0.5 M LiCl solution was added to DMSO, nearly 40% of the SFC5 dissolved; the SFL did not dissolve at all in this solvent. Maximum SFL solubility within the given time of 1 h was approximately 80% in 1 M LiCl/DMSO. Previous reports have indicated that LiCl can cleave hydrogen bonds in cellulose and protein [32,33], thereby increasing the solubility of these polymers in the organic solvent. The observed differences in solubility can be attributed to the difference in the MWD of SFL and SFC. We previously reported on the differences between SFL and SFC, based on MWD and hydrodynamic radius [31]. Although SFL retains the same molecular weight as SF, a significant decrease in molecular weight occurs during the preparation of SFC. As a result, the hydrodynamic radius of SFL is smaller than that of SFC, because non-degraded SFL maintains its original compact structure. Therefore, for a specified dissolution period, the solubility of SFL will be limited by its compact structure, which hinders LiCl penetration. Therefore, we used SFC as the raw material and 1 M LiCl/DMSO as the solvent for further experiments. The molecular weight of SFC, as discussed above, is significantly smaller than that of SF. Therefore, we hypothesized that the solubility of SFC would differ accordingly. In this study, four different SFC samples were prepared according to their dissolution time in a CaCl2 /CH3 CH2 OH/H2 O solution; Table 2 shows the MN , MW , and polydispersity index of each sample. The GFC graphs of each sample are provided in the Supporting Information (Fig. S1). The molecular weight of SFC decreased as the dissolution time increased. In addition, the polydispersity index of SFC also increased as the dissolution time increased, which indicates a broader MWD in those samples dissolved for a longer time in CaCl2 /CH3 CH2 OH/H2 O solution. These differences in molecular weight and its distribution could affect the solubility of SFC in 1 M LiCl/DMSO. To determine the
Table 2 Number average molecular weight (MN ), weight average molecular weight (MW ), and polydispersity index of SFCs. Sample
MN (kDa)
MW (kDa)
Polydispersity index
SFC5 SFC30 SFC60 SFC180
324 286 257 114
398 378 359 219
1.23 1.32 1.40 1.92
Fig. 2. Absorbance of the supernatant after dissolving a predetermined amount of various SFC samples in 1 mL of 1 M LiCl/DMSO solvent. The insoluble portion was precipitated by centrifugation (mean ± SD, n = 3).
highest dope concentration achievable, a predetermined amount of each SFC sample was dissolved in 1 M LiCl/DMSO. Fig. 2 shows the absorbance of the supernatant of each solution, according to the amount of SFC initially added to the solvent. The absence of a further increase in the absorbance indicates that the highest dope concentration has been reached. The highest achievable dope concentration for SFC5, SFC30, SFC60, and SFC180 was 10, 11, 13, and 14 wt%, respectively. Thus, more highly degraded SFC can reach a higher dope concentration in 1 M LiCl/DMSO, perhaps because of its lower molecular weight. 3.2. SF electrospraying A standard technique for electrospraying uses a volatile solvent that is volatized before it reaches the collecting plate. However, this study used DMSO, which has a boiling point that is much higher than room temperature. Therefore, the SF dope solution was electrosprayed toward a coagulant bath instead of the collecting plate, which solidified the SF droplets emitted from the needle tip. Methanol was used as a coagulant because it is a known standard and the most common solvent that can make SF insoluble. During electrospraying of the polymer, the shape and size of the nano- or microparticles greatly depends on several parameters, including the concentration of the polymer solution, the molecular weight of the polymer, the voltage applied, the flow rate, and the tip-tocollector distance. In this study, we focused on the concentration of the dope solution and the MWD of the SF; the applied voltage, flow rate, and tip-to-coagulant bath distance were fixed. 3.2.1. Effect of concentration Fig. 3 shows the effect of SFC concentration on microparticle shape. In the analysis of SFC5, the shape of the microparticles changed from irregular (6 wt%) to oval (9 wt%) and finally became sphere-like (10 wt%) as the SFC concentration increased. Previous studies have indicated that the concentration of the polymer has a high impact on the shape of the particles produced during electrospraying. Recently, Bock et al. [25] reviewed the process for electrospraying of polymers and reported the effect of polymer concentration on the shape of the resulting polymer particles. Polymer concentration is important because it is correlated with polymer chain entanglement. In a dilute polymer solution, there will be no chain entanglement between individual polymer chains. However,
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Fig. 4. Shear viscosity of SFCs in 1 M LiCl/DMSO solvent. The concentration of each sample was 10 wt%.
Fig. 3. SEM images of SFC microparticles according to dissolution time and concentration (scale bar = 200 m).
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when the concentration increases, the polymer chains will start to overlap; the concentration at which this occurs is referred to as the critical chain overlap concentration (Cov ). Therefore, if the concentration of the polymer in the solution is lower than the Cov , no chain entanglement will occur; this is known as the dilute regime. In this condition, electrospraying is impossible or debris is formed. If the concentration is higher than the Cov , three different regimes are possible. The first is the semi-dilute unentangled regime, in which the concentration of the polymer is sufficient for polymer chain overlap, but entanglement is limited. In this regime, particles have an irregular, non-reproducible morphology. The second is designated as the semi-dilute moderately entangled regime, which occurs at higher polymer concentrations, when polymer chains have moderate entanglement, creating dense particles. The semidilute moderately entangled regime is the only regime in which the formation of sphere-like particles can be achieved by electrospraying. Finally, a further increase in the concentration produces fibers or beaded fibers, particularly if the polymer concentration greatly exceeds the Cov . This third regime is known as the semi-dilute highly entangled regime; the process that occurs is referred to as electrospinning. Similar results were also observed in our study. For each SFC sample, irregular shapes were formed at relatively low SFC concentrations, as follows: SFC5 ≤ 8 wt%, SFC30 ≤ 9 wt%, and SFC60 ≤ 11 wt%. No concentration of SFC180 produced regularly shaped particles. These irregular shapes indicate that stable SFC droplets cannot be formed during flight, due to limited
entanglement between the SFC chains; this could be referred to as the semi-dilute unentangled regime. In some samples, however, hemispherical particles were observed (SFC5 at 7 and 8 wt% and SFC30 at 9 wt%), which indicates that the SFC droplets initially had a spherical shape during flight, but were not sufficiently dense to hold that shape and deformed when they collided with the surface of the coagulant bath. Uniform particles cannot be prepared in these conditions. However, when the concentration increases further, more sphere-like particles could be obtained. Spherelike microparticles were observed, along with some bean-like or oval microparticles. When using SFC5 at 10 wt%, SFC30 at 11 wt%, and SFC60 at 12 wt%, the occurrence of sphere-like microparticles increased significantly. At these concentrations, chain entanglement was sufficient to create dense droplets, which could overcome the surface tension of the coagulant. These concentrations represent the semi-dilute moderately entangled regime of SFC. The semi-dilute highly entangled regime was not observed in this study, because a sufficiently concentrated SFC solution could not be prepared (Fig. 2). The diameter of the sphere-like SFC microparticles was approximately 200 m. Interestingly, during the production of the SFC180 microparticles, sphere-like microparticles did not form, even at the maximum concentration. This result indicates that the MWD of SF is also important; we therefore investigated the effects of the MWD of SF. 3.2.2. Effect of dissolution time in calcium chloride/ethyl alcohol/water solvents Examination of the 10 wt% concentration of all SFCs indicated that a similar change, from an irregular to a sphere-like shape, can be observed between SFC180 and SFC60 samples, to SFC30 and SFC5 samples. The MWD of each SFC would cause this result, since the MWD of SFC is greatly affected by the dissolution time in a CaCl2 /CH3 CH2 OH/H2 O solution, as shown in Table 2. A previous study reported that the shear viscosity of SFC in water and formic acid is also greatly affected by the dissolution time [31]. Because the shear viscosity is an important factor in the electrospraying process, we investigated how the shear viscosity of SFC in 1 M LiCl/DMSO affects the final shape of the microparticles. Fig. 4 shows the rheological behavior of 10 wt% SFCs in 1 M LiCl/DMSO solvent. Original SF in the silkworm gland exhibits non-Newtonian behavior, typically shear thinning [34]. In this study, SF in 1 M LiCl/DMSO solvent also exhibited shear thinning, although the extent was very small. Similar shear thinning of SF has been observed in other solvents, such as formic acid [31]. SFC subjected to a longer dissolution time had a lower shear viscosity. The increase in the low-molecular weight SFC would allow for less entanglement
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Fig. 5. Shear viscosity of SFC solutions according to concentration and dissolution time (mean ± SD, n = 3).
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between SFC chains, which would result in a lower shear viscosity. Based on the knowledge that entanglement is the main factor that determines microparticle shape, a decrease in entanglement would have the same effect as a decrease in concentration. Therefore, it can be concluded that both concentration and dissolution time in a CaCl2 /CH3 CH2 OH/H2 O solution can affect the final shape of SFC microparticles. 3.3. Shear viscosity of SF solutions for preparation of sphere-like microparticles This study has demonstrated that both the concentration and the dissolution time in a CaCl2 /CH3 CH2 OH/H2 O solution affect the shape of SF microparticles. Because polymer concentration and molecular weight are the primary factors that determine the viscosity of a polymer solution, their effect on the shear viscosity of SF solutions was investigated. Here, the molecular weight of the SFC was controlled by the dissolution time in a CaCl2 /CH3 CH2 OH/H2 O solution, as shown in Table 2. Fig. 5 presents a summary of the
previously discussed results in the form of a graph that shows concentration versus shear viscosity. Based on the flow rate of the SF solution in the needle, the shear rate during electrospraying was calculated to be 16 s−1 . We compared the shear viscosity at this shear rate with the concentration and the final shape of the SF microparticles in Fig. 5. The shear viscosity of each sample is provided in the Supporting information (Figs. S2–S5). Based on the shape of the SF microparticles, the graph can be divided into three regions. First, where the shear viscosity was low, only irregular particles were formed due to the low degree of entanglement. As the shear viscosity increased, bean-like or oval microparticles were formed, but the degree of entanglement was not yet sufficient to create sphere-like particles. Finally, when the shear viscosity exceeded approximately 0.33 Pa s, solid sphere-like microparticles were formed due to the relatively high degree of entanglement, which resulted in formation of dense droplets. This figure illustrates that the shear viscosity is a critical factor in determining the shape of SFC microparticles. Furthermore, any combination of concentration and dissolution time that produces a particular shear viscosity range would create SF microparticles of similar shape. 3.4. Application of SF microparticles The potential applications of these sphere-like SF microparticles include targeting of biomedical therapies; we therefore investigated their application as cell carriers for cell therapy. In cell therapy, cells are delivered to a target site, where they will proliferate and differentiate, ultimately regenerating the tissue. Initially, bone and cartilage were the main target tissues, but recent work with stem cells has allowed this therapy to be applied to all kinds of tissues. Various materials have been considered as cell carriers such as poly(␣-hydroxyesters), collagen, gelatin, fibrin, chitosan, alginate, dextran, and others [35]. For cell culture experiments, SFC microparticles were prepared using SFC5, which has a non-porous structure and an average diameter of 250 ± 35 m (Fig. 6). Fig. 7 shows the images of growing fibroblasts on SFC microparticles during 4 days of culture. Cell adhesion was observed beginning on day 2, and some cells formed focal adhesions. Usually, cell adhesion on SF substrates is rapid; however, cells were observed to initially adhere to the tissue culture plate on
Fig. 6. SEM images of SFC5 microparticle in (a) low (500×, scale bar = 100 m) and (b) high (30,000×, scale bar = 1 m) magnification.
Fig. 7. SEM images of fibroblast cell culture on SFC5 microparticles (scale bar = 100 m).
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4. Conclusion
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We prepared sphere-like SF microparticles via electrospraying, using a 1 M LiCl/DMSO solvent. We also demonstrated that shear viscosity is the primary parameter that affects the final morphology of SF microparticles under the described conditions; this is also true using other solvent systems such as HFIP and formic acid. The use of a chaotropic solvent and a coagulant bath would reduce the effectiveness of the SF microparticles for the delivery of therapeutic agents. However, the preliminary results of this study showed that SF microparticles have potential for applications in cell delivery. Additional studies are needed to further evaluate the technique and to optimize the application of these microparticles in tissue engineering. Fig. 8. SEM image of fibroblast morphology attached onto SFC5 microparticle (scale bar = 20 m). Inserted image is magnified image of boxed area (scale bar = 1 m).
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This work was supported by a Marine Biomaterials Research Center grant from the Marine Biotechnology Program funded by the Ministry of Land, Transport and Maritime Affairs, Korea, Q4 and by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (NRF-2010-0025378 and NRF2013R1A1A2059239). References
Fig. 9. MTT assay of fibroblast cultured on SFC5 film, Cyclodex1 and SFC5 microparticles for 7 days (mean ± SD, n = 5, * significant at P < 0.01).
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day 1, before migrating to the SFC microparticles. Therefore, no cells were observed on SFC particles on day 1. Cell adhesion proceeded through day 3; the entire surface of the SF microparticles was covered by cells on day 4. Fig. 8 shows the morphology of fibroblast cells on SFC5 microparticle after 4 days of incubation. The smooth surface of SFC5 microparticle is covered with dense and compact cell layer. Cells were also covered with fibrillar network indicating successful production of extracellular matrix (inserted image). To quantify fibroblast adhesion and proliferation, a standard MTT test was performed. SFC5 film and the commercial dextranbased non-porous microcarrier, Cyclodex1 were chosen for comparison. As shown in Fig. 9, SFC5 microparticles showed the highest rate of proliferation among the samples tested. Compared to SFC5 film, the high surface area of SFC5 microparticles allowed much greater cell adhesion and proliferation. SFC5 microparticles showed better cell attachment than the commercial Cyclodex1 microcarrier, due to the higher affinity of fibroblasts to SF than to dextran. After 4 days of culture, further increase of fibroblast on the SFC5 microparticles could not be observed because almost all surface area were already occupied at day 4 (Figs. 7 and 8). Fibroblast cells were used here because they are easy to culture, but SF shows great potential for use with various cell types, such as osteoblasts [6,36], chondrocytes [37,38] and even stem cells [4,39,40]. Thus, SF microparticles show the potential to contribute to the development of cell therapy.
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