Article pubs.acs.org/Biomac
Preparing Silk Fibroin Nanofibers through Electrospinning: Further Heparin Immobilization toward Hemocompatibility Improvement Marília Cestari,† Vinícius Muller,‡ Jean Henrique da Silva Rodrigues,§ Celso V. Nakamura,§ Adley F. Rubira,† and Edvani C. Muniz*,† †
Departamento de Química, Universidade Estadual de MaringáUEM, 87020-900 Maringa, Brazil Centro de Engenharias e Ciências Exatas, Universidade Estadual do Oeste do ParanáUNIOESTE, 85903-000 Toledo, Brazil § Laboratório de Inovaçaõ Tecnológica no Desenvolvimento de Fármacos e Cosméticos, Departamento de Ciências Básicas da Saúde, Universidade Estadual de MaringáUEM, Av. Colombo 5790 87020-900 Maringá, Paraná, Brazil ‡
ABSTRACT: Sodium heparin (HS) was immobilized on the surface of the silk fibroin nanofibers (FS) prepared by electrospinning with the objective of improving the hemocompatibility of the fibers for application as scaffolds in tissue engineering. The nanofiber mats of silk fibroin without (MF-FS) and with (MFFS/HS) immobilized heparin were characterized through scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR), thermogravimetric analyses (TGA), energy dispersive spectroscopy (EDS), contact angle, chemical analysis, and biological tests. The formation of hydrogen bonds between the silk fibroin and heparin was discussed based on FTIR-ATR spectra. The amount of immobilized heparin was quantified through papain/N-acetyl-L-cysteine digestion followed by dimethylmethylene blue complexation. Furthermore, the samples with immobilized HS showed higher hydrophilic capability compared to samples without HS due to lower contact angles. It was possible to verify that the capillary end-to-collector distance of 8.5 cm and flow rate of 0.35 mL h−1 used in the electrospinning process at 20 kV are good conditions for obtaining a small average fiber diameter maintaining the amount of immobilized heparin on MF-FS/HS in ca. 4% w/w. Biological analysis showed that no hemolysis is provoked by MF-FS and MF-FS/HS mat fragments and those such mats are not toxic to Vero cells. However, the MF-FS/HS showed higher cell growth and proliferation than MF-FS, indicating an improvement in the hemocompatibility of the material due to heparin immobilization.
■
INTRODUCTION The fabrication of nanofibers by electrospinning has received great attention due to their versatility to produce submicrometer fibers and nanoscale control of the structure, porosity, and orientation.1 The application of nanofiber matrixes as scaffolds in tissue engineering have shown great results for building and/or the regeneration of various tissues including bone, cartilage, tendons, blood vessels, and heart valves. Scaffolds are three-dimensional (3D) structures that suit the needs of newly growing tissue.2 Many synthetic and natural polymers have been examined to obtain matrixes for application in tissue engineering.3 Silk fibroin is a natural biopolymer that is present in the bark of the cocoons produced by the species Bombyx mori. The silk produced by the silkworm has excellent mechanical properties as well as favorable biocompatibility, environmental stability, controlled proteolytic biodegradability, morphologic flexibility, good permeability to water vapor, and minimal inflammatory reaction.4,5 Because of these properties, the silk fibroin is a material of great potential for the generation of biomaterials for different uses, including devices for tissue engineering.5 The biocompatible, biomimetic, mechanical properties close to the tissue are the main consideration for biopolymers use as based material in scaffolds for cell culture. © 2014 American Chemical Society
In addition, the compatibility of the scaffolds with the blood cells is very important for specific tissue engineering applications such as artery and veins reconstruction, for instance. Thus, various strategies have been proposed to improve the thrombogenicity of biomaterials, such as the incorporation of ionic groups on the polymer surface, changing the surface properties by grafting techniques and immobilization of heparin, functionalized dextrans, or biological compounds.6 In general, immobilization of heparin is the most effective and widely used strategy for improving hemocompatibility through various methods such as coating and mixture grafting.7 Heparin is a highly anionic glycosaminoglycan isolated by extraction from animal tissues, which are rich in mast cells present in pig intestines.5 It is clinically used as an anticoagulant to minimize thrombus formation on artificial organ surfaces. There are two general methods for developing polymeric blood compatible material using heparin. One method uses chemical immobilization of heparin, and the other is focused on the delivery system of heparin.8 The aim of Received: January 26, 2014 Revised: March 21, 2014 Published: March 26, 2014 1762
dx.doi.org/10.1021/bm500132g | Biomacromolecules 2014, 15, 1762−1767
Biomacromolecules
Article
d. Through Thermogravimetry (TGA). Thermogravimetric analyses were carried out using a thermogravimetric analyzer (Netzsch, model STA 409 Luxx PG/4/G, USA) at a rate of 10 °C min−1 under nitrogen atmosphere and a flow rate of 20 mL min−1 in 22−730 °C temperature range. e. Through Contact Angle Measurements. The contact angles, static mode, were measured at room temperature with a Contact Angle Meter (Tantec, Model Micro-Cam, Denmark). The values were obtained by the sessile drop method. The liquid (water or glycerol, 1 μL) was deposited by a syringe into different areas of each sample, and the absorption time of the drop was up to 10 min. At least 3 measures were performed on each sample (n = 3). Quantification of Immobilized Heparin in Nanofiber Mats. A calibration curve based on heparin solutions with known concentrations was built. For that, 400 μL aliquots of each standard heparin solution were mixed with 5.0 mL of a solution of dimethylmethylene blue (DMB). The DMB solution was prepared by dissolving 16 mg of DMB, 3.04 g of glycine, and 2.37 g of NaCl and was made up to 1 L using an aqueous solution of HCl 0.1 mol L−1. For preparing this solution, distilled/deionized water was used. The absorbance reading was performed at a wavelength of λ = 525 nm. The MF-FS/HS were weighed (about 20 ± 0.1 mg) and digested in a phosphate buffer solution (pH 6.8) containing 0.05% (w/w) papain and 0.096% (w/w) N-acetyl-L-cysteine. For digestion, the samples were incubated with 1 mL of digestion solution for 3 h at 60 °C. For each test, 20 μL of the digestion solution, 300 μL of distilled water, and 3.7 mL of DMB (16 mg of DMB, 3.04 g of glycine, and 2.37 g of NaCl and was made up to 1 L using an aqueous solution of HCl 0.1 mol L−1, pH = 3) were used. The standard heparin solutions were prepared in the range of 10−100 mg mL−1. The absorbance was measured at λ = 525 nm on a UV−vis spectrophotometer (Femto, model 800XI, Brazil). The MF-FS (without heparin) samples were used as controls and were treated in the same way as described above for the assay of heparin content.10 The tests were done in triplicate (n = 3). Biological Assays. a. Through Cytoxicity by Hemolysis. For the hemolysis assay, blood was collected, defibrinated on glass beads, and washed in dextrose saline to remove lysed cells and fibrin. The cells were resuspended in 3% glucose saline solution and exposed in 96 well plates at 1 mg fragments of the silk fibroin nanofibers with and without immobilized heparin and incubated at 37 °C for 120 min. After the incubation period, the material was centrifuged and the absorbance of the supernatant was read at λ = 550 nm. Three independent experiments were performed in duplicate. Wells without the addition of nanofibers were kept as a negative control; to achieve 100% lysis of erythrocytes, Triton X-100 at 1% was used as a positive control, allowing calculation of the percentage of hemolysis. b. Through Cytoxicity by Cell Culturing. This test was performed to evaluate the influence of the silk fibroin nanofibers with and without heparin on the growth of mammalian cells. To this end, Vero cells (epithelial kidney cells of Cercopithecus aethiops) cultivated in DMEM (Dulbecco’s Modified Eagle Medium, Gibco Invitrogen) supplemented with 2 mM L−1 glutamine and 10% fetal bovine serum (FBS) were obtained, quantified, and seeded into 24-well culture plates at a concentration of 2.5 × 105 cells mL−1 and kept in a moist chamber at 37 °C and an atmosphere of 5% CO2. After 12 h of incubation, the culture medium was replaced by serum-free DMEM and fragments of the silk fibroin nanofibers with and without heparin (1 mg) were added, followed by further incubation for 72 h. At the end of the incubation, the amount of grown cells was determined by the method of reduction of MTT viability.11 This colorimetric method is based on the ability of mitochondria of viable cells to reduce MTT (tetrazolium salt) into a purple insoluble compound called formazan. For this assay, the cells were washed thoroughly with phosphate buffered saline (PBS) and incubated in 250 μL of MTT solution (2 mg mL−1) protect from the light at 37 °C. After 4 h of incubation, 750 μL of dimethyl sulfoxide was added to each well to solubilize the formazan crystals, and the spectrophotometric reading was performed at λ = 570 nm in a microplate reader (BioTek−Power Wave XS, USA). Cells cultured in the absence of the silk fibroin nanofibers were used as control and taken as 100% growth.
this work was to join excellent mechanical properties, favorable biocompatibility, environmental stability, proteolytic controlled biodegradability, morphological flexibility, good water vapor permeability, and minimal inflammatory reaction of the silk fibroin with the anticoagulant and thrombogenicity properties of heparin through a new method for heparin immobilization on the surface of fresh nanofibers of fibroin, prepared by electrospinning, to obtain scaffolds with greater hemocompatibility for use in tissue engineering.
■
EXPERIMENTAL SECTION
Materials. Cocoons of silkworm silk were kindly supplied by Cocamar (Maringa-PR, Brazil). Kin Master (Passo Fundo-RS, Brazil) kindly supplied heparin sodium salt (HS, CAS 9041-08-1). Other reactants such as formic acid, ethanol, sodium bicarbonate, and calcium chloride were also utilized in this work. All reactants were used as received without further purification. Procedures. Obtaining Regenerated Silk Fibroin. The cocoons of silkworm silk were degummed twice with NaHCO3 0.5% (w/w) at 100 °C for 30 min and then washed three times with distilled water at 70 °C to remove the sericin. The degummed fibroin solution was dissolved in ethanol/water 20/80 (v/v) CaCl2 containing 5 mol L−1 at 80 °C until completely dissolved (about 30 min). After solubilization, the fibroin solution was dialyzed for 3 days in cellulose acetate membrane against distilled water, filtered, and lyophilized to obtain sponges of regenerated silk fibroin. This procedure was based on the work of Wang et al.5 and Miyaguchi et al.9 Preparation of the Silk Fibroin Scaffolds with Immobilized Heparin. The regenerated silk fibroin was dissolved in 98% (w/w) formic acid in order to obtain the spinning solution. Aiming at uniform nanofiber formation, i.e., without the fibers collapsing or formation of drops (beads), some parameters such as the solution concentration, voltage electric field, and flow rate were fixed at 12% (w/v), 20 kV, and 0.35 mL h−1, respectively, for the electrospinning process. The capillary end-to-collector distance was fixed at 13.0, 10.0, 8.5, or 7.0 cm. The choice of values for these parameters was based on the work of Wang et al.5 A Petri dish (⌀ ≈ 10 cm) containing 5 mg mL−1 heparin dissolved in 50/50 (v/v) water/ethanol solution (ca. 20 mL) was placed upon the grounded collector, so that while the nanofibers were collected, the heparin immobilization occurred on the fibers surface just after contact with the heparin solution. At the end of the formation of nanofibers, the mats were left immersed in the solution containing heparin for a further 24 h. Then, the nanofibers were washed three times with distilled water, frozen, and lyophilized. For control the same procedure was repeated with the 50/50 (v/v) water/ ethanol solution without heparin and also without use of solution (collecting the fibers directly on metallic collector surface). Characterization of the Silk Fibroin Scaffolds before and after Heparin Immobilization. a. Through Scanning Electron Microscopy (SEM). The fibroin nanofibers (MF-FS) and fibroin nanofibers with immobilized heparin (MF-FS/HS) after being coated with gold were morphologically analyzed by scanning electron microscopy (SEM) using a Shimadzu equipment, model SS-550, Japan. The average diameter for each type of fiber was calculated with the SIZE METER software, version 1.1. b. Through Fourier-Transform Infrared Spectroscopy with Attenuated Total Reflectance (FTIR-ATR). The MF-FS and MF-FS/ HS were characterized through infrared spectroscopy. The spectra were obtained on a spectrophotometer FTIR Bomem model MB-100 with ATR apparatus (FTIR-ATR), operating in the 4000−400 cm−1 range with a resolution of 4 cm−1 and 37 acquisitions per minute. Each FTIR-ATR spectrum was obtained after 64 acquisitions. c. Through X-ray Energy Dispersive Spectroscopy (EDS). Chemical composition of the surface of the silk fibroin scaffolds with immobilized heparin was investigated by X-ray photoelectron spectrometer (EDS) with a monochromatic source MgKα coupled to a scanning electron microscope (Shimadzu, model SS-550, Japan) operating at 12 kV and 180 W. 1763
dx.doi.org/10.1021/bm500132g | Biomacromolecules 2014, 15, 1762−1767
Biomacromolecules
Article
Figure 1. Micrographs, obtained by SEM, of mats: samples A, B, C, D, and E, according to Table 1. (F) Average diameter of mats in panels A−E.
■
RESULTS AND DISCUSSION Scanning Electron Microscopy Analysis. From the micrographs and data presented in Figure 1, it can be seen that the capillary end-to-collector distance did not have a significant effect on the average diameter of fibers if changed from 7 to 10 cm. For a given condition (Table 1), the fibers appear to be homogeneous. The formation of some agglomeration points (beads) and little coalescence were observed mainly in samples D and E. This may have an effect on the hydrophobicity of the mat by changing the surface topology. Table 1 shows a tendency to increase fiber diameter, as the fibers are collected in heparin solution, but such an increase is to some extent at the level of (or a slightly higher than) the standard deviation. However, it is clear that when collected directly in the collector surface (without solution, sample A), the average diameter of the as-obtained fibers is lower in comparison to others collected in an ethanol/water (50/50 v/v) solution, even in the absence of heparin. This means that the fibers swell a little in contact with such solution in spite of their insolubility. Obviously, such swelling allowed for better immobilization of heparin.
Table 1. Conditions Used to Obtain the Fibers and the Respective Values of Mean Diameter and Standard Deviation; Flow Rate 0.35 mL h−1 sample
capillary end-tocollector distance (cm)
solution for collecting fibers
average diameter (μm)
standard deviation (μm)
A
13.0
0.16
0.05
B C
10.0 10.0
0.22 0.25
0.06 0.08
D
8.5
0.23
0.06
E
7.0
directly to collector ethanol/water ethanol/ water/ heparin ethanol/ water/ heparin ethanol/ water/ heparin
0.24
0.05
Fourier-Transform Infrared Spectroscopy with Attenuated Total Reflectance (FTIR-ATR). FTIR-ATR spectra for the samples A to C and of heparin are presented in Figure 2. The bands for amide I and amide II groups present in the MF1764
dx.doi.org/10.1021/bm500132g | Biomacromolecules 2014, 15, 1762−1767
Biomacromolecules
Article
Figure 2. FTIR spectra of mats: sample A; (B) MF-FS/HS, sample B; (C) MF-FS/HS, sample C, according to Table 1; and heparin.
Figure 3. TGA curves of samples A and D, according to Table 1; and heparin.
FS/HS fibers (sample C) were attributed taking into account such bands present in the spectrum of MF-FS (Figure 2, spectrum A and B). Table 2 shows the wavenumbers for the
loss. The second region extending to 280−370 °C was associated with the degradation process of amino acid side groups and to cleavage of peptide bonds.13,14 The TGA curve for heparin showed a sharp weight loss at about 250 °C, which was associated with degradation.15 It may also be noted that MF-FS (sample A) completely degraded (does not show residue) at 730 °C, whereas MF-FS/HS (sample D) and heparin presented at that temperature (730 °C) a final residue of ca. 40%. The residue was attributed to sulfate salts generated from sulfated groups present in heparin. Obviously this final residue at 730 °C, as indicated in the curve for MF-FS/HS (sample D), was due to the presence of heparin immobilized on MF-FS/HS fibers. Quantification of Immobilized Heparin. Figure 4 shows the amount of immobilized heparin on MF-FS/HS fibers
Table 2. Wavenumbers of Amide I and Amide II and Other Characteristic Bands Present in the Infrared Spectra of the Fibers (Samples A, B, and C, According to Table 1) and Heparin sample
amide I (cm−1)
amide II (cm−1)
A B C heparin
1657 1622 1622
1520 1513 1514
other characteristic bands (cm−1) SO/C−O−S
1040 1032
992 990
818
amide I and amide II attributions. The wavenumbers of bands attributed the amide I present in the FTIR-ATR spectra of fibers collected in water/ethanol (spectrum B) and in water/ ethanol/heparin solution (spectrum C) decreased from 1657 to 1622 cm−1 (see Table 2) as compared to the respective FTIRATR spectra of fibers collected directly on the collector (without solution, spectrum A). Such a fact can be attributed to the possible change in conformation of the silk fibroin chains, which changed from random coil to β-sheet.5 Silk fibroin macromolecules can rearrange their crystal structure due to changes in hydrogen bonding caused by physical contact with chemicals such as methanol, ethanol, and glutaraldehyde.12 It was expected that the silk fibroin and heparin, when in contact with each other by mixing or immobilization, would form additional hydrogen bonds. Thus, the decrease of wavenumbers assigned to amide I bands present in the FTIR-ATR spectra of Figure 2 allows it to be inferred that the formation of these interactions may have occurred. Thus, there is enough evidence that the changes in the conformation of random coil to β-sheet on fibroin fibers occurred. The presence of a band in the region 1030−1050 cm−1 on the FTIR-ATR spectrum of MF-FS/HS (Figure 2, spectrum C) was attributed to the symmetric stretching vibration of SO groups.5 This result also demonstrates that heparin was effectively immobilized on the surface of fibroin fibers, possibly forming hydrogen bonds among the chains of these polymers. Thermogravimetric Analysis (TGA). Figure 3 shows TGA curves for different mats. Two distinct regions of mass loss were detected from the TGA curves (samples A and D): the first region (which goes up to ca. 100 °C) was attributed to water
Figure 4. Amount of immobilized heparin in mats obtained at different capillary end-to-collector distances for samples C, D, and E, according to Table 1.
obtained at different capillary end-to-collector distance. The amounts of immobilized heparin were 4.2% when the capillary end-to-collector distance was 10.0 cm, 4.1% for 8.5 cm, and 4.6% for 7.0 cm. Therefore, no significant effect of capillary end-to-collector distance on the amount of immobilized heparin was observed. Thus, as the flow rate used was low, enough time for complete solvent evaporation before the fibers achieved the collector (or solution) was observed because there was almost no coalescence among fibers. Also, using this flow rate, the average diameter of fibers was lower, allowing higher contact surface among the fibers to be reached in the solution 1765
dx.doi.org/10.1021/bm500132g | Biomacromolecules 2014, 15, 1762−1767
Biomacromolecules
Article
Contact Angle. Figure 6 shows the contact angle of a glycerol drop deposited on the surface of MF-FS (sample B)
and the heparin conducting to a higher amount of immobilized heparin. The presence of residue at 730 °C in TGA of MF-FS/ HS (sample D, Figure 3) indicates that heparin was effectively immobilized on nanofibers. However, as described in this section, the amount of immobilized heparin obtained from papain/N-acetyl-L-cysteine-digested tests should be higher as considered the residue at 730 °C from TGA analysis. Both TGA and digestive tests were done in triplicate. So, the reason for that difference is not still clear but further studies may give a proper explanation for this fact. X-ray Energy Dispersive Spectroscopy (EDS). The Xray energy dispersive spectroscopy (EDS) technique was mainly used to detect the presence of sulfur atoms in the MF-FS/HS mats compared to MF-FS ones. Figure 5 shows,
Figure 6. Contact angle of glycerol as a function of time for MF-FS (sample B) and MF-FS/HS (sample D) mats, according to Table 1.
and MF-FS/HS (sample D) mats as a function of time, up to 10 min. The MF-FS showed a contact angle that was greater than the MF-FS/HS. This result indicates that after the immobilization of heparin to the surface of the fibers, the mat became more hydrophilic. As the content of heparin in MF-FS/ HS is 4.6% for sample D, it can be pointed out that the heparin is immobilized mainly at the surface. Because of the high charge density,16 heparin possesses high hydrophilic capability. Thus, once immobilized at the surface, even in a small amount, such content was enough to change the hydrophilicity of the mat. Biological Analysis. Through Cytoxicity by Hemolysis. The word hemolysis is defined as a disruption of the erythrocyte membrane, causing the release of hemoglobin and other internal blood cell components to the surrounding liquid. It can be caused in vivo and/or in vitro.17 In this work, hemolytic activity was evaluated in vitro to verify the cytotoxicity of MF-FS (sample B) and MF-FS/HS (sample D). To assess the results, the percentage of hemolysis caused by the fibers was evaluated. If the fibers possess the ability to induce significant hemolysis, they would be considered toxic. If not induced, the fibers would be considered not toxic to cells. Figure 7 shows the hemolysis (in %) for MF-FS (sample B) and for MF-FS/HS (sample D). In both cases, no significant induction of hemolysis of erythrocytes was observed (being less than 1%). Therefore, in the light of these results, it can be stated that the MF-HS and MF-FS/HS are not toxic to blood cells.
Figure 5. EDS profile for MF-FS (sample A) and MF-FS/HS (sample D) mats, according to Table 1.
respectively, the EDS profiles for MF-FS (sample A) and MFFS/HS (sample D) mats. The peaks due to C and O atoms appear in both figures at ca. 0.30 and 0.50 keV, respectively, as expected, but the signal due to S atoms at ca. 1.75 keV is observed only in EDS for MF-FS/HS mat. The S peak is related to the presence of sulfated moieties. In this case, it was due to the immobilized heparin on the MF-FS/HS fibers. The sample A (mat without heparin) was collected directly in the metal collector. The metal collector was covered by an aluminum sheet. The sample D was collected in an ethanol− water solution containing heparin. Thus, the presence of peak of aluminum in the EDS of sample A was due to the residue of aluminum from the sheet, and this did not occurr in the EDS profile of sample D. This result is consistent with others already presented in this article and helps to confirm the heparin immobilization on the surface of the silk fibroin nanofibers.
Figure 7. Hemolysis as blood cells are exposed to MF-FS (sample A) and MF-FS/HS (sample D) mats, according to Table 1. 1766
dx.doi.org/10.1021/bm500132g | Biomacromolecules 2014, 15, 1762−1767
Biomacromolecules
■
Through Cell Culturing. The cell culturing technique was used to check the growth and proliferation of Vero cells exposed to MF-SF (sample B) and MF-FS/HS (sample D) fragments and evaluate whether the biocompatibility of fibers was improved after heparin immobilization on the surface of MF-FS fibers. Control (cells cultured in the absence of the silk fibroin nanofibers) was used for comparison. Figure 8 shows
Article
AUTHOR INFORMATION
Corresponding Author
*(E.C.M.) Fax: +55 44 30114125. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■ ■
ACKNOWLEDGMENTS M.C. thanks the CAPES for the Master scholarship, and E.C.M. thanks CNPq (Proc. 478002/2012-2) for the financial support. REFERENCES
(1) Baji, A.; Mai, Y.; Wong, S.; Abtahi, M.; Chen, P. Compos. Sci. Technol. 2010, 70, 703−718. (2) Mo, X. M.; Xu, C. Y.; Kotaki, M.; Ramakrishna, S. Biomaterials 2004, 25, 1883−1890. (3) Zhou, J.; Cao, C.; Ma, X.; Lin, J. Int. J. Biol. Macromol. 2010, 47, 514−519. (4) Vepari, C.; Kaplan, D. L. Prog. Polym. Sci. 2007, 32, 991−1007. (5) Wang, S.; Zhang, Y.; Wang, H.; Dong, Z. Int. J. Biol. Macromol. 2011, 48, 345−353. (6) Abraham, G. A.; Queiroz, A. A. A.; Roman, J. S. Biomaterials 2002, 23, 1625−1638. (7) Wang, J.; Hu, W.; Liu, Q.; Zhang, S. Colloids Surf., B 2011, 85, 241−247. (8) Lv, Q.; Cao, C.; Zhu, H. Biomaterials 2003, 24, 3915−3919. (9) Miyaguchi, Y.; Hu, J. Food Sci. Technol. Res. 2005, 11 (1), 37−42. (10) Martins, A. F.; Piai, J. F.; Schuquel, I. T. A.; Rubira, A. F.; Muniz, E. C. Colloid Polym. Sci. 2011, 289, 1133−1144. (11) Mosmann, T. J. Immunol. Methods 1983, 65 (1−2), 55−63. (12) Yin, G. B.; Zhang, Y. Z.; Bao, W. W.; Wu, J. L.; Shi, D. B.; Dong, Z. H.; Fu, W. G. J. Appl. Polym. Sci. 2009, 111, 1471−1477. (13) Nogueira, G. M.; Rodas, A. C. D.; Leite, C. A. P.; Giles, C.; Higa, O. Z.; Polakiewicz, B.; Beppu, M. M. Bioresour. Technol. 2010, 101, 8446−8451. (14) Um, I. C.; Kweon, H. Y.; Park, Y. H.; Hudson, S. Int. J. Biol. Macromol. 2001, 29, 91−97. (15) Martins, A. F.; Pereira, A. G. B.; Fajardo, A. R.; Rubira, A. F.; Muniz, E. C. Carbohydr. Polym. 2011, 86, 1266−1272. (16) Coelho, T. C.; Laus, R.; Mangrich, A. S.; Fávere, V. T.; Laranjeira, M. C. M. React. Funct. Polym. 2007, 67, 468−475. (17) Dubruel, P.; Dekie, L.; Christiaens, B.; Vanloo, B.; Rosseneu, M.; Vandekerckhove, J.; Mannisto, M.; Urtti, A.; Schacht, E. Biomacromolecules 2003, 4, 1177−1183.
Figure 8. Growth (in %) as Vero cells are exposed to MF-FS (sample B) and MF-FS/HS (sample D), according to Table 1, and to control.
the results collected on day 3 (after 72 h incubating). In the presence of MF-FS and MF-FS/HS fragments, the cells grew up in the same intensity or more than that observed for the control (counted as 100% growth) so that the silk fibroin mats (with and without immobilized heparin) were considered not toxic. It can also be noted that the cell growth for MF-FS/HS is higher (126%) than that of MF-FS (108%). This result shows that the presence of heparin in the mat increased the cell compatibility because higher cell growth was induced. Thus, it can be pointed out that heparin changes the hydrophilicity at the surface (as shown by contact angle) and allows, possibly, better adherence of cells to the mat surface.
■
CONCLUSIONS Silk fibroin nanofibers were obtained by the electrospinning procedure. At the end of the electrospinning process the fibers were collected in an ethanol/water (50/50 v/v) solution containing heparin, targeting heparin immobilization on fibers. The initial hypothesis of this work was that heparin immobilized on the fibers could increase the biocompatibility of the material. The nanofiber mats of silk fibroin with (MFFS/HS) and without (MF-FS) immobilized heparin were characterized by SEM, FTIR-ATR, TGA, EDS, contact angle, chemical analysis, and biological tests. The results confirmed the heparin immobilization and that such occurs, possibly, through the formation of hydrogen bonds between the silk fibroin and heparin chains. The amount of immobilized heparin in the fibers obtained under different electrospinning conditions was evaluated: it was possible to verify that the capillary end-to-collector distance of 8.5 cm and flow rate of 0.35 mL h−1 at 20 kV showed the best results in the average fiber diameter (smaller diameter) and in the amount of immobilized heparin (ca. 4%). Biological analyses showed that the silk fibroin mats without (MF-FS) and with (MF-FS/HS) immobilized heparin are not toxic to cells and that MF-FS/HS showed higher cell growth and proliferation than MF-FS, indicating an improvement in the hemocompatibility of the material. 1767
dx.doi.org/10.1021/bm500132g | Biomacromolecules 2014, 15, 1762−1767
