An alternative solvent for electrospinning of fibrinogen nanofibers

2.1. Preparation of fibrinogen

The cryoprecipitate was donated by blood bank unit of Imam Khomeini Hospital, Tehran, Iran. Briefly, cryoprecipitate was obtained from fresh frozen plasma (FFP) followed by thawed at 4°C and centrifuged at 5000 g at 4°C for 7 min. For preparation of the fibrinogen, first the cryoprecipitate was thawed at 4°C, then centrifuged at 9000 rpm for 10 min at 4°C. The supernatant was removed and pellet washed three times with water containing 10% cold ethanol. The prepared fibrinogen was then freeze-dried and stored in −20°C.

2.2. SDS- PAGE electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) was used to confirm the polypeptide chains of the fibrinogen molecule. The electrophoresis was performed in 12% acrylamide running gel and a 4% stacking. The electrophoretic bands stained with Coomassie Brilliant Blue R-250.

2.3. Electrospinning of fibrinogen

For electrospinning, the fibrinogen solution at a concentration of 10, 11 and 12wt% was prepared in HFIP (Hangzhou Dingyan, China)/DMEM (Inoclon, Iran) 9/1 (v/v), formic acid (FA), formic acid/acetic acid (FA/AA) with ratios of 75/25, 50/50 and 25/75(v/v). The electrospinning process was performed using electrospinning equipment (Electroris, FNM, Tehran, Iran). The polymer solutions were electrospun at a high voltage of 20 kV with flow rate of 1.0 ml/h. The distance between syringe tip and collector was 12 cm.

2.4. Characterization

The morphology of the electrospun nanofibers was characterized by SEM (model Philips XL-30, Netherlands, at 25 kV). The nanofibers were sputter-coated with gold, and the average diameters of nanofibers were measured via Image J software. The surface of the nanofibers was characterized by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy (Nicolet iS10). The spectroscopic analysis of samples was recorded over a range of 400-4000 cm⁻¹ at a resolution of 4 cm⁻¹. The thermal behavior of fibrinogen nanofibers were studied by simultaneously thermal analysis (TGA–DTA) with an STA 503, (BÄHR, Germany). The samples were heated to 600°C at a rate of 10°C/min under argon flow rate of 3 l/h.

2.5. Statistics analysis

All data are presented as the Mean ± standard deviation (SD). Statistical analyses were performed by using one-way analysis of variance (ANOVA) with a Games-Howell post hoc test and Wilcoxon signed rank test. The significance level was set to p < 0.05 .

3.1. Preparation of fibrinogen

Several modifications on the cryoprecipitate method such as different kind of anticoagulant, various centrifugation speeds and the addition of chemical additives have been described [16]. Here we prepared fibrinogen from cryoprecipitate based on described in materials and methods, then compared with commercial product in terms of purity. It has been known that fibrinogen is composed of three pairs of different polypeptide chains with a molecular weights of 66 kDa, 54 kDa, and 48 kDa, which assigned to Aα, Bβ and γ polypeptide chains respectively [17]. Figure 1 shows SDS-PAGE of prepared fibrinogen in comparison with commercial fibrinogen (cod. F4129, Sigma-Aldrich). According to the SDS-PAGE patterns, the results are similar in terms of purity between prepared fibrinogen and the commercial product. On the basis of these results, it is concluded that the cryoprecipitate method is simple and easy way which can give good fibrinogen yield with admissible purity.

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Fig. 1.

The SDS-PAGE of fibrinogen isolated compared with commercial fibrinogen. Lane 1: Fibrinogen obtained by cryoprecipitate method, Lane 2: commercial product fibrinogen (cod. F4129, Sigma-Aldrich), Lane 3: Molecular weight markers (11–180 kDa) from Avagene (Tehran, Iran).

Several parameters including solution properties (such as concentration, electrical conductivity, dielectric properties, surface tension, viscosity), processing parameters (e.g. applied voltage, flow rate, distance between the collector and the tip) and ambient parameters such as humidity and temperature have effect on the nanofibers [18,19]. In this study, the effect of the volume ratios of the FA/AA solvent and various concentrations of fibrinogen on nanofiber morphology and diameter was investigated. However, the processing parameters were optimised and fixed during electrospinning. As shown in Table 1, when HFIP was used as the solvent, the average diameters were 226 ± 146, 327 ± 171 and 537 ± 370 nm at 10%, 11% and 12wt%, respectively. It was found that fiber diameters increase with increasing fibrinogen concentration ( p < 0.05 ), as reported an increase in solution concentration increases fiber diameter and uniformity [18]. The nature of the solvent such as volatility and polarity (dielectric constant), influences the morphology and diameter of the electrospun fibers [20]. There are also reported that conductivity and dielectric constant are necessary for electrospinning solutions which depend on the solvents [21]. HFIP with a dielectric constant of 16.7 (F/m) [21] has advantages including dissolving various kinds of polymers due to its strong hydrogen bonding properties [22]. However, its toxicity developed serious problems including respiratory system and eyes damage. Also, the low amount of HFIP is toxic for in vitro cell culture [23]. The fibrinogen solution (10wt%) in formic acid did not form any fibers due to its low viscosity, and droplets were observed (Fig. 2(d)). However, with increasing fibrinogen concentrations from 10wt% to 12wt% the morphology of structures changed from droplets/beads to a beaded nanofiber. (Fig. 2(e), (f)). Electrospinning process in the acetic acid solvent due to low electrical conductivity and dielectric constant (6.6) is difficult [23,24]. Moreover, acetic acid alone can’t dissolve fibrinogen. Addition of a highly polar solvent such as formic acid (relatively high dielectric constant 57.2) to acetic acid, which formed a new solvent system with intermediate polarity is suggested in order to increase the conductivity and thus to improve the electrospinning process [24]. For this purpose, different fibrinogen solutions were prepared having an FA/AA solvent ratio of 75/25, 50/50 and 25/75. The solution with a ratio of 25/75 was swollen and did not dissolve completely. Therefore, only the ratios of 75/25 and 50/50 of FA/AA can lead to the formation of nanofibers. According to the Table 1, for FA/AA (75/25) the average fiber diameters were 184 ± 37, 218 ± 53 and 241 ± 70 nm and for ratio of 50/50 was obtained 258 ± 121, 441 ± 253 and 562 ± 288 nm at 10%, 11% and 12wt%, respectively. When the ratio of FA/AA was 75/25, the smallest and most uniform bead-free nanofibers obtained in comparison to the solution with ratio of 50/50 or in HFIP solvent ( p < 0.05 ). The smaller fiber diameter is due to the presence of highly polar solvents such as formic acid [22].

ATR-FTIR spectroscopy was performed to reveal the presence of fibrinogen. ATR-FTIR spectra of fibrinogen in acidic and HFIP solvent are presented in Fig. 3. The spectrum of fibrinogen showed the peak at 1650 cm⁻¹ (amide I for C=O stretching) and 1538 cm⁻¹ (amide II for –NH deformation), which are typical peaks of proteins. Similar results were obtained by other researchers [25,26]. As it shown in Fig. 3, there was no difference in ATR-FTIR spectra of fibrinogen in two solvents.

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Fig. 2.

SEM of fibrinogen nanofibers at various concentrations in HFIP and acidic solvent. (a), (b), (c) are nanofibers of 10%, 11% and 12% in HFIP solvent. (d), (e), (f) are nanofibers of 10%, 11%, and 12% in formic acid. (g), (h), (i) are nanofibers of 10%, 11%, and 12% in formic acid/acetic acid (75/25) and (j), (k), (l) are nanofibers of 10%, 11%, and 12% in formic acid/acetic acid (50/50).

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Fig. 3.

ATR-FTIR spectroscopy of fibrinogen nanofibers. (a) fibrinogen nanofibers in HFIP solvent. (b) fibrinogen nanofibers in acidic solvent.

Many studies have investigated the stability of fibrinogen structure under electrospinning conditions [15,27] but, few studies have examined the thermal properties of fibrinogen electrospun nanofibers. Thermal analyses techniques are beneficial to give essential information about a material’s properties and phase transitions [28]. For thermal stability and degradation of the fibrinogen nanofibers in HFIP and acidic solvent, the TGA and DTA analysis was performed and shown in Fig. 4. The thermogram of derivative thermal gravimetric (DTG) is prepared on the basis of the first derivative of the TG plots to show the decomposition temperature. According to the DTG curve of Fig. 4, there are two weight loss peaks. The first is at around 100°C and the second peak at 315°C, which nearly similar to the DTG results of fibrinogen scaffold obtained by Chuanglong He [26]. The first broad peak in all polymeric curves is mainly due to the evaporation of solvent or water [28–30] which can be confirmed by the DTA curve at the same temperature range. In addition Haiguang Zhao mentioned that the weight loss of fibrinogen before 260°C should be attributed to the water evaporation and dehydration between the protein molecules [31]. The second weight loss phase is likely caused by the decomposition and degradation of fibrinogen. As it shown in Fig. 4 the thermal behaviour of electrospun fibrinogen nanofibers in HFIP solvent was similar to the profile of fibrinogen nanofibers in acidic solvent. These results showed that mix solvent of formic acid/acetic acid had no influence on the structure or stability of fibrinogen nanofibers.

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Fig. 4.

Thermal analysis of fibrinogen nanofibers in HFIP and acidic solvents. (a) Thermal gravimetric analysis (TGA) and differential thermal gravimetric (DTG), (b) Differential thermal analysis (DTA).