Note pubs.acs.org/Biomac
Effect of Various Dissolution Systems on the Molecular Weight of Regenerated Silk Fibroin Qin Wang,† Quan Chen,‡ Yuhong Yang,*,§ and Zhengzhong Shao*,† †
State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Laboratory of Advanced Materials and §Research Centre for Analysis and Measurement, Fudan University, Shanghai 200433, P.R. China ‡ Department of Material Science and Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802, United States S Supporting Information *
1. INTRODUCTION It has been more than one century since silk fibroin (SF), one of main fibrous proteins from silkworm silk fiber, attracted much attention for scientists in the biomedical, chemical, and material fields.1−3 As a natural protein having a broad range of applications from medical to microdevice realms, SF materials have been fabricated from regenerated silk fibroin (RSF) into different products such as fiber, foams, films, gels, powders, and so on.3 Therefore, the development of man-made SF-based material with appropriate strength and toughness from regenerated solutions becomes critical for its broad application potential. To prepare RSF solution, strong hydrogen-bond-destroying solvents, such as inorganic salts,4 fluorinated organic solvents,5 concentrated acids,6 ionic liquids,7 and so on, were commonly used to dissolve the degummed silk fiber (dSF). However, there were huge performance differences among those RSFbased materials even if obtained under the same processing condition.8 Although there might be some accidental factors (defect during spinning,9 etc.), the intrinsic characters of the protein molecule were generally regarded as the main reasons. In addition to the aggregation structure of RSF molecular chain, molecular weight, which was affected by the various factors during the solution-preparing process,10 was also considered to play a key role in the overall performance of such materials.11 The molecular weight of RSF has been puzzling researchers for a long time. The lithium bromide silk viscosity test SNV 95595−1963 (developed by the Swiss Standards Association) was early established to measure the intrinsic viscosity of silk to compare the extent of fibroin degradation. However, this method was seldom used, probably due to the degradation of RSF induced by the dissolving (in lithium bromide) process itself.12 The coexisting of hydrophilic and hydrophobic blocks in the chains results in a tendency for RSF to form micellar structure or aggregation rather than “free” random coil in solution,13,14 which might induce fake value, 450 kDa, even higher than the theoretical molecular weight of the heavy chain.3 Therefore, most of common measurements for determining the molecular weight of macromolecules (proteins) were not suitable for the silk proteins. For example, it appeared to be difficult to use gel electrophoresis to differentiate the molecular weight of silk protein with a quantitative analysis,10,15,16 in particular for the RSF,17 due to the broad band in lane. Static light scattering (SLS)18 and small-angle neutron scattering (SANS)19 were also employed to © 2012 American Chemical Society
determine the molecular weight of RSF via measuring the hydrodynamic radius of the RSF chain. However, the existence of protein−protein associations, even in the presence of LiBr, and the inadequate dust removal made the results inconvincible. In addition, Jackson et al. tried an instrument combining size-exclusion chromatography with viscosity and light scattering detector to measure the molecular weight and its distribution (represented by the polydisperity index) of the Nephila clavipes dragline protein in the major ampullate gland.20 Unfortunately, the special mobile phase, hexafluoroisopropanol (HFIP), limited the applicability. Recently, Um and coworkers used gel permeation chromatography (GPC) combining with fast protein liquid chromatography (FPLC) to compare the molecular weight and its distribution of four RSF samples prepared under different dissolution conditions. Nevertheless, the results drawn from such a case were not fully satisfactory as the FPLC was much more suitable for the globular protein but rather not for the unavoidable aggregation of RSF in the employed solvents.21 In our previous work,14 an effective and convenient method was developed to estimate the molecular weight of SF. The SF is dissolved in its good solvent, 1-allyl-3-methylimidazolium chloride (AmimCl), in which RSF molecular chains behave as “free” chains rather than aggregates. Therefore, the linear viscoelastic moduli after time−temperature superposition (TTS) exhibit Rouse-like behavior characterized by a powerlaw region, where G′ ∝ ω1/2 and G′ and G″ almost coincide at high frequencies, followed by a transition region until the terminal relaxation characterized by G′ ∝ ω2 and G″ ∝ ω1 manifests (where G′ and G″ represent the elastic storage and viscous loss modulus, respectively, obtained from the viscoelastic spectrum, and ω represents the frequency).22 The transition region broadens with a broadening of molecular weight distribution. The amplitude and mode distribution of the viscoelastic modulus allow an evaluation of the molecular weight and its distribution through fitting the linear viscoelastic moduli by the Rouse model.23,24 This opens up the possibility to quantitatively measure the molecular weight and its distribution of SF, which further enables an evaluation of sample preparation and a controllable application of regenerated SF materials. Received: September 20, 2012 Published: December 6, 2012 285
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Figure 1. (a) Linear storage and loss modulus, G′ (closed) and G″ (open), obtained as functions of frequency for 15% dSF (degumming in Na2CO3)/AmimCl solution. The lines are the theroretical curves based on the Rouse model incorporating with Gaussian distribution of molecular weight. (b) Molecular weight distribution of the dSF obtained from fitting viscoelastic moduli with the Rouse model. placed along the borders of the measuring cell to prevent an uptake of moisture. In the dynamic linear viscoelastic measurements, the amplitude of the oscillatory strain was kept small (γ ≤ 10%) to ensure the linearity of the storage and loss modulus, G′(ω) and G″(ω), measured as functions of frequency ω in the range of 1 × 102 to 6.81 × 10−2 rad/s for all of the (R)SF/AmimCl solutions. Although N2 protection and thin layer of low-viscosity silicon were used to prevent moisture absorption, each measurement still needs to be controlled within 1.5 h at 40% relative humidity. The TTS principle was introduced into our study for the moduli of SF/AmimCl solutions measured at four temperatures (0, 10, 20, and 30 °C, respectively), and the master curves were reduced at 30 °C, where aT represented the temperature shift factor.14 2.4. Data Processing of the Rheology. For the polydisperse system, the Rouse-model was revised and the moduli were expressed in terms of conventional molecular weight averages, as eqs 1−3 listed
In the present study, we employed this method to investigate the difference of molecular weight of native SF sample from gland and several SF samples regenerated from different solutions and to clarify the effect of sample preparation, such as degumming and dissolving processes, on the degradation degree of SF molecular chains.
2. EXPERIMENTAL SECTION 2.1. Materials. 2.1.1. Native SF. The native SF sample was harvested from silk glands of the fifth instar larvae Bombyx mori silkworm. The gel-like silk protein was immersed in deionized water several times (each 5−10 min) to remove sericin as much as possible after peeling the epithelial skin of the gland by forceps. The gland SF was lyophilized for further use. 2.1.2. Regenerated SF (RSF). a. The RSF samples from various inorganic salts solutions were prepared by traditional processes:25 (1) The raw silk fiber was first boiled in 0.5 wt % Na2CO3 or NaHCO3 solution twice (each 20 min) to remove sericin. (2) The dSF was then dissolved in the inorganic solvents, which were 9.5 mol/L LiBr aqueous solution,26 CaCl2-EtOH-H2O (1:2:8 mol ratio) solution,27 or 52 wt % NaSCN aqueous solution.28 (3) The prepared SF/inorganic solvents were dialyzed against deionized water for 3 days to remove inorganic ions. (4) The regenerated SF was obtained by filming the SF aqueous solution in polystyrene containers via air-dry under ambient conditions. b. The RSF samples from organic solvents were prepared by two additional steps: (1) the regenerated SF film (regenerated from LiBr solution) was redissolved in either HFIP or 89% formic acid (FA) at room temperature. (2) These two kinds of regenerated SF solutions were refilmed by evaporating the organic solvents. 2.2. Preparation of SF/AmimCl Solutions. The solid native SF, dSF and RSF films were dissolved in AmimCl, with the same procedure described in our previous work.14 To ensure the varied SF materials fully dissolving in AmimCl within a fixed time (1.5 h) and to obtain the semidiluted solution of which the modulus could be described by Rouse model, we carefully selected the concentration of SF/AmimCl solution for further measurement (either 10 or 15%) because the solubilities of different SF materials in AmimCl differ from each other. 2.3. Rheology. Rheological measurements were carried on a stresscontrolled rheometer, Physical MCR-301 (Anton Paar), protected with N2 through a H-PTD200 hood with Peltier heating/cooling system. A thin layer of low-viscosity silicon (η30°C = 10 mPa·s) was
G′ =
i
G″ =
⎡
N
⎣
p=1
⎡
N
∑ ⎢⎢f (i)(ρRT /Mi) ∑
∑ ⎢⎢f (i)(ρRT /Mi) ∑ i
⎣
τip = τiR /p2
p=1
⎤ ⎥ 1 + ω2τip2 ⎥⎦ ω2τip2
ωτip 1+
(1)
⎤ ⎥
ω2τip2 ⎥⎦
(2) (3)
where ρ is the density, R is the gas constant, and T is the absolute temperature, Mi and f(i) are the molecular weight and weight fraction of the ith component, respectively, and τRi (∝ Mi2) and τip are the Rouse relaxation time and characteristic time of the pth mode of the ith component, respectively. The significance of differences in the molecular weight of RSF samples undergoing different regenerated processes was determined by one-way ANOVA on the statistics package in OriginPro 8 using a fixed-effects model and Fischer’s test (F-test) to separate means.
3. RESULTS AND DISCUSSION In our previous work,14 it was found that 15 wt % dSF/AmimCl solution was still in a semidilute unentangled concentration regime, which might be due to the inadequate concentration or the low molecular weight of dSF.29 In such a case, the Rouse model can be suitably used to fit dynamic viscoelastic behavior. As shown in Figure S1 in the Supporting Information, the model well fits the experimental data except at very low frequencies. It is well-known that the dynamic modulus is related to the molecular weight of the solute and the solute fractions with various sizes have different contributions to the overall modulus of a polydisperse system. Therefore, the 286
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conditions was nearly negligible. However, it was worth mentioning that the fitting curves and the experimental data diverged at the low frequencies even after incorporating the Gaussian distribution, suggesting there were fractions with larger relaxation. These fractions with larger molecular size might be attributed to the residual sericins with complex components (24−400 kDa),32 which hardly dissolved in AmimCl and possibly further induced fractional association of protein. The Mw obtained from Rouse fit incorporating the most probable distribution was ∼400 kDa (Figure S2 in the Supporting Information). Although the most probable distribution made the Rouse model fit well in lower frequencies, logically it is not suitable for the RSF and dSF systems, in which the molecular weight has an upper limit, that is, 350 kDa. Therefore, the molecular weight was hypothesized to follow the Gaussian distribution for all of the SFs described below. The Mw as well as the polydisperity index of these RSF and dSF samples was listed in Table 1. (Besides Figure 1a, the
molecular weight distribution has a very large effect on the modulus especially the storage modulus at low frequencies.30 It could be imagined that the SF molecule has been randomly degraded into a polydisperse system during the degumming and dissolving processes, as the native SF should have the definite molecule weight which can be calculated from its amino acid sequence. Herein, to fit the data more precisely, we used the revised Rouse-model (described in Section 2.4). (The detailed method of data fitting is presented in the Supporting Information.) For convenience, the molecular weight of SF was hypothesized to follow the Gaussian distribution. Figure 1a compared the storage and loss modulus for 15 wt % dSF/ AmimCl solution, which was obtained from directly dissolving the dSF (degummed by Na2CO3) into AmimCl and theoretical curves based on Rouse-model with the Gaussian distribution. It showed that after incorporating the Gaussian distribution, the Rouse model fitted well with the dynamic linear viscoelastic data at all frequencies. Figure 1b displayed a relatively broad distribution (from 10 to 330 kDa) of the molecular weight after the fitting. The maximum molecular weight (with a minimum proportion) obtained, 330 kDa, was only a little less than the calculated value of heavy chain of Bombyx mori fibroin (350 kDa),31 suggesting that such method for determining molecular weight of SF is valid and reasonable. The results showed that the number-average molecular weight (Mn) and weight-average molecular weight (Mw) for the dSF obtained by degummed raw silk fiber with Na2CO3 are 70 and 140 kDa, respectively. As mentioned above, SF molecules in the regenerated solution have undergone degumming and dissolving processes. To understand the effect of degumming and dissolving processes on molecular weight of SF separately, we dissolved regenerated SF acquired from arbitrary traditional preparations as well as native SF from gland in AmimCl to evaluate molecular weight through the method previously described. The experimental data (symbols) and the model calculation (curves) for native SF in AmimCl are shown in Figure 2, from which Mw was determined to be ∼310 kDa, intriguingly in accordance with the value of heavy chain of Bombyx mori fibroin, 350 kDa.31 It indicated that the degradation of fibroin during dissolving in AmimCl under the present experimental
Table 1. Mw and Polydisperity (Mw/Mn) of the RSF from Different Preparing Processes (n ≥ 4) degumming reagent NaHCO3 a
Na2CO3
dissolving reagent
Mw (kDa)
Mw/Mn
LiBr HFIPc FAc CaCl2-EtOH-H2O
225 ± 12 179 ± 4 176 ± 3 132 ± 4 128 ± 5
1.8 2.1 2.3 2.1 2.0
b
Mw (kDa)a 142 95 93 64 64
± ± ± ± ±
3 1 4 2 3
Mw/Mn 2.0 2.1 1.7 1.6 1.7
Mean ± standard deviation. bDirectly dissolving degummed silk fiber in AmimCl. cRedissolving the RSF film obtained from LiBr involved preparation. It should be emphasized that the Mw of native SF (from the silk gland) measured with this method was ∼310 kDa. a
dynamic viscoelastic data and data fitting with the Rouse model are shown in Figures S3−S11 in the Supporting Information, respectively.) It should be noticed that the fitting curve was not perfectly matched with the experimental data for some RSF materials (e.g., Figure S6 in the Supporting Information), presumably due to the deviation induced by possible residual sericin and the inconformity to Gaussian distribution. Nevertheless, the results delivered from such fitting (considering to fit both G′ and G″ well) was still appropriate, as stated in detail in the Supporting Information. 3.1. Influence of Degumming Process. The RSF samples undergoing different regenerated processes were also investigated. One-way ANOVA analysis showed that these differences in Mw between RSF degummed with different reagents were highly significant (P < 0.001). Apparently, the degumming process had a greater impact on the molecular chain of SF compared with the dissolving process. The Mw of dSF degummed by Na2CO3 (142 kDa) was generally 40% lower than that degummed by NaHCO3 (225 kDa); even the degumming conditions were kept the same. The huge difference in Mw also exists in the RSF degummed by different reagents. Stronger alkalinity of Na2CO3 may lead to such great destruction of molecular chain.10 In our lab, it has been found that the reconstituted silk fiber wet-spun from the regenerated SF solution of which the raw silk fiber was degummed by NaHCO3 was stronger than that from the one degummed by Na2CO3. This feature suggests that a mild degumming process that induces less degradation of the SF chain is indispensable to
Figure 2. Storage and loss modulus, G′ (closed) and G″ (open), obtained as functions of frequency for 10% native SF/AmimCl solution. The lines are the theroretical curves based on the Rouse model with Gaussian distribution of molecular weight. 287
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4. CONCLUSIONS In addition to the study of the conformation of SF molecular chains, interest has been growing in determining the molecular weight of RSF obtained from different processes with the aim to produce high-performance SF-based materials. For this purpose, several RSF/AmimCl solutions were prepared, and Rouse-like rheological behavior was noted for them, which enabled the determination of molecular weight of SF through fitting the linear viscoelastic data with Rouse-model. This fitting further allows us to investigate the influence of the preparation processes of RSF on the molecular chain of silk protein. Our study showed that the cocoon-degumming approaches had greater impact on the degradation of molecular chain of SF than the dissolving process. The weaker the alkalinity of cocoon-degumming reagent, the milder the effect on SF molecular chain. The degradation degree of SF molecular chains in the common aqueous solutions of inorganic salts could be summarized as in LiBr < CaCl2-EtOH. As organic solvents, FA showed much greater influence on the molecular weight than HFIP. The critical importance of molecular weight to high-performance materials made us believe that the useful information presented in this note enabled the further manufacture and application of RSF-based material.
achieve the SF-based materials with high mechanical performance. 3.2. Influence of Dissolving Process. Concentrated aqueous solution and aqueous−organic solutions of inorganic salts, which were chaotropic solvents, have been extensively used in dissolving silk fiber. To our knowledge, there is almost no quantitative study on the effect of inorganic salts on molecular chain of SF, given that a small amount of qualitative research has been done.8,10,33 We selected commonly used inorganic solvent systems, LiBr-H2O and CaCl2-EtOH-H2O, to prepare two types of RSF solutions and then measured the dynamic viscoelastic behavior of RSF/AmimCl solutions of which the cast RSF film was redissolved in AmimCl, respectively. As previously mentioned, the AmimCl would hardly affect the molecular chain size of SF; therefore, the determined molecular weight (shown in Table 1 and Figures S4−S7 in the Supporting Information) could be considered to represent RSF itself. One-way ANOVA analysis showed that these differences in Mw between RSF dissolving in those different reagents were highly significant (P < 0.001), meaning that the molecular chain of SF was subjected to different degrees of damage in these systems. Of those two inorganic solvents, LiBr-H2O seemed to be milder than CaCl2-EtOHH2O, which was consistent with our previous suggestion.11 Yamada et al. also found that CaCl2-EtOH-H2O caused greater degradation of the heavy chain of fibroin than other solvents.10 In addition, the Mw of RSF dissolved in NaSCN-H2O (the raw silk fiber pretreated by Na2CO3) was simulated as 79 kDa (shown in Figure S12 in the Supporting Information), suggesting that this solvent might also be gentler than CaCl2EtOH-H2O in terms of damaging the fibroin chain. Nevertheless, it was difficult to figure out a clear explanation regarding the degradation of SF in different systems due to the complexity of the dissolving mechanism. Besides the inorganic salt system, we studied the molecular weight of RSF in two commonly used organic solvents, FA and HFIP, in which fibroin molecules mainly exist in the form of helix structure and random coil at room temperature.8 Although these two kinds of silk solution were usually applied to prepare RSF based materials by spinning6,34 or filming,35 there was only qualitatively description on the degradability of those two solvents8 based on the observation of the color change of the solutions, as presented in Figure S13 in the Supporting Information. One-way ANOVA analysis on our experimental data (Table 1 and Figure S8−S11 in the Supporting Information) showed that the significance of differences in Mw between LiBr and HFIP was 0.221; whereas that between LiBr and FA was lower than 0.001. These results demonstrated that (1) dissolving in HFIP only slightly affected the molecular chain of RSF, confirming Lock’s suspicion,36 and (2) dissolving in FA caused greater destruction on the Mw of SF, which might be due to the acidity of FA. Indeed, the random degradation of SF molecules resulted by preparing procedure induced the polydisperity of molecular weight, which was listed in Table 1 as well. It could be seen that the polydisperity of RSF and dSF in each system was close to 2 and decreased slightly with the decreasing of Mw of RSF (down to 65 kDa), possibly because the RSF was degraded from a definite molecular chain, that is, heavy chain of SF with 350 kDa.
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ASSOCIATED CONTENT
S Supporting Information *
Experimental data and theoretical fitting curves based Rouse model for those RSF from various preparing systems and the comparison of photographs of SF/HFIP and SF/FA solutions. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (Z.S.);
[email protected]. cn (Y.Y.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (NSFC 21034003) and 973 Project of Chinese Ministry of Science and Technology (no. 2011CB605700)
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REFERENCES
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