Published on Web 10/19/2004

Dissolution and Regeneration of Bombyx mori Silk Fibroin Using Ionic

Liquids
David M. Phillips,† Lawrence F. Drummy,† Deborah G. Conrady,† Douglas M. Fox,‡ Rajesh R. Naik,†
Morley O. Stone,† Paul C. Trulove,‡ Hugh C. De Long,§ and Robert A. Mantz*,†
Materials & Manufacturing Directorate, Air Force Research Laboratory, 2941 Hobson Way,
Wright-Patterson AFB, Ohio 45433; Chemistry Department, U.S. NaVal Academy, Annapolis, Maryland 21402; and
Chemistry and Life Sciences Directorate, Air Force Office of Scientific Research, Arlington, Virginia 22203
Received July 1, 2004; E-mail: [Link]@[Link]

Natural silk fibers have outstanding mechanical properties that suitability of several other ionic liquids was tested for solubilizing
rival the most advanced synthetic polymers.1 Compared to the silk. BMIM+ Cl-, Br-, I-, and BF4-; 1-butyl-2,3-dimethylimida-
limited number of molecular solvents available today, ionic liquids zolium (DMBIM+) Cl-; and 1-ethyl-3-methylimidazolium (EMIM+)
have taken the spotlight as green, designer solvents, with many Cl-, BF4-, and AlCl4- ionic liquids were evaluated to determine
combinations that offer an unprecedented versatility and tunability. their ability to dissolve silk.
In this work, we test the suitability of ionic liquids for dissolving The cocoon silk utilized in these experiments originated from
and regenerating silkworm (Bombyx mori) silk and examine the silkworms raised on a diet of Silkworm Chow (Mulberry Farms,
structural properties of silk films cast from ionic liquid solutions. Fallbrook, CA). The pupae were extracted from the cocoons intact
A single strand of natural cocoon silk fiber contains two silk two to 7 days after spinning by cutting open the cocoons. This
fibroin cores surrounded by a protective, glue-like sericin coating. method avoids possible contamination and thermal degradation from
The actual fibroin cores consist of two macromolecules, with the industrial process of baking the pupae in the cocoons.
molecular weights of 391 and 26 kDa for the heavy and light chains, For most of the experiments, the sericin was extracted from the
respectively.2 The crystalline regions of the heavy chain occur silk prior to solubilizing in the ionic liquids. However, for the
primarily in the repeated GAGAGS amino acid motifs. These BMIM Br, BMIM I, and BMIM BF4 experiments, the sericin
regions assume a hydrogen bonded, antiparallel, β-sheet structure remained on the fibers, which were dried under vacuum. In the
in the natural cocoon fiber.3,4 The hydrogen bonding and the BMIM Cl and DMBIM Cl experiments, the sericin was stripped
hydrophobic nature of these crystalline regions make dissolution in a 0.05 M Na2CO3 and 0.05 M Na2EDTA solution with 9%
of silk a formidable task.5 (w/w) cocoons at 65 °C for a day. The silk was rinsed thoroughly
Multiple methods have been utilized to prepare silk fibroin and lyophilized prior to solubility testing. In the EMIM Cl
solutions. These generally involve stripping the sericin in a experiments, the sericin was stripped in a 0.2 M Na2CO3 solution
Na2CO3 wash, rinsing and drying the silk, and dissolving the by boiling for 2 h. These cocoons were rinsed thoroughly and dried
silk in a high-concentration, aqueous lithium salt solution or a
at 100 °C in a vacuum oven overnight prior to solubility testing.
CaCl2/ethanol/water solution.6 These solutions are subsequently
The dissolution experiments were conducted under an inert
dialyzed to remove the salt and have a shelf life of about 1 week
atmosphere of N2 (BMIM and DMBIM) or He (EMIM) due to the
before the onset of gelation. Stable fibroin solutions can be prepared
hygroscopic nature of the ionic liquids. To determine the solubility
by lyophilizing the dialyzed solutions and dissolving the resulting
of the silk in each of the ionic liquids, silk was added slowly to
silk powder in another solvent such as 1,1,1,3,3,3-hexafluoro-2-
the ionic liquid melt while providing agitation. The resultant ionic
propanol or hexafluoroacetone.7
liquid/silk solutions were clear with an amber color and quite
Ionic liquids have been used to successfully dissolve other
viscous above 10% (w/w).
biological macromolecules such as cellulose.8,9 Swatloski et al.
The temperatures of the ionic liquid solutions were maintained
found that the ionic liquid 1-butyl-3-methylimidazolium chloride
with a temperature-controlled oil bath at 100 °C. Attempts to
(BMIM Cl) can be used to prepare solutions of up to 25% (w/w)
cellulose.8 They also reported that cellulose was soluble in BMIM dissolve the silk fibroin in BMIM Cl using a microwave were
Br and BMIM SCN but with less than half the degree of solubility unsuccessful due thermal decomposition of the silk. The solubility
of BMIM Cl. The success of BMIM Cl has been attributed to the results listed in Table 1 are samples dissolved using an oil bath as
ability of the chloride anion to disrupt the hydrogen bonding present the heat source.
between the cellulose chains. The solubility of silk fibroin in ionic liquids depends on the
Swatloski et al. also found that the solution preparation method identity of both the cation and anion, with the anion having a much
greatly affected the solubility.8 In particular, they used microwave larger effect. The more the cation and anion are able to participate
heating on a BMIM Cl/cellulose solution to dissolve 25% (w/w) in hydrogen bonding, the greater the solubility of the silk fibroin.10
cellulose, as compared to a 10% (w/w) cellulose solution heated Presumably, the ionic liquid disrupts the hydrogen bonding present
in an oil bath at 100 °C. However, they did not report the peak in the β-sheets. The BMIM Br, BMIM I, and BMIM BF4 were
temperature that was reached during the microwave dissolution or tested with both silk and sericin. Sericin is soluble in BMIM Br
analyze for cellulose degradation. and I but not in BMIM BF4.
The ability of BMIM Cl to disrupt hydrogen bonding makes it The solubility of the silk fibroin in BMIM Cl was also examined
an attractive solvent for silk fibroin. In addition to BMIM Cl, the with wide-angle X-ray scattering (WAXS) by examining the crystal
structure. In Figure 1a, the data from a 12.24% (w/w) silk solution
† Air Force Research Laboratory.
‡ U.S. Naval Academy.
sealed between Kapton windows show a broad amorphous halo
§ Air Force Office of Scientific Research. centered near a 2θ of 20°, indicating that amorphous silk, but no
14350 9 J. AM. CHEM. SOC. 2004, 126, 14350-14351 10.1021/ja046079f CCC: $27.50 © 2004 American Chemical Society
 COMMUNICATIONS

Table 1. Saturated Solubility by Weight for Silk in Ionic Liquids of any cocoon silk fiber structure by optical inspection in BMIM
anion Cl indicates that the silk has dissolved.
cation Cl- Br- I- BF4- AlCl4-
With the solubility of silk confirmed, a 9.51% (w/w) silk in
BMIM Cl solution at 100 °C was used to cast films on both silicon
BMIM+ 13.2% 0.7%a 0.2%a 0.0%a c
wafers and glass slides. The BMIM Cl was removed by rinsing
DMBIM+ 8.3% c c c c
EMIM+ 23.3% c c 0.0% 0.0%b with either acetonitrile or methanol. Raman analysis indicated that
both the acetonitrile and methanol rinses removed the BMIM Cl.
a Both sericin and fibroin added to the solvent; only the sericin is soluble.
Attempts to rinse the BMIM Cl with water resulted in dissolution
With the exception of BF4-, these solutions are not saturated. b Solvent is
an EMIM Cl/EMIM AlCl4 mixture with a 1.0:0.7 molar ratio. c System not of the silk film. Figure 2 shows optical microscopy images of silk
tested. films. The acetonitrile-rinsed film is white in color due to light
scattering and has a visible surface topography. The methanol-rinsed
film is not smooth but is transparent. This film is birefringent with
50 µm domains, whereas the acetonitrile-rinsed film is not
birefringent. Neither film shows residual fiber structure, further
supporting our assertion that the silk was solubilized.
The crystal structures of films cast on silicon wafers were
analyzed with both WAXS and laser Raman at 514 nm. Figure 3
compares both films with a cocoon fiber. Figure 3a shows the
WAXS data for the three samples. It is clear that the methanol-
rinsed film exhibits a high degree of crystallinity that is similar to
the cocoon fiber. This is in agreement with previous results that
have shown methanol treatment of silk induces the β-sheet
Figure 1. WAXS crystallinity analysis for (a) 12.24% (w/w) silk in BMIM
Cl (labeled “Amorphous Silk/BMIM”) at 30 °C after cooling from 100 °C structure.11 In contrast, the acetonitrile-rinsed film only has a small
compared with crystalline cocoon silk and (b) pure BMIM Cl at 30 °C. degree of crystallinity. The Raman data in Figure 3b indicate that
The BMIM Cl disrupts the β-sheet crystalline structure in the silk. the crystal structure for both films and the cocoon fiber are indeed
β-sheet, with peaks at 1229 and 1084 cm-1.12
In conclusion, BMIM Cl, DMBIM Cl, and EMIM Cl are able to
disrupt the hydrogen bonding in silkworm silk fibroin to form solu-
tions that have no residual resemblance to the original cocoon fibers.
WAXS results confirm that the crystal structure of silk fibroin in
BMIM Cl is eliminated after heating to 100 °C. The structure of
films cast from silk in BMIM Cl solutions is highly dependent on
the rinse treatment; acetonitrile yields a convoluted surface structure
with little crystallinity, and methanol yields a transparent film with
a high degree of crystallinity. Our results confirm that ionic liquids
Figure 2. Reflectance optical microscopy images of films on glass slides are viable solvent systems for dissolving and regenerating silk
cast from 9.51% (w/w) silk in BMIM Cl and rinsed with (a) acetonitrile
and (b) methanol. The acetonitrile-rinsed film has a convoluted surface and
fibroin.
scatters light. The methanol-rinsed film appears to be clear and is
Acknowledgment. Funding for this research was provided by
birefringent. Neither film has any residual fiber structure.
the Air Force Office of Scientific Research. We gratefully
acknowledge the National Research Council for providing post-
doctoral fellowships at the Air Force Research Laboratory (D.M.P.
and L.F.D.).

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J. AM. CHEM. SOC. 9 VOL. 126, NO. 44, 2004 14351