VOLUME 4, NUMBER 3, MARCH 2004 © Copyright 2004 by the American Chemical Society
Virus-Based Fabrication of Micro- and Nanofibers Using Electrospinning Seung-Wuk Lee and Angela M. Belcher* Department of Materials Science and Engineering and Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Chemistry and Biochemistry, The UniVersity of Texas at Austin, Austin, Texas 78712 Received October 20, 2003; Revised Manuscript Received December 18, 2003
ABSTRACT Long rod-shaped M13 viruses were used to fabricate one-dimensional (1D) micro- and nanosized diameter fibers by mimicking the spinning process of the silk spider. Liquid crystalline virus suspensions were extruded through the micrometer diameter capillary tubes in a crosslinking solution of glutaraldehyde. Resulting fibers were 10−20 µm in diameter. AFM imaging verified that the molecular long axis of the virus fibers was parallel to the fiber long axis. M13 viruses were suspended in 1,1,1,3,3,3-hexafluoro-2-propanol and were then electrospun into fibers. After blending with a highly water soluble polymer, polyvinyl pyrolidone (PVP), M13 viruses were spun into continuous uniform virusblended PVP (virus-PVP) nanofibers. Resulting virus-PVP electrospun fibers maintained their ability to infect bacterial hosts after resuspending in buffer solution.
Efforts to mimic the unique structures and specific functions of natural systems have provided various useful tools and materials in nanoscience.1-15 Biosystems produce highly programmed, self-assembled, self-templated structures.5-15 For example, a small percentage of protein in abalone shell nucleates a CaCO3 protein composite that is 3000 times tougher than pure CaCO3.1 By mimicking the biomineralization process in nature, our group has shown that protein sequences, selected using a genetically engineered peptide virus library, could specifically bind to and nucleate desired materials.3,4,7,8,10 The one-pot synthetic route provided by these genetically programmed viruses results in the selfassembly of highly ordered nanocrystalline composite materials.4 Recently, much research has been focused on producing nanosized one-dimensional materials such as nanorods, * Corresponding author. E-mail:
[email protected]. 10.1021/nl034911t CCC: $27.50 Published on Web 02/20/2004
© 2004 American Chemical Society
nanowires, and nanofibers.17-24 Naturally, silk spiders and silk worms spin highly engineered continuous fibers by passing aqueous liquid crystalline protein (fibroin) solution through their spinneret.17,18 Once the fibroin solution is released to the air, it hardens into a flexible and highly oriented semicrystalline fiber that is stronger than any other polymer fiber spun.17 Among synthetic routes investigated, nanofiber fabrication using electrospinning has been an efficient means of generating high surface-to-volume ratios of materials that may possibly be used as highly sensitive sensors and functional membranes.23-29 Electrospinning uses high electric fields allowing for the fabrication of narrow fibers with diameters ranging from tens of nanometers to micrometers. The continuous fibers can be easily converted into nonwoven fabrics, which may be useful for synthesizing novel membranes due to their high surface-to-volume ratios. Here we report two methods of fabricating virus-based
Figure 1. A schematic diagram illustrating the virus fiber fabrication process using wet-spinning and electrospinning.
composite fibers that mimic the spinning process of silk spiders. M13 viruses were spun into micro- and nanofibers using wet-spinning and electrospinning, respectively, which are illustrated in Figure 1. The resulting fibers showed nematic ordered morphologies due to flowing forces. In addition, the viruses were blended with polyvinyl pyrolidone (PVP) to improve processing ability. The resulting virusblended PVP fibers were continuous and were transformed into nonwoven fabrics that retained their ability to infect bacterial hosts. These hybrid materials, including flexible fibers and mats, have possible applications including biomedical and tissue engineering as well as templates to biocontrolled-synthesis of electronic or optical materials.8,10 In addition, further genetic engineering of pVIII major and pIX minor coat proteins of M13 virus might provide multiple hybrid functions of virus-based fibers.34 Anti-streptavidin M13 bacteriophage (virus), possessing an engineered peptide sequence N′-WDPYSHLLQHPQ-C′ as a fused protein into the pIII coat protein, was used as a basic building block to fabricate the micro- and nanoscale fibers with or without conjugation with R-phycoerythrin (eBioscience, CA).7 The M13 virus suspension (∼100 mg/ mL in tris-buffered saline (pH 7.5)) was extruded through a
∼20 µm capillary tube into a 37.3% aqueous glutaraldehyde solution. This cross-linked suspension was air-dried and formed microfibers. A polarized optical microscopy (POM) image (Figure 2A), taken using an Olympus IX51 polarized optical microscope (Olympus, Japan), showed the fibers were birefringent, indicating the liquid crystalline ordered structure of the fibers. Scanning electron microscope images (SEM; JEOL 6320 FEGSEM, Japan) (Figure 2B), of the fibers showed that the fibers had 10-20 µm diameters and were composed of several bundle-like fibers which were propagated to the fiber long axis. The parallel orientation of the individual virus building blocks was observed using an Atomic Force Microscope (AFM; Veeco Nanoscope, CA). An AFM image (Figure 2C) showed close-packed M13 viruses, with a long axis parallel to the long axis of the fibers. Due to the flow field, the smectic ordered structure in the suspension was disrupted and a smectic-to-nematic transition occurred. Because the suspension was immediately exposed to a cross-linking solution after release from the capillary, the nematic ordered viruses were cross-linked to each other and formed nematic ordered fibers. Fluorescent viral microfibers were fabricated after binding of the anti-streptavidin virus with R-phycoerythrin conjugated streptavidin prior to spinning.7 A uniform fluorescent field observed (Figure 2D) throughout the fibers supported the nematic ordered structure observed in POM and AFM. Various volatile organic solvents were tested as electrospinning solvents as a possible mechanism to electrospin the low viscosity M13 virus suspension. Most of the electrospinning solvents tested, such as methanol, ethanol, DMF, acetone, and trifluoroethanol, formed a slurry-like aggregation when added to virus pellets and suspensions. However, 1,1,1,3,3,3,hexafluoro-2-propanol (HFP; Alfa Aeser, MA) successfully formed a homogeneous virus suspension when the virus pellets were dissolved. To fabricate nanofibers, capillary tubes (20 µL volume, ∼0.5 mm in diameter, 6.5 cm long, Drummond Scientific Co., PA) were filled with virus suspension (∼92 mg/mL in HFP), and a graphite rod
Figure 2. M13 virus fiber fabricated by wet-spinning process (A) POM image (scale bar: 100 µm), (B) SEM image (scale bar: 20 µm), and (C) AFM image and schematic diagram inset of nematic oriented morphology of virus fiber (scale bar: 1 µm). (D) fluorescence micrograph of virus-phycoerythrin conjugated fibers fabricated by wet-spinning. 388
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Figure 3. (A) POM image of electrospun virus-only fibers, (B) SEM image of electrospun virus-only fibers. (Scale bars: 5 µm).
(0.5 mm in diameter) was inserted into one end of the capillary tube using a syringe pump (Harvard Apparatus, Inc., MA) at a feeding rate of 3-6 µL/min. A 20-30 kV potential was applied by a high voltage source across air (Glassman High Voltage, NJ). Electrospun fibers were collected onto metal plates (10 cm in diameter), grounded and coveredby aluminum foil. The distance between the capillary and collector was 10-15 centimeters. A POM image (Figure 3A) verified that these continuous electrospun fibers were highly birefringent under the cross polars, indicating that fibers were composed of highly crystalline ordered structures. A relatively broad distribution of diameter was observed, ranging from a few micrometers to tens of nanometers in diameter. SEM images (Figure 3B) showed that most of the fibers and bundles branched to form smaller fibers. Due to the toxicity of HFP to the M13 virus, infectibility of M13 virus in HFP solution was dramatically decreased, showing no infectibility. Because intact virus structures were rarely observed from TEM observation, virus fibers spun using HFP could be composed of the fragment of virus and dissembled subunits. To improve processing ability and preserve the intact viral structure and infecting ability, the M13 virus suspension was blended with a highly water soluble polymer, PVP (MW: 1,300,000; Alfa Aesar, MA). The suspensions were blended in ratios of 1:1, 1:2, 1:3, and 1:4 between virus suspension in TBS (∼100 mg/mL) and PVP solution (25%, (w/w)) in water. Due to the low viscosity and high surface tension of the aqueous suspensions, 1:1 and 1:2 suspensions, when electrospun, deposited droplets of virus-blended PVP suspension. The electrospinning of the 1:3 suspension resulted in sporadic fiber formation, with typical results being a mix of bead and string type fibers, which is normally observed in low viscous or low concentration solutions in the electrospinning process.29-31 Continuous M13 virus-blended PVP fibers were fabricated from the electrospinning of the 1:4 suspension. A photograph of electrospun fibers (Figure 4A) showed that electrospun fibers could be transformed to any desired shape of nonwoven fabrics using a mask. An SEM image showed that the resulting fibers were continuous and formed homogeneous rope shapes (Figure 4B). Distribution of the diameter was relatively narrow with the diameter ranging from 100 to 200 nanometers. The electrospun fibers observed using polarized optical microscopy exhibited nematic-like birefringency (Figure 4C). The fibers showed maximum brightness when the fibers were oriented at 45 degrees to the cross polars and were extinct when the Nano Lett., Vol. 4, No. 3, 2004
Figure 4. Electronspun fiber of M13 virus-blended with PVP. (A) Photograph of nonwoven fiber spun through the mask inscribed with the word “NANO”, (B) SEM image (scale bar: 1 µm) and (C) POM image. (D) Fluorescence micrographs of PVP blended with virus-phycoerythrin fibers fabricated by electrospinning.
fibers were oriented parallel with cross polars. When the viruses were conjugated with R-phycoerythrin using a streptavidin linker, fluorescence images could be observed using fluorescence microscopy (Figure 4D). After dissolving the electrospun fibers in 3 mL of TBS, the suspension was tested for infection ability of the M13 virus. The resuspended virus suspension was still able to infect the bacterial host. This result supports the notion that both the protein structure and genetic information were stable in these virus-based fibers. Furthermore, we believe this is the first electrospinning of an organism to form oriented pseudo-infinite fiber structures. The incorporation of R-phycoerythrin into the virus fibers demonstrates that any molecule could be incorporated and organized in these 1D fibers. This suggested that many other materials including small molecules, drugs, proteins, and semiconducting and magnetic materials can also be electrospun into virus-based fibers and mats to form continuous materials over a pseudo-infinite length scale. In summary, virus-based micro- and nanofibers were fabricated using wet-spinning and electrospinning processes. M13 viruses in the wet-spun fibers were aligned parallel to the fiber long axis. Electrospun fibers, composed of fragments of M13 viruses and its subunits, were also fabricated by suspending M13 viruses in HFP. By blending the viruses with PVP, fibers were formed with the intact structure of the viruses. These fibers demonstrated that novel biomaterials can be fabricated from a programmed organism to extend the dimension of engineered viruses into fibers useful in nucleating semiconductor nanofibers or in selectively binding any type of desired materials.3,4,7,10 Additionally, uniform nanofibers fabricated by blending nanomaterial-conjugated M13 virus with PVP might provide useful biological functions and highly sensitive catalytic functions in future biomedical applications and biosensors. 389
Acknowledgment. This work was supported by the ARO, NSF, and Institute for Collaborative Biotechnologies. S.-W. Lee thanks Dr. Esther Ryan, Dr. Christine Flynn, and Eric Krauland for assistance in editing this paper for clarity.
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