Justin A. Jones. Randolph V. Lewis Laboratory. Aqueous Based Processing of Recombinant. Spider Silks. Page 2. Fiber. Film. Hydrogel. Lyogel. Sponge.
Aqueous Based Processing of Recombinant Spider Silks Justin A. Jones
Randolph V. Lewis Laboratory
A
B C
Fiber Film Hydrogel Lyogel Sponge Adhesive
Aqueous Based Films
FlYS3
80kDa
E. coli
MaSp1 + MaSp2
65kDa ea.
Goat
Aqueous Based Films
Aqueous Based Films: Mechanical Properties 250
200
150
100
50
0
Stress (Mpa)
Strain (%)
Aqueous-Based Recombinant
HFIP-Based Recombinant
Collagen X-Linked
Polylactic Acid
B. mori Silk Fibroin
B. mori: Jiang, C.; Wang, X.; Gunawidjaja, R.; Lin, Y.-H.; Gupta, M. K.; Kaplan, D. L.; Naik, R. R.; Tsukruk, V. V. Adv. Funct. Mater. 2007, 17, 2229–2237. Collagen: Mallika, P.; Himabindu, A.; Shailaja, D. J. Appl. Polym. Sci. 2006, 101, 63–69. PLA: Brandrup, J.; Immergut, E. H.; Grulke, E. A. Polymer handbook, 4th edition; Wiley: New York; Chichester, 2004.
2D WAXD of Aqueous Based MaSp1 Films A: As-poured film
• Stretched film (B) shows substantial increase in β-sheet and clear alignment parallel to fiber axis.
B: 2.5X Stretched in 80:20 MeOH:Water
1H-13C
CP-MAS of Aqueous Based MaSp1 Films
Protein Powder
As-poured film
Stretched film
• Protein powder: β-sheets • As-poured film: helical • Stretched films: β-sheets
Aqueous Based Fibers
Aqueous Based Fibers
• • • • •
4 touch screen control monitors along fiber path. 3 godet system allowing for 2 independent stretches. Multi-fiber spinning. Variable temperature. Real-time video monitoring of fiber properties.
Aqueous Based Fibers
Dope Not Sonicated: 12.5% w/v M4, 40X objective
Dope Sonicated: 5% w/v M4, 40X objective.
• Dope processing • Sonicating suspension improves fiber • Reduces qty of silk required to form fiber
Aqueous Based Fiber Comparison 400
350
300
250
200
150
100
50
0 Teule (J. Mat. Sci. 2007)
Teule Silkworm (Spider 6) 2012)
Stress (Mpa)
(PNAS Teule Silkworm (Spider 6-GFP) (PNAS 2012)
Strain (%)
Lewis Aqueous Method
ETB (MJ/m3)
• Aqueous fibers compare to or are better than other rSSP fibers in literature. • No optimization of process.
Aqueous Based Fibers 12% w/v Aqueous Dope, MaSp1:MaSp2
Energy to Break (MJ/m3) + S.D.
Avg. Stress (MPa) + S.D.
Avg. Strain (mm/mm) + S.D.
30:70 (1.5X/1.5X)
8.788 ± 16.194
53.009 ± 28.403
0.115 ± 0.170
50:50 (2.5X/2.5X)
52.22 ± 21.055
144.481 ± 36.101
0.438 ± 0.193
80:20 (2.5X/2.5X)
16.224 ± 4.615
101.359 ± 32.380
0.199 ± 0.049
90:10 (2.5X/2.0X)
33.770 ± 33.552
192.224 ± 51.470
0.281 ± 0.260
0:100 (2.0X/2.0X)
30.369 ± 19.764
145.006 ± 24.108
0.465 ± 0.181
Maxima Values
85.76
272.27
0.64
• Varying protein ratio (rMaSP1 to rMaSP2) alters the mechanical properties of the fiber. • Varied ratios require different stretches to achieve maximum properties.
Aqueous Based Fibers
100 cycles to 50% load and then testing to failure • Aqueous fibers able to withstand repeated loadings. • Multiple loadings demonstrate nearly identical stress-strain.
Aqueous Based Material
Aqueous Coatings with Antibiotic
Silicone Wafer with Kanamycin D ≈ 22mm
Stainless Steel with Kanamycin D ≈ 20mm
• Can coat and functionalize silicone and stainless steel with antibiotics.
Silicone Catheters with Kanamycin L ≈ 2.5mm
rSSP coating with azole Candida albicans
1
3 C: Control rSSP without azole 1: 1.0X concentration of azole 2: 1.5X concentration of azole 3: 2.0X concentration of azole
2
• Can functionalize silk protein with azole and coat silicone.
CHO Cells Control
MaSP1 silk film + RGD
Collagen film
MaSP1 silk film
• CHO cells grow well on both RGD functionalized MaSP1 and MaSP1 films produced from our aqueous method.
Drug Release 0.3
6% Hydrogel 0.25
6% Hydrogel2 12% Hydrogel 0.2
O.D. 580
1.5 mL 12% Hydrogel 1.5 mL 12% Hydrogel2
0.15
0.1
0.05
0 1
2
4
5
6
7
8
11
12
13
14
15
17
18
19
20
Time (Days)
• Methyl violet diffuses from hydrogels beyond two weeks.
Conclusions • Solubilization method developed for a variety of rSSP that are otherwise insoluble in water. • Additives can enhance/change solubility and ability to process dopes and/or form gels, fibers, films and foams.
• Films and fibers produced from this aqueous method have equal or better mechanical properties and protein structures to those produced using HFIP. • Aqueous based material can be functionalized with antibiotics, antimycotics, heparin and drug analogues. • Biocompatiblity is preserved with this aqueous method when compared to HFIP derived material as indicated by E. coli and CHO cell growth.
Acknowledgments Funding: • Utah Science and Technology Research initiative (USTAR) • National Science Foundation • Department of Defense: Office of Naval Research
Individuals: • • • • • • •
•
Chauncey Tucker (Aq. and films) Kyle Berg (Aq. and fibers) Thomas Harris (Coatings and films) Cole Petersen (Piriform) Dr. Paula Oliviera (MaSp1) Dr. Sreevidhya Tarakkad Krishnaji (FlYS and FlAS) Dr. Jeffery Yarger, Arizona State University (Structural Determinations) Dr. Jon Takemoto (Antimycotic)
