1. Electrospinning of hybrid polymers to mimic spider dragline silk Lim Yao Chong 4S2 Low Rui Hao 4S2 Tracey Atkinson AOS Patrick Steiner AOS

2. Background Spider Dragline Silk It is the material that makes up the main “axels” of orb-weaver spider webs. It has a High tensile strength and High extensibility.

3. Background Spider Dragline Silk It has a composite structure of: 20% crystalline regions 80% highly elastic substances Extensible regions of the spider dragline silk connect crystalline regions to produce the amazing properties of the spider silk.

4. Keratin: a material that provides strength in biomaterials such as nails, bird beaks, horns, etc. Biodegradable Has same beta-sheet composition as spider silk Elastin: A material that provides elasticity to artery walls, lung tissue, skin, ligaments, etc. Biodegradable More elastic than spider silk

5. A polymer is dissolved in a volatile solvent and placed in a syringe. The solution is charged with a high voltage. The high voltage creates an electric field that causes the polymer to be spun out in thin threads (nanofibers) to a collector plate. A fibrous mat is formed.

6. Objectives To create fibrous electrospun mats with blended fibers, part keratin part elastin, to mimic the high tensile strength and extensibility of spider dragline silk. Blended fibers: parallel syringes method (physical mixture)

7. Hypothesis By combining Elastin and Keratin spun under optimal conditions into blended fibres in electrospun mats, a mat with tensile strength and extensibility similar to that of spider silk will be produced.

8. Materials Polyethyleneoxide(PEO) 1 M hydrochloric acid, Elastin Powder, Elastin Products Company Inc. Keratin, Advanced Scientific and Chemical Inc. Urea Powder, Sigma Aldrich

9. Variables Independent: The parameters of the method including electrospinning method variables: distance to collector plate flow rate of jet needle gauge and chemical variables of the mat: concentration of the spun solution ratio of Elastin to Keratin Dependent: tensile strength extensibility of the fibrous mat produced from the Electrospinning.

10. Variables Controlled:  Polymers used  Syringe pumps used  Solvents used  Solution size spun (2 mL)  Power source  Syringes used (5 mL)  Material coating collector plate (aluminium foil)  Spin time (20 min)  Voltage (20kV)  Collector plate size (25cmx25cm)

11. Methodology Phases Phase 1 Preparation Phase 2 Optimizing Spinning parameters of each Polymer Phase 3 Prove Hypothesis by varying ratio of Elastin to Keratin through Flow rate

12.  To dissolve Keratin and Elastin in suitable solvents to be used in Electrospinning  To determine spin time of the respective solutions of Keratin and Elastin (estimation) Phase 1 Preparation

13.  Optimize the conditions for electrospinning keratin and elastin individually distance to collector plate flow rate of jet Concentration of solution Ratio of Keratin/Elastin to PEO  The optimal conditions found will be kept constant in Phase 3 of the experiment Phase 2 Optimizing spinning parameters for individual polymers

14. Example: 10%, 70:30 1.Add 0.157 grams of powdered keratin to 2 ml of aqueous solution containing 8M urea. and stir until the powder has completely dissolved. 2.Add 0.067 grams of polyethylene oxide (PEO) to the keratin solution and stir until the PEO has completely dissolved. 3.Place 2ml of the solution in a 5ml syringe with a 22 gauge needle. 4.Set the voltage applied to 20kV, and the flow rate to 0.6 ml h- 1. Place the collecting plate 15cm from the syringe tip. 5.After starting the electrospinning, leave the set-up running for 20 minutes for sufficient deposition before stopping. Phase 2 Optimizing spinning parameters for individual polymers

15. A suitable amount of powdered keratin, followed by powdered polyethylene oxide (PEO) is dissolved in 8M urea solution to produce a polymer solution of intended concentration and keratin:PEO ratio 2mL of the polymer solution is placed in a 5mL syringe with a 22 gauge needle, which is placed in a syringe pump. The anode is connected to the needle, while the cathode is connected to the collector plate. The flow rate of the solution is set at a suitable amount, the needle is placed at a suitable distance from the collector plate, and a suitable voltage is applied to the setup. Start the syringe pump and let the experiment run for 20 minutes to ensure sufficient deposition of polymer. Examine the polymer mat under a microscope to check for beading and polymer thickness. Repeat the experiment under different conditions to compare results.

16. Phase 3 Determining optimal ratio of keratin to elastin  Spin the optimal parameters of Keratin and Elastin  Measure tensile strength of “optimal hybrid mat”  Repeat the experiment with different flow rates of keratin and elastin

17. Progress  Pure keratin dissolved in urea solution could not be spun. Keratin crystals were formed instead.  As a result, we added PEO to the solution in order to increase the viscosity of the solution in order to create a continuous jet, resulting in fibre formation.

18. Data Analysis  Distance from collector plate 10cm, 15cm, 20cm Affects results as sufficient distance is needed for evaporation of solvent, but if too far, jet will not be able to reach collector. All experiments were conducted under same voltage and solution concentration, to ensure same force of jet eruption.

19. Data Analysis 10cm, 6.48% 15cm, 6.48%

20. Data Analysis  10cm: Fibrous mat formed, but with outgrowth of fibres from mat  15cm: Flat fibrous mat formed.  20cm: Jet of polymer solution erupted from syringe, but was too far from collector plate and did not reach it – no mat formed.  Conclusion: 15cm is the optimal distance from the collector plate.

21. Data Analysis  Concentration of polymer solution 5%, 7%, 10%, 20% Increased concentration increases viscosity Allows for continuous jet and fibre. Too much prevents jet eruption from solution through syringe needle.

22. Data Analysis Image of fibrous matConcentrationRemarks 5% (of keratin+PEO solute by weight) Large amount of beadings, as well as droplets of solutions (i.e. failed) 7%Even mat formed. Fibres have more beading than in 10%, but thinner fibres. 10%Even mat formed. Fibres had least amount of beading. Thicker fibres than 7% 20%No mat was formed, only a strand of polymer. Thickest fibres

23. Data Analysis 5%

24. Data Analysis 7%

25. Data Analysis 10%

26. Data Analysis 20%

27. Data Analysis Image of fibrous matConcentrationRemarks 5% (of keratin+PEO solute by weight) Large amount of beadings, as well as droplets of solutions (i.e. failed) 7%Even mat formed. Fibres have more beading than in 10%, but thinner fibres. 10%Even mat formed. Fibres had least amount of beading. Thicker fibres than 7% 20%No mat was formed, only a strand of polymer. Thickest fibres

28. Data Analysis  Least beading seen in 10% solution.  The 20% solution was very viscous and did not result in a mat, but instead a strand of polymer from the syringe to the collector plate.  Conclusion: A keratin+PEO solution of concentration 10% is the optimal.

29. Data Analysis  Least beading seen in 10% solution.  The 20% solution was very viscous and did not result in a mat, but instead a strand of polymer from the syringe to the collector plate.  Conclusion: A keratin+PEO solution of concentration 10% is the optimal.

30. Results Elastin  Spun at: Distance:10 cm from the collector plate Voltage:16 kV Flow rate: 14.4 mL/h  These solutions also both contained: sodium chloride (increase conductivity) PEO (increase viscosity-5% weight concentration)

31. Results Elastin

32. Aluiji, A., Ferrero, F., Mazzuchetti, G., Tonin, C., Varesano, A., Vineis, C.(2008) Structure and properties of keratin/PEO blend nanofibers. European Polymer Journal. 44. 2465-2475. Awazu, K., Ishii, K., Kanai, T., Natio, Y., Yashihashi-Suzuki(2004). Matrix-assisted laser desorption/ionization of protein samples containing a denaturant at high concnetratin using a mid-infrared free electron laster (MIR-FEL). International Journal of Mass Spectrometry. 15. 49-46. Buttafoco, L., Dijkstra, P.J., Engbers-Buijtenhuijs, P., Feijen, J., Kolkman, N.G., Poot, A.A., Vermes, I.(2006). Electrospinning of collage and elastin for tissue engineering applications. Biomaterials. 27. 224-234 Bhardwaj, N., Kundu, S.C.(2009). Electrospinning: A fascinating fiber fabrication technique. Biotechnology Advances. 10.1016. Gosline, J.M., Guerette, P.A., Ortlepp, C.S., Savage, K.N.(1999). The mechanical design of spider silks: from fibroin sequence to mechanical function. The Journal of Experimental Biology. 202, 3295-3303

33. Thank you Q&A