Direct-Write of 3D Biomolecule Microstructures into Hydrogel Materials

1. Direct-Write of 3D Biomolecule Microstructures into Hydrogel Materials Stephanie Seidlits, Jason Shear, Christine Schmidt University of Texas at Austin

2. ECM Components 3D Architecture Alberts, MBOC, 4th ed. Bochaton-Piallat, Ophtamol Vis Sci, 2000 Engineered in vivo-like Scaffolds Submicron Features Spatial Control Esch, J. Neurosci, 1999 Teixeira, JCS, 2003

3. Neuronal Cell Body Extending Neurite 3D Hydrogel Support Crosslinked Protein Structures Scaffold design

4. Hyaluronic acid • Nonimmunogenic • Angiogenesis and --wound healing • Enzymatically biodegradable • Photocrosslinkable --------------hydrogels (Leach, 2003, Biotech Bioeng) • Not cell adhesive GMHA Reaction ring opening product hyaluronic acid glycidylmethacrylate transesterification product Adapted from Derouet, 2002, Eur Poly J

5. www.fsu.edu/microscopyprimer Multiphoton excitation • Advantages of MPE photocrosslinking: • Inherently 3D • Subcellular resolution • Complex geometries --possible • Heating effects minor ----and localized • Can fabricate---------------structures of natural -----materials www.cellscience.biorad.com

6. Resolution and biocompatibility of MPE photocrosslinking 1 µm Kaehr, PNAS, 2004

7. Crosslink protein structures directly into saturated GMHA hydrogels using multiphoton excitation Make GMHA hydrogels and soak in photosensitizer and protein solution Wash to remove unreacted solution MPE crosslinking in 3D

8. MPE crosslinking in 3D

9. BSA-FITC structures in Texas Red-GMHA hydrogels Pyramid base = 50 μm

10. Z=40 µm Z=20 µm 50 µm 50 µm Z=0 µm 50 µm 50 µm 50 µm 50 µm 3D protein structures in HA hydrogels Z=40 µm Z=20 µm Z=0 µm

11. Scanning Electron Microscopy 2 µm 200 nm 2 µm

12. F F F F + + + + + + + B B avidin avidin bsa Avidin-biotin functionalization Strategy 1: Strategy 2:

13. Strategy 1: Avidin structures Avidin BSA 150 µm 150 µm ~30 ms exposure time

14. 50 µm 50 µm Strategy 2: BSA structures modified with avidin BSA + Avidin-fluorescein BSA + unlabeled avidin ~3 ms exposure time

15. 50 µm MPE crosslinking within hydrogels provides: • Flexibility to incorporate multiple bioactive molecules in arbitrary patterns • Inherent 3D geometry and biomolecule presentation • Submicron sized features

16. Acknowledgements Principle Investigators: • Dr. Christine Schmidt • Dr. Jason Shear Undergraduates: • Rebecca Rosenberger • Kat McCarty • Jill Heisler Lab Members: • Curt Deister • Bryan Kaehr • Rex Nielson • Rest of Schmidt and Shear Lab Members Funding: NSF-IGERT NSF BES-0500969 and BES-021744 Welch Foundation

17. IGERT: Cellular and Molecular Imaging for Diagnostics and Therapeutics The University of Texas at Austin Novel Contrast Agent Development Novel Imaging and Spectroscopy System Development Diagnostic and Vital Imaging Spectroscopy Imaging to Monitor Therapeutics Integrative Graduate Education & Research Training Program Nicholas Peppas, Sc.D., Director Jennifer Brodbelt, Ph.D., Co-Principal InvestigatorChristine Schmidt, Ph.D., Co-Principal Investigator http://www.bme.utexas.edu/igert/

18. A) B) Figure 4. 3D MPE crosslinked BSA structures within hydrogels. A) Optical transmission images of 200 mg/mL BSA (2% BSA-FITC) photocrosslinked in a 2 wt% GMHA hydrogel with 6 mM FAD using a pulsed TiS laser at 740 nm. Each image was taken in the same xy plane and a different z plane (the first image is focused at the top of the pyramid and the last at the bottom). C) Confocal microscope image of 200 mg/mL BSA (2% BSA-FITC) crosslinked using 6 mM FAD within a 100 mg/mL PEGDA hydrogel. Images are from different planes spanning a total depth of 80 m. Note: concentrations given are those in the uncrosslinked solution. Scale bars = 20 µm.

21. Crosslinking with FAD • Two Mechanisms: • Type I: triplet state abstract hydrogen to create radicals • Type II: creates singlet oxygen (Spikes 1999 Photochem Photobio) • Photocrosslinkable amino acids: Tyr, Trp, His, Cys…

22. Avidin-Biotin - Au NP

23. Advantages of MPE Polymerization