© Copyright 2021 
 
Christopher R. Fellin 
  
 Stimuli-Responsive Triblock Copolymer Hydrogels for 
Extrusion-Based Additive Manufacturing 
 
 
 
 
Christopher Fellin 
 
 
 
A dissertation 
 
submitted in partial fulfillment of the 
 
requirements for the degree of 
 
 
 
Doctor of Philosophy 
 
 
 
University of Washington 
 
2021 
 
 
 
Reading Committee: 
 
Alshakim Nelson, Chair 
 
Forrest Michael 
 
Christine Luscombe 
 
 
 
Program Authorized to Offer Degree: 
 
Department of Chemistry 
 
 
 
 University of Washington 
 
 
 
 
Abstract 
 
 
 
 
 Stimuli-Responsive Triblock Copolymer Hydrogels for 
Extrusion-Based Additive Manufacturing 
 
 
 
 
Christopher Fellin 
 
 
 
Chair of the Supervisory Committee: 
Dr. Alshakim Nelson 
Department of Chemistry 
 
 
 
Additive manufacturing (AM) is a rapidly expanding field that has revolutionized a number of 
diverse fields including medicine, construction, aerospace, and robotics. The hardware of AM has 
seen a dramatic improvement over the years, and there currently exists a vast array of technologies 
with unique advantages for different applications. Extrusion-based printers, specifically direct-ink 
write (DIW), are one such AM technology that has seen extensive use due to its relatively low cost 
and fast print speeds. Despite the rapid growth of AM, research has mostly focused on the 
 adaptation of existing materials for AM applications- leaving material development lagging 
behind. This has provided an opportunity for materials scientists and engineers to develop novel 
materials specifically designed for AM applications.  Hydrogels are one class of materials that has 
garnered significant attention for DIW AM. These soft materials mimic the extracellular matrices 
of cellular environments and have seen extensive use as cell laden inks for 3D bioprinting.  Stimuli-
responsive hydrogels respond to environmental cues, enabling both effective printing and 
providing post-print functionality. This thesis focuses on the development of a multi-stimuli-
responsive  hydrogel platform based on poly(alkyl glycidyl ether) triblock copolymers. Chapter 1 
includes an overview of the field of AM and stimuli-responsive hydrogels for extrusion-based AM. 
Chapter 2 describes the development of the initial temperature- and shear- responsive triblock 
copolymer hydrogel platform. Chapter 3 further expands this platform through the photo-chemical 
crosslinking of the hydrogel network and its implementation for the additive manufacturing of 
catalytically active living materials (AMCALM). Lastly, Chapter 4 continues to advance the 
functionality the platform through the addition of thiol-reactive monomers for post-
functionalization of the hydrogel material. 
 
 
i 
 
 
TABLE OF CONTENTS 
 
List of Figures ................................................................................................................................ vi 
List of Tables ................................................................................................................................. xi 
List of Schemes ............................................................................................................................. xii 
CHAPTER 1: Introduction ......................................................................................................... 16 
1.1 Introduction to Additive Manufacturing ....................................................................... 16 
1.2 Hydrogel Inks for DIW AM ......................................................................................... 17 
1.3 Triblock Copolymer Hydrogels for DIW AM .............................................................. 18 
1.4 Developing Stimuli-Responsive Functional Hydrogels for DIW AM ......................... 23 
1.5 Conclusion .................................................................................................................... 24 
1.6 References ..................................................................................................................... 26 
CHAPTER 2: Tunable Temperature- and Shear-Responsive Hydrogels Based on Poly(alkyl 
glycidyl ether)s ............................................................................................................................ 34 
2.1 Abstract ............................................................................................................................... 34 
2.2 Introduction ......................................................................................................................... 34 
2.3 Materials and Methods ........................................................................................................ 37 
2.3.1 Materials ...................................................................................................................... 37 
2.3.2 Homopolymer Synthesis ............................................................................................... 37 
2.3.3 Triblock Copolymer Synthesis ..................................................................................... 38 
2.3.4 UV-Vis Spectroscopy.................................................................................................... 39 
ii 
 
2.3.5 Rheological Measurements .......................................................................................... 39 
2.3.6 Direct-Write 3D Printing of Hydrogels ....................................................................... 40 
2.3.7 Cell Viability Study ...................................................................................................... 40 
2.4 Results and Discussions ...................................................................................................... 41 
2.4.1 Synthesis of Homopolymers and Characterization of LCST Response........................ 41 
2.4.2 Synthesis of Triblock Copolymers and Characterization of LCST Response .............. 43 
2.4.3 Triblock Copolymer Phase Diagrams.......................................................................... 44 
2.4.4 Triblock Copolymer Hydrogel Rheology ..................................................................... 46 
2.4.5 Direct-Write 3D Printing ............................................................................................. 48 
2.4.6 Cell Viability ................................................................................................................ 48 
2.5 Conclusion .......................................................................................................................... 49 
2.6 References ........................................................................................................................... 50 
CHAPTER 3: Poly(alkyl glycidyl ether) Hydrogels for Harnessing the Bioactivity of 
Engineered Microbes .................................................................................................................. 64 
3.1 Abstract ............................................................................................................................... 64 
3.2 Introduction ......................................................................................................................... 65 
3.3 Materials and Methods ........................................................................................................ 67 
3.3.1 Materials ...................................................................................................................... 67 
3.3.2 Yeast Strains ................................................................................................................. 68 
3.3.3 Synthesis of Polymer 1 ................................................................................................. 69 
3.3.4 Synthesis of Polymer 2 ................................................................................................. 69 
iii 
 
3.3.5 Preparation of Synthetic Complete Media ................................................................... 70 
3.3.6 Preparation of Hydrogel Solution................................................................................ 70 
3.3.7 Preparation of Yeast-Laden Hydrogel Ink ................................................................... 71 
3.3.8 Rheometrical Characterization .................................................................................... 71 
3.3.9 Additive Manufacturing (Direct-Write 3D Printing) of a Cuboidal-Lattice................ 72 
3.3.10 Microscopy and Imaging ........................................................................................... 72 
3.3.11 Cell Viability Assay .................................................................................................... 73 
3.3.12 α-Factor Production .................................................................................................. 73 
3.3.13 α-Factor Detection and Quantification ..................................................................... 74 
3.4 Results and Discussions ...................................................................................................... 76 
3.4.1 Synthesis and Functionalization of the Triblock Copolymer ....................................... 76 
3.4.2 Rheology of the Functionalized Triblock Copolymer Hydrogel .................................. 77 
3.4.3 Direct-Write 3D Printing of Triblock Copolymer Dimethacrylate Hydrogels ............ 78 
3.4.4 Incorporation of Yeast Cells and Cell Viability ........................................................... 78 
3.4.5 α-Factor Production with 3D Printed AMCALMs ...................................................... 79 
3.5 Conclusion .......................................................................................................................... 80 
3.6 References ........................................................................................................................... 82 
CHAPTER 4: The Post-Functionalization of Multi-Stimuli-Responsive Hydrogels for 
Direct-Ink Write Additive Manufacturing ............................................................................... 92 
4.1 Abstract ............................................................................................................................... 92 
4.2 Introduction ......................................................................................................................... 93 
iv 
 
4.3 Materials and Methods ........................................................................................................ 95 
4.3.1 Materials .................................................................................................................. 95 
4.3.2 Synthesis of HEPDS ................................................................................................. 96 
4.3.3 Synthesis of PDS-UM ............................................................................................... 96 
4.3.4 Preparation of PGE-MA/PDS-UM Hydrogel Solution............................................ 97 
4.3.5 Hydrogel Sample Preparation ................................................................................. 97 
4.3.6 PGE-MA Swelling Studies ....................................................................................... 97 
4.3.7 2-Pyridothione Calibration ...................................................................................... 98 
4.3.8 2-Pyridothione Release Studies ............................................................................... 98 
4.3.9. Direct-write 3D Printing of PGE-MA/PDS-UM Hydrogels ................................... 99 
4.4 Results and Discussion ....................................................................................................... 99 
4.4.1. Temperature Dependent Hydrogel Swelling ........................................................... 99 
4.4.2. Formulation and Rheological Characterization of PGE-MA/PDS-UM Hydrogels
......................................................................................................................................... 100 
4.4.3. Reactivity of PGE-MA/PDS-UM Hydrogels Toward Thiols ................................ 102 
4.4.4. Thiol Containing Molecular Library .................................................................... 103 
4.4.4. 3D Printing and Post-Functionalization of PGE-MA/PDS-UM Hydrogels ......... 105 
4.5 Conclusion ........................................................................................................................ 105 
4.5 References ......................................................................................................................... 107 
Appendix A ................................................................................................................................ 117 
v 
 
Tunable Temperature- and Shear-Responsive Hydrogels Based on Poly(alkyl glycidyl 
Ether)s ................................................................................................................................. 117 
Appendix B ................................................................................................................................ 138 
Poly(alkyl glycidyl ether) Hydrogels for Harnessing the Bioactivity of Engineered Microbes
 ............................................................................................................................................. 138 
Appendix C ................................................................................................................................ 143 
The Post-Functionalization of Multi-Stimuli-Responsive Hydrogels for Direct-Ink Write 
Additive Manufacturing ...................................................................................................... 143 
 
vi 
 
LIST OF FIGURES 
Figure 1.1 Visualization of the seven 3D printing methods according to the ASTM……………..33 
Figure 1.2 Cartoon representation of the fully hydrated coil to collapsed globule transition of 
amphiphilic homopolymers in dilute solutions (1 wt%) as the ΔGmix becomes positive with 
increasing temperature…………………………………………………………………………...33 
Figure 1.3 Pyridyl disulfide methacrylate was incorporated into a hydrogel network by 
copolymerization with poly(ethylene oxide) methyl ether methacrylate. The thiol-reactive 
hydrogel platform was functionalized with biotin and RGD-SH to selectively “catch” Extravidin 
and attach HUVEC cells, followed by release with DTT…………………………………………34 
Figure 2.1 Graphical representations of (a) the temperature-dependent equilibrium between 
unimers (low temperature) and a flower micelle network (high temperature) and (b) the shear 
stress-induced breaking of physical crosslinks for the poly(alkyl glycidyl ether)-block-
poly(ethylene oxide)-block-poly(alkyl glycidyl ether) triblock copolymer platform. Representative 
photographs that demonstrate the macroscale (c) thermo-responsive sol (5 °C) to gel (25 °C) 
transition and (d) extrusion activated shear-responsive properties of a 20 wt% hydrogel of polymer 
9 (0.41 mm inner diameter nozzle) ................................................................................................57 
Figure 2.2 Temperature-concentration phase diagrams summarizing the solution and hydrogel 
states of triblock copolymers of the same total block length containing (a) 5.0 iPGE units (polymer 
5), (b) 7.9 iPGE units (polymer 8), (c) 10.2 iPGE units (polymer 6), and (d) 15.2 iPGE units 
(polymer 3). The green/blue/red shaded areas indicate formulations existing as transparent/opaque 
liquids (□/■), viscous solutions ( / ), and gels (○/●), respectively. Transparent formulations are 
clear with no turbidity. Opaque formulations are turbid and optically cloudy. Their physical states 
were determined using the vial inversion method .........................................................................61 
Figure 2.3 Temperature-concentration phase diagrams summarizing the solution and hydrogel 
states of triblock copolymers with increasing DP (a) 12.8 (polymer 7), (b) 16.1 (polymer 8), (c) 
19.7 (polymer 9), and (d) 25.3 (polymer 10). The green/blue/red shaded areas indicate 
transparent/opaque liquids (□/■), viscous solutions ( / ), and gels (○/●), respectively. 
Transparent formulations are clear with no turbidity. Opaque formulations are turbid and optically 
cloudy. Their physical states were determined using the vial inversion method ...........................62 
Figure 2.4 Rheological experiments (a) Oscillatory stress experiment indicating yield stress. Yield 
stress values = 569, 1200, and 1252 for 20 wt% solutions of polymer 9, 10, and 3, respectively. 
(b) Dynamic oscillatory temperature ramp displaying elastic (G’, filled) and viscous (G’’, open) 
moduli. Tgel = 18.72 °C (c) Viscosity vs shear rate experiment depicting shear-thinning behavior 
by a decrease in viscosity with increasing shear rate and (d) Cyclic strain experiment 
demonstrating rapid recovery of hydrogel elastic modulus (black circles) from periods of high 
(100%) to low (1%) oscillatory strain (red circles). Arrows indicate reference axis; elastic/viscous 
moduli (left axis) and oscillatory strain (right axis).......................................................................63 
vii 
 
Figure 2.5 3D printed structures of a 15-layer (a) University of Washington initials and (b-c) 
benzene ring with a 0.41 mm inner diameter nozzle at 8.0 mm s-1. This structure was printed with 
a formulation of polymer 9 at 20 wt% using a pneumatic direct-write 3D printer (scale bars: 1 cm)
........................................................................................................................................................64 
Figure 2.6 Evaluation of a LIVE/DEAD® assay performed on three polymer 9 hydrogels at 
different concentrations (11.25, 15, 20 wt%). (a) Composite channel confocal microscopy image 
of encapsulated HeLa cells (11.25 wt%) (scale bar: 1000 µm). ....................................................64 
Figure 3.1 A graphical overview of the three key stimuli responses of polymer 2 hydrogels 
necessary for AMCALM applications. (a) The temperature responsive feature of the hydrogels 
enabled facile loading of the hydrogel material at 5 °C. (b) The shear-stress response facilitated 
the formation of complex three-dimensional objects at 21 °C. (c) UV-light (365 nm) initiated the 
polymerization of the methacrylate end-groups and chemical crosslinking of the polymer 2 
hydrogel. (d) The chemical structure of polymer 2. The letter designations (z = 6.4, y = 7.4, x = 
182) refer to the average number of isopropyl glycidyl ether, ethyl glycidyl ether, and ethylene 
oxide repeat units, respectively ......................................................................................................88 
Figure 3.2 Rheometrical experiments for a 20 wt% formulation of polymer 2. (a) Dynamic 
oscillatory temperature ramp displaying elastic (G’, filled) and viscous (G’’, open) moduli. Tgel = 
15.61 °C. (b) Cyclic strain experiment demonstrating rapid recovery of hydrogel elastic modulus 
(black circles) from periods of high (100%) to low (1%) oscillatory strain (red circles). Arrows 
indicate reference axis; elastic/viscous moduli (left axis) and oscillatory strain (right axis) ........89 
Figure 3.3 (a) A rheological UV-cure experiment using a 20 mm parallel plate geometry. The 
hydrogel was equilibrated for 120 s before being subjected to 5 mW cm-2 of 365 nm UV light for 
420 s at a constant strain (1%) and frequency (1 Hz). (b-c) A 3D printed, proof of concept cuboid 
structure (1.9 cm by 1.9 cm by 1.2 cm). This structure was printed from a pneumatic direct write 
3D printer at 5 mm s-1 and 20 psi using a 0.41 mm inner diameter nozzle (scale bar: 1 cm) ......90 
Figure 3.4 (a) A sample composite image of live cells (green channel) and dead cells (red channel) 
after 7 days of incubation (scale bar: 200 µm). (b) Side and (c) top-down view of the 3D-printed, 
yeast-laden hydrogel after two rounds of α-factor synthesis in SC media (scale bar 1 cm) ..........90 
Figure 3.5 Immobilized yeast within the hydrogel matrix were incubated in SC media for 72 h at 
30 °C with automated shaker agitation. The media was collected at the end of the incubation period 
and exposed to the receiver strain in a separate tube, and the resulting fluorescence was measured. 
The cuboidal lattice was reused in a subsequent incubation with fresh media to produce an 
additional batch of α-factor ............................................................................................................91 
Figure 3.6 Fluorescence values obtained from the quantification of α-factor produced from the 
wild type (control) AMCALMs and the secreting strain AMCALMs. Round 1 indicates the first 
incubation period of 72 h, while Round 2 indicates the reemployment of the same printed materials 
in a second, subsequent 72 h batch reaction in fresh SC media ....................................................92 
viii 
 
Figure 4.1 A graphical representation of the LCST-driven temperature dependent swelling 
behavior of PGE-MA 
hydrogels…………………………………………………………………………………..……114 
 
Figure 4.2 (a) Swelling ratio (%) vs Time (h) for PGE-MA/PDS-UM samples at 5 (blue 
squares), 25 (black triangles), and 37 °C (red circles). (b) Demonstration of reversible swelling 
from 5 °C to 37 °C over ten days………………………………………………………………114 
 
Figure 4.3 (a) Absorbance profiles and (b) calibration curve of 2-pyridothione (0.1-0.5 mM) in 
deionized water…………………………………………………………………………………115 
Figure 4.4 Percent release of 2-pyridothione (%) vs time (h) for (a) as-cured and (b) preswollen 
PGE-MA/PDS-UM samples at 5 (blue squares), 25 (black triangles), and 37 °C (red circles)..115 
Figure 4.5 A 3D printed robot figurine was printed using a custom made, pneumatic DIW 
printer with a 0.41mm inner diameter nozzle at 5.0 mm/s. The fluorescent tag FITC-SH was 
conjugated to the object and the figurine was swelled at (a) 5°C and (b) 37 °C to demonstrate the 
thiol-reactivity and temperature dependent swelling of the PGE-MA/PDS-UM system (scale 
bars: 1cm)……………………………………………………………………………….………117 
Figure A1. Homopolymer Cloud Point Measurements…………………………………………124 
Figure A2. Tcp of (a) Polymer 1, (b) Polymer 2, (c) Polymer 3, and (d) Polymer 4……………124 
Figure A3. Tcp of (a) Polymer 5, (b) Polymer 6, (c) Polymer 7, (d) Polymer 8, (e) Polymer 9, and 
(f) Polymer 10……………………………………………………………………………..……125 
Figure A4. Allyl glycidyl ether triblock brittle hydrogel ............................................................126 
Figure A5. Cell Viability data for 11.25, 15, and 20 wt% polymer 9 hydrogels in supplemented 
DMEM .........................................................................................................................................126 
Figure A6. Confocal microscopy images of encapsulated HeLa cells in a 11.25 wt% formulation 
of polymer 9 in supplemented DMEM (a) green fluorescence channel indicating live cells (b) red 
fluorescence channel indicating dead cells (c) composite channel (scale bar: 1000 µm) ...........126 
Figure A7. Confocal microscopy images of encapsulated HeLa cells in a 15.0 wt% formulation of 
polymer 9 in supplemented DMEM (a) green fluorescence channel indicating live cells (b) red 
fluorescence channel indicating dead cells (c) composite channel (scale bar: 1000 µm) ...........127 
Figure A8. Confocal microscopy images of encapsulated HeLa cells in a 20.0 wt% formulation of 
polymer 9 in supplemented DMEM (a) green fluorescence channel indicating live cells (b) red 
fluorescence channel indicating dead cells (c) composite channel (scale bar: 1000 µm) ...........127 
Figure A9. 1H NMR of n-propyl glycidyl ether ..........................................................................128 
Figure A10. 1H NMR of poly(methyl glycidyl ether)2.1k ............................................................128 
Figure A11. 1H NMR of poly(ethyl glycidyl ether)3.0k ...............................................................129 
ix 
 
Figure A12. 1H NMR of poly(ethyl glycidyl ether)9.5k ...............................................................129 
Figure A13. 1H NMR of poly(ethyl glycidyl ether)24.5k ..............................................................130 
Figure A14. 1H NMR of poly(allyl glycidyl ether)3.3k ................................................................130 
Figure A15. 1H NMR of poly(allyl glycidyl ether)5.8k ................................................................131 
Figure A16. 1H NMR of poly(allyl glycidyl ether)11.7k ...............................................................131 
Figure A17. 1H NMR of poly(isopropyl glycidyl ether)4.0k ........................................................132 
Figure A18. 1H NMR of poly(isopropyl glycidyl ether)7.7k ........................................................132 
Figure A19. 1H NMR of poly(isopropyl glycidyl ether)16.0k .......................................................133 
Figure A20. 1H NMR of poly(n-propyl glycidyl ether)3.2k .........................................................133 
Figure A21. 1H NMR of poly(n-propyl glycidyl ether)8.0k .........................................................134 
Figure A22. 1H NMR of Polymer 1 ............................................................................................134 
Figure A23. 1H NMR of Polymer 2 ............................................................................................135 
Figure A24. 1H NMR of Polymer 3 ............................................................................................135 
Figure A25. 1H NMR of Polymer 4 ............................................................................................136 
Figure A26. 1H NMR of Polymer 5 ............................................................................................136 
Figure A27. 1H NMR of Polymer 6 ............................................................................................137 
Figure A28. 1H NMR of Polymer 7 ............................................................................................137 
Figure A29. 1H NMR of Polymer 8 ............................................................................................138 
Figure A30. 1H NMR of Polymer 9 ............................................................................................138 
Figure A31. 1H NMR of Polymer 10 ..........................................................................................139 
Figure B1. Graphical representation of the temperature induced sol-gel transition of polymer 2
......................................................................................................................................................140 
Figure B2-B3. Viscosity vs Shear Rate and Oscillatory Yield Stress experiments for a 20 wt % 
solution of Polymer 1 ...................................................................................................................140 
Figure B4. Confocal microscopy images of live/dead assay results at day 1 of incubation in SC 
media. The first column shows the dead cells (red channel), while the second column shows the 
live cells (green channel). The third column is an overlay of the live and dead cell channels ....141 
Figure B5. Confocal microscopy images of live/dead assay results at day 3 of incubation in SC 
media. The first column shows the dead cells (red channel), while the second column shows the 
live cells (green channel). The third column is an overlay of the live and dead cell channels. ...142 
x 
 
Figure B6. Confocal microscopy images of live/dead assay results at day 7 of incubation in SC 
media. The first column shows the dead cells (red channel), while the second column shows the 
live cells (green channel). The third column is an overlay of the live and dead cell channels. ...143 
Figure A7. 1H NMR of Polymer 1 ..............................................................................................144 
Figure B8. 1H NMR of Polymer 2 ..............................................................................................144 
Figure C1. Rheological characterization of PGE-MA/PDS-UM hydrogels. (a) Dynamic 
oscillatory temperature ramp displaying storage (G’, filled) and loss (G’’, open) moduli. (b) 
Viscosity vs shear rate experiment depicting shear-thinning behavior (c) Cyclic strain experiment 
demonstrating rapid recovery of hydrogel storage modulus (black circles) from periods of high 
(100%) to low (1%) oscillatory strain (red circles). (d) Dynamic oscillatory UV-Cure experiment 
demonstrating photochemical crosslinking upon exposure to 365 nm UV light. ........................149 
Figure C2. 1H NMR of HEPDS ..................................................................................................150 
Figure C3. 1H NMR of PDS-UM................................................................................................151 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
xi 
 
 
 
 
LIST OF TABLES 
Table 2.1 Characteristics of poly(alkyl glycidyl ether) homopolymers ........................................58 
Table 2.2 Characteristics of poly(alkyl glycidyl ether)-block-PEO-block-poly(alkyl glycidyl ether) 
triblock copolymers .......................................................................................................................60 
Table 4.1 Percent release of 2-pyridothione (%) at different temperatures (5, 25, 37 °C), swelling 
statuses (as cure/preswollen), and time points (24/96 h)………………………………………...116 
Table 4.2 Conjugation of thiol-containing molecules to PGE-MA/PDS-UM hydrogels. The 
percent release of 2-pyridothione was determined via UV-Vis spectroscopy at 24 and 96 h. The 
pH (3, 5, and 7.4) and temperature (5, 25, 37 °C) were modified to suppress auto-oxidation of the 
conjugating molecule and the 2-pyridothione leaving group……………………………………116     
xii 
 
LIST OF SCHEMES 
Scheme 2.1 Synthesis of poly(alkyl glycidyl ether)s wherein R represents methyl, ethyl, allyl, 
isopropyl, or n-propyl groups ........................................................................................................57 
Scheme 2.2 Synthesis of ABA Triblock Copolymers, wherein R represents ethyl, allyl, and 
isopropyl groups.............................................................................................................................59 
Scheme 3.1 Synthesis of the ABA triblock copolymer (polymer 1) and functionalization with 
methacrylic anhydride (polymer 2). The letter designations (z = 6.4, y = 7.4, x = 182) refer to 
degree of polymerization for isopropyl glycidyl ether, ethyl glycidyl ether, and ethylene oxide, 
respectively ....................................................................................................................................89 
Scheme C1...................................................................................................................................148 
Scheme C2...................................................................................................................................148 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
xiii 
 
ACKNOWLEDGEMENTS 
 
First and foremost, I would also like to thank my graduate advisor, Dr. Alshakim Nelson. 
You have been a pillar of support and encouragement throughout my years as a graduate student. 
Your guidance has helped mold me into the scientist I am today. You allowed me to explore my 
own interests and intuitions, while always providing the necessary grounding to bring me back 
down to earth. I could not have asked for a better mentor and boss- supportive without being 
overbearing and hands-off without being unavailable. When the time comes for me to assume the 
role of mentor, I will strive for the best with you in mind. 
Additionally, I could not have done this without the supportive collaboration of the entire 
Nelson lab. Dylan and Ryan, you were integral to my transition into graduate school, and I 
always appreciated your patience and willingness to answer any and all questions that popped 
into my head. Trevor and Amrita, you are the best of lab friends, and I could not have made it 
through without you. Jenn you have become such a good friend over the last few months, and I 
know you will do great things. And Pat- we came into grad school together, we lived together, 
and now we leave together. Cheers. 
I would like to thank my committee members Dr. Forrest Michael, Dr. Christine Luscombe, 
and Dr. Cole DeForest for their support as well as collaborators Dr. Eric Seibel, Dr. Len Nelson, 
and Shawn Swanson. Working with the UnTape team has been a highlight of my graduate school 
career and I am blessed to have had the opportunity.  
To all my amazing friends: none of this would be possible without you. Some of you I have 
known for years, others I have only met recently. Ben, Andrew, Trevor, Camille, Ashley, Sarah, 
xiv 
 
Azra, Maire, Claire, Jarred, all of my friends from home and many many more. All of you helped 
make my graduate school experience one to remember.  Whether it’s late-night chemistry 
socials, learning how to salsa dance, or arguing about fantasy football at 2 in the morning, I will 
always cherish our experiences together. And a special, special shoutout to Cece Martin. You 
have made the past six months truly unforgettable.  
Last but definitely not least, I would like to thank my family. Mom, Dad, Bryan, Eric, and 
Amanda, this would not be possible without your constant friendship and support. You are the 
best family I could ask for and I would not be the man I am today without you. Thanks for 
always giving me a safe place to come home to. Here’s to more Fellin family vacations, Harry 
Potter marathons, and ice cream deserts.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
xv 
 
 
 
CHAPTER 1: INTRODUCTION 
1.1 INTRODUCTION TO ADDITIVE MANUFACTURING  
 Additive manufacturing (AM), also known as 3D printing, is a relatively recent 
manufacturing technique that utilizes a layer-by-layer process to build complex three-dimensional 
objects.1–3 This is in contrast to more traditional subtractive processes where material is removed 
from a bulk object until the final shape is obtained. AM is advantageous in a number of ways 
including the ability to rapidly manufacture custom designed prototypes,4 lower costs and minimal 
waste,5 and enhanced object complexity.6  
There are three main components to additive manufacturing: Software, hardware, and 
materials. Software programs such as OpenSCAD and Fusion 360 facilitate the development of 
computer-aided design (CAD) files that create the virtual image of the final printed object. From 
here, the CAD files are transferred to a printer (the hardware) and the additive manufacturing 
process begins. The ASTM Standard has identified seven main AM printer technologies: binder 
jetting, directed energy deposition, material extrusion, material jetting, powder bed fusion, sheet 
lamination, and vat photopolymerization (Figure 1.1).7,8 Each technology is unique and suitable 
for their own targeted applications. Two of the most common printing techniques for polymeric 
soft materials are material extrusion and vat photopolymerization. Extrusion-based printers such 
as Fused-Deposition Modeling (FDM) and Direct-Ink Write (DIW) eject material through a nozzle 
and gradually build the object through layer-by-layer deposition. Vat-photopolymerization 
methods such as Stereolithography (SLA) and Digital Light Processing (DLP) utilize a light source 
to selectively cure a photopolymerizable resin until the final print is obtained. Extrusion-based 
printers are typically cheaper, less time consuming, and offer straightforward opportunities for 
17 
 
multi-material prints.9 Meanwhile, vat-photopolymerization methods can achieve much better 
resolution prints but are typically more expensive and time consuming than their extrusion-based 
counterparts.10,11 AM hardware is an incredibly innovative field that continues to push the 
boundaries of AM implementation into ever expanding commercial and academic applications. 
The last component of AM is the material used to construct the printed object. Historically, 
engineers have developed inks by applying existing materials to the field of AM. Engineers have 
adapted all sorts of common materials such as metals, ceramics, and plastics towards this end.12 
Poly(lactic acid), or PLA, is an excellent example of this phenomenon- first discovered in 1932 
and commonly used to manufacture plastic films, bottles, and medical devices, PLA has become 
one of the most common materials for FDM printing due to its relatively low melting point (130-
180 °C) and ease of printing.13 While this was an adequate approach during the early days of AM, 
it no longer serves as a suitable model for the development of novel AM inks because many 
existing materials do not meet the unique rheological requirements necessary for effective 
printing.9 Over the past few decades, research has begun to specialize in the development of 
materials explicitly designed for AM applications. 
1.2 HYDROGEL INKS FOR DIW AM  
Hydrogels— three  dimensional networks of macromolecules held together by chemical or 
physical crosslinks— are one class of material that has garnered significant research interest for 
AM applications. These materials have carved out a niche role as cell-laden inks in 3D bioprinting 
due to their inherently high water content and their ability to replicate some of the chemical and 
physical attributes of extracellular matrices.14–16 This has led to groundbreaking advancements in 
the fields of tissue engineering17–22 and immobilized-cell bioreactors.23–25 
18 
 
The viscoelastic nature of hydrogel materials makes them excellent candidates for 
extrusion-based AM. For DIW methods, researchers have identified three key viscoelastic 
parameters that help describe the relative printability of hydrogel inks; viscosity (η),26 yield stress 
(σ),27–30 and tan δ.31  Ideally, hydrogel inks will possess some form of variable viscosity in order 
to properly eject from the printhead nozzle.32,33 This requirement is typically met via shear-
thinning behavior wherein the viscosity of the material decreases as shear force increases. These 
materials are proposed to undergo a “plug-flow” extrusion as the material travels through the high 
shear environment of the printhead nozzle.34,35  
Many hydrogel inks are derived from naturally occurring biopolymers such as alginate,36 
gelatin,37 and chitosan,38 among others. These materials are advantageous insofar as they are 
biocompatible, chemically versatile, and inherently functional.39 However, most biologically 
derived hydrogels are quite difficult to print and face significant engineering hurdles in order to 
effectively achieve proper print conditions. Some may require a heating jacket or a cooling bed or 
must be crosslinked immediately after printing each layer in order to avoid deformation.40–43 
Others require the combination of multiple materials in order to achieve suitable printing 
properties.44–46 Synthetic polymers on the other hand, can be specifically designed for AM due to 
their inherent compositional and architectural control. 
1.3 TRIBLOCK COPOLYMER HYDROGELS FOR DIW AM 
 Our group is interested in developing synthetic triblock copolymer hydrogels for DIW AM. 
Triblock copolymers are an advantageous platform for hydrogel inks because they can be 
synthetically designed to exhibit two key stimuli-responsive features necessary for efficient 
extrusion-based AM: a sol-gel temperature response and shear-thinning behavior. A sol-gel 
temperature response— wherein the material exists as a non-viscous solution at low temperatures 
19 
 
(5 °C) but returns to its gel morphology at elevated temperatures (25 °C)— allows for 
homogenization of additives such as cells or drugs and facile loading of the hydrogel ink in the 
liquid state. Meanwhile, shear-thinning behavior facilitates the extrusion of the material as rod-
like filaments and the formation of 3D printed objects. 
One of the most utilized hydrogels for DIW methods is an ABA triblock copolymer 
composed of a central, temperature-responsive poly(propylene oxide) (PPO) ‘B’ block and two 
flanking, hydrophilic poly(ethylene oxide) (PEO) ‘A’ blocks known as Pluronic F127. The 
hydrophobic nature of the PPO block at ambient temperatures leads to aggregation of the polymer 
in order to reduce the surface area of the PPO block exposed to the aqueous environment.47–49 
Polymers of this compositional architecture, i.e. central temperature-responsive and flanking 
hydrophilic blocks, create spherical micelles.50,51 As the concentration of the polymer increases, 
the material transitions from a dilute solution of individual micelles into a packed hydrogel 
network of micelles, held together by weak van der waals forces in a body-centered cubic 
arrangement. Because these micelles are only held together by physical, transient crosslinks, the 
material will yield under stress as the network becomes temporarily disrupted.48,49,52 This manifests 
itself at the macroscopic level as a shear-thinning behavior. Under ambient conditions, the material 
exists as a free-standing gel, but upon exposure to shear force the viscosity of the material drops 
and it begins to flow. 
 Pluronic F127 hydrogels also demonstrate a sol-gel temperature response. This behavior is 
governed by a phenomenon unique to amphiphilic polymers known as a lower critical solution 
temperature, or LCST. Generally, the solubility of a polymer in aqueous solutions is determined 
by the free energy of mixing, or ΔGmix, as seen in equation 1.1. 
 
20 
 
𝐄𝐪𝐮𝐚𝐭𝐢𝐨𝐧 𝟏. 𝟏      Δ𝐺𝑚𝑖𝑥 =  Δ𝐻𝑚𝑖𝑥 − 𝑇Δ𝑆𝑚𝑖𝑥  
 
The hydrophilic regions of the polymer enthalpically favor a mixed state (negative ΔHmix)  
due to hydrogen bonding between these domains and water molecules. At the same time, it is 
entropically unfavorable (negative ΔSmix) for water to solvate the PPO block due to the 
hydrophobic effect.53–55 This balance between an enthalpically favored mixed state, and an 
entropically favored demixed state, leads to a temperature dependent solubility. At low 
temperatures, the enthalpic forces control the system, the  ΔGmix is negative, and the polymer is 
fully solvated. As temperature increases, the positive total entropy change (-TΔSmix) dominates the 
system, ΔGmix becomes positive, and phase separation occurs.56,57 This can be observed 
macroscopically for F127 by a change in the viscoelastic properties of the material with 
temperature. At elevated temperatures (25-37 °C), the polymer chains aggregate due to the LCST-
like behavior of the polymer and the materials exists in the hydrogel state. However, upon exposure 
to low temperature environments (5 °C), the material assumes a liquid morphology as solvation of 
the polymer becomes favorable.  
 Wang and coworkers developed a similar temperature- and shear- responsive triblock 
polymer hydrogel by utilizing the LCST behavior of poly(N-isopropyl acrylamide)- or PNIPAm.58 
Compared to F127, this polymer possesses the inverse compositional architecture; temperature 
responsive (PNIPAm) ‘A’ blocks and a central hydrophilic (PEO) ‘B’ block. Because the 
temperature responsive blocks are located at the ends of the polymer chains, they self-assemble to 
form flower-like micelles. This architecture leads to a number of different macro and nanoscale 
properties. Instead of relying solely on micelle packing to induce hydrogel formation, these 
polymers bridge individual micelles and require pull-out of the polymer chains to disrupt the 
21 
 
hydrogel network, leading to differences in viscoelasticity and gel formation.50,51 Additionally, 
this architectural design positions the ends of the polymer in the central, hydrophobic segment of 
the micelle. This is contrary to Pluronic F127 micelles, where the chain ends are located on the 
hydrophilic portion of the polymer and are exposed to aqueous environments. Wang and 
coworkers took advantage of this property by attaching the hydrogen bonding moiety ureido 
pyrimidinone (UPy) to the ends of the polymer chains. These polymers self-assemble upon an 
increase in temperature as the PNIPAm chains dehydrate, creating micelles and a hydrophobic 
microenvironment for the UPy groups to hydrogen bond together. Thus, the hydrogel is 
crosslinked both by the hydrophobic association of the PNIPAm chains and by dimerization of the 
UPy moieties induced by hydrogen bonding. This triblock copolymer hydrogel proved to be 
extrudable (shear-thinning), self-healable, thermo-reversible, and injectable.  
  Poly(alkyl glycidyl ethers) are another class of temperature-responsive polymers that have 
recently been incorporated into triblock copolymer hydrogels. Watanabe and coworkers first 
investigated the LCST behavior of various poly(alkyl glycidyl ether) homopolymers.59 Methyl, 
ethyl, and ethoxy ethyl glycidyl ether monomers were polymerized using anionic ring opening 
polymerization (AROP) methods and the LCST of the resulting polymers were determined via 
cloud point measurement. In dilute solutions (1 wt%) and low temperatures, amphiphilic 
homopolymers exist as fully solvated coils. As the temperature increases and the ΔGmix becomes 
positive, the polymer chains phase separate completely into hydrophobic globules (Figure 
1.2).7,53,55,56  
This phase separation behavior can be observed and quantified as the cloud point 
temperature (Tcp) using UV-Vis spectroscopy to monitor a change in absorbance values. Watanabe 
and coworkers correlated increasing Tcp with a decrease in hydrophobicity of the polymer side 
22 
 
chains i.e. poly(methyl glycidyl ether) > poly(ethoxy ethyl glycidyl ether) > poly(ethyl glycidyl 
ether). Nelson and coworkers exploited the temperature dependent behavior of poly(alkyl glycidyl 
ether)s to develop a novel triblock copolymer platform for DIW AM.60 Similar to the design by 
Wang and coworkers, these triblocks contained a central PEO hydrophilic block, and two 
temperature-responsive poly(isopropyl glycidyl ether) ‘A’ blocks. The resulting triblock 
copolymer was capable of forming thermo-reversible, shear-thinning hydrogels that were utilized 
as inks for DIW AM. 
 The temperature- and shear- thinning response of these triblock copolymer hydrogels make 
them ideal inks for DIW AM; the sol-gel temperature response allows for facile loading and the 
dispersion of additives in the liquid state while the shear-thinning response facilitates the formation 
of hydrogel filaments. However, the same property that enable these behaviors, i.e. the physical 
crosslinking of polymer chains, make them poor hydrogels for many post-print applications. The 
shear response that facilitates extrusion also leaves the printed constructs vulnerable to 
deformation, with objects often unable to withstand physical touch. Additionally, these polymers 
will tend to become diluted over time in aqueous media, leading to dissolution of the hydrogel 
over short periods of time. This leaves the hydrogel unsuitable for biomedical applications such as 
tissue engineering, where the printed construct must be submerged in media in order to sustain cell 
growth and proliferation.  
 With this in mind, Nelson and coworkers developed a modified Pluronic F127 based 
hydrogel that enabled post-print curing of the printed structure in order to create a robust hydrogel 
able to withstand dissolution and deformation.29,61 In this work, Pluronic F127 was functionalized 
with methacrylate end groups- a common moiety used in free radical polymerizations. The 
resulting hydrogel retained both the temperature- and shear- responsive properties that enable 
23 
 
loading and extrusion of the material. Upon exposure to 365 nm UV-light, the modified polymer 
could undergo photo-chemical crosslinking induced by free radical polymerization of the 
methacrylate end groups. The physical, transient polymer crosslinks became permanent, chemical 
ones, leading to a much more robust material. This platform was utilized in a number of biomedical 
applications including the tissue engineering of 3D printed vascular tubes and the creation of 
immobilized cell bioreactors.62–64  
1.4 DEVELOPING STIMULI-RESPONSIVE FUNCTIONAL HYDROGELS FOR DIW AM 
Additive manufacturing also opens the door for the incorporation of functional materials 
as 3D printed constructs, although the field of triblock copolymer hydrogels has not been 
thoroughly investigated in this regard. These hydrogels respond to environmental stimuli such as 
temperature, pH, or small molecules after printing, enabling tunable control over behaviors such 
as swelling and drug release. A great example of this technique was demonstrated by Gracias and 
coworkers, who developed a dual-gel platform for the 4D printing of bioinspired constructs.65 By 
alternating segmental patterns of a temperature responsive PNIPAm hydrogel and an inert 
polyacrylamide (PAAM) hydrogel, this group was able to take advantage of the differential 
swelling behavior of each layer to create a range of multi-functional shape-changing structures. 
Meanwhile, Magdassi and coworkers developed a pH responsive hydrogel for DLP AM.66 The 
hydrogel was formulated using an acrylic acid monomer, a poly(ethylene glycol) diacrylate 
crosslinker, and TPO nanoparticles. Complex three-dimensional objects were printed and 
demonstrated pH responsive swelling. Sulforhodamine B, a pH independent fluorescent dye, was 
used to model a small molecule hydrophilic drug. These materials can be utilized to enable drug 
delivery at specific locations throughout the body where local pH is more basic. Recent work by 
Howon Lee uses a light responsive hydrogel and  multi-material microstereolithography to develop 
24 
 
cephalopod inspired artificial chromatophores.67 Polydopamine nanoparticles, a common 
photothermal agent, were incorporated into a PNIPAm hydrogel matrix to induce light-driven 
contraction as the temperature of the material heats above its LCST. This behavior yielding printed 
objects capable of individual and independent light sensing and actuation. Lastly, Sanyal and 
coworkers demonstrated an elegant technique for the modular “catch and release” of proteins and 
cells using a small molecule sensitive hydrogel.68 Pyridyl disulfide methacrylate was incorporated 
into a hydrogel network by copolymerization with poly(ethylene oxide) methyl ether methacrylate 
and a PEO based crosslinker. The pyridyl disulfide handle is capable of effectively and reversibly 
conjugating thiol-containing molecules and biomolecules, lending thiol reactive properties to the 
resulting hydrogel. Micropatterned hydrogel interfaces were fabricated and used to reversibly 
conjugate, or catch, biomolecules and cells onto their surfaces. These conjugated bonds could be 
subsequently cleaved, or released, in the presence of dithiothreitol (DTT). Successful conjugation 
of a biotin containing ligand enabled selective binding of the protein extravidin even in the 
presence of other proteins such as bovine serum albumin. By conjugating the cell binding protein 
RGD to the hydrogel surface, the group was able to successfully demonstrate facile cellular 
attachment with high levels of cell viability (Figure 1.3).  
1.5 CONCLUSION 
 The following chapters will discuss the design and development of a multi-stimuli 
responsive triblock copolymer hydrogel for DIW AM. Chapter 2 investigates how the structure of 
the triblock copolymer impacts the temperature- and shear- responsive properties of the hydrogel. 
Chapter 3 builds on this platform by modifying the polymer with reactive methacrylate chain ends, 
enabling post-print photo-chemical crosslinking and the adaptation of this platform for the additive 
manufacturing of catalytically active living materials. Finally, chapter 4 incorporates pyridyl 
25 
 
disulfide monomers into a multi-stimuli responsive hydrogel network for the post-
functionalization of 3D printed objects. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
26 
 
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(67)  Han, D.; Wang, Y.; Yang, C.; Lee, H. Multimaterial Printing for Cephalopod-Inspired 
Light-Responsive Artificial Chromatophores. ACS Appl. Mater. Interfaces 2021, 13 
(11), 12735–12745. 
 
(68)  Gevrek, T. N.; Cosar, M.; Aydin, D.; Kaga, E.; Arslan, M.; Sanyal, R.; Sanyal, A. 
Facile Fabrication of a Modular “Catch and Release” Hydrogel Interface: Harnessing 
Thiol-Disulfide Exchange for Reversible Protein Capture and Cell Attachment. ACS 
Appl. Mater. Interfaces 2018, 10 (17), 14399–14409. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
32 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.2. Cartoon representation of the fully hydrated coil to collapsed globule transition of 
amphiphilic homopolymers in dilute solutions (1 wt%) as the ΔGmix becomes positive with 
increasing temperature 
Figure 1.1. Visualization of the seven 3D printing methods according to the ASTM. 
33 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.3 Pyridyl disulfide methacrylate was incorporated into a hydrogel network by 
copolymerization with poly(ethylene oxide) methyl ether methacrylate. The thiol-reactive 
hydrogel platform was functionalized with biotin and RGD-SH to selectively “catch” 
Extravidin and attach HUVEC cells, followed by release with DTT.  
34 
 
CHAPTER 2: TUNABLE TEMPERATURE- AND SHEAR-
RESPONSIVE HYDROGELS BASED ON POLY(ALKYL 
GLYCIDYL ETHER)S 
2.1 ABSTRACT 
Herein, we describe the synthesis, characterization, and direct-write 3D printing of triblock 
copolymer hydrogels that have a tunable response to temperature and shear stress. In aqueous 
solutions, these polymers utilize the temperature-dependent self-association of poly(alkyl glycidyl 
ether) ‘A’ blocks and a central poly(ethylene oxide) (PEO) segment to create a physically cross-
linked three-dimensional network. The temperature response of these hydrogels was dependent 
upon composition, chain length, and concentration of the ‘A’ block in the copolymer. Rheological 
experiments confirmed the existence of sol-gel transitions and the shear-thinning behavior of the 
hydrogel. The temperature- and shear-responsive properties enabled direct-write 3D printing of 
complex objects with high fidelity. Hydrogel cytocompatibility was also confirmed by 
incorporating HeLa cells into select hydrogels resulting in high viabilities over 24 h. The tunable 
temperature response and innate shear-thinning properties of these hydrogels, coupled with 
encouraging cell viability results, present an attractive opportunity for additive manufacturing and 
tissue engineering applications. 
2.2 INTRODUCTION  
Hydrogels are water-swollen, three-dimensional networks of molecules or molecular 
assemblies that are useful in a range of applications that include drug delivery and tissue 
engineering.1–4 These hydrophilic networks are bound by chemical or physical cross-links between 
the molecules.5 Stimuli-responsive hydrogels belong to a unique class of materials that adapt to 
35 
 
environmental cues such as pH,6,7 temperature,8,9 light,10,11 and mechanical impetus.12–14 
Temperature and shear stress are examples of two important stimuli responses that enable effective 
drug delivery,15–17 additive manufacturing,18–20 and tissue engineering.21–23 A thermally reversible 
sol-gel response—wherein, the hydrogel liquefies upon cooling—allows for facile loading of the 
hydrogel ink and homogenous dispersion of heat-insensitive drugs or additives in the liquid state.24 
Furthermore, shear-thinning hydrogels facilitate the formation of 3D printed objects and protect 
encapsulated cells from the damaging shear stress of the syringe nozzle.25,26 These materials exist 
in the gel state under ambient conditions, but experience a drop in viscosity when activated by 
shear stress. The extruded filaments are proposed to undergo a “plug-flow” as the material travels 
through the nozzle.27  
Multi-stimuli responsive hydrogels can be formulated from aqueous solutions of amphiphilic 
ABA triblock copolymers comprised of at least one block that exhibits a temperature-dependent 
aqueous solubility known as a lower critical solution temperature (LCST). The Lewis group 
reported the 3D printing of one such hydrogel composed of commercially available Pluronic F127, 
which is a poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) triblock 
copolymer.28 It was demonstrated that the temperature response of Pluronic F127 hydrogels is 
driven by the LCST of the poly(propylene oxide) (PPO) ‘B’ block.29–31   
We have recently reported a similar ABA triblock copolymer that incorporates poly(isopropyl 
glycidyl ether) (PiPGE) ‘A’ blocks as hydrogel inks for 3D printing.32 These poly(isopropyl 
glycidyl ether)-block-poly(ethylene oxide)-block-poly(isopropyl glycidyl ether) triblock 
copolymers utilize the LCST of the PiPGE ‘A’ blocks to form hydrogels in aqueous solutions. 
These hydrogels exhibit a similar dual-stimuli response to temperature and shear stress as F127 
but require lower polymer concentrations. Based on this research, we envisioned a poly(alkyl 
36 
 
glycidyl ether)-block-poly(ethylene oxide)-block-poly(alkyl glycidyl ether) triblock copolymer 
platform with a tunable temperature response. The inherent architectural design of the triblock 
would retain the temperature- and shear- responsive properties demonstrated in our previous work 
but enable additional freedom to alter the temperature response through the incorporation of 
comonomers, adjustment of chain length, and alteration of polymer concentration. We theorize 
that these triblock copolymers exist as individually solvated unimers at low temperatures, but form 
networks of physically crosslinked flower micelles at higher temperatures as the solvent becomes 
increasingly unfavorable for the poly(alkyl glycidyl ether) ‘A’ blocks.33–36 The dynamic nature of 
the physical crosslinks between micelles enable a reversible response to shear stress, facilitating 
extrusion of these hydrogels from a syringe (Figure 2.1).37  
In order to further examine the versatility of this platform as temperature- and shear-
responsive materials, we investigated a series of poly(alkyl glycidyl ether) homopolymers and 
poly(alkyl glycidyl ether)-block-poly(ethylene oxide)-block-poly(alkyl glycidyl ether) triblock 
copolymers and their corresponding hydrogels. Aqueous solutions of these polymers were 
examined via UV-Vis spectroscopy to identify trends in cloud point temperature (Tcp), while phase 
diagrams were employed to map the physical state of the triblock copolymers at different 
concentrations and temperatures. A series of rheological tests were conducted to further 
characterize the dual-stimuli responsive behavior of these hydrogels. Investigations into the 
printability and cytocompatibility of the platform indicate a promising potential for use in additive 
manufacturing and tissue engineering. 
 
37 
 
2.3 MATERIALS AND METHODS 
2.3.1 Materials  
All chemicals and solvents were purchased from Sigma-Aldrich or Fisher Scientific and used 
without further purification unless noted otherwise. Isopropyl glycidyl ether (iPGE, 98%), ethyl 
glycidyl ether (EGE, 98%), allyl glycidyl ether (AGE, 99%, Acros), methyl glycidyl ether (MGE, 
85%, TCI America), and n-propyl glycidyl ether (Synthesis detailed in Supporting Information) 
were dried over CaH2 for 24 h, distilled into a flask containing butyl magnesium chloride (2 M in 
tetrahydrofuran, THF), re-distilled, and stored under N2 atmosphere. Poly(ethylene oxide) (PEO, 
Mn 8000 g mol
-1) was dried under vacuum overnight prior to use. Dry THF was obtained using 
neutral alumina using a Pure Process Technology solvent purification system. A potassium 
naphthalenide solution (1M) was prepared by dissolving naphthalene (3.2 g) in THF (25 mL), 
adding potassium (0.975 g), and storing under N2 atmosphere. 
1H NMR spectra were obtained on 
a Bruker Advance 300 or 500 MHz spectrometer. Gel permeation chromatography was performed 
using a Waters chromatograph equipped with two 10 µm Malvern columns (300 mm x 7.8 mm) 
connected in series with increasing pore size (1,000, 10,000 Å), using chloroform (Optima, 0.1% 
v/v trimethylamine) as the eluent, and calibrated with poly(ethylene oxide) standards (400 to 40, 
000 g mol-1). The relative molecular weights were measured in chloroform using poly(ethylene 
oxide) standards and a refractive index detector (flow rate: 1 mL min-1).  
2.3.2 Homopolymer Synthesis  
All alkyl glycidyl ether homopolymers were synthesized by utilizing the same synthetic 
procedure with different monomer feed ratios. The following poly(isopropyl glycidyl ether) 
synthetic scheme will serve as an example. The initiator 4-methyl benzyl alcohol (0.122 g, 1 mmol) 
38 
 
was added to an oven-dried, 100 mL round-bottom flask. Potassium naphthalenide solution (1M 
in THF) was titrated until a light green paste was formed. The reaction flask was evacuated 
overnight to drive off the remaining THF. Isopropyl glycidyl ether (6.49 mL, 51.56 mmol) was 
added to the dried mixture of deprotonated initiator, and the reaction was stirred at 70 °C for 45 h. 
The polymer solution was quenched using a degassed 1% v/v AcOH in MeOH solution and 
dialyzed against MeOH for 3 d (3 solvent exchanges) using Spectra/Por regenerated cellulose 
tubing (MWCO 1.0 kDa) that was pre-soaked in water. The dialyzed polymer solution was 
concentrated under reduced pressure and dried in a vacuum oven for 24 h to afford a viscous, pale 
yellow liquid (0.8 g). 1H NMR (500 MHz, CDCl3): δ 1.13-1.14 (m, -O-CH-(CH3)2), 2.34 (s, -Ph-
CH3), 3.43-3.89 (m, (-O-CH2-CH(CH2-O-CH-(CH3)2)-O-), 4.50 (s, -Ph-CH2-O-), 7.13-7.14 (d, 
CH3-Ph-CH2-, J = 7.5 Hz), 7.21-7.22 (d, CH3-Ph-CH2-, J = 8 Hz). Molecular weights were 
determined by 1H NMR spectroscopy by comparing the integration values of the methyl benzylic 
protons (2.33 ppm) to the alkyl glycidyl ether protons (1.13-1.25 ppm) or the methylene (5.2 ppm) 
and vinyl (5.9 ppm) protons of allyl glycidyl ether. 
2.3.3 Triblock Copolymer Synthesis  
ABA triblock copolymers were synthesized via anionic ring-opening polymerization. All 
copolymers were initiated from PEO (Mn = 8,000 g mol
-1). The following procedure for P(iPGE-
stat-EGE)2.2k-block-PEO8k-block-P(iPGE-stat-EGE)2.2k (Polymer 9) will serve as an example for 
a typical triblock copolymer synthesis. PEO (10 g, 1.25 mmol) was added to the reaction vessel 
and dried under vacuum overnight. Dry THF (100 mL) was added under an Ar atmosphere and 
heated to 50 °C to facilitate dissolution of the macroinitiator. Once sufficiently dissolved, a 
potassium naphthalenide solution (1M in THF) was titrated into the flask until the solution 
remained a slight green color, indicating full deprotonation of PEO hydroxyl end groups. Isopropyl 
39 
 
glycidyl ether (4.07 g, 35 mmol) and ethyl glycidyl ether (3.57 g, 35 mmol) were added to begin 
polymerization. The reaction continued for 24 h at 65 °C and was subsequently quenched with a 
degassed solution of 1% v/v AcOH in MeOH. In the polymerizations with allyl glycidyl ether, 
reactions were performed at 30 °C to avoid allyl-vinyl isomerization as reported by Lynd and co-
workers.38 The reaction mixture was then precipitated into cold hexane. The polymer was collected 
via centrifugation (4400 rpm, 10 min) and the supernatant decanted. The product was washed twice 
with additional hexane and collected again in the same manner. The isolated polymer solution was 
dried in a vacuum oven for at least 24 h to afford an off-white solid (13.6 g). 1H NMR (500 MHz, 
CDCl3): δ 1.13-1.15 (m, -O-CH-(CH3)2), 1.15-1.19 (t, -O-CH2-CH3, J = 7.0 Hz), 3.47-3.79 (m -O-
CH2-CH2-O- and -O-CH2-CH(CH2-O-CH2-CH3)-O- and -O-CH2-CH(CH2-O-CH-(CH3)2)-O-). 
The method used to calculate the degree of polymerization (DP) can be found in the Supporting 
Information.  
2.3.4 UV-Vis Spectroscopy   
Cloud point temperatures (Tcp) were determined by UV-Vis spectroscopic measurements on 
an Agilent 8453 UV Visible Spectrometer. Temperature-dependent absorbance values were 
acquired every 0.5 °C at λ = 600 nm with a two-minute equilibration time for all samples. The 
polymers were dissolved in water and equilibrated at 5 °C to afford final sample concentrations of 
1 wt%. The Tcp was reported as the temperature at 50% of the absorbance increase. 
2.3.5 Rheological Measurements  
Dynamic oscillatory rheological experiments were performed on a TA Instruments Discovery 
HR-2 rheometer equipped with a 20 mm parallel-plate geometry unless otherwise specified. 
Samples were equilibrated in an ice bath for at least 10 min, then carefully loaded onto the Peltier 
40 
 
plate at 5 °C. A pre-shear experiment was applied to ensure bubbles were eliminated from the 
sample cell. The sample was equilibrated at 21 °C for 8 min. Strain sweep experiments were 
performed, and all studies were conducted using a strain value in the linear viscoelastic regime. 
Temperature ramp experiments were performed at 1 Hz from 5 to 50 °C at 2 °C min-1. Cyclic strain 
tests (frequency 1 Hz) were performed at 21 °C using alternating strains of 1% for 5 minutes and 
100% for 3 minutes per cycle. Viscosity versus shear rate experiments were performed at 21 °C. 
The gel yield stress values were measured under oscillatory strain (frequency: 1 Hz, 21 °C) starting 
with an initial strain of 0.01% and converted to applied oscillatory stress.  
2.3.6 Direct-Write 3D Printing of Hydrogels  
A modified pneumatic direct-write 3D printer was assembled based on a Tronxy P802E 
3D Printer kit, from Shenzen Tronxy Technology Co. The hydrogel ink was cooled to 5 °C and 
poured into a Nordson Optimum 10 cc fluid dispensing barrel equipped with a Metcal conical (410 
µm inner diameter) precision tip nozzle. The loaded syringe was warmed to ambient temperature 
and pressurized using nitrogen gas (20 psi) to extrude the gel from the nozzle at 8.0 mm s-1. The 
printer was controlled with an Arduino using the Marlin firmware. The G-code file was produced 
with Slic3r software. 
2.3.7 Cell Viability Study 
Three solutions of polymer 9 (15, 20, and 26.67 wt%) in Dulbecco’s Modified Eagle’s 
Medium (DMEM) supplemented with 10% phosphate buffer solution (PBS) and 1% 
streptomycin/penicillin were equilibrated at 5 °C for 3 d. HeLa cells were grown to confluency at 
37 °C. The cells were rinsed with PBS, detached from culture plates using trypsin (0.05%) and 
EDTA (0.53 mM), and mixed with supplemented DMEM to afford a final concentration of 10 x 
41 
 
106 cells/mL. To utilize the thermally reversible gel-to-sol transition, cell media (20 µL) was 
combined with polymer solution (60 µL) on ice; hydrogels were formed with final concentrations 
of 11.25, 15, and 20 wt%. The cell/polymer media solution (60 µL) was pipetted onto a culture 
plate, immersed in supplemented DMEM, and incubated at 37 °C for 24 h. The sample was cooled 
on an ice block and mixed with a DMEM solution containing LIVE/DEAD® reagents calcein (2 
µM) and ethidium homodimer-1 (4 µM). This solution was placed in an incubator for 30 min at 
37 °C, imaged on a Leica SP8X Confocal Microscope, and analyzed using the Fiji image 
processing package.  
2.4 RESULTS AND DISCUSSIONS 
2.4.1 Synthesis of Homopolymers and Characterization of LCST Response  
Living anionic polymerization is an effective method39–42 for the ring-opening 
polymerization43,44 of glycidyl ether derivatives to afford polymers of controlled molecular weight 
and dispersity. All poly(alkyl glycidyl ether)s in this investigation were synthesized via the 
initiation from an alcohol using potassium naphthalenide as the base. Watanabe and co-
workers45,46 previously demonstrated that poly(alkyl glycidyl ether)s show LCST behavior in 
aqueous solutions. Thus, we first synthesized alkyl glycidyl ether homopolymers derived from 
methyl, ethyl, allyl, isopropyl, or n-propyl glycidyl ether monomer (Scheme 2.1) in order to 
characterize their LCST responses to confirm these results for comparison to the triblock 
copolymers to be discussed later in this section. 
Polymers with molecular weights ranging from 2.1 kg mol-1 to 24.5 kg mol-1 were 
synthesized, and 1 wt% aqueous solutions of these polymers were subjected to UV-Vis 
spectroscopic cloud point measurements to quantify the LCST (Table 2.1, Figure A1). The LCST 
transition was governed by a change in the system’s free energy of mixing, ΔGmix, from negative 
42 
 
to positive upon an increase in temperature.47,48 This was due to the amphiphilic nature of the 
polymer in an aqueous environment. The oxygen atoms in the poly(alkyl glycidyl ether)s acted as 
hydrogen bond acceptors with water that enthalpically favored a mixed state. Meanwhile, the alkyl 
portions of the polymer contributed to an unfavorable entropic driving force associated with the 
hydrophobic effect.49–51 The balance between an enthalpically favored mixed state and an 
entropically favored demixed state led to a temperature-dependent solubility. At lower 
temperatures, the enthalpic forces of the polymer dominated the system, which yielded a negative 
ΔGmix and a transparent solution. As the temperature increased, the negative total entropy change 
dominated the system and the free energy of mixing became positive. Phase separation occurred 
as the polymer chains underwent a coil-to-globule transition, and the solution became turbid.48,49,52 
The Tcp values of the poly(alkyl glycidyl ether)s studied were found to abide by these 
thermodynamic principles and correlated inversely with the number of carbons in the R group. 
Hydrophilic monomers, wherein R = ethyl and methyl, displayed LCST behavior at approximately 
10.8 and 45 °C, respectively. As the number of carbons in the R group increased to three (R = 
isopropyl, n-propyl, and allyl), the large entropy term dominated at all temperatures and yielded 
water-insoluble polymers. We also observed that the Tcp of the alkyl glycidyl ether homopolymers 
did not show any dependence on molecular weight, remaining nearly constant from 3.0 to 24.5 kg 
mol-1 for poly(ethyl glycidyl ether) (PEGE). These values were consistent with the results of Satoh 
and Watanabe et al.46,53 However, we noted a difference in the Tcp observed for poly(methyl 
glycidyl ether) compared to a previous report by Watanabe and co-workers,46 which was attributed 
to the hydrophobicity of the methyl benzyl end group that had a significant effect upon the 
solubility of the homopolymer as a consequence of its low molecular weight.  
43 
 
2.4.2 Synthesis of Triblock Copolymers and Characterization of LCST Response  
The incorporation of different alkyl glycidyl ethers into an ABA triblock copolymer 
comprised of poly(alkyl glycidyl ether) ‘A’ blocks and a PEO ‘B’ block afforded a platform of 
polymers with a tunable temperature response. Poly(alkyl glycidyl ether)-block-PEO-block-
poly(alkyl glycidyl ether) triblock copolymers were synthesized via anionic ring opening 
polymerization in THF using PEO as the macroinitiator (Mn = 8,000 g mol
-1) and potassium 
naphthalenide as a base (Scheme 2.2).   
The block length of each glycidyl ether segment was controlled by varying the molar ratio of 
monomer to PEO macroinitiator. Statistical copolymers were afforded by the addition of two 
different alkyl glycidyl ether monomers simultaneously. Specifically, EGE and AGE were co-
polymerized to form polymer 4, while EGE and iPGE were co-polymerized to form polymers 5-
10. The degree of polymerization (DP) was less than the theoretical value, which suggested that 
homopolymer impurities or unreacted monomers were present in the reaction mixture but were 
removed during purification. The low dispersity values (≤ 1.16) of all synthesized triblock 
copolymers were consistent with controlled ring-opening polymerizations. The LCSTs of these 
polymers (Table 2.2) were quantified in a manner similar to the homopolymers discussed 
previously in this section (Figures A2 and A3). 
The thermodynamic principles that governed the LCST response of the homopolymers was 
also applicable to the triblock copolymers. However, the temperature-dependent aqueous 
solubility of the copolymer as a whole must be taken into consideration, as opposed to the 
solubility of the individual blocks.53–57 Although the AGE and the iPGE homopolymers (Table 
2.1) did not exhibit LCST behavior, the ABA triblock copolymers containing poly(allyl glycidyl 
ether) (PAGE) ‘A’ blocks (polymer 2) or PiPGE ‘A’ blocks (polymer 3) had Tcp values of 29.7 °C 
44 
 
and 24.2 °C, respectively. Polymer 1, which had PEGE ‘A’ blocks, exhibited a Tcp at 60.0 °C. The 
statistical co-polymerization of EGE and iPGE enabled further tuning of the LCST behavior. We 
successfully decreased the Tcp of the copolymer from 60.0 °C (polymer 1) to 46.2 °C (polymer 5), 
40.3 °C (polymer 8), and 32.4 °C (polymer 6) by replacing EGE units in the ‘A’ blocks with more 
hydrophobic iPGE monomers (at similar molecular weights). 
In addition to molecular composition, the molecular weight of the ‘A’ blocks can also be 
exploited to tune the LCST response of the copolymer platform. To probe this relationship, the 
phase transition temperature of polymers 7-10, with approximately a 1:1 ratio between EGE and 
iPGE—and with molecular weights of 1.4k, 1.8k, 2.2k, and 2.8k, respectively—were investigated. 
We observed that the Tcp of the triblock copolymer and chain length of the glycidyl ether blocks 
were inversely related, as the hydrophobic character of the polymer increased with the molecular 
weight of the poly(alkyl glycidyl ether) segments. We successfully tuned the temperature response 
of the triblock copolymer in water from 49.3 °C (polymer 7) to 24.1 °C (polymer 10) by changing 
the molecular weight of the A block.  
2.4.3 Triblock Copolymer Phase Diagrams  
At low concentrations (1-5 wt%), the triblock copolymer solutions became turbid as the 
temperature increased, due to the cloud point transition of these polymers. However, as the 
polymer concentration increased (≥ 5 wt%), this phase separation mechanism gave way to the 
formation of a three-dimensional hydrogel network and a sol-gel transition. To better understand 
the role of temperature, polymer concentration, chain length, and composition in the formation of 
the polymer hydrogels, qualitative phase diagrams were produced for each triblock copolymer. 
Polymer solutions (2-30 wt%) were prepared and incubated at temperatures that ranged from 5 to 
50 °C. Their physical states were determined using the vial inversion method and recorded as a 
45 
 
transparent/opaque liquid, viscous solution, or gel. Transparent formulations were clear, with no 
turbidity present, whereas opaque solutions were turbid and optically cloudy in appearance. 
In the same manner that the hydrophobicity of the copolymers was utilized to tune the LCST 
response, we exploited the inherent differences in hydrophobic character of the alkyl glycidyl ether 
monomers to alter the sol-gel transition temperature. Polymer 1 afforded solutions that only 
became a gel at temperatures >50 °C and concentrations >30 wt% as a consequence of the 
hydrophilicity of the PEGE block. Relative to polymer 1, polymers 5, 8, 6, and 3 had a greater 
hydrophobic character due to the presence of iPGE units in the copolymer. We successfully 
formulated solutions of polymers 5, 8, 6, and 3 that gelled at progressively lower temperatures and 
concentrations relative to polymer 1 (Figure 2.2).  
For example, 20 wt% solutions of polymers 5, 8, 6, and 3 exhibited a sol-gel transition at 45, 
30, 25, and 10 °C, respectively. The solubility of polymer 3 (with PiPGE ‘A’ blocks) significantly 
decreased, such that it was not fully soluble at 30 wt%. The AGE-containing polymers afforded 
brittle hydrogels (Figure A4) and were not further investigated. 
The sol-gel transition of these polymers in aqueous solution was also affected by molecular 
weight and polymer concentration. The sol-gel transition temperature was tuned by altering the 
molecular weight of the glycidyl ether ‘A’ blocks (Figure 2.3). As the polymer chain length 
increased, the hydrophobicity of the polymer also increased. For polymers 7-10 at identical 
concentrations in water (20 wt%), the sol-gel transition temperature decreased as the molecular 
weight was increased (40, 30, 20, and 15 °C, respectively). Thus, a 25 °C range in gelation 
temperature was accomplished with the inclusion of 12.5 additional glycidyl ether repeat units per 
block. The polymer concentration also served an important role in the tunability of the gelation 
temperature. The sol-gel transition of each hydrogel decreased as the polymer concentration 
46 
 
increased (Figure 2.3). For example, the sol-gel transition temperature of polymer 7 was altered 
from 40 °C to 25 °C as the concentration was increased from 20 to 30 wt%. 
Although the gelation temperature of these solutions cannot be directly predicted by the Tcp 
values, we observed that they were dictated by the polymer composition, molecular weight, and 
concentration in aqueous media. These results are consistent with that reported by Georgiou and 
co-workers, whom obtained58–61 similar results for their thermo-responsive methacrylate-based 
terpolymers. Similarly, Pluronic F127 has a reported62 sol-gel transition (14-40 °C) that is 
dependent upon concentration, but has a Tcp above 100 °C.
63 These findings stand in contrast to 
the PNiPAAm-block-PEO-block-PNiPAAm triblock copolymers studied by Teodorescu and 
coworkers, wherein the cloud point measurements and the vial inversion gelation temperatures 
occurred at similar temperatures.64,65  
2.4.4 Triblock Copolymer Hydrogel Rheology  
Temperature- and shear- responsive hydrogels are useful for direct-write 3D printing as 
fugitive inks66,67 and in the fabrication of composites hydrogels.68 Rheometric characterization of 
the viscoelastic behaviors of hydrogels can serve as a screening protocol for the suitability of the 
hydrogels as inks for direct-write 3D printing.28,69 Specifically, yield stress values can be used to 
compare the relative extrudability of hydrogels from a nozzle during the printing process. The 
hydrogel ink maintains a gel state below its yield stress, but the gel network is broken at stresses 
above this value causing the ink to flow and a steep drop in modulus. The phase diagrams in 
Figures 2.2 and 2.3 were utilized to identify the optimal hydrogels to investigate as inks for direct-
write 3D printing. The selected hydrogels were gels at ambient temperature but reversibly 
transformed into the ‘liquid’ or ‘viscous solution’ state upon cooling. 
47 
 
 Oscillatory stress ramp experiments (Figure 2.4a) were used to determine the yield stress of 
these select hydrogel formulations. The 20 wt% solutions of polymers 9, 10, and 3 had yield stress 
values of 569, 1200, and 1252 Pa, respectively. Hydrogels derived from polymer 9 were found to 
be particularly well-suited for direct-write 3D printing, whereas the yield stress for hydrogels of 
polymers 10 and 3 were too high and the materials were more difficult to extrude. Thus, a 20 wt% 
formulation of polymer 9 was chosen for all further rheological experiments, as well as the direct-
write 3D printing of the structures presented later in this section.  
Temperature-dependent sol-gel transitions in hydrogel inks is advantageous for the addition 
and homogeneous distribution of additives and the transfer of these inks into the printer cartridge 
(syringe). The temperature-dependent viscoelastic characterization (Figure 2.4b) confirmed the 
presence of a sol-gel transition as defined by the intersection of elastic (G’) and viscous (G”) 
moduli (the rheometrical gelation temperature (Tgel) = 18.72 °C). At temperatures below the Tgel, 
the G” exceeded the G’ and the polymer solution was free-flowing; above the Tgel, the G’ exceeded 
the G” and the solution became a gel with a maximum G’ of 33.3 kPa. Additional rheological 
characterization demonstrated the shear-thinning and self-healing properties of these hydrogels 
governed by the physical cross-linking of the low-Tg poly(alkyl glycidyl ether) blocks. The shear-
thinning behavior was reflected by the decrease in viscosity with increasing shear rate (Figure 
2.4c). A dynamic oscillatory strain experiment (Figure 2.4d) revealed a rapid and reversible 
response of the hydrogel G’ and G” moduli to consecutive cycles of high (100%) and low (1%) 
strain. During periods of high strain, the G” was greater than the G’, which indicated that the 
material was able to flow. Whereas the sample was a gel when subjected to periods of low strain, 
as indicated by the larger value for G’ relative to G”. Also, G’ was nearly fully recovered after 
every cycle with a low degree of mechanical hysteresis. 
48 
 
2.4.5 Direct-Write 3D Printing  
As a demonstration printability of our hydrogel platform, a hydrogel ink comprised of 20 wt% 
polymer 9 was extruded by a direct-write 3D printer. The hydrogel was loaded into the printer 
syringe in its liquid state at 5 °C and was then warmed to ambient temperature. The hydrogel ink 
was extruded through a 0.41 mm inner diameter nozzle at 20 psi and an 8.0 mm s-1 print speed. 
During the direct-write 3D printing process, multiple layers are sequentially printed in a layer-by-
layer manner. The printed objects shown in Figure 2.5 had 15 layers and exhibited excellent shape 
fidelity with no sagging or deformation of the individual layers. 
2.4.6 Cell Viability  
The cytocompatibility of hydrogels based on poly(alkyl glycidyl ether) block copolymers 
were investigated using HeLa cells. Hydrogels derived from polymer 9 were formulated using 
DMEM supplemented with 10% FBS and 1% streptomycin/penicillin. The hydrogels in media 
exhibited a temperature-dependent sol-gel transition—wherein the sample became liquid upon 
cooling in an ice bath. This temperature-responsive feature enabled the uniform incorporation and 
distribution of HeLa cells in the liquid state prior to warming the solution to its gel state. The 
HeLa-embedded hydrogels were incubated at 37 °C for 24 h at final polymer concentrations of 
11.25, 15, and 20 wt%. Cell viability was quantified via LIVE/DEAD® assay and confocal 
microscopy (Figure 2.6). These tests were performed in triplicate (Figure A5- 
A8) and afforded viabilities of 91% (± 4.10%), 93% (± 6.27%), and 84% (± 18.24%), respectively. 
The results of these experiments indicate acceptable cell viability at all concentrations tested and 
demonstrate a promising potential for use in cell encapsulation applications such as cell 
therapeutics and tissue engineering. 
 
49 
 
2.5 CONCLUSION 
In summary, we have developed a highly tunable, biocompatible ABA triblock copolymer 
platform that utilized the hydrophobic self-association of poly(alkyl glycidyl ether) ‘A’ blocks in 
aqueous solutions to form three-dimensional networks composed of flower micelles that 
responded to both thermal and shear stimuli. The identity of the alkyl side-chain (ethyl, 
isopropyl, n-propyl, and allyl) on the poly(alkyl glycidyl ether) block played a significant role in 
the temperature-responsive behaviors. Interestingly, the 3-carbon alkyl groups afforded 
homopolymers that did not exhibit any temperature response, but yielded ABA triblock 
copolymers which clearly exhibited temperature-dependent gelation. Copolymers comprised of 
ethyl and isopropyl glycidyl ethers were investigated to further tune the temperature response. 
The gelation temperature of this platform can be readily tuned between 10 and 45 C by altering 
the molecular composition, molecular weight, and concentration of the polymer. The temperature 
and shear-responsive nature of the triblock copolymer hydrogels were characterized via phase 
diagrams and rheology. We also demonstrated that these polymers were suitable as inks for 
direct-write 3D printing to afford free-standing, multi-layered 3D constructs. These stimuli-
responsive hydrogels represent a promising platform to develop new materials for additive 
manufacturing and tissue engineering. 
 
 
 
 
50 
 
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56 
 
 
 
 
 
 
 
 
 
 
Figure 2.1 Graphical representations of (a) the temperature-dependent equilibrium between 
unimers (low temperature) and a flower micelle network (high temperature) and (b) the shear 
stress-induced breaking of physical crosslinks for the poly(alkyl glycidyl ether)-block-
poly(ethylene oxide)-block-poly(alkyl glycidyl ether) triblock copolymer platform. 
Representative photographs that demonstrate the macroscale (c) thermo-responsive sol (5 °C) to 
gel (25 °C) transition and (d) extrusion activated shear-responsive properties of a 20 wt% 
hydrogel of polymer 9 (0.41 mm inner diameter nozzle). 
 
 
Scheme 2.1 Synthesis of poly(alkyl glycidyl ether)s wherein R represents methyl, ethyl, allyl, 
isopropyl, or n-propyl groups. 
57 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table 2.1 Characteristics of poly(alkyl glycidyl ether) homopolymers 
R Group Mn (g mol
-1)a Ðb Tcp (°C)c 
Methyl 2.1k 1.14 45.0 
Ethyl 3.0k 1.09 10.0 
Ethyl 9.5k 1.12 11.1 
Ethyl 24.5k 1.14 10.8 
Allyl 3.3k 1.18 ─ d 
Allyl 5.8k 1.10 ─ d 
Allyl 11.7k 1.15 ─ d 
Isopropyl 4.0k 1.08 ─ d 
Isopropyl 7.7k 1.11 ─ d 
Isopropyl 16.0k 1.22 ─ d 
n-propyl 3.2k 1.16 ─ d 
n-propyl 8.0k 1.16 ─ d 
aNumber average molecular weight (Mn) was determined by 
1H NMR Spectroscopy. 
bDispersity (Ð) was determined by GPC. cCloud point temperatures (Tcp) was determined by 
UV-Vis Spectroscopy. dThe Tcp of these homopolymers could not be determined due to 
aqueous insolubility at all temperatures.  
 
 
 
58 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Scheme 2.2 Synthesis of ABA Triblock Copolymers, wherein R represents ethyl, allyl, and 
isopropyl groups 
 
 
 
 
 
 
 
 
 
 
 
 
59 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table 2.2 Characteristics of poly(alkyl glycidyl ether)-block-PEO-block-poly(alkyl 
glycidyl ether) triblock copolymers 
 Side Chain (R1/R2) Mn (g mol
-1)a DP (z/y)a Ðb Tcp (°C)
c 
Polymer 1 Ethyl 1.5k-b-8k-b-1.5k 14.8 1.10 60.0 
Polymer 2 Allyl 1.4k-b-8k-b-1.4k 12.4 1.11 29.7 
Polymer 3 Isopropyl 1.8k-b-8k-b-1.8k 15.2 1.16 24.2 
Polymer 4 Ethyl/Allyl 1.7k-b-8k-b-1.7k 5.6/9.6 (15.2) 1.11 43.1 
Polymer 5 Ethyl/Isopropyl 1.7k-b-8k-b-1.7k 10.6/5.0 (15.6) 1.11 46.2 
Polymer 6 Ethyl/Isopropyl 1.7k-b-8k-b-1.7k 5.4/10.2 (15.6) 1.16 32.4 
Polymer 7 Ethyl/Isopropyl 1.4k-b-8k-b-1.4k 6.5/6.3 (12.8) 1.15 49.3 
Polymer 8 Ethyl/Isopropyl 1.8k-b-8k-b-1.8k 8.2/7.9 (16.1) 1.12 40.3 
Polymer 9 Ethyl/Isopropyl 2.2k-b-8k-b-2.2k 9.8/9.9 (19.7) 1.11 31.3 
Polymer 10 Ethyl/Isopropyl 2.8k-b-8k-b-2.8k 12.6/12.7 (25.3) 1.14 24.1 
aNumber average molecular weight (Mn) and degree of polymerization (DP) were determined by 
1H NMR Spectroscopy. bDispersity (Ð) was determined by GPC. cCloud point temperature (Tcp) 
was determined by UV-Vis Spectroscopy. 
60 
 
 
 
 
 
 
 
 
Figure 2.2 Temperature-concentration phase diagrams summarizing the solution and hydrogel 
states of triblock copolymers of the same total block length containing (a) 5.0 iPGE units 
(polymer 5), (b) 7.9 iPGE units (polymer 8), (c) 10.2 iPGE units (polymer 6), and (d) 15.2 iPGE 
units (polymer 3). The green/blue/red shaded areas indicate formulations existing as 
transparent/opaque liquids (□/■), viscous solutions (/), and gels (○/●), respectively. 
Transparent formulations are clear with no turbidity. Opaque formulations are turbid and 
optically cloudy. Their physical states were determined using the vial inversion method. 
 
 
 
 
 
 
 
 
 
 
 
 
61 
 
 
 
 
 
 
 
 
 
 
Figure 2.3 Temperature-concentration phase diagrams summarizing the solution and hydrogel 
states of triblock copolymers with increasing DP (a) 12.8 (polymer 7), (b) 16.1 (polymer 8), (c) 
19.7 (polymer 9), and (d) 25.3 (polymer 10). The green/blue/red shaded areas indicate 
transparent/opaque liquids (□/■), viscous solutions (/), and gels (○/●), respectively. 
Transparent formulations are clear with no turbidity. Opaque formulations are turbid and 
optically cloudy. Their physical states were determined using the vial inversion method. 
 
62 
 
 
 
 
 
 
 
 
 
 
Figure 2.4 Rheological experiments (a) Oscillatory stress experiment indicating yield stress. 
Yield stress values = 569, 1200, and 1252 for 20 wt% solutions of polymer 9, 10, and 3, 
respectively. (b) Dynamic oscillatory temperature ramp displaying elastic (G’, filled) and 
viscous (G’’, open) moduli. Tgel = 18.72 °C (c) Viscosity vs shear rate experiment depicting 
shear-thinning behavior by a decrease in viscosity with increasing shear rate and (d) Cyclic 
strain experiment demonstrating rapid recovery of hydrogel elastic modulus (black circles) 
from periods of high (100%) to low (1%) oscillatory strain (red circles). Arrows indicate 
reference axis; elastic/viscous moduli (left axis) and oscillatory strain (right axis). 
 
 
 
 
 
 
 
63 
 
 
 
 
 
 
 
 
 
 
 
Figure 2.5 3D printed structures of a 15-layer (a) University of Washington initials and (b-c) 
benzene ring with a 0.41 mm inner diameter nozzle at 8.0 mm s-1. This structure was printed 
with a formulation of polymer 9 at 20 wt% using a pneumatic direct-write 3D printer (scale bars: 
1 cm). 
 
 
 
Figure 2.6 Evaluation of a LIVE/DEAD® assay performed on three polymer 9 hydrogels at 
different concentrations (11.25, 15, 20 wt%). (a) Composite channel confocal microscopy 
image of encapsulated HeLa cells (11.25 wt%) (scale bar: 1000 µm). 
 
 
64 
 
CHAPTER 3: POLY(ALKYL GLYCIDYL ETHER) HYDROGELS 
FOR HARNESSING THE BIOACTIVITY OF ENGINEERED 
MICROBES 
3.1 ABSTRACT 
Herein, we describe a method to produce yeast-laden hydrogel inks for direct-write 3D 
printing cuboidal lattices for immobilized whole-cell catalysis. A poly(alkyl glycidyl ether)-based 
triblock copolymer was designed to have three important features for this application: (1) a 
temperature response which allowed for facile processing of the material; (2) the shear response 
which facilitated the extrusion of the material through a nozzle; and (3) a UV-light induced 
polymerization which enabled the post extrusion chemical crosslinking of network chains, and the 
fabrication of robust printed objects. These three key stimuli responses were confirmed via 
rheometrical characterization. A genetically engineered yeast strain with an upregulated α-factor 
production pathway was incorporated into the hydrogel ink and 3D printed. The immobilized yeast 
cells exhibited adequate viability of 87.5% within the hydrogel. The production of the up-regulated 
α-factor was detected using a detecting yeast strain and quantified at 268 nM (s = 34.6 nM) over 
72 h. The reusability of these bioreactors was demonstrated by immersion of the yeast-laden 
hydrogel lattice in fresh SC media and confirmed by the detection of similar amounts of up-
regulated α-factor 259 nM (s = 45.1 nM). These yeast-laden materials represent an attractive 
opportunity for whole-cell catalysis of other high-value products in a sustainable and continuous 
manner. 
65 
 
3.2 INTRODUCTION  
Whole-cell biocatalysis is a standard practice across a wide range of industries wherein cells 
are used to transform molecular precursors into a product of interest (antibiotics, drugs, vitamins, 
insulin, vaccines, etc.). These reactions are generally employed as a batch process wherein the 
cells and the necessary molecular precursors are combined into a single reaction vessel, stirred for 
several days, and then the product is isolated from the complex mixture.1–10  However, batch cell 
reactors are costly and time consuming because they require sterile conditions, can have low 
yields, and purification protocols are labor and cost intensive. Immobilized-cell bioreactors, 
wherein metabolically active cells are trapped within a material such as a hydrogel, offer an 
alternative method that can simplify the isolation of the product, minimize product inhibition or 
toxicity, and allow recycling of the cells.11 
We have previously reported additive manufactured catalytically active living materials 
(AMCALM) as a platform for immobilized-cell bioreactors.12 In contrast to previous reports 
wherein calcium alginate beads13–16 or electrospun fibers17–19 were used to encapsulate microbes 
for fermentation, our approach utilized customized lattices of yeast-laden hydrogels that were 
printed using a direct-write 3D printer. Additive manufacturing (or 3D printing) is a fabrication 
process that utilizes layer-by-layer material deposition to construct three-dimensional geometries 
according to a computer-aided design (CAD) model.20–27 As such, 3D printing is well-suited for 
manufacturing living materials28–33 with spatial and geometrical organization of cells. Hydrogel-
based materials are particularly attractive as cell-laden inks for direct-write 3D printing because 
these materials can recapitulate some of the chemical and physical attributes of the extracellular 
matrix that exists in biofilms, living tissue, and other naturally occurring microenvironments.31,34–
66 
 
36 These features could include the presence of ligands or functional groups,37–40 or the stiffness of 
the hydrogel.41,42    
There were three important features of the yeast-laden hydrogel ink that were developed for 
AMCALMs: (1) a temperature response, wherein the material exhibited a reversible gel-to-sol 
transition upon cooling, that enabled homogeneous dispersion of the yeast cells within the gel and 
facile loading of the hydrogel into a syringe; (2) a shear-thinning response that facilitated the 
extrusion of the hydrogel ink from a nozzle to enable the layer-by-layer formation of three-
dimensional objects; and (3) an irreversible photo-response wherein polymerizable groups 
chemically cross-linked the hydrogel into a robust structure. Yeast-laden hydrogel lattices were 
printed and then utilized in a continuous batch process for the fermentation of glucose to produce 
ethanol. 
Herein, we demonstrate a new polymer hydrogel for the encapsulation and direct-write 3D 
printing of yeast-laden hydrogel lattices that can be used for the production of a polypeptide. The 
poly(glycidyl ether)-based ABA triblock copolymer (polymer 2) has similar features (temperature-
, shear-, and photo-responses, Figure 3.1) to the Pluronic-based triblock copolymer that was 
reported previously.12 Furthermore, a genetically-engineered yeast strain with an upregulated α-
factor production pathway was incorporated into the hydrogel to demonstrate the production of an 
extracellularly excreted polypeptide from the yeast-laden hydrogel. While α-factor is a yeast 
hormone that does not have any known therapeutic effects, the polypeptide is commonly utilized 
in the expression and recovery of recombinant proteins.43,44 When the α-factor leader sequence is 
appended to the genetic code for an engineered protein, the yeast cell utilizes its native cellular 
machinery to secrete the protein beyond the cell membrane.45–50 In our experiments, we not only 
67 
 
demonstrate that the secreted α-factor can be detected in the liquid media surrounding the hydrogel 
lattice, but also the reusability of the yeast-laden hydrogel lattices. 
3.3 MATERIALS AND METHODS 
3.3.1 Materials  
All chemicals and solvents were purchased from Sigma-Aldrich or Fisher Scientific and used 
without further purification unless noted otherwise. Isopropyl glycidyl ether (iPGE, 98%) and 
ethyl glycidyl ether (EGE, 98%) were dried over CaH2 for 24 h, distilled into a flask containing 
butyl magnesium chloride (2 M in tetrahydrofuran, THF), re-distilled, and stored under N2 
atmosphere. Poly(ethylene oxide) (PEO, Mn 8000 g mol
-1) was dried under vacuum overnight prior 
to use. Dry THF was obtained using neutral alumina using a Pure Process Technology solvent 
purification system. A potassium naphthalenide solution (1M) was prepared by dissolving 
naphthalene (3.2 g) in THF (25 mL), adding potassium (0.975 g), and storing under N2 atmosphere. 
1H NMR spectra were obtained on a Bruker Advance 300 or 500 MHz spectrometer. Gel 
permeation chromatography was performed using a Waters chromatograph equipped with two 10 
µm Malvern columns (300 mm x 7.8 mm) connected in series with increasing pore size (1,000, 
10,000 Å), using chloroform (Optima, 0.1% v/v trimethylamine) as the eluent, and calibrated with 
poly(ethylene oxide) standards (400 to 40, 000 g mol-1). The relative molecular weights were 
measured in chloroform using poly(ethylene oxide) standards and a refractive index detector (flow 
rate: 1 mL min-1). Drop-out Mix Complete without Yeast Nitrogen Base D9515) and Yeast 
Nitrogen Base (Y2025) were purchased from US Biological Life Sciences. Sytox Green nucleic 
acid strain (5 mM solution in DMSO; Invitrogen) was purchased from Thermo Fisher Scientific. 
68 
 
3.3.2 Yeast Strains  
For microscopy and live/dead viability tests, the strain yJS001 was used (genotype SO992 
mfa2::pTEF1_mCherry(kanR)). This strain constitutively expressed mCherry protein, which 
facilitated characterization using fluorescence microscopy. Saccharomyces cerevisiae strain 
SO992 was used as the control strain in the α-factor production experiments. This strain is a 
derivative of W303 (genotype MATa ura3 leu2 trp1 his3 can1R ade). 
As the α factor-producing strain, we employed a genetically modified MAT-α yeast strain 
(‘secreting strain’) expressing the S. cerevisiae gene MFα1 (YPL187W) on a constitutively-
expressed promoter pGPD (natively expressing YPL197W). The MFα1 gene expresses 3 copies 
of the α-factor peptide, a 13-amino acid peptide natively used as mating hormone from the MAT-
α to the MAT-A strains.  
To quantify the α-factor secreted from the yeast immobilized in the hydrogels, we employed 
a genetically modified MAT-A yeast strain that expresses the fluorescence protein yeVenus driven 
by the pFUS1 promoter, which is downstream the MAPK signaling pathway, activated by α-factor 
detection (‘detecting strain’). The MAT-A native strain was engineered by deleting BAR1 (native 
α-factor protease) and integrating the POG1 gene on a constitutively-expressed pGPD promoter to 
avoid α-factor-induced growth arrest. 
Yeast transformations were carried out using a standard lithium acetate protocol. Yeast cells 
were made competent by growing 50 mL cultures in rich media to log growth phase, then spinning 
down the cells and washing with H20. Next, linearized DNA, salmon sperm donor DNA, 50% 
polyethylene glycol and 1M LiOAc were combined with 50 mL of competent cells and the mixture 
was heat shocked at 42 °C for 15 min. The cells were then spun down, supernatant was removed, 
69 
 
and they were resuspended in H2O and then plated on selective agar media. Transformations were 
done into MATa W303-1A and MATα W303-1B background strains. 
3.3.3 Synthesis of Polymer 1  
The ABA triblock copolymer was synthesized via anionic ring-opening polymerization. PEO 
(Mn = 8,000 g mol
-1, 20 g, 2.5 mmol) was added to the reaction vessel and dried under vacuum 
overnight. Dry THF (250 mL) was added under an Ar atmosphere and heated to 50 °C to facilitate 
dissolution of the macroinitiator. Once sufficiently dissolved, a potassium naphthalenide solution 
(1M in THF) was titrated into the flask until the solution remained a slight green color, indicating 
full deprotonation of PEO hydroxyl end groups. Isopropyl glycidyl ether (4.94 g, 42.5 mmol) and 
ethyl glycidyl ether (4.34 g, 42.5 mmol) were added to begin polymerization. The reaction 
continued for 24 h at 65 °C and was subsequently quenched with a degassed solution of 1% v/v 
AcOH in MeOH. The reaction mixture was then precipitated into cold hexane. The polymer was 
collected via centrifugation (4400 rpm, 10 min) and the supernatant decanted. The product was 
washed twice with additional hexane and collected again in the same manner. The isolated polymer 
solution was dried in a vacuum oven for at least 24 h to afford polymer 1 as an off-white solid 
(27.5 g). 1H NMR (500 MHz, CDCl3): δ = 1.15-1.17 (m, -O-CH-(CH3)2), 1.17-1.23 (t, -O-CH2-
CH3, J = 7.0 Hz), 3.47-3.81 (m -O-CH2-CH2-O- and -O-CH2-CH(CH2-O-CH2-CH3)-O- and -O-
CH2-CH(CH2-O-CH-(CH3)2)-O-). 
3.3.4 Synthesis of Polymer 2  
Polymer 1 (20g, 1.81 mmol) was dissolved in dry THF (250 mL) under a nitrogen atmosphere 
until complete dissolution of the polymer. Triethylamine (2.45 mL, 18.1 mmol) was added to 
increase the reactivity of the polymer hydroxyl chain ends and the mixture was heated at 65 °C for 
70 
 
30 min. Methacrylic anhydride (26.9 mL, 181 mmol) was then added and the reaction mixture was 
stirred for 16 h at 65 °C. After this time, the reaction was quenched with a degassed solution of 
1% v/v AcOH in MeOH. The reaction mixture was then precipitated into cold ether. The polymer 
was collected via centrifugation (4400 rpm, 10 min) and the supernatant decanted. The product 
was washed twice with additional ether, once with hexane, and collected again by centrifugation. 
The isolated polymer was dried in a vacuum oven for 24 h to afford polymer 2 as an off-white 
solid (16.7g). The degree of functionalization (𝑓n) was determined by comparing the integrations 
of the methacrylate vinyl (6.12 and 5.55 ppm) and methyl (1.94 ppm) protons, as well as the PEO 
chain-end methylene protons (5.08-5.15 ppm), to their theoretical values. For example, 1.99 (vinyl, 
actual)/2 (vinyl, theoretical) X 100 ≈ 100% functionalization of chain ends. These integration 
values were referenced to the total alkyl glycidyl ether protons for each polymer chain (1.12-1.20 
ppm, 121 H).   1H NMR (300 MHz, CDCl3): δ = 1.12-1.20 (m, -O-CH-(CH3)2) and -O-CH2-CH3), 
1.94 (s, CH3C(CO2)=CH2), 3.39-3.89 (m, -O-CH2-CH2-O- and -O-CH2-CH(CH2-O-CH2-CH3)-O-
, and -O-CH2-CH(CH2-O-CH-(CH3)2)-O-), 5.08-5.15 (m, -CH2-CH-O-(C=O)), 5.48 (s, H-CH=C), 
6.18 (s, H-CH=C). 
3.3.5 Preparation of Synthetic Complete Media 
The SC media (1L) was prepared by dissolving drop-out mix (2 g), yeast nitrogen base (6.7 
g), and glucose (20 g) in Milli-Q water. The resulting solution was sterilized by filtration through 
a 0.2 μm nylon filter. 
3.3.6 Preparation of Hydrogel Solution 
Polymer 2 was dissolved in sterile, deionized water at a concentration of 20 wt % polymer. 
The resulting polymer solution was cooled overnight at 5 °C to facilitate hydrogel formation via 
71 
 
LCST behavior.  After homogenization of the solution at low temperature, the solution was 
warmed to 21 °C to induce the formation of a gel state. Hydrogels used to print the proof-of-
concept models in Figure 3.3 were mixed with the photo-radical initiator 2-hydroxy-2-
methylpropiophenone (10 μL) and centrifuged (4400 rpm, 10 min) to remove bubbles.  
3.3.7 Preparation of Yeast-Laden Hydrogel Ink  
Three To prepare yeast-laden hydrogels, the aforementioned hydrogel solution was cooled to 
5 °C in a refrigerator. At this temperature, the hydrogel solution underwent gel-to-sol transition, 
affording a free-flowing liquid. Yeast cells were added from an overnight liquid culture, at a 
concentration of 107 cells per gram of hydrogel while the gel was in its solution state. The resulting 
solution was mixed thoroughly and allowed to equilibrate at 5 °C until all of the bubbles present 
in the solution were removed. Finally, the hydrogel was warmed to 21 °C to undergo a sol to gel 
transition, resulting in a shear-responsive gel. 
3.3.8 Rheometrical Characterization 
For rheometrical characterization of the hydrogels, dynamic oscillatory experiments were 
performed on a TA Instruments Discovery Hybrid Rheometer-2 (DHR-2) equipped with a Peltier 
temperature controller. The instrument operates by applying a known displacement (strain) and 
measuring the material’s resistance (stress) to the force. Rheometry experiments were conducted 
by depositing hydrogel between the rheometer base plate and 20 mm parallel plate geometry with 
a final gap of 1 mm. Samples were equilibrated in an ice bath for at least 30 min and then were 
carefully loaded onto the Peltier plate at 5 °C and a preshear experiment was applied to eliminate 
the bubbles from the sample. The sample was equilibrated at 21 °C for 8 min before each run. The 
gel yield stress values were measured under oscillatory strain (frequency: 1 Hz, 21 °C) starting 
72 
 
with an initial strain of 0.01% and converted to applied oscillatory stress. Viscosity versus shear 
rate experiments were performed in the range of shear rates between 0.01 and 100 Hz. Cyclic shear 
thinning experiments (frequency = 1 Hz) were performed at 21 °C using alternating strains of 1% 
for 5 min and 100% for 3 min per cycle, to investigate the shear-thinning and recovery behavior 
of the hydrogels. Temperature ramp experiments were performed at 1 Hz from 5−50 °C at 2 °C 
min-1. Photocuring was performed using a fully integrated smart swap LED photo curing 
accessory. A 120 s dwell time (frequency: 1 Hz) elapsed before the UV lamp (365 nm LED with 
an irradiation intensity of 5 mW cm-2) was turned on for 420 s at a constant oscillatory strain (1%). 
3.3.9 Additive Manufacturing (Direct-Write 3D Printing) of a Cuboidal-Lattice 
A modified pneumatic direct-write 3D printer was assembled based on a Tronxy P802E 3D 
Printer kit, from Shenzen Tronxy Technology Co. The printer was controlled with an Arduino 
using the Marlin firmware. All CAD models were designed in OpenSCAD. G-code commands for 
the printer were generated using Slic3r. The resulting G-code was modified using Python to 
introduce required commands for the dispensing of the hydrogel via pneumatic pressure. All 
printing was performed using a 20 wt % hydrogel ink with an extrusion air pressure of 20 psi, a 
print speed of 5 mm s-1, and a 0.41 mm inner diameter CML Supply conical nozzle attachment.  
 The dimensions for the 3D printed lattices are 1.9 cm by 1.9 cm by 1.2 cm and each cube 
weighed between 1.8 and 2.0 g. Upon completion of the 3D printing, the cubes were irradiated 
under 365 nm light (at 3.4 mW cm-2) for 3 min to cure and chemically fix the structures. 
3.3.10 Microscopy and Imaging 
Optical imaging: Images of the 3D-printed hydrogels were captured using an iPhone XS. 
Confocal imaging: Confocal microscopy images were taken using a Leica TCS SP5 II laser 
73 
 
scanning confocal microscope. All images were taken with a dry 20x objective. MCherry protein 
fluorescence was excited with a 561 nm laser at 5% laser power, and emission was scanned from 
569 to 700 nm. Sytox green viability dye was excited with a 488 nm laser at 5% laser power, and 
emission was scanned from 500 to 550 nm. Samples were sequentially scanned, and the output 
images were processed using ImageJ Java software.  
3.3.11 Cell Viability Assay 
The yeast-laden hydrogel was prepared as described above. This gel was cooled to 5 °C to 
induce a gel-to-sol transition, which was then poured and spread into a sterile petri dish to produce 
a thin film (~0.5 mm) of the yeast-laden hydrogel. The sample was irradiated with 365 nm UV 
light for 3 min. This film was then incubated in fresh SC media at 30 °C, and periodically agitated. 
The media was exchanged every 24 h to ensure fresh nutrient delivery to the embedded cells. At 
the imaging intervals described in this work, a small square of the film (5 mm × 5 mm) was cut 
and removed for staining. Sytox Green stain stock solution was diluted to 5 μM, and 20 μL of the 
dye solution was exposed to hydrogel sample for 5 min. The sample was then washed with SC 
media and imaged using a Leica SP5 confocal microscope. Constitutively expressed mCherry 
protein fluorescence from the yeast cells was measured to indicate live cells, while the Sytox dye 
fluorescence indicated the presence of dead cells.  
3.3.12 α-Factor Production 
α-Factor production from 3D printed yeast-laden cuboidal lattices was quantified as follows. 
A yeast-laden hydrogel ink containing 107 yeast cells per gram of hydrogel was direct-write 3D 
printed and photo-cured (3 min) with 365 nm irradiation. The lattices were then washed with SC 
media, and placed into 50 mL falcon tubes. The tubes were then filled with 10 mL of SC media 
74 
 
for fermentation and placed in a 30 °C shaker (225 rpm) for incubation. After a period of 72 h, the 
lattices were removed from the reactors and the media was collected. The fermentation media was 
filtered with a 0.2 μm nylon filter. The up-regulated yeast strain experiments were performed in 
triplicate alongside a wild-type yeast-laden lattice. 
To determine the reusability of the lattices in subsequent fermentation cycles, the same lattices 
from the first round of α-factor were again washed individually in sterile SC media. The lattices 
were then placed into new falcon tubes containing 10 mL of SC media, and incubated at the same 
conditions for an additional 72 h. After incubation, the samples were collected and prepared for 
characterization in the same manner as mentioned above. 
3.3.13 α-Factor Detection and Quantification 
To quantify α-factor synthesis from within the yeast-laden hydrogel, we collected the SC 
media that was used to submerge the hydrogel sample after 72 h. We collected 9 mL of media for 
each experimental condition and replicate; each sample was split into six 1.5 mL Eppendorf tube 
and then dehydrated in a Savant SpeedVac Plus SC110A Concentrator for 12 h at Medium 
dehydration speed. Subsequently, we added 100 μL of molecular graded water into each 
dehydrated sample, resuspended, and collected the resulting 600 μL into a single sample. The final 
sample was run again through the Speed-Vac for 12 h at Medium speed and resuspended into 100 
μL of molecular graded water. This process concentrated the initial sample to 90-fold its original 
concentration. 
We experimentally tested for α-factor synthesis using detecting strains with a standard 
cytometer assay protocol. The detecting strains were grown overnight for 20 h in SC media, then 
diluted to 30 events/μL again in SC media. After 3 h, we inoculated the concentrated samples from 
the yeast-laden hydrogels into different vials, plus two extra vials: one kept as control (3 μL of 0.1 
75 
 
M Sodium Acetate) and one was inoculated with 10 μM 98% HPLC-pure α-factor peptide in 0.1 
M Sodium Acetate obtained from Zymo Research (Irvine, CA, USA). Samples were collected for 
cytometer measurement 8 h after induction, and median fluorescence values were computed from 
the resulting histogram. 
Fluorescence intensity of the detecting strain was measured with a BD Accuri C6 flow 
cytometer equipped with a CSampler plate adapter using excitation wavelengths of 488 and 640 
nm and an emission detection filter at 533 nm (FL1 channel). A total of 10,000 events above a 
400,000 FSC-H threshold (to exclude debris) were recorded for each sample with and core size of 
22 mm using the Accuri C6 CFlow Sampler software. Cytometry data were exported as FCS 3.0 
files and processed using custom Python scripts to obtain the median FL1-A value at each data 
point. 
We used a model fitted to data to estimate the amount of α-factor detected by the 
detecting strain starting from its median fluorescence value response. The model 
interpolates a titration curve of an α-factor-detecting strain that is equivalent to the one used 
in this study, except its synthesis yeGFP fluorescent protein instead of yeVenus.  The 
interpolation follows a standard Hill activation function: 
𝑦 = 𝐾
𝑢𝑛
𝜑 + 𝑢𝑛
+ 𝑦0 
 
where u represents the α-factor input concentration in nM, and y the median fluorescence 
output. To account for the different fluorescent protein, we used the control and the 10  μM 
α-factor median fluorescent outputs from this experiment to re-fit K and y0.  
To estimate the amount of α-factor synthesized by our strains in the hydrogel, we 
inverted the formula above to get: 
76 
 
𝒖 = √𝝋
𝒚 − 𝒚𝟎
𝑲 + 𝒚𝟎 − 𝒚
𝒏
 
 
3.4 RESULTS AND DISCUSSIONS 
3.4.1 Synthesis and Functionalization of the Triblock Copolymer  
ABA triblock copolymers of poly(isopropyl glycidyl ether-stat-ethyl glycidyl ether)-block-
poly(ethylene oxide)-block-poly(isopropyl glycidyl ether-stat-ethyl glycidyl ether) afford shear-
thinning hydrogels that have a tunable sol-gel transition temperature based on the ratio of ethyl 
and isopropyl glycidyl ether monomers (EGE and iPGE, respectively), ‘A’ block chain length, and 
concentration.51  When these polymers were dissolved in aqueous media, flower-like micelles52 
were expected to form based on the design of this ABA triblock copolymer--hydrophobic ‘A’ 
blocks flanking a hydrophilic ‘B’ block. Hydrogels based on this ABA triblock copolymer 
platform (20 wt%) were suitable inks for direct-write 3D printing and were able to create self-
supporting hydrogel structures.52 However, the physical cross-links present in this system were 
insufficient to maintain the integrity of the 3D printed object when subjected to either excess 
aqueous media or to mechanical loads. Thus, chemically cross-linked hydrogels are necessary to 
improve the robustness of the 3D printed objects. 
Polymer 1 was synthesized (Scheme 3.1) via living anionic ring-opening polymerization53–56 
from a poly(ethylene oxide) (PEO) macroinitiator (Mn = 8,000 g mol
-1) with an EGE:iPGE ratio 
of 1.15:1 to afford triblock copolymers with narrow dispersity (Ð = 1.11). Polymerizable 
methacrylate groups were introduced onto the chain-ends of polymer 1 to afford polymer 2 
(Scheme 3.1). We hypothesized that the resulting polymer hydrogel would maintain its thermal- 
77 
 
and shear-responses pre-extrusion, and then photochemically cross-link post-extrusion. The 
degree of chain-end functionalization (𝑓n) was determined by comparing the integrations of the 
methacrylate vinyl (6.12 and 5.55 ppm) and methyl (1.94 ppm) protons, as well as the PEO chain-
end methylene protons (5.08-5.15 ppm), to their theoretical values and was found to be 
quantitative. The polymer was dissolved water (20 wt %) and 2-hydroxy-2-methylpropiophenone 
was added as the photo-radical initiator (0.1 wt%) for polymerization of the methacrylate groups.  
3.4.2 Rheology of the Functionalized Triblock Copolymer Hydrogel 
The viscoelastic properties of a 20 wt% solution of polymer 2 in water were characterized 
using a rheometer. The temperature-dependent viscoelastic behavior of the gel was confirmed by 
the presence of a sol-gel transition as defined by the intersection of the elastic (G’) and viscous 
(G”) moduli (15.61 °C, Figure 3.2a). The solution maintained a non-viscous, liquid-like 
morphology at 5 °C and became a self-supporting hydrogel network by 21 °C. The temperature-
dependent sol-gel transition was also confirmed visually as shown in the photographs in Figure 
B1.  
Hydrogels based on polymer 2 also exhibited shear-thinning behavior. The viscosity of the 
material decreased with increasing applied shear rate, which is indicative of a shear thinning 
hydrogel. An oscillatory strain sweep experiment afforded a yield stress value of 1.33 kPa (Figure 
B2-B3). A dynamic oscillatory strain experiment (Figure 3.2b) demonstrated the strain dependent 
viscoelastic behavior of the gel upon repeated cycles of high (100%) and low (1%) strain. Initially 
under low strain, the material exhibited a gel-like morphology as indicated by greater values for 
G’ relative to G”. During periods of high strain, G” exceeded G’, which indicated that the gel had 
a higher viscous character consistent with the material in its sol state. The material rapidly 
78 
 
recovered to its gel state when the strain was reduced to 1% and exhibited very little mechanical 
hysteresis.  
The post-extrusion UV cure of the gel was simulated under constant strain (1%) and frequency 
(1 Hz). After 120 s of equilibration time, the hydrogel was subjected to 5 mW cm-2 of 365 nm UV 
light for 420 s. The G’ of the hydrogel increased from 21.56 kPa to 94.23 kPa within 75 s of UV 
exposure, and the G” decreased from 1.88 kPa to 0.47 kPa in the same time frame. These changes 
to the G’ and G” were indicative of a rapid chemical crosslinking of the hydrogel network via 
photo-induced radical polymerization of the methacrylated chain ends (Figure 3.3a).  
3.4.3 Direct-Write 3D Printing of Triblock Copolymer Dimethacrylate Hydrogels 
Utilizing these three stimuli responses, a proof of concept cuboidal lattice was 3D printed 
using a pneumatic direct write 3D printer at 5 mm s-1 and 20 psi with a 0.41 mm inner diameter 
conical nozzle. The temperature response allowed for facile processing of the hydrogel into the 
syringe at 5 °C in its liquid state. The hydrogel was then warmed to ambient temperature and 
extruded. The shear-response facilitated the extrusion of the hydrogel as rod-like filaments.  A 
cuboidal lattice structure, with dimensions of 1.9 cm by 1.9 cm by 1.2 cm, was cured post-extrusion 
using a custom-made UV box equipped with two 365 nm A19 UV lamps for 180 s at 3.4 mW cm-
2 (Figure 3.3b-c). 
3.4.4 Incorporation of Yeast Cells and Cell Viability  
To ensure the viability of encapsulated yeast cells within the polymer 2 hydrogel, 
Saccharomyces cerevisiae constitutively expressing mCherry fluorescent protein was inoculated 
into a solution of polymer 2 at 5 °C and mixed to make a homogenous mixture. A film of the 
resulting yeast-laden solution was cast at the same temperature, subsequently warmed to 21 °C to 
79 
 
induce gelation, and cured to create a physically robust hydrogel film. This yeast-laden film 
appeared transparent after processing due to the relatively low loading concentration of yeast 
cells. A small sample of the yeast-laden hydrogel film was extracted, stained, and imaged 
periodically over seven days. Sytox green staining results showed that cells remained 87.5% 
viable within the cast hydrogel at the end of the week of imaging, as seen in Figures 3.4a and 
Figures B4-B6. Significant cell colony growth was observed by both confocal microscopy and 
optical imaging. This growth was also observed visually, as indicated by the opacity of the 3D-
printed hydrogel structure in Figure 3.4b-c. These results suggest that the poly(alkyl glycidyl 
ether)-based hydrogels are comparable in their ability to house and promote yeast cell viability 
over time to our previously employed12 F127-based hydrogels. 
3.4.5 α-Factor Production with 3D Printed AMCALMs  
An engineered yeast strain with upregulated α-factor production was used to demonstrate 
AMCALM lattices that produced a polypeptide. The pGPD promoter present in the secreting yeast 
strain allowed the constitutive expression of the MFα1 gene, and thus, continuous production of 
the α-factor polypeptide. After the reaction, the aqueous reaction media was exposed to the 
detecting strain that fluoresced in the presence of α-factor.  
These lattices were incubated in SC media for a period of 72 h (Figure 3.5), producing an 
average of 268 nM (s = 34.6 nM) of α-factor (Figure 3.6). The induced fluorescence response from 
the cuboidal lattice with the secreting strain was 4.40 times greater that of the control strain 
cuboidal lattice (without upregulated α-factor), which indicated that the up-regulation of the α-
factor pathway was successful in the secreting strain. These results provide evidence that 
AMCALMs were suitable for the production of polypeptides, and that the products could readily 
diffuse out from the hydrogel matrix into the surrounding media. 
80 
 
One advantage of using an immobilized yeast platform for polypeptide synthesis is the 
potential to reuse the printed cuboidal lattices. We investigated the reusability of the poly(alkyl 
glycidyl ether)-based hydrogel living materials for the continued production of α-factor in 
subsequent batch reactions. When the printed AMCALMs were removed from their first 72 h 
incubation, and placed into fresh media, they continued to produce an average of 259 nM (s = 45.1 
nM) of α-factor after a second 72 h cycle (Figure 3.6). The second batch production was directly 
comparable to the output achieved during the first batch reaction, which suggests that these 
immobilized cell bioreactors could be useful for continuous whole-cell catalysis. 
3.5 CONCLUSION 
In conclusion, we developed an ABA triblock copolymer based on a poly(alkyl glycidyl ether) 
that is suitable for 3D printing yeast-laden hydrogels for whole-cell catalysis. These polymer 
hydrogels exhibit a temperature, shear, and UV-light responsive behaviors that are integral to the 
preparation of the hydrogel ink and subsequent printing. The post-extrusion, chemical crosslinking 
of the polymer micelles induced by UV-light is particularly important to fabricate robust forms 
that do not degrade or dissolve over time. 
The poly(alkyl glycidyl ether) based hydrogels also proved to have a negligible effect on the 
viability and biological activity of the encapsulated yeast cells. The engineered yeast-laden living 
materials were shown to be capable of producing an average of 263.5 nM of α-factor during two 
consecutive batch reactions, exhibiting no significant reduction in efficiency between the two 
cycles.  
In this work, we demonstrated that polypeptides can be produced using the AMCALM 
platform. This result, combined with the prominence of the α-factor leader sequence in 
81 
 
recombinant protein design and production, suggests that that these materials could be employed 
in the whole-cell catalysis of other higher-value molecules in a sustainable and continuous manner. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
82 
 
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87 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.1 A graphical overview of the three key stimuli responses of polymer 2 hydrogels 
necessary for AMCALM applications. (a) The temperature responsive feature of the hydrogels 
enabled facile loading of the hydrogel material at 5 °C. (b) The shear-stress response facilitated 
the formation of complex three-dimensional objects at 21 °C. (c) UV-light (365 nm) initiated the 
polymerization of the methacrylate end-groups and chemical crosslinking of the polymer 2 
hydrogel. (d) The chemical structure of polymer 2. The letter designations (z = 6.4, y = 7.4, x = 
182) refer to the average number of isopropyl glycidyl ether, ethyl glycidyl ether, and ethylene 
oxide repeat units, respectively. 
88 
 
 
 
 
 
Scheme 3.1 Synthesis of the ABA triblock copolymer (polymer 1) and functionalization with 
methacrylic anhydride (polymer 2). The letter designations (z = 6.4, y = 7.4, x = 182) refer to 
degree of polymerization for isopropyl glycidyl ether, ethyl glycidyl ether, and ethylene oxide, 
Figure 3.2 Rheometrical experiments for a 20 wt% formulation of polymer 2. (a) Dynamic 
oscillatory temperature ramp displaying elastic (G’, filled) and viscous (G’’, open) moduli. Tgel = 
15.61 °C. (b) Cyclic strain experiment demonstrating rapid recovery of hydrogel elastic modulus 
(black circles) from periods of high (100%) to low (1%) oscillatory strain (red circles). Arrows 
indicate reference axis; elastic/viscous moduli (left axis) and oscillatory strain (right axis). 
89 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.3 (a) A rheological UV-cure experiment using a 20 mm parallel plate geometry. The 
hydrogel was equilibrated for 120 s before being subjected to 5 mW cm-2 of 365 nm UV light for 
420 s at a constant strain (1%) and frequency (1 Hz). (b-c) A 3D printed, proof of concept cuboid 
structure (1.9 cm by 1.9 cm by 1.2 cm). This structure was printed from a pneumatic direct write 
3D printer at 5 mm s-1 and 20 psi using a 0.41 mm inner diameter nozzle (scale bar: 1 cm). 
Figure 3.4 (a) A sample composite image of live cells (green channel) and dead cells (red channel) 
after 7 days of incubation (scale bar: 200 µm). (b) Side and (c) top-down view of the 3D-printed, 
yeast-laden hydrogel after two rounds of α-factor synthesis in SC media (scale bar 1 cm).
90 
 
 
 
 
 
 
Figure 3.5 Immobilized yeast within the hydrogel matrix were incubated in SC media for 72 h at 
30 °C with automated shaker agitation. The media was collected at the end of the incubation 
period and exposed to the receiver strain in a separate tube, and the resulting fluorescence was 
measured. The cuboidal lattice was reused in a subsequent incubation with fresh media to 
produce an additional batch of α-factor. 
91 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.6 Fluorescence values obtained from the quantification of α-factor produced from the 
wild type (control) AMCALMs and the secreting strain AMCALMs. Round 1 indicates the first 
incubation period of 72 h, while Round 2 indicates the reemployment of the same printed materials 
in a second, subsequent 72 h batch reaction in fresh SC media. 
92 
 
CHAPTER 4: THE POST-FUNCTIONALIZATION OF MULTI-
STIMULI-RESPONSIVE HYDROGELS FOR DIRECT-INK 
WRITE ADDITIVE MANUFACTURING 
4.1 ABSTRACT 
 Herein, we describe the development of a multi-stimuli-responsive hydrogel platform 
capable of post-functionalization with thiol-bearing molecules as inks for DIW 3D printing. The 
poly(alkyl glycidyl ether) methacrylate (PGE-MA) hydrogels maintain three key stimuli-
responses necessary for extrusion based additive manufacturing: a sol-gel temperature response, 
shear-thinning behavior, and the ability to photochemically crosslink. In addition, the chemically 
crosslinked hydrogels demonstrated a temperature dependent swelling consistent with LCST 
behavior. Pyridyl disulfide urethane methacrylate (PDS-UM) monomers were introduced to the 
network as a thiol-reactive handle for post-functionalization of the hydrogel. The reactivity of 
PGE-MA/PDS-UM hydrogels were investigated at different temperatures (5, 25, 37 °C) and 
swelling statuses (as cured/preswollen) using glutathione as a reactive probe. To illustrate the 
versatility of the PGE-MA/PDS-UM platform, a number of additional thiol-containing probes 
such as proteins, polymers, and small molecules were conjugated to the hydrogel network at 
various temperatures, pH’s, and concentrations. In a final demonstration of the multi-stimuli-
responsive hydrogel platform, a customized DIW 3D printer was used to fabricate a robot 
figurine that was subsequently conjugated with a fluorescent tag and displayed the ability to 
change in size with environmental temperature. 
93 
 
4.2 INTRODUCTION 
Hydrogels are water-swollen networks of macromolecules bound by chemical or physical 
crosslinks. Their soft nature and inherently high water content (typically 70-99%) mimic the 
natural environment of living tissues, making them ideal for tissue engineering1–3 and wound 
healing applications.4,5 Stimuli-responsive hydrogels react to environmental cues such as 
temperature,6 light,7 or mechanical stress8,9 to induce a chemical or physical change in material 
properties. These hydrogels have recently been adopted in the field of additive manufacturing, a 
technique that utilizes computer aided hardware and software for the sequential deposition of a 
material to fabricate three-dimensional objects.10,11 Direct-ink write (DIW) additive manufacturing 
is one such method that can use shear-thinning hydrogels to facilitate flow of the material through 
fine resolution nozzles.12–15 Certain stimuli-responses, such as shear-thinning behavior, enable 
effective printing while others create objects with advanced functionality after printing such as 
actuation,16–18 shape memory,19–21 and pH dependent drug release.22,23    
Stimuli-responsive triblock copolymers can afford versatile gel-based inks for DIW 3D 
printing.24–29 Our group has investigated and developed poly(alkyl glycidyl ether)-based hydrogels 
that exhibit temperature-, shear-, and light-responsive properties.30–32 The sol-gel temperature 
response enables facile homogenization of additives while the shear-thinning response facilitates 
the extrusion of the material from the printhead nozzle and generates the 3D printed object. The 
material can then be chemically cross-linked using photo-initiated free radical polymerization to 
create robust objects capable of withstanding dissolution and deformation.  
The conjugation of molecules to hydrogel materials under mild conditions with high 
specificity offers a straightforward method of introducing additional functionality to these 
systems.33–38 For instance, the Anseth group developed a PEG/protein hydrogel with orthogonal 
94 
 
alkene functionality for the conjugation of photopatterned biomolecules. Thiol-ene click chemistry 
was used to modify the existing hydrogel with the cell-adhesion tripeptide motif Arg-Gly-Asp 
(RGD) for cell attachment with spatial control.39 Alternatively, azide-alkyne click reactions were 
utilized by Sanyal and coworkers for the post-functionalization of dendron-polymer conjugate 
hydrogels. While some of the alkynes were utilized as crosslinking points to form the hydrogel, 
residual alkyne groups were available for the covalent attachment of additional azide-containing 
molecules including fluorescent dyes (4-azido-N-ethyl-1,8-naphthalimide and BODIPY) and 
biotin for protein immobilization.40 Adapting such techniques to the post-functionalization of 3D 
printed systems could enable facile modification of hydrogel materials for a multitude of post-print 
applications. 
Pyridyl disulfide is a versatile motif used in a number of applications as a reactive handle 
for thiol-containing molecules.41 The efficient thiol-pyridyl disulfide exchange enables 
conjugation of small-to-large molecule reactants including peptides,42–44 proteins,45–47 and even 
RNA48–50 with high specificity under mild conditions. Additionally, the 2-pyridothione leaving 
group has a convenient absorbance spectrum that allows for methodical tracking of conjugation 
rates by monitoring the evolution of the 343 nm absorbance peak via UV-Vis spectroscopy.  
While the utilization of pyridyl disulfides in polymeric systems has been investigated, its 
use as a  reactive functionality towards the post-functionalization of hydrogels has not been 
thoroughly explored. One recent example includes work by Sanyal and co-workers to post-
functionalize PEG hydrogels with thiolated biotin to immobilize TRITC−extravidin proteins and 
RGD to immobilize HUVEC cells.51  
Herein, we report the post-functionalization of multi-stimuli-responsive hydrogels for DIW 
3D printing. Methacrylated poly(isopropyl-stat-ethyl glycidyl ether)1.6k-block-poly(ethylene 
95 
 
oxide)8.0k-block-poly(isopropyl-stat-ethyl glycidyl ether)1.6k (PGE-MA) based hydrogels 
demonstrated temperature-dependent equilibrium swelling ratios (ESRs) consistent with lower 
critical solution temperature (LCST) behavior. Pyridyl disulfide urethane methacrylate (PDS-UM) 
monomers were successfully incorporated into PGE-MA hydrogels as a platform for post-
functionalization with thiol-containing molecules. Glutathione (GSH) was used as a small 
molecule probe to measure the conjugation kinetics of PGE-MA/PDS-UM hydrogels at different 
temperatures (5, 25, 37 °C) and swelling states (as cured/preswollen). Other thiol-containing 
molecules (small molecules, polymer, and protein) were also conjugated onto PGE-MA hydrogels. 
To demonstrate the potential for these materials, we PGE-MA/PDS-UM hydrogels were DIW 3D 
printed and post-print functionalized with fluorescein thiol (FITC-SH). The differential swelling 
in response to environmental temperatures was also confirmed for these printed objects. 
4.3 MATERIALS AND METHODS 
4.3.1 MATERIALS 
 All chemicals and solvents were purchased from Sigma-Aldrich, Fisher Scientific, or 
Oakwood Chemical and used without further purification unless noted otherwise. CYCLO(ARG-
GLY-ASP-D-PHE-CYS) (RGD-SH) was purchased from AstraTech. RGD-PEG-SH was 
purchased from Biochempeg Scientific. Isopropyl glycidyl ether (iPGE, 98%) and ethyl glycidyl 
ether (EGE, 98%) were dried over CaH2 for 24 h, distilled into a flask containing butyl magnesium 
chloride (2 M in tetrahydrofuran, THF), re-distilled, and stored under N2 atmosphere. 
Poly(ethylene oxide) (PEO, Mn 8000 g mol-1) was dried under vacuum overnight prior to use. 
Dry THF was obtained using neutral alumina using a Pure Process Technology solvent purification 
system. A potassium naphthalenide solution (1M) was prepared by dissolving naphthalene (3.2 g) 
in THF (25 mL), adding potassium (0.975 g), and storing under N2 atmosphere. 1H NMR spectra 
96 
 
were obtained on a Bruker Advance 300 or 500 MHz spectrometer. All absorbance spectra were 
collected on either a Varian Cary 5000 UV-Vis-NIR or Agilent 8453 UV-Vis spectrophotometer. 
4.3.2 SYNTHESIS OF HEPDS 
Hydroxyethyl pyridyl disulfide was synthesized (Scheme C1) according to literature 
procedures. Briefly, dipyridyl disulfide (5 g, 23 mmol) and glacial acetic acid (0.6 mL) were 
dissolved in MeOH (60 mL). A solution of 2-mercaptoethanol (1.41 mL, 20 mmol) in MeOH (25 
mL) was added dropwise to the reaction solution at over 30 min and the solution was allowed to 
stir at RT for 24 h. The solvent was then removed under reduced pressure and the product was 
redissolved in minimal ethyl acetate. The solution was washed with deionized water (3x), dried 
with anhydrous sodium sulfate, and filtered. After concentrating again under reduced pressure, 
the product was purified via silica gel column chromatography using an ethyl acetate/hexane 
mixture (17:3) as the elution solvent. The product was obtained as a light-yellow oil (1.58 g, 
38% yield). 1H NMR (499 MHz, CDCl3): δ = 8.52-8.50 (d, 1H), 7.60-7.57 (t, 1H), 7.42-7.40 (d, 
1H), 7.17-7.14 (t, 1H), 3.82-3.80 (t, 2H), 2.97-2.95 (t, 2H). 
4.3.3 SYNTHESIS OF PDS-UM 
Pyridyl disulfide-urethane methacrylate (PDS-UM) was synthesized (Scheme C2) using 
modified literature procedures52. HEPDS (1.58 g, 8.44 mmol) was dissolved in dry THF (5 mL) 
and added to a flame dried reaction flask. 2-isocyanoethyl methacrylate (1.43 mL, 10.12 mmol) 
and dibutyltin dilaurate (19.75 μL) were subsequently added under inert N2 atmosphere and the 
reaction mixture was allowed to stir at RT for 20 h. The solvent was then removed under reduced 
pressure. The impure product was redissolved in DCM and was washed with deionized water and 
brine. The product was purified via silica gel column chromatography using an ethyl 
97 
 
acetate/hexane mixture (2:3) as the elution solvent. The product was obtained as a white powder 
(2.41 g, 88% yield). 1H NMR (499 MHz, CDCl3): δ = 8.48-8.47 (d, 1H), 7.70-7.62 (m, 2H), 7.11-
7.08 (t, 1H), 6.12 (s, 1H), 5.60 (s, 1H), 4.99 (s, 1H), 4.34-4.32 (t, 2H), 4.24-4.21 (t, 2H), 3.51-3.47 
(q, 2H), 3.05-3.03 (t, 2h), 1.95 (s, 3H). 
4.3.4 PREPARATION OF PGE-MA/PDS-UM HYDROGEL SOLUTION 
For the formulation of a 5 g batch of hydrogel, PGE-MA was first dissolved in deionized 
water at a concentration of 20 wt % polymer. The resulting polymer solution was cooled overnight 
at 5 °C to facilitate complete dissolution of the polymer via LCST behavior.  Using the 
temperature-responsive sol-gel transition of the polymer, a 0.088 M solution of PDS-UM in MeOH 
(100 μL) and the photo-radical initiator 2-hydroxy-2-methylpropiophenone (5 μL) were added to 
the sol state at 5 °C and homogenized using a vortex mixer for 30 s. The hydrogel was centrifuged 
(4400 rpm, 10 min) to remove bubbles. 
4.3.5 HYDROGEL SAMPLE PREPARATION 
Hydrogel solutions were poured into nine disk molds (r = 5 mm, h = 5 mm) and cured 
under 365 nm UV light for 15 min. The nine cured disks were washed with water to remove any 
unreacted polymer from the sample surface.  
4.3.6 PGE-MA SWELLING STUDIES 
The swelling rates of PGE-MA hydrogels were studied at three different temperatures (5, 
25, and 37 °C) and three different swelling states (dry, as cured, and preswollen).  Three hydrogel 
samples were placed in scintillation vials containing deionized water at their respective 
temperatures and weighed at time points from 0-48 h. The swelling ratio was determined using the 
following equation:  
98 
 
𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑅𝑎𝑡𝑖𝑜 =
𝑊𝑡 − 𝑊𝑖
𝑊𝑖
 
Where Wt refers to the weight of the hydrogel sample at time point t and Wi  refers to the weight 
of the initial dried or as cured sample. Swelling reversibility studies were conducted in a similar 
manner. Three hydrogel samples were placed in scintillation vials containing deionized water in a 
37 °C environment. The samples were allowed to equilibrate and weighed after 1 day. The samples 
were returned to the scintillation vials and placed in a 5 °C environment and weighed again after 
1 day. This procedure was repeated five times for a total of 10 days. 
4.3.7 2-PYRIDOTHIONE CALIBRATION 
A calibration curve was established by dissolving various concentrations of 2-pyridothione 
(0.01 – 0.5 mM) in deionized water and measuring the corresponding absorbance values at λ = 343 
nm. A linear regression analysis of the absorbance vs concentration plot produced a linear fit with 
an R2 value of 0.999. 
4.3.8 2-PYRIDOTHIONE RELEASE STUDIES 
The release rates of PGE-MA/PDS-UM hydrogels were studied at three different 
temperatures (5, 25, and 37 °C) and three different swelling states (dry, as cured, and preswollen).  
GSH was chosen as the free-thiol reactant molecule at concentrations typically found in tumor 
cells (10 mM). Three hydrogel samples were placed in scintillation vials containing a GSH solution 
(7 mL, 10 mM in deionized water) at their respective temperatures. Periodically over the course 
of 96 h, aliquots were taken for UV-Vis analysis. The percent release was determined using the 
absorbance at λ = 343 nm corresponding to the released 2-pyridothione and the following equation: 
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑟𝑒𝑙𝑒𝑎𝑠𝑒 (%) =
𝐴𝑏𝑠𝑡
𝑀𝑐 ∗  
0.0006𝑔 𝑃𝐷𝑆 − 𝑈𝑀
1𝑔 𝐻𝑦𝑑𝑟𝑜𝑔𝑒𝑙 ∗
1
342.44
𝑔
𝑚𝑜𝑙
∗ 1000 ∗
1
7 + 𝛿 𝑚𝐿 ∗ 1000 ∗ 5.4776
∗ 100 
99 
 
Where Abst refers to the absorbance of the reaction solution at time t and Mc refers to the mass of 
the as cured samples. Any water added from the hydrogel samples (𝛿) was taken into account and 
added to the initial volume (7 mL). 
4.3.9. DIRECT-WRITE 3D PRINTING OF PGE-MA/PDS-UM HYDROGELS 
A modified pneumatic direct-write 3D printer was assembled based on a Tronxy P802E 
3D Printer kit, from Shenzen Tronxy Technology Co. The CAD model was designed on Fusion 
360 and the G-code file was produced with Slic3r software. The hydrogel ink was cooled to 5 °C 
and poured into a Nordson Optimum 10 cc fluid dispensing barrel equipped with a Metcal conical 
(410 µm inner diameter) precision tip nozzle. The loaded syringe was warmed to ambient 
temperature and pressurized using nitrogen gas (20 psi) to extrude the gel from the nozzle at 5.0 
mm s-1. The printer was controlled with an Arduino using the Marlin firmware.  
4.4 RESULTS AND DISCUSSION 
4.4.1. TEMPERATURE DEPENDENT HYDROGEL SWELLING 
PGE-MA triblock copolymers were synthesized via anionic ring-opening polymerization 
of ethyl or isopropyl glycidyl ether initiated from poly(ethylene glycol) using potassium 
naphthalenide as a base and functionalized using methacrylic anhydride. The lower critical 
solution temperature (LCST) driven sol-gel transition is ideal for material processing, while the 
shear-thinning response enables effective extrusion-based additive manufacturing. Photo-chemical 
crosslinking of the material through UV-light initiated polymerization of methacrylate chain ends 
creates robust hydrogels capable of post-print bioreactor applications. However, the permanent 
fixation of the network removes the sol-gel and shear-thinning response of the material, as the 
polymer chains are now covalently bound and no longer have the freedom to disassemble at lower 
100 
 
temperatures or under high shear environments. Interestingly, chemically crosslinked polymers 
such as F12753–55 or poly(N-isopropyl acrylamide) (PNIPAm)56–58 are known to exhibit volume 
phase transitions as a result of LCST behavior. Therefore, we hypothesized that PGE-MA 
hydrogels would maintain their temperature-responsive behavior after crosslinking in the form of 
temperature dependent equilibrium swelling ratios (ESR) (Figure 4.1).  
To investigate this behavior, crosslinked hydrogel samples (20 wt% PGE-MA in deionized 
water)  were placed in aqueous solutions at 5, 25, and 37 °C and their swelling ratios were recorded 
periodically over 48 h (Figure 4.2a). At low temperatures (5 °C), the hydrogels experience 
enhanced swelling, achieving an ESR of 53.0 ± 0.8%. Meanwhile, at room (25 °C) or elevated (37 
°C) temperatures, the material reaches an ESR of 28.5 ± 1.3 and 8.6 ± 0.8%, respectively. This is 
consistent with LCST behavior: As the temperature of the environment decreases, the system 
absorbs a higher degree of water as the polymer becomes more hydrophilic and the glycidyl ether 
blocks become fully solvated. With increasing temperatures, the polymer chains contract as they 
become more hydrophobic, and the material equilibrates at lower swelling ratios. All hydrogel 
samples reached final ESR values after 24 h of incubation. 
Similar to the reported sol-gel transition, the ESR of the crosslinked hydrogel is reversible 
and showed no loss in mass or change in ESR over 5 cycles of alternating between 5 and 37 °C  
(Figure 4.2b).  Hydrogel samples originally equilibrated at 5 °C experienced a drop in ESR when 
placed in high (37 °C) temperature environments. Once placed back into low temperature 
solutions, the hydrogel samples recover back to their highly swollen state.  
4.4.2. FORMULATION AND RHEOLOGICAL CHARACTERIZATION OF PGE-MA/PDS-UM HYDROGELS 
PDS-UM monomers were synthesized using a modified procedure52 and incorporated into 
PGE-MA hydrogels to create a hydrogel that can be post-functionalized with thiol-containing 
101 
 
molecules (Scheme C1-C2). PDS monomers are naturally hydrophobic and often require 
fabrication in organic solvents51,59,60  or functionalization onto hydrophilic polymers61,62 in order 
to prepare PDS containing hydrogels. One unique aspect of amphiphilic triblock copolymers such 
as PGE-MA is their ability to self-assemble into flower-like micelles and encapsulate hydrophobic 
molecules such as PDS-UM.63–67 The inherent sol-gel temperature transition of PGE-MA 
hydrogels was used to efficiently dissolve and homogenize the PDS-UM monomer at a molar ratio 
of 1:10 (PDS-UM:PGE-MA). The PGE-MA/PDS-UM formulation was optimized by visually 
comparing different concentration of PDS-UM to ensure maximum loading within the micelles 
while maintaining good solubility.  
Rheological characterization of PGE-MA/PDS-UM hydrogels confirmed that the 
incorporation of PDS-UM did not negatively impact the temperature-, shear-, or light-responsive 
properties of the material (Figure C1). The temperature-dependent viscoelastic character of the 
material was confirmed by the presence of a sol-gel transition (T = 14.32 °C) as defined by the 
crossover between the solid-like character of the material (storage modulus, G’) and the liquid-
like character of the material (loss modulus, G”) (Figure C1a). The material exhibited shear-
thinning behavior as characterized by a negative correlation between viscosity and shear rate, 
illustrating the shear-induced flow required for extrusion-based printing methods (Figure C1b). A 
dynamic oscillatory strain experiment demonstrated the rapid and reversible response of the 
material to periods of high (100%) and low (1%) strain (Figure C1c). At low strain, such as those 
experienced by the hydrogel before and after printing, the material exists as a gel with G’ values 
greater than G”. Under periods of high strain, such as those experienced during extrusion, G” 
values were greater than G’ indicating the material was able to flow. Finally, the photochemical 
crosslinking of the hydrogel network was confirmed by increasing G’ values upon exposure to 365 
102 
 
nm UV light (Figure C1d). This indicates a rapid polymerization of methacrylate chain ends, the 
formation of chemical crosslinks, and the stiffening of the hydrogel network. 
4.4.3. REACTIVITY OF PGE-MA/PDS-UM HYDROGELS TOWARD THIOLS 
Given the temperature-responsive behavior of PGE-MA/PDS-UM hydrogels, we further 
investigated the effect of the thermally responsive swelling behavior upon the reactivity of the 
PDS-UM toward thiols. We hypothesized that the LCST response of the PGE-MA network will 
afford fully solvated polymer chains at lower temperatures and the exposed PDS-UM monomer 
would be more reactive. To test this hypothesis, as-cured PGE-MA/PDS-UM hydrogel samples 
were placed in pre-equilibrated solutions of glutathione (GSH) at 5, 25, and 37 °C. The conjugation 
of GSH with the PDS-UM monomer can be tracked by monitoring the absorbance peak of released 
2-pyridothione at 343 nm. A calibration curve was established by dissolving 2-pyridothione in 
aqueous solutions at various concentrations (0.1-5 mM, Figure 4.3).  
An aliquot of each solution was taken at various time points over the course of 96 h and 
investigated by UV-Vis spectroscopy. The percent release was calculated relative to the theoretical 
concentration of 2-pyridothione, assuming complete reaction of all PDS-UM active sites and 
plotted versus time (Figure 4.4a). Contrary to our hypothesis, the release rates of 2-pyridothione 
from PGE-MA/PDS-UM hydrogels were faster at smaller ESRs and higher temperatures, and the 
release rates were slower at greater ESRs and lower temperatures (Table 4.1). For instance, the 
percent release of 2-pyridothione at 24 h for samples equilibrated at 37 °C was 10.4 % higher than 
at 25 °C and 23.8 % higher than at 5 °C. The largest difference in percent release was observed 
throughout the first 24 h of incubation, with near quantitative release of 2-pyridothione at all 
temperatures within 96 h. 
103 
 
These results suggest that as-cured PGE-MA/PDS-UM hydrogels do, in fact, exhibit 
temperature-dependent release profiles, albeit with an inverse correlation between ESR and release 
rate. Interestingly, when hydrogel samples are preswollen before placement in reactive GSH 
solutions (Figure 4.4b), the magnitude of 2-pyridothione release is significantly diminished. Not 
only were percent release values lower during the early stages of release (24 h = 46.2 vs 60.8, 53.0 
vs 74.2 and 50.8 vs 84.6 for 5, 25, and 37 °C, respectively) but the final maximum release values 
never broke 73 %. Additionally, the temperature dependent release profiles observed for as cured 
samples are minimized, if not entirely removed. For instance, samples equilibrated at 25 °C had 
the highest percent release of 2-pyridothione at 24 h, 2.2 % greater than samples at 37 °C and 6.8 
% more than samples at 5 °C.  These results suggest that the active swelling of the hydrogel 
network has a major impact on the magnitude and temperature dependency of release kinetics for 
PGE-MA/PDS-UM hydrogels. 
4.4.4. THIOL CONTAINING MOLECULAR LIBRARY 
To demonstrate the versatility of our PGE-MA/PDS-UM platform, a number of different 
thiol-containing molecules were conjugated to the hydrogels. The percent release of 2-
pyridothione after 24 and 96 h for each compound was recorded in Table 4.2. Cysteamine and 2-
mercaptoethanol (2-MCE) were chosen as model compounds to demonstrate the ability of our 
platform to conjugate molecules in the presence of polar hydrogen bonding functionalities (-NH2 
and -OH). RGD-SH and RGD-PEG-SH (Mn = 5,000) were included due to their cell adhesive 
capabilities as well as to demonstrate the ability to conjugate large polymeric compounds. 
Similarly, BSA (Mn = 66kD) with a single free cysteine reacted with up to 12.9% of the available 
PDS-UM sites. The larger macromolecules, BSA and PEG conjugated significantly less (12.9 % 
and 30.9 %, respectively) than the smaller molecular probes GSH/RGD (97.9 % and 80.4 %, 
104 
 
respectively). This can be attributed to the large increase in molecular weight (1-2 orders of 
magnitude) for the same number of available thiols (1) per molecule.  
Meanwhile, 2,2- (Ethylenedioxy)diethanethiol (EDT) and pentaerythritol tetra(3-
mercaptopropionate) (PET) were incorporated to demonstrate the conjugation of multi-functional 
small molecules. A stoichiometric amount of EDT (2x thiol) and PET (4x thiol) relative to thiol 
equivalent (0.5 and 0.25 molar equivalents) were added to the reaction solution. Conjugation 
values of 77.4 and 33.2 % suggest that one molecule of EDT/PET is reacting with multiple PDS-
UM reactive sites, as a 1:1 conjugation ratio would yield values of 50 and 25%, respectively. 
Because EDT and PET are both small molecules and cannot be expected to bridge the gap between 
multiple micelles, these results further suggest that multiple PDS-UM monomers are located 
within the hydrophobic core of a single micelle. 
Lastly, we investigated the effect of pH upon the reaction of thiols with PGE-MA/PDS-
UM. Bagiyan and co-workers previously reported that thiols can undergo spontaneous auto-
oxidation in the presence of molecular oxygen in aqueous solutions. According to their results, the 
rate of auto-oxidation to disulfides is affected by both pH and temperature. For instance, the auto-
oxidation rate of cysteamine was reportedly 15 - 20 times larger at a pH of 8.5 - 9.0 than 4 – 5, and 
6 times larger at 45 °C compared to 15 °C. It was therefore necessary to modulate both the pH and 
temperature of our reaction solutions to suppress auto-oxidation of both the conjugating molecule 
as well as the 2-pyridothione leaving group. GSH, a naturally acidic compound containing two 
carboxylic acid moieties, undergoes efficient conjugation at pH  3 and 37 °C. Cysteamine 
hydrochloride, EDT, PET, RGD, and RGD-PEG-SH were reacted in solutions with the pH 5, while 
BSA and 2-MCE were conjugated in a phosphate buffer solution (1x PBS, pH 7.4). These 
compounds were therefore incubated at 25 and 5 °C, respectively, to account for the increasing 
105 
 
basicity of the reaction solutions. All molecules were able to achieve efficient conjugation under 
these reaction conditions. 
4.4.4. 3D PRINTING AND POST-FUNCTIONALIZATION OF PGE-MA/PDS-UM HYDROGELS 
As a final demonstration, we 3D printed hydrogel objects that were post-print 
functionalized with a fluorescent dye. A customized DIW 3D printer was used to extrude hydrogel 
ink in a patternwise manner to create the robot figurine shown in Figure 4.5. The sol-gel 
temperature response allowed for homogenization of the PDS-UM monomer, as well as facile 
loading of the material into the printer syringe. The shear-thinning response facilitated the 
formation of rod-like filaments and enabled effective printing of the material into the computer-
generated object. The post-print photochemical crosslinking allowed for handling and swelling of 
the object without deformation or dissolution. A thiolated fluoresceine dye (FITC-SH) was 
synthesized according to literature procedures68 and conjugated to the material via thiol-reactive 
PDS-UM groups (Figure 4.5). The figure demonstrated high fluorescence intensity with no 
observable decline after successive equilibrations with fresh buffer solution. And finally, the 
temperature dependent ESRs allow the printed object to grow/shrink based on environmental 
conditions. The printed figure achieved an upper limit size of 5.2 x 7.1 cm at 5 °C (Figure 4.5a), 
and a minimum size of 4.75 x 6.34 cm at 37 °C (Figure 4.5b).  
4.5 CONCLUSION 
In conclusion, we developed a multi-stimuli-responsive hydrogel platform capable of post-
functionalization with thiol-containing molecules for DIW additive manufacturing. Crosslinked 
PGE-MA hydrogel samples demonstrated temperature dependent equilibrium swelling ratios 
consistent with LCST behavior i.e., higher degrees of swelling at lower temperatures. PDS-UM 
106 
 
monomers were incorporated into the network, providing the hydrogel with a thiol-reactive handle 
for post-functionalization. The reactivity of PGE-MA/PDS-UM hydrogels was investigated at 
different temperatures (5, 25, 37 °C) and swelling statuses (as cured/preswollen). The conjugation 
rates were found to increase with temperature and were faster in as cured samples. The versatility 
of the platform was demonstrated by conjugating a diverse set of thiol-containing probes to the 
hydrogel including peptides, proteins, polymers, and multi-functional small molecules at various 
temperatures, pH values, and concentrations. Lastly, the multi-stimuli-responsive properties of the 
PGE-MA/PDS-UM system were demonstrated by post-functionalizing a 3D printed robot figurine. 
The sol-gel temperature response, shear-thinning behavior, and photochemical crosslinking 
properties enabled efficient DIW printing. The printed figurine was post-functionalized with the 
thiolated fluorescent tag, FITC-SH, and displayed changes in size induced by the temperature 
dependent swelling of the PGE-MA hydrogel. 
 
 
 
 
 
 
 
 
 
 
107 
 
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Hydrophobic Comonomers on the Volume Phase Transition Temperatures and Thermal 
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Poly(N-Isopropylacrylamide)-Based Thermoresponsive Composite Hydrogels for 
Biomedical Applications. Polymers. MDPI AG March 1, 2020, p 580. 
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112 
 
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Chem. 2017, 292 (40), 16539–16553. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
113 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 4.1 A graphical representation of the LCST-driven temperature dependent swelling 
behavior of PGE-MA hydrogels. 
(a) (b) 
Figure 4.2 (a) Swelling ratio (%) vs Time (h) for PGE-MA/PDS-UM samples at 5 (blue squares), 
25 (black triangles), and 37 °C (red circles). (b) Demonstration of reversible swelling from 5 °C 
to 37 °C over ten days. 
114 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 4.4 Percent release of 2-pyridothione (%) vs time (h) for (a) as-cured and (b) preswollen 
PGE-MA/PDS-UM samples at 5 (blue squares), 25 (black triangles), and 37 °C (red circles). 
 
 (a) (b) 
Figure 4.3 (a) Absorbance profiles and (b) calibration curve of 2-pyridothione (0.1-0.5 mM) in 
deionized water. 
(b) (a
) 
115 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Temperature (°C) Swelling status 24h (%) 96h (%) 
5 
5 
25 
25 
37 
37 
Preswollen 
As cured 
Preswollen 
As cured 
Preswollen 
As cured 
46.2 ± 6.0 
60.8 ± 6.4 
53.0 ± 4.2 
74.2 ± 8.8 
50.8 ± 10.1 
84.6 ± 6.6 
71.6 ± 3.6 
93.1 ± 8.2 
72.6 ± 4.7 
98.5 ± 13.0 
66.1 ± 5.5 
102.3 ± 10.2 
Molecule % Release (24 h) % Release (96 h) Equivalents pH Temperature (°C) 
GSH 
Cysteamine 
EDT 
PET 
RGD-SH 
RGD-PEG-SH 
2-MCE 
BSA 
79.3 
80.9 
80.4 
30.3 
71.6 
24.3 
87.5 
10.1 
97.9 
77.0 
77.5 
33.2 
80.4 
30.9 
90.2 
12.9 
40 
40 
0.5 
0.25 
5 
5 
40 
1 
3 
5 
5 
5 
5 
5 
7.4 
7.4 
37 
25 
25 
25 
25 
25 
5 
5 
Table 4.1 Percent release of 2-pyridothione (%) at different temperatures (5, 25, 37 °C), swelling 
statuses (as cure/preswollen), and time points (24/96 h).  
 
Table 4.2 Conjugation of thiol-containing molecules to PGE-MA/PDS-UM hydrogels. The 
percent release of 2-pyridothione was determined via UV-Vis spectroscopy at 24 and 96 h. The 
pH (3, 5, and 7.4) and temperature (5, 25, 37 °C) were modified to suppress auto-oxidation of the 
conjugating molecule and the 2-pyridothione leaving group.    
116 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
(a) (b) 
Figure 4.5 A 3D printed robot figurine was printed using a custom made, pneumatic DIW printer 
with a 0.41mm inner diameter nozzle at 5.0 mm/s. The fluorescent tag FITC-SH was conjugated 
to the object and the figurine was swelled at (a) 5°C and (b) 37 °C to demonstrate the thiol-
reactivity and temperature dependent swelling of the PGE-MA/PDS-UM system (scale bars: 1cm). 
117 
 
APPENDIX A 
Tunable Temperature- and Shear-Responsive Hydrogels Based on Poly(alkyl glycidyl Ether)s 
 
N-propyl Glycidyl Ether Synthesis: N-propyl glycidyl ether was synthesized according to 
literature from epichlorohydrin (34.4 mL, 0.44 mol) and 1-propanol (32.9 mL, 0.44 mol) in 
hexanes at reflux for 6 h.  Potassium hydroxide pellets (56.11 g, 1 mol) were used as base with 
tetrabutylammonium bromide (1.77g, 5.5 mmol) as a phase transfer catalyst.  The crude product 
was filtered, washed three times with brine, concentrated under reduced pressure, and purified 
via vacuum distillation to afford a yellow oil (15 g, 30% yield). 1H NMR (300 MHz, CDCl3): δ 
0.91-0.95 (t, 3H, J = 7.5 Hz), 1.55-1.67 (sext, 2H, J = 7.5 Hz), 2.60-2.62 (dd, 1H, J = 4.5, 8.5 
Hz) 2.78-2.81 (dd, 1H, J = 7.0, 8.5) 3.12-3.17 (m, 1H) 3.35-3.52 (m, 2H) 3.69-3.74 (dd, 1H, J = 
5, 19.5) 
 
General synthesis of poly(alkyl glycidyl ethers): All alkyl glycidyl ether homopolymers were 
synthesized by utilizing the same synthetic procedure with different monomer feed ratios. The 
following poly(isopropyl glycidyl ether) synthetic scheme will serve as an example. The 
initiator, 4-methyl benzyl alcohol (0.122 g, 1 mmol) was added to an oven dried, 100 mL round 
bottom flask. Potassium naphthalenide solution (1M in THF) was titrated until a light green paste 
was formed. The reaction flask was evacuated overnight to drive off the remaining THF. 
Isopropyl glycidyl ether (6.49 mL, 51.56 mmol) was added to the dried mixture of deprotonated 
initiator, and the reaction was stirred at 70 °C for 45 h. The polymer solution was quenched 
using a degassed 1% v/v AcOH in MeOH solution and dialyzed against MeOH for 3 d (3 solvent 
exchanges) using Spectra/Por regenerated cellulose tubing (MWCO 1.0 kDa) that was pre-
118 
 
soaked in water. The dialyzed polymer solution was concentrated under reduced pressure and 
dried in a vacuum oven for 24 h to afford a viscous, pale yellow liquid (0.8 g). 
 
General synthesis of triblock copolymers: ABA triblock copolymers were synthesized via 
anionic ring-opening polymerization. All copolymers were initiated from PEO (Mn = 8,000 g mol
-
1). The following procedure for (PiPGE-stat-PEGE)2.2k-b-PEO8k-b-(PEGE-stat-PiPGE)2.2k 
(Polymer 9) will serve as an example for a typical triblock copolymer synthesis. PEO (10 g, 1.25 
mmol) was added to the reaction vessel and dried under vacuum overnight. Dry THF (100 mL) 
was added under an argon atomosphere and heated to 50 °C to facilitate dissolution of the 
macroinitator. Once sufficiently dissolved, a potassium naphthalenide solution (1M in THF) was 
titrated into the flask until the solution remained a slight green color, indicating full 
deprotonation of PEO hydroxyl end groups. This was followed by the simultaneous addition of 
isopropyl glycidyl ether (4.07 g, 35 mmol) and ethyl glycidyl ether (3.57 g, 35 mmol) to begin 
polymerization. The reaction continued for 24 h at 65 °C and was subsequently quenched with a 
degassed solution of 1% v/v AcOH in MeOH. In the polymerizations with allyl glycidyl ether, 
the reactions were performed at 30 °C in order to avoid allyl-vinyl isomerization as reported by 
Lynd and co-workers.32 The reaction mixture was then precipitated into cold hexane. The 
polymer was collected via centrifugation (4400 rpm, 10 min) and the supernatant decanted. The 
product was washed twice with additional hexane and collected again in the same manner. The 
isolated polymer solution was dried in a vacuum oven for at least 24 h to afford an off-white 
solid (13.6 g). 
 
Calculation of Triblock Copolymer Mn 
119 
 
Number average molecular weight was calculated using 1H NMR and an example for a 
copolymer of PEGE-stat-PiPGE-b-PEG-b-PEGE-stat-PiPGE is given. The Mn of PEG is 8k Da 
which corresponds to approximately 182 ethylene glycol units per macroinitiator. If we use 
Polymer 9 and Figure A22 as the example spectrum then the number of ethyl and isopropyl 
glycidyl ether units can be calculated by finding the PEG to PEGE and PEG to PiPGE ratio. The 
integration for the peaks from 1.16-1.20 ppm correspond to the methyl group on PEGE and is 
usually set to 3.00.  The peaks from 1.13-1.6 ppm corresponding to the PiPGE methyl groups 
integrate to 5.82. The PEG, PEGE, and PiPGE ether backbone protons manifest as peaks from 
3.2-3.9 ppm and integrate to a value of 48.486. Since the PEGE and PiPGE ether backbone peaks 
overlap in the 3.5 ppm region, their contribution to the overall integration must be subtracted 
through the following steps. 
First it is necessary to calculate the relative integrations of the iPGE and EGE methyl protons. 
The integration values for the methyl groups of iPGE and EGE are divided by the number of 
protons they represent (6 and 3, respectively). 
 
5.82 𝐼𝑛𝑡𝑒𝑔𝑟𝑎𝑡𝑒𝑑 i𝑃𝐺𝐸 𝐶𝐻3 𝑃𝑟𝑜𝑡𝑜𝑛𝑠
6 𝐴𝑐𝑡𝑢𝑎𝑙 𝑖𝑃𝐺𝐸 𝐶𝐻3 𝑃𝑟𝑜𝑡𝑜𝑛𝑠
= 0.97 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 i𝑃𝐺𝐸 𝐼𝑛𝑡𝑒𝑔𝑟𝑎𝑡𝑖𝑜𝑛 
3.00 𝐼𝑛𝑡𝑒𝑔𝑟𝑎𝑡𝑒𝑑 𝐸𝐺𝐸 𝐶𝐻3 𝑃𝑟𝑜𝑡𝑜𝑛𝑠
3 𝐴𝑐𝑡𝑢𝑎𝑙 𝐸𝐺𝐸 𝐶𝐻3 𝑃𝑟𝑜𝑡𝑜𝑛𝑠
= 1.00 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝐸𝐺𝐸 𝐼𝑛𝑡𝑒𝑔𝑟𝑎𝑡𝑖𝑜𝑛 
 
These values are then used to calculate the number of relative EGE and iPGE backbone protons. 
This is done by multiplying the relative integrations by the number of backbone protons.  
 
0.97 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 i𝑃𝐺𝐸 𝐼𝑛𝑡𝑒𝑔𝑟𝑎𝑡𝑖𝑜𝑛 ∗ 6 i𝑃𝐺𝐸 𝐵𝑎𝑐𝑘𝑏𝑜𝑛𝑒 𝑃𝑟𝑜𝑡𝑜𝑛𝑠 = 5.82 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 i𝑃𝐺𝐸 𝐵𝑎𝑐𝑘𝑏𝑜𝑛𝑒 𝑃𝑟𝑜𝑡𝑜𝑛𝑠 
1.00 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝐸𝐺𝐸 𝐼𝑛𝑡𝑒𝑔𝑟𝑎𝑡𝑖𝑜𝑛 ∗ 7 𝐸𝐺𝐸 𝐵𝑎𝑐𝑘𝑏𝑜𝑛𝑒 𝑃𝑟𝑜𝑡𝑜𝑛𝑠 = 7 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝐸𝐺𝐸 𝐵𝑎𝑐𝑘𝑏𝑜𝑛𝑒 𝑃𝑟𝑜𝑡𝑜𝑛𝑠 
120 
 
 
The contribution of EGE protons can now be subtracted from the total integration, and the 
corresponding number of repeat units determined, using the calculation shown below.  
 
182 𝐸𝑡ℎ𝑦𝑙𝑒𝑛𝑒 𝐺𝑦𝑙𝑐𝑜𝑙 𝑈𝑛𝑖𝑡𝑠
(48.486 𝑇𝑜𝑡𝑎𝑙 𝐵𝑎𝑐𝑘𝑏𝑜𝑛𝑒 − 7 𝐸𝐺𝐸 − 5.82 i𝑃𝐺𝐸)
4 𝑃𝐸𝐺 𝐶𝐻2
∗ 1.0 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝐸𝐺𝐸 𝐼𝑛𝑡𝑒𝑔𝑟𝑎𝑡𝑖𝑜𝑛 = 20.4 𝐸𝐺𝐸 𝑅𝑒𝑝𝑒𝑎𝑡 𝑈𝑛𝑖𝑡𝑠 
 
To calculate the number of iPGE repeat units we need only to multiple the EGE repeat units by 
the relative iPGE integration  
20.4 𝐸𝐺𝐸 𝑅𝑒𝑝𝑒𝑎𝑡 𝑈𝑛𝑖𝑡𝑠 ∗ 0.97 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 i𝑃𝐺𝐸 𝐼𝑛𝑡𝑒𝑔𝑟𝑎𝑡𝑖𝑜𝑛 = 19.8 i𝑃𝐺𝐸 𝑅𝑒𝑝𝑒𝑎𝑡 𝑈𝑛𝑖𝑡𝑠 
 
1H NMR annotation for Poly(methyl glycidyl ether) (500 MHz, CDCl3): 2.34 (s, -Ph-CH3), 
3.35-3.93 (m, (-O-CH2-CH(CH2-O-CH3)-O-), 4.50 (s, -Ph-CH2-O-), 7.14-7.15 (d, CH3-Ph-CH2-, 
J = 7.5 Hz), 7.21-7.22 (d, CH3-Ph-CH2-, J = 8.0 Hz) 
1H NMR annotation for Poly(ethyl glycidyl ether) (500 MHz, CDCl3): δ 1.16-1.20 (t, -O-
CH2-CH3, J = 7.2 Hz), 2.34 (s, -Ph-CH3), 3.47-3.63 (m, (-O-CH2-CH(CH2-O-CH2-CH3)-O-), 
4.50 (s, -Ph-CH2-O-), 7.12-7.15 (d, CH3-Ph-CH2-, J = 8.1 Hz), 7.20-7.23 (d, CH3-Ph-CH2-, J = 
8.1 Hz) 
1H NMR annotation for Poly(allyl glycidyl ether (500 MHz, CDCl3): 2.34 (s, -Ph-CH3), 3.45-
3.83 (m, (-O-CH2-CH(CH2-O-CH2-CH=CH2)-O-), 3.98-3.99 (d, -O-CH2-CH=CH2)-O-, J = 5.5 
Hz) 4.49 (s, -Ph-CH2-O-) 5.14-5.16 (d, -O-CH2-CH=CH2)-O-, 10.0 Hz) 5.24-5.27 (d, -O-CH2-
CH=CH2)-O-, J = 17.0 Hz) 5.85-5.92 (m, -O-CH2-CH=CH2)-O-) 7.13-7.14 (d, CH3-Ph-CH2-, 8.0 
Hz) 7.20-7.21 (d, CH3-Ph-CH2-, 8.0 Hz) 
121 
 
1H NMR annotation for Poly(n-propyl glycidyl ether) (500 MHz, CDCl3): δ 0.89-0.92 (t, -O-
CH2-CH2-CH3, J = 7.0 Hz), 1.54-1.61 (sext, -O-CH2-CH2-CH3 J = 7.0 Hz), 2.34 (s, -Ph-CH3), 
3.38-3.93 (m, (-O-CH2-CH(CH2-O-CH2-CH2-CH3)-O-), 4.50 (s, -Ph-CH2-O-),  7.13-7.15 (d, 
CH3-Ph-CH2-,  J = 7.5 Hz), 7.21-7.22 (d, CH3-Ph-CH2-,  J = 7.5 Hz) 
1H NMR annotation for Polymer 1 (500 MHz, CDCl3): δ 1.17-1.20 (t, -O-CH2-CH3, J = 4.2 
Hz) 3.47-3.65 (m -O-CH2-CH2-O- and -O-CH2-CH(CH2-O-CH2-CH3)-O-)  
1H NMR annotation for Polymer 2 (500 MHz, CDCl3):  3.47-3.65 (m, -O-CH2-CH2-O- and -
O-CH2-CH(CH2-O-CH2-CH=CH2)-O-), 3.99 (s, -O-CH2-CH=CH2)-O-), 5.14-5.16 (d, -O-CH2-
CH=CH2)-O-, J = 10 Hz), 5.24-5.28 (d, -O-CH2-CH=CH2)-O-, J = 17.5 Hz). 5.85-5.92 (m, -O-
CH2-CH=CH2)-O-). 
1H NMR annotation for Polymer 3 (500 MHz, CDCl3): δ 1.12-1.15 (m, -O-CH-(CH3)2), 3.47-
3.65 (m, -O-CH2-CH2-O- and -O-CH2-CH(CH2-O-CH-(CH3)2)-O-) 
1H NMR annotation for Polymer 4 (500 MHz, CDCl3):  δ 1.16-1.22 (t, -O-CH2-CH3), 3.48-
3.66 (m, -O-CH2-CH2-O- and -O-CH2-CH(CH2-O-CH2-CH=CH2)-O- and -O-CH2-CH(CH2-O-
CH2-CH3)-O-), 3.98-3.99 (dd, -O-CH2-CH=CH2)-O-, J = 4.0 Hz), 5.14-5.16 (d, -O-CH2-
CH=CH2)-O-, J = 10 Hz), 5.24-5.28 (d, -O-CH2-CH=CH2)-O-, J = 17.5 Hz), 5.85-5.92 (m, -O-
CH2-CH=CH2)-O-)  
 
 
 
 
 
 
 
 
122 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A1. Homopolymer Cloud Point Measurements 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A2. Tcp of (a) Polymer 1, (b) Polymer 2, (c) Polymer 3, and (d) Polymer 4 
a) b) 
c) d) 
 
123 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A3. Tcp of (a) Polymer 5, (b) Polymer 6, (c) Polymer 7, (d) Polymer 8, (e) Polymer 9, and 
(f) Polymer 10 
 
 
 
 
 
 
 
a) b) 
c) d) 
e) f) 
124 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A4. Allyl glycidyl ether triblock brittle hydrogel 
 
 
 
 
Figure A5. Cell Viability data for 11.25, 15, and 20 wt% polymer 9 hydrogels in supplemented 
DMEM 
 
 
 
 
 
 
 
 
 
 
 
Figure A6. Confocal microscopy images of encapsulated HeLa cells in a 11.25 wt% formulation 
of polymer 9 in supplemented DMEM (a) green fluorescence channel indicating live cells (b) red 
fluorescence channel indicating dead cells (c) composite channel (scale bar: 1000 µm) 
 
 
 
 11.25 wt% 15 wt% 20 wt% 
Trial 1 .936404 .956416 .985594 
Trial 2 .944649 .966234 .638673 
Trial 3 .90142 .963687 .910025 
Trial 4 .926465 N/A N/A 
Trail 5 .842851 N/A N/A 
    
Average .910358 .928779 .844764 
Standard Deviation .041081 .062681 .182436 
(a) (b) (c) 
1000 μm 
125 
 
 
 
 
 
 
 
 
 
 
 
Figure A7. Confocal microscopy images of encapsulated HeLa cells in a 15.0 wt% formulation 
of polymer 9 in supplemented DMEM (a) green fluorescence channel indicating live cells (b) red 
fluorescence channel indicating dead cells (c) composite channel (scale bar: 1000 µm) 
 
 
 
 
 
 
 
 
 
 
 
Figure A8. Confocal microscopy images of encapsulated HeLa cells in a 20.0 wt% formulation 
of polymer 9 in supplemented DMEM (a) green fluorescence channel indicating live cells (b) red 
fluorescence channel indicating dead cells (c) composite channel (scale bar: 1000 µm) 
 
 
 
(a) (b) (c) 
1000 μm 
1000 μm 
(a) (b) (c) 
126 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A9. 1H NMR of n-propyl glycidyl ether 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A10. 1H NMR of poly(methyl glycidyl ether)2.1k 
 
 
 
 
 
 
 
 
 
Figure A10. 1H NMR of poly(methyl glycidyl ether)2.1k 
 
1H NMR (CDCl3, 500 MHz) 
poly(methyl glycidyl ether)2.1k 
CDCl3 
TMS 
1H NMR (CDCl3, 300 MHz) 
n-propyl glycidyl ether 
 
TMS 
CDCl3 
127 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Figure A11. 1H NMR of poly(ethyl glycidyl ether)3.0k 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A12. 1H NMR of poly(ethyl glycidyl ether)9.5k 
 
 
1H NMR (CDCl3, 500 MHz) 
poly(ethyl glycidyl ether)9.5k 
H2O 
CDCl3 
TMS 
1H NMR (CDCl3, 500 MHz) 
poly(ethyl glycidyl ether)3.0k 
CDCl3 
TMS 
128 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A13. 1H NMR of poly(ethyl glycidyl ether)24.5k 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A14. 1H NMR of poly(allyl glycidyl ether)3.3k 
 
 
1H NMR (CDCl3, 300 MHz) 
poly(ethyl glycidyl ether)24.5k 
H2O 
CDCl3 
TMS 
1H NMR (CDCl3, 500 MHz) 
poly(allyl glycidyl ether)3.3k 
H2O 
CDCl3 
TMS 
129 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A15. 1H NMR of poly(allyl glycidyl ether)5.8k 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A16. 1H NMR of poly(allyl glycidyl ether)11.7k 
 
1H NMR (CDCl3, 500 MHz) 
poly(ethyl glycidyl ether)9.5k 
H2O 
CDCl3 
TMS 
1H NMR (CDCl3, 500 MHz) 
poly(ethyl glycidyl ether)11.7k 
H2O 
CDCl3 
TMS 
130 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A17. 1H NMR of poly(isopropyl glycidyl ether)4.0k 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A18. 1H NMR of poly(isopropyl glycidyl ether)7.7k 
 
 
 
1H NMR (CDCl3, 500 MHz) 
poly(isopropyl glycidyl ether)4.0k 
H2O 
CDCl3 
TMS 
1H NMR (CDCl3, 500 MHz) 
poly(isopropyl glycidyl ether)7.7k 
CDCl3 
TMS 
131 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A19. 1H NMR of poly(isopropyl glycidyl ether)16.0k 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A20. 1H NMR of poly(n-propyl glycidyl ether)3.2k 
 
1H NMR (CDCl3, 500 MHz) 
poly(isopropyl glycidyl ether)16.0k 
H2O 
CDCl3 
TMS 
H2O 
1H NMR (CDCl3, 500 MHz) 
poly(n-propyl glycidyl ether)3.2k 
CDCl3 
TMS 
132 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A21. 1H NMR of poly(n-propyl glycidyl ether)8.0k 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A22. 1H NMR of Polymer 1 
 
 
CDCl3 
1H NMR (CDCl3, 500 MHz) 
Polymer 1 
TMS 
1H NMR (CDCl3, 500 MHz) 
poly(n-propyl glycidyl ether)8.0k 
CDCl3 
TMS 
133 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A23. 1H NMR of Polymer 2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A24. 1H NMR of Polymer 3 
 
H2O 
CDCl3 
TMS 
1H NMR (CDCl3, 500 MHz) 
Polymer 3 
CDCl3 
1H NMR (CDCl3, 500 MHz) 
Polymer 2 
134 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A25. 1H NMR of Polymer 4 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A26. 1H NMR of Polymer 5 
 
 
1H NMR (CDCl3, 500 MHz) 
Polymer 4 
CDCl3 
TMS 
H2O 
1H NMR (CDCl3, 500 MHz) 
Polymer 5 
TMS 
CDCl3 
H2O 
135 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A27. 1H NMR of Polymer 6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A28. 1H NMR of Polymer 7 
 
 
TMS 
1H NMR (CDCl3, 500 MHz) 
Polymer 6 
CDCl3 H2O 
1H NMR (CDCl3, 500 MHz) 
Polymer 7 
CDCl3 
TMS 
H2O 
136 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A29. 1H NMR of Polymer 8 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A30. 1H NMR of Polymer 9 
 
 
 
1H NMR (CDCl3, 500 MHz) 
Polymer 9 
CDCl3 
TMS 
H2O 
1H NMR (CDCl3, 500 MHz) 
Polymer 8 
CDCl3 
TMS 
H2O 
137 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure A31. 1H NMR of Polymer 10 
 
 
 
 
 
 
 
 
 
 
 
 
 
1H NMR (CDCl3, 500 MHz) 
Polymer 10 
CDCl3 
TMS 
H2O 
138 
 
APPENDIX B 
Poly(alkyl glycidyl ether) Hydrogels for Harnessing the Bioactivity of Engineered Microbes   
 
Figure B1. Graphical representation of the temperature induced sol-gel transition of polymer 2. 
 
 
 
 
 
 
 
 
Figure B2-B3. Viscosity vs Shear Rate and Oscillatory Yield Stress experiments for a 20 wt % 
solution of Polymer 1 
 
139 
 
 
Figure B4. Confocal microscopy images of live/dead assay results at day 1 of incubation in SC 
media. The first column shows the dead cells (red channel), while the second column shows the 
live cells (green channel). The third column is an overlay of the live and dead cell channels. 
 
 
 
140 
 
 
Figure B5. Confocal microscopy images of live/dead assay results at day 3 of incubation in SC 
media. The first column shows the dead cells (red channel), while the second column shows the 
live cells (green channel). The third column is an overlay of the live and dead cell channels. 
 
 
 
141 
 
 
Figure B6. Confocal microscopy images of live/dead assay results at day 7 of incubation in SC 
media. The first column shows the dead cells (red channel), while the second column shows the 
live cells (green channel). The third column is an overlay of the live and dead cell channels. 
 
 
 
142 
 
Figure A7. 1H NMR of Polymer 1 
Figure B8. 1H NMR of Polymer 2 
 
1H NMR (CDCl3, 500 MHz) 
Polymer 1  
CDCl3 
H2O 
CDCl3 
1H NMR (CDCl3, 300 MHz) 
Polymer 2 
143 
 
APPENDIX C 
The Post-Functionalization of Multi-Stimuli-Responsive Hydrogels for Extrusion-Based Additive 
Manufacturing 
 
Synthesis of PGE  
The ABA triblock copolymer was synthesized via anionic ring-opening polymerization 
according to previous literature reports from our group. Briefly, PEO (Mn = 8,000 g mol-1, 10 g, 
1.25 mmol) was added to the reaction vessel and dried under vacuum overnight. Dry THF (150 
mL) was added under an Ar atmosphere and heated to 50 °C to facilitate dissolution of the 
macroinitiator. Once sufficiently dissolved, a potassium naphthalenide solution (1M in THF) was 
titrated into the flask until the solution remained a slight green color, indicating full 
deprotonation of PEO hydroxyl end groups. Isopropyl glycidyl ether (2.90 g, 25 mmol) and ethyl 
glycidyl ether (2.17 g, 21.25 mmol) were added to begin polymerization. The reaction continued 
for 24 h at 65 °C and was subsequently quenched with a degassed solution of 1% v/v AcOH in 
MeOH. The reaction mixture was then precipitated into cold hexane. The polymer was collected 
via centrifugation (4400 rpm, 10 min) and the supernatant decanted. The product was washed 
twice with additional hexane and collected again in the same manner. The isolated polymer 
solution was dried in a vacuum oven for at least 24 h to afford PGE as an off-white solid (14.14 
g). 1H NMR (500 MHz, CDCl3): δ = 1.15-1.17 (m, -O-CH-(CH3)2), 1.17-1.23 (t, -O-CH2-CH3, 
J = 7.0 Hz), 3.47-3.81 (m -O-CH2-CH2-O- and -O-CH2-CH(CH2-O-CH2-CH3)-O- and -O-
CH2-CH(CH2-O-CH-(CH3)2)-O-). 
Synthesis of PGE-MA 
144 
 
PGE (7 g, 0.624 mmol) was dissolved in dry THF (50 mL) under a nitrogen atmosphere until 
complete dissolution of the polymer. Triethylamine (0.86 mL, 6.24 mmol) was added to increase 
the reactivity of the polymer hydroxyl chain ends and the mixture was heated at 65 °C for 30 
min. Methacrylic anhydride (9.3 mL, 62.4 mmol) was then added and the reaction mixture was 
stirred for 24 h at 65 °C. After this time, the reaction was quenched with MeOH. The reaction 
mixture was then precipitated into cold ether. The polymer was collected via centrifugation 
(4400 rpm, 10 min) and the supernatant decanted. The product was dried in a vacuum oven for 
24 h. The polymer was collected, redissolved in MeOH, and precipitated into ether once more. 
The isolated polymer was dried in a vacuum oven for 24 h to afford PGE-MA as an off-white 
solid (4.64 g). The degree of functionalization (𝑓n) was determined by comparing the 
integrations of the methacrylate vinyl (6.12 and 5.55 ppm) and methyl (1.94 ppm) protons, as 
well as the PEO chain-end methylene protons (5.08-5.15 ppm), to their theoretical values. For 
example, 1.99 (vinyl, actual)/2 (vinyl, theoretical) X 100 ≈ 100% functionalization of chain ends. 
These integration values were referenced to the total alkyl glycidyl ether protons for each 
polymer chain (1.12-1.20 ppm, 121 H).   1H NMR (300 MHz, CDCl3): δ = 1.12-1.20 (m, -O-
CH-(CH3)2) and -O-CH2-CH3), 1.94 (s, CH3C(CO2)=CH2), 3.39-3.89 (m, -O-CH2-CH2-O- 
and -O-CH2-CH(CH2-O-CH2-CH3)-O-, and -O-CH2-CH(CH2-O-CH-(CH3)2)-O-), 5.08-5.15 
(m, -CH2-CH-O-(C=O)), 5.48 (s, H-CH=C), 6.18 (s, H-CH=C). 
Rheological Measurements. Dynamic oscillatory rheological experiments were performed on a 
TA Instruments Discovery HR-2 rheometer equipped with a 20 mm parallel-plate geometry unless 
otherwise specified. Samples were equilibrated in an ice bath for at least 10 min, then carefully 
loaded onto the Peltier plate at 5 °C. A pre-shear experiment was applied to ensure bubbles were 
eliminated from the sample cell. The sample was equilibrated at 21 °C for 8 min. Strain sweep 
145 
 
experiments were performed, and all studies were conducted using a strain value in the linear 
viscoelastic regime. Temperature ramp experiments were performed at 1 Hz from 5 to 50 °C at 2 
°C min-1. Cyclic strain tests (frequency 1 Hz) were performed at 21 °C using alternating strains of 
1% for 5 minutes and 100% for 3 minutes per cycle. Viscosity versus shear rate experiments were 
performed at 21 °C. The gel yield stress values were measured under oscillatory strain (frequency: 
1 Hz, 21 °C) starting with an initial strain of 0.01% and converted to applied oscillatory stress.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Scheme C1. Synthesis of hydroxyethyl pyridyl disulfide  (HEPDS) 
Scheme C2. Synthesis of pyridyl disulfide urethane methacrylate (PDS-UM) 
 
146 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
a) b) 
c) d) 
Figure C1. Rheological characterization of PGE-MA/PDS-UM hydrogels. (a) Dynamic 
oscillatory temperature ramp displaying storage (G’, filled) and loss (G’’, open) moduli. (b) 
Viscosity vs shear rate experiment depicting shear-thinning behavior (c) Cyclic strain experiment 
demonstrating rapid recovery of hydrogel storage modulus (black circles) from periods of high 
(100%) to low (1%) oscillatory strain (red circles). (d) Dynamic oscillatory UV-Cure experiment 
demonstrating photochemical crosslinking upon exposure to 365 nm UV light. 
 
147 
 
 
Figure C2. 1H NMR of HEPDS (CDCl3,499 MHz) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1H NMR (CDCl3, 499 MHz) 
HEPDS 
148 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure C3. 1H NMR of PDS-UM (CDCl3,499 MHz) 
 
1H NMR (CDCl3, 499 MHz) 
PDS-UM