Photolabile Linkers: Exploiting Labile Bond Chemistry to Control Mode and Rate of Hydrogel Degradation and Protein Release

Article abstract of DOI:10.1021/jacs.9b11564

Photolabile moieties have been utilized in applications ranging from peptide synthesis and controlled protein activation to tunable and dynamic materials. The photochromic properties of nitrobenzyl (NB) based linkers are readily tuned to respond to cytocompatible light doses and are widely utilized in cell culture and other biological applications. While widely utilized, little is known about how the microenvironment, particularly confined aqueous environments (e.g., hydrogels), affects both the mode and rate of cleavage of NB moieties, leading to unpredictable limitations in control over system properties (e.g., rapid hydrolysis or slow photolysis). To address these challenges, we synthesized and characterized the photolysis and hydrolysis of NB moieties containing different labile bonds (i.e., ester, amide, carbonate, or carbamate) that served as labile crosslinks within step-growth hydrogels. We observed that NB ester bond exhibited significant rates of both photolysis and hydrolysis, whereas, importantly, the NB carbamate bond had superior light responsiveness and resistance to hydrolysis within the hydrogel microenvironment. Exploiting this synergy and orthogonality of photolytic and hydrolytic degradation, we designed concentric cylinder hydrogels loaded with different cargoes (e.g., model protein with different fluorophores) for either combinatorial or sequential release, respectively. Overall, this work provides new facile chemical approaches for tuning the degradability of NB linkers and an innovative strategy for the construction of multimodal degradable hydrogels, which can be utilized to guide the design of not only tunable materials platforms but also controlled synthetic protocols or surface modification strategies.

Full text of DOI:10.1021/jacs.9b11564

Subscriber access provided by LUNDS UNIV
Article
Photolabile linkers: exploiting labile bond chemistry to control
mode and rate of hydrogel degradation and protein release
Paige J. LeValley, Raghupathi Neelarapu, Bryan Sutherland,
Srimoyee Dasgupta, Christopher J Kloxin, and April M. Kloxin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b11564 • Publication Date (Web): 09 Feb 2020
Downloaded from pubs.acs.org on February 9, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 35
Journal of the American Chemical Society
1
2
3
4
5
Photolabile linkers: exploiting labile bond chemistry to control mode and rate of hydrogel
degradation and protein release
6
7
8
Paige J. LeValley,a Raghupathi Neelarapu,b Bryan P. Sutherland,a Srimoyee Dasgupta,c
Christopher J. Kloxin,a,c* April M. Kloxina,c,*
9
a. Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, United
States
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
b. Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, United States
c. Department of Material Science and Engineering, University of Delaware, Newark, DE 19716, United States
Abstract
Photolabile moieties have been utilized in applications ranging from peptide synthesis and
controlled protein activation to tunable and dynamic materials. The photochromic properties of
nitrobenzyl (NB) based linkers are readily tuned to respond to cytocompatible light doses and are
widely utilized in cell culture and other microbiological applications. While widely utilized, little is
known about how the microenvironment, particularly confined aqueous environments (e.g.,
hydrogels), affects both the mode and rate of cleavage of NB moieties, leading to unpredictable
limitations in control over system properties (e.g., rapid hydrolysis or slow photolysis). To address
these challenges, we synthesized and characterized the photolysis and hydrolysis of NB moieties
containing different labile bonds (i.e., ester, amide, carbonate, or carbamate) that served as labile
crosslinks within step-growth hydrogels. We observed that NB ester bond exhibited significant
rates of both photolysis and hydrolysis, whereas, importantly, the NB carbamate bond had
superior light responsiveness and resistance to hydrolysis within the hydrogel microenvironment.
Exploiting this synergy and orthogonality of photolytic and hydrolytic degradation, we designed
concentric cylinder hydrogels loaded with different cargoes (e.g., model protein with different
fluorophores) for either combinatorial or sequential release, respectively. Overall, this work
provides new facile chemical approaches for tuning the degradability of NB linkers and an
innovative strategy for the construction of multimodal degradable hydrogels, which can be utilized
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 35
1
2
3
4
5
6
to guide the design of not only tunable materials platforms but also controlled synthetic protocols
or surface modification strategies.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 3 of 35
Journal of the American Chemical Society
1
2
3
4
Introduction
5
6
7
8
Materials with light responsive properties, especially hydrogels, are useful for a multitude
of applications including self-healing,1 biosensing,2 drug delivery,3 polymer structure,4 and tissue
engineering5. Photolabile materials are widely used because they offer on-demand spatial and
temporal control over system properties for designing tunable platforms, building upon seminal
works that have utilized photolabile groups as cages to control the activity of small molecules or
proteins.6–8 Photolabile chemistries are commonly integrated into the material design as linkers
or pendant groups for modulating structure,9,10 surface modifications,11,12 mechanical
properties13,14 matrix degradation,15,16 or biomolecule presentation,17,18 providing opportunities for
probing how the timing and magnitude of changes in these properties influence downstream
responses. Nitrobenzyl (NB) based linkers are one of the most commonly used classes of
photodegradable linkers within this field. The popularity of NB linkers can be attributed, in part, to
their responsiveness to cytocompatible doses of light and the observed in vitro and in vivo
biocompatibility of their cleavage products when attached to polymers, which are essential
properties in designing materials for biological applications.19,20 However, in these applications,
variations in the cleavage behavior of NB linkers have emerged that appear to be
microenvironment specific, motivating the need for new fundamental studies that inform and
enable innovative material designs.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Initial seminal studies incorporating NB linkers within such materials, particularly
hydrogels, focused on tuning the absorbance of NB moieties and the rate of photocleavage
through modifications to the NB ring, which alter the molar absorptivity, quantum yield, and
ultimately half-life of the moiety upon irradiation.21,22 In general, substitution of a methyl group in
the benzylic position leads to enhancements in photocleavage kinetics along with the added
benefit of producing less reactive byproducts compared with the NB without a benzylic
substitution.23 Other modifications that have been investigated include the addition of methoxy
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 35
1
2
3
4
5
6
7
8
9
groups to the ring structure of the NB, which broadly increases absorbance of long wavelengths
of light but also results in lower quantum yields in comparison to their unmodified counterparts.22,24
Further, methods for installing orthogonal reactive handles have been investigated to provide
mechanisms for installation of NB groups onto polymer backbones or surfaces in addition to
conjugation to other molecules.19,25 Utilizing these strategies, combinations of different NB
moieties have been integrated within the crosslinks to bias the degradation of hydrogels21 and
within pendant groups to control the release of small molecules22 or peptides.26 Alternatively, such
NB groups have been combined to act as a photocage and photocleavable linker, respectively,
to control peptide presentation within hydrogels.26 While methods for controlling the
photodegradation properties of NB-based materials through ring substitutions are now well
established, less is known about how the degradation properties of these materials are impacted
by the chemical nature of the photocleavable bond itself.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NB linkers with ester (NB-ester) or amide (NB-amide) photocleavable bonds are widely
used for the design of responsive materials and have been observed to have variable degradation
properties in different microenvironments. Indeed, while helpful in guiding the initial design for
photodegradable applications, many solution-based studies (i.e., small molecule studies in
organic solvents owing to solubility) do not accurately represent the hydrogel microenvironment
and, consequently, can lack predictivity. For example, solution based studies comparing the
photodegradation rate of an NB-ester to NB-amide with the same ring substituents found that the
NB-amide degraded at a faster rate than the NB-ester; however, the photodegradation rate of
NBs immobilized onto a solid-phase resin was observed to decrease for NB-amides but not with
NB-esters.23,27 Within the biomaterials community, similar trends have been observed when these
linkers were incorporated as crosslinks within hydrogel networks, where switching from the NB-
ester to the NB-amide moiety within the crosslinks of poly(ethylene glycol) (PEG) hydrogels has
been observed to decrease the rate of photodegradation 3- to 10-fold.28–31 Interestingly, significant
ACS Paragon Plus Environment

Page 5 of 35
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
decreases in rate were not reported for NB-amide moieties used to tether hydrophobic small
molecules to the hydrogel network; this potentially points to the importance of the nature of the
group proximate to the cleavable bond (e.g., hydrophobic vs. hydrophilic).21,22
9
In addition to these complexities associated with photodegradation of NB groups in
different microenvironments, the labile bond also has been observed to impact their hydrolytic
stability in some applications, which can significantly impact the use of these molecules in
systems requiring long term stability in aqueous microenvironments. While hydrolysis of NB
linkers often is not characterized, NB-esters incorporated within step-growth PEG-based
hydrogels have been observed to exhibit appreciable hydrolysis under physiological pH even over
short times (hours to days), limiting user control over the properties of these systems at longer
time scales (e.g., in situ tuning of structure, modulus, release over many days to weeks).30,32
Cumulatively, these examples show the importance of the microenvironment in the
photodegradation behaviors of NB moieties and the potential tunability associated with these
groups for modulating properties within different chemical, material, and biological contexts.
Broadly accessible strategies for addressing these disparities in degradation behaviors
within hydrogels are needed for both fundamental insights and improved tools for materials
design. To address this, in this work, we present a simple and innovative approach for tuning the
rates of hydrolysis and photolysis of NB-linked step-growth hydrogels through modifications to
labile bond chemistry. Specifically, we establish alternatives to the NB-ester and NB-amide linking
moieties to address the limitations presented by these two linkers while enhancing the toolbox of
light responsive chemistries that can be employed within such polymeric materials. Four different
NB labile bond chemistries (NB-ester, NB-amide, NB-carbonate, and NB-carbamate) were
synthesized and characterized as crosslinks within well-defined hydrogel networks formed with
strain-promoted azide-alkyne cycloaddition (SPAAC) crosslinking chemistry (Figure 1A-B). We
hypothesized that the NB-carbonate and NB-carbamate linkers would impart increased stability
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 35
1
2
3
4
5
6
7
8
9
in water while maintaining the rate of photolysis, based on the known mechanisms of cleavage
for these alcohol-based nitrobenzyl groups.33,34 Both, the photolytic and hydrolytic behavior of the
four different NB linkers was investigated, establishing key differences in susceptibility to stimuli
and the timescales of these different degradation mechanisms (Figure 1C-D). To employ the
resulting orthogonal versus dual modes of degradation, sequential or combinatorial release of
encapsulated model cargoes (e.g., a protein labeled with complementary fluorophores) was
demonstrated with concentric cylinder hydrogels integrating different labile linkers for quantitative
comparison with reaction kinetics. These studies provide new and accessible chemical
approaches for independent control of photolysis and hydrolysis and unique insights into the effect
of NB labile bond chemistry on material degradation for guiding the design of photolabile systems
for a range of applications, from soft materials and nanoparticles to peptides and proteins.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Overview of approach. (A) Nitrobenzyl (NB) moieties were synthesized to have various labile bond
chemistries, X: carbamate (blue), ester (orange), carbonate (green), or amide (pink). (B) Hydrogel networks formed
with NB containing crosslinks were degraded over short time scales by light irradiation or over long time scales by
incubation in aqueous conditions. (C) Hydrogels were polymerized using a SPAAC between PEG-4-DBCO and
photodegradable PEG-bis-NB-azide (Az) to form triazole linkages. (D) Upon irradiation with 365 nm light the
incorporated NB moiety undergoes an irreversible photocleavage event that breaks hydrogel crosslinks, leading to
hydrogel erosion. The cleavable bond, X, dictates the rate of photodegradation and the functionality of the cleavage
products formed, Y. (E) Additionally, the nature of the cleavable bond influences the ability of the hydrogel to degrade
in aqueous environments; here, a hydroxyl is shown, which would be generated upon hydrolysis of NB-ester, NB-
carbonate, and NB-carbamate linkers, whereas an amine would be generated upon hydrolysis of NB-amide linkers.
The functionality of the hydrolysis cleavage product is represented by, Z.
ACS Paragon Plus Environment

Page 7 of 35
Journal of the American Chemical Society
1
2
3
4
Results and Discussion
5
6
Synthesis of PEG-NB crosslinkers
7
8
9
In addition to synthesizing a NB-ester-azide (see SI, Figure S4), we designed and successfully
synthesized two new NB linkers: a NB-carbonate or NB-carbamate labile linker possessing azide
functionality. For the synthesis of the NB-carbonate-azide, 3-azidopropyl (4-nitrophenyl)
carbonate was reacted with ethyl 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoate to
form the ester protected product, which was subsequently deprotected to yield the desired azide
functionalized carbonate-linked NB moiety (Scheme 1, SI, and Figure S9). A slightly modified
procedure was utilized to synthesize the NB-carbamate-azide, owing to safety concerns with
forming the 3-azidopropyl (4-nitrophenyl) carbamate. 4-nitrophenyl chloroformate was first
reacted with ethyl 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoate followed by the
addition of 3-azidopropan-1-amine to yield the ester protected carbamate-linked product, which
was subsequently cleaved to yield the desired azide-functionalized carbamate-linked NB moiety
(Scheme 2, SI, and Figure S12).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme
1.
Synthesis
of
4-(4-(1-(((3-azidopropoxy)carbonyl)oxy)ethyl)-2-methoxy-5-
nitrophenoxy)butanoic acid (NB-carbonate-Az)
OH
O
NO2
O
O
O
N3
NO2
N3
O
O
NO2
O
O
O
DMAP, CH2Cl2, RT
Yield = 95%
O
O
O
O
O
O
O
O
N3
TFA
NO2
THF-H2O (2:1), 60 ºC
Yield = 77%
O
OH
O
O
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 35
1
2
3
4
5
6
7
8
9
Scheme
2.
Synthesis
of
4-(4-(1-(((3-azidopropoxy)carbamoyl)oxy)ethyl)-2-methoxy-5-
nitrophenoxy)butanoic acid (NB-carbamate-Az)
NO2
O
OH
NO2
O
O
O
NO2
Cl
O
NO2
O
Et3N, CH2Cl2, RT
Yield = 96%
O
O
O
O
O
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CH2Cl2, RT
Yield = 88%
H2N
N3
O
O
O
N
H
N3
O
N
H
N3
NO2
LiOH
NO2
THF-H2O (2:1), RT
Yield = 74%
O
O
O
OH
O
O
O
O
These linkers, along with a NB-ester and NB-amide, were attached to linear PEG diamine
monomers via amide coupling (see SI) to form diazide end-functionalized linear photodegradable
PEG (PEG-bis-NB-azide) (Figure S14 – S17) to be used as crosslinks in hydrogel networks. The
successful synthesis of NB moieties with carbonate and carbamate labile bonds represents two
new linker chemistries that, to our knowledge, have yet to be characterized as crosslinks within
hydrogel matrices. While utilized here for hydrogel formation, the design of these photolabile
moieties with an azide and a carboxylic acid handle provides opportunities for facile conjugations
to other amine or hydroxyl presenting molecules, including proteins and peptides.
Photolytic behavior of NB containing hydrogels
The linear viscoelastic properties of the hydrogels can be utilized to monitor both the
polymerization and degradation behaviors of these networks, where the storage modulus
correlates directly with bond formation and cleavage, respectively. Well-defined step-growth
hydrogels were formed using a SPAAC reaction between cyclooctyne end-functionalized four-
arm PEG (PEG-4-DBCO, Figure S18) and PEG-bis-NB-azide (Figure 1B). SPAAC is a
biologically orthogonal reaction chemistry that is widely used for both materials synthesis and
ACS Paragon Plus Environment

Page 9 of 35
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
bioconjugation reactions. The polymerization time and storage modulus of the four different
hydrogel formulations (i.e., containing either an ester, an amide, a carbonate, or a carbamate
labile bond) were determined using dynamic time sweep measurements conducted in situ on a
rheometer within the linear viscoelastic regime for these materials. PEG-4-DBCO and PEG-bis-
NB-azide precursor solutions at a 1:1 stochiometric ratio of cyclooctyne to azide and final polymer
concentration of approximately 5 wt% were mixed to form hydrogels. To ensure that degradation
results were not convoluted by different starting moduli, the four hydrogel formulations
investigated were slightly adjusted such that the final storage moduli of all samples were
statistically the same prior to degradation (Figure S26).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The effect of NB labile bond chemistry on the rate of photodegradation was investigated by
monitoring the storage modulus of hydrogels during irradiation with 365 nm light at an intensity of
4 mW cm-2 (Figure S25). Within the time scales of interest, we assumed that bond cleavage was
strictly due to irradiation, eventually leading to complete hydrogel degradation (i.e., when the
hydrogel can no longer store stress).35,36 Based on rubber elasticity theory and hydrogel network
connectivity, the storage modulus of the hydrogel is directly related to the crosslink density of the
network and therefore bond cleavage, which can be modeled using first-order reaction kinetics
ꢅ
(i.e., storage modulus (G¢) ∝ crosslink density (rx) ∝ ꢀꢁꢂ ).30,37 This relationship between the
effective first order rate constant and the storage modulus of the network affords the opportunity
to fit linearized rheological data to determine the effective first order rate constant for the hydrogel.
Specifically, the natural log of the normalized modulus was plotted versus time and fit using linear
regression over the first two minutes of light irradiation to determine the effective first order
photodegradation constant for each linker within the hydrogel (Figure S27). To obtain the
photodegradation rate, k, for the NB moiety within the hydrogels, the effective first order rate
constant was divided by two as a decrease in crosslinking density occurs due to a photolytic
ꢃꢄꢄ
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 35
1
2
3
4
5
6
7
8
9
cleavage event on either side of the elastically active chain between nodes within the crosslinked
network (e.g., PEG-bis-NB).38 Importantly, using this approach we found that the fastest rate of
photodegradation occurred in hydrogels formed with the NB-carbamate linker followed by the NB-
ester, the NB-carbonate, and finally the NB-amide linkers (Figure 2B).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Rate of small molecule and hydrogel degradation in response to light. (A) The NB moiety
incorporated within the hydrogel backbone undergoes a β-elimination reaction upon 365 nm light
irradiation. Four different photocleavable bonds, X, were investigated, all of which lead to different
cleavage products, Y (as confirmed by NMR and mass spectrometry, Figure S21 – S24 and Table S1 –
S4). (B) Small molecule NBs (I0 = 10 mW cm-2 resulting in an Iavg = 7.8 mW cm-2) and photolabile
hydrogels (I0 = 4 mW cm-2 resulting in an Iavg = 3.9 mW cm-2) were irradiated with 365 nm light to
determine the first order photodegradation rate and quantum yield. The molar absorptivity was
determined using the absorbance of each small molecule NB moiety at 365 nm. First order rate constants
were determined for each of the four NB groups with different cleavable bonds in solution and within
hydrogels (Figure S20 and S27) and used to calculate the quantum yield. All formulations (NB-ester vs.
NB-Amide vs. NB-carbonate vs. NB-carbamate) were found to be statistically different except that
determined for the ester and carbonate in solution and for the amide and carbonate in hydrogels (p =
0.05). For full statistical analysis see Tables S5, S7, and S8. The data shown illustrate the mean (n = 3)
with error representing standard error.
The determined rate constants for the cleavage of the NB-ester and NB-amide moieties
integrated within the crosslinks of hydrogels follow trends observed in literature, with the NB-
amide bond degrading slower than the NB-ester bond within the same hydrogel
microenvironment. Specifically, from previous work published by our group, the photodegradation
of NB-amide bonds contained within hydrogel crosslinks were determined to have a
ACS Paragon Plus Environment

Page 11 of 35
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
photodegradation rate that was ~2.8x slower than an identically formed hydrogel containing NB-
ester crosslinks, consistent with the results found here.30,31 To compare the photodegradation
properties between NBs in solution and those integrated within the crosslinks of hydrogels, the
photodegradation kinetics of the small molecule NB groups were assessed through solution-
based NMR studies performed in deuterated water containing 5% DMSO and 1x PBS salts, both
used to improve solubility of NB moieties where the latter mimics hydrogel conditions. As the
PEG-NB-amide-azide was synthesized using a NB-amide-Fmoc, followed by deprotection and
amide coupling of 4-azidobutanoic acid, a NB-amide-acetyl was synthesized as an analog, which
has been used as a model NB-amide in previous studies and is more representative of the NB-
amide within the crosslink than the Fmoc derivative.23 Similar to previous reports, we observed
that the rate of the NB-amide was fastest in solution followed by the NB-carbamate, NB-ester,
and NB-carbonate (Figure 2B, Figure S20).23 Interestingly, while the NB-ester, NB-carbonate,
and NB-carbamate linkers exhibited no significant differences in photodegradation rate between
the solution and hydrogels, the NB-amide linker had a significantly reduced photodegradation rate
upon incorporation into hydrogels (>5x, Figure 2B). Of note is the NB-carbamate, which exhibited
a photodegradation rate that was significantly faster than all 3 of the other photolabile bonds within
the hydrogel environment, demonstrating the utility in exploiting NB labile bond chemistry for
tuning degradation properties.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Beyond providing a new reactive handle for rapid photocleavage, the observation of rapid
degradation of the NB-carbamate linker relative to the NB-amide linker within hydrogels provides
some new insight into the origin of disparities for photocleavage of the NB-amide in solution
versus in hydrogel microenvironments. As noted earlier, when the NB-amide has been utilized as
a linker in a confined environment, such as through attachment to resin in peptide synthesis23 or
utilization as a crosslink within a hydrogel,31 the rate of photodegradation is significantly slowed.
This observed decrease in photodegradation rate has been hypothesized by others as an artifact
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 35
1
2
3
4
5
6
7
8
9
of light scattering, shadowing effects, and/or swelling/solvation properties of the support.23 As
demonstrated by our data above and consistent with previous reports on resin,23 the NB-amide
exhibited slowed photodegradation when crosslinked within hydrogels in comparison with solution
based photodegradation. Of note, amide bond stability has been directly linked to bond rotation
and distortion,39 where confinement within a crosslink of a hydrogel network may limit activation
of the amide linkage. Additionally, while the incorporation of the NB-amide within hydrogel
crosslinks leads to slowed photodegradation, using it to tether hydrophobic molecules to a
hydrogel network has been observed to not drastically decrease the photodegradation rate
compared to what has been previously reported in solution.22,23 In mechanistic studies, it has been
reported that destabilization of the reaction intermediates that the NB moiety goes through upon
the light initiated photoisomerization (Norrish type II) reaction leads to slowed photodegradation
and is related to the labile bond.33 In this broader context, we speculate that a combination of the
microenvironment effects contribute to the observed disparity in the rate of NB-amide degradation
in the hydrogel vs. in free solution: confinement exerted on the amide bond upon being integrated
into the hydrogel network leads to changes in bond conformation and hydration that may increase
the thermodynamic driving force required for the photoisomerization reaction. Regardless of
mechanism, our data demonstrate quantitatively what has been observed anecdotally to date: a
significantly slowed rate of NB-amide photodegradation upon incorporation within the crosslinks
of hydrogels, which remains a consistent a challenge for their use in specific applications.
One additional observation is that the NB-amide hydrogels did not undergo complete hydrogel
erosion within 30 min of continuous irradiation, whereas all hydrogels with the other NB labile
bonds did completely degrade. Specifically, the NB-amide hydrogel storage modulus never
crossed over the loss modulus upon light irradiation (Figure S25), and a hydrogel layer was still
observed upon lifting the rheometer plate. We postulated that this incomplete degradation was
caused by a shift in the rate-limiting reaction intermediate due to the participation of the amide in
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 13 of 35
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
the Norris type-II reaction.33,40 To test this and examine a potential means for rescuing the
photodegradability of the NB-amide, we utilized a carbonyl scavenger (semicarbazide), which has
been reported in the literature as an effective method to overcome such decreases in
photodegradation rate.7,23 Notably, with this approach, we observed full degradation of the amide-
linked hydrogel in the presence of the carbonyl scavenger, with complete degradation within a
timeframe commensurate with the photodegradation rate observed at early irradiation time
(Figure S28). This ability of an immobilized NB-amide to fully degrade in the presence of a
carbonyl scavenger has been observed in literature and was hypothesized by others to be caused
by the scavenger aiding in the photocleavage mechanism.23 More broadly, our new study here
demonstrates a method for achieving complete photodegradation of NB-amide crosslinked
hydrogels through the utilization of a carbonyl scavenger. As a whole, our investigations of the
rates of photolysis demonstrate how different NB labile bond chemistries can be used to tune the
effective rates of photodegradation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Hydrolytic behavior of NB containing hydrogels
Hydrolysis of the NB labile bond is another method by which these hydrogels can degrade in
response to a stimulus over longer times (e.g., days to months). Understanding the rate of
hydrolysis of NB containing hydrogels is critical for establishing relevant timescales for tuning of
network properties either solely by photolysis, hydrolysis, or dually with both, as desired for the
application of interest. To investigate any differences in hydrogel responses to aqueous
environments, we conducted an accelerated hydrolysis test: hydrogels were incubated in PBS pH
= 10, and the hydrogel volume was measured over 72 h (Figure 3).41–43 Previous reports have
demonstrated the relevance of accelerated hydrolysis tests for predicting trends in hydrolysis
under physiological conditions, supporting the relevance of this approach in assessing the long
term stability of hydrolytically sensitive hydrogels.43 Under these conditions, hydrogels undergo
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 35
1
2
3
4
5
6
7
8
9
bulk degradation that leads to an increase in hydrogel volume (rx ∝ Q = V/V0) until a single chain
no longer spans the percolated network and the hydrogel is fully degraded (e.g., hydrogel
undergoes reverse gelation). For NB-ester hydrogels, a rapid swelling followed by complete
degradation was observed within 1 h in the incubation buffer, as expected based upon the known
susceptibility of ester bonds to hydrolysis (Figure 3). NB-ester hydrogels exhibited the fastest
degradation rate, followed by that of the NB-carbonate and NB-amide. Pivotally, no significant
degradation of the NB-carbamate hydrogels was observed under these conditions, with only
minimal volume changes after an initial equilibrium swelling, similar to the behavior observed in
the non-degradable control hydrogels.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Hydrolysis of NB-linked hydrogels. (A) The cleavable bond (X) of the NB group dictates the
rate of hydrogel hydrolysis and the products formed, where Z1 and Z2 represent the products formed on
the NB ring and cleaved chain, respectively. (B) Hydrogel volume was monitored over 72 h as a semi-
quantitative measure of the rate of hydrolysis, where hydrogels were incubated in PBS pH = 10 for
accelerated hydrolysis. Hydrogels crosslinked with ether-linked PEG-bis-azide served as a non-
degradable (ND) control. The empty shapes indicate complete hydrogel degradation which occurred at
< 1 h for NB-ester, < 2 h for NB-carbonate, and < 12 h for NB-amide. The volume was normalized to the
equilibrium swollen hydrogel volume at t = 0 h. The data shown illustrate the mean (n = 3) with error bars
representing standard error.
ACS Paragon Plus Environment

Page 15 of 35
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
The hydrolysis chemistry of ester, amide, carbonate, and carbamate labile bonds has been
well established in literature;44–46 as expected, the ester labile bond exhibited both rapid
photodegradation and hydrolysis, whereas the amide labile bond showed a slower
photodegradation rate and increased hydrolytic stability, comparatively.30,31 Excitingly, the
carbonate and carbamate labile bonds offer degradation properties that differ from the NB labile
bond chemistries commonly used within hydrogels. The carbamate labile bond is especially
appealing with both a rapid photodegradation rate and prolonged hydrolytic stability. To confirm
that the trends observed using the accelerated hydrolysis test would hold under physiological
conditions, hydrogels were incubated in PBS (pH = 7.4, containing 1% penicillin/streptomyocin
and 0.2% fungizone) at 37 ºC with 5% CO2, and the volume was monitored for more than one
month (Figure S30). Indeed, the trends observed from the accelerated hydrolysis test held, with
the NB-ester degrading within 21 days, the NB-carbonate hydrogels degrading within 49 days,
and the NB-amide, NB-carbamate, and non-degradable formulations following trends observed
in the accelerated hydrolysis test. These data further support the use of accelerated hydrolysis
conditions to quickly assess the long term aqueous stability of these NB hydrogel formulations.
Understanding the interplay between labile bond chemistry and degradation properties is
important when designing photodegradable hydrogel-based platforms for various applications,
where both the photolytic and hydrolytic properties of the different NB-linkers can be utilized to
engineer systems that degrade over multiple time scales based upon the stimulus applied.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Dual and sequential protein release from concentric cylinder hydrogel utilizing different NB linkers
Materials that respond to multiple stimuli across various time scales are of interest for a wide
range of applications from controlled surface modifications to tunable cell scaffolds. One
application of broad interest is the design of materials that can deliver multiple therapies in either
a combined or sequential manner. For example, different temporal combinations of nivolumab
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 16 of 35
1
2
3
4
5
6
7
8
9
(anti-CTLA4) and ipilimumab (anti-PD1) have proven beneficial for the treatment of advanced
melanoma.47 As a proof-of-concept toward such combination therapies, dually functional
concentric cylinder hydrogels were formed to take advantage of the different NB linker hydrolytic
and photolytic properties for creating either a combined or sequential release profile. Specifically,
NB-ester and NB-carbamate hydrogel precursor solutions were loaded with a model protein
labeled with different fluorophores (BSA-Alexa Fluor (AF) 488 and BSA-AF647, respectively), and
hydrogels were formed sequentially as concentric cylinders using custom designed molds (Figure
S31). These two formulations were chosen because they have similar photolytic behavior, but
opposite hydrolytic behavior, providing a mechanism for creating combined or sequential release
profiles. Here, BSA labeled with complementary fluorophores was selected as model cargoes for
quantitative comparison of release profiles to each other and to hydrogel degradation kinetics,
where encapsulated BSA is known to serve as an effective surrogate for and model of bioactive
therapeutic proteins.48
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
First, the similar photodegradation rates of the NB-ester and NB-carbamate linkers were
utilized to create a combined release profile of BSA-AF488 and BSA-AF647 upon 365 nm light
irradiation (I0 = 10 mW cm-2), monitored using confocal microscopy. The fluorescent intensity
within each of the hydrogel regions was monitored over time and found to decrease at similar
rates within both hydrogel layers (Figure 4A). As these are optically thick constructs, the
hydrogels degrade by surface erosion, which is observed when looking at the z-projections of the
hydrogel over time where the relative height of each layer was observed to decrease after each
irradiation period (Figure 4B).
ACS Paragon Plus Environment

Page 17 of 35
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Combinatorial protein release from dually functional concentric cylinder hydrogels. NB-ester
(BSA-AF488 labeled) and NB-carbamate (BSA-AF647 labeled) dually functional concentric cylinder
hydrogels were formed and subsequently irradiated with light (365 nm, I0 = 10 mW cm-2) to demonstrate
quantitatively the combined release of multiple components dictated by photodegradation kinetics of the
respective linkers. (A) The normalized intensity of each region of the concentric cylinder hydrogels was
monitored over time and observed to decrease at a similar rate for both layers. (B) Representative
images of the surface (top) and side (bottom, 200 µm z-stack) of concentric cylinder hydrogels at 0 min
(left), 7.5 min (middle), and 12.5 min (right). The data shown illustrate the mean (n = 3) with error bars
representing standard error. No statistical differences observed between compositions at each time point
(p = 0.05). Scale bars 500 µm.
Next, the sequential release of these model cargoes from the concentric cylinder hydrogels
was investigated by deploying the opposite hydrolysis behavior exhibited by the NB-ester and the
NB-carbamate linkers. The hydrogels were first incubated in PBS pH = 10 for 60 min, degrading
the NB-ester layer and releasing BSA-AF488. The remaining hydrogel was subsequently
irradiated with 365 nm light (I0 = 10 mWcm-2) to degrade the NB-carbamate layer and release
BSA-AF647, and the fluorescent intensity within each layer was monitored over the time course
of the experiment as before. Importantly, the fluorescent intensity of the NB-ester layer decreased
over the 60 min of incubation within basic buffer conditions, releasing the BSA-AF488, followed
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 18 of 35
1
2
3
4
5
6
7
8
9
by rapid release of the BSA-AF647 from the degrading NB-carbamate layer upon irradiation with
light (Figure 5). Note, on average, a small segment of hydrogel at the edge of the NB-ester layer
remained at 60 min for the hydrogel samples tested, leading to the average normalized
fluorescent intensity above zero prior to the light irradiation; this arises from edge effects where
the two formulations (NB-ester and NB-carbamate) mixed causing a zone with different
degradation properties.49
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
These proof-of-concept experiments demonstrated the utility of understanding the modes and
rates of degradation of different NB linkers within hydrogels and their applicability for controlling
the release of encapsulated cargoes. Previous studies have utilized individual NB moieties, with
alterations in the number and nature of substitutions to the NB ring or NB moieties, or an NB
moiety in combination with other photolabile groups (i.e., coumarins) to control the release of
tethered small molecules22,34 and bioactive proteins18,25,50 or encapsulated cells.21,22,51 Here, we
demonstrated a facile method for biasing release of encapsulated cargoes through simple
processes. Selection of the labile bond afforded on (pre-programmed) / off control via hydrolysis
and externally tunable control via photolysis and can be readily translated for use with other NB
ring structures and cargoes (e.g., encapsulated nanoparticles or cells). Through full
characterization of both photodegradation and hydrolysis of the NB-linkers, design of material
systems with tailorable properties can be achieved for investigating various phenomena in both
chemical and biological arenas, whether it be surface modification of nanoparticles or tunable
hydrogel scaffolds. For example, in drug delivery applications, like that used to guide our proof-
of-concept experiment and of large interest within the biomaterials and polymer communities,
layered hydrogels with different NB linkers could be utilized to release multiple therapeutic
proteins on different time scales. In particular, the unique materials and designs presented here
provide a mechanism to bias the release of encapsulated unmodified proteins using both
endogenous and exogenous stimuli, with great potential for enabling combination therapies with
ACS Paragon Plus Environment

Page 19 of 35
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
increased efficacy toward addressing the systemic toxicity that has limited clinical translation of
these therapeutic regimes. Additionally, these materials could be designed for 3D hydrogel cell
culture applications to study the transition from a healthy to diseased state or influence of matrix
properties on cell phenotype and behavior, harnessing the different timescales of degradation
that can be achieved with individual or combinations of the NB linkers.5 The dual modes of
degradation and tailorable degradation rates afforded by these NB groups with different labile
bonds also could be integrated into workflows, within polymers, or on substrates for tunable
synthetic strategies, structure-properties, or surface chemistries. More broadly, the findings of
this work demonstrate that different NB labile bonds exhibit different degrees of hydrolytic stability
and has significant implications for the testing and use of NB groups in any aqueous
microenvironment, where interest in their translation into the human body continues to grow.52
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Sequential protein release from dually functional concentric cylinder hydrogels. NB-ester (BSA-
AF488 labeled) and NB-carbamate (BSA-AF647 labeled) concentric cylinder hydrogels were formed and
subsequently incubated under accelerated hydrolysis conditions (PBS pH = 10) for 60 min followed by
irradiation with light (365 nm, I0 = 10 mW cm-2). (A) The ester hydrogel layer (green) was observed to
degrade during incubation in basic conditions, whereas the carbamate layer (purple) was not observed
to degrade until light irradiation. (B) Representative images of the hydrogel at 0 min (left), 60 min (middle),
and 75 min (right). The data shown illustrate the mean (n = 3) with error bars representing standard error
(p = 0.05). Scale bars 500 µm.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 20 of 35
1
2
3
4
Conclusion
5
6
7
8
In summary, new NB linkers containing a carbonate or carbamate labile bond were
synthesized and incorporated into step-growth hydrogels, and their degradation properties
compared to established NB linkers with an ester or amide labile bond, addressing limitations of
these existing systems in terms of relative rates of hydrolysis and photolysis, respectively. The
photodegradation rate of NB-amide moieties in solution versus within hydrogels was found to
differ, highlighting the importance of characterizing degradation properties within the
microenvironment the moieties will be utilized. Critically, we observed that the NB-carbamate
exhibited fast photodegradation kinetics within the hydrogel microenvironment and hydrolytic
stability while the NB-ester hydrolytically degraded over the experimental time frame of an
accelerated hydrolysis test and hydrolysis test conducted in physiological conditions. Concentric
cylinder hydrogels with NB-ester and NB-carbamate linker layers were fabricated to exploit the
synergistic degradation modes and rates of these two linkers, allowing quantitative simultaneous
release of proteins with either irradiation or sequential hydrolysis and irradiation. This molecular
approach to the design of new NB linkers in conjunction with an in-depth understanding of their
photolysis and hydrolysis behaviors within aqueous hydrogel environments provides numerous
opportunities for the design of innovative degradable materials systems with tunable and
controlled properties engineered for applications ranging from surface modifications to tissue
engineering.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Associated Content
Supporting information contains experimental and synthetic procedures and additional
characterization data (NMR, rheology, and statistical analysis).
ACS Paragon Plus Environment

Page 21 of 35
Journal of the American Chemical Society
1
2
3
4
Author Information
5
6
Corresponding Authors: *cjk@udel.edu and *akloxin@udel.edu
7
8
9
Acknowledgments
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The authors would like to thank Dr. Kristi Kiick and Dr. John Koh for helpful conversations
regarding hydrogel design and photolabile chemistry. We would like to additionally thank Dr.
Megan Smithmyer and Mr. Joseph Spohn for help in the design of the concentric circle hydrogel
molds, formed with a 3D printer generously donated by AutoDesk. The research presented here
was supported by the Delaware COBRE programs with grants from the NIGMS (P20GM104316
and P30GM110758-02 for core instrument support) and the Delaware Bioscience Center for
Advanced Technology (CAT) grant (12A00448), and the W.M. Keck Foundation. We would
additionally like to thank support provided through the University of Delaware NMR and Mass
Spectrometry Laboratories.
References
(1)
(2)
(3)
(4)
Habault, D.; Zhang, H.; Zhao, Y. Light-Triggered Self-Healing and Shape-Memory
Polymers. Chem. Soc. Rev. 2013, 42 (17), 7244–7256.
Wei, M.; Gao, Y.; Li, X.; Serpe, M. J. Stimuli-Responsive Polymers and Their Applications.
Polym. Chem. 2017, 8 (1), 127–143.
Alvarez-Lorenzo, C.; Bromberg, L.; Concheiro, A. Light-Sensitive Intelligent Drug Delivery
Systems. Photochem. Photobiol. 2009, 85 (4), 848–860.
Chatani, S.; Kloxin, C. J.; Bowman, C. N. The Power of Light in Polymer Science:
Photochemical Processes to Manipulate Polymer Formation, Structure, and Properties.
Polym. Chem. 2014, 5 (7), 2187–2201.
(5)
Ruskowitz, E. R.; DeForest, C. A. Photoresponsive Biomaterials for Targeted Drug Delivery
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 22 of 35
1
2
3
4
and 4D Cell Culture. Nat. Rev. Mater. 2018, 3, 17087.
5
6
7
8
(6)
(7)
(8)
Kharkar, P. M.; Kiick, K. L.; Kloxin, A. M. Designing Degradable Hydrogels for Orthogonal
Control of Cell Microenvironments. Chem. Soc. Rev. 2013, 42 (17), 7335–7372.
Bochet, C. G. Photolabile Protecting Groups and Linkers. J. Chem. Soc. Perkin Trans. 1
2002, No. 2, 125–142.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Griffin, D. R.; Borrajo, J.; Soon, A.; Acosta-Vélez, G. F.; Oshita, V.; Darling, N.; Mack, J.;
Barker, T.; Iruela-Arispe, M. L.; Segura, T. Hybrid Photopatterned Enzymatic Reaction
(HyPER) for in Situ Cell Manipulation. ChemBioChem 2014, 15 (2), 233–242.
Lee, J.; Hee Ku, K.; Kim, J.; Jun Lee, Y.; Gyu Jang, S.; Kim, B. J. Light-Responsive, Shape-
Switchable Block Copolymer Particles. J. Am. Chem. Soc. 2019, 141 (38), 15348–15355.
(9)
(10) Li, Z.; Davidson-Rozenfeld, G.; Vázquezvaź quez-Gonzaíez, M.; Fadeev, M.; Zhang, J.;
Tian, H.; Willner, I. Multi-Triggered Supramolecular DNA/Bipyridinium Dithienylethene
Hydrogels Driven by Light, Redox, and Chemical Stimuli for Shape-Memory and Self-
Healing Applications. J. Am. Chem. Soc. 2018, 140 (50), 17691–17701.
(11) van der Vlies, A. J.; Barua, N.; Nieves-Otero, P. A.; Platt, T. G.; Hansen, R. R. On Demand
Release and Retrieval of Bacteria from Microwell Arrays Using Photodegradable Hydrogel
Membranes. ACS Appl. Bio Mater. 2019, 2, 266–276.
(12) Ramanan, V. V.; Katz, J. S.; Guvendiren, M.; Cohen, E. R.; Marklein, R. A.; Burdick, J. A.
Photocleavable Side Groups to Spatially Alter Hydrogel Properties and Cellular
Interactions. J. Mater. Chem. 2010, 20 (40), 8920–8926.
(13) Houck, H. A.; Blasco, E.; Du Prez, F. E.; Barner-Kowollik, C. Light-Stabilized Dynamic
Materials. J. Am. Chem. Soc. 2019, 141 (31), 12329–12337.
(14) Rosales, A. M.; Vega, S. L.; DelRio, F. W.; Burdick, J. A.; Anseth, K. S. Hydrogels with
Reversible Mechanics to Probe Dynamic Cell Microenvironments. Angew. Chemie Int. Ed.
2017, 56 (40), 12132–12136.
ACS Paragon Plus Environment

Page 23 of 35
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
(15) LeValley, P. J.; Tibbitt, M. W.; Noren, B.; Kharkar, P.; Kloxin, A. M.; Anseth, K. S.; Toner,
M.; Oakey, J. Immunofunctional Photodegradable Poly(Ethylene Glycol) Hydrogel
Surfaces for the Capture and Release of Rare Cells. Colloids Surfaces B Biointerfaces
2019, 174, 483–492.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(16) Norris, S. C. P.; Delgado, S. M.; Kasko, A. M. Mechanically Robust Photodegradable
Gelatin Hydrogels for 3D Cell Culture and: In Situ Mechanical Modification. Polym. Chem.
2019, 10 (23), 3180–3193.
(17) Kohman, R. E.; Cha, S. S.; Man, H. Y.; Han, X. Light-Triggered Release of Bioactive
Molecules from DNA Nanostructures. Nano Lett. 2016, 16 (4), 2781–2785.
(18) Gawade, P. M.; Shadish, J. A.; Badeau, B. A.; DeForest, C. A. Logic-Based Delivery of
Site-Specifically Modified Proteins from Environmentally Responsive Hydrogel
Biomaterials. Adv. Mater. 2019, 31 (33), 1902462.
(19) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Photodegradable Hydrogels for
Dynamic Tuning of Physical and Chemical Properties. Science (80-. ). 2009, 324, 59–63.
(20) Kloxin, A. M.; Tibbitt, M. W.; Kasko, A. M.; Fairbairn, J. A.; Anseth, K. S. Tunable Hydrogels
for External Manipulation of Cellular Microenvironments through Controlled
Photodegradation. Adv. Mater. 2010, 22 (1), 61–66.
(21) Griffin, D. R.; Kasko, A. M. Photodegradable Macromers and Hydrogels for Live Cell
Encapsulation and Release. J. Am. Chem. Soc. 2012, 134 (31), 13103–13107.
(22) Griffin, D. R.; Kasko, A. M. Photoselective Delivery of Model Therapeutics from Hydrogels.
ACS Macro Lett. 2012, 1 (11), 1330–1334.
(23) Holmes, C. P. Model Studies for New O-Nitrobenzyl Photolabile Linkers: Substituent
Effects on the Rates of Photochemical Cleavage. J. Org. Chem. 1997, 62 (8), 2370–2380.
(24) Salerno, C. P.; Cleaves, H. J. A Simple Synthesis of Photolabile Α‐Methyl Nitrobenzyl
Compounds. Synth. Commun. 2004, 34 (13), 2379–2386.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 24 of 35
1
2
3
4
5
6
7
8
(25) Griffin, D. R.; Schlosser, J. L.; Lam, S. F.; Nguyen, T. H.; Maynard, H. D.; Kasko, A. M.
Synthesis of Photodegradable Macromers for Conjugation and Release of Bioactive
Molecules. Biomacromolecules 2013, 14 (4), 1199–1207.
9
(26) Deforest, C. A.; Tirrell, D. A. A Photoreversible Protein-Patterning Approach for Guiding
Stem Cell Fate in Three-Dimensional Gels. Nat. Mater. 2015, 14, 523–531.
(27) Mikkelsen, R. J. T.; Grier, K. E.; Mortensen, K. T.; Nielsen, T. E.; Qvortrup, K. Photolabile
Linkers for Solid-Phase Synthesis. ACS Comb. Sci. 2018, 20 (7), 377–399.
(28) DeForest, C. A.; Anseth, K. S. Cytocompatible Click-Based Hydrogels with Dynamically
Tunable Properties through Orthogonal Photoconjugation and Photocleavage Reactions.
Nat. Chem. 2011, 3 (12), 925–931.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(29) McKinnon, D. D.; Brown, T. E.; Kyburz, K. A.; Kiyotake, E.; Anseth, K. S. Design and
Characterization of a Synthetically Accessible, Photodegradable Hydrogel for User-
Directed Formation of Neural Networks. Biomacromolecules 2014, 15 (7), 2808–2816.
(30) Kharkar, P. M.; Kiick, K. L.; Kloxin, A. M. Design of Thiol- and Light-Sensitive Degradable
Hydrogels Using Michael-Type Addition Reactions. Polym. Chem. 2015, 6 (31), 5565–
5574.
(31) Kharkar, P. M.; Scott, R. A.; Olney, L. P.; LeValley, P. J.; Maverakis, E.; Kiick, K. L.; Kloxin,
A. M. Controlling the Release of Small, Bioactive Proteins via Dual Mechanisms with
Therapeutic Potential. Adv. Healthc. Mater. 2017, 6 (24), 1700713.
(32) Claaßen, C.; Claaßen, M. H.; Gohl, F.; Tovar, G. E. M.; Borchers, K.; Southan, A.
Photoinduced Cleavage and Hydrolysis of O-Nitrobenzyl Linker and Covalent Linker
Immobilization in Gelatin Methacryloyl Hydrogels. Macromol. Biosci. 2018, 18 (9),
1800104.
(33) Šolomek, T.; Mercier, S.; Bally, T.; Bochet, C. G. Photolysis of Ortho-Nitrobenzylic
Derivatives: The Importance of the Leaving Group. Photochem. Photobiol. Sci. 2012, 11
ACS Paragon Plus Environment

Page 25 of 35
Journal of the American Chemical Society
1
2
3
4
(3), 548–555.
5
6
7
8
(34) Shi, Y.; Truong, V. X.; Kulkarni, K.; Qu, Y.; Simon, G. P.; Boyd, R. L.; Perlmutter, P.;
Lithgow, T.; Forsythe, J. S. Light-Triggered Release of Ciprofloxacin from an in Situ
Forming Click Hydrogel for Antibacterial Wound Dressings. J. Mater. Chem. B 2015, 3 (45),
8771–8774.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(35) Tibbitt, M. W.; Kloxin, A. M.; Sawicki, L. A.; Anseth, K. S. Mechanical Properties and
Degradation of Chain and Step-Polymerized Photodegradable Hydrogels. Macromolecules
2013, 46 (7), 2785–2792.
(36) Kloxin, A. M.; Kloxin, C. J.; Bowman, C. N.; Anseth, K. S. Mechanical Properties of
Cellularly Responsive Hydrogels and Their Experimental Determination. Adv. Mater. 2010,
22 (31), 3484–3494.
(37) Treloar, L. R. . The Physics of Rubber Elasticity; Oxford University Press: Oxford, 1975.
(38) Zhong, M.; Wang, R.; Kawamoto, K.; Olsen, B. D.; Johnson, J. A. Quantifying the Impact
of Molecular Defects on Polymer Network Elasticity. Science (80-. ). 2016, 353, 1264–
1268.
(39) Aubé, J. A New Twist on Amide Solvolysis. Angew. Chemie Int. Ed. 2012, 51 (13), 3063–
3065.
(40) Rinnová, M.; Nováková, M.; Kašička, V.; Jiráček, J. Side Reactions during Photochemical
Cleavage of an Alpha-Methyl-6-Nitroveratryl-Based Photolabile Linker. J. Pept. Sci. 2000,
6 (8), 355–365.
(41) Shen, J.; Burgess, D. J. Accelerated in Vitro Release Testing of Implantable PLGA
Microsphere/PVA Hydrogel Composite Coatings. Int. J. Pharm. 2012, 422, 341–348.
(42) Coutinho, D. F.; Sant, S. V.; Shin, H.; Oliveira, J. T.; Gomes, M. E.; Neves, N. M.;
Khademhosseini, A.; Reis, R. L. Modified Gellan Gum Hydrogels with Tunable Physical
and Mechanical Properties. Biomaterials 2010, 31 (29), 7494–7502.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 26 of 35
1
2
3
4
5
6
7
8
(43) Browning, M. B.; Cereceres, S. N.; Luong, P. T.; Cosgriff-Hernandez, E. M. Determination
of the in Vivo Degradation Mechanism of PEGDA Hydrogels. J. Biomed. Mater. Res. Part
A 2014, 102A (12), 4244–4251.
9
(44) Brown, R. S.; Bennet, A. J.; Slebocka-Tilk, H. Recent Perspectives Concerning the
Mechanism of H3O+- and Hydroxide-Promoted Amide Hydrolysis. Acc. Chem. Res. 1992,
25 (11), 481–488.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(45) Day, J. N. E.; Ingold, C. K. Mechanism and Kinetics of Carboxylic Ester Hydrolysis and
Carboxyl Esterification. Trans. Faraday Soc. 1941, 37, 686–705.
(46) Dittert, L. W.; Higuchi, T. Rates of Hydrolysis of Carbamate and Carbonate Esters in
Alkaline Solution. J. Pharm. Sci. 1963, 52 (9), 852–857.
(47) Maverakis, E.; L; Cornelius, L. A.; Bowen, G. M.; Phan, T.; Patel, F. B.; Fitzmaurice, S.;
He, Y.; Burrall, B.; Duong, C.; Kloxin, A. M.; Sultani, H.; Wilken, R.; Martinez, S. R.; Patel,
F. Metastatic Melanoma – A Review of Current and Future Treatment Options. Acta Derm.
Venereol. 2015, 95 (5), 516–524.
(48) Rehmann, M. S.; Skeens, K. M.; Kharkar, P. M.; Ford, E. M.; Maverakis, E.; Lee, K. H.;
Kloxin, A. M. Tuning and Predicting Mesh Size and Protein Release from Step Growth
Hydrogels. Biomacromolecules 2017, 18 (10), 3131–3142.
(49) Aziz, A. H.; Wahlquist, J.; Sollner, A.; Ferguson, V.; DelRio, F. W.; Bryant, S. J. Mechanical
Characterization of Sequentially Layered Photo-Clickable Thiol-Ene Hydrogels. J. Mech.
Behav. Biomed. Mater. 2017, No. 65, 454–465.
(50) Azagarsamy, M. A.; Marozas, I. A.; Spaans, S.; Anseth, K. S. Photoregulated Hydrazone-
Based Hydrogel Formation for Biochemically Patterning 3D Cellular Microenvironments.
ACS Macro Lett. 2016, 5 (1), 19–23.
(51) Azagarsamy, M. A.; Anseth, K. S. Wavelength-Controlled Photocleavage for the
Orthogonal and Sequential Release of Multiple Proteins. Angew. Chemie - Int. Ed. 2013,
ACS Paragon Plus Environment

Page 27 of 35
Journal of the American Chemical Society
1
2
3
4
52 (51), 13803–13807.
5
6
7
8
(52) Raman, R.; Hua, T.; Gwynne, D.; Collins, J.; Tamang, S.; Zhou, J.; Esfandiary, T.; Soares,
V.; Pajovic, S.; Hayward, A.; Langer, R.; Traverso, G. Light-Degradable Hydrogels as
Dynamic Triggers for Gastrointestinal Applications. Sci. Adv. 2020, 6 (3), eaay0065.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 28 of 35
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 29 of 35
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Overview of approach. (A) Nitrobenzyl (NB) moieties were synthesized to have various labile bond
chemistries, X: carbamate (blue), ester (orange), carbonate (green), or amide (pink). (B) Hydrogel
networks formed with NB containing crosslinks were degraded over short time scales by light irradiation or
over long time scales by incubation in aqueous conditions. (C) Hydrogels were polymerized using a SPAAC
between PEG-4-DBCO and photodegradable PEG-bis-NB-azide (Az) to form triazole linkages. (D) Upon
irradiation with 365 nm light the incorporated NB moiety undergoes an irreversible photocleavage event that
breaks hydrogel crosslinks, leading to hydrogel erosion. The cleavable bond, X, dictates the rate of
photodegradation and the functionality of the cleavage products formed, Y. (E) Additionally, the nature of
the cleavable bond influences the ability of the hydrogel to degrade in aqueous environments; here, a
hydroxyl is shown, which would be generated upon hydrolysis of NB-ester, NB-carbonate, and NB-carbamate
linkers, whereas an amine would be generated upon hydrolysis of NB-amide linkers. The functionality of the
hydrolysis cleavage product is represented by, Z.
ACS Paragon Plus Environment