Short Soluble Coumarin Crosslinkers for Light-Controlled Release of Cells and Proteins from Hydrogels

Article abstract of DOI:10.1021/acs.biomac.5b00950

Materials that degrade or dissociate in response to low power light promise to enable on-demand, precisely localized delivery of drugs or bioactive molecules in living systems. Such applications remain elusive because few materials respond to wavelengths

Full text of DOI:10.1021/acs.biomac.5b00950

Article
pubs.acs.org/Biomac
Short Soluble Coumarin Crosslinkers for Light-Controlled Release of
Cells and Proteins from Hydrogels
Caroline de Gracia Lux,†,⊥ Jacques Lux,†,⊥ Guillaume Collet,† Sha He,§ Minnie Chan,‡ Jason Olejniczak,‡
Alexandra Foucault-Collet,† and Adah Almutairi*,†,§,∥
§
‡
†Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of NanoEngineering, Department of Chemistry and
∥
Biochemistry, and Center for Excellence in Nanomedicine and Engineering, University of California at San Diego, 9500 Gilman
Drive, La Jolla, California 92093-0600, United States
S
* Supporting Information
ABSTRACT: Materials that degrade or dissociate in response to low
power light promise to enable on-demand, precisely localized delivery of
drugs or bioactive molecules in living systems. Such applications remain
elusive because few materials respond to wavelengths that appreciably
penetrate tissues. The photocage bromohydroxycoumarin (Bhc) is
efficiently cleaved upon low-power ultraviolet (UV) and near-infrared
(NIR) irradiation through one- or two-photon excitation, respectively.
We have designed and synthesized a short Bhc-bearing crosslinker to
create light-degradable hydrogels and nanogels. Our crosslinker breaks
by intramolecular cyclization in a manner inspired by the naturally
occurring ornithine lactamization, in response to UV and NIR light,
enabling rapid degradation of polyacrylamide gels and release of small
hydrophilic payloads such as an ∼10 nm model protein and murine
mesenchymal stem cells, with no background leakage.
and degradation rate. Research in the field can be divided into
two main categories: phototriggered ligand presentation and
payload release from photodegradable scaffolds. Shoichet et al.
reported examples of chemical hydrogel patterning by photo-
controlled immobilization of biomolecules.41,42 This strategy
allows light-programmed cellular growth and migration in 3D
agarose matrices. The Anseth group also pioneered this field
with the first photodegradable hydrogels bearing nitrobenzyl
ether linkers in PEG-based gels, allowing light-mediated control
over cell culture environments.34
The vast majority of reported light-triggerable biomedical
matrices for controlled delivery systems, biomolecular pattern-
ing, or programmable scaffolds13,39,43−46 respond to UV light,
which does not penetrate tissues deeply due to high scattering
and absorption by hemoglobin and melanin.47 The next
generation of light-responsive hydrogels should employ
chemistries responsive to NIR light (650−900 nm), which
can penetrate tissues deeply with the high spatial and temporal
resolution of multiphoton excitation while causing minimal
damage. Extensive work has been done on hydrogels bearing
ortho-nitrobenzyl (ONB) protecting groups,34,39,48,49 while only
a few coumarin-based hydrogels have been reported.37,43,50−53
They are better candidates for two-photon triggered degrada-
tion because of coumarins’ superior two-photon action cross
INTRODUCTION
■
Light-responsive materials promise remote control over the
availability of molecules in living systems.1−6 As optogenetics
has transformed neuroscience,7 broadening the range of species
releasable using light should bring this precise form of light
control to a wider range of biological research. In therapeutic
delivery, potentially the most important field for the application
of such materials, triggered release from reservoirs would
stabilize therapeutics and reduce toxicity and unwanted side
effects by lowering dosage and avoiding nonspecific activity.
Despite remarkable advances in photoresponsive systems, few
light-responsive materials have yet been designed that would
allow control over the availability of bioactive hydrophilic
molecules (e.g., proteins, siRNA, and pharmacological
agents).8−15 Very recently, the first example of light-triggered
́
in vivo presentation of bioligands was reported. Garcia et al.
used light-regulated activation of RGD peptides on hydrogels
to promote cell adhesion and vascularization while avoiding
inflammation.16
Responsive hydrogels have sparked interest due to their
unique stability in aqueous media and compatibility with
delivery of large cargo such as proteins and cells.17−20 They
show promise in a wide range of biomaterial applications such
as tissue engineering (scaffolds, matrices for cell-culturing,
wound healing), diagnostics, and drug delivery.21−25 Among
the pool of available stimuli (temperature,26 pH,27−29
enzymes,30,31 redox,29,32 electric signal,33 or light13,34−40),
light uniquely offers spatiotemporal control over gel structure
Received: July 15, 2015
Revised: September 4, 2015
© XXXX American Chemical Society
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sections52 (∼2 GM and 1 GM at 740 nm for 6-bromo-7-
hydroxycoumarin-4-ylmethyl acetate and Bhc-Glu, respec-
tively,54 vs less than 0.1 GM for ONB).54,55 In addition,
coumarin degradation yields alcohols, which are more
biocompatible and less reactive than the aldehydes or ketones
generated by ONB.54
Here, we report the synthesis, characterization, and study of
UV- and NIR-degradable polyacrylamide (PAA)-based bulk
hydrogels and hydrogel nanoparticles (nanogels) incorporating
short ornithine-based crosslinkers containing the Bhc photo-
cage (Scheme 1b,c). We incorporated ornithine, a non-
Our crosslinker design features a Bhc photocage through a
carbamate linkage for maximal efficiency and hydrolytic
stability. Halogenation of the coumarin lowers the pKa of the
phenol, promoting formation of the more strongly absorbing
and water-soluble phenolate under physiological pH.54,63 The
presence of this heavy atom is also believed to promote
intersystem crossing to the reactive triplet state. This greater
efficiency means that systems incorporating the photocage can
be triggered using low-power lasers that do not cause tissue
damage. Finally, carbamate-linked coumarins have been
reported to be more stable than ester linkages against
hydrolysis.54
We designed a short crosslinker, rather than light-responsive
PEG-based crosslinkers, for maximal stability and to inhibit
burst release and payload leakage. In addition, our light-
degradable crosslinker is versatile and can be incorporated into
other materials besides PAA, such as biodegradable polymeric
scaffolds (hyaluronic acid, alginic acid, chitosan, or polylactic
acid).
a
Scheme 1
NIR-triggered release of proteins from nanogels has not yet
been shown; control over protein availability within such
structures requires a tight mesh and thus a short crosslinker. To
our knowledge, this is the first report of short water-soluble
NIR-degradable crosslinker used in bulk or nanogels for
efficient encapsulation and on-demand release of hydrophilic
payloads such as proteins (∼10 nm) and cells with minimal
background leakage.
EXPERIMENTAL SECTION
■
General Procedures and Instrumentation. All chemicals were
obtained from commercial sources and were used without further
purification. All reactions were carried out under argon in oven-dried
glassware unless otherwise noted. Flash column chromatography
purification was performed using a Teledyne Isco Combiflash
Companion with RediSep Rf prepacked silica. Mass determination
of synthesized compounds and byproducts obtained from crosslinker
degradation was performed with a HPLC-MS Agilent 160 Infinity
(binary pump, UV−vis 1260 DAD, 6120 Quadrupole LC/MS ESI
source) with a RP-18 column. 1H NMR spectra were acquired using a
Bruker 600 MHz NMR spectrometer and 13C NMR spectra were
acquired using a Bruker 150 MHz NMR spectrometer. UV−vis spectra
were collected using a Shimadzu UV-3600 UV−vis spectrophotom-
eter. Irradiation with 365 nm UV light was performed either using a
Luzchem LZC-ORG photoreactor equipped with an 8UV-A lamp
(316−400 nm range, resolved peaks: 313, 351 (broad), 365, 406 nm, 8
W maximum intensity, measured power: 1 mW·cm−2) or an
OmniCure S2000 Curing System (30 cm from light source; power
setting: 15%; measured power density: 1 mW·cm−2). NIR excitation
was performed using a Ti:sapphire laser (Mai Tai HP, Spectra
Physics). GFP expressing murine mesenchymal stem cells (GFP-
MSCs) and mMSC-cell culture media were provided by Cyagen
Biosciences. Nanogel size, PDI, and count rate measurements were
determined using a Malvern Zetasizer Nano ZS dynamic light-
scattering instrument and direct visualization of the nanogels was
imaged by transmission electron microscopy (TEM; FEI Spirit,
operating at 120 kV). Bhc-PNP was prepared according to the
previously reported procedure.64
a
(a) Unlike the 20 standard amino acids, ornithine is not carried by
tRNA during protein synthesis because it undergoes lactamization in
activated esters under biologically relevant conditions. (b) Chemical
structures of the bromo-hydroxycoumarin-based crosslinkers 1 and 2.
R = triethylene glycol (TEG) to increase water solubility. (c) Light-
induced degradation of PAA gels encapsulating hydrophilic payload via
UV- and NIR-triggered cleavage of cross-links (upon Bhc photo-
cleavage) through intramolecular cyclization. Payload (gray circle) is
encapsulated by copolymerizing acrylamide with light-responsive
bis(acrylamide) crosslinker 2. R = TEG. Detailed mechanism of Bhc
photocleavage is described in Scheme S1.
proteinogenic amino acid not supported by tRNA because of
its rapid fragmentation by lactamization (Scheme 1a).56,57 The
unrestricted access of water throughout the hydrogel side steps
the inefficient uncaging of Bhc in hydrophobic polymeric
nanoparticles58 and allows high photosolvolysis quantum yield.
This approach would allow low-intensity and, therefore, more
cell-compatible exposure to UV or NIR light to initiate light-
triggered degradation and release, all while stably retaining
cargo in the absence of irradiation (Scheme 1c). Several recent
examples of live cells and bioactive protein release using
biocompatible UV irradiation have been reported.39,46,59−62
However, our system uses the lowest UV power density ever
reported for such platforms (365 nm, 1 vs 10 mW/cm2
generally reported).34,39,43,46,52,61,62
Synthesis of 3. Fmoc-Orn(Boc)-OH (1.28 g, 2.8 mmol) was stirred
in a mixture of TFA (1.8 mL) and DCM (9 mL) for 1 h. Solvents were
removed under reduced pressure. TFA was removed by redissolution
of the residue in DCM and evaporation, repeated three times. The
residual white solid foam was redissolved in DCM (45 mL) with DIEA
(10.8 mL, 62 mmol, 20 equiv). A solution of Bhc-PNP64 (1.35 g, 2.8
mmol, 1 equiv) in DCM (50 mL) was added dropwise to this solution,
and the reaction mixture was stirred overnight. The organic mixture
was extensively washed with brine and HCl (2.5 mM, pH 3−4) and
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dried over MgSO4. After filtration, addition of diethyl ether to the
organic layer induced the precipitation of the product (white solid, 864
mg). The filtrate was evaporated and redissolved in DCM. Similar
treatment gave an additional 365 mg, for an overall yield of 1.23 g
(65%), Alternatively, the product was recovered from the DCM phase
without ether precipitation (85% yield, with residual DIEA). 1H NMR
(600 MHz, DMSO-d6) δ (ppm) 7.97 (s, 1H), 7.89 (d, J = 7.5 Hz, 2H),
7.72 (d, J = 7.5 Hz, 2H), 7.67 (d, J = 8.0 Hz, 1H), 7.59 (t, J = 5.6 Hz,
1H), 7.41 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.28 (s, 1H),
6.29 (s, 1H), 5.43 (s, 2H), 5.28 (s, 2H), 4.27 (m, 2H), 4.22 (m, 1H),
3.94 (m, 1H), 3.42 (s, 3H), 3.04 (q, J = 6.5 Hz, 2H), 1.74 (m, 1H),
1.60 (m, 1H), 1.51 (m, 2H). MS (ESI) m/z: [M − H]− calcd for
C33H30BrN2O10, 693.1; found, 693.0.
Synthesis of 4. To a stirred solution of 3 (680 mg, 0.98 mmol),
DMAP (24 mg, 0.20 mmol) and 2-hydroxyethylacrylamide (0.4 mL,
0.45 g, 3.9 mmol) in DCM (9 mL) was added dropwise a solution of
DCC (202 mg, 0.98 mmol) in DCM (4 mL) at 0 °C. The reaction
mixture was stirred overnight at RT. The crude mixture was washed
three times with water and once with brine. The organic layer was
removed under reduced pressure and the gray solid residue (960 mg)
was used in the next step without further purification. MS (ESI) m/z:
[M + H]+ calcd for C38H39BrN3O11, 792.2; found, 792.1.
Synthesis of 7. Over a solution of tetraethylene glycol monomethyl
ether (3.74 g, 17.9 mmol) in acetone (530 mL) was added dropwise
15 mL of Jones reagent ([CrO3] = 2.4 M) at 0 °C. The reaction was
stirred at room temperature for 2 h followed by the addition of
isopropyl alcohol (8 mL). Afterward, saturated sodium chloride (320
mL) was added to the mixture and stirred for 1 h before the removal of
acetone under reduced pressure. Then, the aqueous mixture was
extracted with DCM (4 × 200 mL). Organic layers were combined,
then dried over MgSO4 and finally removed under reduced pressure.
1
Yield: 3.69 g (93%). H NMR (600 MHz, CDCl3) δ 4.14 (s, 2H),
3.75−3.74 (m, 2H), 3.69−3.68 (m, 4H), 3.65−3.62 (m, 4H), 3.58−
3.56 (m, 2H), 3.39 (s, 3H).
Synthesis of 8. 3-Amino-1,2-propane diol (3.09 g, 34 mmol) in
DMF (100 mL) was added dropwise at 0 °C to a stirred solution of
tert-butyl(chloro)diphenylsilane (9.8 mL, 37.4 mmol) and imidazole
(2.78 g, 40.8 mmol) in DMF (200 mL). The reaction mixture was
stirred overnight at RT. DMF was removed under reduced pressure
and the residue was dissolved in DCM. The organic layer was washed
with brine, dried over MgSO4 and finally removed under reduced
pressure. The product was purified by flash chromatography on silica
1
gel (DCM/methanol, 100:0 to 90:10). Yield: 4.14 g (37%). H NMR
(600 MHz, CDCl3) δ 7.63 (d, J = 7.5 Hz, 4H), 7.39 (t, J = 7.5 Hz,
2H), 7.36 (t, J = 7.5 Hz, 4H), 3.86−3.82 (m, 1H), 3.63−3.61 (m, 2H),
2.95−2.85 (m, 2H), 1.04 (s, 9H). MS (ESI) m/z: [M + H]+ calcd for
C19H28NO2Si, 330.2; found, 330.1.
Synthesis of 5. Crude 4 (960 mg) was dissolved in DMF (10 mL)
with piperidine (94.1 mg, 1.1 mmol), stirred for 1 h 30 min and
monitored by LCMS. Upon completion, the reaction mixture was
filtered (1 μm filter) and the solvent was removed under reduced
pressure. DCM was added, and the precipitate formed (remaining
DCU salts) was filtered out (0.45 μm filter). The product was purified
by flash chromatography on silica gel (DCM/methanol). Yield: 331
Synthesis of 9. After dissolution of 8 (3.52 g, 10.6 mmol) in EtOAc
(75 mL), a solution of K2CO3 (14.79 g, 107 mmol) in water (75 mL)
was added, and the resulting mixture was stirred vigorously at 0 °C.
Acryloyl chloride (1.73 mL, 21.2 mmol) was then added dropwise to
the reaction at 0 °C. After a few minutes, the reaction mixture was
allowed to reach RT and was stirred until completion of the reaction,
monitored by LCMS. After 2 h, the organic and aqueous layers were
separated. The aqueous phase was extracted twice with EtOAc.
Organic layers were combined, washed once with water, then dried
over MgSO4, and finally removed under reduced pressure. Yield: 3.78
g (92%). 1H NMR (600 MHz, CDCl3) δ 7.64 (d, J = 7.0 Hz, 4H), 7.45
(t, J = 7.5, 7.0 Hz, 2H), 7.40 (t, J = 7.5, 7.0 Hz, 4H), 6.25 (d, J = 17.5
Hz, 1H), 6.01 (dd, J = 17.5, 9.6 Hz, 1H), 5.79 (bs, 1H), 5.64 (d, J = 9.6
Hz, 1H), 3.86 (bs, OH), 3.70−3.67 (m, 1H), 3.65−3.60 (m, 2H),
3.30−3.26 (m, 1H), 3.10−3.09 (m, 1H), 1.08 (s, 9H). ES-MS: M + Na
= 406.2 g·mol−1 (calcd = 406.2 g·mol−1). MS (ESI) m/z: [M + Na]+
calcd for C22H29NNaO3Si, 406.2; found, 406.2.
1
mg (60%, over two steps). H NMR (600 MHz, DMSO) δ (ppm)
8.22 (t, J = 5.2 Hz, 1H), 7.97 (s, 1H), 7.55 (t, J = 5.7 Hz, 1H), 7.28 (s,
1H), 6.28 (s, 1H), 6.20 (dd, J = 17.1, 10.1 Hz, 1H), 6.08 (dd, J = 17.1,
1.8 Hz, 1H), 5.58 (dd, J = 10.1, 1.8 Hz, 1H), 5.43 (s, 2H), 5.28 (s,
2H), 4.08 (m, 2H), 3.42 (s, 3H), 3.38 (m, 2H), 3.30 (m, 1H), 3.02 (q,
J = 6.1 Hz, 2H), 1.59 (m, 1H), 1.52−1.39 (m, 3H). MS (ESI) m/z: [M
+ H]+ calcd for C23H29BrN3O9, 570.1; found, 570.2.
Synthesis of 6. After dissolution of 5 (57 mg, 0.1 mmol) in EtOAc
(1.5 mL), a solution of K2CO3 (138.1 mg, 1 mmol) in water (1.5 mL)
was added, and the resulting mixture was stirred vigorously at 0 °C.
Acryloyl chloride (16.2 μL, 0.2 mmol) was then added dropwise to the
reaction at 0 °C. After a few minutes, the reaction mixture was allowed
to reach RT and was stirred until completion of the reaction,
monitored by LC-MS. After 30 min, the organic solvent was removed
under reduced pressure and DCM was added to the crude. The
organic layer was washed extensively with water and evaporated under
Synthesis of 10. A mixture of 7 (3.64 g, 9.5 mmol), 9 (1.06 g, 4.7
mmol), and DMAP (0.116 g, 0.95 mmol) was dissolved in DCM (160
mL). A solution of DCC (1.1 g, 5.3 mmol) in DCM (20 mL) was
added dropwise at 0 °C, and the reaction mixture was stirred overnight
at RT. DCU salts were filtered off, and the solvent was removed under
reduced pressure. The product was purified by flash chromatography
1
reduced pressure to give a white solid (63 mg, 100%). H NMR (600
MHz, DMSO-d6) δ (ppm) 8.48 (d, J = 7.5 Hz, 1H), 8.18 (t, J = 5.7
Hz, 1H), 7.97 (s, 1H), 7.56 (t, J = 5.7 Hz, 1H), 7.28 (s, 1H), 6.31 (dd,
J = 17.1, 10.1 Hz, 1H), 6.28 (s, 1H), 6.20 (dd, J = 17.1, 10.1 Hz, 1H),
6.10 (td, J = 16.7, 2.2, 1.8 Hz, 2H), 5.64 (dd, J = 10.1, 1.8 Hz, 1H),
5.59 (dd, J = 10.1, 2.2 Hz, 1H), 5.43 (s, 2H), 5.28 (s, 2H), 4.33 (m,
1H), 4.15−4.06 (m, 2H), 3.42 (s, 3H), 3.38 (q, J = 11.6, 5.7 Hz, 2H),
3.03 (q, J = 12.8, 6.6 Hz, 2H), 1.80−1.72 (m, 1H), 1.68−1.58 (m,
1H), 1.51−1.44 (m, 2H).
Synthesis of 1. Compound 6 (63.4 mg, 0.1 mmol) was stirred in
DCM (2 mL) and TFA (0.2 mL) for 1 h. TFA was removed under
high vacuum. TFA was removed by redissolution of the residue in
DCM and evaporation, repeated three times, yielding 59 mg of whitish
solid foam (100%). 1H NMR (600 MHz, DMSO-d6) δ (ppm) 8.48 (d,
J = 7.4 Hz, 1H), 8.18 (t, J = 5.4 Hz, 1H), 7.87 (s, 1H), 7.55 (t, J = 5.4
Hz, 1H), 6.91 (s, 1H), 6.31 (dd, J = 17.1, 10.1 Hz, 1H), 6.20 (dd, J =
17.1, 10.1 Hz, 1H), 6.18 (bs, 1H), 6.10 (td, J = 17.1, 1.8 Hz, 2H), 5.64
(dd, J = 10.1, 1.8 Hz, 1H), 5.59 (dd, J = 10.1, 1.8 Hz, 1H), 5.25 (s,
2H), 4.33 (m, 1H), 4.15−4.06 (m, 2H), 3.37 (m, 2H), 3.03 (q, J =
12.7, 6.6 Hz, 2H), 1.78−1.72 (m, 1H), 1.66−1.60 (m, 1H), 1.50−1.43
(m, 2H). 13C NMR (150 MHz, DMSO) δ (ppm) 25.76, 28.24, 37.55,
51.82, 60.77, 62.92, 103.15, 106.14, 108.48, 110.48, 125.27, 125.90,
128.33, 130.98, 131.42, 151.06, 153.73, 155.24, 157.38, 159.63, 164.68,
164.83, 171.79. HRMS (ESI) m/z: [M + Na]+ calcd for
C24H26BrN3NaO9, 602.0745; found, 602.0744.
1
on silica gel (Hex/EtOAc, 100:0 to 0:100). Yield: 2.11 g (76%). H
NMR (600 MHz, CDCl3) δ 7.64 (d, J = 7.0 Hz, 4H), 7.43 (d, J = 7.5
Hz, 2H), 7.39 (d, J = 7.5, 7.0 Hz, 4H), 6.29 (bs, 1H), 6.25 (d, J = 17.1
Hz, 1H), 6.03 (dd, J = 17.1, 10.1 Hz, 1H), 5.61 (d, J = 10.1 Hz, 1H),
5.14−5.11 (m, 1H), 4.16−4.04 (m, 2H), 3.82−3.78 (m, 2H), 3.68−
3.61 (m, 10H), 3.55−3.51 (m, 2H), 3.35 (s, 3H), 1.05 (s, 9H). MS
(ESI) m/z: [M + H]+ calcd for C31H46NO8Si, 588.3; found, 588.2.
Synthesis of 11. To a solution of 10 (716 mg, 1.2 mmol) in THF
(9 mL) was added a solution of TBAF hydrate (637 mg, 2.4 mmol) in
THF (10 mL) at 0 °C. After a few minutes, the reaction mixture was
allowed to reach RT and was stirred until completion of the reaction,
monitored by LCMS. THF was removed under reduced pressure, and
the product was purified by flash chromatography on silica gel (DCM/
methanol, 100:0 to 95:5). Yield: 303 mg (71%). 1H NMR (600 MHz,
CDCl3) δ 6.50 (bs, NH), 6.32 (d, J = 16.7 Hz, 1H), 6.15 (dd, J = 16.7,
11.0 Hz, 1H), 5.68 (d, J = 11.0 Hz, 1H), 4.24−4.15 (m, 4H), 4.04−
4.00 (m, 1H), 3.73−3.63 (m, 12H), 3.56−3.55 (m, 2H), 3.37 (s, 3H).
MS (ESI) m/z: [M + H]+ calcd for C15H28NO8, 588.3; found, 588.2.
Synthesis of 12. To a stirred solution of 3 (410.6 mg, 0.47 mmol),
DMAP (115.4 mg, 0.20 mmol), and 11 (165.0 mg, 0.47 mmol) in
DCM (6 mL) was added dropwise DCC (194.9 mg, 0.94 mmol)
dissolved in DCM (4 mL) at 0 °C. The reaction mixture was stirred
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overnight at RT. DCU salts were filtered and the solvent were
removed under reduced pressure. The product was purified by flash
chromatography on silica gel (DCM/MeOH, 100:0 to 95:5). Yield:
284 mg (59%). 1H NMR (600 MHz, CDCl3) δ (ppm) 7.72 (d, J = 7.5
Hz, 2H), 7.65 (bs, 1H), 7.57 (d, J = 7.5 Hz, 2H), 7.55 (bs, 1H), 7.37
(t, J = 7.5 Hz, 2H), 7.28 (t, J = 7.5 Hz, 2H), 7.11 (s, 1H), 6.89−6.77
(m, 1H), 6.35 (s, 1H), 6.26 (t, J = 15.8 Hz, 1H), 6.13 (m, 1H), 5.88
(m, 1H), 5.59 (m, 1H), 5.19 (m, 4H), 4.38 (m, 4H), 4.26−4.11 (m,
5H), 3.59 (m, 12H), 3.49 (bs, 5H), 3.32 (s, 3H), 3.24 (m, 2H), 1.88
(m, 1H), 1.88 (m, 1H), 1.60 (m, 2H).
H]+), and m/z = 187 (byproduct from hydrolysis, [M + H]+). Area
under the peak corresponding to each intermediate was measured and
these integrations were then normalized. A similar experiment was
done with nonirradiated samples.
Preparation of Photodegradable Hydrogels. Hydrogels at 2.7
mol % cross-linking density were formed by dissolving 4.65 mg (5.7
μmol) of Bhc crosslinker 2 and acrylamide (15 mg, 211 μmol) with
fluorescein-labeled BSA (0.5 mg) in 100 mM sodium phosphate
buffer, pH 8.5 (100 μL). A total of 1 μL of 50% ammonium persulfate
(APS) in 100 mM sodium phosphate buffer, pH 8.5, was added,
followed by the addition of TMED (1 μL). The resulting mixture was
gently mixed in a 1 mL syringe and left at room temperature for 1 h.
After this time, hydrogels were stiff enough to be manipulated with a
spatula and were used for protein release experiments. Empty
hydrogels were also prepared and analyzed as blanks.
Protein Release Study. The hydrogel was added to a 20 mL
scintillation vial and washed in PBS 1× (pH 7.4, 10 mL) under mild
swirling (14 h, rt, in the dark). Washing solution was removed and
replaced by fresh PBS (5 mL). Hydrogel was incubated at 37 °C. At
each time point, complete solution was removed and replaced by fresh
PBS (5 mL). The irradiated samples were irradiated at each time point
(15 min UV, 1.0 mW/cm2). On each collected sample, the amount
(mi, μg) of FTC-BSA released was assessed by micro-BCA protein
assay kit (Thermo Scientific). Percentage released at each point was
Synthesis of 13. Compound 12 (370.7 mg, 0.36 mmol) was
dissolved in DMF (5.1 mL) with piperidine (30.7 mg, 0.36 mmol),
stirred for 3 h until completion of the reaction (LCMS monitoring).
DMF was removed under reduced pressure and the product was
purified by flash chromatography on silica gel (DCM/methanol, 100:0
1
to 80:20). Yield: 261 mg (90%). H NMR (600 MHz, CDCl3) δ
(ppm) 7.68 (s, 1H), 7.13 (s, 1H), 6.86−6.66 (m, 1H), 6.35 (bs, 1H),
6.28 (d, J = 16.7 Hz, 1H), 6.15 (m, 1H), 6.03 (m, 1H), 5.63 (m, 1H),
5.26 (m, 2H), 5.21 (m, 2H), 4.45−4.10 (m, 4H), 3.73−3.61 (m, 12H),
3.54−3.45 (m, 4H), 3.49 (s, 3H), 3.35 (bs, 3H), 3.23 (m, 2H), 1.78
(m, 1H), 1.63 (m, 3H). MS (ESI) m/z: [M + H]+ calcd for
C33H47BrN3O15, 804.2; found, 804.0.
Synthesis of 14. After dissolution of 13 (260 mg, 0.32 mmol) in
EtOAc (10 mL), a solution of K2CO3 (446.5 mg, 3.23 mmol) in water
(10 mL) was added, and the resulting mixture was stirred vigorously at
0 °C. Acryloyl chloride (54.3 μL, 0.67 mmol) was then added
dropwise to the reaction at 0 °C. After a few minutes, the reaction
mixture was allowed to reach RT and was stirred until completion of
the reaction, monitored by LCMS. Portion-wise additions of acryloyl
chloride (108 μL) were needed to reach complete removal of the
MOM groups. After 4 h, the organic and aqueous layers were
separated. The aqueous phase was extracted twice with EtOAc.
Organic layers were combined, washed twice with water and brine,
then dried over MgSO4 and finally removed under reduced pressure.
Yield: 268.6 mg (97%). 1H NMR (600 MHz, CDCl3) δ (ppm) 7.66 (s,
1H), 7.12 (s, 1H), 7.08−6.95 (m, 1H), 6.34−6.00 (m, 7H), 5.65 (m,
1H), 5.59 (m, 1H), 5.27 (m, 2H), 5.21 (m, 2H), 4.65−4.20 (m, 5H),
3.73−3.59 (m, 12H), 3.51 (m, 2H), 3.48 (s, 3H), 3.36 (m, 3H), 3.33
(bs, 3H), 3.24 (m, 2H), 1.89 (m, 1H), 1.79 (m, 1H), 1.61 (m, 2H).
MS (ESI) m/z: [M + H]+ calcd for C36H49BrN3O16, 858.2; found,
858.0.
calculated by mi/Σmi × 100%, with mi = ci × Vi + Σmi−1
.
Cell Culture. Mouse mesenchymal stem cells stably expressing
GFP (Cyagen, OriCell, strain C57BL/6, mMSC-GFP, Cat. No.
MUBMX-0110) were cultured in OriCell mouse mesenchymal stem
cell growth medium (Cyagen, Cat. No. MUXMX-90011) supple-
mented according to provider with cell-qualified fetal bovine serum,
glutamine, and penicillin-streptomycin. Cells were routinely cultured
at 37 °C in a humidified incubator in a 95% air/5% CO2 atmosphere
and passaged by detaching cells with 0.25% trypsin−0.05% EDTA (w/
v) solution (Gibco Invitrogen).
Preparation of Hydrogels Encapsulating mMSCs-GFP.
Hydrogels at 2.9 mol % cross-linking density were formed by
dissolving 5 mg (6.1 μmol) of Bhc crosslinker 2 and acrylamide (15
mg, 211 μmol) in sterile DMEM, pH 9.1 (75 μL). pH reached back
7.4 upon saturation with CO2 for 5 min. A total of 1 μL of 50% APS in
DMEM, pH 7.4, was added, followed by the addition of TMED (1
μL). The resulting mixture was combined with fibronectin (4 μL, 1
mg/mL) and GFP-MSCs solution (50000 cells, 21 μL DMEM) and
gently vortexed for a few seconds. The solution was aliquoted in 12
samples (8 μL) in two Lab-Tek 8 chambers, and the hydrogels were
allowed to polymerize for 45 min in the incubator (37 °C, 5% CO2).
Then, 100 μL of mMSC-cell culture media (wash #1) was added, and
the hydrogels were allowed to equilibrate for 40 min (37 °C, 5% CO2).
Wash #1 was replaced by fresh mMSC-cell culture media (100 μL,
wash 2) for further equilibration (37 °C, 5% CO2). Washes #1 and #2
allow us to remove cells that are weakly entrapped at the gel surface in
order to evaluate the accurate number of light triggered cell released
from the gel.
GFP-mMSCs Release Study. Cell culture media was replaced by
PBS 1× (pH 7.4, 100 μL). Hydrogels (N = 6) were exposed to UV
light (365 nm, OmniCure S2000 Curing System, 30 cm from light
source, 1 mW/cm2) for 15 min intervals. After each exposure time,
additional cell culture media was added (100 μL) and the hydrogels
were incubated 1.5 h (37 °C, 5% CO2). Before each irradiation, the
supernatant solution was moved to a new well and cells were allowed
to settle for few hours before counting the cells released by microscopy
(irradiated fractions 1−6, Figure S9). Nonirradiated hydrogels (N = 6)
were studied in parallel (control fractions 1−6, Figure S9). A total of
six irradiations were performed on the hydrogels, and the final
incubation time was 10 h.
Synthesis of 2. After dissolution of 14 (260 mg, 0.30 mmol) in
DCM (6.8 mL), TFA (1.3 mL) was added, and the resulting mixture
was stirred 1 h at room temperature. Additional TFA (0.3 mL) was
added to reach the completion of the reaction. After 2 h of reaction,
the solvents were removed under reduced pressure to give the target
molecule as a pale yellow solid. Yield: 239.2 mg (97%). 1H NMR (600
MHz, CDCl3) δ (ppm) 7.62 (s, 1H), 7.23−7.08 (m, 1H), 6.98 (s, 1H),
6.34−6.21 (m, 5H), 6.00−5.86 (m, 1H), 5.75 (d, 1H, J = 9.6 Hz), 5.69
(dd, 1H, J = 14.5 Hz, 10.5 Hz), 5.30−5.16 (m, 3H), 4.44 (m, 3H),
4.26−4.14 (m, 3H), 3.78−3.52 (m, 14H), 3.37 (bs, 3H), 3.28 (m,
2H), 1.94 (m, 1H), 1.81 (m, 1H), 1.65 (m, 2H). HRMS (ESI) m/z:
[M + Na]+ calcd for C34H44BrN3NaO15, 836.1848; found, 836.1847.
Crosslinker 1 Photolysis and Degradation. 1H NMR Study. A 1
mg/mL solution of compound 1 in deuterated PBS 1× (pH 7.4)/
DMSO-d6 (95:5) was placed into a NMR tube and irradiated using a
Luzchem LZC-ORG photoreactor equipped with 8UV-A lamp, for 15
min (1 mW·cm−2). The crosslinker solution was incubated at 37 °C
1
and H NMR (600 MHz) spectra were taken periodically to estimate
the degree of crosslinker degradation as the percent of the cleaved
ester bonds.
HPLC-MS Study. A 0.1 mg/mL solution of compound 1 in PBS 1×
(pH 7.4)/DMSO (95:5) was placed into a quartz semimicrocuvette
and irradiated using a Luzchem LZC-ORG photoreactor equipped
with 8UV-A lamp, for 15 min (1 mW·cm−2). The solution was
incubated at 37 °C and aliquots (10 μL) were removed periodically
and injected into HPLC-MS to determine the presence of the
intermediates by integrating the peaks of the single ions, m/z = 580 (1,
[M + H]+), m/z = 284 (uncaged crosslinker, [M + H]+), m/z = 169
(lactam byproduct obtained by the intramolecular cyclization, [M +
Preparation of Photodegradable Nanogels. Nanogels at 1.7
mol % cross-linking density were formed by dissolving 12 mg (13
μmol) of Bhc crosslinker 2 and acrylamide (61.3 mg, 862 μmol) with
or without 10 nm iron oxide (II,III) magnetic nanoparticles (50 μL of
a 5 mg/mL solution in H2O) in 10 mM Tris-HCl, pH 8 (250 μL). The
resulting solution was added into 9% docusate sodium (AOT; 1 g)
D
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solution in hexane (10 mL). After vortexing for 1 min, 16 μL of 50%
APS in 10 mM Tris-HCl, pH 8, was added. The reaction mixture was
vortexed for 1 min and then filtered through a 0.22 μm PVDF filter.
After portionwise addition of 500 μL of 5% tetramethylethylenedi-
amine (TMEDA) in hexane (10 μL × 50), the particle solution was
left on a shaker at 60 rpm for 30 min, followed by removal of hexane
by rotary evaporation at 40 °C. Nanogels were washed with acetone
(10 mL × 3) and collected each time by centrifugation at 5000g for 10
min. The white precipitate was then dispersed in 1 mL of PBS 1× (pH
7.4).
Dynamic Light Scattering. All DLS experiments were done in
triplicate.
Degradation upon UV Exposure. In a typical experience, 25 μL of
the nanogel solution mentioned above was diluted with PBS 1× (pH
7.4, 100 μL) in a UV transparent disposable cuvette. Then the solution
was irradiated in the Luzchem LZC-ORG photoreactor for 15 min. A
nonirradiated mixture was studied in parallel. Time course of nanogels
degradation was monitored as a function of incubation time at 37 °C.
Degradation upon NIR Exposure. In a typical experience, 8 μL of
the nanogel solution mentioned above was diluted with PBS 1× (pH
7.4, 50 μL). Then the solution was irradiated at 740 nm with a
Ti:sapphire laser for 1 h in a quartz cuvette (50 μL, 3 mm, Z = 15
mm). A nonirradiated mixture was studied in parallel. Time course of
nanogels degradation was monitored as a function of incubation time
at 37 °C.
Transmission Electron Microscopy of Nanogels. Samples
before and after irradiation/incubation were diluted five times with DI
water and one droplet of the diluted solution for each sample was
dropcast onto a Pelco carbon-coated 400 square mesh copper grid
(Ted Pella, inc.). The grids were naturally dried in air for 3−5 h before
imaging.
Figure 1. 1H NMR and HPLC-MS analysis confirm that crosslinker 1
degrades exclusively via lactamization upon light-triggered depro-
tection (15 min UV irradiation, 1 mW/cm2) in PBS (1×, pH 7.4)/
DMSO-d6 (95:5) at 37 °C. (a) Potential products formed by
photolysis of the light-cleavable crosslinker 1. (b) 1H NMR spectra of
1, following irradiation, as a function of incubation time. Integration of
C and c were slightly off because the intensity of the signal decreases
due to water signal suppression, required to avoid domination of the
spectra by the water residual signal (at 4.79 ppm). (c) Kinetics of light-
triggered degradation of 1 (15 min UV irradiation) or by hydrolysis, as
determined by 1H NMR spectroscopy. (d) HPLC-MS analysis
confirms degradation of 1 via lactamization following irradiation
([M + H]+ = 116 was not detected).
RESULTS AND DISCUSSION
■
We combined ornithine, which spontaneously forms a six-
membered lactam upon deprotection, with a coumarin-based
photocage to yield light-degradable crosslinkers 1 and 2
(Scheme 1). Absorption spectra of 1 and 2 were recorded in
diluted organic and aqueous solutions to determine their molar
absorption coefficients (Table 1 and Figure S3).
Table 1. Absorption Spectral Data of 1 and 2
a
b
compound
solvent
λmax
ε
was then introduced through a carbamate linkage. The first
acrylamide group was then added by DCC coupling with N-(2-
hydroxyethyl)acrylamide. For ease of synthesis, the crude
intermediate was treated with piperidine to remove the Fmoc
protecting group. The second acrylamide moiety was attached
by addition of acryloyl chloride using Schotten−Baumann
conditions. In the last step, TFA treatment removed the
methoxymethyl acetal protecting group (MOM) to give the
target crosslinker structure 1. 1H and 13C NMR spectra of 1 are
reported in the Supporting Information (Figures S1 and S2).
1
1
1
2
2
2
DMSO
330
372
371
330
373
371
10846
14889
10274
14696
18454
27119
PBS (1×, pH 7.4)/DMSO (95:5)
DMEM
DMSO
PBS (1×, pH 7.4)/DMSO (95:5)
DMEM
a
b
Maximum absorption wavelengths. Molar absorption coefficient
(M−1·cm−1). Concentrations ranged from 3.10−6 to 8.10−5 M.
1
Before investigating hydrogel stability and light-triggered
degradation at the bulk and nanoscale, model compound 1 was
exposed to UV light to confirm degradation via lactamization.
Detailed characterization of the photodegradation reaction was
performed using 1H NMR spectroscopy and HPLC-MS
(Figure 1). Both techniques show (1) high stability of the
crosslinker at 37 °C in the absence of irradiation, (2) near
complete light-triggered degradation within less than 24 h of
incubation at 37 °C, and (3) formation of only lactam products.
Synthesis of Model Crosslinker 1. The ornithine-inspired
light-degradable crosslinker 1 was obtained via a five-step
synthesis (Scheme 2; global yield: 39%). Briefly, commercially
available Fmoc-Orn(Boc)-OH was first treated with TFA to
selectively remove the Boc protecting group. The Bhc moiety
1H NMR Spectroscopic Degradation Study. H NMR
spectroscopy confirmed that crosslinker 1 degrades exclusively
by lactamization following photocleavage in deuterated PBS
(1×, pH 7.4)/DMSO-d6 (95:5). Degradation products were
identified and degradation kinetics were quantified after
incubation at 37 °C with or without UV irradiation (Figures
1a−c and S4 and Table 2). Upon irradiation, all resonance
signals from the protons of the Bhc (7.8, 6.6, 6.1, and 5.3 ppm)
disappeared, indicating complete removal of the photocage
(Figure 1, 0 h); nonsoluble cleaved Bhc residues typically
precipitate and are no longer detected. In addition, the shift
from 3.2 to 3.1 ppm is characteristic of the methylene protons
in α position of the amine (D′ → D). In the nonirradiated
sample, no shift of signal D′ is observed (Figure S4), indicating
E
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light-triggered degradation, we needed a more hydrophilic
crosslinker (compound 2, Scheme 3) to formulate hydrogels
Scheme 2. Synthesis of Ornithine-Inspired Light-Degradable
Crosslinker 1
a
Scheme 3. Synthesis of Ornithine-Inspired Light-Degradable
a
Crosslinker 2
a
Reagents and conditions: (a) TFA/DCM; Bhc-PNP, DIEA, DCM/
DMF, o.n., 65%; (b) N-(2-hydroxyethyl)acrylamide, DCC, DMAP,
DCM/DMF, o.n.; (c) piperidine, DMF, 1.5 h, 60% over 2 steps; (d)
acryloyl chloride, K2CO3, EtOAc/H2O, 30 min, 100%; (e) TFA/
DCM, 1 h, 100%. R = methoxymethyl acetal (MOM).
Table 2. Mechanism of Degradation and Half-Life (t1/2) of 1
Incubated at 37 °C, as Determined by H NMR
1
a
Reagents and conditions: (a) CrO3/H2SO4, acetone, 2 h, 93%; (b)
tert-butyl(chloro)diphenylsilane, imidazole, DMF, o.n., 37%; (c)
acryloyl chloride, K2CO3, EtOAc/H2O, 2 h, 92%; (d) 7, DCC,
DMAP, DCM, o.n., 76%; (e) tetrabutylammonium fluoride hydrate,
THF, 3 h, 71%; (f) 3, DCC, DMAP, DCM, o.n., 59%; (g) piperidine,
DMF, 3 h, 90%; (h) acryloyl chloride, K2CO3, EtOAc/H2O, 4 h, 97%;
(i) TFA, DCM, 2 h, 97%.
a
b
time
lactamization (%)
hydrolysis (%)
t1/2
UV
45 h
45 h
82.3 5.7
0
0
12 h
20 d
no UV
13.8 2.5
a
% lactamization = ∫ d/(∫ D + ∫ d) ≈ ∫ f/(∫ F + ∫ f) ≈ ∫ e/(∫ E + ∫ e).
% hydrolysis = ∫ f/(∫ F + ∫ f) ≈ ∫ e/(∫ E + ∫ e). ∫ refers to the
integration below each respective peak.
b
and to assess the feasibility of NIR/UV-triggered hydrogel and
nanogel degradation. Without modifying the overall length of
the crosslinker (14 carbons (2) vs 13 (1)), a triethylene glycol
(TEG) arm was introduced within the ornithine-inspired
structure. Briefly, commercially available tetraethylene glycol
monomethyl ether was first treated with Jones reagent mixture
to oxidize the terminal alcohol to form carboxylic acid 7. In
parallel, silyl ether 8 was obtained from 3-amino-1,2-propane-
diol using tert-butyl(chloro)diphenylsilane and imidazole. The
first acrylamide moiety was attached by addition of acryloyl
chloride to 8. The remaining alcohol in 9 was then coupled to
the TEG chain of 7 by DCC coupling to obtain TBDPS-
protected TEG compound 10. Finally, similar procedures as
used in the synthesis of 1 were followed to synthesize 2
from 10.
The key and unique feature of the reported light-degradable
hydrogels (bulk and nano) is the short length between the two
acrylamide functional groups of crosslinker 2 (15.4 Å, as
measured by ChemBio3D after MM2 energy minimization).
Upon copolymerization with acrylamide (2−3 mol % cross-
linking density), the obtained hydrogels are stable in the
absence of irradiation and degrades in a rapid and controlled
fashion upon irradiation. We found that these polymerization
conditions yielded a suitable mesh size for the quantitative
entrapment and release of proteins or other hydrophilic
payloads with a hydrodynamic diameter of >7 nm.66,67
Light-Triggered BSA Release Kinetics. Fluo-BSA-loaded
bulk hydrogels were prepared by copolymerizing 2 and
acrylamide in sodium phosphate buffer (100 mM, pH 8.5) in
that the Bhc group remains intact. New resonance signals (e
and f) correspond to the formation of N-(2-hydroxyethyl)
acrylamide, upon the cleavage of the ester linkage. The shift
from 3.1 to 3.4 ppm (D → d) confirmed the formation of
lactam derivatives after irradiation and incubation.65
HPLC-MS Degradation Study. After irradiation, no
remaining intermediate (deprotected 1, [M + H]+ = 284) is
detected in the media within 20 h (Figure 1d). This result is in
agreement with the time needed to completely degrade
ornithine-based dimers when exposed to UV light.65 Moreover,
only lactam derivatives ([M + H]+ = 169) were formed from
the irradiated crosslinker, as no primary amine intermediates
resulting from ester hydrolysis ([M + H]+ = 187) were
1
detected. Interestingly, both H NMR and HPLC-MS are in
agreement with no hydrolysis of the ester bound but an
exclusive degradation by cyclization. No degradation species
were detected in nonirradiated crosslinker (Figure S5).
To validate that our hydrogel degrades in response to NIR
light, we irradiated our crosslinker for 1 h at 740 nm (1 W) and
1
incubated at RT overnight. H NMR spectroscopy confirmed
the efficiency of photocleavage (shift from 3.2 to 3.1 ppm,
indicating the formation of a free amine; only around 5% of
protecting group signal remains; red rectangles, Figure S6a).
The extent of lactamization was calculated to be near 30%,
which is close to the percentage obtained upon UV irradiation
at RT (Figure S6b).
Synthesis of Crosslinker 2. While crosslinker 1 was a
model compound used to validate the mechanism of fast and
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the presence of the protein. To confirm light-induced
degradation and release, both irradiated and nonirradiated
gels were incubated in PBS (1×, pH 7.4) at 37 °C, and the
amount of protein released over time in the supernatant was
measured by microbicinchoninic acid assay (micro-BCA assay;
Figure 2b). BCA analysis of the solution used to wash the
(measured by infrared camera, Figure S7). No significant
heating by water absorption is expected with a femtosecond
laser operating at a water-transparent wavelength70 (740 nm)
with low powers. As a proof of concept, we formulated
acrylamide-based hydrogels encapsulating murine mesenchymal
stem cells expressing green fluorescent protein (GFP-mMSCs)
in bicarbonate-buffered cell culture medium (Dulbecco’s
modified Eagle’s medium, DMEM). Taking advantage of the
sodium bicarbonate−carbon dioxide system, 2, was solubilized
in DMEM (pH 9, after competing the CO2 by bubbling argon
for 1 h, Figure S8). Neutral pH was recovered prior to cell
addition simply by bubbling the solution with CO2 for a few
minutes. After formation and equilibration of the hydrogels in
mMSC cell-culture medium, some were exposed to UV light for
six periods of 15 min (365 nm, 1 mW/cm2) and incubated 1.5
h between each exposure. The medium was changed between
each irradiation and the number of collected cells was counted
in both irradiated and nonirradiated hydrogels (Figures 2c and
S9). Quantifying nonencapsulated cells in the wash revealed
that the encapsulation efficiency was above 92% ( 1.8%, n =
12). In addition, mMSCs were released 15× faster when
hydrogels were exposed to repeated UV irradiation. Non-
irradiated hydrogels did not release appreciable numbers of
cells (Figure 2c).
While this is not the first report of light-triggered cell release,
our design for release at low power is an important property for
biologically relevant applications.39
Light-Triggered Release of Iron Oxide Nanoparticles
from Nanogels. Using the same chemistry, crosslinker 2 was
also employed to synthesize light-degradable nanogels. Because
they can efficiently encapsulate, transport, and release hydro-
philic payload in a preprogrammed manner, nanogels are ideal
carriers for the delivery of small therapeutic agents or
biomacromolecules, combining small size with large surface
area, efficient swelling, and high loading.71−74 Empty nanogels
were formulated by means of inverse emulsion redox
polymerization at 1.7 mol % crosslinker density in Tris HCl
(1×, pH 8.0) to verify stability and determine light-triggered
degradation kinetics. No change in hydrodynamic diameter and
PDI was observed in the absence of irradiation, confirming that
light-responsive nanogels (200 nm) are stable for at least 1
week at 37 °C (Figures 3a,d,e and S10). Conversely, brief
exposure to UV (15 min) or NIR (1 h) triggers disassembly
within a few hours of incubation (Figure 3b−f).
After 5 h, only nonirradiated nanogels yielded reliable DLS
data; the difficulty in analyzing irradiated nanogels is a result of
aggregated or sedimented particles and small fragments. At the
first stage of degradation, photocage cleavage leads to nanogel
swelling due to increased hydrophilicity from unmasking free
amines. Further incubation of the loosened network led to an
additional increase in diameter in combination with diffusion of
free hydrophilic PAA chains. No swelling was observed in the
absence of light, indicating stability of the polymeric network at
this crosslinking density. This absence of initial swelling, likely a
result of the crosslinker’s chemical structure and short length, is
crucial to prevent burst release and payload leakage. Polymer
mesh morphology and chemical composition are known to
affect release characteristics.75,76 In these hydrogels, hydro-
phobic coumarin moieties covalently attached to the network
reduce the initial penetration of water, and the short crosslinker
limited mesh space available for diffusion preirradiation.
Finally, we investigated whether nanogels release contents in
response to light. PAA nanogels containing crosslinker 2 and
Figure 2. (a) Accelerated hydrogel erosion and degradation upon UV
irradiation. (b) UV irradiation-dependent release of Fluo-BSA from
hydrogels analyzed by micro-BCA assay. Hydrogels were irradiated
(365 nm, 15 min, 1 mW·cm−2) prior to incubation for each time point.
Results are presented as means SEM; n = 3. (c) UV irradiation-
triggered release of encapsulated GFP-mMSCs. Hydrogels (n = 6)
were irradiated (15 min, 365 nm, 1 mW/cm2), and the released cells
were counted after each incubation interval (1.5 h). Results are
presented as means SEM; n = 6.
hydrogels indicated that 80% of proteins were entrapped in the
gel. This is in agreement with the gels’ small pore size capable
of efficiently retaining BSA-size payload (7 nm).68 Similar BSA
release kinetics were obtained with hydrogels prepared from
acid-labile acetal crosslinkers (comparable linker length, cross-
linking ratio, and gelation conditions).27 Light acceleration of
hydrogel degradation and BSA release were clearly observed
(Figure 2a). Upon irradiation, the hydrogel released 100% of
the entrapped protein within 10−20 h, and the hydrogel was no
longer visible (fully degraded) in the media after 20 h
incubation. Conversely, the nonirradiated hydrogel retained
its integrity and more than 80% of the payload over the same
amount of time.
Light-Triggered Release of GFP-mMSCs from Hydro-
gels. Despite substantial challenges, hydrogels hold great
potential as cell delivery implants for regenerative medicine.69
This work demonstrates the ability of crosslinker 2 to build cell
reservoir matrices for the encapsulation and on-demand
delivery of therapeutic cells. No appreciable increase in
temperature was observed after 15 min of UV irradiation
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Figure 4. Nanogels fall apart within 5 h upon UV or NIR irradiation.
Representative TEM microphotographs of nanogels after 5 h
incubation at 37 °C in phosphate buffer (pH 7.4, 10 mM), either
(a) nonirradiated or (b) UV-irradiated (365 nm, 15 min, 1 mW·cm−2)
or NIR-irradiated (740 nm) for (c) 1 h (1 W, 1.9 × 106 mW·cm−3)
and (d) 2 h (0.5 W, 9.5 × 105 mW·cm−3).
Figure 3. Upon UV and NIR exposure, nanogel particles first swell
then degrade. DLS profiles of (a) nonirradiated, (b) UV-, and (c)
NIR-irradiated nanogels (15 min, 365 nm, 1 mW·cm−2 and 1 h, 740
nm, 1 W, 1.9 × 106 mW·cm−3, respectively). Individual DLS
parameters of nonirradiated and irradiated nanogels over time: (d)
hydrodynamic diameter, (e) PDI, and (f) mean count rate. Results are
presented as means SEM; n = 3.
encapsulation and light-activated release of hydrophilic pay-
loads as small as 7 nm in diameter. As these materials also
degrade in response to NIR light, they hold promise for
spatiotemporal control over protein and cell presentation and
delivery in cultured and engineered tissues as well as within
living tissue. The next step in this line of research is to use this
crosslinker to develop light-degradable hydrogels from
biodegradable materials in place of PAA, such as methacrylated
hyaluronic acid (HA), alginic acid, chitosan, or polylactic acid,
all known to be enzyme-degradable. Our design may find
application where complete on-demand degradation is of high
importance, especially to avoid surgical removal of materials
following cell delivery.79
superparamagnetic iron oxide nanoparticles (SPIONs) were
prepared following the same emulsion technique. SPIONs are
effective contrast agents for magnetic resonance imaging (MRI)
and are widely used for clinical oncology imaging and
therapy.77,78 Hydrophilic SPIONs (Fe3O4, 10 nm) display
high electron density, facilitating detection of nanogels by
TEM. TEM micrographs of these nanogels show that nanogels
are spherical, dispersible in water, and efficiently encapsulate
iron oxide particles (Figure S11). No precipitation in acetone of
nonencapsulated SPIONs was observed during the formulation.
As expected from the crosslinker design, UV- or NIR-
irradiated nanogels completely fell apart and aggregated within
5 h, corroborating degradation kinetics, as determined by DLS
(Figure 3). Nonirradiated nanogels were intact after 5 h (8 h
incubation in Figure S12), with iron oxide particles located only
inside nanogels (Figure 4a). After UV or NIR irradiation, intact
particles are no longer visible; only fragments of material
coexist with iron oxide aggregates (Figure 4b−d).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
mac.5b00950.
■
S
1H and 13C NMR of crosslinker 1, absorbance spectra in
DMEM of crosslinkers 1 and 2, 1H NMR and HPLC-MS
photolysis and degradation studies, photographs and
thermal images, and release experiments, DLS, TEM
(PDF).
CONCLUSION
■
Two new ornithine-based photodegradable coumarin-bearing
short crosslinkers were synthesized and used to formulate PAA-
based bulk hydrogels and nanogels encapsulating protein
(BSA), cells (GFP-mMSCs), and iron oxide nanoparticles.
Both the crosslinker and the resulting hydrogels are stable in
PBS (pH 7.4) in the absence of irradiation with minimal or no
leakage of their contents, depending on the payload size.
However, upon irradiation with low-power UV or NIR light,
the gels fully degrade into short fragments through intra-
molecular cyclization. As expected from the molecular design of
the crosslinker, these hydrogels are capable of efficient
AUTHOR INFORMATION
Corresponding Author
*E-mail: aalmutairi@ucsd.edu.
■
Present Address
⊥UT Southwestern Medical Center, 5323 Harry Hines Blvd.,
Dallas, TX 75390-8896
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank the NIH (R01EY024134) for funding. NMR data
was acquired at the UCSD Skaggs School of Pharmacy and
Pharmaceutical Sciences NMR facility.
ABBREVIATIONS
■
BCA, bicinchoninic acid; Bhc, bromohydroxycoumarin; Bhc-
Glu, N-(6-bromo-7-hydroxycoumarin-4-yl)methoxycarbonyl-L-
glutamic acid; Bhc-PNP, (6-bromo-7-(methoxymethoxy)-2-
oxo-2H-chromen-4-yl)methyl(4-nitrophenyl) carbonate; BSA,
bovine serum albumin; DCC, N,N′-dicyclohexylcarbodiimide;
DCM, dichloromethane; DLS, dynamic light scattering; DIEA,
N,N-diisopropylethylamine; DMAP, 4-dimethylaminopyridine;
DMEM, Dulbecco’s modified Eagle’s medium; DMF, dime-
thylformamide; DMSO, dimethyl sulfoxide; EtOAc, ethyl
acetate; Fluo-BSA, fluorescein-labeled bovine serum albumin;
GM, Goeppert−Mayer; kcps, kilo counts per second; HA,
hyaluronic acid; HPLC, high-performance liquid chromatog-
raphy; mMSCs-GFP, mice mesenchymal stem cells expressing
green fluorescent protein; MOM, methoxymethyl acetal; MS,
mass spectrometry; NIR, near-infrared; o.n., overnight; ONB,
ortho-nitrobenzyl; PBS, phosphate buffer saline; PDI, poly-
dispersity index; PEG, polyethylene glycol; PMMA, poly-
(methyl methacrylate); PNP, para-nitrophenyl; TBDPS, tert-
butyldiphenylsilane; TEG, triethylene glycol; TEM, trans-
mission electron microscopy; TFA, trifluoroacetic acid; tRNA,
trans-ferribonucleic acid; UV, ultraviolet; Zavg, Z average
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