Photoresponsive coumarin polyesters that exhibit cross-linking and chain scission properties

Article abstract of DOI:10.1021/ma400584y

The synthesis and properties of a new class of photoresponsive coumarin polyesters are described. Incorporation of the coumarin chromophore in the polymer chain provides interesting properties such as polymer chain cross-linking upon irradiation at 350 nm and chain un-cross-linking when irradiated at 254 nm. In addition, irradiation at 254 nm also results in polymer chain scission. The cross-linking, un-cross-linking, and chain scission properties were studied by ssNMR, ATR-IR, and GPC measurements. These properties enable the fabrication of 2D surfaces having complementary micropatterned features. Also, initial biocompatibility profiles of the polymers and their irradiation products were demonstrated using MTT assays.

Full text of DOI:10.1021/ma400584y

Article
pubs.acs.org/Macromolecules
Photoresponsive Coumarin Polyesters That Exhibit Cross-Linking
and Chain Scission Properties
Murthy V. S. N. Maddipatla,† Daniel Wehrung,‡ Chuan Tang,† Weizheng Fan,† Moses O. Oyewumi,‡
Toshikazu Miyoshi,† and Abraham Joy*,†
†Department of Polymer Science, The University of Akron, Akron, Ohio 44325, United States
‡College of Pharmacy, Northeast Ohio Medical University, Rootstown, Ohio 44272, United States
S
* Supporting Information
ABSTRACT: The synthesis and properties of a new class of
photoresponsive coumarin polyesters are described. Incorpo-
ration of the coumarin chromophore in the polymer chain
provides interesting properties such as polymer chain cross-
linking upon irradiation at 350 nm and chain un-cross-linking
when irradiated at 254 nm. In addition, irradiation at 254 nm
also results in polymer chain scission. The cross-linking, un-
cross-linking, and chain scission properties were studied by
ssNMR, ATR-IR, and GPC measurements. These properties enable the fabrication of 2D surfaces having complementary
micropatterned features. Also, initial biocompatibility profiles of the polymers and their irradiation products were demonstrated
using MTT assays.
Scheme 1. Photochemical Reactions of Coumarin
Compounds
INTRODUCTION
■
The design of stimuli responsive polymers is an exciting and
challenging area of polymer science.1 Polymers that are
responsive to an appropriate wavelength of light have the
advantage of spatial and temporal control of the output.2 Such
photoresponsive polymers have demonstrated very interesting
properties such as reversible bending of an azobenzene liquid
crystal polymer film,3 light activation of shape memory
polymers,4 modulation of refractive index,5 release of
biomolecules,6 change of phase behavior,7 light activated
mechanophores,8 formation of surface topologies,9 and
activation of cell penetrating peptides.10 Photoresponsive
polyesters, polycarbonates, and other bioresorbable polymers
with potential biological applications have also been designed.11
Along this direction, we recently reported alkoxyphenacyl-
based polycarbonates that undergo controlled chain scission.12
Hydrogels that release model therapeutics by photoirradiation
at multiple wavelengths13 and the use of upconverting
nanoparticles embedded in photosensitive hydrogels for the
release of biomolecules have also been recently described.14
This report expands on the functional repertoire of polymers
that are both photoresponsive and bioresorbable. We describe a
class of coumarin-based photoresponsive polyesters in which
the chromophore is embedded in the polymer chain.
Coumarin-based molecules have been examined as therapeu-
tics,15 protecting groups,16 phototriggers for biological
molecules,17 and photoresponsive units on polymeric sys-
tems.18 Upon irradiation at ∼350 nm, coumarin derivatives
such as 7-methoxycoumarin undergo [2 + 2] cycloaddition to
yield cyclobutane dimers (Scheme 1). This reaction has been
observed both in solution19 and in the solid state.20 The
cycloaddition reaction is reversible when irradiated at a shorter
wavelength.18,21 In addition to the above reversible cyclo-
addition reactions, some coumarin derivatives such as esters or
phosphates substituted at the 4-position undergo photorelease
at 350 nm, which is the basis for coumarin-based photo-
triggers.17,22
Similarly, there are several examples of coumarin-based
polymers that show cycloaddition reactions.23 For example,
formation of single chain nanoparticles from coumarin
containing methacrylate polymers and bending of hydrogen
bonded coumarin polymers have been reported by Zhao.24
High refractive index coumarin based polyacrylates and
siloxanes have been designed by Hampp.25 Shi has shown the
Received: March 19, 2013
Revised: May 16, 2013
© XXXX American Chemical Society
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Scheme 2. Synthesis of Photoresponsive Monomer and Polymers
Chemical shift was calibrated using CH signal of adamantine at 29.46
ppm as an external reference. Each spectrum was obtained by 10 240
scans at ambient temperature. Gaussian peaks were applied for spectral
fitting. The signal intensity obtained by CP method highly relies on
CP efficiency.
GPC Analysis. GPC was performed on two TSK-GEL Super H
3000 columns and one TSK-GEL Super H 4000 column in series and
analyzed using a TOSOH EcoSEC HLC-8320 instrument. Samples
were dissolved in CHCl3 with a constant flow rate of 0.4 mL/min.
Molecular weights were obtained relative to PS standards.
ATR-IR and UV Analysis. IR spectra of the free-standing solid
films were recorded on a Shimadzu MIRacle 10 ATR-FTIR. UV was
recorded on a Shimadzu UV-1800 UV spectrophotometer.
AFM was obtained on DI MultiMode SPM AFM (Tapping mode).
Tensile modulus was measured in the Applied Polymer Research
Center at The University of Akron (Dynamic Mechanical Analysis).
Specimens were cut into 14 mm long, 9.75 mm wide, and 0.06 mm
thick samples. The samples were measured under uniaxial tension at
0.04 mm/s.
Fluorescence Microscopic Images. Polymers CA11 (4) and
CAQ (5) were flow coated on precleaned microscopic glass slides and
kept under vacuum for 24 h. Macroscopic template (1 cm × 1 cm, 200
mM sized patterns) placed on top of the glass slides and irradiated at
254 nm (and 350 nm) wavelength light for 40 min. The irradiated
samples were kept in CHCl3/MeOH (30:70) for 30 min and then
rinsed quickly in CHCl3/MeOH (70:30) for 2−3 min. These glass
slides were kept under vacuum for 24 h before taking the fluorescence
images. The optical images were obtained by automated fluorescence
microscope with an Olympus IX81 microscope (Olympus, Center
Valley, PA) equipped with a computer-controlled translation stage
(Prior Scientific, Rockland, MA) and Metamorph software. The
images of the excited samples were obtained using DAPI (4′,6-
diamidino-2-phenylindole) filter.
Synthesis of Monomer and Polymers (Scheme 2). 1: To a
stirring solution of resorcinol (10 g, 91 mmol, 1 equiv) and 4-
chloromethyl acetoacetate (17 g, 103 mmol, 1.14 equiv) in toluene
(150 mL) was added p-toluenesulfonic acid (3.6 g, 19 mmol, 0.21
equiv). The solution was connected to a Dean−Stark apparatus and
heated to reflux at 110 °C for 45 min. The reaction mixture was
concentrated and purified by column chromatography (EtOAC:hexane
1:9) to yielded a white solid 1 (12 g, 65% yield).
photo-cross-linking and photocleavage of a hyperbranched
polyester functionalized with peripheral 4-methylcoumarin
units.26 Coumarin end-functionalized polycaprolactones have
been designed by Priestley as a means to stabilize PCL
nanoparticles, which have potential as a fluorescent drug
carrier.27 Most of the above examples have the coumarin unit as
pendant groups, and there are much fewer reports of the
incorporation of the coumarin chromophore in the backbone.
Shea et al. have prepared coumarin-bridged polysilsesquioxanes
which undergo photoinduced deformation.28 Kasko et al. have
reported coumarin containing PEG macromers in which the
coumarin unit sensitizes o-nitrobenzyl groups for photo-
degradation.29 We envisioned that such in-chain coumarin
incorporated polymers would possess dual photoresponsive
properties of polymer chain cross-linking and chain scission. In
this report, we show that the coumarin-based polyesters
described here exhibit chain cross-linking at 350 nm and that
the cross-linked materials can be un-cross-linked by 254 nm
irradiation. Irradiation at 254 nm also leads to polymer chain
scission by photoinduced cleavage of the ester bond.
EXPERIMENTAL SECTION
■
Materials. Unless otherwise noted, solvents were purchased from
Fisher Scientific and were used as received. Acetone was dried over
activated 4 Å molecular sieves. Sodium bisulfate, potassium carbonate,
18-crown-6, p-toluenesulfonic acid, dichloromethane (extra dry), 4 Å
molecular sieves, 3-bromo-1-propanol, and adipic acid were purchased
from Acros Organics, ethyl 4-chloroacetoacetate and hydroquinone
bis(2-hydroxyethyl) ether were purchased from Alfa Aesar, and
diisopropylcarbodiimide (DIC) was purchased from Oakwood
Chemicals and used without further purification.
NMR Spectroscopy. Solution: 1H NMR spectra were recorded on
a Varian Mercury 300 MHz or Varian 500 MHz spectrometer. 13C
NMR spectra were recorded at 125 MHz on a Varian 500 MHz
spectrometer. Chemical shifts were recorded in ppm (δ) relative to
TMS. Solid state: 13C CPMAS NMR spectra were obtained using
BRUKER Avance III 300 NMR spectrometer equipped with a 4 mm
double resonance CP/MAS NMR probe. The magic angle spinning
(MAS) frequency was set to 12 000 5 Hz. The 1H 90° pulse length
2: To a stirring solution of water (350 mL) was added 7-hydroxy-4-
(chloromethyl)coumarin (1, 2.95 g, 14 mmol). The reaction mixture
was refluxed for 3 days, filtered while hot, and cooled to room
temperature over 12 h to yield off-white needles (2). The product was
1
is 3.75 μs. High-power H TPPM decoupling with a field strength of
65 kHz is applied during signal acquisition. Cross-polarization (CP)
contact time and recycle delay were 1.5 ms and 2 s, respectively.
B
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Table 1. Physical Properties of the Polymers CA11 and CAQ
a
b
% cross-linking
modulus, E (MPa)
a
a
c
c
d
e
polymer
Mn (g/mol)
PDI
Td (°C)
Tg (°C)
34−36
254 nm
350 nm
before hν
after hν
302
413
CA11 (4)
20 800
79 400
1.9
2.4
335
365
12
4
80
42
68
CAQ143 (5)
6−8
132
a
b
c
d
e
GPC analysis. Measured by uniaxial tension. Upon irradiation for 40 min. Tensile modulus of the polymer measured before irradiation. Tensile
modulus of the polymer measured after irradiation at 350 nm for 40 min.
Scheme 3. Irradiation of Coumarin Polyesters at 350 nm Leads to Polymer Chain Cross-Linking and Irradiation at 254 nm
Results in the Reverse Un-Cross-Linking and/or Ester Cleavage Reactions
filtered and connected to vacuum overnight (2.7 g, quantitative). The
product was used further without purification.
to the medium) solutions for 24 h. The treatment solutions were
aspirated, and the cells were washed with PBS (100 μL) before MTT
dye was added and incubated at 37 °C for 4 h. Aspiration of the dye
solution was followed by solubilizing the MTT dye in DMSO (100
μL) and glycine buffer (0.1 M, 15 μL). The absorbance of each well
was immediately measured using a microplate reader (Spectramax-
340PC, Bucher Biotec AG, Basel, Switzerland) at 570 nm. The
absorbance values obtained were plotted as a percentage of untreated
(control) cells.
c. Macrophage Activation Studies. Macrophage activation was
assessed based on reactive oxygen species (ROS) production using the
fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-
DA). RAW 264.7 cells were plated (5 × 104 cells/well) in a black 96-
well plate and allowed to recover overnight before being exposed to
treatment solutions containing varying concentrations of the polymers
(dissolved in DMSO and added to the medium) for 16 and 24 h. Cells
treated with hydrogen peroxide (100 μM) were used as the positive
control. Afterward, the treatment solutions were aspirated, and the cell
monolayer was rinsed with PBS before DCFH-DA was added and
incubated for 60 min (37 °C). The monolayer was again rinsed twice
with PBS before the fluorescence intensity was measured (excitation
485 nm, emission 528 nm). Fluorescence intensity measurements were
normalized per microgram of protein in each well. The protein content
was assessed using the Pierce BCA Protein Assay Kit (Fisher Scientific,
Pittsburgh, PA).
d. Biocompatibility after Polymer Irradiation. Cellular viability
and macrophage activation were also assessed after the polymer
(CA11) was irradiated with UV light (350 nm for 20 min and 254 nm
for 30 min). Cellular viability was measured after incubation (37 °C,
24 h) RAW cells with the polymer treatment solutions (dissolved in
DMSO and added to the medium) using the MTT assay. The extent
of macrophage activation was assessed by measuring the level of nitrite
production using untreated cells as negative controls. Briefly, RAW
264.7 cells were plated (5 × 104 cells/well) and allowed to recover
overnight before receiving treatment solutions containing the
irradiated CA11 samples. Afterward, the level of nitrite in the growth
media was measured using the Griess reagent system (Cayman, Ann
Arbor, MI). Lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO)
was used as the positive control, and all data points were expressed as a
percentage of LPS-treated cells.
3: To a stirring solution of anhydrous acetone (15 mL), 2 (1.0 g, 5.2
mmol, 1 equiv), potassium carbonate (2 g, 14.5 mmol, 2.8 equiv), and
3-bromo-1-propanol (1.5 g, 10.8 mmol, 2.1 equiv) was added 18-C-6
(0.7 g, 2.65 mmol, 0.5 equiv). The mixture was stirred for 5 min and
then transferred into a microwave reactor (CEM, Discover). The
reaction mixture was refluxed at 55 °C for 40 min (power: 20−30
mW) and filtered. The solvent was removed under reduced pressure to
yield a light yellow solid. Purification by column chromatography
(EtOAC:hexane 4:1) yielded a white solid (1.18 g, 91%) (3).
CA11 (4). In a two-neck round-bottom flask, 3 (250 mg, 1 mmol, 1
equiv), adipic acid (146 mg, 1 mmol, 1 equiv), and 4-(N,N-
dimethylamino)pyridinium-4-toluenesulfonate (DPTS, 117 mg, 0.4
mmol, 0.25 equiv) were taken. The flask was vacuum backfilled 3 times
with N2. Dichloromethane (2.5 mL) was added while cooling the flask
at 0 °C. After 15 min, N,N-diisopropylcarbodiimide (DIC, 470 mL, 3
mmol, 3 equiv) was added, and the reaction was allowed to stir at
room temperature for 48 h. The polymer was precipitated in mixture
of alcohols (MeOH:EtOH:PrOH 1:1:1). The polymer was collected,
centrifuged, and dried under vacuum to yield a white solid 4 (310 mg,
85%).
CAQ (5). In a two-neck round-bottom flask, 3 (62.5 mg, 0.25 mmol,
0.25 equiv), hydroquinone bis(2-hydroxyethyl) ether (149 mg, 0.75
mmol, 0.75 equiv), adipic acid (146 mg, 1 mmol, 1 equiv), and DPTS
(117 mg, 0.4 mmol, 0.25 equiv) were taken. The flask was vacuum
backfilled three times with N2. Dichloromethane (2.5 mL) was added
while cooling the flask at 0 °C. After 15 min, DIC (470 mL, 3 mmol, 3
equiv) was added, and the reaction was allowed to stir at room
temperature for 72 h. The polymer was precipitated in mixture of cold
alcohols (MeOH:EtOH:PrOH 1:1:1). The polymer was collected,
centrifuged, and dried under vacuum to yield a white solid 5 (252 mg,
88%).
Biocompatibility Studies. a. Cell Culture. Macrophage cells
(RAW 264.7) were obtained from ATCC (Manassas, VA). The cells
were cultured in humidified incubations (37 °C, 5% CO2) in complete
growth media comprised of Dulbecco’s Modified Eagle Medium
(DMEM), 10% fetal bovine serum (FBS), and antibiotics (100 μg/mL
streptomycin and 100 units/mL penicillin).
b. MTT Assay. Macrophage cells (RAW 264.7) were used for all
biocompatibility studies. Briefly, 5 × 104 cells were plated per well in a
96-well plate and allowed to recover overnight. The cells were exposed
to varying concentrations of polymer (dissolved in DMSO and added
C
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Figure 1. GPC traces of the polymers CA11 (A, B) and CAQ (C, D) after irradiation at 254 nm (A, C) and 350 nm (B, D).
Figure 2. UV−vis spectra of the polymers CA11 (A, B) and CAQ (C, D) after irradiation at 254 nm (A, C) and 350 nm (B, D).
homopolyester 4 (CA11). Similarly, a copolymer of the
coumarin diol and the bis(hydroxyethyl)hydroquinone (5)
(CAQ) was also synthesized and studied. Incorporation of a
second diol in the polymer facilitates modulation of the
mechanical properties and the efficiency of photoresponse of
the polymers. These polymers were characterized by 1D/2D
NMR (Supporting Information, Figures S1−S6), attenuated
total reflectance IR (ATR-IR), and gel permeation chromatog-
raphy (GPC). The polymers have UV absorption spectra from
250 to 370 nm with a λmax at 322 nm (molar extinction
coefficient, ε = 7.1 × 103 M−1 cm−1). They possess high
RESULTS AND DISCUSSION
■
The photoactive coumarin monomer 3 was synthesized as
shown in Scheme 2. A published protocol17 was used to
synthesize 7-hydroxy-4-(hydroxymethyl)coumarin (2). In order
to facilitate polymer synthesis, 2 was chain extended to yield (7-
(hydroxypropoxy)-4-(hydroxymethyl)coumarin (3) by micro-
wave-assisted irradiation of 2 with 1-bromo-3-propanol and
K2CO3 in 91% yields. Compared to published thermal methods
of alkylation of phenols,30 this microwave-assisted alkylation
reaction improved the yield for 3 from 25% to 91%. The diol
(3) was subsequently reacted with adipic acid under
carbodiimide/DPTS-catalyzed conditions to provide the
D
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Figure 3. ssNMR (left) and ATR-IR (right) spectra of the polymer (CA11) before irradiation (A, blue), after irradiation at 350 nm (B, red), and
after irradiation at 350 nm followed by 254 nm irradiation (C, green).
thermal stability (Td ∼ 335−365 °C) and low glass transition
temperatures (Tg ∼ 5−35 °C, Table 1).
Higher efficiency of polymer chain scission is observed at 254
nm, and this observation is analogous to work showing higher
release efficiency of phenylalanine ester in coumarins and
quinolones at 254 nm compared to 350 nm.22a These
photocleavage and photo-cross-linking reactions at two differ-
ent wavelengths were also monitored by UV−vis spectroscopy
(Figure 2). Chain scission due to 254 nm irradiation does not
change the overall chromophore structure in either CA11 or
CAQ, so the UV spectra do not change (Figure 2A,C).
However, cross-linking at 350 nm leads to decrease in the
coumarinyl band (322 nm) as shown in Figure 2B,D.23a
To examine the details of photo-cross-linking and test the
reversibility of the cross-linked polymer, solvent-cast films
(thickness ∼70 μm) of polymer CA11 were irradiated at 350
nm and followed by attenuated total reflectance IR (ATR-IR)
and solid-state 13C NMR (ssNMR). Irradiation at 350 nm
results in cross-linking as indicated by the appearance of a new
peak at 44 ppm, which corresponds to the resonance for the
cyclobutane ring.31 Additionally, cross-linking decreases the
intensity of the signal at 149 ppm, which corresponds to the
C−C double bond of the coumarin (Figure 3B, red spectrum).
On the other hand, irradiation of the cross-linked polymer
(CA11) film at 254 nm initiates the reverse reaction, as
indicated by a decrease of the peak intensity at 44 ppm and
increase of the peak at 149 ppm (Figure 3C, green spectrum).
Approximately 34% of the polymer was cross-linked upon
irradiation at 350 nm for 5 min and 18% of it un-cross-linked
after irradiation at 254 nm (see Supporting Information for
details, Figure S7). These reactions can also be followed by
ATR-IR which showed the disappearance and reappearance of
CC stretching frequency (1620 cm−1) at the expense of
cyclic −C−C− bending frequency (1508 cm−1) (Figure 3 and
Figure S8).21,32
Irradiation of CA11 at 254 nm (2 mg/mL; CHCl3:MeOH
99:1; Rayonet reactor; 14 mW/cm2 for 8 lamps, degassed with
argon for 10 min; caution: irradiation of CHCl3 without degassing
can generate toxic phosgene) leads to polymer chain scission by
cleavage of the ester group at the fourth position on coumarin
(Scheme 3), and gel permeation chromatography (GPC)
analysis showed that within 40 min of irradiation the Mn of the
homopolymer CA11 decreases from 21K to 4K (Figure 1A).
Alternatively, irradiation of the homopolymer CA11 at 350 nm
(22 mW/cm2, 16 lamps) leads primarily to polymer cross-
linking (∼82%) (Figure 1B). The area under the GPC curve
reflects the total concentration of soluble sample; decreasing
area under the curve therefore is an indirect measure of sample
loss through the formation of insoluble cross-linked chains.
The copolymer CAQ that contains a lower mol % of
coumarin than the homopolymer CA11 upon irradiation at 254
nm for 40 min resulted in a decrease of molecular weight from
80K to 6K (Figure 1C). Irradiation of polymer CAQ at 350 nm
resulted in polymer cross-linking, and from the area under the
peaks, it was estimated that 42% of the chains undergo cross-
linking and become insoluble material (Figure 1D). The
difference in cross-linking efficiency can largely be attributed to
the amounts of coumarin in polymer CA11 (50 mol %) and
polymer CAQ (12.5 mol %), although the large difference in
Mn between CA11 and CAQ may have an effect of polymer
chain dynamics, which in turn may affect the photoresponse. At
350 nm the coumarin polyesters predominantly display a cross-
linking reaction, which is also seen in coumarin-based small
molecules and polymers. In the current coumarin polyesters
CA11 and CAQ, irradiation at 350 nm also results in polymer
chain scission due to photorelease of the ester substituent.
However, contrary to small molecule phototriggers, polymer
chain scission at 350 nm is the minor reaction. We attribute this
difference to a higher local concentration of coumarin in our
polymers when compared to the low concentrations of
reported phototriggers.
There is a need for materials that demonstrate variable
modulus33 under specific stimuli, and these coumarin polyesters
may have potential for such applications. Therefore, this aspect
was examined by cross-linking the polymers by irradiation at
350 nm. As determined from tensile tests, the homopolymer
CA11 has a Young’s modulus of 68 MPa, and after irradiation at
E
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350 nm, the modulus increases to 302 MPa. Similarly, the
Young’s modulus of the copolymer CAQ increased from 132 to
413 MPa (Table 1) after cross-linking.
Fluorescence images of polymer films patterned with masks
having 200 μm circular patterns proved that these patterns are
not limited to the microscale but can be translated to feature
sizes that are relevant for tissue engineering and cell biology
applications (Figure 5). (The fluorescence is due to the
coumarin polymer.)
Micropatterned surfaces and devices have the potential to be
useful for peripheral nerve guidance devices and for the
investigation of cell−cell and cell−material interactions.34
Photoresponsive polymers that are also biodegradable, such
as the ones described here, may be useful to fabricate guidance
conduits that resorb after nerve regrowth, although more work
needs to be carried out to show that this is the case. The dual
photochemical behavior of these coumarin-based polymers
enables the fabrication of complementary micropatterned
surfaces from these materials. Polymer CA11 was coated onto
a silicon wafer (∼100−150 nm thickness) and irradiated either
at 350 or 254 nm through a 1000 mesh TEM grid for 40 min
and washed thoroughly with a CHCl3/MeOH solvent mixture.
As shown in Figure 4, complementary TEM grid patterns are
obtained by 350 and 254 nm irradiation. Irradiation at 350 nm
Figure 5. Fluorescence microscopic images of the polymers (A) CA11
and (B) CAQ after pattern transfer using 350 nm light.
The hydrolytic degradation of these coumarin polyesters
were investigated by incubating thin films of the polymers in
phosphate buffered saline (PBS, pH 7.4) for 10 weeks. During
this time frame, CA11 showed a decrease in Mn from 11K to
6K (Figure S11). However, during the same time frame CAQ
did not show any appreciable loss of molecular weight. This
stability of CAQ can be attributed to the increased hydro-
phobicity due to the aromatic groups and the higher Mn relative
to CA11.
The biocompatibility assessment of the coumarin polyesters
was evaluated in a macrophage cell line (RAW 264.7) exposed
to varying concentrations of the polymers CA11 and CAQ
dissolved in DMSO and added to the complete culture media.
Data from 24 h cell viability, as determined by MTT assay,
showed high cell viability for both polymers (Figure 6A) and is
comparable to poly(lactic acid-co-glycolic acid) (PLGA; 50:50),
a commonly used biomaterial (p > 0.05; Figure 6A). Even at
the highest polymer concentration tested (50 mg/mL), cellular
viability remained above 90%. We also investigated the
biocompatibility of microparticles of CA11 irradiated at 254
nm (30 min) and 350 nm (20 min). Under these conditions,
photodegradation is maintained at a low concentration, which
is a more likely representation of the in vivo conditions where
the degraded polymer concentration will be much lower than
the original polymer concentration.
Figure 4. AFM images of the photopatterns of the polymer CAQ.
AFM image of the photomasked polymer surface after 350 nm
irradiation (A, C). AFM image of the photomasked polymer surface
after 254 nm irradiation (B, D).
The biocompatibility of photoirradiated CA11 was examined
by incubation of the products of microparticle photoirradiation
(dissolved in DMSO and added to media) with RAW cells.
These studies showed high cell viability for the photoirradiated
products, which is comparable to the nonirradiated CA11 and
also to PLGA (Figure 6B; p > 0.05).
In related studies, macrophage activation of CA11 and CAQ
was assessed based on production of reactive oxygen species
(ROS) after incubation of varying concentrations of the
polymers with RAW cells. At 24 h time points, ROS production
in cells exposed to the polymers was comparable to untreated
cells as well as to PLGA (p > 0.05; Figure 7A). Data points
cross-links the exposed areas and the masked regions of
polymer are removed by solvent washing (Figure 4A,C).
Conversely, irradiation at 254 nm results predominantly in
oligomeric chains due to polymer scission and washing of the
residual oligomers with suitable solvents results in the patterns
shown in Figure 4B,D. The depth of polymer etching is
dependent on the time of irradiation, and under these
conditions the polymer degraded to a depth of ∼40 nm.
Using the 254 nm Rayonet source, this corresponds to a
penetration of about 1 nm/min of irradiation. These results
highlight the utility of this polymer system to fabricate
complementary micropatterns using two different wavelengths.
Confocal microscopic images of these micropatterns show
similar micrometer-sized images (Figures S9 and S10). The
height/depth profiles from the confocal microscope reveal
complementary images resulting from 254 and 350 nm
irradiations.
(mean
SD; n = 4) are expressed as fluorescence intensity
(FLR INT) normalized per microgram of protein. Each data
point is plotted as a percentage of the positive control
(hydrogen peroxide, 100 μM). ROS levels in polymer-treated
cells were comparable to untreated control cells. Similarly,
macrophage activation (monitored by nitrite production) of
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photoirradiated CA11 polymer was comparable to untreated
control cells. The trend indicated that photoirradiation of CA11
polymer did not adversely affect the observed biocompatibility
(p > 0.05; Figure 7B). (Each data point is plotted as a
percentage of the positive control (LPS, 10 μg/mL). Nitrite
levels in polymer-treated cells were comparable to untreated
(control) cells.)
CONCLUSIONS
■
In conclusion, the synthesis, characterization, and photo-
chemical behavior of two novel coumarin-based polyesters are
reported here. These photoresponsive polymers exhibit dual
photoresponsive properties: cross-linking upon 350 nm
irradiation and polymer chain scission and un-cross-linking at
254 nm irradiation. The photochemical behavior was studied by
ssNMR, IR, UV, and GPC. In addition, we have shown that
complementary micropatterned surfaces can be fabricated by
irradiation at 350 and 254 nm. These coumarin polyesters are
mechanically robust and stable in the absence of light.
Preliminary studies with macrophage RAW 267.4 cells show
that these polymers and their irradiation products are
biocompatible, and the cell viability is comparable to that for
PLGA. The properties of these polymers may be useful for
printing 2D and 3D devices and for controlled release devices.
Figure 6. (A) Biocompatibility evaluation (mean
SD; n = 4) of
ASSOCIATED CONTENT
* Supporting Information
■
polymers 4 (CA11) and 5 (CAQ) based on cellular viability following
incubation (37 °C; 24 h) with RAW 264.7 cells. (B) Cellular viability
of RAW 264.7 cells following incubation (37 °C, 24 h) with
photoirradiated CA11 polymer (mean SD; n = 7).
S
1H and 13C NMR details of the monomer and polymers,
solution and ssNMR experiment details, GPC analysis,
photopatterning protocols, confocal microscope images, and
biocompatibility studies are provided. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: abraham@uakron.edu (A.J.).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors express their gratitude to The University of Akron
and NEOMED for startup funding.
■
REFERENCES
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