Low power, biologically benign NIR light triggers polymer disassembly

Article abstract of DOI:10.1021/ma201850q

Near infrared (NIR) irradiation can penetrate up to 10 cm deep into tissues and be remotely applied with high spatial and temporal precision. Despite its potential for various medical and biological applications, there is a dearth of biomaterials that are responsive at this wavelength region. Herein we report a polymeric material that is able to disassemble in response to biologically benign levels of NIR irradiation upon two-photon absorption. The design relies on the photolysis of the multiple pendant 4-bromo7-hydroxycoumarin protecting groups to trigger a cascade of cyclization and rearrangement reactions leading to the degradation of the polymer backbone. The new material undergoes a 50% M_w loss after 25 s of ultraviolet (UV) irradiation by single photon absorption and 21 min of NIR irradiation via two-photon absorption. Most importantly, even NIR irradiation at a biologically benign laser power is sufficient to cause significant polymer disassembly. Furthermore, this material is well tolerated by cells both before and after degradation. These results demonstrate for the first time a NIR sensitive material with potential to be used for in vivo applications.

Full text of DOI:10.1021/ma201850q

ARTICLE
pubs.acs.org/Macromolecules
Low Power, Biologically Benign NIR Light Triggers
Polymer Disassembly
Nadezda Fomina, Cathryn L. McFearin, Marleen Sermsakdi, Josꢀe M. Morachis, and Adah Almutairi*
Skaggs School Pharmacy and Pharmaceutical Sciences, Department of NanoEngineering, Materials Science and Engineering and
Biomedical Sciences Programs, University of California at San Diego, La Jolla, California 92093, United States
S Supporting Information
b
ABSTRACT: Near infrared (NIR) irradiation can penetrate up
to 10 cm deep into tissues and be remotely applied with high
spatial and temporal precision. Despite its potential for various
medical and biological applications, there is a dearth of bioma-
terials that are responsive at this wavelength region. Herein we
report a polymeric material that is able to disassemble in
response to biologically benign levels of NIR irradiation upon
two-photon absorption. The design relies on the photolysis of
the multiple pendant 4-bromo7-hydroxycoumarin protecting
groups to trigger a cascade of cyclization and rearrangement
reactions leading to the degradation of the polymer backbone. The new material undergoes a 50% Mw loss after 25 s of ultraviolet
(UV) irradiation by single photon absorption and 21 min of NIR irradiation via two-photon absorption. Most importantly, even
NIR irradiation at a biologically benign laser power is sufficient to cause significant polymer disassembly. Furthermore, this material
is well tolerated by cells both before and after degradation. These results demonstrate for the first time a NIR sensitive material with
potential to be used for in vivo applications.
’ INTRODUCTION
that degrades into small fragments upon NIR light exposure,
which can then be easily excreted, with less long-term risks.
Therefore, we designed a linear synthetic polymer with multiple
pendant light-sensitive triggering groups in such a way that once
these groups are cleaved, a cascade of cyclization and rearrange-
ment reactions is triggered, leading to backbone degradation.48
The first proof-of-concept polymer utilized a commercially avail-
able and well-studied o-nitrobenzyl (ONB) triggering group,
which was attached to the polymer backbone via a diamine linker.
ONB groups are widely used in synthetic chemistry as protecting
groups for alcohols and amines, which can be readily removed
with UV light. They were also shown to photolyze upon NIR
irradiation via two-photon excitation, although their two-photon
uncaging cross sections (a quantitative measure of the efficiency
of a molecule to simultaneously absorb two photons of light
and convert that energy into a chemical reaction) are very low.71
Consequently, several hours of continuous NIR irradiation at
high laser power were required to trigger the polymer degrada-
tion. Another drawback of the ONB triggering group is potential
toxicity of nitrosobenzaldehyde, the product of photocleavage
of ONB groups. Clearly, the reported polymer required further
modification in order to become a practically useful material.
The material we are reporting here utilizes another well-known
photocleavable group, 4-bromo7-hydroxycoumarin (Bhc), which
Smart polymeric materials are presently one of the main fo-
cuses in biomedical materials research. These types of materials
respond to subtle environmental changes in a controlled, predic-
table way, which makes them useful tools for tissue engineering,1À10
implants1,11À14 and wound healing,15 drug delivery9,16À19 and
biosensors.20À25 Various internal and external triggers, such as
pH,26À29 specificenzymes,30À36 temperature,4,37À41 ultrasound,42À44
magnetic field17,45,46 and light18,45,47À56 are being explored.
Optical stimulus is especially attractive as it can be remotely
applied for a short period of time with high spatial and temporal
precision. A large number of light-degradable materials (micelles,57,58
polymeric nanoparticles59 and bulk hydrogels60,61) have been
reported recently. However, most of the materials reported res-
pond to NIR light by undergoing a hydrophobicity switch62À65
and the photodegradation products are high molecular weight
linear or cross-linked polymer fragments that may be difficult to
clear from the body. Additionally, most of the reported light-
degradable materials respond efficiently to UV irradiation. Near
infrared (NIR) light can penetrate up to 10 cm deep into tissue66
with less damage and absorption or scattering and is more
desirable for in vivo applications.67À69 Despite these advantages,
only a handful of organic materials reported to date can respond
to high power NIR light due to the inefficient two photon
absorption process. None are able to respond to low power
NIR light which is important to biological applications because
it causes less photodamage to tissue and cells.70 For in vivo
applications, it would be more advantageous to have a material
Received: August 16, 2011
Revised:
September 17, 2011
Published: September 30, 2011
r
2011 American Chemical Society
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has a much higher two-photon uncaging cross-section,72À74
and produces no toxic byproducts upon cleavage. Introduction
of the new triggering group drastically increases the sensitivity of
the material to NIR light, reducing the exposure time required to
produce appreciable polymer degradation to a few minutes.
Moreover, we show that laser power as low as 200 mW is suffi-
cient to trigger polymer fragmentation. To our knowledge, this is
the only polymeric material specifically designed to disassemble
into small fragments in response to biologically benign levels of
NIR irradiation.
3.62À3.53 (m, 4H), 3.51 (s, 3H), 3.61À3.00 (m, 6H), 2.32 (s, 3H),
0.9 (s, 18H), 0.06 (s, 12H) ppm.
13C NMR (100 MHz, CDCl3): 160.38, 156.33, 155.17, 154.93,
154.26, 149.20, 143.20, 135.54, 133.43, 127.68, 115.60, 112.57, 106.66,
104.14, 95.25, 64.26, 62.56, 62.18, 60.57, 60.39, 56.82, 48.33, 47.29,
35.60, 26.04, 18.51, À5.13 ppm.
HRMS: measured mass, 873.2780; theoretical mass, 873.2784; com-
position, C39H59N2O10BrSi2Na.
Compound 6. Compound 5 (0.11 g,) was dissolved 15 mL of
MeOH and 2 mL of DCM, Amberlyst 15 was added and reaction was
stirred at room temperature for 2 h. The catalyst was filtered off, sol-
vents were removed on rotovap and the residue was purified by flash-
chromatography on silica gel with hexanes/ethyl acetate (70%/30%-
0%/100%). Yield: 0.059 g (74%).
’ EXPERIMENTAL SECTION
General Methods and Instrumentation. 2,6-Bis(hydroxymethyl)-
p-cresol and 4-bromoresorcinol were purchased from Acros Organics and
used as received. 4,5-Dimethoxy-2-nitrobenzyl alcohol and N,N-di-
methylethylenediamine was purchased from Sigma-Aldrich and used
as received. Amberlyst 15 (dry resin) was purchased from Supelco.
Adipoyl chloride was purchased from Aldrich and purified by vacuum
distillation. All reactions requiring anhydrous conditions were per-
formed under a nitrogen atmosphere. Flash chromatography was per-
formed using a CombiFlash Companion system. 1H NMR spectra were
acquired using a Joel 500 MHz spectrometer or a Varian 400 MHz
spectrometer. 13C NMR spectra were acquired using a Varian spec-
trometer operated at 100 MHz. UV spectra were collected using a
Shimadzu UV-3600 UVÀvis-NIR Spectrophotometer. Degradation of
the monomers containing Bhc and ONB triggering groups (designated
BhcM and ONBM) was monitored by an Agilent 1200 HPLC equipped
with PDA and MSD detectors and a C18 column with 0.1% formic
acid/H2O and 0.1% formic acid/acetonitrile as eluents at a flow rate of
0.3 mL/min. The molecular weights of the polymers, BhcP and ONBP
(where P denotes polymer), were measured relative to polystyrene
standards using a Waters e2196 Series HPLC system equipped with RI
and PDA detectors and a Waters Styragel HR 2 size-exclusion column
with 0.1% LiBr/DMF as eluent and flow rate of 1 mL/min at 37°C. For
irradiation with UV light, a Luzchem LZC-ORG photoreactor equipped
with 8 UV-A lamps (350 nm maximum intensity, 8 W) was used. A Ti:
sapphire laser (Mai Tai HP, Spectra Physics) with ∼100 fs pulse widths
and 80 MHz repetition rate generated light for NIR irradiation. For
monomer and polymer degradation by NIR, 2.5 W (4 kW/cm2) of
750 nm (for ONBM and ONBP) and 740 nm (for BhcM and BhcP)
light was focused into the solution using a EFL 33.0 mm lens. Low
power irradiation experiments used 200 mW (0.32 kW/cm2) of 740 nm
light (2.5 nJ/pulse for the laser repetition rate) attenuated with a wave
plate/polarizer combination.
1H NMR (400 MHz, CDCl3): 7.73À7.67 (m, 1H), 7.18À7.04 (m,
3H), 6.36À6.26 (m, 1H), 5.30À5.24 (m, 4H), 4.51À4.43 (m, 4H),
3.69À3.56 (m, 4H), 3.51 (s, 3H), 3.20À3.03 (m, 6H), 2.31 (s, 3H) ppm.
13C (100 MHz, CDCl3): 160.84, 156.69, 154.14, 149.68, 145.04,
136.27, 133.22, 130.16, 127.69, 110.75, 109.57, 106.91, 104.05, 95.69,
95.21, 64.20, 62.38, 60.56, 56.81, 55.50, 46.94, 35.18, 20.94 ppm.
HRMS: theoretical mass, 645.1054; measured mass, 645.1050; com-
position, C27H31BrN2O10Na.
BhcM. Compound 6 (0.12 g, 0.137 mmol) was dissolved in 1 mL of
DCM, and 1 mL of TFA was added. The reaction mixture was stirred at
room temperature and monitored by TLC (ethyl acetate/hexane =7/3).
After the reaction was completed, solvents were removed on high vacuum,
and crude product was purified by flash-chromatography on silica gel with
hexanes/ethyl acetate (70%/30% to 0%/100%). Yield: 0.05 g (61%).
1H NMR (400 MHz, DMSO): 7.68À7.64 (m, 1H), 7.38À7.27 (m,
2H), 7.00À6.97 (m, 1H), 6.36À6.27 (m, 1H), 5.32À5.27 (m, 6H),
3.64À3.53 (m, 4H), 3.19À3.05 (m, 6H), 2.38À2.35 (m, 3H) ppm.
13C (100 MHz, DMSO): 159.71, 157.47, 154.97, 153.81, 150.75,
142.80, 134.47, 134.08, 128.50, 126.18, 110.51, 108.35, 106.23, 103.23,
62.03, 57.70, 46.60, 46.05, 35.06, 34.37, 33.81, 20.89 ppm.
HRMS: theoretical mass, 601.0787; measured mass, 601.0792; com-
position, C25H27BrN2O9Na.
BhcP. Monomer 6(0.2 g, 0.32 mmol) and adipoylchloride(0.046mL,
0.32 mmol) were dissolved in 2 mL of DCM under nitrogen, and
pyridine (0.156 mL, 1.92 mmol) was added to the reaction mixture
dropwise over 10 min. The polymerization was allowed to proceed
overnight at room temperature. The reaction mixture was concentrated
on a rotovap to 0.2 mL and precipitated into 5 mL of cold EtOH,
yielding waxy polymer product 7. Compound 7 was dissolved in 0.5 mL
of DCM, and 0.5 mL of TFA was added. The solution was stirred for
30 min at room temperature. The solvents were removed on rotovap.
The oligomers were removed by repeated precipitation of the polymer
solution in DCM into cold MeOH. Yield: 63% (white solid).
1H NMR (500 MHz, CDCl3): 7.62 (s, 1H), 7.23À7.13 (m, 2H), 6.67
(s, 1H), 6.31 (s, 1H), 5.29 (s, 2H), 5.00 (s, 4H), 3.65À3.46 (m, 4H),
3.17À3.04 (m, 6H), 2.29 (s, 6H), 1.64 (s, 3H) ppm.
Compounds 2 and 3 were synthesized according to a previously pub-
lished procedure.75 Compound 9 was synthesized according to a pre-
viously published procedure.74 Compounds 4 and 10 were synthesized
according to a previously published procedure.73 Their 1H NMR spectra
were in agreement with the published data and the experimental details
are provided in the Supporting Information. The synthesis of ONBM
and ONBP is described in a previous publication.48
13C (100 MHz, DMSO): 159.60, 157.51, 153.75, 150.48, 145.01,
129.16, 128.30, 110.47, 108.41, 106.21, 103.16, 61.94, 60.74, 60.57,
35.00, 33.40, 33.29, 32.94, 24.04, 23.93, 23.78, 23.71, 20.61, 20.37 ppm.
UV and NIR Degradation of ONBM and BhcM. Solutions of
ONBM and BhcM in PBS pH 7.4 (1 mg/mL), with 4-hydroxy-benzoic
acid-n-hexyl ester as an internal standard, were placed in quartz semi-
micro spectrophotometer cells (10 mm path length) and irradiated with
UV light for certain periods of time. For NIR irradiation experiments, the
solutions of ONBM and BhcM were placed in 50 μL quartz cells with
3 mm path length and irradiated at 740 and 750 nm, respectively. The
irradiated solutions were injected into HPLC and chromatograms at
280 nm were recorded. The fraction of the remaining caged compounds
was calculated by integrating the peaks of ONBM and BhcM relative to
the peak of the internal standard.
Compound 5. Compound 3 (0.5 g, 0.89 mmol) in 10 mL DCM was
added dropwise over 30 min to a solution of N,N-dimethylethylenedia-
mine in 15 mL of DCM and 5 mL of DMF at 0°C. After 30 min the sol-
vents and excess of N,N-dimethylethylenediamine were removed on
rotovap and reaction mixture was dissolved in 4 mL of dry DMF and
Et3N (0.8 mL) and compound 4 were added. The reaction was stirred at
room temperature for 1 h, after that the solvents were removed on
rotovap and the residue was purified by flash-chromatography on cyano-
modified silica gel with hexanes/ethyl acetate (100%/0% to 0%/100%)
as eluent. Yield: 0.39 g (51%).
1H NMR (500 MHz, CDCl3): 7.70 (s, 1H), 7.17À7.12 (m, 3H), 6. 31
(s, 1H), 5.31 (s, 2H), 5.28À5.25 (m, 2H), 4.63À4.58 (m, 4H),
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Scheme 1. Synthetic Route to BhcM and BhcP and the Structures of ONBM and ONBP
UV and NIR Degradation of ONBP and BhcP. For UV
degradation of the polymers, solutions of ONBP and BhcP (0.1 mg/
mL) in a mixture of acetonitrile and PBS 7.6 (9:1 and 7:3, respectively)
were placed into quartz semimicro spectrophotometer cells (10 mm
path length) and irradiated with UV light inside a photoreactor for
certain periods of time. The irradiated solutions were incubated at 37°C
for 96 h. The solvents were removed on vacuum. The organic residue
was dissolved in DMF and injected into GPC. In the NIR irradiation
experiments, for each data point three separate solutions containing
ONBP or BhcP were irradiated for the given time and combined for
incubation at 37°C followed by solvent removal and dissolution in DMF
to achieve acceptable signal-to-noise ratio in GPC.
route to BhcP shown in Scheme 1. We started with commercially
available 2,6-bis(hydroxymethyl)-p-cresol, 1, and selectively pro-
tected the benzylic alcohols with TBDMSCl in the presence of
imidazole to obtain compound 2 in 87.5% yield. Activated
carbonate 3 was obtained in 85% yield by reacting compound
2 with PNPCl in the presence of DMAP and Et3N in DCM. N,N-
Dimethylethylene diamine was reacted with compound 3 at
a stoichiometric ratio of 3 to 1 to achieve conversion of only
one amino group of the diamine into a carbamate. Excess N,N-
dimethylethylene diamine was removed and the coumarin
derivative 4 was added into the reaction mixture to obtain
compound 5 in 51% yield. The TBDMS protecting groups were
removed with Amberlyst-15 (74% yield). Monomer 6 was co-
polymerized with adipoyl chloride in DCM in the presence of
pyridine to afford polymer 7. Finally, the MOM protective
groups were removed in DCM/TFA solution to afford the final
polymer, BhcP. Low molecular weight oligomers were removed
by precipitating the polymer into ice-cold MeOH. The combined
’ RESULTS AND DISCUSSION
Synthesis of BhcM and BhcP. In order to install the
7-hydroxy-4-bromocoumarin triggering group we modified the
previously published scheme for ONBP48 resulting in a synthetic
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Figure 1. Disappearance of ONBM and BhcM upon UV irradiation (A) and NIR (B) irradiation.
Scheme 2. Degradation Mechanism of a Light-Sensitive Polymer Incorporating a QuinoneÀMethide Moietya
a TG = triggering group.
yield of BhcP after the polymerization and deprotection steps
was 63%. The molecular weight (Mw) of BhcP was deter-
mined by GPC to be 31 500 Da (PDI = 1.09) relative to PS
standards.
BhcM was obtained in 61% from compound 6 by removing
the MOM protective group in a mixture of TFA and DCM.
Degradation of ONBM and BhcM. To compare the rates of
cleavage of the ONB and Bhc triggering groups, solutions of
ONBM and BhcM were first exposed to 350 nm light for certain
time periods and injected into the HPLC. The formation of
nitrosobenzaldehyde and 4-bromo-7-hydroxycoumarin con-
firmed the photolysis of ONBM and BhcM. Figure 1A shows
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Figure 2. GPC chromatograms of ONBP (A) and BhcP (B) after UV exposure for 0, 10, 20, 60, or 300 s and incubation at 37°C for 96 h.
Figure 3. Decrease of the Mw of ONBP and BhcP as a function of exposure time to UV light (A) and NIR light (B).
the percentage of remaining monomer, calculated relative to an
internal standard, as a function of UV exposure time for ONBM
and BhcM. The rate of photolysis of BhcM was 10 times higher
compared to ONBM, consistent with the previous reports of
one-photon uncaging quantum yields of other alcohols and
amines.72À74 Comparing the red and blue traces in Figure 1A,
50% of the Bhc groups were cleaved after 3.2 min of irradiation,
while 30.18 min irradiation was required to cleave 50% of ONB
groups. The same 10-fold difference in the rates of triggering
group cleavage was observed upon NIR irradiation of the
monomers. Figure 1B shows 50% of Bhc groups were cleaved
after 34 min versus 370 min for ONB groups. The Bhc protecting
group shows a higher two photon absorption due to the in-
creased π-conjugation length which leads to a higher dipole mo-
ment induced by the electric field of a light wave.76 Additionally,
the introduction of halogen atoms enhances intersystem crossing
and therefore improves the photolysis quantum yield.74 Conse-
quently, a large difference in the two-photon uncaging cross
sections of the two triggering groups could be expected. How-
ever, it is difficult to predict by how much the cleavage rate will
change when switching from one caging group to the other, since
the uncaging efficiency is affected by many factors, such as the
structure of the leaving group, the solvent and the wavelength
and the power of the laser used in the experiment. The reported
two-photon uncaging of acetic acid by Bhc and ONB-protected
esters at 740 nm was 1.99 GM and 0.03 GM, respectively (66-
fold difference) and 0.42 GM and 0.01 GM at 800 nm (42-fold
difference).74 Uncaging of L-glutamic acid by Bhc ester was only
0.95 GM.74 It should also be mentioned that in our experiments
the ONBM and BhcM were irradiated with 750 and 740 nm
of light, respectively, to account for the difference in the two-
photon absorption maxima of the two groups. However, given
the very short pulse widths of our laser, this difference in wave-
length is likely not a large factor in the differences between our
degradation rates and previously reported uncaging cross sections.
Degradation of ONBP and BhcP. Scheme 2 shows the
mechanism of degradation of light-sensitive polymers contain-
ing a quinone-methide self-immolative moiety.75,77,78 The degra-
dation starts when a triggering group is cleaved upon irradiation
with either UV or NIR light, releasing an amino group. N,N-
Dimethylethylene diamine linker cyclizes, unmasking an unstable
phenol. The quinone-methide rearrangement of the phenol
results in the cleavage of the polymer backbone.
Degradation of the polymers containing ONB and Bhc trig-
gering groups was studied in acetonitrile: PBS pH 7.6 (9:1 and
7:3, respectively). These combinations of solvents were found
suitable to fully dissolve the polymers. The solutions were ir-
radiated with UV light for 0, 10, 20, 60, and 300 s, incubated at
37°C for 96 h and analyzed by GPC. As we have shown pre-
viously for ONBP,48 the polymer degradation is complete in
4 days in neutral pH. The chromatograms of ONBP and BhcP
after UV exposure are shown in Figure 2. For both polymers, the
GPC traces shift to longer elution times after irradiation and
shorter fragments are formed. However, much shorter irradiation
times are required to produce a significant reduction in the
molecular weight of BhcP compared to ONBP. Plotting the
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Figure 4. 1H NMR spectra of BhcP in DMSO-d6: D2O (6:1) before (A) and after (B) UV exposure.
Figure 5. GPC chromatograms of ONBP (A) and BhcP (B) after exposure to NIR for 0, 5, 15, 30, or 60 min and incubation at 37°C for 96 h.
percent change in the molecular weight of the polymers as a
function of irradiation time (Figure 3A) reveals that BhcP de-
grades 10 times faster than ONBP, as could be expected from the
monomers’ degradation rates. Thus, Mw of BhcP decreases by
50% after 25 s of UV irradiation compared to 300 s in the case of
ONBP. In the control experiment, the solutions of ONBP and
BhcP not exposed to UV or NIR irradiation were incubated at
37°C for 96 h. The molecular weights of the polymers remained
unchanged, demonstrating that backbone fragmentation is con-
trolled exclusively by the removal of the triggering groups and no
dark hydrolysis takes place during this time.
In order to confirm the degradation mechanism of BhcP, the
polymer was exposed to UV irradiation in DMSOd6: D2O (6:1)
Figure 6. GPC chromatograms of BhcP after exposure to low energy
NIR irradiation for 60 min and incubation at 37°C for 96 h.
and incubated for 72 h at 37°C. The expected degradation
products, 2,6-bis(hydroxymethyl)-p-cresol and 1,3-dimethyl-2-
imidazolidinone, were identified in the 1H NMR spectrum of the
and appearance of the low molecular weight fragments were
partially degraded BhcP (Figure 4). The methyl group of cresol
(t) appears at 1.52 ppm, shifted downfield compared to the
methyl groups of cresol incorporated into the polymer (k, 1.44
ppm). The aromatic protons (s) appear as a sharp singlet at 6.26
ppm. The signal from the benzylic protons (r) is obscured by the
signal from D2O. The signals of 1,3-dimethyl-2-imidazolidinone
appear very distinctly at 2.01 and 2.61 ppm. The peak assign-
ments were confirmed by taking the spectra of the pure 2,6-bis-
(hydroxymethyl)-p-cresol and 1,3-dimethyl-2-imidazolidinone
in DMSOd6: D2O (6:1).
Having confirmed that BhcP degrades in the way it was de-
signed to, we moved to the degradation experiments using NIR
light. The polymer was irradiated for 5, 15, 30, and 60 min and
incubated for 96 h at 37°C. The GPC chromatograms after 96 h
of incubation are shown in Figure 5. Similar to UV irradiation, a
significant drop in the intensity of the high molecular weight peak
observed. In comparison with ONBP, much shorter irradiation
times were required to produce significant fragmentation of
BhcP. Figure 2B shows 50% molecular weight loss was achieved
after 21 min of NIR irradiation of BhcP, while for ONBP 1 h of
continuous irradiation only resulted in 20% weight loss. At-
tempted irradiation of ONBP for more than 60 min to achieve
50% molecular weight loss resulted in evaporation of acetonitrile,
which caused precipitation of ONBP from solution. Neverthe-
less, comparison of the degradation profiles of ONBP and BhcP
after 60 min of irradiation confirms significantly improved NIR
sensitivity of the polymer containing the Bhc triggering group.
Even though NIR irradiation is considered more benign than
UV wavelengths, there is a certain energy threshold above which
photodamage will occur. Watanabe et al demonstrated that laser
energies between 2 nJ/pulse and 4 nJ/pulse did not produce
any damage to living cells.70 Therefore, we attempted NIR light
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degradation of BhcP within this range (200 mW, corresponding
to 2.5 nJ/pulse) to further demonstrate the practicality of using
this material for in vivo applications. Exposure of the BhcP
solution to low power NIR irradiation for 60 min resulted in
the 29% drop in the molecular weight (Figure 6). This further
illustrates the improvement achieved by using Bhc instead of
ONB as a triggering group considering that after an hour of irra-
diation of ONBP at full laser power there was only a 20% decrease in
molecular weight of the polymer. Furthermore, this experiment
confirms that the polymer degradation is caused by the two-photon
absorption process and not simply by possible heat generated by the
laser, since at 200 mW heat generation is less likely.79
We investigated the cytotoxicity of BhcP by incubating various
concentrations of it with cells andmonitoring the cellular metabolic
activity via a MTT assay. Measurements taken before and after
irradiation show that the polymer and its degradation products are
well tolerated by cells (Figure S1, Supporting Information).
It has been reported that absorption properties and photolysis
quantum yields of coumarin triggering groups are strongly af-
fected by the polarity and hydrophobicity of the medium. For
example, the quantum yield of a Bhc-protected galactose deriva-
tive in 10 mM KMops, pH 7.2 containing 25% acetonitrile was
two times lower than in 10 mM KMops, pH 7.2 containing 0.1%
DMSO.73 Therefore, we did not expect that BhcP would main-
tain its high light sensitivity in bulk, since in this case the polymer
backbone would create a local hydrophobic environment. There-
fore, we envision further applications of this material in hydrogel
systems where unrestricted access of water will allow for high
photolysis quantum yields.
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S
Supporting Information. Details for the synthesis of com-
b
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’ AUTHOR INFORMATION
Corresponding Author
*E-mail: aalmutairi@ucsd.edu.
’ ACKNOWLEDGMENT
We thank the NIH Directors New Innovator Award (1 DP2
OD006499-01) and King Abdul Aziz City of Science and
Technology (KACST) for financially supporting this study.
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