Drug-loaded, bivalent-bottle-brush polymers by graft-through ROMP

Article abstract of DOI:10.1021/ma1021506

Graft-through ring-opening metathesis polymerization (ROMP) using ruthenium N-heterocyclic carbene catalysts has enabled the synthesis of bottle-brush polymers with unprecedented ease and control. Here we report the first bivalent-brush polymers; these materials were prepared by graft-through ROMP of drug-loaded poly(ethylene glycol) (PEG) based macromonomers (MMs). Anticancer drugs doxorubicin (DOX) and camptothecin (CT) were attached to a norbornene-alkyne-PEG MM via a photocleavable linker. ROMP of either or both drug-loaded MMs generated brush homo- and copolymers with low polydispersities and defined molecular weights. Release of free DOX and CT from these materials was initiated by exposure to 365 nm light. All of the CT and DOX polymers were at least 10-fold more toxic to human cancer cells after photoinitiated drug release while a copolymer carrying both CT and DOX displayed 30-fold increased toxicity upon irradiation. Graft-through ROMP of drug-loaded macromonomers provides a general method for the systematic study of structure-function relationships for stimuli-responsive polymers in biological systems.

Full text of DOI:10.1021/ma1021506

10326 Macromolecules 2010, 43, 10326–10335
DOI: 10.1021/ma1021506
Drug-Loaded, Bivalent-Bottle-Brush Polymers by Graft-through ROMP
Jeremiah A. Johnson, Ying Y. Lu, Alan O. Burts, Yan Xia, Alec C. Durrell, David A. Tirrell,*
and Robert H. Grubbs*
Division of Chemistry and Chemical Engineering, California Institute of Technology,
1200 E. California Boulevard, Pasadena, California 91125, United States
Received September 15, 2010; Revised Manuscript Received November 8, 2010
ABSTRACT: Graft-through ring-opening metathesis polymerization (ROMP) using ruthenium N-hetero-
cyclic carbenecatalysts has enabledthe synthesisofbottle-brushpolymerswithunprecedentedeaseand control.
Here we report the first bivalent-brush polymers; these materials were prepared by graft-through ROMP of
drug-loaded poly(ethylene glycol) (PEG) based macromonomers (MMs). Anticancer drugs doxorubicin (DOX)
and camptothecin (CT) wereattachedtoa norbornene-alkyne-PEG MMvia a photocleavablelinker. ROMPof
either or both drug-loaded MMs generated brush homo- and copolymers with low polydispersities and defined
molecular weights. Release of free DOX and CT from these materials was initiated by exposure to 365 nm light.
All of the CT and DOX polymers were at least 10-fold more toxic to human cancer cells after photoinitiated
drug release while a copolymer carrying both CT and DOX displayed 30-fold increased toxicity upon irra-
diation. Graft-through ROMP of drug-loaded macromonomers provides a general method for the systematic
study of structure-function relationships for stimuli-responsive polymers in biological systems.
Introduction
variety of norbornene-terminated MMs to yield brush homo-
and copolymers that have high DPns and low PDIs.27-31 These
Recent advances in catalysis and polymer synthesis have
allowed the preparation of new materials with unprecedented
functional and structural diversity and blurred the line between
small-molecule and polymer synthesis.1,2 Ring-opening metath-
esis polymerization (ROMP) using fast-initiating ruthenium
catalysts (e.g., 1, Figure 1) is particularly suited for the synthesis
of diverse side-chain functional polymers with controllable mo-
lecular weights (Mn) and low polydispersities (PDI).3-6
studies have set a benchmark for efficiency in brush polymer
synthesis and led us to explore the application of graft-through
ROMP to the synthesis of novel bivalent-brush polymers (Figure 1)
with one branch comprised of a hydrophilic solubilizing polymer
and the other a drug molecule covalently attached through a
degradable linker. Brush polymers and other branched polymeric
architectures (dendrimers, hyperbranched polymers, dendro-
nized polymers, etc.) possess features, such as multivalency and
nanoscopic size, which make them attractive for in vivo drug
delivery applications.32-34 Branched structures of sufficient size
display extended in vivo circulation times in comparison to their
linear analogues-an advantageous feature for passive tumor
targeting via the enhanced permeation and retention effect
(EPR effect).35,36 Dendrimers are the most extensively studied
branched polymers in this regard; their monodisperse, globular
structures resemble those of proteins and render them attractive
for biological applications.37,38 Despite the promise of dendri-
mers, synthetic challenges limit their utility in therapeutic appli-
cations. It is difficult to prepare dendrimers larger than ∼10 nm
due to the steric hindrance which must be overcome when
functionalizing the periphery of a high-generation dendritic
Discrete bottle-brush polymers (brush polymers) are typically
comprised of a linear polymer backbone connected at each
monomer unit to a polymeric side chain. Brush polymers with
two polymer side-chains attached to each monomer unit of a
polymer backbone (centipede-brushes) have been reported; these
materials can possess disparate polymer domains within the same
polymer structure.7-9 Synthetic approaches to all types of brush
polymers fall into “graft-to,” “graft-from,” or “graft-through”
categories; each approach has advantages and disadvantages.10
For example, graft-to and graft-from strategies, whereby linear
polymers are coupled to the backbone of a linear polymer or are
grown from the backbone of a linear macroinitiator respectively,
are suited to most polymerization methods but suffer from
sterically limited grafting densities for even the most efficient
coupling and polymerization reactions.11-20 The alternative
graft-through approach, which involves polymerization of well-
defined monofunctional macromonomers (MMs), ensures quan-
titative grafting density but requires a polymerization method
capable of propagation under conditions of very low monomer
concentration and high steric hindrance.21-26 Thus, most brush
polymers made by graft-through approaches have significant
amounts of MM impurities due to incomplete conversion; few
examples of functional systems of high degree of polymerization
(DPn) have been reported.27,28
ꢀ
structure. To overcome this limitation, the Frechet group has
appended linear poly(ethylene glycol) (PEG) chains to dendri-
mers to increase their size, water solubility, and biocompatibility
while retaining their inherent multivalency.39-41 These “PEGylated”
dendrimers have proven remarkably effective for treatment of
cancer in mice via controlled delivery of doxorubicin (DOX)39
and camptothecin (CT).41 Though large polymers may be pre-
ferable in certain applications, several reports suggest that non-
degradable polymers for drug delivery applications must be no
larger than ∼10 nm to ensure complete renal clearance.35,42
A
synthetic approach capable of rapidly generating branched poly-
meric structures of easily variable sizes is highly desirable.
Here we introduce a new bivalent-brush polymer structure for
use in chemotherapy delivery. Figure 1 depicts a schematic of our
Recently, our group and others have shown the utility of
ROMP using catalyst 1 for the graft-through polymerization of a
*Corresponding authors.
pubs.acs.org/Macromolecules
Published on Web 12/02/2010
r 2010 American Chemical Society

Article
Macromolecules, Vol. 43, No. 24, 2010 10327
Figure 1. Schematic depiction of bivalent macromonomer (MM) and bivalent-brush polymer described in this work. Mes = mesityl.
design; a water-soluble PEG side chain and a drug molecule are
attached to a polynorbornene backbone via a branch point. The
drug is attached via a degradable linker that allows controlled
release in response to an appropriate stimulus. We expect the PEG
chains to extend into solution effectively shielding the hydropho-
bic drug þ polynorbornene core; the structure resembles that of a
unimolecular micelle. We reasoned that these bivalent-brush poly-
mers might exhibit similar drug-delivery attributes when com-
pared to PEGylated dendrimers but may be easier to synthesize,
especially over a wide range of nanoscale sizes and with greater
functional diversity, using graft-through ROMP of a PEGylated-
norbornene MM. Here we demonstrate the power of this ap-
proach for the preparation of water-soluble polynorbornene-
g-PEG brush polymers and copolymers that have DOX and CT
covalently bound near the core through a photocleavable linker.
Brief ultraviolet (UV, 365 nm) irradiation of these brush polymers
releases the respective drug molecules in unmodified form; we
demonstrate the utility of these systems for photoregulated che-
motherapy delivery in human cancer cell culture.
with compressed air to remove dust. The sample solution was
passed through a 0.4 μm Teflon syringe filter directly into the
cuvette; the cuvette was capped and placed in the DLS for particle
sizing. At least three measurements were made per sample and
average hydrodynamic diameters were calculated by fitting the
DLS correlation function using the CONTIN routine (ISDA
software package from Brookhaven instruments). Nuclear mag-
netic resonance (NMR) experiments were performed on either a
Mercury 300 MHz spectrometer, an INOVA 500 MHz spectrom-
eter, or an INOVA 600 MHz spectrometer. Varian VNMRJ
and MestReNove NMR 5.3.2 software were used to obtain and
analyze the NMR spectra, respectively. Analytical high-perfor-
mance liquid chromatography mass spectrometry (HPLC-MS
or LC-MS) data was obtained using an Agilent 1100 series
HPLC system equipped with a variable wavelength ultraviolet-
visible (UV-vis) detector andanAgilent 1100 VL LC/MSD mass
spectrometer. Separation was achieved using a 9.4 ꢀ 50 mm
Agilent Zorbax XDB-C18 column with mobile phase gradients of
0.1% acetic acid in water and acetonitrile. Experiments were per-
formed at room temperature with a flow rate of 1.0 mL/min. Pre-
paratory HPLC was performed on an Agilent 1100 series HPLC
system with an Agilent 1200 series automated fraction collector
and an 1100 series variable wavelength detector. Separation was
achieved using a 9.4 ꢀ 250 mm Agilent Eclipse XDB-C18 column
with 0.1% acetic acid in water and acetonitrile mobile phase.
Experiments were performed at room temperature with a flow
rate of 5 mL/min. High-resolution mass spectrometry data was
obtained on an Agilent 6200 series accurate-mass time-of-flight
(TOF) LC/MS. Matrix assisted laser desorption ionization mass
spectrometry (MALDI) measurements were performed by the
California Institute of Technology mass spectrometry facility using
a Voyager De_Pro TOF mass spectrometer (Applied Biosystems)
fitted with a 355 nm YAG laser from Blue Ion Technologies. In
a typical experiment, 1.0 mg of polymer sample was dissolved in
100 μL of THF and diluted 10-fold with the MALDI matrix,
dithranol (10 mg/mL in THF). To each sample was added 0.1 μL
of saturated NaI in ethanol and 0.35 μL of the sample-matrix
mixture was spotted on a MALDI plate for analysis. The Voyager
De_Pro was operated in linear mode with an accelerating voltage
of 20 000 V, grid voltage of 95.2%, guide wire 0.03%, extraction
delay time 250 ns, acquisition mass range 800-5000 Da, and laser
rep rate 20 Hz. The instrument was calibrated externally using a
Sequazyme Mass Standard Kit supplied by Applied Biosystems.
Brush polymer purification was performed by centrifugal filtration
through 30 kDa molecular-weight cut off (MWCO) Amicon
Ultra-15 centrifugal filter units (Millipore Inc.). Photolysis experi-
ments were performed using a Multiple Ray Lamp (UVP) fitted
with an 8 W, longwave, filtered blacklight bulb (365 nm). Sample
Materials and Methods
All reagents and solvents were purchased from Aldrich or
VWR chemical companies and were used as supplied unless
otherwise noted. Ruthenium catalyst 1,43 3-azidopropyl-1-amine
(note: care must be taken when working with small-molecule, <6
carbons per azide, azides. In this work, 3-azidopropyl-1-amine is
used as a ∼1 M solution in toluene and never isolated),44 and
3-(tert-butyldimethylsilyloxymethyl)-2-nitrobenzoic acid 845 were
prepared according to literature procedures. Degassed dichloro-
methane (DCM), tetrahydrofuran (THF), and dimethyl sulfoxide
(DMSO) solvents were passed through solvent purification col-
umns prior to use.46 Doxorubicin hydrochloride (DOX-HCl) was
purchased from Axxora LLC.
Gel permeation chromatography (GPC) was performed using
two I-series Mixed Bed Low MW ViscoGel columns (Viscotek)
connected in series with a DAWN EOS multiangle laser light
scattering (MALLS) detector (Wyatt Technology) and an Opti-
lab DSP differential refractometer (Wyatt Technology). Experi-
ments were performed at room temperature using 0.2 M LiBr in
N,N-dimethylformamide (DMF) eluant at a flow rate of 1 mL/
min. Molecular weights were calculated from dn/dc values that
were obtained assuming 100% mass elution from the columns.
Dynamic light scattering (DLS) measurements were made at
room temperature using a Brookhaven ZetaPALS DLS instru-
ment. Samples were dissolved in nanopure water at a concentra-
tion of ∼1 mg/mL. A fresh, clean, polystyrenecuvettewas washed

10328 Macromolecules, Vol. 43, No. 24, 2010
Johnson et al.
vials were placed as close as possible to the light source and
irradiated for the desired time before analysis by LC-MS.
CDCl3): δ 6.18 (s, 2H), 3.33 (t, J = 7.3 Hz, 2H), 3.29 (d, J = 2.4
Hz, 2H), 3.14 (s, 2H), 2.62-2.46 (m, 4H), 2.12 (t, J = 2.4 Hz,
1H), 1.53-1.30 (m, 5H), 1.30-1.14 (m, 5H), 1.10 (d, J = 9.8 Hz,
1H). 13C NMR (300 MHz, CDCl3): δ 177.9, 137.7, 82.2, 71.2,
48.3, 47.7, 45.0, 42.6, 38.5, 38.0, 29.5, 27.6, 26.7, 26.6. TOF
HRMS: calcd for C18H24N2O2 [M þ H]þ, 301.1911; found,
301.1951.
Norbornene-Hexanol (2). A solution of 6-amino-1-hexanol
(3.0 g, 25.6 mmol) and cis-5-norbornene-exo-2,3,-dicarboxylic
anhydride (4.0 g, 24.4 mmol) in toluene (50 mL) was added to a
dried, 150 mL round-bottom flask fitted with a Dean-Stark trap
andplacedinanoilbathpresetto140°C for 24 h while stirring. The
reaction mixture was transferred to a silica gel column primed using
10% ethyl acetate in hexanes (10% EtOAc/hexane). A 300 mL
portion of 10% EtOAc/hexanes was flushed through the column
before elution of the product using 50% EtOAc/hexanes (TLC
Rf = 0.3, 50% EtOAc/hexanes, KMnO4 stain). Removal of sol-
vent by rotary evaporation yielded 6.0 g of 2 as a colorless oil
(94%). 1H NMR (300 MHz, CDCl3): δ 6.00 (s, 2H), 3.26 (t, J =
6.4Hz, 2H), 3.13(t, J = 7.3, 2H), 2.93(s, 2 H), 2.38(s, 2H), 1.31-
Norbornene-Acid-Alkyne (5). Succinic anhydride (134 mg,
1.34 mmol) was combined with amine 4 (382 mg, 1.28 mmol) in
DCM (13 mL) and the resulting solution was stirred for 1 h at
room temperature before transferring to a silica gel column.
Elution with 60% EtOAc/hexanes (TLC Rf = 0.2, stain with
bromocresol green solution) gave the purified acid 5 (364 mg,
71%) as a mixture of amide rotamers after concentration on a
rotary evaporator. 1H NMR (300 MHz, CDCl3): δ 9.25 (b, 1H),
6.24 (s, 2H), 4.15 (d, J = 2.4 Hz, 1.2H), 4.01 (d, J = 2.3 Hz,
0.8H), 3.50-3.31 (m, 4H), 3.22 (s, 2H), 2.82-2.50 (m, 6H), 2.29
(t, J = 2.3 Hz, 0.3H), 2.17 (t, J = 2.5 Hz, 0.7H), 1.68-1.40 (m,
5H), 1.39-1.21 (m, 5H), 1.16 (d, J = 9.8 Hz, 1H). 13C NMR
(300 MHz, CDCl3): δ 178.2, 177.0, 171.6, 171.1, 137.8, 78.9,
78.4, 72.8, 71.8, 47.8, 47.1, 46.6, 45.1, 42.7, 38.5, 38.4, 37.5, 34.6,
29.4, 29.3, 28.1, 28.0, 27.9, 27.5, 27.2, 26.5, 26.2. TOF HRMS:
calcd for C22H27N2O5 [M - H]-, 399.1920; found, 399.1941.
Norbornene-Alkyne-N-Hydroxysuccinimidyl (NHS)-Ester
(6). DCM (10 mL) was added to a flask containing N-(3-
(dimethylamino)propyl)-N0-ethylcarbodiimide hydrochloride
(EDCI, 262 mg, 1.36 mmol), N-hydroxysuccinimide (157 mg,
1.36 mmol), 4-(dimethylamino)pyridine (DMAP, 11.1 mg, 0.091
mmol), and 5 (364 mg, 0.91 mmol). The resulting solution was
stirred under argon at room temperature for 20 h. The mixture
was transferred to a silica gel column. Elution with 70% EtOAc/
hexanes (TLC Rf = 0.2, stain with anisaldehyde solution and/or
visualize under UV light) gave norbornene 6 (339 mg, 75%) after
concentration on a rotary evaporator. 1H NMR (300 MHz,
CDCl3): δ 6.23 (s, 2H), 4.16 (d, J = 2.4 Hz, 1.2 H), 3.98 (d,
J = 2.2 Hz, 0.8 H), 3.49-3.26 (m, 4H), 6.23 (s, 2H), 2.95 (t, J =
6.9 Hz, 2H), 2.79 (s, 4H), 2.70 (t, J = 6.9 Hz, 2H), 2.62 (s, 2H),
2.29 (t, J = 2.4 Hz, 0.3H), 2.17 (t, J = 2.5 Hz, 0.7H), 1.68-1.40
(m, 5H), 1.39-1.20 (m, 5H). 13C NMR (300 MHz, CDCl3): δ
178.0, 169.6, 169.3, 169.0, 168.4, 137.8, 78.9, 78.4, 77.5, 76.7, 72.9,
71.8, 47.7, 46.9, 46.6, 45.1, 42.7, 38.5, 38.3, 37.4, 34.4, 28.1, 28.0,
27.8, 27.6, 27.5, 27.3, 26.5, 26.2, 25.6. TOF HRMS: calcd for
C26H32N3O7 [M þ H]þ, 498.2241; found, 498.2203.
1.15 (m, 5H), 1.14-0.95 (m, 4H), 0.92 (t, J = 7.4 Hz, 1H). 13
C
NMR (300 MHz, CDCl3): δ 177.9, 137.5, 61.8, 47.5, 44.8, 42.4,
38.3, 32.2, 27.4, 26.4, 25.0. TOF HRMS: calcd for C15H22NO3
[M þ H]þ, 264.1600; found, 264.1612.
Norbornene-aldehyde (3). A three-neck round-bottom flask
containing a stir bar was equipped with a vacuum adaptor and
two 150 mL addition funnels each capped with a rubber septum.
The flask was flame-dried under vacuum, cooled to room tem-
perature, and backfilled with argon. A positive argon pressure
(using a mercury bubbler) was maintained through the course of
the reaction. DCM (58 mL) was added to the flask via cannula
followed by oxalyl chloride (3.21 mL, 37.36 mmol). The solution
was cooled to -76 °C using an acetone/dry ice bath. One of the
addition funnels was charged with DCM (7.3 mL) and DMSO
(5.31 mL, 74.72 mmol) while alcohol 2 (6.60 g, 24.90 mmol)
dissolved in DCM (43 mL) was added to the other. The DMSO/
DCM solution was added dropwise to the flask containing
oxalyl chloride over 15 min while stirring. After the addition,
the solution was stirred for 15 min at -76 °C. The solution of 2
in DCM was then added dropwise over 20 min while stirring.
The addition funnel was washed twice with 5 mL of DCM and
the reaction mixture was stirred for 30 min at -76 °C. Triethy-
lamine (20.83 mL, 149.4 mmol) and DCM (3.7 mL) were com-
bined in the washed addition funnel that previously held 2 and
this solution was added dropwise over 15 min to the flask during
which time a thick white precipitate formed. After the addition
the mixture was stirred for 10 min before warming to room tem-
perature and transferring to a separatory funnel. The mixture
was washed twice with 50 mL of 1 M HCl and once with brine,
dried over Na2SO4 and concentrated on a rotary evaporator.
The crude product was purified by silica gel column chroma-
tography (30% EtOAc/hexanes, TLC Rf = 0.25, stain with ani-
saldehyde solution) to yield 3 (5.83 g, 89%) as a colorless oil. 1H
NMR (300 MHz, CDCl3): δ 9.52 (s, 1H), 6.08 (s, 2H) 3.23 (t, J =
7.3 Hz, 2H), 3.02 (s, 2H), 2.46 (s, 2H), 2.22 (td, J = 7.2, 1.4 Hz,
2H), 1.52-1.22 (m, 5H), 1.21-1.05 (m, 2H), 0.99 (d, J = 9.8 Hz,
1H). 13C NMR (300 MHz, CDCl3): δ 202.0, 177.7, 137.6, 47.6,
44.9, 43.3, 42.5, 38.1, 27.3, 26.2, 21.3. TOF HRMS: calcd for
C15H19NO3 [M þ H]þ, 262.1443; found, 262.1438.
Norbornene-Alkyne-PEG(3000) Macromonomer (7). O-(2-
Aminoethyl)poly(ethylene glycol) (100 mg, 33.3 μmol) and 6
(17.4 mg, 35 μmol) were dissolved in anhydrous DMF (1 mL)
and the resulting solution was stirred at room temperature for
4 h. The reaction mixture was added dropwise to diethyl ether
(20 mL) to precipitate 7 as a white solid which was collected by
centrifugation and decanting of the ether before redissolving in
DCM (1 mL). This process of precipitation, centrifugation, and
redissolving was repeated five times. On the fifth iteration, the
precipitate was dried under vacuum to afford macromonomer
7 as a white powder (78.1 mg, 69%). GPC (0.2 M LiBr in DMF)
3300 Da, PDI 1.10. MALDI mass spectrum and NMR are shown
in the Supporting Information (Figures S1-S3).
N-(3-Azidopropyl)-3-(tert-butyldimethylsilyloxymethyl)-2-ni-
trobenzamide (9). EDC (92.4 mg, 0.48 mmol) was added to a
suspension of acid 8 (100 mg, 0.32 mmol) and DMAP (3.9 mg,
0.032 mmol) in DCM (4.0 mL). The suspension became a clear
solution within 2 min indicating formation of a soluble acyli-
sourea intermediate. At this time, 3-azidopropyl-1-amine (1.0 M
in toluene, 482 μL, 0.48 mmol) was added dropwise to the reac-
tion mixture. The resulting solution was stirred overnight at
room temperature under an argon atmosphere. The reaction
mixture was diluted with 100 mL EtOAc and washed three times
with 1.0 M HCl (50 mL), three times with sat. NaHCO3 (50 mL),
and once with brine (50 mL). The organic layer was then dried
over MgSO4, filtered, and concentrated on a rotary evaporator.
The resulting white solid was passed through a silica plug using
Norbornene-Alkyne-Amine (4). Aldehyde 3 (1.0 g, 3.83
mmol) and propargyl amine (258 μL, 4.0 mmol) were dissolved
in methanol (10 mL) in a round-bottom flask. The mixture was
stirred at room temperature under argon atmosphere for 30 min
to form an imine intermediate (reaction monitored by TOF-
LC/MS: calcd for imine C18H22N2O2 [M þ H]þ, 299.1754; found,
299.1856). The reaction mixture was cooled to 0 °C using an
ice bath; NaBH4 (232 mg, 6.13 mmol) was carefully added. The
ice bath was removed and the mixture was stirred for 3 min before
quenching with 100 mL of saturated NaHCO3(aq.). The mixture
was transferred to a separatory funnel and washed five times with
DCM (100 mL). The organic fractions were combined and dried
over Na2SO4, filtered, and concentrated on a rotary evaporator.
The resulting oil was purified by silica gel chromatography (2%
MeOH/CH2Cl2, TLC Rf = 0.2, stain with ninhydrin solution)
to yield 4 as a colorless oil (836 mg, 73%). 1H NMR (300 MHz,

Article
Macromolecules, Vol. 43, No. 24, 2010 10329
Scheme 1. Synthesis of PEG-Norbornene-Alkyne Macromoner 7
50% EtOAc/hexanes and evaporated to dryness to give 9 (101 mg,
80%) as a white crystalline solid (128 mg, 80%). 1H NMR (300
MHz, CDCl3): δ7.75 (d, J = 6.4 Hz, 1H), 7.52 (t, J= 7.7Hz, 1H),
7.41 (d, J = 6.2 Hz, 1H), 6.46 (b, 1H), 4.78 (s, 2H), 3.65-3.15 (m,
4H), 1.84 (p, J = 6.6 Hz, 2H), 0.92 (s, 9H), 0.09 (s, 6H). 13C NMR
(300 MHz, CDCl3): δ 165.8, 146.4, 135.4, 131.2, 130.7, 129.9,
126.5, 60.8, 49.3, 37.8, 28.4, 25.0, 18.3, 5.6. TOF HRMS: calcd for
C17H28N5O4Si [M þ H]þ, 394.1911; found, 394.1900.
THF) was added dropwise via a rubber septum using a gastight
syringe. The reaction was stirred overnight during which time
a white precipitate formed. The reaction mixture was diluted
with EtOAc (100 mL) and washed once with water (50 mL),
twice with 1.0 M HCl (25 mL), and once with brine (50 mL). The
organic layer was dried over MgSO4, filtered, and concentrated
on a rotary evaporator. The solid residue was purified by col-
umn chromatography (100% EtOAc, TLC Rf = 0.2, visualize
under UV light) to give CT-NBOC-N3 as a white solid (106 mg,
90%). 1H NMR (600 MHz, CDCl3): δ 8.41 (s, 1H), 8.26 (d, J =
8.6 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.86 (ddd, J = 8.4, 6.9, 1.4
Hz, 1H), 7.72-7.68 (m, 2H), 7.56 (t, J = 7.7 Hz, 1H), 7.43 (dd,
J = 7.6, 1.3 Hz, 1H), 7.34 (s, 1H), 6.33 (t, J = 5.8 Hz, 1H), 5.59
(d, J = 17.1 Hz, 1H), 5.34 (d, J = 17.1 Hz, 1H), 5.32-5.19 (m,
4H), 3.46 (q, J = 6.5 Hz, 2H), 3.42 (t, J = 6.5 Hz, 2H), 2.27 (dt,
J = 14.9, 7.5 Hz, 1H), 2.21-2.11 (m, 1H), 1.85 (p, J = 6.5 Hz,
2H), 1.01 (t, J = 7.5 Hz, 3H). 13C NMR (300 MHz, CDCl3): δ
167.1, 165.5, 157.2, 153.0, 151.9, 148.5, 146.9, 146.3, 145.4,
131.9, 131.8, 131.5, 131.0, 130.4, 129.5, 129.3, 128.5, 128.3,
128.2,128.1, 120.3, 96.1, 78.5, 67.0, 65.3, 50.0, 49.2, 45.0, 37.8,
31.8, 28.4, 7.6; TOF HRMS: calcd for C32H28N7O9 [M þ H]þ,
654.1949; found, 654.2010.
N-(3-Azidopropyl)-3-(hydroxymethyl)-2-nitrobenzamide (10).
Compound 9 (101 mg, 0.26 mmol) was dissolved in tetrahydro-
furan (3 mL) in a round-bottom flask which was subsequently
cooled to 0 °C. Tetrabutylammonium fluoride (1.0 M in THF,
0.385 mL, 0.39 mmol) was added dropwise and the mixture was
stirred for 15 min. The solution was diluted with EtOAc (50 mL)
and washed three times with 1.0 M HCl (25 mL) and once with
brine (50 mL). The organic layer was dried over MgSO4, filtered,
and passed through a silica plug to give pure 10 (56 mg, 78%) as
a white solid. 1H NMR (300 MHz, acetone): δ 7.98 (b, 1H), 7.82
(d, J = 4.6 Hz, 1H), 7.69-7.56 (m, 2H), 4.71 (s, 2H), 3.63-3.28
(m, 4H), 1.88 (p, J = 6.8 Hz, 2H). 13C NMR (300 MHz, acetone):
δ 205.4, 165.2, 135.3, 130.8, 130.1, 126.9, 59.4, 48.8, 36.9. TOF
HRMS: calcd for C11H13N5O4 [M þ H]þ, 280.1046; found,
280.1067.
DOX-NBOC-N3. A suspension of 10 (45 mg, 0.16 mmol) in
THF (2 mL) and triethylamine (25 μL, 0.18 mmol) was treated
with 4-nitrophenyl chloroformate (35 mg, 0.18 mmol). TLC and
1H NMR confirmed complete conversion to carbonate 11 with-
in 15 min. The reaction mixture was transferred to a short silica
gel column and eluted with 70% EtOAc. UV active fractions
with Rf = 0.4 were combined and dried on a rotary evaporator.
The resulting white solid, 11 (40 mg, 90 μmol), was immediately
CT-NBOC-N3. The following reaction (see Scheme 2) was
a modified literature procedure for the preparation of 20-
O-acylcamptothecins.47 (S)-(þ)-Camptothecin (CT, 62.7 mg,
0.18 mmol) and DMAP (70.1 mg, 0.57 mmol) were suspended
in DCM (5 mL) under argon atmosphere. Triphosgene (19.6 mg,
0.066 mmol) was added and the mixture was stirred for 30 min at
room temperature. Alcohol 10 (55.2 mg, 0.2 mmol, in 2 mL

10330 Macromolecules, Vol. 43, No. 24, 2010
Scheme 2. Synthesis of Clickable, Photocleavable Drugs CT-NBOC-N3 and DOX-NBOC-N3
Johnson et al.
Scheme 3. Click Coupling of 7 to Photocleavable Drug Derivatives
dissolved in anhydrous DMF (1 mL). DOX-HCl (53 mg,
91 μmol) and anhydrous N,N-diisopropylethylamine (DIPEA,
17 μL, 99 μmol) were added and the resulting solution was stirred
overnight at room temperature. The reaction mixture was diluted
with 50 mL EtOAc and washed twice with 0.1 M HCl (20 mL),
once with H2O (20 mL), and once with brine (20 mL) before drying
over magnesium sulfate, filtration, and concentration on a rotary
evaporator. The resulting red solid was purified by column chro-
matography. The column was eluted first with 3% MeOH/CH2Cl2
and then with 5% MeOH/CH2Cl2 to give DOX-NBOC-N3 as a
red solid (73 mg, 95%). 1H NMR (600 MHz, DMSO-d6): δ 8.77 (t,
J = 5.6 Hz, 1H), 7.96-7.87 (m, 2H), 7.67-7.61 (m, 3H), 7.57 (dd,
J = 6.9, 2.1 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 5.42 (s, 1H), 5.20 (s,
1H), 5.01 (dd, J = 37.8, 13.7 Hz, 2H), 4.92 (m, 1H), 4.81 (t, J = 6.0
Hz, 1H), 4.69 (d, J = 5.7 Hz, 1H), 4.54 (d, J = 6.0 Hz, 2H),
4.14-4.09 (m, 1H), 3.98 (s, 3H), 3.71-3.60 (m, 1H), 3.42-3.39 (m,
1H), 3.38-3.35 (m, 2H), 3.24-3.19 (m, 2H), 2.96 (q, J = 18.3 Hz,
2H), 2.12 (dt, J = 14.5, 9.0 Hz, 2H), 1.83 (dd, J = 12.8, 9.2 Hz,
1H), 1.69 (p, J = 6.7 Hz, 2H), 1.09 (d, J = 6.5 Hz, 4H). 13C NMR
(500 MHz, CD2Cl2): δ 213.1, 186.1, 164.5, 160.4, 155.3, 154.7,
154.0, 146.7, 135.0, 134.6, 132.8, 132.7, 130.6, 130.5, 130.4, 130.1,
126.7, 120.0, 118.7, 117.9, 110.8, 110.7, 99.8, 75.9, 68.7, 68.6,
66.6, 64.7, 61.2, 55.8, 48.6, 46.4, 37.0, 34.8, 33.2, 28.9, 27.7, 15.8.
TOF HRMS: calcd for C39H40N6O16 [M - H]-, 847.2423; found,
847.2418.
General Macromonomer Synthesis by Copper-Catalyzed Azi-
de-Alkyne Cycloaddition (CuAAC) Click Chemistry. Drug
azide, CT-NBOC-N3 or DOX-NBOC-N3, (1.01 equiv to alkyne)
was combined with norbornene-PEG-alkyne 7 (100 mg,
29.4 μmol) in a 2 mL HPLC vial and THF (0.5 mL) was added.
A spatula tip of sodium ascorbate was added followed by a 1.0 M
solution of CuSO4 in H2O (88 μL, 3 equiv to alkyne). The mixture
was flushed with argon, sealed with a septum, and stirred until
completion (as monitored by LC-MS) which was typically ∼1 h.
After the required time, the drug-loaded macromonomer was
purified by preparative HPLC (linear gradient of 95:5 water-0.1%
AcOH:MeCN to 5:95 water-0.1% AcOH-MeCN over 12 min).
The fractions containing pure MM were combined and concen-
trated on a rotary evaporator. The resulting residue was dissolved
in DCM, dried over Na2SO4, filtered, and dried under vacuum
to give pure macromonomer CT-MM or DOX-MM (typical
yield ∼75 mg, ∼70%). MALDI and 1H NMR spectra are shown

Article
Macromolecules, Vol. 43, No. 24, 2010 10331
Scheme 4. Synthesis and Structure of Poly(norbornene)-PEG Brush Polymers with DOX or CT Attached via a Photocleavable NBOC Linker.
Pyr = pyridine.
in the Supporting Information (Figures S4-S7). The synthesis is
depiced in Scheme 3.
Cell Culture. Human breast cancer cell line MCF-7 (ATCC,
HTB-22) was cultured at 37 °C under a humidified atmosphere
of 5% CO2. The cells were grown in Eagle’s minimum essential
medium (EMEM, ATCC, 30-2003) supplemented with 10%
fetal bovine serum (Gibco, 10437028), 1% antibiotics (100 U/mL
penicillin and 100 μg/mL streptomycin, Gibco, 105140122), and
10 μg/mL bovine insulin (Sigma, I0516). The cells were continu-
ously maintained in the culture medium and subcultured every
3-4 days.
Drug Treatment and Cell Viability Assay. MCF-7 cells were
seeded at 10,000 cells/well in a 96-well plate and allowed to
attach for 20 h before drug treatment. Prior to drug exposure,
the culture medium was removed and the cells were washed once
with warm phosphate-buffered saline (PBS). Then, fresh media
with drug concentrations ranging from 0 to 100 μM (based on
dry weight of polymer dissolved in H2O) were added to the ap-
propriate wells. After recovering for 10 min at 37 °C, one plate
of cells was submitted to UV light (Multiple Ray Lamp with
filtered blacklight bulb, 365 nm) for 10min while the control plate
was kept in the dark. The cells were subsequently incubated in a
cell culture incubator for 24 h. The medium was removed and the
cells were washed twice with warm PBS before fresh drug-free
medium was added to each well. The cells were incubated for
another 24 h before analysis by the MTT cell proliferation assay
(ATCC, 30-1010K). Cells werewashedonce withwarmPBS and
incubated with fresh medium containing MTT reagent for 3 h at
37 °C. Detergent was added to solubilize the purple formazan
crystals formed by proliferating cells. Absorbance at 570 nm was
measured on a Safire II (Tecan) plate reader. Data were fit to a
sigmoidal function to determine the half-maximum inhibitory
concentration (IC50).
General ROMP Polymerization. Macromonomer DOX-MM
or CT-MM (20 mg, ∼5 μmol), or a combination of the two, was
added to a 2 mL vial containing a stir bar. The vial was capped
with a septum and placed under vacuum for 5 min and then pur-
ged with argon. DCM was added followed by a freshly prepared
solution of catalyst 1 in DCM (1 mg 1/mL DCM, amount added
to give the desired MM:1) such that the total concentration of
MM was 0.05 M. The mixture was stirred at room tempera-
ture under argon for 90 min after which time the reaction became
noticeably viscous. One drop of ethyl vinyl ether was added to
quench the polymerization and the vial was placed under vacuum
to remove volatiles. The resulting polymer film was dissolved in
deionized water (15 mL) and transferred to a centrifugal filter
tube (30 kDa MWCO). The tube was spun at 4000 rpm until
all of the solvent had passed through the filter except for ∼1 mL
(typically ∼45 min). More water was added (14 mL) and this
process was repeated at least 5 times to remove any remaining MM.
After the last centrifugation, the 1 mL solution of brush polymer in
water was transferred to a weighed glass vial and lyophilized to
dryness. Typical yields after purification were ∼15 mg (75%).
1
Representative H NMR spectra are shown in the Supporting
Information (Figures S8 and S9).
LC-MS Methods. Two methods were used for analytical
LC-MS experiments; acetonitrile (MeCN) percentage was varied.
Method A was a linear gradient of 5% MeCN to 95% MeCN over
5 min followed by a 2 min hold at 95% MeCN to flush the column.
Method B began at 5% MeCN and ran to 70% MeCN linearly over
5 min followed by a 2 min flush at 95% MeCN. Method A was used
for the DOX loaded polymers (pDOX02 and the copolymer) and
method B for pCT03. The concentration of CT and/or DOX in
photolyzed samples was estimated from free CT and DOX calibra-
tion curves, respectively. The calibration curves were generated as
follows. A 1 mM solution of CT in DMSO was serially diluted with
DMSO to generate 100 μM, 10 μM, and 1 μM solutions. In a
similar fashion, a 1 mM solution of DOX-HCl in water was serially
diluted with water to generate solutions of known concentration.
Each of these samples was analyzed by LC-MS with wavelength
detection set at 368 and 500 nm for CT and DOX respectively.
Method A was used in both cases. The area under the absorbance
curve for each run was calculated and plotted against the concen-
tration of drug (Supporting Information, Figure S10). Linear fitting
of the resulting calibration curve gave an extinction coefficient that
was used to estimate the concentration of drug released in photo-
lysis experiments.
Results and Discussion
Synthesis of Norbornene-PEG-Alkyne MM. Graft-
through ROMP reduces the problem of brush polymer
synthesis to design of an appropriate, strained alkene MM;
a bivalent-brush is derived from a bivalent norbornene MM
(Figure 1). Using a branched MM avoids the need for copo-
lymerization of two different monomers, one PEG-MM
and one drug-loaded monomer; a high drug loading is main-
tained and issues arising from different propagation rates be-
tween MM and small-molecule monomers are negated. Toward
this end, we prepared norbornene-imide derivative 6 (Schemes 1
and 4) which carries two orthogonally addressable functional
groups, an N-hydroxysuccinimidyl (NHS) ester and an alkyne.

10332 Macromolecules, Vol. 43, No. 24, 2010
Johnson et al.
Table 1. GPC Characterization of pDOX and pCT Brush Polymer
Samples and Random Copolymer pDOX50-pCT50
b
c
sample
pDOX01
pDOX02
pDOX03
pCT01
pCT02
pCT03
pCT04
pCT05
MM:1a DPn Mn (GPC, kDa) PDI
Dh
10
50
100
15
9
58
96
15
30
75
107
135
101
33.7
227
352
55.4
111
276
394
499
393
1.07
1.05 12 (2)
1.04 15 (2)
1.09
1.17
1.38
1.61
1.70
6.2 (0.5)
7.1 (0.5)
25
8.7 (0.9)
n.d.e
n.d.
100
150
200
100
n.d.
d
pDOX50-pCT50
1.13 15 (1)
a Ratio of MM to catalyst (i.e., theoretical DPn). b DPn observed
derived from Mn(GPC)/Mn(MM). c Hydrodynamic diameter measured
by dynamic light scattering (DLS) using the CONTIN fitting algorithm.
Reported values are the average of three experiments with error shown
in parentheses. d pDOX50-pCT50 carries approximately 50 DOX and
50 CT based on MM stoichiometry prior to ROMP. e Dh values not
determined for these samples due to high polydispersity.
Figure 2. MALDI spectra for PEG-norbornene 7 before (black trace)
and after (red trace) CuAAC click coupling of CT-NBOC-N3 to give
CT-MM. The observed mass shift of 653 Da agrees with the calculated
mass of CT-NBOC-N3 and confirms successful attachment of the
photocleavable drug moiety.
The NHS-ester of 6 was efficiently coupled to water-soluble
PEG-NH2 (Mn = 3 kDa) to give PEG-MM 7 (Figure 2).
We independently prepared DOX- and CT- nitrobenzyloxy-
carbonyl-azide analogues (DOX-NBOC-N3 and CT-NBOC-
N3, Scheme 2 and Figure 2) that allow for drug attachment via
copper-catalyzed azide-alkyne cycloaddition (CuAAC) click
chemistry48-50 and controlled drug release in response to long
wavelength UV irradiation (∼365 nm). CuAAC coupling of 7
to either drug-azide proceeded in high yield to give the desired
drug-loaded PEG-MMs (DOX-MM and CT-MM). The
MALDI spectra of CT-MM and its alkyne precursor 7
confirmed the expected mass increase after CuAAC coupling
(Figure 2).
Figure 3. Representative GPC traces for brush polymer samples. Black
and red chromatograms correspond to crude ROMP reaction mixtures.
The brush polymers display narrowly dispersed, monomodal molecular
weight distributions. The GPC trace for purified pDOX02 is shown in
orange, indicating that it is possible to remove trace MM impurity.
As demonstrated above, graft-through ROMP allows
for rapid access to brush polymers of controlled, variable
molecular weights. We envisioned this methodology also being
useful for preparing multiple-drug-loaded brush polymers via
copolymerization of appropriate MMs. For example, treat-
ment of equimolar mixtures of DOX-MM and CT-MM with
catalyst 1 in DCM yielded copolymer pDOX50-pCT50 which
exhibited a narrow, monomodal MW distribution (Table 1,
Figure 3). Combination of a variety of therapeutic moieties
within the same polymer system and controlled release using
external, and perhaps different, stimuli will enable study and
discovery of synergistic drug effects and design of synchro-
nized drug releasing systems.
UV Photolysis Experiments. To demonstrate controlled
release of DOX and CT from these brush polymer scaffolds in
response to 365 nm UV light we irradiated aqueous solutions of
the polymers for various times from 30 s to 10 min and moni-
tored the progress of photorelease by high-performance liquid
chromatography connected in series to a single wavelength UV
detector and an electrospray mass spectrometer (LC-MS).
The resulting chromatograms for brush polymer pDOX02
(∼1.1 μM of bound DOX in H2O) before and after irradiation
are shown in Figure 4a. With increasing irradiation time, the
polymer absorbance at 500 nm is diminished and a new peak is
observed at ∼3.1 min; the mass of the species giving rise to this
new peak is 542.30 Da which corresponds to that of free DOX-
Hþ. The yield for photocleavage in this time was ∼50% based
on integration of the polymer and free DOX peaks.
ROMP of Drug-Loaded, PEGylated MMs. Treatment of
either MM with 1 in methylene chloride (DCM) for 90 min
under N2 yielded polymers (pDOX and pCT) with low PDIs
and Mn dependent on the ratio of MM to 1 (Table 1). The
pDOX brushes were characterized by PDIs on the order
of 1.1 as previously reported for graft-through ROMP poly-
merizations using catalyst 1.27,28 The PDI values for pCT
samples were low for DPn below ∼30 but higher at high
DPn and the overall attainable DPn was limited to ∼150. For
in vivo delivery of nondegradable polymers, hydrodynamic
radii of <5-10 nm are often desirable;35,42 graft-through
ROMP of either MM is highly controlled within this size
domain (Table 1). For the higher DPn CT-MM polymeriza-
tions, we hypothesize that the presence of potential chelating
moieties (quinoline and pyrrole) in CT may interfere with
catalyst initiation and propagation especially at high DPn.
Nevertheless, the success of the graft-through ROMP poly-
merizations for both MMs attests to the remarkable functional-
group tolerance of catalyst 1. Figure 2 shows gel-permeation
chromatography (GPC) traces of brush polymer samples
pCT03 and pDOX02 without purification, confirming a mono-
modal MW distribution and a very high conversion (>95%).
All of the polymer samples were highly soluble in water (>100
mg/mL); trace MM was removed by passage of an aqueous
solution of polymer through a 30 kDa cutoff centrifuge filter
to give pure brush polymer (Figure 3, red trace). The purified
samples were lyophilized to dryness and redissolved in water
prior to subsequent experiments.

Article
Similar data for pCT03 (∼2.6 μM bound CT in H2O) are
Macromolecules, Vol. 43, No. 24, 2010 10333
We recorded the UV-vis absorption spectra for pDOX01,
pCT01, and pDOX50-pCT50 in water to verify that CT and
DOX were present (Figure 5a). The spectra of pCT01 and
pDOX01 display broad absorption bands at wavelengths
above 300 nm that result from bound CT or DOX, respec-
tively. The spectrum of the copolymer shows both bands.
This information, along with the monomodal GPC trace
(Figure 3) and photolysis data (Figure 5b) suggests that
copolymer pDOX50-pCT50 does indeed carry both drug
molecules bound to the same polymer chain (rather than a
mixture of two homopolymers which would likely result in
broadening of the GPC trace). LC-MS traces for the copoly-
mer (∼1.5 μM in H2O) both before and after irradiation are
shown in Figure 5b. Absorption was monitored at two wave-
lengths, 368 and 500 nm, to detect CT and DOX respectively.
As expected, UV irradiation induced release of both drugs
from the copolymer; to our knowledge, this is the first example
of a polymer system capable of releasing two covalently bound
anticancer drugs (DOX and CT) in response to a controlled
external stimulus. A recent report by Shen and co-workers
suggests that materials capable of releasing both DOX and CT
will display synergistic cytotoxicity when compared to either
drug alone.51
shown in Figure 4b. After 10 min irradiation we observed ∼64%
release of free CT along with two minor peaks labeled “/” in
Figure 4b. CT is a common target for drug delivery because it
is highly active against cancer cells but insoluble and un-
stable in neutral, aqueous solution.47 We believe that these
two peaks may represent degradation products of CT that
result from hydrolysis (open lactone form) or photochemical
degradation; however we have been unable to generate the
same chromatogram by simply photolyzing free CT in solu-
tion due to its insolubility and we did not observe a molecular
ion in the LC-MS that corresponds to the mass of the open
lactone form (the major peak at ∼4.1 min corresponds to
the therapeutically active lactone form of CT). These experi-
ments show that the pCT brush polymers effectively solubi-
lize their CT payload and allow for drug release even in
aqueous solution where the CT cargo is insoluble.
Cell Culture Studies. To confirm that these drug-bound,
PEG-based brush polymers were inherently nontoxic, and
that photoinitiated drug release did indeed yield sufficient
amounts of chemotherapeutic agent to kill cancer cells, we
performed cell viability experiments using MCF-7 human
breast cancer cells. Cells were treated with aqueous solutions
of either free drug or the corresponding drug-loaded brush
polymer at various concentrations and irradiated for 10 min
using 365 nm light or kept in the dark. The cells were then
incubated in the dark for 24 h, washed twice, and incubated for
another 24 h in fresh, drug-free growth medium. After this time,
cell viability was assessed using the MTT assay (see Methods
and Materials for details). Representative data are shown in
Figure 6a-c. In parts a and b of Figure 6, both free CT and
DOX controls, with and without UV irradiation, gave simi-
lar dose-response curves with IC50 values of ∼1.2 μM and
∼4.9 μM, respectively. These data suggest that UV irradiation
at 365 nm for 10 min is not by itself toxic to the cells nor is it
detrimental to the drug toxicity. On the other hand, polymer
samples pCT01 and pDOX02 without UV irradiation were
nontoxic to cells at concentrations greater than 10 times
those of the free drugs (39 μM and 105 μM, respectively) indi-
cating that the PEG brush polymers effectively shield the
toxic effects of CT and DOX prior to drug release. We were
pleased to find that irradiation of the drug-bound polymers
led to greatly increased cytotoxicity (IC50 = 2.2 μM and
8.7 μM for pCT01 and pDOX02, respectively) compared
to the nonirradiated samples suggesting that photoreleased
Figure 4. HPLC-MS traces of aqueous brush polymer solutions be-
fore and after 365 nm UV irradiation for various times. pDOX02 and
pCT03 yield free DOX and CT, respectively. LC-MS method A (see
Methods and Materials) was used for pDOX02 while method B was
used for pCT03. Inset mass spectra, obtained from the free DOX and
CT peaks, show strong signals at m:z ratios that correspond to the
molecular ions of DOX and CT.
Figure 5. (A) UV-vis absorption of pDOX01, pCT01, and copolymer pDOX50-pCT50. (B) HPLC traces of an aqueous solution of pDOX50-pCT50
before and after 365 nm UV irradiation for 10 min.

10334 Macromolecules, Vol. 43, No. 24, 2010
Johnson et al.
Figure 6. (A-C) Viability of MCF-7 human breast cancer cells treated with free DOX and CT and drug-loaded brush polymers both with and without
UV irradiation. Data points were fit to a sigmoidal function and the half-maximum inhibitory concentrations (IC50) are shown. The x-axis labels refer
to the concentration of both free and polymer-conjugated drug. (D) Table of therapeutic factors for each brush polymer formulation. These values
represent the fold-increase in toxicity after irradiation and drug release.
CT and DOX were therapeutically effective. Figure 6c com-
pares the toxicity of copolymer pDOX50-pCT50 with a 1:1
mixture of pDOX02 and pCT01 before and after irradiation.
The copolymer was nontoxic prior to irradiation at concen-
trations less than 100 μM whereas the mixture of both poly-
mers appeared to be toxic at lower concentration (29 μM).
UV induced drug release, however, led to a similar IC50 for
both systems (3.2 μM for pDOX50-pCT50 and 2.2 μM for
the mixture). Figure 6d shows the therapeutic factors (X)
for these materials: a measure of the increase in cytotox-
icity after photoinduced drug release. All of the polymers
studied showed at least a 12X increase in toxicity upon drug
release; these results are encouraging and suggest the utility
of these brush polymer systems for in vivo drug delivery
applications.
system combined with the versatility of graft-through ROMP will
enable incorporation of new cleavable linkers into bivalent-brush
polymers.
Acknowledgment. We thank Dr. S. Virgil for helpful discus-
sion and advice. We also thank the Beckman Institute for a post-
doctoral fellowship for JAJ. UV-vis experiments were performed
in the Beckman Institute Laser Center. This work was supported
by the National Institutes of Health (NIH, R01-GM31332), the
MRSEC program of the National Science Foundation (NSF)
under award number DMR-0520565, and the NSF Center for
Chemical Innovation (Powering the Planet, CHE-0802907 and
CHE-0947829).
Supporting Information Available: Figures showing MALDI
and H NMR spectral data for polymer samples and liquid
1
Conclusions
chromatography calibration curves. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
To our knowledge, this report is the first example of simul-
taneous photoregulated release of DOX and CT and the first
example of bivalent-brush polymers capable of controlled
release of anticancer drugs (for other examples of photorelease
of anticancer drugs see refs 52-54). The graft-through approach
ensures that the weight percentage of drug loaded onto the brush
polymers is the same as the weight percentage of drug on the MM
(because of 100% grafting density) and is independent of DPn
and conversion. Thus, pCT and pDOX polymers carry 8.5% CT
and 12.6% DOX by weight, respectively. These values could
be increased by shortening the length of the PEG side chain
prior to ROMP or designing an MM linked to more than one
drug molecule. The synthesis of these materials was facilitated
by the graft-through ROMP paradigm and we expect this ap-
proach to prove useful for the synthesis of a range of other func-
tional multivalent-brush polymer systems. We are also develop-
ing clickable linkers with alternate drug release mechanisms; one
limitation of this system is the requirement for UV light to initiate
drug release. Though long-wavelength UV (UVA) is used for
photochemotherapeutic treatment of various cancers and skin
disorders,55-61 there is need for new photocleavable groups with
longer absorption wavelengths and high two-photon cross sec-
tions to increase tissue penetration.62,63 The modularity of this
References and Notes
(1) Hawker, C. J.; Wooley, K. L. Science (Washington, DC) 2005, 309,
1200–1205.
(2) Hawker, C. J.; Fokin, V. V.; Finn, M. G.; Sharpless, K. B. Aust. J.
Chem. 2007, 60, 381–383.
(3) Kolonko, E. M.; Pontrello, J. K.; Mangold, S. L.; Kiessling, L. L. J.
Am. Chem. Soc. 2009, 131, 7327–7333.
(4) Conrad, R. M.; Grubbs, R. H. Angew. Chem., Int. Ed. 2009, 48,
8328–8330.
(5) Clark, P. M.; Dweck, J. F.; Mason, D. E.; Hart, C. R.; Buck, S. B.;
Peters, E. C.; Agnew, B. J.; Hsieh-Wilson, L. C. J. Am. Chem. Soc.
2008, 130, 11576–11577.
(6) Rawat, M.; Gama, C. I.; Matson, J. B.; Hsieh-Wilson, L. C. J. Am.
Chem. Soc. 2008, 130, 2959–2961.
(7) Yuan, Y.-Y.; Du, Q.; Wang, Y.-C.; Wang, J. Macromolecules 2010,
43, 1739–1746.
(8) Li, C.; Ge, Z.; Fang, J.; Liu, S. Macromolecules 2009, 42, 2916–2924.
(9) Li, A.; Lu, Z.; Zhou, Q.; Qiu, F.; Yang, Y. J. Polym. Sci., Part A:
Polym. Chem. 2006, 44, 3942–3946.
(10) Zhang, M.; Mueller, A. H. E. J. Polym. Sci., Part A: Polym. Chem.
2005, 43, 3461–3481.
(11) Jiang, X.; Lok, M. C.; Hennink, W. E. Bioconjugate Chem. 2007,
18, 2077–2084.

Article
Macromolecules, Vol. 43, No. 24, 2010 10335
(12) Tsarevsky, N. V.; Bencherif, S. A.; Matyjaszewski, K. Macromo-
lecules 2007, 40, 4439–4445.
(13) Lutz, J.-F.; Boerner, H. G.; Weichenhan, K. Macromolecules 2006,
39, 6376–6383.
(14) Allen, M. J.; Wangkanont, K.; Raines, R. T.; Kiessling, L. L.
Macromolecules 2009, 42, 4023–4027.
(15) Cheng, G.; Boeker, A.; Zhang, M.; Krausch, G.; Mueller, A. H. E.
Macromolecules 2001, 34, 6883–6888.
(39) Lee, C. C.; Gillies, E. R.; Fox, M. E.; Guillaudeu, S. J.; Frechet,
J. M. J.; Dy, E. E.; Szoka, F. C. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 16649–16654.
(40) Guillaudeu, S. J.; Fox, M. E.; Haidar, Y. M.; Dy, E. E.; Szoka,
F. C.; Frechet, J. M. J. Bioconjugate Chem. 2008, 19, 461–469.
(41) Fox, M. E.; Guillaudeu, S.; Frechet, J. M. J.; Jerger, K.; Macaraeg,
N.; Szoka, F. C. Mol. Pharm. 2009, 6, 1562–1572.
(42) Grayson, S. M.; Godbey, W. T. J. Drug Targeting 2008, 16, 329–
356.
(43) Love, J. A.; Morgan, J. P.; Trnka, T. M.; Grubbs, R. H. Angew.
(16) Lu, H.; Wang, J.; Lin, Y.; Cheng, J. J. Am. Chem. Soc. 2009, 131,
13582–13583.
(17) Morandi, G.; Pascual, S.; Montembault, V.; Legoupy, S.; Delorme,
N.; Fontaine, L. Macromolecules 2009, 42, 6927–6931.
(18) Neugebauer, D.; Sumerlin, B. S.; Matyjaszewski, K.; Goodhart, B.;
Sheiko, S. S. Polymer 2004, 45, 8173–8179.
Chem., Int. Ed. 2002, 41, 4035–4037.
(44) Vercillo, O. E.; Andrade, C. K. Z.; Wessjohann, L. A. Org. Lett.
2008, 10, 205–208.
(45) Johnson, J. A.; Finn, M. G.; Koberstein, J. T.; Turro, N. J.
(19) Sumerlin, B. S.; Neugebauer, D.; Matyjaszewski, K. Macromole-
cules 2005, 38, 702–708.
(20) Gao, H.; Matyjaszewski, K. J. Am. Chem. Soc. 2007, 129, 6633–
6639.
(21) Hadjichristidis, N.; Pitsikalis, M.; Iatrou, H.; Pispas, S. Macromol.
Macromolecules 2007, 40, 3589–3598.
(46) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.;
Timmers, F. J. Organometallics 1996, 15, 1518–1520.
(47) Zhao, H.; Lee, C.; Sai, P.; Choe, Y. H.; Boro, M.; Pendri, A.; Guan,
S.; Greenwald, R. B. J. Org. Chem. 2000, 65, 4601–4606.
(48) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004–2021.
Rapid Commun. 2003, 24, 979–1013.
(22) Tsukahara, Y.; Mizuno, K.; Segawa, A.; Yamashita, Y. Macro-
molecules 1989, 22, 1546–1552.
(23) Tsukahara, Y.; Tsutsumi, K.; Yamashita, Y.; Shimada, S. Macro-
(49) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596–2599.
molecules 1990, 23, 5201–5208.
(24) Dziezok, P.; Sheiko, S. S.; Fischer, K.; Schmidt, M.; Moller, M.
Angew. Chem., Int. Ed. 1998, 36, 2812–2815.
(25) Neiser, M. W.; Okuda, J.; Schmidt, M. Macromolecules 2003, 36,
5437–5439.
(50) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
3057–3064.
(51) Shen, Y.; Jin, E.; Zhang, B.; Murphy, C. J.; Sui, M.; Zhao, J.;
Wang, J.; Tang, J.; Fan, M.; Van Kirk, E.; Murdoch, W. J. J. Am.
Chem. Soc. 2010, 132, 4259–4265.
(26) Neiser, M. W.; Muth, S.; Kolb, U.; Harris, J. R.; Okuda, J.;
Schmidt, M. Angew. Chem., Int. Ed. 2004, 43, 3192–3195.
(27) Xia, Y.; Kornfield, J. A.; Grubbs, R. H. Macromolecules 2009, 42,
3761–3766.
(52) Agasti, S. S.; Chompoosor, A.; You, C.-C.; Ghosh, P.; Kim, C. K.;
Rotello, V. M. J. Am. Chem. Soc. 2009, 131, 5728–5729.
(53) Kim, H.-C.; Hartner, S.; Behe, M.; Behr Thomas, M.; Hampp
Norbert, A. J. Biomed. Opt. 2006, 11, 34024.
(28) Xia, Y.; Olsen, B. D.; Kornfield, J. A.; Grubbs, R. H. J. Am. Chem.
Soc. 2009, 131, 18525–18532.
(29) Li, Z.; Zhang, K.; Ma, J.; Cheng, C.; Wooley, K. L. J. Polym. Sci.,
Part A: Polym. Chem. 2009, 47, 5557–5563.
(30) Li, Z.; Ma, J.; Cheng, C.; Zhang, K.; Wooley, K. L. Macromole-
(54) Choi, S. K.; Thomas, T.; Li, M.-H.; Kotlyar, A.; Desai, A.; Baker,
J. R., Jr. Chem. Commun. (Cambridge, U.K.) 2010, 46, 2632–2634.
(55) Krutmann, J. J. Photochem. Photobiol., B 1998, 44, 159–164.
(56) Bethea, D.; Fullmer, B.; Syed, S.; Seltzer, G.; Tiano, J.; Rischko,
C.; Gillespie, L.; Brown, D.; Gasparro, F. P. J. Dermatol. Sci. 1999,
19, 78–88.
cules 2010, 43, 1182–1184.
(31) Le, D.; Montembault, V.; Soutif, J. C.; Rutnakornpituk, M.;
Fontaine, L. Macromolecules 2010, 43, 5611–5617.
(32) Duncan, R. Nat. Rev. Drug Discovery 2003, 2, 347–360.
(33) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.;
Langer, R. Nat. Nanotechnol. 2007, 2, 751–760.
(34) Mammen, M.; Chio, S.-K.; Whitesides, G. M. Angew. Chem., Int.
Ed. 1998, 37, 2755–2794.
(35) Fox, M. E.; Szoka, F. C.; Frechet, J. M. J. Acc. Chem. Res. 2009, 42,
1141–1151.
(36) Matsumura, Y.; Maeda, H. Cancer Res. 1986, 46, 6387–6392.
(37) Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.;
Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Polym. J. (Tokyo, Jpn.)
1985, 17, 117–132.
(57) Breuckmann, F.; Gambichler, T.; Altmeyer, P.; Kreuter, A. BMC
Dermatol. 2004, 4.
(58) Diffey, B. Phys. Med. Biol. 2006, 51, R229–R244.
(59) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Nat. Rev.
Cancer 2003, 3, 380–387.
(60) Dolmans, D. E. J. G. J.; Kadambi, A.; Hill, J. S.; Flores, K. R.;
Gerber, J. N.; Walker, J. P.; Borel Rinkes, I. H. M.; Jain, R. K.;
Fukumura, D. Cancer Res. 2002, 62, 4289–4294.
(61) Wozniak, M. B.; Tracey, L.; Ortiz-Romero, P. L.; Montes, S.;
Alvarez, M.; Fraga, J.; Herrera, J. F.; Vidal, S.; Rodriguez-Peralto,
J. L.; Piris, M. A.; Villuendas, R. Br. J. Dermatol. 2009, 160, 92–102.
(62) Buckup, T.; Southan, A.; Kim, H. C.; Hampp, N.; Motzkus, M.
J. Photochem. Photobiol., A 2010, 210, 188–192.
(38) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. Rev.
(Washington, DC) 1999, 99, 1665–1688.
(63) Haertner, S.; Kim, H.-C.; Hampp, N. J. Polym. Sci., Part A:
Polym. Chem. 2007, 45, 2443–2452.