A photochemical approach for controlled drug release in targeted drug delivery

Article abstract of DOI:10.1016/j.bmc.2011.12.020

Photochemistry provides a unique mechanism that enables the active control of drug release in cancer-targeting drug delivery. This study investigates the light-mediated release of methotrexate, an anticancer drug, using a photocleavable linker strategy based on o-nitrobenzyl protection. We evaluated two types of the o-nitrobenzyl-linked methotrexate for the drug release study and further extended the study to a fifth-generation poly(amidoamine) dendrimer carrier covalently conjugated with methotrexate via the o-nitrobenzyl linker. We performed the drug release studies by using a combination of three standard analytical methods that include UV/vis spectrometry, ¹H NMR spectroscopy, and anal. HPLC. This article reports that methotrexate is released by the photochemical mechanism in an actively controlled manner. The rate of the drug release varies in response to multiple control parameters, including linker design, light wavelength, exposure time, and the pH of the medium where the drug release occurs.

Full text of DOI:10.1016/j.bmc.2011.12.020

Bioorganic & Medicinal Chemistry 20 (2012) 1281–1290
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A photochemical approach for controlled drug release in targeted drug delivery
Seok Ki Choi a,b, , Manisha Verma c, Justin Silpe c, Ryan E. Moody d, Kenny Tang c, Jeffrey J. Hanson c,
⇑
James R. Baker Jr. a,b,
⇑
a Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan, Ann Arbor, MI 48109, USA
b Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA
c College of Literature, Science and The Arts, University of Michigan, Ann Arbor, MI 48109, USA
d College of Engineering, University of Michigan, Ann Arbor, MI 48109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Photochemistry provides a unique mechanism that enables the active control of drug release in cancer-
targeting drug delivery. This study investigates the light-mediated release of methotrexate, an anticancer
drug, using a photocleavable linker strategy based on o-nitrobenzyl protection. We evaluated two types
of the o-nitrobenzyl-linked methotrexate for the drug release study and further extended the study to a
fifth-generation poly(amidoamine) dendrimer carrier covalently conjugated with methotrexate via the o-
nitrobenzyl linker. We performed the drug release studies by using a combination of three standard ana-
lytical methods that include UV/vis spectrometry, 1H NMR spectroscopy, and anal. HPLC. This article
reports that methotrexate is released by the photochemical mechanism in an actively controlled manner.
The rate of the drug release varies in response to multiple control parameters, including linker design,
light wavelength, exposure time, and the pH of the medium where the drug release occurs.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 26 October 2011
Revised 6 December 2011
Accepted 13 December 2011
Available online 20 December 2011
Keywords:
o-Nitrobenzyl linker
Photocleavage
Methotrexate
Dendrimer
Controlled release
Drug delivery
1. Introduction
to overcome such a therapeutic limitation, targeted delivery pro-
vides a route for facilitating the MTX uptake by a cancer cell, and
Targeted delivery plays an essential role in the detection, diag-
nosis and treatment of life-threatening diseases, including can-
cers.1–3 A challenging aspect facing the delivery strategy relates
to the timely control of the drug release after uptake by the tar-
geted cell.1,4–7 Most of the release mechanisms currently being ex-
plored involve chemical and enzymatic reactions which are
triggered passively under the influence of specific internal cellular
factors (Fig. 1). We have been interested in designing an orthogonal
release approach in which an external tool such as light8–13 or
ultrasound14,15 is applied to actively trigger the drug release. The
present study aims to investigate a photon-based external ap-
proach for the release of methotrexate (MTX) as the model cancer
drug.
as a consequence, for enhancing its therapeutic index.4,6,7,22 This
Nanoscale carrier
Targeting ligand
Drug
Receptor
Cancer cell
Uptake
MTX belongs to a class of antifolate molecules and constitutes
one of the clinically approved anticancer drugs.1,16,17 It is a potent
inhibitor (Ki = 0.058 nM16) of dihydrofolate reductase (DHFR18,19
)
localized in the cytoplasm. Despite its proven therapeutic value in
the treatment of certain cancers20 and rheumatoid arthritis,21
MTX suffers from its non-selective cytotoxicity that contributes to
lower its therapeutic index.17 As the approach frequently applied
Drug release
Endosome; Lysosome
⇑
Corresponding authors. Tel.: +1 734 615 0618; fax: +1 734 615 0621 (S.K.C.);
tel.: +1 734 647 2777; fax: +1 734 615 2990 (J.R.B.).
Figure 1. A proposed schematic illustrating the concept of cancer targeting drug
delivery. The drug release can be controlled by the mechanism triggered by an
endogenous factor (low pH, reduction, enzymes), or an external tool such as light.
E-mail addresses: skchoi@umich.edu (S.K. Choi), jbakerjr@umich.edu (J.R. Baker).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.020

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S. K. Choi et al. / Bioorg. Med. Chem. 20 (2012) 1281–1290
strategy relies on a macromolecular system designed in a rational
way such that the therapeutic payloads are carried by a system that
is conjugated with a targeting ligand for binding to the cancer
cell.5,6,23–27 Such delivery strategy has already led to a number of
successful applications for MTX5,28 and for other anti-cancer thera-
peutic agents represented by cisplatin,6 doxorubicin,29 and paclit-
axel.4,23 In each of these cases, the drug molecule is delivered by a
nanometer-sized carrier based on dendritic macromolecules,22 lip-
osomes,30 polymers,3 and metallic nanoparticles.6 Despite the rapid
progress achieved in this field, there are certain technical aspects
that deserve further optimization—in particular, in the method of
drug release. Most of the current release methods rely on either a
chemical or enzymatic cleavage reaction of the linker that tethers
the drug molecule.4,6,7 Such methods are incorporated by an es-
ter-based or amide-based linker which is cleaved hydrolytically at
the acidic subcellular compartments, such as the endosomes and
lysosomes (pH 4–5), where the drug carriers are internalized.6,7 In
addition, there are other specialized linkers based on di-sulfide,4
indolequinone,31 and nitroheterocycle32 which are cleavable differ-
ently through bioreductive mechanisms. Despite the differences
among such release mechanisms, it is common that the drug re-
lease occurs passively only in response to environmental and path-
ophysiological factors.
Linker
O
OH
N
Linker
O
O2N
O
hν
O
Drug
OCH3
OCH3
O
Drug
aci-Nitro form
H
O
Linker
O
Linker
O
N
ON
O
O
O
OCH3
OCH3
Drug
Drug OH
Figure 2. A schematic for photon-based cleavage of an o-nitrobenzyl (ONB) linker
as the mechanism that enables the controlled drug release.
A synthetic method for photolinker 1 and its MTX conjugate 3 is
described in Scheme 1. Synthesis of 1 was achieved by the O-alkyl-
ation reaction of 2-nitro-5-hydroxybenzyl alcohol with a bromo-
acetamide-containing spacer, 6. The linker 1 was then derivatized
to 7 by reacting with bromoacetyl chloride such that its resultant
bromoacetate moiety is used for the conjugation with MTX by the
O-alkylation reaction at the carboxylate site of the L-glutamate do-
This study aims to investigate the orthogonal method, which al-
lows the active release of drug molecules through application of an
external trigger. We employed photochemistry as the orthogonal
means to control the release of MTX. Our approach is based on the
concept of photocaging,33 in which a biologically active molecule
(ligand, drug) is temporarily inactivated by protecting it with a
photocleavable group (‘photocaged’). This caged molecule releases
its parent species in an actively controlled manner only when its
photosensitive protective group is cleaved by UV irradiation. The fo-
cus of this release method has mainly been on chemical and biolog-
ical problems, such as the spatiotemporal control of cell signaling
processes,34–36 and it was only recently applied to drug deliv-
ery.8,11–13 The photocleavable linkers that are applicable for the cur-
rent application are comprised of those based on o-nitrobenzyl
(ONB),33,35,37 coumarin,35,36 xanthene,38 and benzophenone.39 Fig-
ure 2 illustrates a schematic for controlled drug release triggered
by a photochemical mechanism where a drug molecule attached
to the photocleavable linker is released upon UV irradiation.8 In
the present study, we selected the ONB group as the core of the
photocleavable linker and developed linker chemistry that is appli-
cable for the drug attachment and conjugation to poly(amidoamine)
(PAMAM) dendrimer as the delivery platform. Here, we report the
photochemical mechanism of MTX release with the ONB-linker
strategy and analyze a set of key basic parameters that determine
the kinetics of the drug release.
main. After the coupling reaction, the product 8 was isolated by
flash silica column chromatography as a mixture of two regioisom-
ers,
the
a
- and
c-ester (a/c
= 37/63 on the basis of 1H NMR data; only
c-isomer is shown). Their separation was not attempted be-
cause each isomer is able to release the MTX payload. The N-Boc
group in 8 was deprotected by the TFA treatment, yielding a MTX-
linker 3 that contains a free primary amine at the linker terminus.
Synthesis of the other MTX-linker 4 was completed by adopting
the synthetic approach as described earlier but with minor modifi-
cations (Scheme 2). In this synthetic process, the benzyl alcohol of
the linker 2 was preactivated to its methanesulfonate derivative,
and it was then used for the O-alkylation reaction with MTX.8 The
product, MTX-linker 9, was treated with TFA, yielding the MTX-lin-
ker 4.
2.2. Design of PAMAM dendrimer conjugated with MTX through
the photocleavable linker
In our photochemical approach to drug delivery, we chose a fifth
generation (G5) PAMAM dendrimer (mean diameter ꢀ5.4 nm43
)
conjugated with a folic acid (FA) ligand as the cancer-targeting sys-
tem that carries the photocaged MTX (Scheme 3). The notion that
cancer cells are targetable by use of the folate ligand has been well
established in the field of targeted delivery. Such cancer targeting
relies on folate receptor (FAR)-mediated cellular uptake by a cancer
cell with the up-regulated level of the folate receptor.5,6,23,44,45
Scheme 3 describes the synthesis of the dendrimer conjugate 11, a
FAR-targeting G5 PAMAM dendrimer conjugated with MTX through
the photocleavable linker. First, a precursor dendrimer, G5-GA 10
(G5-glutaric acid; Mn = 42730 g molÀ1, PDI = Mw/Mn ꢀ1.046),8 was
preactivated by an EDC/NHS method, in which the carboxylic acid
present on the dendrimer surface was converted to the amine-reac-
tive N-hydroxysuccinimide ester. Second, the MTX-linker 4and a
FAR-targeting ligand (FA-ethylenediamine46) were covalently cou-
pled to the preactivated dendrimer through the amide formations.
The dendrimer conjugate 11 was produced and purified by dialysis
using membrane tubing (MWCO 10 kDa) to remove unreacted reac-
tants and reagents. The purity of this conjugate 11 was determined
by analytical HPLC (P96%), and its molar mass was determined by
MALDI-TOF mass spectrometry (54,000 g molÀ1). The average num-
ber of the photocaged MTX and the FA ligands attached to the
dendrimer was determined using the method described
2. Results and discussion
2.1. Design of methotrexate (MTX)-photolinker conjugates
The two photolinker molecules 1 and 2 used in this study are
shown in Figure 3. The core of each linker is based primarily on
the ONB group,40–42 where its benzylic alcohol serves as the phot-
olabile site for its covalent conjugation with an MTX molecule. The
structure of each linker also contains a primary amine located at
the end opposite to the benzylic position, and this terminal amine
serves as the site to covalently anchor the photocaged drug mole-
cule to a PAMAM dendrimer carrier. The two linkers are quite
homologous in their structure and bifunctional design, but they
are different in the pattern of aromatic substitutions at the ONB
group. We introduced such variation in order to evaluate the
significance of the ONB core in the photochemical drug release
and to identify an optimal substitution pattern for the ONB core.

S. K. Choi et al. / Bioorg. Med. Chem. 20 (2012) 1281–1290
1283
Figure 3. Structures of bifunctional o-nitrobenzyl (ONB) molecules 1, 2 and two methotrexate (MTX)-ONB conjugates 3, 4, derived from the linkers.
O
i
ii
Br
H2N
NHBoc
NHBoc
N
H
6
5
O2N
HO
O2N
iii
H
N
H
N
NHBoc
O
NHBoc
O
O
Br
O
O
O
N
O
O
1
7
iv-v
N
O
OH
O
γ
O
O
NH2
N
N
H
O
N
H
NHR
α
N
N
O
O2N
8: R = Boc
3: R = H
H2N
Scheme 1. Synthesis of a methotrexate (MTX)-ONB linker3. Reagents and conditions: (i) bromoacetyl chloride, i-Pr2NEt, CH2Cl2, 0 °C; (ii) 2-nitro-5-hydroxybenzyl alcohol,
K2CO3, MeCN, reflux; (iii) bromoacetyl chloride, i-Pr2NEt, CH2Cl2, 0 °C to rt; (iv) methotrexate, Cs2CO3, DMF, rt, 24 h (23% isolation yield); (v) TFA, CH2Cl2, rt.
elsewhere8,25 after analysis of the data obtained from MALDI mass
spectrometry,5,25 1H NMR spectroscopy,47 and UV/vis spectrome-
try.8,25 The dendrimer conjugate 11 (G5-FA4-MTX8) contains on
average four copies of the folate ligand and eight copies of 4 per den-
drimer molecule.
at 310 nm (
at the longer wavelength 340 nm (
e
= 9575 MÀ1 cmÀ1), while the other linker, 2, has kmax
= 2750 MÀ1 cmÀ1). This spec-
e
tral difference is attributable mainly to the effect of an additional
methoxy group present at the ONB system 2. Such a bathochromic
shift is consistent with observations reported elsewhere48,49 that
the addition of an electron-rich methoxy group into the ONB sys-
tem leads to longer kmax and influences the quantum yield of
ONB photolysis as well. The UV spectral data suggest such kmax
of 310–340 nm as the preferred range of light that should provide
an optimal level of photoactivation for the two ONB linkers or their
MTX conjugates.
2.3. Photolysis of ONB linkers
The electronic absorption properties of the two ONB linkers 1
and 2 were studied by measuring their UV/vis absorption spectra
in aqueous medium, as provided in Figure 4a. The linker 1 has kmax

1284
S. K. Choi et al. / Bioorg. Med. Chem. 20 (2012) 1281–1290
O
OH
O2N
HO
O
NHBoc
vi-viii
i-v (Lit.)
N
H
O
OCH3
OCH3
2
Vanillin
O
O
OH
O2N
O
O
NHR
O
N
H
γ
NH2
N
H
OCH3
α
N
N
O
N
N
N
H2N
9: R = Boc
4: R = H
Scheme 2. Synthesis of a methotrexate (MTX)-ONB linker 4. Reagents and conditions: (i) Ethyl bromoacetate, K2CO3, DMF, rt; (ii) NaOH, THF, MeOH, H2O, rt; (iii) concd HNO3,
AcOH, 0 °C to rt; (iv) N-Boc-1,2-diaminoethane, DCC, DMAP, DMF, 0 °C to rt; (v) NaBH4, THF, MeOH, rt; (vi) methanesulfonyl chloride, Et3N, CHCl3, 0 °C to rt; (vii) Cs2CO3, NaI,
methotrexate, DMF, rt; (viii) TFA, CHCl3, rt, 15 min.
O
O
~5.4 nm
(NH
2)
HN
OH
108
110
ii-iv
i
Generation 5
PAMAM dendrimer
10 (G5-GA)
O
MeO
O
O
R1
H
N
N
H
O
O
NO2
8
H
11
N
R2
R1CO2H = MTX
R2CO2H = FA
O
N
H
4
O
Scheme 3. Synthesis of a folate receptor-targeting PAMAM dendrimer conjugated
with methotrexate (MTX) 11. The dendrimer contains folic acid (FA) as the targeting
ligand, and also carries MTX tethered at the photolabile ONB linker. Reagents and
conditions: (i) glutaric anhydride, Et3N, MeOH, rt, 24 h; (ii) NHS, EDC, DMAP, DMF,
rt, 36 h; (iii) 4 (MTX-ONB linker, 15 mol eq per dendrimer), FA-CONHCH2CH2NH2,
5 mol eq per dendrimer), Et3N, DMF, rt, 36 h; (iv) ethanolamine; then dialysis
(MWCO 10 kDa) against phosphate-buffered saline (PBS) and deionized water.
Photolysis of the two MTX-linker conjugates 3 and 4 was stud-
ied by exposing each conjugate dissolved in an aqueous solution
(30 lM) to the UV-A (365 nm) or UV-B (312 nm) light source.
The UV/vis absorption spectra for each drug conjugate and its irra-
diation time course are shown in Figure 4b and c. Prior to irradia-
tion, each MTX-linker conjugate shows strong absorption features
at the range of a 250–400 nm wavelength, and such absorption re-
sults from the contribution of its ONB linker and MTX as well
(Fig. 4a; absorption peaks: 310 nm,
e
spectra were taken for each conjugate, and these showed signifi-
cant changes in the absorption peaks that are assigned to the
ONB linker. A rapid decrease in absorbance at around 310 nm
was observed along with a concomitant increase at around
e
= 27535 MÀ1 cmÀ1; 380 nm,
Figure 4. (a) UV/vis absorption spectra of methotrexate (MTX, 11
lM in PBS), and
= 7990 MÀ1 cmÀ1). Following UV exposure, UV/vis absorption
two photocleavable linkers 1 (26 M) and 2 (33 M), each in an aqueous medium
l
l
(0.5% MeOH/H2O); (b and c) UV/vis spectral traces of MTX-ONB conjugates 3 and 4
after exposure to UV-B (312 nm for 3), or UV-A (365 nm for 4) light as a function of
irradiation time (t = 0, 1, 3, 5, 7, 10, 15 min). Each plot in the inset (b and c) shows
the change in the absorption of the irradiated solution at the indicated wavelength
that led to ONB-associated spectral changes.

S. K. Choi et al. / Bioorg. Med. Chem. 20 (2012) 1281–1290
1285
linker photolysis. First, the drug release is time-dependent in a
way that it was faster at early time points (up to 7 min). This result
from the HPLC analysis is consistent with that from the earlier UV
spectrophotometric analysis (Fig. 4). Second, the drug release is
wavelength-dependent. The UV exposure at 312 nm led to a
slightly faster release than at 365 nm before reaching a maximal
level at 7–10 min. However, lack of a much larger difference be-
tween the two wavelengths might be attributable to the broad
range of the absorptivity, from 300 to 370 nm, displayed by the
photolinker 2 (Fig. 4a). Third, the drug release is linker-dependent,
such that the efficiency of MTX release is greater for conjugate 4
than for conjugate 3. Comparison of the two conjugates after max-
imal UV exposure (15 min) at 312 nm shows that 4 released MTX
(68%) at the rate ꢀtwo-fold greater than 3 (33%). In summary,
we investigated a number of external and internal factors, includ-
ing the linker structure, light wavelength, and irradiation time, and
were able to understand the significance of the role played by each
factor in photochemical drug release.48,49
2.4. pH effect on linker photolysis
Our earlier investigation focused on the photochemical release
of MTX performed at neutral pH. While certainly important, we
also recognized that a study spanning a range of pHs was war-
ranted given that pH clearly has physiological relevance in the
development of targeted drug delivery strategies. For example, a
tumor cell is characteristically more acidic than a normal healthy
cell,50 and subcellular compartments such as early endosomes
and lysosomes where drug carriers are taken up and temporarily
reside are acidic (pH <5–6) relative to the neutral cytosolic
medium.51,52
First, we performed photolysis experiments for the ONB linker 2
and determined whether the pH of the medium influenced the
photocleavage rate (Fig. S1, Supplementary data). In this experi-
ment, each photolytic reaction leads to the release of one water
molecule (R1OH = H2O) per linker molecule40 in a mechanism that
involves the fragmentation of the ONB group to the nitrosobenzal-
dehyde derivative. The experiments were performed by exposure
to longer wavelength light (365 nm), and the reaction progress
after each exposure was monitored by 1H NMR spectroscopy. Fig-
ure S1a illustrates a spectral region of interest selected for 1H
NMR spectra acquired for the photolysis of 2 in methanol-d4
(1.2 mM) as a function of irradiation time. Proton signals H3 and
H6, both assigned to the ONB aromatic system, decrease in their
intensities. In addition, several new signals are generated over time
as the photolysis products, including the aldehyde CH(@O) and its
hydrate form CH(OH)2. Integration of the area under each signal for
the remaining 2 was quantified relative to an internal reference
and was used to determine the rates of photolysis.
Figure S1b compares the time-courses for the photolytic reac-
tions performed in various media. The result from methanol-d4
shows that approximately 50% of the linker was cleaved within
the 10-min period of irradiation. Notably, the results obtained from
aqueous solutions performed at different pH conditions (pH 7.4,
9.0, and 5.0) show that the photolysis of 2 is significantly affected
by the pH value of the medium. It is clear that the linker cleavage
was faster at an acidic or basic solution than at the physiological
condition (pH 7.4). The value of half-life t1/2 (the exposure time re-
quired to afford 50% of linker cleavage) estimated from each curve
allows us to summarize the order of the photolysis rate as follows:
pH 9 (5 min) P pH 5 (8 min) > pH 7.4 (15 min). Such pH depen-
dency observed in this NMR study is consistent with other obser-
vations that relate to the photolysis of ONB-tethered small
molecules,42 including glycine53 and urea.48 We attribute the ori-
gin of this pH effect primarily to the photochemical mechanism
that underlies the fragmentation of the ONB linker (Fig. 2). The
Figure 5. UV light-mediated release of methotrexate (MTX) from the MTX-ONB
conjugate 4 (30 M in 2% MeOH/H2O) by exposure to UV-A (365 nm) light. (a) HPLC
traces of the irradiated solutions as a function of UV exposure time (t = 0, 1, 3, 5, 7,
10, 15 min); (b) plots for the photochemical release of MTX from MTX-ONB
conjugates 3 and 4 as a function of time exposed to UV-B (312 nm) or UV-A
l
(365 nm) light. In the release kinetics for 3 (30
lM in 2% MeOH/H2O), the drug
release refers to the amount of MTX-glycolic acid.
370 nm. Such spectral features were reported similarly in the pho-
tolysis of the ONB linker 2 (quantum efficiency
= 0.298). This
U
spectral change is attributable to the photocleavage of the ONB lin-
ker, yielding a 2-nitrosobenzaldehyde-derived product, and as a
consequence, triggering the MTX release. The UV/vis time course
for each linker cleavage is illustrated by plotting the absorption
at 366 or 370 nm as a function of irradiation time (inset). Each plot
indicates faster increase in absorbance up to 7 min followed by
slower increase. This spectrometric study suggests that the drug
release occurs in a nonlinear manner and more rapidly in earlier
time points.
The kinetics of the photochemical drug release for conjugate 4
was further investigated by using analytical reversed phase HPLC,
as illustrated in Figure 5. HPLC traces acquired after UV exposure
show growth of a peak assigned to the free MTX (tr = 6.1 min) as
a function of exposure time. The area under curve (AUC) for this
peak was integrated to determine the percent amount of MTX re-
leased relative to the initial amount of 4, and the rate of drug re-
lease is presented in Figure 5b. This figure also summarizes other
release rates acquired from the MTX-linker conjugates 3 and 4
after UV exposure at 312 nm. The release results for 4 are charac-
terized by several notable features that pertain to the kinetics of

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S. K. Choi et al. / Bioorg. Med. Chem. 20 (2012) 1281–1290
areas) relative to an internal reference as a function of irradiation
time. In addition to linker fragmentation, those proton signals as-
signed to MTX (Hb, Hc) split to more peaks in response to the irra-
diation, suggesting the release of free MTX molecules. The rate of
MTX release acquired in methanol-d4 is plotted in Figure 6b, show-
ing that approximately 40% of the drug release is achieved within
30 min of irradiation. Such drug release of 4 occurs apparently
more slowly than the cleavage of the linker 2, as determined earlier
(Fig. S1b). This lowered release rate is perhaps closely associated
with the strong absorption of the applied UV light by the tethered
drug molecule. The MTX molecule has strong absorption at 310–
380 nm by its pteridine chromophore ( Fig. 4a), and, therefore,
the photoactivation of the ONB core might be competitively inhib-
ited (Fig. 4c).
The kinetics of the drug release using the MTX-linker conjugate
4 (1.2 mM) was studied at variable pH conditions (7.4, 9.0, and 5.0),
and the results are summarized in Figure 6b. Half life (t1/2) for the
drug release takes 630 min at pH 5, but is two-fold longer
(P70 min) at pH 7.4 or 9. In general, the release studies were per-
formed at the mM concentration of 4, and the rate was slower than
that determined at a much lower concentration (30 lM) by the UV/
vis and HPLC method ( Fig. 5). In addition, this result diverges
slightly from the pH trend we observed with the photolinker 2,
in which the rate of linker cleavage was almost similar at pH 5
and 9, but lower at pH 7.4 (Fig. S1). One reason for this difference
may be the nature of the molecular species that is released as a re-
sult of the linker fragmentation. Upon photolysis, the ONB linker 2
releases the water molecule, but the MTX-ONB conjugate 4 re-
leases MTX in a manner that its (L)-glutamyl carboxylic acid serves
as the leaving group. Prior studies also showed mixed release pro-
files in response to pH variation that are dependent on the nature
of the leaving group.53–55 Here, the release profile of MTX appears
to correlate with the pH trend observed for glycine53 or car-
bamylcholine54 which was pH 5.5 > pH 9.0 > pH 7.4; it was not
consistent with the release profile of N-methyl-D
-aspartate55
O
MeO
O
O
MTX
Figure 6. Photolysis of an ONB linker from the MTX-ONB conjugate 4 by exposure
to UV-A light. (a) 1H NMR spectral traces acquired after UV irradiation of 4 in
MeOH-d4 as a function of exposure time (t = 0–80 min); (b) pH effect on the rate of
linker cleavage from 4 in the aqueous solution at pH 7.4 (50% CD3CN/PBS), 9.0 (20%
H
N
N
H
O
NO2
8
O
4
CD3CN/D2O), or 5.0 (20% CD3CN/D2O). The percent amount of intact
4 was
determined by the peak integration method applied to the ONB-associated protons
Hd, He, and Hf. Each data point represents a mean value from this analysis.
11
H
N
FA
O
N
H
O
hν (365 nm)
pH 7.4 or 4.6
fragmentation mechanism is mediated by the formation of several
charged species such as the aci-nitro form, and their rates of forma-
tion are likely to be sensitive to the pH variation.40,42
MeO
O
CH(OH)2
NO
2.5. pH effect on MTX release
O
H
N
N
H
We proceeded to determine whether the pH of the medium also
influences the rate of drug release in lieu of water release, as stud-
ied earlier with the photolinker itself. This time, we studied the
kinetics of the linker cleavage, using the MTX-linker conjugate 4
at variable pH conditions ( Fig. 6). The progress of the reaction
was monitored by 1H NMR spectroscopy taken after exposure to
the UV light. Figure 6a shows a spectral region of interest for the
1H NMR spectra acquired from the experiments in methanol-d4
(1.2 mM). Each spectral segment shows a subset of proton signals,
including Hf, Hg, and Hh, that are assigned to the ONB linker. These
ONB protons decrease in the peak intensities (and integration
O
+
H
N
FA
N
O
MTX (released)
H
4
O
Figure 7. Light-controlled release of methotrexate (MTX) from 11, a fifth gener-
ation PAMAM dendrimer conjugated with MTX through the photocleavable linker.
The drug release was controlled by the irradiation of 11 (0.1 mg/mL, 1.96 lM) in the
PBS buffer (pH 7.4), or acetate buffer (pH 4.6).

S. K. Choi et al. / Bioorg. Med. Chem. 20 (2012) 1281–1290
1287
Figure 8. Photochemical MTX release from the dendrimer-MTX conjugate 11 (G5-FA4-MTX8). UV/vis spectra (left) and anal. HPLC traces (right) are shown for the release
study performed at pH 7.4 (a) or 4.6 (b). Each spectral or chromatographic overlay is plotted as a function of UV exposure time (t = 0–15 min). ⁄ This peak has an MTX-like UV/
vis absorption profile, and its identity might be associated with MTX as the tautomer.
which was pH 4 > pH 7 > pH 10. Overall, the photochemical release
of MTX is sensitive to pH variation.
2.6. MTX release from its PAMAM dendrimer conjugate
Light-controlled release of MTX was investigated with the ONB-
linked MTX attached to PAMAM dendrimer (11, G5-FA4-MTX8) by
exposing the dendrimer conjugate to UV-A light (Fig. 7). We stud-
ied the rate of drug release at two different pH conditions, 7.4 (PBS
buffer) and 4.6 (acetate buffer). The progress of the release was
monitored using a combination of UV/vis spectrometry and analyt-
ical HPLC, as illustrated in Figure 8. Conjugate 11 has a strong
absorption features at 300–380 nm when measured in the PBS
solution (Fig. 8a; see the UV/vis curve at t = 0 min). Such absorption
features are attributable to a weighted combination of three inde-
pendent chromophores comprising the folate ligand (280 nm,
e
Figure 9. A summary for the amount of MTX released plotted as a function of
exposure time at two different pH conditions. MTX was released from the
dendrimer-MTX conjugate 11 by exposure to 365 nm light, and its amount was
quantified by the AUC analysis for each of the HPLC traces shown in Figure 8.
= 25545 MÀ1 cmÀ1; 347 nm, = 6676 MÀ1 cmÀ1), the MTX, and
e
the ONB linker 2. The UV/vis time course for the photolysis at pH
7.4 is characterized by large increases in the absorbance around
300 nm and small increases above 350 nm. The large increases
were also observed in the photolysis of the MTX-linker conjugate4
that lacks the FA ligand (Fig. 4c). The photolysis reaction performed
at the acetate buffer (pH 4.6) led to smaller spectral changes at 280
and 350 nm (Fig. 8b). Thus, the rate of spectral changes suggests
the contribution of the pH effect on the rate of drug release.
We further determined the rate of MTX release by using the
HPLC results, as illustrated with the overlaid HPLC traces (Fig. 8).
The relative amount of MTX released was determined by the AUC
method, and it is plotted as a function of UV exposure time
( Fig. 9). At the end of the irradiation (15 min), the drug was

1288
S. K. Choi et al. / Bioorg. Med. Chem. 20 (2012) 1281–1290
Table 1
A summary for the rate of drug (ligand) release at three different pH conditions.
Compound
Molecule released
t1/2 (min) or relative ratea
pH 5 pH 7.4
MeOH
pH 9
2
4
11
H2O
MTX
MTX
Glycine
Carbamoylcholine
NMDA
8
8
15
P70
15
+
+
++
5
P70
na
++
++
630
na
na
na
na
630
>15
+++
+++
+
Glycine (N-ONB)53
Carbamoylcholine (N-ONB)54
NMDA (O-ONB)55
++++
a
Relative order: ++++ (faster) to + (slower); na: not available.
released at 47% (pH 7.4) and 34% (pH 4.6). The results indicate a
slightly faster release in the neutral condition from the PAMAM
dendrimer conjugate 11. This pH effect is the opposite of what
we observed with the MTX-linker molecule 4, where the drug re-
lease occurred faster under acidic conditions. These inconsistent
trends suggest that there is no direct correlation between the
MTX-release rate associated with the ONB-MTX molecule alone,
and its much larger, dendrimer-tethered form. Table 1 compares
photorelease profiles for various ONB-linked compounds studied
here at various pH conditions along with the trends reported for
other photocaged molecules53–55 in literature. The photolysis rates
appear to be pH dependent, but such dependency also varies by the
types of the compounds. This irregularity may be attributable to
the differential contribution of multiple variables that include: (i)
delivery applications in vitro or perhaps in vivo—in particular, those
therapeutic and diagnostic applications that require non-invasive or
spatiotemporal drug/probe activation.
4. Materials and methods
General synthetic methods, details of synthesis (1–4), and cop-
ies of their spectral data are provided in the Supplementary data.
4.1. Synthesis of PAMAM dendrimer conjugate 11 (Scheme 3)
Generation 5 PAMAM dendrimer was purchased as a 17.5%
(wt/wt) methanol solution (Dendritech, Inc., Midland, MI), and
purified by dialysis (MWCO 10 kDa) prior to use as described else-
where.5, 8,25, 47 The average number of primary amines per dendri-
mer molecule (#NH2 per dendrimer ꢁ 110) was determined by
potentiometric titration.5 Glutaric acid-derivatized dendrimer 10
was prepared as described earlier.8,25 The average number of glu-
taric acid molecules attached on the surface was determined by
the NMR method (Supplementary data, page S16) where the inte-
gration area for the proton signals of glutaric acid was compared
to that of select dendrimer protons.
Preparation of a fifth generation PAMAM dendrimer conjugated
with folic acid and MTX 11 was performed according to the dendri-
mer conjugation method described elsewhere.8,25 First, the glutaric
acid-derivatized dendrimer 10 (25 mg) was activated to its NHS es-
ter form by treatment with 4-dimethylaminopyridine (9 mg,
pH-dependent molar absorptivity (e) of the ONB-linked drug con-
jugate (e.g., pH 7 > pH 5 in Figure 8); (ii) the leaving group (drug-
released) effect53–55; and (iii) the pH-responsive configurational
changes of the PAMAM dendrimer.1,44 To our knowledge, such
mechanistic aspects have not yet been thoroughly investigated,
and remain to be better understood.
In summary, we demonstrated the photochemical mechanism
of MTX release with the G5 PAMAM dendrimer conjugated with
the photocaged MTX 11. The drug release was achieved at both a
neutral and an acidic condition, with a slightly greater rate at the
neutral condition. One specific aspect of the photochemical appli-
cations we are currently interested in is to investigate the mecha-
nism of drug action following cellular entry of nanoparticles
conjugated with MTX. Despite extensive investigations of these
nanoparticles as a delivery system to target cancer cells, the ther-
apeutic action of these conjugates following cellular entry is poorly
understood.1,5,17,28 In particular it is unclear whether the therapeu-
tic activity requires release of the MTX. This light-controlled ap-
proach for the drug release will provide evidence whether MTX
must be released to be fully able to inhibit the activity of its en-
zyme target DHFR (dihydrofolate reductase), and the growth of
cancer cells. We will report the results of this application in due
course.
73.8
ethyl-3-(3-dimethylaminopropyl)carbodiimide
(15 mg, 78.2
vation for 36 h at rt, FA-ethylernediamine46 (10; 1.5 mg, 3.0
mol), and triethylamine (4.3 L, 30.9 mol), each
l
mol), N-hydroxysuccinimide (9 mg, 78.2
lmol), and 1-
hydrochloride
lmol) in anhydrous DMF (10 mL) at rt. After preacti-
lmol),
4ÁTFA salt (9.2
l
l
l
dissolved in a minimal volume of DMF, were added to the preacti-
vated dendrimer solution. After stirring at rt for 36 h in the dark,
the reaction mixture was treated with ethanolamine (7.4
lL,
123 mol). After stirring for additional 2 h, the conjugation reac-
l
tion was quenched by adding water (2 mL), and the mixture was
concentrated in vacuo. The residue was diluted with water
(15 mL) and dialyzed by using membrane tubing (MWCO 10 kDa,
SpectrumÒ Labs, Inc.) against deionized water (4 L), a phosphate-
buffered saline solution (1 Â 4 L), and deionized water (3 Â 4 L)
over 3 days. The dialyzed solution contained in the tubing was col-
lected and lyophilized, yielding 11 as pale yellow fluffy solid
(19 mg). 1H NMR (400 MHz, D2O/DMSO-d6): d = 8.55 (br), 8.46
(s), 8.0–7.8 (br m), 7.60 (m), 7.55 (br), 7.05 (br), 6.70 (br m), 6.55
(br), 6.41 (br), 5.35 (br), 3.30 (m), 3.20–3.0 (br s), 2.7–2.5 (br m),
2.40–2.30 (br), 2.20–2.0 (br m), 1.60 (br) ppm. The number ratio
between FA and MTX attached per dendrimer was determined by
the analysis of 1H NMR signals for FA and MTX (d = 8.46,
6.7 ppm, respectively) and for the ortho-nitrobenzyl linker
(d = 7.05, 5.35 ppm): the ratio = [(number of MTX) Ä (number of
FA) = NMTX Ä NFA = 2.1]. An average molecular weight of 11 was
3. Conclusion
The present study describes an ONB-based linker strategy for the
light-controlled release of MTX. We demonstrated the release of
MTX from both an ONB-tethered MTX molecule, and a folate recep-
tor-targeting PAMAM dendrimer carrying the photocaged drug.
Certain factors are considered to be important for controlling the
drug release, including light wavelengths, exposure time, the sub-
stitution patterns of the photolabile ONB core, and the pH values
of the media where the drug release occurs. In this study, we dem-
onstrated that UV light serves as an effective trigger mechanism,
and it could be applied in an active manner orthogonal to other pas-
sive release approaches. We believe that this photochemical ap-
proach is generally applicable for other anticancer drugs and other

S. K. Choi et al. / Bioorg. Med. Chem. 20 (2012) 1281–1290
1289
determined by MALDI TOF on the basis of a peak intensity at m/
z = 54000 gmolÀ1. Increment in the molecular weight of 11 relative
monitored by UV/vis spectrometry. The analytical HPLC was also
performed for each aliquot (700 L) which was taken out at a spe-
cific time point as indicated in Figure 8.
l
to 10 (m/z = 40200 gmolÀ1 8
)
is attributed to the molecular weight
contributed from both FA, MTX, and hydroxyethylamine (HEA) at-
tached to 10:
(10) À 5800
D
wt (unit, gmolÀ1) = [54000 (11) À 40200
Acknowledgments
(HEA)] = 8000 = [(MWof
FA) Â NFA + (MW
of
4) Â NMTX] = [483 Â NFA + 735 Â NMTX]. The mean number for FA
and MTX attached per dendrimer was thus calculated by solving
the two equations: 4.1 (FA) and 8.2 (MTX).
This work was supported by the National Cancer Institute, Na-
tional Institutes of Health under award 1 R01 CA119409. The
author (S.K.C.) thanks Undergraduate Research Opportunity Pro-
gram (UROP) at University of Michigan for its support, and also
for the UROP fellowship (J.S.). We thank Mr. Ankur Desai for his
help in the HPLC analysis.
4.2. Photolysis experiments of 3 and 4 (Figs. 4 and 5)
Photolysis experiments were carried out using SpectrolineÒ UV
bench lamps (XX-15A; power = 1.1 mW/cm2), either at the UV-B
(312 nm) or UV-A (365 nm) wavelength. As a representative pho-
tolysis method, the linker 3 was dissolved in an aqueous medium
A. Supplementary data
Supplementary data (A full description of synthetic details for
compounds 1–4, copies of their 1H NMR, and mass spectra, full
1H NMR spectral traces for Figure S1 and 76, and copies of GPC
and MALDI TOF traces for 10–11.) associated with this article can
be found, in the online version, at doi:10.1016/j.bmc.2011.12.020.
(30 lM, 2% MeOH/H2O), and the solution (20 mL) was loaded onto
a glass Petri dish. The linker solution was exposed to the UV lamps
irradiated at the distance of ꢀ5 cm at 312 nm over up to 15 min.
Progress of the photolysis was monitored by UV/vis spectrometry,
and for the analysis, each aliquot (700 lL) was taken out during the
exposure at a specific time point as indicated in Figure 4. Photolysis
of the linker 4 was performed similarly in the same aqueous med-
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