Far-red light activatable, multifunctional prodrug for fluorescence optical imaging and combinational treatment

Article abstract of DOI:10.1021/jm5000722

We recently developed photo-unclick chemistry , a novel chemical tool involving the cleavage of aminoacrylate by singlet oxygen, and demonstrated its application to visible light-activatable prodrugs. In this study, we prepared an advanced multifunctional prodrug, Pc-(L-CA4)₂, composed of the fluorescent photosensitizer phthalocyanine (Pc), an SO-labile aminoacrylate linker (L), and a cytotoxic drug combretastatin A-4 (CA4). Pc-(L-CA4)₂ had reduced dark toxicity compared with CA4. However, once illuminated, it showed improved toxicity similar to CA4 and displayed bystander effects in vitro. We monitored the time-dependent distribution of Pc-(L-CA4)₂ using optical imaging with live mice. We also effectively ablated tumors by the illumination with far-red light to the mice, presumably through the combined effects of photodynamic therapy (PDT) and released chemotherapy drug, without any sign of acute systemic toxicity.

Full text of DOI:10.1021/jm5000722

Article
pubs.acs.org/jmc
Open Access on 04/02/2015
Far-Red Light Activatable, Multifunctional Prodrug for Fluorescence
Optical Imaging and Combinational Treatment
Moses Bio,† Pallavi Rajaputra,† Gregory Nkepang,†,‡ and Youngjae You*,†,‡
†Department of Pharmaceutical Sciences and ‡Department of Chemistry and Biochemistry, University of Oklahoma Health Sciences
Center, Oklahoma City, Oklahoma 73117, United States
S
* Supporting Information
ABSTRACT: We recently developed “photo-unclick chem-
istry”, a novel chemical tool involving the cleavage of
aminoacrylate by singlet oxygen, and demonstrated its
application to visible light-activatable prodrugs. In this study,
we prepared an advanced multifunctional prodrug, Pc-(L-
CA4)2, composed of the fluorescent photosensitizer phthalo-
cyanine (Pc), an SO-labile aminoacrylate linker (L), and a
cytotoxic drug combretastatin A-4 (CA4). Pc-(L-CA4)2 had
reduced dark toxicity compared with CA4. However, once
illuminated, it showed improved toxicity similar to CA4 and
displayed bystander effects in vitro. We monitored the time-
dependent distribution of Pc-(L-CA4)2 using optical imaging with live mice. We also effectively ablated tumors by the
illumination with far-red light to the mice, presumably through the combined effects of photodynamic therapy (PDT) and
released chemotherapy drug, without any sign of acute systemic toxicity.
Optical imaging is a valuable tool for tracking fluorescent or
luminescent molecules. The ability to optically image the light-
activatable prodrug is useful for determining an illumination
time when the prodrug is at its maximum concentration at the
target site. It can be utilized in real time at a low cost. If the
prodrug accumulates in the target sites, we can use optical
imaging to detect the target areas, as well as to treat the disease.
We expected that we could optically image our photo-
unclickable prodrug in vivo using a fluorescent photosensitizer.
Thus, we could track the prodrugs and then treat the tumor
with the combined effects of photodynamic therapy (PDT) and
local chemotherapy (Figure 1a). The released drug could
damage the cancer cells that survived the initial PDT damage
through bystander effects (Figure 1b).
We generated Pc-(L-CA4)2, an advanced multifunctioning
CA4 prodrug, for both fluorescence optical imaging and
combination therapy with PDT and released CA4. We chose
phthalocyanine (Pc) because Pc is a fluorescent photosensitizer
that can generate both fluorescence and singlet oxygen.12−14
Although fluorescence emission and SO generation are
competing processes, Pc has uniquely balanced yields of both
functionality (i.e., Si-Pc: Φ1O2 = 0.22 and Φf = 0.4) with a high
molar extinction coefficient (ε).15,16 Its brightness (BT) is
greater than that of CMP (e.g., ε = 150,000 M−1 cm−1 at 675
nm, BT = 6000 M−1 cm−1 for Pc vs ε = 5000 at 690 nm, BT =
50 M−1 cm−1 for CMP).17,18 We prepared Pc-(NCL-CA4)2 as
its pseudo-prodrug. This pseudo-prodrug is similar to Pc-(L-
INTRODUCTION
■
The use of visible and near-infrared (NIR) light, penetrable to
deep tissue, is an attractive method of spatiotemporally
controlling drug release from various drug delivery forms,
such as prodrugs, liposomes, polymers, and other nano- and
macrodelivery systems.1 However, because of its lower energy,
it is difficult to directly cleave a chemical bond using such light.
Thus, novel mechanisms using lower energy light to trigger the
release of biologically active compounds have been a major
topic of interest.2 The photodynamic process and the unique
chemistry of singlet oxygen (SO) were adopted to mediate
lower energy light release of drugs.3−11 SO is formed during the
photodynamic process and reacts with electron-rich olefins to
form unstable dioxetanes. These dioxetanes decompose to
release drugs.
Aminoacrylate was an ideal SO-cleavable linker.8 We named
the cleavage of aminoacrylate by SO “photo-unclick chemistry”,
and demonstrated the release of the anticancer drug
combretastatin A-4 (CA4) from its prodrug using this method.
We prepared CMP-L-CA4, a CA4 prodrug that can be activated
by far-red light (690 nm), by combining a SO-labile
aminoacrylate linker, a core-modified porphyrin (CMP)
photosensitizer, and CA4.10 The prodrug released CA4 and
enhanced cytotoxicity upon illumination. The released CA4
damaged cancer cells, demonstrating our system’s ability to
generate bystander effects in vitro. We found that the antitumor
effects of CMP-L-CA4 were superior to the effects of its
pseudo-prodrug CMP-NCL-CA4 (NCL = noncleavable linker).
The pseudo-prodrug cannot release CA4 even after illumina-
tion in vivo.
Received: January 14, 2014
Published: April 2, 2014
© 2014 American Chemical Society
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potential = 11.64
1.38 mV, 16.81
1.67 mV and mean
diameter = 71.96 1.34 nm, 75.07 1.45 nm, respectively).
To visualize the formation of the polymeric micelles, we used
transmission electron micrographs (TEM). TEM images of the
micelles showed consistent particle sizes (61−78 nm for Pc-(L-
CA4)2 and 65−80 nm for Pc-(NCL-CA4)2 micelles). The
prodrug concentrations in the micelles were 211 and 210 μM,
respectively. The stability of the micelles was monitored by
their particle sizes and zeta potentials at 4 °C under dark
conditions. These values remained within 95% of the initial
values for up to 21 days.
Effects of Pc-(L-CA4)2 and Pc-(NCL-CA4)2 on Tubulin
Polymerization. CA4 is known to inhibit tubulin polymer-
ization by binding to the colchicine binding pocket of
tubulin.24,25 Because the bulky groups (Pc-L and Pc-NCL)
are attached to CA4, we expected lower inhibitory activity of
tubulin polymerization. We determined the effects of these
prodrugs using the tubulin polymerization assay, in which
fluorescence emission increases as tubulins polymerize (Figure
3a). The polymerization enhancer PCX and polymerization
inhibitor CA4 were used as positive controls. Consistent with
our data on the previous CA4 prodrug CMP-L-CA4,10 both Pc-
(L-CA4)2 and Pc-(NCL-CA4)2 had significantly (p < 0.02)
lower inhibitory activity (23% and 17%, respectively) than the
parent drug CA4 (100%, Figure 3b).
Dark and Phototoxicity. Due to the dramatic reduction of
the inhibition of tubulin polymerization, it was expected that
Pc-(L-CA4)2 and Pc-(NCL-CA4)2 would have lower dark
toxicity (cytotoxicity without illumination) than CA4. Using
MCF-7 cells, we found that the dark toxicity of the prodrugs
decreased by 19- and 101-fold [IC50D values = 9, 173, and 916
nM for CA4, Pc-(L-CA4)2 and Pc-(NCL-CA4)2, respectively].
Pc-L and Pc-NCL reduced the cytotoxicity, presumably by
interfering with its binding to tubulin (Figure 3). However,
illumination enhanced the toxicity of both the prodrug and the
pseudo-prodrug [IC50P = 6 nM and 34 nM for Pc-(L-CA4)2
and Pc-(NCL-CA4)2, respectively].
Bystander Effects by Pc-(L-CA4)2. The difference in the
magnitudes of phototoxicity and dark toxicity effects may be
the result of each prodrug using a different mechanism. While
Pc-(NCL-CA4)2 can only kill cells via the PDT effects of SO,
Pc-(L-CA4)2 can potentially kill cells by both PDT effects and
released CA4. To prove this mechanistic difference, we tested
the bystander effects after the illumination. After treating the
cells in wells of 24-well plates with the prodrug or pseudo-
prodrug, one-half of each well was exposed to light. At 48 h
post-illumination, live cells were stained with Calcein AM, and
the center of each well was imaged using a fluorescence
microscope.
Because the lifetime of SO in aqueous medium/biological
systems is very short (∼40 ns),26 SO generated in the
illuminated half of the well cannot kill the cells in the
unilluminated half. SO should decay before reaching the other
half of the well (diffusion distance of SO = ∼20−200 nm).27,28
However, the released CA4 can induce bystander effects
because it can diffuse to the unilluminated half of the well. As
we expected, bystander effects were found in the Pc-(L-CA4)2-
treated wells: cells in the nonilluminated side were damaged as
much as cells in the illuminated side (Figure 5b). However,
wells treated with Pc-(NCL-CA4)2 had cell damage only in the
illuminated halves of the wells (Figure 5c). This clearly
supported our hypothesis that illuminated Pc-(L-CA4)2 releases
Figure 1. (a) Multifunctional prodrug for optical imaging and
combined treatment with PDT and local chemotherapy. (b) Bystander
effects from the released drugs can kill cancer cells that survive PDT
damage. [The lifetime of SO is short (submicrosecond scale). Thus,
direct cell damage by SO occurs only during illumination. The light
dose used for imaging will be much lower than the light dose used for
treatment. Thus, we do not expect any significant damage during
optical imaging.]
CA4)2 in structure, but cannot release CA4 upon illumination.
It will mimic the PDT effects of Pc-(L-CA4)2, but cannot
induce damage from released CA4. We evaluated the cytotoxic
effects of these two prodrugs with and without illumination, the
inhibition of tubulin polymerization, the in vitro bystander
effects, tumor localization using optical imaging, and the
antitumor effects.
RESULTS AND DISCUSSION
■
Synthesis. We developed a synthetic scheme using high-
yield reactions, such as esterification, nucleophilic substitution,
and the yne-amine reaction, to make the process easily
adaptable to other alcohol-containing drugs (Scheme 1). CA4
was esterified at room temperature to yield compound 1.
Alkylation of CA4 gave compound 2. A nucleophilic
substitution reaction of silicon phthalocyanine dichloride (Pc-
Cl2) yielded compound 3. Pc-(L-CA4)2 was synthesized
through a click (yne-amine) reaction of compounds 1 and 3.
Under the basic condition, Pc-(NCL-CA4)2 was synthesized by
N-alkylation of compounds 2 and 3. Overall, the synthesis was
straightforward and all steps gave high yields (>73% each step).
Formulation in PEG−PLA Polymeric Micelle. We
formulated the prodrugs using PEG−PLA [poly(ethylene
glycol)-poly(D,L-lactide)] copolymer micelles to take advantage
of the enhanced permeability and retention (EPR) effect to
enhance the delivery to tumor.19 The nanosized PEG−PLA
polymer micelle was expected to provide three advantages: (1)
passive targeting to tumors via the EPR effect,20,21 (2)
prolonged circulation in the plasma, and (3) solubilization of
the nonpolar prodrug. The biodegradable and nontoxic PEG−
PLA micelle of paclitaxel (PCX) was approved by the FDA.22,23
PEG−PLA polymer micelles of Pc-(L-CA4)2 and Pc-(NCL-
CA4)2 were readily prepared. The zeta potentials and mean
diameters of the micelles of Pc-(L-CA4)2 and Pc-(NCL-CA4)2
were determined by dynamic light scattering (DLS) (zeta
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a
Scheme 1. Synthesis of Pc-(L-CA4)2 and Pc-(NCL-CA4)2
a
Reagents and conditions: (i) propynoic acid, DCC, DMAP, CH2Cl2, room temp, 24 h; (ii) 1,3-dibromopropane, anhydrous K2CO3, acetone, reflux,
12 h; (iii) 2-(piperazin-1-yl)ethanol, pyridine, toluene, reflux, 4 h; (iv) 1, anhydrous THF, 30 min; (v) 2, anhydrous K2CO3, acetone, reflux, 12 h.
Figure 2. (a) Particle size distribution and TEM images (inset) of micelles of (a) Pc-(L-CA4)2 and (b) Pc-(NLC-CA4)2.
CA4 that can induce bystander effects, which is consistent with
data from our previous study with CMP-L-CA4.10
to tumors by EPR effects, we prepared PEG−PLA polymer
micelles of Pc-(L-CA4)2 or Pc-(NCL-CA4)2. As a control
formulation, we also made solutions of these prodrugs in 5%
Cremophor EL, which does not produce EPR effects. Balb/c
mice with SC tumors (colon-26 cells, 4−6 mm in length) were
injected retro-orbitally with 2 μmol/kg of the prodrug. Then,
the mice were imaged at various postinjection time points
Preclinical Optical Imaging in Live Mice. One of our
major goals was to make Pc-(L-CA4)2 detectable in tissues
using fluorescence optical imaging. We expected that the
fluorescent photosensitizer Pc would provide sufficient
fluorescence emission for this goal. To deliver the prodrugs
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Because the illumination for optical imaging could
theoretically generate SO and thus release CA4 from Pc-(L-
CA4)2, we monitored the body weight change and tumor
growth pattern of the mice imaged with Pc-(L-CA4)2. We did
not see any significant impact on mouse body weight or tumor
growth since the light dose used for imaging was negligible
[675 filter (660−690 nm) at ∼1.5 μW/cm2 for 2 s (3.0 × 10−6
J/cm2)]. The treatment used a (1.2 × 108)-fold higher light
dose than the dose used for imaging.
Antitumor Efficacy. We expected that the new prodrug Pc-
(L-CA4)2 would show better antitumor effects than our
previous prodrug CMP-L-CA4, because Pc has superior light
absorption properties and the prodrug releases two CA4
instead of one. We used BALB/c mice with SC tumors to
evaluate the antitumor effects of the prodrugs with illumination.
Twenty-four hours post-retro-orbital injection of 1 or 2 μmol/
kg of the prodrug, the tumor was illuminated by a 690 nm
diode laser for 30 min at 100 mW/cm2 (180 J/cm2) or 200
mW/cm2 (360 J/cm2). These treatment conditions were
chosen based on data from pilot studies. Six groups were
used to assess the antitumor effects of the prodrug and pseudo-
prodrug: G1, negative control; G2, [CA4 (1 μmol/kg) + no
hv]; G3, [Pc-(NCL-CA4)2 (1 μmol/kg) + hv (100 mW/cm2)];
G4, [Pc-(L-CA4)2 (1 μmol/kg) + hv (100 mW/cm2)]; G5,
[Pc-(L-CA4)2 (2 μmol/kg) + hv (100 mW/cm2)]; and G6,
[Pc-(L-CA4)2 (2 μmol/kg) + hv (200 mW/cm2)]. Antitumor
effects were monitored by measuring tumor volume (Figure 7a
and b). [The tumor growth curves of G5 and G6 were nearly
identical to G4 until day 15. So, these curves were omitted from
Figure 7a for clarity (Figure S2).]
We found outstanding antitumor effects in the mice treated
with Pc-(L-CA4)2, G4−G6. After 24 h illumination, all tumors
shrank to a nonmeasurable size and remained so for almost 15
days (Figure 7a). Mice in group G4 experienced tumor growth
only after day 16 (Figure S2). All mice in G4−G6 lived until
day 30, while tumor size of all 4 mice in the control group (G1)
reached >800 mm3 in 12 days (Figure 7b). The PDT effects
resulting from Pc-(NCL-CA4)2 treatment (G3) had a
significant impact on tumor size until day 3 (p < 0.05).
However, after day 3, the tumors grew back at a rate similar to
the tumors in the control group G1. Throughout the
observation period, Pc-(L-CA4)2 treatment yielded significantly
better antitumor effects (p < 0.01), than Pc-(NCL-CA4)2
treatment. Thus, it seemed that the PDT effects alone might
not be sufficient to produce such a robust antitumor effect as
seen in group G4. We hypothesized that our findings stemmed
from the contribution of the released CA4 in addition to PDT
effects. Interestingly, no mice in G1−G6 experienced a
significant decrease in body weight (Figure S3).
Figure 3. Effects of 3 μM each of PCX, CA4, Pc-(L-CA4)2, and Pc-
(NCL-CA4)2 on tubulin polymerization: (a) one data set of
representative kinetic traces (data from two more experiments are
found in Figure S1, Supporting Information) and (b) inhibition of
tubulin polymerization by CA4, Pc-(L-CA4)2, and Pc-(NCL-CA4)2
after 1 h incubation ( SD of three experiments).
Figure 4. Dark and phototoxicity of Pc-L-(CA4)2 and Pc-NCL-
(CA4)2.
The histological data were consistent with the antitumor
effects (Figure 7c). After 24 h of treatment, tumors were
collected and stained with H&E to visualize the tissue damage.
While mice treated with Pc-(NCL-CA4)2 experienced tissue
damage only on the skin, mice receiving Pc-(L-CA4)2 also
experienced direct tumor damage. In fact, the volume of tumors
treated with Pc-(L-CA4)2 shrank to about 1/8 of the volume of
the tumors in the control group.
(Figure 6 and Figure S4). As anticipated, we could clearly see
the fluorescence emissions from the two prodrugs in live mice.
The imaging data revealed two important findings. First, the
images from the mice that received the polymer micelles of
both prodrugs showed “hot spots” in tumors, with a peak at
around 24 h postinjection, presumably due to the EPR effects
of the nanosized polymer micelles. These hot spots were more
evident in the mice who received injections of Pc-(L-CA4)2.
Second, the “hot spots” resulting from formulations with the
polymer micelles persisted longer than those “hot spots”
resulting from formulations with Cremophor solutions. The
polymer micelles appeared to delay the clearance of the
prodrugs from the system.
CONCLUSION
■
We successfully demonstrated a multifunctional photounclick-
able prodrug that can be visualized by optical imaging, and
ablates tumors with a combination of PDT and local
chemotherapy. The prodrug Pc-(L-CA4)2 and its pseudo-
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Figure 5. Fluorescence live cell images of the center of each well treated with (a) vehicle (diluting solution without a prodrug), (b) 25 nM Pc-(L-
CA4)2, and (c) 25 nM Pc-(NCL-CA4)2. The left half of each well was illuminated with a 690 nm diode laser (11 mW/cm2 for 15 min). At these
concentrations, Pc-(L-CA4)2 and Pc-(NCL-CA4)2 did not produce any significant dark toxicity.
Figure 6. Fluorescence optical images of the mice after retro-orbital injection of 2 μmol/kg of prodrug: (a) Pc-(L-CA4)2 in polymer micelles, (b) Pc-
(NCL-CA4)2 in polymer micelles, (c) Pc-(L-CA4)2 in 5% Cremophor solution, and (d) Pc-(NCL-CA4)2 in 5% Cremophor solution. BG:
background image before prodrug injection. Scale bar unit: fluorescence arbitrary unit.
prodrug Pc-(NCL-CA4)2 were prepared in high yields through
a facile and flexible scheme. The cytotoxicity of these prodrugs
was lower than that of the parent drug CA4, but both prodrugs
showed enhanced cytotoxicity upon illumination.
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sensitizer within the prodrug. Using optical imaging, we were
able to noninvasively generate PK information about the
prodrug without causing any observable acute toxicity to mice.
Our multifunctional prodrug strategy includes (1) activation by
the clinical translatable far-red light (or NIR), (2) the unique
combination of PDT and local chemotherapy, and (3) the dual
function of optical imaging and treatment with one prodrug.
We anticipate that this strategy will be applicable to various
drug delivery forms, clinically approved drugs, and advanced
drug delivery systems targeted at tumors.
EXPERIMENTAL SECTION
■
Synthesis. CA4,29 compound 1,9 and compound 210 were
synthesized as reported previously. The purity of the biologically
evaluated compounds Pc-(L-CA4)2 and Pc-(NCL-CA4)2 was
confirmed to be >95% by high-performance liquid chromatography
(HPLC) (Figures S9 and S10).
Compound 3. 1-(2-Hydroxyethyl)-piperazine (1.91 g, 14.70 mmol)
and pyridine (2.5 mL) were added to a solution of silicon(IV)
phthalocyanine dichloride (Pc-Cl2, 1 g, 1.63 mmol) in 50 mL toluene.
The reaction mixture was refluxed for 12 h. The solvent was then
removed under reduced pressure. The residue was dissolved in
CH2Cl2 and washed with water. The solvent of the combined organic
layers was removed by evaporation, and the crude was recrystallized
with CHCl3/n-hexane (1:4 v/v) to give a blue solid compound 3 (1.22
1
g, 94%). H NMR (300 MHz, CD2Cl2) δ −1.19 (t, J = 5.8 Hz, 4H),
−0.82 (t, J = 5.8 Hz, 1H), 0.28 (m, 8H), 1.75 (m, 8H), 8.30−8.40 (m,
8H, Pc-Hβ), 9.55−9.69 (m, 8H, Pc-Hα); 13C NMR (300 MHz,
CD2Cl2) δ 123.4, 131.0, 135.9, 149.2; HRMS ESI (m/z): [M + H]+
calculated for C44H43N12O2Si, 799.3401; found, 799.3395.
Pc-(L-CA4)2. Compound 1 (46 mg, 0.13 mmol) and compound 3
(50 mg, 0.06 mmol) were dissolved in 20 mL dry THF, and the
solution was stirred at room temperature for 15 min. The solvent was
removed under reduced pressure to yield the crude product, which was
then recrystallized from CHCl3/n-hexane (1:5 v/v) to give Pc-(L-
Figure 7. Antitumor effects. (a) Tumor growth curves, drug IV
administration: once a day on day −1, illumination 24 h postdrug
administration [hv#: 100 mW/cm2 for 30 min (180 J/cm2) or 200
mW/cm2 for 30 min (360 J/cm2)], 5 mice per group except the
control group (4 mice). Error bars represent SE. In inset: the order of
tumor size was G1 > G3 > G4 during day 1 to day 4 [** p < 0.01 (G1
vs G3, from day 1 to day 4) and ## p < 0.01 (G3 vs G4, from day 1 to
day 4)]. (b) Kaplan−Meier plot of response to treatment. (c) H&E
staining of tumors 24 h post-illumination from (i) control mice or
mice treated with (ii) Pc-(NCL-CA4)2, or (iii) Pc-(L-CA4)2. Prodrug
administration and illumination conditions were same as for those in
(a). VT, viable tumor; DT, damaged tumor; and DS, damaged skin.
1
CA4)2 (83 mg, 87%). H NMR (300 MHz, CD2Cl2)) δ −1.96 (t, J =
5.6 Hz, 4H), −0.55 (t, J = 5.0 Hz, 4H), 0.29 (br s, 8H), 2.20 (br s,
8H), 3.67 (s, 12H), 3.72 (s, 6H), 3.77 (s, 6H), 4.34 (d, J = 12.8 Hz,
2H), 6.45 (d, J = 4.9 Hz, 4H), 6.62 (s, 4H), 6.84 (d, J = 8.1 Hz, 2H),
6.96 (s, 2H), 7.00 (s, 2H), 7.09 (d, J = 12.8 Hz, 2H), 8.38−8.43 (m,
8H, Pc-Hβ), 9.65−9.70 (m, 8H, Pc-Hα); 13C NMR (300 MHz,
CD2Cl2) δ 55.8, 56.6, 60.4, 81.8, 105.9, 111.8, 123.5, 123.9, 126.6,
128.7, 129.1, 129.8, 131.2, 132.5, 135.8, 137.2, 140.3, 149.3, 151.1,
152.2, 153.0, 167.2; HRMS ESI (m/z): [M + H]+ calculated for
C86H83N12O14Si, 1535.5921; found, 1535.5914.
Pc-(NCL-CA4)2. Anhydrous K2CO3 (68 mg, 0.500 mmol) and
compound 3 (200 mg, 0.25 mmol) were added to a solution of
compound 2 (218 mg, 0.50 mmol) in 10 mL dry DMF. The reaction
mixture was stirred at room temperature for 12 h. The K2CO3 was
removed by suction filtration and the solvent was removed under
reduced pressure. The crude product was then recrystallized from
CHCl3/n-hexane (1:5 v/v) to give Pc-(NCL-CA4)2 (302 mg, 80%).
1H NMR (300 MHz, CD2Cl2) δ −1.96 (t, J = 5.6 Hz, 4H), −0.78 (t, J
= 5.8 Hz, 4H), 0.41 (br s, 8H), 1.50 (m, 2H), 1.90 (m, 2H), 2.18 (m,
2H), 2.49 (m, 2H), 3.32 (s, 4H), 3.60 (br s, 8H), 3.65 (s, 12H), 3.75
(s, 6H), 3.80 (s, 6H), 6.46 (m, 4H), 6.51 (m, 4H), 6.74 (m, 2H), 6.81
(m, 2H), 6.92 (m, 2H), 8.38 (m, 8H, Pc-Hβ), 9.66 (m, 8H, Pc-Hα);
13C NMR (300 MHz, CD2Cl2) δ 55.8, 60.4, 105.1, 111.4, 113.6, 115.2,
117.5, 123.4, 126.7, 126.8, 126.9, 130.9, 132.8, 135.9, 149.1; HRMS
ESI (m/z): [M + H]+ calculated for C86H91N12O12Si, 1511.6649;
found, 1511.6612.
Preparation of Pc-(L-CA4)2 and Pc-(NCL-CA4)2 Micelles. Briefly, 3
mg of Pc-(L-CA4)2 or Pc-(NCL-CA4)2 was dissolved in 1.3 mL of
THF. Ten mg of mPEG−PLA (cat #AK09, vendor: Polyscitech) was
dissolved in 1 mL of THF. 600 μL of the 3 mg of Pc-(L-CA4)2 or Pc-
(NCL-CA4)2 dissolved in 1.3 mL THF was added to the mPEG-PLA-
THF mixture. The volume of the resulting mixture was reduced to 300
μL under reduced pressure. The 300 μL of the mixture of Pc-(L-CA4)2
The mechanisms of cell damage of Pc-(L-CA4)2 and Pc-
(NCL-CA4)2 combined with illumination should be different.
While the use of Pc-(NCL-CA4)2 and illumination killed cancer
cells through PDT effects, the use of Pc-(L-CA4)2 and
illumination combined these PDT effects with local chemo-
therapy through the released CA4. This was supported by the
bystander effects demonstrated in vitro. Through the use of
optical imaging, we found that both prodrugs were detected at
the therapeutic dose within tumors. Optical imaging also
provided the information about the PK profiles of the prodrugs
so that we could find the optimal time point for illumination.
As expected, the antitumor effects in mice treated with Pc-(L-
CA4)2 were dramatically better than in mice treated with Pc-
(NCL-CA4)2. Treatment with either Pc-(NCL-CA4)2 or CA4
produced minimal antitumor effects, suggesting that the
outstanding antitumor effects of Pc-(L-CA4)2 may have been
a result of the synergistic effects of PDT and chemotherapy.
In addition to confirming that our current SO-activatable
prodrug strategy provides improved antitumor efficacy, we
demonstrated the innovative use of a fluorescent photo-
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or Pc-(NCL-CA4)2 and mPEG−PLA was added to 3 mL of distilled
water dropwise while stirring. The mixture was stirred for 3 h, after
which the organic solvent was evaporated under reduced pressure at
40 °C. The resultant solution was filtered through a 0.2 μm filter. The
concentration of Pc-(L-CA4)2 or Pc-(NCL-CA4)2 micelles was
determined by diluting the micelles in THF: the absorbance was
measured by absorbance of Pc group. The concentration was
calculated from the molar extinction coefficient (EC) of Pc-(L-
CA4)2 or Pc-(NCL-CA4)2 at 675 nm in THF (EC of Pc-(L-CA4)2 =
205,000; Pc-(NCL-CA4)2 = 206,110 M−1 cm−1) using the Beer−
Lambert law. The concentrations of Pc-(L-CA4)2 or Pc-(NCL-CA4)2
micelles were determined to be 211 and 210 μM, respectively. Freshly
prepared Pc-(L-CA4)2 and Pc-(NCL-CA4)2 micelles were used for all
the experiments.
Cremophor Solution. 2.0 mM stock solutions of Pc-(L-CA4)2 and
Pc-(NCL-CA4)2 prepared in THF were further diluted with 5%
Cremophor EL in PBS to achieve appropriate concentrations.
Characterization of Pc-(L-CA4)2 and Pc-(NCL-CA4)2 Micelles. The
Pc-(L-CA4)2 and Pc-(NCL-CA4)2 micelles in an aqueous solution
were characterized by measuring their hydrodynamic diameter and
zeta potential via dynamic light scattering (DLS). The size
measurement was carried out at a concentration of 1.0 mg/mL of
Pc-(L-CA4)2 or Pc-(NCL-CA4)2. Pc-(L-CA4)2 and Pc-(NCL-CA4)2
micelles were also imaged by transmission electron microscopy
(TEM) at 20,000× operating at 80 kV. TEM samples were prepared
by depositing 20 μL of diluted Pc-(L-CA4)2 and Pc-(NCL-CA4)2
micelle solution on a 300 mesh copper TEM grid with a carbon film.
The sample grid was air-dried before the measurements were taken.
Tubulin Polymerization Assay. A fluorescence-based tubulin
polymerization assay was conducted using a kit supplied by
Cytoskeleton, Inc. (cat # BK011P). The basic principle is that an
increase in fluorescence will occur as a fluorescence reporter is
incorporated into microtubules during the course of polymerization.
The assay was performed following the experimental procedure
described in version 2.1 of the tubulin polymerization assay kit manual.
Briefly, a drug in DMSO stock solution was added to a mixture of
tubulin and GTP in a buffer solution, to give a final concentration of 3
μM. The reaction mixture was incubated at 37 °C. Fluorescence was
monitored (excitation = 360 nm and emission = 450 nm) every 2 min
for 1 h. PCX and CA4 were included in the assay as positive controls,
as well as a vehicle-only negative control.
Dark and Phototoxicity. The cytotoxicity of Pc-(L-CA4)2 and Pc-
(NCL-CA4)2 was determined with and without illumination. MCF-7
cells were maintained in minimum essential medium (α-MEM)
supplemented with 10% bovine growth serum, 2 mM L-glutamine, 50
units/mL penicillin G, 50 μg/mL streptomycin, and 1.0 μg/mL
fungizone. MCF-7 cells (5000 cells/well) were seeded on 96-well
plates in the medium and incubated for 24 h at 37 °C in 5% CO2.
Stock Pc-(L-CA4)2 and Pc-(NCL-CA4)2 micelle solutions (200 μM)
were prepared in distilled water. The stock solutions were further
diluted with medium to obtain the necessary final concentrations. The
diluted solution (10 μL) was then added to each well (190 μL). The
plates were incubated for 24 h and then removed from the incubator.
For the phototoxicity study: The uncovered plate was illuminated for
30 min using a diode laser (690 nm, 5.6 mW/cm2). To ensure
uniformity of the light during the illumination, each plate was shaken
gently on an orbital shaker (Lab-line, Barnstead International). For the
dark toxicity study: Plates were kept in the dark for 30 min and then
returned to the incubator. After 3 days, cell viability was determined
using an MTT assay. Briefly, a 10 μL solution of MTT (10 mg in 1 mL
PBS buffer) was added to each well and the plate was incubated for 4 h
at room temperature or 37 °C. Then, the MTT solution was removed
and the cells were dissolved in 200 μL of DMSO. The absorbance of
each well was measured at 570 nm with background subtraction at 650
nm. Cell viability was quantified by measuring the absorbance of the
treated wells compared to that of the untreated control wells, and
expressed as a percentage.
water. 200 μM of the stock solutions were added to wells (1 mL) to
obtain appropriate final concentrations of both Pc-(L-CA4)2 and Pc-
(NCL-CA4)2 at 25 nM. After 24 h incubation, the plates were
illuminated from the bottom with a 690 nm diode laser at 11 mW/cm2
for 15 min. During illumination, half of each well was blocked with
black masking tape (cat# T743-1.0, vendor: Thorlabs). The
illuminated plates were incubated for an additional 48 h. Then, a
Calcein AM live cell staining assay (cat # 4892-010-K, vendor:
Molecular, Probes Tervigen) was performed. The cells were washed
once with 1 mL Calcein AM wash buffer, then 250 μL fresh wash
buffer and 250 μL working reagent were added to the wells. The cells
were incubated for 30 min. Fluorescent images were obtained with an
Olympus IX51 inverted microscope with a green fluorescence channel
to visualize live cells. All images were taken at 10× magnification.
In Vivo Optical Imaging. The concentrations of Pc-(L-CA4)2 and
Pc-(NCL-CA4)2 micelles in aqueous solution were determined by
diluting 10 μL of the formulation stock in 1 mL of THF and
measuring the absorbance of phthalocyanine. The concentration was
calculated from the EC of Pc-(L-CA4)2 at 672 nm in THF (EC of Pc-
(L-CA4)2 = 205,000; Pc-(NCL-CA4)2 = 206,110 M−1 cm−1) using the
Beer−Lambert law.
We used four- to six-week-old BALB/c mice to investigate the
biodistribution and tumor targeting ability of the polymeric micelles.
The mice were shaved before the imaging experiments, and were
imaged using the IVIS Imaging system. The mice were injected with
Pc-(L-CA4)2 and Pc-(NCL-CA4)2 in the micelle formulation (2
μmol/kg, i.v.). As a comparison, the prodrugs were also evaluated in
the Cremophor solution. Fluorescence images were taken 0, 3, 6, 12,
24, 48, and 72 h after retro-orbital injection. Before taking the images,
the mice were anesthetized in an acrylic chamber with a 2.5%
isoflurane/air mixture. The following parameters were used to acquire
images with Living Image software: fluorescence mode, exposure time:
2 s, binning: medium, F/Stop: 2, excitation: 675 filter (660−690 nm),
and emission: 720 filter (710−730 nm). During post processing, image
counts were adjusted to 3 × 104 as minimum and 6.0 × 104 a.u. as
maximum color scale.
Antitumor Efficacy Study. Four- to six-week-old BALB/c mice
(18−20 g) were used for the murine tumor model. The mice were
implanted SC with 2 × 106 colon 26 cells in PBS (100 μL) on the
lower back of the neck. Tumor growth was monitored using digital
calipers. The longest axis of the tumor (1) and the axis perpendicular
to l (w) were used to calculate tumor volume (lw2/2). Mice with
tumors 5−6 mm in diameter were used for the experiments.
We used stock solutions of Pc-(L-CA4)2 and Pc-(NCL-CA4)2
micelles in aqueous solution and further dilutions to achieve final
doses as follows: [CA4, (1 μmol/kg each)], [Pc-(NCL=CA4)2, (1
μmol/kg each) ], [Pc-(L-CA4)2, (1 μmol/kg each)], and [Pc-(L-
CA4)2, (2 μmol/kg each)]. To each mouse, 200 μL of sample was
injected via IV once on day −1. Twenty-four hours later mice were
anesthetized by IP injection of 80 mg/kg ketamine and 6 mg/kg
xylazine. Tumors were illuminated with a 690 nm diode laser at 100
mW/cm2 or 200 mW/cm2 for 30 min −180 or 360 J/cm2, respectively.
Tumor size was measured every day after the treatment.
Histology Study (H&E Staining). To evaluate antitumor effect, mice
from various groups were euthanized 24 h after laser illumination and
tumors were collected. The specimens were fixed in 10% buffered
formalin, embedded in paraffin, and ∼4 mm diameter tissue sections
were stained with hematoxylin and eosin following a standard
procedure at the tissue pathology core facility at OUHSC. The
sections were viewed and photographed by bright-field microscopy at
4× magnification.
Statistical analysis. Student’s t test was used for statistical analysis.
P values less than 0.05 were considered significant.
ASSOCIATED CONTENT
■
S
* Supporting Information
Supporting Information includes detailed descriptions of
general experimental conditions, additional tubulin polymer-
ization data, extended tumor growth curves, body weight
Bystander Effect. Colon-26 cells were seeded at 5000 cells/well on
24-well plates and incubated for 24 h. Stock Pc-(L-CA4)2 and Pc-
(NCL-CA4)2 micelle solutions (200 μM) were prepared in distilled
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Journal of Medicinal Chemistry
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AUTHOR INFORMATION
Corresponding Author
*Tel: 1-405-271-6593, ext. 47473. E-mail: youngjae-you@
ouhsc.edu.
■
Author Contributions
M. B. and Y. Y. designed the project and wrote the manuscript.
M.B. and G.N. prepared the prodrugs, and M.B. and P.R.
carried out the biological evaluations of the compounds.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The helpful discussion with Drs. Michael Ihnat and Sukyung
Woo are appreciated. We thank the Peggy and Charles
Stephenson Cancer Center at the University of Oklahoma
Health Sciences Center, Oklahoma City, OK, and an
Institutional Development Award (IDeA) from the National
Institute of General Medical Sciences of the National Institutes
of Health under grant number P20 GM103639 for the use of
the Molecular Imaging Core facility that provided services for
the optical in vivo imaging and Cancer Tissue Pathology Core
facility that provided services for the H&E staining. We also
thank the Oklahoma Medical Research Foundation imaging
core facility that provided the services for the transmission
electron micrographs. This research was supported by the DoD
[Breast Cancer Research Program] under Award Number
W81XWH-09-1-0071. Views and opinions of and endorse-
ments by the authors do not reflect those of the U.S. Army or
the DoD.
ABBREVIATIONS
■
CA4, combretastatin A-4; DCC, N,N′-dicyclohexylcarbidiimide;
DMAP, 4-dimethylaminopyridine; DS, damaged skin; DT,
damaged tumor; Pc, silicon (iv) phthalocyanine; L, amino-
acrylate linker; NCL, noncleavable linker; PDT, photodynamic
therapy; PEG, poly(ethylene glycol); PLA, ploy(D,L-lactide);
ROS, reactive oxygen species; SO, singlet oxygen; VT, viable
tumor
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