Mitochondria-targeted aggregation induced emission theranostics: Crucial importance of: In situ activation

Article abstract of DOI:10.1039/c6sc02236g

Tissue selective targeting and specific suborganellular localization combined with an efficient pathology associated enzymatic activation of drugs in drug delivery systems may exhibit a clear advantage over conventional cancer treatment. Here, a mitochond

Full text of DOI:10.1039/c6sc02236g

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DOI: 10.1039/C6SC02236G
Journal Name
ARTICLE
Mitochondria‐Targeted Aggregation Induced Emission
Theranostics: Crucial Importance of In Situ Activation
Received 00th January 20xx,
Accepted 00th January 20xx
Weon Sup Shin,‡a Min Goo Lee,‡b Peter Verwilst,‡a Joung Hae Lee,c Sung Gil Chi,*b and Jong Seung
Kim,*a
DOI: 10.1039/x0xx00000x
Tissue selective targeting and specific suborganellular localization combined with an efficient pathology associated
enzymatic activation of drugs in drug delivery systems may exhibit a clear advantage over conventional cancer treatment.
www.rsc.org/
Here,
a mitochondria targeted aggregation induced emission (AIE) fluorophore further conjugated with an
NAD(P)H:quinone oxidoreductase‐1 (NQO1) cleavable masking unit showed preferential uptake in cancer cells and was
selectively activated, resulting in bright AIE fluorescence and apoptosis via the caspase pathway, triggered by
mitochondrial dysfunction. In vivo experimental data further support the conclusions from in vitro experiments, clearly
showing the dependence of the therapy’s success on both the suborganelle localization and specific in situ activation. And
the site specific and enzyme dependent activation and aggregation was further supported by in vivo and ex vivo imaging.
As a whole, the data comprised in this work represent a strong argument for the further development of this type of novel
anticancer drugs.
mitochondria exhibit intricately interwoven signal transduction
pathways, crucial to the survival and metastatic potential of
cancer cells.4 Therefore, interrupting the metabolism of this
Introduction
Despite astounding advances in the field of cancer treatment
over the last decades, cancer therapeutics, often exhibiting
suboptimal narrow therapeutic indexes, are inherently
associated with severe side effects due to a lack of specificity
for cancerous tissues and treatment often results in the
development of drug resistance, as cancer cells are likely to be
repeatedly exposed to non‐lethal concentrations of drugs
during the course of cancer treatment.1
In a quest to develop more specific cancer drugs, thus
diminishing side effects and widening the therapeutic window
of the drugs, targeted drug delivery has emerged as an
efficient way to deliver drugs, utilizing specific cellular
receptors, overexpressed on certain tumor tissues and other
cancer‐associated physiological differences.2 In recent years
the specific targeting of not just the cancerous tissues, but the
precise delivery of a cytotoxic agent at the subcellular level has
been shown to hold great promise to further decrease side
effects and combat multi‐drug resistance.3
organelle represents an elegant and efficient target for novel
cancer therapies. Mitochondria exhibit significant cancer‐
associated dysfunctions, amongst them, the reduced activity of
ATP synthase results in an increased membrane potential and
the increased production of reactive oxygen species (ROS),4
representing yet another mechanism by which cancer cells
may acquire drug resistance following redox adaptation.5
The transmembrane potential of mitochondria, with a
negatively charged interior, attracts delocalized lipophilic
cations, with the triphenylphosphonium group as the
prototypical example, resulting in their electrophoretic
transmembrane migration and upconcentration with reported
concentration enhancements up to 500‐fold.6 As cancerous
cells contain mitochondria with increased mitochondrial
potential and thus significantly higher mitochondrial
upconcentration of delocalized lipophilic cations, the
conjugation of these cations to cytotoxic agents opens the
path towards increasing tumor specificities of anticancer
agents, and thus the mitochondria represent a sensitive
suborganellar targeting site with a convenient targeting
mechanism.3,6
Mitochondria, widely known as the cell’s powerhouse, are
vital subcellular organelles in both normal and malignant cells.
Upon the cells evolution into a pathological state, the
Key to developing more specific anticancer agents is not
only exerting control over the tissue and organelle specificity
of anticancer drugs, but also the presence of a mechanism to
verify the delivery of the novel drugs, and whereas several
strategies exist, the use of fluorescence is particularly
interesting as a safe, non‐invasive method, generally requiring
relatively inexpensive experimental setups. Conventional
a. Department of Chemistry, Korea University, Seoul, 136‐701, Korea. E‐mail:
jongskim@korea.ac.kr
b. School of Life Sciences and Biotechnology, Korea University, Seoul, 136‐701,
Korea.E‐mail: chi6302@korea.ac.kr
c. Korea Research Institute of Standards and Science, Daejon 305‐600, Korea.
† Electronic Supplementary Informaꢀon (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
‡ These authors contributed equally to this work
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The combination of both tissue and/or organelle selective
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delivery and tissue specific enzyme DgOaI:t1e0d.103a9c/tCiv6aSCti0o2n236oGf
prodrugs dramatically increases the selectivity of a drug
conjugate and in the current work we utilize this strategy on
an AIE scaffold. Here, an AIE fluorophore is decorated with
both a tumor and organelle targeting triphenylphosphonium
moiety and a quinone based trigger,12 allowing the selective
activation of the AIE self‐assembly by the NQO1 enzyme. The
proposed mechanism is depicted in Scheme 1 where the non‐
P
P
NQO1
NADH
O
O
O
O
O
O
O
O
O
HO
OH
O
Fluorescence OFF
O
fluorescent prodrug
1 is targeted towards and upconcentrated
P
P
O
OH
P
in the mitochondria of cancer cells. The aggregates of
1
Aggregation
undergo rapid reduction of the quinone scaffold and
subsequent cyclisation releases the active form of the AIE dye,
signalled by fluorescence due to the elimination of a photo‐
induced electron transfer process between the AIE scaffold
and the quinone trigger. The AIE aggregates, specifically
localised to the mitochondria, subsequently induce the
disruption of major metabolic pathways, finally culminating in
the selective apoptosis of the targeted cells.
OH
O
O
O
OH
O
O
O
OH
O
HO
O
O
P
P
OH
O
λex 340 nm
λem 470 nm
Fluorescence ON
Fluorescence OFF
Scheme 1. Proposed mechanism of the reaction of 1 with NQO1
approaches to this combination of therapy and diagnosis,
known as small molecule based theranostic cancer treatment,7
entail the covalent linking of a flurophore or profluorophore to
the drug. A more atom‐efficient way to achieve a theranostic
platform, is using a drug, that upon delivery becomes
fluorescent itself. Aggregation induced emission (AIE)
fluorophores, a particular class of fluorophores, exhibiting
increased fluorescence upon aggregation, as opposed to most
fluorophores associated with quenched fluorescence upon
aggregation, due to restricted intermolecular rotations in the
self‐assembled state,8 are ideally suited for this purpose.
Furthermore, the specific aggregation of this type of
fluorophore at mitochondrial sites has recently been shown to
result in cell death due to disrupted mitochondrial
metabolism, whilst signaling the event by fluorescence
originating from those mitochondria.9
Results and discussion
A second strategy to increase the specificity of anticancer
drugs towards malignant tissues is the use of enzyme
activatable prodrugs. Making use of the differential expression
of various enzymes in cancerous cells and microenvironments,
these masked cytotoxic drugs are predominately activated in
the target tissues, thus lowering the potential of side‐effects
resulting from non‐specific drug uptake and thus potentially
widening the therapeutic window.10
NAD(P)H:quinone oxidoreductase‐1 (NQO1) is an enzyme
expressed throughout the cell and its expression is
upregulated as a response to stress. The enzyme acts as a two‐
electron reductase towards various toxins such as quinone
xenobiotics and the superoxide anion with concomitant
consumption of either NADH or NADPH.11 In view of its stress
induced upregulation it is not surprising that the enzyme is
overexpressed in various cancerous tissues11 with ratios up to
50 fold in the case of human liver carcinoma.11a Furthermore,
it is theorized that NQO1 upregulation may be an early event
in the pathological transformation of normal cells to cancerous
cells.11
Scheme 2. Synthetic route towards 1.
2 | J. Name., 2012, 00, 1‐3
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Synthesis. As depicted in Scheme 2, the NQO1 trigger
6
was
View Article Online
DOI: 10.1039/C6SC02236G
(a)
(b)
synthesized in two steps according to a previously reported
▬ 16 min
▬ 14 min
▬ 12 min
▬ 10 min
▬ 8 min
▬ 6 min
▬ 4 min
▬ 2 min
▬ 0 min
600
400
200
0
600
400
200
0
470 nm
▬ 16 min
▬ 14 min
▬ 12 min
▬ 10 min
▬ 8 min
▬ 6 min
▬ 4 min
▬ 2 min
▬ 0 min
procedure.13 The triphenylphosphonium‐containing targeting
moiety
5 was also obtained efficiently as previously
described.12g The dibrominated tetraphenylethene precursor
4
was prepared via the McMurry coupling of 4‐
bromoacetophenone, resulting in a cis/trans ratio of 44/56
(data not shown), as previously reported.14 The cis/trans
mixture was subsequently used as the scaffold for a Suzuki
coupling reaction with (2‐hydroxyphenyl)boronic acid, yielding
400
500
600
400
500
600
Wavelength (nm)
Wavelength (nm)
(c)
(d)
600
400
200
0
600
450
300
150
0
the π‐extended tetraphenylethene
targeted NQO1 activatable AIE fluorophore
reference compounds and , lacking the NQO1 triggering
unit and the mitochondria targeting unit, respectively, were
available through the esterification of using a carbodiimide
were
3. Finally, the mitochondria
400
200
0
1, as well as
8
9
400
500
600
3
0
10
20
30
40
coupling reagent. The identities of all compounds 1‐6
confirmed by 1H NMR, 13C NMR and ESI‐MS data as provided in
the supporting information (SI) (Fig. S1‐10). The purity of
Time (min)
Fig. 1 Fluorescence spectrum of a 20 μM solution of 1 (a) in PBS (pH 7.4) during a 15
min period upon reduction by 40 μM sodium dithionite. (b) in MeOH during a 15 min
period upon reduction by 40 μM sodium dithionite. (λex = 340 nm, slit = 5/5 T = 25 °C)
(c) Fluorescence response assays for compound 1 (20 µM) and various biological
reductants, including thiol reductants (100 µM) and amino acids (100 µM), in
phosphate buffered saline (0.5% DMSO) at pH 7.4. Bars represent the final fluorescence
intensity at 470 nm (I470) after a 12‐h incubation. Excitation was effected at 340 nm
with the excitation and emission slit widths both set at 5 nm. (d) Time course of
changes in fluorescence intensity at 470 nm for 40 min in the presence of sodium
dithionite.
compounds 1, 8 and 9 was further determined by HPLC to be
92.4%, 85.8% and 95.0%, respectively (Fig. S11).
DFT calculations. The relative energies of the frontier orbitals
of both cis and trans isomers of the AIE core (Fig. S12a) have
been calculated by DFT calculations using the B3LYP hybrid
functional at the 6‐31G(d) level of theory in the gas phase. The
calculations revealed relatively small differences in energy
between them (Fig. S12b), with energies for the HOMO being ‐
5.31 and ‐5.37 eV, for the cis and trans isomer respectively and
for the LUMO being ‐1.43 and ‐1.44 eV, for the cis and trans
isomer respectively. This can be rationalised by a relatively
small contribution of the ortho‐substituted phenylacetate
pendent arms, as can be seen from the electron distributions
of the frontier molecular orbitals (Fig. S12c).
potential interference from NAD(P)H fluorescence.12f The
addition of sodium dithionite (40 μM) to the solution of
1 (20
μM) in aqueous PBS resulted in rapid appearance of an AIE
band centered at 470 nm (Fig. 1a and 1d). By contrast, the
release of the fluorophore in MeOH did not result in a
significant fluorescence increase (Fig. 1b), confirming the AIE
origin of the fluorescence, as MeOH solvation of the released
fluorophore prohibits the AIE effect. Probe
1 demonstrated
Solution studies. The absorption and emission spectra of
1, 8
and excellent selectivity profile, showing a more than 200‐fold
increase in fluorescence upon the addition of sodium
dithionite whilst no or negligible amounts of fluorescence
increase could be observed upon the addition of other
biologically relevant thiols and amino acids (Fig. 1c). The
selective triggering by sodium dithionite was further
demonstrated by HPLC analysis, showing the complete
and were recorded in methanol with increasing amounts of
9
water (Fig. S13). Whereas the absorption was largely
independent of the solvent composition, a large solvent
dependent fluorescence increase was observed for
8 only,
resulting from efficient aggregation induced fluorescence at
higher water concentrations. It is interesting to note that
compounds
exhibit significant increases in fluorescence at higher
concentrations of water, despite having a similar polarity as
1 and 9, both bearing the NQO1 trigger did not
conversion of 1 to 8 (Fig. S14).
8.
Subcellular localization. The two‐photon cross section of
probe in water was determined to be 0.47 GM (data not
Taking the DFT calculation results of the quinone trigger into
account (Fig. S12), this observation is supported by the relative
energies of the frontier orbitals of both the trigger and the AIE
core (both cis and trans isomers), where the NQO1 trigger
quenches the fluorescence of the AIE unit by an oxidative
electron transfer (OeT)‐type photoinduced electron transfer
(PeT) effect, as previously reported for a naphtalimide‐based
NQO1 specific fluorophore.12f
8
shown), providing a convenient means to visualize the
fluorophore by means of two‐photon excitation confocal
microscopy. Colocalization studies using the NQO1 positive
A549 cell line clearly demonstrate the lipophilic delocalized
cation’s efficiency to bestow mitochondria targeting upon the
AIE probe (Fig. 2). Here both triphenylphosphonium bearing
compounds
1 and 8 demonstrate efficient intracellular AIE
To assess the behaviour of conjugate
1 upon activation of
emission originating from the mitochondria (Fig. 2a‐d and 2e‐
h), whereas virtually no fluorescence was observed in the
absence of this subcellular targeting agent (Fig. 2 i‐l).
the trigger, the fluorescence of as a solution in PBS
1
(phosphate buffered saline) was monitored after the addition
of sodium dithionite, as an enzyme free model system without
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DOI: 10.1039/C6SC02236G
(a)
(b)
Probe
Mito-tracker
(b)
Bright field
(c)
Merge
▲ 1
■ 8
● 9
(a)
(e)
(i)
(d)
(h)
(l)
1.0
0.5
0.0
1
8
9
NQO1
Tubulin
*
*
*
30 μm
30 μm
30 μm
30 μm
0 0.5 1
2
5
10 20 50
Does (μM) / 48 h
(f)
(j)
(g)
(k)
(c)
(d)
(e)
Normal cells
293T
NQO1 (low) Cancer cells
U2OS HCT116
3
2
1
0
3
3
2
1
0
30 μm
30 μm
30 μm
30 μm
2
1
0
Mock
1
8
9
Mock
1
8
9
Mock
1
8
9
(f)
(g)
(h)
30 μm
30 μm
30 μm
30 μm
NQO1 (high) Cancer cells
*
NHA-tert
HeLa
A549
Fig. 2 Confocal microscopy images of A549 cells treated with 1, 8 and 9. A549 cell after
incubation with 20 μM of 1, 8 and 9 for 12 h, co‐stained with 200 nm Mito‐tracker deep
red. The red fluorescence is from Mito‐tracker deep red, λex = 633 nm, λem = 650‐700
nm, and the green fluorescence is from the probes, λex = 740 nm, λem = 450‐550 nm.
Cell images were obtained using two photon excitation wavelengths of 740 nm and
emission wavelengths of 450−550 nm, green signal, respectively.
*
3
2
1
0
3
2
1
0
3
2
1
0
*
Mock
1
8
9
Mock
1
8
9
Mock
1
8
9
Fig. 3 Effect of 1 on tumor cell viability and its association with expression status of
NQO1. (a) Cytotoxicity (A549 cells), as determined by a WST‐1 assay, after 48h of
incubation with increasing amounts of probes 1, 8 and 9. (b) Immunoblot assay
showing NQO1 expression levels in six human cells tested in this study (c‐h) Three
NQO1 low‐expressing cells (c‐e), including 293T embryonic kidney cells and three NQO1
high‐expressing cells (f‐h) were treated with 20 μM of 1, 8, and 9 for 48 h. Cell viability
was determined using aWST‐1 assay. * P < 0.005; ** P < 0.001; Cell viability data
represent the mean and s.d. of triplicate determinations from a single experiment that
has been repeated one or two times with similar outcomes.
In vitro dependency of cytotoxicity on NQO1. The dose
dependence of the cytotoxicity of 1, 8 and 9 in A549 cells,
expressing high levels of the NQO1 enzyme, was determined
using a WST‐1 assay. In contrast to the non‐targeted reference
compound
and showed elevated cytotoxicity (Fig. 3a), which reflects a
conclusion in line with the subcellular localization studies
reported in Fig. 2. To address whether provokes a NQO1‐
9, triphenylphosphonium appended molecules 1
8
1
associated cytotoxic effect, we performed cell viability assays
using human embryonic 293T cells and five human cancer cell
lines of various tissue origins, which express differential levels
and caspase‐3, and this effect was noticeably blocked in
NQO1‐depleted cells (Fig. 4c). Consistently, flow cytometric
analysis of the apoptotic sub‐G1 fraction showed that probe
of NQO1 protein (Fig. 3b) at a 20 µM dose of probes
Intriguingly, treatment of resulted in no or only slightly
1, 8 and 9.
1
’s induction of apoptosis is suppressed by siNQO1 in a dose‐
dependent manner (Fig. 4a). In addition, a cell fractionation
assay also revealed that stimulates the cytoplasmic release
1
decreased viability of 293T (normal kidney), U2O3
(osteosarcoma), and HCT116 (colon cancer) cells (Fig. 3c‐e),
which have low NQO1 expression levels, whereas it caused a
considerable decrease in cell viability of NHA (glioma), HeLa
(cervical cancer), and A549 (lung cancer) cells, associated with
1
of cytochrome C in a NQO1‐dependent manner (Fig. 4d).
Together, these results strongly suggest that NQO1‐dependent
aggregation of
dysregulation of membrane permeability, leading to
cytochrome C release and subsequent caspase activation.
1 within the mitochondria may trigger
high NQO1 expression levels (Fig. 3f‐h). As anticipated,
displayed a strong cytotoxic effect on all cell types we tested,
indiscriminate of NQO1 levels and pathological state, while
failed to show any significant effect. Therefore, this finding
supports that exerts its cytotoxic effect on human cancer
8
Next, the intracellular aggregation of
1 and its dependency
9
on NQO1 was determined by monitoring the relation between
the fluorescent intensity of the AIE fluorophore and NQO1
depletion and overexpression. In A549 cells, harboring high
NQO1 levels, knockdown of the endogenous NQO1 expression
using increasing doses of siNQO1 transfection resulted in a
dose‐associated reduction in fluorescence intensity (Fig. 5a
and 5b). Consistently, ectopic overexpression of NQO1 using
FLAG‐NQO1 transfection in HCT116 cells led to a drastic dose‐
associated increase in the observed AIE fluorescence (Fig. 5c
1
cells in a highly NQO1‐dependent fashion.
To further define the NQO1‐dependeny of
1
cytotoxicity, we
activity in A549 cells,
using small interfering RNA (siRNA)‐mediated knockdown of
NQO1. As shown in Fig. 4b, the viability‐decreasing activity of
was attenuated by siNQO1 transfection in a dose‐dependent
manner, while effects of and were not affected by siNQO1.
tested the NQO1 depletion effect on
1
1
8
9
and 5d). Thus, these results clearly indicate probe
1’s
An immunoblot analysis of cleaved PARP and caspases was
performed to investigate the induction of apoptosis as a
aggregation and fluorescence induction are highly dependent
on the NQO1 expression in tumor cells.
mechanism in the NQO1‐dependency on the cytotoxicity of
Probe strongly activated the cleavage of PARP, caspase‐9,
1.
1
4 | J. Name., 2012, 00, 1‐3
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knockdown. Identical numbers of siControl or siNQO1 cells (1 ×
View Article Online
(a)
1 (20 μM)
107) were injected subcutaneously into tDhOeIf:l1a0n.1k03o9f/Cn6uSdCe02m23ic6eG.
-
+
+
+
+
siNQO1
0
0
10
20
40
Both cells generated visible tumors of similar mass 8 days
32
16
8
subsequent to injection, and
1 was administered by tail vein
Sub G1
1.45 %
9.52 %
7.10 %
3.63 %
1.51 %
injection (2 mg/kg/d) once per three days for 2 weeks. The
tumor growth was monitored regularly up to 60 days.
Compared with siControl tumors, showing a dramatic response
0
to
1 (79% reduction), siNQO1 tumors displayed a greatly
■ siControl
■ siNQO1 20 pmol
■ siNQO1 40 pmol
(b)
(c)
(d)
A549
A549
Cytosol
Mito.
attenuated response (28% reduction), indicating that
suppresses tumor growth in a highly NQO1‐dependent manner
in vivo (Fig. 6a). Subsequently, to assess the potential of probe
1
3
2
1
0
-
-
-
+
+
+
1
(20 μM /48 h)
0
20 40
0
20 40
siNQO1 (pmol)
Cl-PARP
1
(20 μM /48 h)
- + - + - + - +
- - + + - - + +
*
siNQO1 (pmol)
Cytochrome C
COX4
Cl-caspase-9
Cl-caspase-3
NQO1
*
*
1
to cause tumor size regression in HCT116 cell based tumors,
either transfected with a control plasmid (pcDNA) or a NQO1
expression plasmid (1
107 cells) were injected
NQO1
Tubulin
Tubulin
Mock
1
8
9
×
Fig. 4 The NQO1‐dependency of 1 cytotoxicity. (a) Flow cytometric analysis of apoptosis
showing a NQO1 depletion effect on 1 induction of apoptosis. A549 cells transfected
with siNQO1 were incubated with 1 as indicated and flow cytometric analysis was
carried out to determine the percentages of sub‐G1 fraction. (b) Attenuation of 1
cytotoxicity by siRNA‐mediated knockdown of NQO1 expression. A549 cells transfected
with either siControl or siNQO1 were incubated with 1, 8, or 9 (10 M, 24 hours). Cell
viability was determined by a WST‐1 assay. Each bar represents the average of
subcutaneously in the flank of nude mice. Tumor growth
curves clearly implicate the vital importance on both the
presence of the NQO1 enzyme and
tumor size regression, when treated 10‐days post tumor
xenograft inoculation. After approximately 35 days post tumor
1 on the efficiency of the
triplicate measurements; *; P < 0.001. (c) NQO1‐dependency of 1 activation of xenograft inoculation a clear divergent behavior could be
apoptosis. Cells transfected with siRNAs were incubated with 1 (20 μM). After 48 hours,
an immunoblot assay was performed to measure the cleaved PARP, caspase‐3, and
growth, and only the combination of both the presence of the
caspase‐9 levels. Cl: cleaved. (d) NQO1‐dependency of 1 on stimulation of cytochrome
C release. Cells were transfected with siRNAs and treated 1 as indicated, and lysates
from the cytosolic and mitochondrial fractions were subjected to immunoblot assay of
cytochrome C. COX4 and tubulin were used as markers for the mitochondria and
cytosol fractions, respectively.
observed, with the control treatments exhibiting unrestricted
enzyme and
1 displaying cancer size regression. Treatment
thus ultimately resulted in an approximately 4‐fold difference
in tumor size, 40 days after the start of the treatment (Fig. 7a).
In vivo and ex vivo tissue distribution of aggregated 1. Finally,
to prove the result of the cancer growth curves was caused by
In vivo tumor growth suppression and tumor growth
reduction. We examined the effect of probe
1 on the tumor
the specific delivery and unique activation of probe
1 in NQO1
growth and its dependency on NQO1 expression, using
xenograft tumors of A549 cells with and without NQO1
positive tumors, the mice bearing HCT116 cells, with and
without expression of NQO1 (see also Fig. 7B) were subjected
to in vivo fluorescence studies 25 days after inoculation with
HCT116 cells and 10 days after the start of probe
1 based
(a)
A549 NQO1 (high)
(b)
therapy. As can clearly be seen in Fig. 8a, the probe was
specifically upconcentrated in the tumor tissue, and the
activation in the case of the NQO1 positive cells, clearly
resulted in a higher degree of probe fluorescence and thus
insitu aggregation. The same mice were subsequently
40
▬ Mock
A549
+
40
30
20
10
0
▬ 0
30
20
10
0
▬ 10
▬ 20
▬ 40
1
(10 μM / 24 h)
-
+
+
-
+
-
+
-
siControl
+
-
-
500
600
10 20 40 siNQO1 (pmol)
NQO1
Tubulin
(a)
(b)
Mock
1
Con 0 10 20 40
Concentration of siNQO1 (pmol)
■ siControl + Mock
▲ siNQO1 + 1
siControl siControl siNQO1
6
4
2
0
● siControl + 1
(C)
HCT116 NQO1 (low)
(d)
40
▬ Mock
HCT116
40
▬ 0
▬ 1
▬ 2
▬ 4
30
30 20
10
1
(10 μM / 24 h)
-
+
-
+
+
-
+
+
+
*
*
pcDNA
-
-
-
20
0
500
600
1
2
4
Flag-NQO1 (μg)
Flag-NQO1
Tubulin
10
0
12 24 36 48 60
days
0
Con 0
1 2 4
Concentration of flag-NQO1 (μg)
Fig. 6 Mouse tumor xenograft assay showing the effect of probe 1 on in vivo tumor
growth (A549 cells). (a) Inhibition of tumor growth by 1 and its dependency of NQO1.
The data represents the mean ± SD (n = 5 per group; **, P < 0.01). A549 cells
transfected with either siNQO1 or siControl were injected subcutaneously in the right
flanks of nude mice. 1 was administered by tail vein injection (2 mg/kg/d) at day 8 and
subsequently once per three days for 2 weeks. The tumor growth was monitored
periodically, and the volume (V) was calculated by using the modified ellipsoidal
formula: V = 1/2 × length × (width)2. (b) Representative photographs of xenograft
tumor bearing mice, 5 weeks after administration of 1.
Fig. 5 The fluorescence induction of 1 is highly dependent on the NQO1 expression
level. (a) A siNQO1 dose‐associated decrease in 1‐induced fluorescence intensity in
A549 cells. Cells transfected with increasing doses of siNQO1 were incubated with 1 (10
μM). The fluorescence of cell lysates was determined after 24 h incubation. (b)
Immunoblot assay of siNQO1‐mediated NQO1 knockdown in A549 cells. (c) A NQO1‐
associated increase in 1‐induced fluorescence intensity in HCT116 cells. Cells
transfected with increasing doses of FLAG‐NQO1 plasmid were incubated with 1 (10
μM). (d) Immunoblot assay of FLAG‐NQO1 levels in HCT116 cells.
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spectra were recorded in CDCl3 on a VariaVniew4A0rt0icle OMnlHinze
instrument. All chemical shifts are reported in ppm values
HCT116
(a)
(b)
DOI: 10.1039/C6SC02236G
■ Mock-pc-stable
Mock
NQO1
1
● Mock-NQO1-stable
▲ 1-pc-stable
pc
pc
NQO1
using the peak of TMS as an internal reference. Reverse‐phase
HPLC experiments were conducted using an Young Lin HPLC
system (YL9100) equipped with a VDSpher 100 C18‐E column
(5µm, 250 × 4.6 mm) operated at a flow rates of 1 mL/min
using a mobile phase consisting of a binary gradient of solvent
A (water with 0.5% v/v TFA) and solvent B (acetonitrile with
0.5% v/v TFA). ESI mass spectrometric analyses were carried
out using an LC/MS‐2020 Series (Shimadzu) instrument.
▼ 1-NQO1-stable
8
6
4
2
0
*
*
10 20
Days
40 50
0
30
Fig. 7 The effect of 1 on the tumor size (HCT116 cells). (A) Growth response of
xenograft tumors derived from control (pcDNA) or NQO1‐expressing HCT116 cells to
prodrug 1 (n=5 per group; error bars indicate ±SD; ** p‐value<0.01). HCT116 cells
transfected with either pcDNA or NQO1 expression plasmid were injected
subcutaneously in the right flanks of nude mice. 1 was administered by tail vein
injection (4 mg/kg) at day 10 and subsequently once per five days for 2 weeks. The
tumor growth was monitored periodically, and the volume (V) was calculated by using
the modified ellipsoidal formula: V = 1/2 × length × (width)2. (B) Representative
photographs of xenograft tumors at day 30 after probe 1 injection.
DFT calculations. Density functional theory calculations were
performed using B3LYP15 at the 6‐31G(d) level of theory,16
using the Gaussian G09W software package.17 Gaussian input
file generation and molecular orbital visualization were
performed using the Gabedit 2.4.8 software package.18 The
structure optimization was performed from several starting
geometries to ensure a global minimum was reached.
euthanized and the fluorescence of the excised organ tissues
was recorded, confirming the superior fluorescence originating
from the NQO1 positive cell based tumor. The excellent tissue
selectivity, showing no or non‐significant levels of fluorescence
originating from either the reticulo‐endothelial system (liver
and spleen) or the renal system (kidneys) as well as the heart,
provides further evidence towards the excellent tissue specific
Human normal and cancer cell lines. Human cancer cell lines
and the embryonic kidney 293T cells were purchased from
American Type Culture Collection (Rockville, MD, USA) or
Korea Cell Line Bank (Seoul, South Korea). The cells were
maintained in Dulbecco's modified Eagle's medium
supplemented with 10% FBS (GIBCO BRL) at 37 °C in a
humidified atmosphere with 5% CO2.
and enzyme selective activation of probe 1.
Expression plasmids, siRNA, and transfection. The NQO1
expression vector was purchased from Addgene (MA, USA).
Transfections were performed using Lipofectamine 2000
Experimental section
General synthetic methods. The reagents used in this study
were purchase from Alfa‐Aesar, Aldrich, TCI, Carbosynth,
Ducsan, and Acros and used without further purification. Silica
(Invitrogen).
siRNA
duplex
against
NQO1
(5`‐
GAGAGTTTGCTTACACTTA‐3`) was synthesized by Dharmacon
Research. Transfection of siRNA was performed using a siRNA‐
Oligofectamine mixture or electroporation (Neon transfection
system; Invitrogen).
gel 60 (Merck, 0.040−0.063 mm) was used for column
chromatography and Merck 60 F254 silica gel plates were used
for analytical thin‐layer chromatography. 1H and 13C NMR
Immunoblotting assay. Immunoblot analyses were performed
using antibodies specific for NQO1 (SC‐16464), cleaved PARP
(5625), cleaved caspase‐3 (9664), cleaved caspase‐9 (7237),
COX‐4 (4850), and cytochorome C (4280), purchased from
Santa Cruz Biotechnology or Cell Signaling Technology. Anti‐
Flag (F3165) and anti‐tubulin antibodies were obtained from
Sigma and Invitrogen, respectively. Briefly, cells were washed
twice in ice‐cold PBS and lysed in a radioimmunoprecipitation
assay buffer containing 50 mM tris∙HCl (pH 7.5), 150 mM NaCl,
0.1% SDS, 1% Triton X‐100, 1% sodium deoxycholate, and
protease inhibitor mixture. After sonification, the lysate was
centrifuged, and supernatant was recovered and loaded on an
8% SDS–polyacrylamide gel for electrophoresis.
1
(a)
(b)
HCT116
( )
+
( )
-
NQO1
NQO1
1
NQO1
( ) ( )
+
-
( ) ( )
+
+
106
106
5 x
5 x
4
3
2
1
0
4
3
2
1
0
( ) ( )
+
-
( ) ( )
+
+
Flow cytometric analysis of apoptosis. Cells transfected with
Fig. 8 The in vivo and ex vivo imaging of HCT116 bearing mice 40 days after the start of
the treatment (see also fig. 7). (a) Representative images of mice bearing HCT116 cells,
pretreated with control plasmid (left) and NQO1 expression plasmid (right) under white
light (top) and the represenatative in vivo fluorescent images of probe 1 accumulation
in xenograft tumors (λex = 430 ‐ 480 nm, λem = 490 ‐ 550 nm). (b) Dissected organs and
tumors (up) and their fluorescent images (down) of probe 1‐injected mice (λex = 430 ‐
480 nm, λem = 490 ‐ 550 nm).
siNQO1 or siControl were treated with
1 (20 μM). After 24 h
treatment, the cells were fixed with 70% ethanol and
resuspended in 1 ml of PBS containing 50 mg/ml RNase and 50
mg/ml propidium iodide (Sigma). The assay was performed on
a FACScan flow cytometer (Becton Dickinson, San Jose, CA)
and the apoptotic profile was analyzed using Modfit software
(Becton Dickinson).
6 | J. Name., 2012, 00, 1‐3
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Separation of cytosolic and mitochondria fractions. 6.96 – 6.88 (m, 1H), 3.88 – 3.70 (m, 3H), 3.03 (dV,ieJw=Ar4tic.7le6OnHlinze,
DOI: 10.1039/C6SC02236G
Separation of cytosolic and mitochondria fractions was 2H), 2.27 (q, J = 7.28 Hz, 2H), 2.10 (d, J = 18.36 Hz, 3H), 1.87 –
performed using the Mitochondria Isolation Kit (Thermo 1.80 (m, 6H), 1.76 – 1.47 (m, 8H), 1.36 (s, 3H), 1.27 (s, 3H), 0.96
Scientific, USA). Briefly, the cells were washed with cold PBS – 0.83 (m, 3H) ppm. 13C NMR (CDCl3, 100 MHz): δ 12.26, 12.29,
and lysed by 5‐cycle freeze‐thawing in Mitochondria Isolation 12.83, 12.86, 14.34, 14.43, 14.49, 23.83, 24.07, 24.21, 24.23,
Reagent A buffer, containing 10 mM phenylmethylsulfonyl 25.46, 28.72, 28.83, 29.66, 29.78, 29.83, 29.95, 31.76, 33.67,
fluoride (PMSF). After ice incubation for 10 min and 33.70, 34.84, 38.07, 38.12, 47.57, 47.65, 53.75, 118.01, 118.06,
Mitochondria Isolation Reagent B buffer addition, the lysates 118.86, 118.92, 123.94, 123.06, 123.09, 128.45, 126.53, 126.62,
were centrifuged at 700 g for 10 min following inverting after 126.76, 126.78, 126.87, 127.87, 127.91, 128.00, 128.27, 128.33,
treatment of the Mitochondria Isolation Reagent C buffer. The 128.39, 128.55, 130.65, 130.77, 130.83, 130.89, 131.00, 131.06,
supernatant was transferred to a new tube, centrifuged at 131.42, 131.46, 131.47, 131.53, 131.55, 131.59, 131.62, 131.66,
°
12,000 g for 15 min at 4 C, and collected as the cytosolic 133.75, 133.78, 133.85, 133.88, 134.53, 134.59, 134.64, 134.68,
fraction. After addition of 500 μL of Mitochondria Isolation 153.23, 135.23, 135.24, 135.26, 135.55, 135.57, 135.66, 135.77,
Reagent C, the pellet was centrifuged at 12,000 g for 5 min and 138.44, 138.48, 138.72, 138.74, 140.89, 140.93, 140.95, 142.92,
collected as the mitochondrial fraction.
142.98, 143.04, 143.07, 143.11, 143.15, 143.71, 143.80, 147.59,
147.60, 147.84, 147.85, 152.41, 152.46, 171.59, 171.64, 171.93,
Animal Studies. Four‐week‐old immunodeficient nude mice 171.99, 187.55, 187.58, 190.91, 190.96 ppm. ESI‐MS: m/z calcd
(nu/nu) mice (Orient Bio Inc.) were maintained in pressurized for C76H68O6P (M+H) 1107.47; detected 1107.25.
ventilated cages. Cells transfected with siNQO1 or siControl (1
× 107) were injected subcutaneously into five mice.
1
was Synthesis of 2. A solution of
6
(109 mg, 0.43 mmol) in DCM
HCl (180 mg,
3 (300 mg, 0.58 mmol), and DMAP (141 mg,
administered by tail vein injection (2 mg/kg/d) for once per was slowly added dropwise to a solution of EDC
‧
three days for 2 weeks. HCT116 cells (1.0 × 107) transfected 1.16 mmol),
with empty (pcDNA) or NQO1 expression plasmid were 1.16mmol) in CH2Cl2 (30 mL) at 0 °C. After 12 h the mixture was
injected subcutaneously into the right flank of mice. When washed with 1N aqueous HCl, the organic layer was dried with
tumor growth reached a detectable size (approx. 20 mm3), the MgSO4, filtered and concentrated in vacuo. Purification by
mice were treated with compound
1 (4ꢀmg/kg) or vehicle PBS column chromatography yielded 243 mg of the title product
by tail vein injection once every 5 days for 20 days. Tumor (56 % yield). 1H NMR (CDCl3, 400 MHz): δ 7.35 – 7.30 (m, 4H),
growth was monitored periodically, and volume (V) was 7.29 – 7.18 (m, 6H), 7.17 – 7.08 (m, 13H), 6.98 – 6.91 (m, 3H),
calculated by using the modified ellipsoidal formula: V = 1/2 × 3.04 (d, J = 6.24 Hz, 2H), 2.12 (d, J = 15.12 Hz, 3H), 1.86 (t, J =
length × (width)2. All animal studies were performed with the 14.04 Hz, 6H), 1.38 (s, 3H), 1.30 (s, 3H) ppm. 13C NMR (CDCl3,
approval of Korea University Institutional Animal Care and Use 100 MHz): 12.32, 12.35, 12.88, 12.92, 14.41, 14.52, 14.56,
Committee and Korea Animal Protection Law.
23.92, 28.77,28.89, 31.85, 38.14, 38.18, 116.04, 116.19, 121.00,
121.04, 123.98, 123.02, 126.61, 126.65, 126.87, 127.00, 127.93,
In Vivo and ex Vivo Fluorescent Imaging. To study tumor 127.96, 128.05, 128.15, 128.43, 128.46, 128.51, 128.58, 128.61,
target specificity and organ distribution of Probe in vivo and 128.65, 129.25, 129.27, 130.38, 130.47, 131.08, 131.15, 131.54,
1
ex vivo, stably expressed empty (pcDNA) or NQO1 HCT116 cells 131.58, 131.65, 131.69, 131.72, 132.39, 132.42, 134.73, 135.33,
were injected subcutaneously into the mice. When tumor 135.55, 135.69, 135.84, 138.60, 138.63, 138.86, 140.91, 140.92,
growth reached a detectable size (approx. 20 mm3), the mice 141.21, 141.30, 143.00, 143.03, 143.23, 143.23, 143.35, 143.52,
were treated with probe
injection once every 5 days for 20 days. After 20 days of probe 152.76, 171.70, 171.76, 187.68, 191.06 ppm. ESI‐MS: m/z calcd
injections, mouse were sacrificed and in vivo fluorescence for C52H44O5 (M+Na) was 771.31; detected 771.30, (M+K)
1 (4ꢀmg/kg) or vehicle PBS by tail vein 143.70, 143.79, 143.84, 147.67, 147.68, 152.51, 152.55, 152.72,
1
intensity of the tumors and organs of the control and injected 787.28, detected 787.25.
mice were measured using Maestro2 (excitation and emission;
500 and 650 nm, respectively). The fluorescence images and Synthesis of 3. To a solution of
4 (1.0 g, 2.05 mmol), 2‐
autofluorescence were then unmixed with commercial
hydroxyphenylboronic acid (1.13 g, 8.19 mmol) in DME (40 mL)
software (Maestro software ver.2.4, CRi, Woburn, MA, USA) and 2M Na2CO3 20 mL in a 100 mL two‐neck round bottom
using the multiexcitation spectral analysis function.
flask, 24 mg of tetrakis(triphenylphosphine)palladium(0) was
added. The mixture was stirred at room temperature for 1 h,
and subsequently heated under reflux conditions for 12 h.
5 (45 After completion of the reaction, the mixture was washed with
Synthesis of 1. EDC
‧
HCl (41 mg, 0.26
(100 mg, 0.13 mmol),
mmol) was added to a solution of
3
mg, 0.12 mmol) and DMAP (32 mg, 0.26 mmol) in CH2Cl2 (20 a 1N HCl solution, the organic layer was dried with MgSO4,
mL) at room temperature. After 12 h the mixture was washed filtered and concentrated in vacuo. The reissue was purified by
1
with an aqueous 1N HCl solution, the organic layer was dried column chromatography. H NMR (CDCl3, 400 MHz): δ 7.25 –
with MgSO4, filtered and concentrated in vacuo. Purification by 7.08 (m, 23H), 6.96 – 6.91 (m, 4H) ppm. 13C NMR (100 MHz,
column chromatography yielded 114 mg (78 % yield) of the CDCl3) δ 116.09, 121.11, 126.96, 127.93, 128.05, 128.58,
1
title product. H NMR (CDCl3, 400 MHz): 7.86 – 7.75 (m, 9H), 129.32, 130.43, 131.59, 132.42, 135.53, 141.17, 143.41, 143.52,
7.70 – 7.65 (m, 5H), 7.37 – 7.19 (m, 7H), 7.17 – 7.01 (m, 16H),
This journal is © The Royal Society of Chemistry 20xx
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152.60 ppm. ESI‐MS: m/z calcd for C38H28O2 (M+Na) 539.20, cleavable linker was essential to the pathology specific toxicity.
View Article Online
DOI: 10.1039/C6SC02236G
The crucial dependence of the self‐aggregation process and
detected 539.25, (M+K) 555.17, detected 555.25.
the associated cytotoxicity cleaved
1 on the expression levels
Synthesis of 4. This compound was synthesized, according to a of NQO1 was further demonstrated both in vitro and in vivo by
previously reported procedure,19 with a 77 % yield.
NQO1 expression modulation. The cytotoxicity was
determined to originate from the caspase‐dependent
Synthesis of 5. This compound was synthesized, according to a induction of apoptosis subsequent to cytochrome C release by
previously reported procedure,12g with a 98 % yield.
membrane permeabilization. Probe demonstrated an in vivo
tumor growth suppression of 79%, a result highly dependent
Synthesis of 6. This compound was synthesized, according to a on the expression of NQO1, as gene knockdown resulted in a
1
previously reported procedure,13 with a 84 % yield.
significantly attenuated growth inhibition effect (28% growth
reduction) in A549 tumor xenografts. The tumor size
Synthesis of 7. This compound was synthesized, according to a regression at
4 mg/kg every five days also clearly
previously reported procedure,13 with a 98 % yield.
demonstrated the efficiency of the probe in HCT116 tumor
xenografts with observed continuous tumor growth in the
Synthesis of 8. EDC
solution of (152 mg, 0.20 mmol),
DMAP (49 mg, 0.40mmol) in CH2Cl2 (20 mL) at room tumors and probe
‧
HCl (63 mg, 0.40 mmol) was added to a control samples and NQO1 absent cell line xenograft and a
(68 mg, 0.18 mmol) and clear tumor size reduction in the presence of NQO1 expressing
treatment. Tissue distribution of the
2
5
1
temperature. After 12 h the mixture was washed with an aggregated AIE fluorophores both in vivo and ex vivo
aqueous 1N HCl solution, the organic layer was dried with demonstrated a remarkable tumor specificity. The results
MgSO4, filtered and concentrated in vacuo. Purification by obtained in this study fully demonstrate the potential of this
column chromatography yielded 160 mg (72%) of the title novel cytotoxic drug, specifically acting on the disruption of
compound. 1H NMR (CDCl3, 400 MHz): δ 7.76 – 7.57 (m, 15H), mitochondrial function and integrity for NQO1 expressing
7.50 (d, J = 7.88 Hz, 1H), 7.44 (d, J = 8.24 Hz, 1H), 7.32 – 7.21 tumors.
(m, 6H), 7.10 – 6.96 (m, 17H), 6.82 – 6.69 (m, 2H), 3.73 – 3.46
(m, 3H), 2.20 (t, J = 7 Hz, 1H), 2.02 (t, J = 6.96 Hz, 1H), 1.61 –
1.26 (m, 10H), 0.97 – 0.83 (m, 3H) ppm. 13C NMR (CDCl3, 100
MHz): 14.37, 23.87, 24.15, 31.80, 33.75, 34.88, 117.47, 117.92,
117.96, 118.77, 118.81, 119.28, 123.94, 126.37, 126.65, 127.46,
127.83, 127.89, 128.44, 128.69, 128.71, 130.33, 130.64, 130.67,
130.77, 130.80, 131.17, 131.40, 131.62, 131.66, 133.70, 133.73,
133.80, 133.83, 134.79, 135.23, 135.26, 135.63, 136.87, 137.41,
140.35, 140.64, 141.46, 141.76, 142.09, 143.26, 143.99, 144.04,
147.90, 154.44, 155.49, 171.96, 172.33 ppm. ESI‐MS: m/z calcd
for C62H52O3P (M+H) was 875.36; detected 875.35
Acknowledgements
This work was supported by a CRI project (No. 2009‐0081566,
J.S.K.) and a NRF grant (NRF‐20158A2A1A01005389, S.G.C.)
from the National Research Foundation of Korea and a Korea
University Grant (P.V.).
Notes and references
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Synthesis of 9. EDC
solution of compound
‧
HCl (60 mg, 0.38 mmol) was added to a
(100 mg, 0.19 mmol), (145 mg, 0.58
3
6
mmol) and DMAP (47 mg, 0.38 mmol) in CH2Cl2 (30 mL) at
room temperature. After 12 h the mixture was washed with an
aqueous 1N HCl solution, the organic layer was dried with
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2
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47.45, 123.61, 123.62, 126.95, 138.76, 139.21, 142.20, 143.09,
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8 | J. Name., 2012, 00, 1‐3
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