Self-assembly of Fluorescent Dehydroberberine Enhances Mitochondria-Dependent Antitumor Efficacy

Article abstract of DOI:10.1002/chem.201801112

Selective imaging and inducing mitochondrial dysfunction in tumor cells using mitochondria-targeting probes has become as a promising approach for cancer diagnosis and therapy. Here, we report the design of a fluorescent berberine analog, dehydroberberine (DH-BBR), as a new mitochondria-targeting probe capable of self-assembling into monodisperse organic nanoparticles (DTNPs) upon integration with a lipophilic counter anion, allowing for enhanced fluorescence imaging and treatment of tumors in living mice. X-ray crystallography revealed that the self-assembly process was attributed to a synergy of different molecular interactions, including π–π stacking, O???π interaction and electrostatic interaction between DH-BBR and counter anions. We demonstrated that DTNPs could efficiently enter tumor tissue following intravenous injection and enhance mitochondrial delivery of DH-BBR via an electrostatic interaction driven anion exchange process. Selective accumulation in the mitochondria capable of emitting strong fluorescence and causing mitochondrial dysfunction was achieved, enabling efficient inhibition of tumor growth in living mice. This study demonstrates promise for applying lipophilic anions to control molecular self-assembly and tune antitumor activity of mitochondria-targeting probes, which can facilitate to improve cancer treatment in vivo.

Full text of DOI:10.1002/chem.201801112

A Journal of
Accepted Article
Title: Self-assembly of Fluorescent Dehydroberberine Enhances
Mitochondria-Dependent Antitumor Efficacy
Authors: Ruibing An, Zhanni Gu, Haifeng Sun, Yuxuan Hu, Runqi Yan,
Deju Ye, and Hong Liu
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Chem. Eur. J. 10.1002/chem.201801112
Link to VoR: http://dx.doi.org/10.1002/chem.201801112
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Self-assembly of Fluorescent Dehydroberberine Enhances
Mitochondria-Dependent Antitumor Efficacy
Ruibing An+,[a] Zhanni Gu+,[b] Haifeng Sun+,[b] Yuxuan Hu,[a] Runqi Yan,[a] Deju Ye,*[a] and Hong Liu*[b]
Abstract: Selective imaging and inducing mitochondrial dysfunction
in tumor cells using mitochondria-targeting probes has become as a
promising approach for cancer diagnosis and therapy. Here, we report
the design of a fluorescent berberine analog, dehydroberberine (DH-
BBR), as a new mitochondria-targeting probe capable of self-
assembling into monodisperse organic nanoparticles (DTNPs) upon
integration with a lipophilic counter anion, allowing for enhanced
fluorescence imaging and treatment of tumors in living mice. X-ray
crystallography revealed that the self-assembly process was
attributed to a synergy of different molecular interactions, including π-
π stacking, Oꞏꞏꞏπ interaction and electrostatic interaction between
DH-BBR and counter anions. We demonstrated that DTNPs could
efficiently enter into tumor tissue following intravenous injection and
enhance mitochondrial delivery of DH-BBR via an electrostatic
interaction driven anion exchange process. Selective accumulation in
the mitochondria capable of emitting strong fluorescence and causing
mitochondrial dysfunction was achieved, enabling efficient inhibition
of tumor growth in living mice. This study demonstrates promise for
applying lipophilic anions to control molecular self-assembly and tune
antitumor activity of mitochondria-targeting probes, which can
facilitate to improve cancer treatment in vivo.
activity,[8] and regulating cellular process.[9] In addition, they can
generally use the enhanced permeation and retention (EPR)
effect to facilitate the delivery into tumors, ultimately improving the
imaging and treatment of tumors in vivo.[10]
Recently, mitochondria-targeting theranostic probes that
enable selective imaging and induction of mitochondrial
dysfunction have attracted increased attention to advance cancer
therapy.[11] These probes generally use delocalized lipophilic
cations (DLCs) to trigger selective delivery of imaging and
therapeutic moieties into mitochondria.[12] One of the most used
DLCs is triphenylphosphonium (TPP), which is directly
conjugated to the imaging and antitumor cargos, affording TPP-
based mitochondria-targeting theranostic probes capable of
imaging and killing cancer cells.[13] Another intriguing approach is
the direct use of DLCs-based organic fluorophores (e.g.,
rhodamine, coumarin) to build mitochondria-targeting theranostic
probes through covalently coupling with an antitumor drug.[13-14] In
spite of encouraging results have been demonstrated, both of
these two kinds of probes require tedious chemical conjugation of
either TPP or DLCs-based fluorophore with the drug, which may
interrupt the interactions between the drugs and the molecular
targets in cancer cells, potentially reducing the antitumor
efficacy.[15] Therefore, the development of new small molecule
DLCs-based compounds with their structure-inherent abilities of
mitochondria-targeting, fluorescence imaging and inducing
mitochondrial dysfunction without additional chemical conjugation
could offer an alternative approach to enhance the mitochondria-
dependent antitumor efficacy.
Introduction
Self-assembly of small molecules into nanoarchitectures has
emerged as
a
promising approach to build functional
nanomaterials capable of tuning imaging signals,[1] controlling
drug delivery,[2] and improving therapeutic efficacy.[3] Tremendous
efforts have been made to develop small molecule compounds,
including amino acids,[4] peptides,[5] and other organic
compounds,[6] with the propensity for self-assembly into organic
nanostructures by different intermolecular interactions (e.g., π-π
stacking, hydrophobic interactions, ionic attraction, and hydrogen
bond). These organic assemblies have the advantages of
structural diversity, high biodegradability and tunable functionality,
As a natural isoquinoline alkaloid, berberine (BBR) is a widely
used traditional Chinese medicine for the treatment of many
diseases, such as diabetes, cardiovascular disease, hyperlipemia,
and inflammation.[16] BBR has a cationic structure, which has also
been used as a DLCs-based compound capable of targeting to
mitochondria, emitting weak green fluorescence and inducing
mitochondrial dysfunction in tumor cells.[17] However, the potency
against tumor cells is not very efficient,[16b] substantially limiting its
application as an antitumor drug. Moreover, although BBR
contains a multi-aromatic ring structure for emitting fluorescence,
the fluorescence quantum yield in aqueous solution (0.0021) is
too low for cancer imaging, presumably because of its partial π-
conjugated structure (Figure 1a). Thus, the direct application of
BBR as a mitochondria-targeting probe for fluorescence imaging
and treatment of tumors is very limited. There is still lack of studies
to design BBR analogues as efficient mitochondria-targeting
probes with improved fluorescence property and antitumor activity.
offering wide applications in biology and medicine, such as
7]
molecular imaging of enzyme activity,[1b,
tuning antitumor
[a]
[b]
R. An, Y. Hu, R. Yan, Pro. D. Ye
State Key Laboratory of Analytical Chemistry for Life Science,
School of Chemistry and Chemical Engineering, Nanjing University,
Nanjing, 210023, China.
E-mail: dejuye@nju.edu.cn
Z. Gu, Dr. H. Sun, Pro. H. Liu
Herein, we report the design of
a new BBR analog,
State key Laboratory of Drug Research and Key Laboratory of
Receptor Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203,
China.
dehydroberberine (DH-BBR, Figure 1a), as a new mitochondria-
targeting probe for fluorescence imaging and treatment of tumors
in vivo. We showed that DH-BBR contains a planar and full π-
conjugated multi-ring structure, substantially improving the
fluorescence properties and cytotoxicity toward cancer cells
compared to that of BBR. The subsequent incorporation of a
lipophilic anion tetraphenylborate (TPB) with DH-BBR to trigger
E-mail: hliu@simm.ac.cn
[
+]
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201801112
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efficient molecular self-assembly into
mono-dispersed
DLC characteristic of DH-BBR could promote mitochondrial
accumulation. We further compared the cytotoxicity of DH-BBR
and BBR against both HeLa and MDA-MB-231 tumor cells using
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. The results showed that DH-BBR exhibited higher
cytotoxicity than BBR, with an IC50 value of ~22.14 and ~32.87
μM against HeLa and MDA-MB-231 cells, respectively. In contrast,
more than 50% cells were still alive after incubation with 50 μM
BBR for 48 h (Figure S3). These results demonstrated that DH-
BBR was a more appropriate mitochondria-targeting probe than
BBR in terms of enhanced fluorescence properties and
cytotoxicity.
nanoparticles (DTNPs) was further demonstrated, which could
significantly enhance the delivery of fluorescent DH-BBR into
mitochondria of tumor cells, producing much stronger
mitochondrial fluorescence than that of DH-BBR. Moreover,
DTNPs also showed enhanced cytotoxicity against tumor cells
over normal cells via a mitochondria-dependent apoptosis
pathway, allowing for efficient inhibition of tumor growth without
obvious side effects in vivo. This study has demonstrated the
great potential of using self-assembly approach to engineer
mitochondria-targeting small molecules for improved cancer
imaging and therapy.
Results and Discussion
Figure 1a shows the molecular structure of fluorescent
mitochondria-targeting compound DH-BBR and the general
procedure for the preparation of DTNPs using the lipophilic anion-
driven self-assembly approach. DH-BBR is designed to possess
a full π-conjugated four-ring structure in relative to BBR,
contributing to a longer fluorescence emission wavelength and
higher fluorescence quantum yield for tumor cell imaging.
Moreover, DH-BBR also possesses a planar structure, which can
provide strong molecular interactions (e.g., π-π stacking) to
trigger efficient self-assembly into DTNPs. Figure 1b illustrates
the mechanism of action by which DTNPs selectively interact with
tumor cells via a proposed electrostatic interaction driven anion
exchange process. Upon delivery into tumor tissue via the EPR
effect, the tumor cell membranes, which are full of negative
charges, can trigger DTNPs degradation, liberating the free DH-
BBRꞏTPB ion-pair near the membrane. Subsequent anion
exchange between TPB- in the ion-pair and anions on the cell
membrane can facilitate cation DH-BBR+ entry into cells, while the
anion TPB- is blocked extracellularly due to the repulsion
interactions. After entering into tumor cells, the DLCs feature of
DH-BBR+ can prompt their accumulation in the mitochondria,
leading to mitochondrial dysfunction and release of cytochrome c
(cyt c) into the cytosol. This subsequently activates caspase-3,
triggers cell apoptosis, and ultimately induces cell death. Normal
Figure 1. General design of lipophilic anion-driven self-assembly of
mitochondria targeting fluorescent cation DH-BBR for enhanced cancer
treatment. (a) The chemical structure of BBR, DH-BBR and the general
procedure to form DTNPs through self-assembly of DH-BBRꞏTPB ion-pair. (b)
Illustration of the mechanism of DTNPs selectively interact with tumor cells to
enhance cellular uptake and cytotoxicity via
apoptosis process.
a mitochondria-dependent
We then prepared the organic ion-pair DH-BBRꞏTPB, using the
ion-exchange approach shown in Scheme S2. As expected, DH-
BBRꞏTPB showed an increased partition coefficient (Log P =
2.75) compared to that of either DH-BBR+Cl- (-0.70) or BBR+Cl- (-
1.71) (Table S2). This increased lipophilicity could help DH-
BBRꞏTPB to self-assemble into DTNPs in aqueous solution.
Dynamic light scattering (DLS) analysis revealed the presence of
cell membranes are less negative than those of tumor cells[18]
;
therefore, uptake into normal cells through the proposed
electrostatic interaction-driven anion exchange process is
reduced, thus exerting weak cytotoxicity. Hence, DTNPs show
enhanced and selective cytotoxicity toward tumor cells.
mono-dispersed nanoparticles in water, with
a
mean
hydrodynamic size of ~110 nm (Figure 2a). This was also verified
by both transmission electron microscopy (TEM) and atomic force
microscopy (AFM) analysis, showing spherical nanoparticles with
an average diameter of ~ 100 nm (Figures. 2b & c). In contrast,
neither DH-BBR+Cl- nor Na+TPB- could self-assemble into
nanoparticles under the same conditions (Figure S4), suggesting
that the combination of cation DH-BBR+ and lipophilic anion TPB-
is essential to form DTNPs.
To clarify the molecular interactions for the self-assembly, a
single crystal structure of DH-BBRꞏTPB was obtained using X-ray
crystallography. A perspective view of DH-BBRꞏTPB is shown in
Figure 2d, and the crystal refinement data are summarized in
Table S3. The crystal packing structure of DH-BBRꞏTPB shows
two different stacking modes between DH-BBR cations along axis
b (Figure 2e & Figure S5). In mode one, the two DH-BBR cations
We first synthesized DH-BBR according to the approach
outlined in Scheme S1. Owing to the formation of a prolonged π-
conjugated coplanar structure, DH-BBR showed bathochromic
shifts of UV-Vis absorption (λabs = 453 nm) and fluorescence
emission (λem = 578 nm) relative to that of BBR (λabs/λem = 423/548
nm) in phosphate-buffered saline (PBS) buffer (pH 7.4) (Table S1).
More importantly, DH-BBR also showed a significantly higher
fluorescence quantum yield (Φf = 0.15) than that of BBR (0.0021)
in PBS buffer, emitting much higher fluorescence in tumor cells
(Figure S1). Subsequent colocalization studies with Mito-tracker
or Lyso-tracker demonstrated that DH-BBR accumulated mainly
in the mitochondria of HeLa cells (Figure S2), suggesting that the
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take an offset π-π stacking between the four aromatic rings from
each other, with an interplanar distance of 3.38 Å (red dash lines,
Figure 2e). Each cation adopts an antiparallel arrangement that
can reduce the steric/electrostatic repulsions. In mode two, the
two DH-BBR cations take a head-to-head stacking between the
aromatic ring A and 1,3-dioxolo ring from each other, offering two
strong Oꞏꞏꞏπ interactions (O2ꞏꞏꞏπ = 3.31 Å, green dashed lines).
These two different, strong intermolecular interactions promote
DH-BBR cations to pack together closely, forming supramolecular
chains. These are further connected by adjacent lipophilic TPB
anions through electrostatic interactions between N+ in DH-BBR+
and B- in TPB- (blue dashed lines, Figure 2f). Moreover, slipped
π-π stacking between one phenyl ring of TPB- and two aromatic
rings of DH-BBR+ (rings C and D) also occurs (magenta dashed
lines, interplanar distance of 3.78 Å), which can provide additional
molecular interactions to connect DH-BBR cations together
(Figure S6). As such, DH-BBRꞏTPB is prone to self-assemble into
nanoparticles through the synergy of π-π stacking, Oꞏꞏꞏπ
interaction and electrostatic interaction.
(DMEM) showed that DTNPs were very stable under
physiologically relevant conditions (Figure S8), attributing to the
small critical micelle concentration (CMC; 2.86 μM, Figure S9)
and negative zeta potential (-8.5 mV). Moreover, DTNPs were
also very stable either in PBS buffer with different pH (5.0-7.4) or
in water with the addition of varying NaCl concentrations (Figures
S10 & S11).
Figure 3. Evaluation of the uptake of DTNPs in cells using fluorescence imaging.
(a) Fluorescence imaging of Hela cells following incubation with DH-BBR (20
μM), DTNPs (20 μM), or HUVEC cells incubated with DTNPs (20 μM) for 2 h.
(b) Colocalization studies of HeLa cells following incubation with DTNPs
(pseudo green) and the Mito-Tracker Red (pseudo red, up) or Lyso-Tracker Red
(pseudo red, down). Cells were incubated with 200 nM the Mito-Tracker Red or
Lyso-Tracker Red for 20 min and then incubated with DTNPs (20 μM) for 1 h.
The colocalization of green fluorescence from the DH-BBR+ with red
fluorescence from the Mito-Tracker Red but not Lyso-Tracker Red suggests that
DH-BBR+ accumulates mainly in the mitochondria.
Figure 2. Nanocharacterization of DTNPs. (a) DLS analysis, (b) TEM and (c)
AFM images of DTNPs (20 μM) in H2O. (d) Single crystal structure of DH-
BBRꞏTPB. (e) The packing structure of DH-BBRꞏTPB revealed by X-ray
crystallography shows two different stacking modes between DH-BBR cations
along axis b. Red dash lines indicate the offset π-π stacking between two
adjacent DH-BBR cations, and green dash lines indicate the Oꞏꞏꞏπ interactions
between another two DH-BBR. The TPB- anions are omitted for clarity. (f) The
packing structure of DH-BBRꞏTPB shows the presence of both slipped π-π
stacking (magenta dash lines) and electrostatic interactions (blue dash lines)
between DH-BBR+ and TPB-. The phenyl rings in TPB- are shown green, and
the B atoms are shown magenta. All H atoms in the structures were omitted for
clarity.
The uptake of DTNPs into HeLa tumor cells was then studied
after optimizing the incubation conditions. DTNPs could enter into
HeLa cells in both incubation concentration- and time-dependent
manners (Figure S12), producing bright fluorescence in HeLa
cells after incubation with 20 μM DTNPs for 2 h (Figure 3a). The
strong fluorescence was also verified by flow cytometric analysis,
revealing that the fluorescence intensity in HeLa cells reached
maximum upon incubation with DTNPs (20 μM) for 2 h (Figure
S13). In contrast, the incubation of HeLa cells with DH-BBR+Cl-
(20 μM) for 2 h produce much weaker intracellular fluorescence
(Figure 3a). We then employed the high performance liquid
chromatography (HPLC) assay to quantify the cellular uptake of
DH-BBR+. We found that the amount of DH-BBR+ in each HeLa
cell following incubation with 20 μM DTNPs for 2 h was ~4.50 fmol,
which was more than 2-fold larger than that with DH-BBR+Cl-
(~1.89 fmol/cell) (Table 1). These results demonstrated that
DTNPs had a remarkably improved ability to deliver DH-BBR+ into
HeLa cells compared to that of DH-BBR+Cl-. The subsequent
colocalization study with either Mito-Tracker Red or Lyso-Tracker
Red revealed that the green fluorescence of DH-BBR+ overlapped
well with the red fluorescence of Mito-Tracker (Figures 3b & S14),
The self-assembly of DH-BBRꞏTPB into DTNPs in PBS buffer
resulted in a considerable red-shift (λabs = 480 nm) and broader
UV-vis absorption compared with that of DH-BBR+Cl- (Figure S7a).
DTNPs also showed significantly increased scattering around
500-650 nm, further confirming the formation of nanoparticles in
PBS buffer. This red-shift in the UV-vis absorption could be
attributed to the J-aggregation of DH-BBR cations in DTNPs,
which caused remarkable fluorescence quenching (Figure S7a).
The quenched fluorescence could be recovered when DTNPs
were degraded in DMSO (Figure S7b). The investigation of
stability of DTNPs (20 μM) in either PBS (1×, pH 7.4), serum-PBS
(PBS containing 10% mouse serum), or cell culture medium
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suggesting that DH-BBR+ was distributed mainly in the
mitochondria. It was notable that the DTNPs-induced
mitochondrial fluorescence in HeLa cells with S or G2/M phase
arrest was significantly higher than that in G0/G1 phase (Figure
S15), correlating well to the higher mitochondrial membrane
potential (Δψm) observed in S or G2/M phase.[19] These results
suggested that DTNPs was able to monitor the changes in
mitochondria during cell cycle progression.
fluorescence was substantially reduced when free phosphates
(10, 20, 40, and 60 mM) were added into the culture medium to
outcompete the phosphates on cell membrane (Figure S20),
confirming the important roles of cell membrane anions in
triggering cellular uptake of DTNPs.
Notably, the incubation of human umbilical vein endothelial
cells (HUVEC) with DTNPs showed significantly reduced
intracellular fluorescence relative to that of HeLa cells (Figure 3a),
indicating less uptake of DTNPs into HUVEC cells. This could be
attributed to the fact that normal cells generally possess much
fewer anions on their membranes,[18b,24] thus limiting the proposed
electrostatic interaction-driven anion exchange process to induce
cellular uptake. Additionally, the relatively less mitochondrial
activity in normal cells could also lead to lower intracellular
fluorescence.[25]
Table 1. Quantification of the amount of DH-BBR+ in HeLa cells following
incubation with DH-BBR+Cl- or DTNPs.
Cellular uptake of DH-BBR+ (fmol/cell) a
Probe
10 μM, 1 h
10 μM, 2 h
20 μM, 2 h
DH-BBR+Cl-
0.85
1.18
1.89
DTNPs
3.72
3.97
4.50
a
Calculated based on the HPLC assay using a standard curve. HeLa cells
(~1× 106) were incubated with DH-BBR+Cl- or DTNPs for 1 or 2 h, and then
lysed with RIPA buffer. The amount of DH-BBR+ in the cell lysates were
analyzed by HPLC, and normalized to each cell.
To investigate the mechanism involved in the cellular uptake of
DTNPs, we first incubated HeLa cells with 20 μM DTNPs at 4 ºC
for 2 h. Interestingly, there was little difference in intracellular
fluorescence compared with that at 37 ºC, suggesting that the
uptake of DTNPs was not energy-dependent (Figure S16). We
then pretreated HeLa cells with different endocytosis inhibitors,
such as chlorpromazine (CPZ),[20] Filipin III,[21] and 5-(N-ethyl-N-
isopropyl) amiloride (EIPA),[22] which also had little effect on the
intracellular fluorescence ( Figure S17). These results suggested
that the intracellular delivery of DTNPs did not rely on the
endocytosis/phagocytosis pathway. Furthermore, HPLC analysis
of both culture medium and lysed HeLa cells following incubation
with 20 μM DTNPs for 2 h showed that the majority of cationic
DH-BBR+ was taken up by HeLa cells, while the anionic TPB- was
distributed mainly extracellularly (Figure S 18). Bearing in mind
that cancer cell membranes are characterized by negative
charges, we proposed that the uptake of DTNPs might be related
to an electrostatic interaction-driven anion exchange process
(Figure 1b).[23] When DTNPs arrive at the surface of cell
membranes, the hydrophilic anions, such as phosphates, can
interact with the DH-BBR+ cations in DTNPs and cause
nanoparticle degradation. The subsequent anion exchange
between the TPB- in DH-BBRꞏTPB ion-pair and anions on the cell
membrane can facilitate DH-BBR+ entry into cells, ultimately
accumulating in the mitochondria, while the repulsion interaction
between membrane-bound anions and TPB- will prevent the
uptake of TPB-. As such, this process can help to bypass energy-
dependent endocytosis, leading to fast and enhanced cellular
uptake. Indeed, time-course fluorescence images of live HeLa
cells revealed that the fluorescence appeared at cell membranes
in only 1 min, and rapidly became brighter in the cytoplasm within
3 min (Figure S19 & Movie S1). In contrast, the intracellular
Figure 4. Investigation of the cytotoxicity against tumor cells. (a) Cell viability of
indicated cells following incubation with varying concentrations of DTNPs for 48
h. Values were mean ± SD (n = 3). (b) The quantitative measurement of
mitochondria membrane potential (∆ψm) of HeLa cells using mitochondria
staining dye JC-10. HeLa cells were untreated (control), treated with DTNPs (20
μM) for 4 h or a mitochondria uncoupling agent FCCP (20 μM) for 30 min, and
then incubated with JC-10 (10 μM) for additional 20 min. The intensities of green
fluorescence (IGreen, monomer of JC-10) and red fluorescence (IRed, aggregate
of JC-10) were measured on a microplate reader, respectively. The ∆ψm was
obtained from the IRed/IGreen, and normalized to the control HeLa cells. Values
were mean ± SD (n = 3, **P < 0.01, ***P< 0.001). (c) Immunostaining of cyt c in
untreated HeLa cells (control) or HeLa cells treated with DTNPs (20 μM) for 6
h. Scale bar: 20 μm. (d) Comparison of the relative caspase-3 activity in
untreated HeLa cells (control) or HeLa cells following treatment with DTNPs (20
μM) for 6 h. Values were mean ± SD (n = 3, ****P < 0.0001).
The cytotoxicity of DTNPs toward both tumor cells and normal
cells was then evaluated using the MTT assay. As shown in
Figure 4a, DTNPs exhibited high cytotoxicity against all the six
tested tumor cells, including HeLa, HCT116, MCF-7, MDA-MB-
213, MDA-MB-435, and MDA-MB-468 cells, with IC50 values of
~3.31, 3.48, 5.89, 4.05, 2.54, and 2.44 μM, respectively. The IC50
value of DTNPs toward MDA-MB-231 cells was approximately 8-
fold smaller than that of DH-BBR+Cl- (~32.87 μM), while Na+TPB-
alone showed almost no cytotoxicity (Figure S21). These results
suggested that the cation DH-BBR+ and anion TPB- combination
played a synergistic role in tuning the cytotoxicity against tumor
cells. In contrast, DTNPs showed much lower cytotoxicity against
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HUVECs cells, with more than 60% cells remaining alive after
incubation with DTNPs (50 μM) for 48 h (Figure 4a), consistent
with their reduced uptake in HUVEC cells (Figure 3a).
injection, once every two days for a total of ten times. As shown
in Figure 5a, the tumor volumes in the mice receiving i.v. injection
of DTNPs (10 mg/kg) were significantly reduced compared with
those receiving either DH-BBR (10 mg/kg) or saline as the control.
The average tumor volume in the DTNPs-treated mice at day 20
was ~58% smaller than that treated with saline (~5-fold vs ~12-
fold). Mice treated with DTNPs did not show an obvious change
in body weight compared with those treated with saline, implying
minimal side effects of DTNPs in the mice (Figure 5b). The
significant difference in tumor size was also confirmed by
photograph of tumors resected from mice after treatment for 20
days (Figure 5c). Hematoxylin-eosin (H&E) and terminal
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
staining of tumor tissue slices resected from DTNPs-treated mice
showed significant cell death and apoptosis in the tumor tissues,
while tumor tissues resected from saline-treated mice showed
negligible cell death and apoptosis (Figures 5d & 5e). The obvious
tumor cell apoptosis could be attributed to the efficient uptake of
DTNPs into tumors, which was revealed by the remarkable
fluorescence in DTNPs-treated tumor tissues (Figure 5f). These
results indicated that DTNPs assembled from the DH-BBRꞏTPB
ion-pair were able to efficiently enter into tumors and inhibit tumor
growth in living mice. To the best of our knowledge, this is the first
time to show the in vivo antitumor activity of BBR-based
compounds. However, Figure 5a also showed that the tumor
remained growing during the course of therapy with DTNPs, albeit
the growth rate was significantly reduced. It is still very
challenging to eliminate the tumors in vivo using DTNPs only. In
We next investigated the potential mechanism of DTNPs to
induce tumor cell death. We found that the fluorescence of DTNPs
in HeLa cells was significantly reduced when the incubation was
prolonged over 4 h (Figures. S12a & S13), which was also
observed when HeLa cells were pretreated with a mitochondrial
uncoupling agent p-triflouromethoxyphenylhydrazone (FCCP)
(Figure S22). The reduced fluorescence could be attributed to the
decline of membrane potential in mitochondria, which was
confirmed by the subsequent measurement of the Δψm with a
commercial JC-10 dye. It was revealed that DTNPs could
apparently reduce the Δψm to a level similar to that induced by
FCCP (Figures 4b & S23). In addition, immunostaining of cyt c in
DTNPs-treated HeLa cells showed an obvious distribution of cyt
c in the cytosol, different to that observed in non-treated viable
cells, in which the cyt c was distributed mainly in the mitochondria
(Figure 4c). These results suggested that the selective
accumulation of cation DH-BBR+ from DTNPs in the mitochondria
of cancer cells could efficiently trigger mitochondrial dysfunctions,
resulting in the release of DH-BBR+ and cyt c to cytosol. As cyt c
is a proapoptotic inducer that can activate caspase cascades and
induce cell apoptosis,[26] caspase-3 activity and apoptosis events
in DTNP-treated HeLa cells were next examined. The results in
Figure 4d showed that the caspase-3 activity in HeLa cells was
significantly increased upon treatment with DTNPs, which could
trigger significant cell apoptosis, as revealed by the flow
cytometric analysis of DTNPs-treated HeLa cells with Annexin
V/propidium iodide (PI) staining (Figure S24). These results
demonstrated that DTNPs could induce severe cell death through
a mitochondria-dependent apoptosis pathway.
future,
a
combination chemotherapy with DTNPs and
immunotherapy with antibodies (e.g., Anti-PD-L1, vaccines)[27]
may largely improve the therapeutic effect against cancers.
Conclusions
In conclusion, we report the study of using a lipophilic anion
TPB-driven self-assembly of a novel mitochondria targeting
fluorescent compound, DH-BBR, into organic nanoparticles
DTNPs for cell imaging and improved antitumor efficacy. We
employed X-ray crystallography to reveal that the formation of
DTNPs was caused by a synergy of strong molecular interactions,
including π-π stacking, Oꞏꞏꞏπ interaction and electrostatic
interaction. The subsequent fluorescence imaging showed that
DTNPs could remarkably enhance the uptake of DH-BBR into
tumor cells through an electrostatic interaction-mediated anion
exchange process, inducing significant mitochondrial dysfunction
and cell apoptosis. Moreover, DTNPs could enhance the delivery
of DH-BBR into tumors following i.v. administration, resulting in
efficient inhibition of tumor growth with negligible side effects in
living mice. The results reported here revealed that the use of
lipophilic anions to trigger self-assembly of DH-BBR into
nanoparticles was promising for cancer treatment, which could
serve as a general approach to enhance the delivery of DLC-
based small molecules into tumors, thus improving antitumor
efficacy.
Figure 5. Antitumor efficiency of DTNPs in vivo. (a,b) The change of tumor
volumes (a) and body weight (b) of HeLa tumor-bearing nude mice following i.v.
injection with saline, 10 mg/kg DH-BBR or 10 mg/kg DTNPs for 20 days. Values
were mean ± SD (n = 5, ***P < 0.001). Red arrows in (a) indicated injection days.
(c) Photograph of HeLa tumors resected from mice after indicated treatment at
day 20. (d,e,f) H&E staining (d), TUNEL staining (e) and fluorescence imaging
(f) of tumor tissue slices resected from HeLa tumor-bearing mice following
treatment with saline or DNTPs. The mice were treated with saline or DTNPs
(10 mg/kg) every other day, and last for 20 days. The tumors were resected
from mice on day 20.
Encouraged by the enhanced cellular uptake and cytotoxicity,
we next examined the antitumor activity in nude mice bearing
subcutaneous HeLa tumors. Mice were administered with saline,
DH-BBR+Cl- or DTNPs (10 mg/kg) in saline through intravenous
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10.1002/chem.201801112
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PBS for three times, the cells were then incubated with 20 μM DH-BBR or
DTNPs for another 1 h. The fluorescence images of the cells were
captured on the fluorescent microscope.
Experimental Section
General Materials and Methods. All chemicals and biological reagents
were purchased from commercial suppliers and used without further
purification. Analytical thin-layer chromatography (TLC) was HSGF 254
(150–200 μm thickness, Yantai Huiyou Company, China). Nuclear
magnetic resonance (NMR) spectra were performed on a Bruker AMX-400
and AMX-500 NMR. Chemical shifts were reported in parts per million
(ppm, δ) downfield from tetramethylsilane (TMS). Proton coupling patterns
were described as singlet (s), doublet (d), triplet (t), quartet (q), multipet
(m) and broad (br). Low- and high-resolution mass spectra (MS and
HRMS) were given with electrospray ionization (ESI) and electric ionization
(EI) produced by Finnigan MAT-95. Single crystal x-ray diffraction data
were collected using Bruker-AXS SMART-APEXII CCD diffractometer.
HPLC analysis was conducted on Thermo Fisher U3000 HPLC systems.
Absorption spectra were recorded an Ocean Optics UV-vis spectrometer.
Cytotoxicity assay (MTT). The cells were seeded in flat-bottomed 96-well
plates (5000 cells/well) and incubated at 37°C for 24 h. Varying
concentration of BBR, DH-BBR or DTNPs in DMEM medium were then
added. After being incubation for 48 h, 50 μL MTT solution (1 in PBS)
was then added into each well. The cells were kept at 37°C for 4 h, and
the solution in each well was then removed carefully. The resulting purple
formazan crystals in the wells were dissolved by addition of 100 μL DMSO.
The absorbance (OD) at 550 nm in each well was acquired on a microplate
reader (Tcan). The absorbance of blank cells (OD control) were used as
the control, and the percentage of cell viability in each treatment was
calculated by dividing OD to OD control.
Flow cytometry analysis. Approximately 1× 106 HeLa cells in 2 mL
DMEM medium were seeded onto 6-well plates. After being incubation at
37 °C overnight, the medium was replaced with 1 mL of fresh medium
containing 20 μM DTNPs. and the cells were incubated for 10 min, 0.5, 1,
2, 4 and 6 h. After being washed with PBS (×1) for three times, trypsin (300
μL) was added into each well, and kept at 37 °C for 5-10 min to detach the
cells. The cell pellets were then collected after centrifugation at 1000 rpm
at 4 °C for 4 min. After being washed with cold PBS buffer, the cell pellets
were resuspended in 1 mL cold PBS, and the flow cytometry analysis was
conducted on a Beckman FC500 flow cytometer with excitation at 488 nm.
For each analysis, 10000 cells were used.
Fluorescence spectra were collected on
a HORIBA Jobin Yvon
Fluoromax-4 fluorescence spectrometer. Absolute quantum yield was
measured by calibrated integrating sphere system (Integrating Sphere F-
3018, HORIBA Jobin Yvon). Fluorescence imaging assays were
conducted on a Leica SP5 confocal laser scanning microscope or an
Olympus IX73 fluorescent inverted microscope. Dynamic light scattering
analysis and Zeta potential analysis were conducted on a 90 Plus/ BI-MAS
equipment (Brook haven, USA). Flow cytometric analysis were conducted
on a Coulter FC-500 flow cytometer (Beckman Coulter, USA). MTT assay
was performed on a microplate reader (Tcan). The in vivo MR imaging
experiments were performed on a 1.0 T small animal MRI system (Bruker
ICON).
Quantitative analysis of DH-BBR in HeLa cells. Approximately 1× 106
HeLa cells in 2 mL DMEM medium were seeded onto 6-well plates. After
being incubation at 37 °C overnight, the medium was replaced with 1 mL
fresh DMEM containing 10 or 20 μM DH-BBR or DTNPs. The cells were
incubated for 1 or 2 h. Then, the medium was carefully removed and the
cells were washed with cold PBS three times. The cells were trypsinized,
collected, and the cell number were counted. After being lysed with 50 μL
RIPA (Radio-Immunoprecipitation Assay) buffer, the cell lysates were
collected, mixed with 100 μL cold MeOH, and centrifuged at 14,000×g at
4 °C for 10 min. The supernatant was collected, and then injected into an
HPLC system for analysis. The amount of DH-BBR in each well was then
measured using the standard curve of DH-BBR, with results summarized
in Table 1 and Figure S15.
Synthesis. DH-BBR and DH-BBRꞏTPB were synthesized according to the
procedure in Schemes S1 and S2. The structure characterizations of them
were described in the Supporting Information.
Single crystal structure determination. The crystal of DH-BBRꞏTPB was
obtained by slow evaporation of the CH3OH-CH2Cl2 solution of DH-
BBRꞏTPB at r.t.. A single crystal with dimensions of 0.12  0.1  0.1 mm3
was used for structural determination, and the structure refinement data
were collected on a Bruker-AXS SMART-APEXII CCD diffractometer using
graphite-monochromated Mo K radiation (λ = 0.710 73 Å) at room
temperature. The crystallographic data for complex DH-BBRꞏTPB are
listed in Table S3. The refinement details could be found in the cif file
(CCDC 1588249) of DH-BBRꞏTPB, which are provided free of charge by
Mitochondrial membrane potential assay. HeLa cells (~5 × 104) were
seeded to a glass-bottom cell culture dish, and kept growing overnight.
DTNPs (20 μM) or FCCP (20 μM) in FBS free medium were then added,
and the cells were kept incubation at 37 °C for 4 h for DTNPs or 30 min for
FCCP. After removal of the medium, the cells were washed with cold PBS
three times. The mitochondria staining dye JC-10 (10 μM) in fresh medium
was then added, and the cells were kept incubation for additional 20 min.
Then, the medium was removed, and the cells was washed with cold PBS
buffer for three times. The fluorescence images of cells were then acquired
on a Leica TCS SP5 confocal laser scanning microscope. A green laser
(488 nm) with the emission filter from 500-540 nm was used to detect the
green fluorescence of the monomer of JC-10 dye, and a yellow-green laser
(543 nm) with the emission filter from 570-650 nm was used to detect the
red fluorescence of the aggregates of JC-10 dye. In addition, the
mitochondrial membrane potential was measured using a microplate
assay. HeLa cells were seeded in 96-well plates with a cell density of 1 ×
104 cells per well. After being incubation at 37 °C overnight, cells were then
incubated with FCCP (20 μM) for 30 min or DTNPs (20 μM) for 4 h. After
removal of the medium, the cells were washed with cold PBS three times.
JC-10 (10 μM) in fresh medium was then added, and the cells were kept
incubation for additional 20 min. Then, the medium was removed, and the
cells was washed with cold PBS buffer for three times. Both the monomer
The
Cambridge
Crystallographic
Data
Centre
via
www.ccdc.cam.ac.uk/data_request/cif.
Cell cultures. HeLa, HCT116, MCF-7, MDA-MB-213, MDA-MB-435 and
MDA-MB-468 tumor cells were grown in high glucose DMEM medium
(Gibco) supplemented with 10% (v/v) fetal bovin serum (FBS) and 1% (v/v)
penicillin/steptomycin (PS). All cells were cultured in a 5% CO2 humidified
incubator at 37 ºC.
Fluorescence imaging and colocalization analysis. HeLa cells (~5 
104) were seeded to a glass-bottom cell culture dish, and kept growing
overnight. Varying concentrations of BBR, DH-BBR or DTNPs (5, 10, 20
μM) in FBS free DMEM medium were added into cells and incubated at 37
ºC for 10 min, 0.5, 1, 2, 4 and 6 h. After incubation, the medium was
removed and the cells were washed with cold PBS for three times.
Fluorescence images were then acquired using an Olympus IX73
fluorescent inverted microscope.
For Co-localization studies, HeLa cells were incubated with 200 nM
MitroTracker
(LysoTracker™ Red DND-99) for 20 min. After being washed with cold
(MitoTracker™
Red
CMXRos)
or
LysoTracker
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10.1002/chem.201801112
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and aggregates of JC-10 in each well were measured on a Tecan
microplate reader. The green fluorescence of JC-10 (monomer) at 535 nm
was measured with excitation at 485 nm, and the red fluorescence JC-10
(aggregates) at 596 m was measured with excitation at 540 nm. The
aggregate/monomer ratio in each well was calculated and used to
determine the mitochondrial membrane potential Δψm. Each measurement
was repeated for three times, and the data were expressed as mean ±
standard deviation (SD).
sectioned into 10-μm slices. The H&E staining and TUNEL staining were
performed according to the manufacturer’s instructions (KeyGen Biotech.
Co. Ltd., Nanjing, China). The images were acquired on an IX73 optical
microscope equipped with a color camera.
Statistical analysis. Statistical comparisons between two groups were
determined by Student’s t-test. p < 0.05 was considered to be statistical
significance. The statistical tests were run with Prism 6 (Prism GraphPad
Software, Inc., San Diego).
Immunofluorescence staining of cyt c. Immunofluorescence staining of
cyt c was performed using a recombinant rabbit monoclonal anti-cyt c
primary antibody and an Alexa Fluor 594-conjugated donkey anti-rabbit
secondary antibody (abcam) according to the protocol described by the
manufacture. Briefly, HeLa cells were treated with 20 μM DTNPs for 6 h,
fixed by polyformaldehyde, followed by staining with anti-cyt c primary
antibody and secondary antibody. After being staining, the nucleus was
further stained with DAPI, and the fluorescence images were then acquired.
Acknowledgements
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (81620108027, 21632008,
21775071, 91229204 and 81220108025), the Major Project of
Chinese National Programs for Fundamental Research and
Development (2015CB910304) and National S&T Major Projects
(2014ZX09507002-001).
Measurement of Caspase-3 activity. HeLa cells were seeded in 96-well
plates with a cell density of 1 × 104 cells per well, and incubated at 37 °C
overnight. Then, the culture medium was replaced with 100 μL FBS free
medium containing 20 μM DTNPs. After incubation at 37 °C for 6 h, the
caspase-3 activity in each well was measured using the Caspase-Glo@ 3
assay (Promega) according to the standard protocol described by the
manufacture. Briefly, 100 μL of Caspase-Glo@ 3 reagent was added to
each well. The plate was then gently mixed, and incubated under dark at
r.t. for 30 min. The resulting luminescence was then measured on a Tecan
microplate reader. Data were then background-corrected, normalized, and
the caspase-3 activity was expressed as normalized increased fold of
luminescence intensity compared to the untreated control cells.
Keywords: self-assembly • dehydroberberine • mitochondria •
cell imaging • antitumor
[1]
a) G. Liang, H. Ren, J. Rao, Nat. Chem. 2010, 2, 54-60; b) Y. Gao,
J. Shi, D. Yuan, B. Xu, Nat. Commun. 2012, 3, 1033; c) D. Ye, A. J.
Shuhendler, L. Cui, L. Tong, S. S. Tee, G. Tikhomirov, D. W. Felsher,
J. Rao, Nat. Chem. 2014, 6, 519-526; d) Y. Yuan, S. Ge, H. Sun, X.
Dong, H. Zhao, L. An, J. Zhang, J. Wang, B. Hu, G. Liang, ACS
nano 2015, 9, 5117-5124; e) D. Zhang, G. B. Qi, Y. X. Zhao, S. L.
Qiao, C. Yang, H. Wang, Adv. Mater. 2015, 27, 6125-6130.
Endocytosis inhibition studies. The inhibitors (chlorpromazine (CPZ) for
clathrin-mediated endocytosis, filipin III for caveolae-mediated endocytosis
and ethylisopropyl amiloride (EIPA) for micropinocytosis) that function at
different endocytotic processes were used to determine the possible
pathways of cellular uptake. The HeLa cells were treated with or without
each inhibitor (30 μM CPZ, 5 μg/mL filipin III or 100 μM EIPA) for 30 min,
and then treated with DTNPs (20 μM) for another 2 h. After being washed
with cold PBS three times, the fluorescence images were acquired using
a fluorescence microscope. The resulting intracellular fluorescence were
quantified for the comparison.
[2]
a) A. G. Cheetham, P. Zhang, Y. A. Lin, L. L. Lock, H. Cui, J. Am.
Chem. Soc. 2013, 135, 2907-2910; b) P. Huang, D. Wang, Y. Su,
W. Huang, Y. Zhou, D. Cui, X. Zhu, D. Yan, J. Am. Chem. Soc. 2014,
136, 11748-11756; c) Y. Yuan, L. Wang, W. Du, Z. Ding, J. Zhang,
T. Han, L. An, H. Zhang, G. Liang, Angew. Chem. Int. Ed. 2015, 54,
9700-9704; d) J. Zhang, Y. C. Liang, X. Lin, X. Zhu, L. Yan, S. Li, X.
Yang, G. Zhu, A. L. Rogach, P. K. Yu, P. Shi, L. C. Tu, C. C. Chang,
X. Zhang, X. Chen, W. Zhang, C. S. Lee, ACS Nano 2015, 9, 9741-
9756; e) Y. Cai, H. Shen, J. Zhan, M. Lin, L. Dai, C. Ren, Y. Shi, J.
Liu, J. Gao, Z. Yang, J. Am. Chem. Soc. 2017, 139, 2876-2879; f)
A. G. Cheetham, R. W. Chakroun, W. Ma, H. Cui, Chem. Soc. Rev.
2017, 46, 6638-6663; g) Z. Zhang, L. Shi, C. Wu, Y. Su, J. Qian, H.
Deng, X. Zhu, ACS Appl. Mater. Interfaces 2017, 9, 29505-29514.
a) Y. Kuang, B. Xu, Angew. Chem. Int. Ed. 2013, 52, 6944-6948; b)
J. Shi, B. Xu, Nano Today 2015, 10, 615-630; c) Y. Huang, G. Zhang,
F. Hu, Y. Jin, R. Zhao, D. Zhang, Chem. Sci. 2016, 7, 7013-7019;
d) Y. Wen, W. Zhang, N. Gong, Y. F. Wang, H. B. Guo, W. Guo, P.
C. Wang, X. J. Liang, Nanoscale 2017, 9, 14347-14356.
Evaluation of antitumor activity in living mice. Animal care and
euthanasia were carried out with the approval of the institutional animal-
use and –care regulations of the Model Animal Research Center of Nanjing
University (MARC). Six-week-old female nude mice were injected
subcutaneously with 4 × 106 HeLa cells to establish xenograft HeLa tumors
in the right forelimb axilla. The tumors were allowed to grow to a mean
volume of ~250 mm3 for the antitumor activity studies. The mice were
divided into three groups randomly (n = 5). Mice were administered
intravenously with saline, 10 mg/kg DH-BBR or 10 mg/kg DTNPs every
other day, and last for 20 days. The tumor volume and body weight for
each mouse were measured in every other day. The tumor volume was
calculated as (tumor length) × (tumor width)2/2, and the relative tumor
volumes were calculated as V/V0 (V0 was the tumor volume when the
treatment was initiated).
[3]
[4]
a) L. Adler-Abramovich, L. Vaks, O. Carny, D. Trudler, A. Magno, A.
Caflisch, D. Frenkel, E. Gazit, Nat. Chem. Biol. 2012, 8, 701-706; b)
F. Clerici, E. Erba, M. L. Gelmi, S. Pellegrino, Tetrahedron Lett.
2016, 57, 5540-5550; c) K. Tao, A. Levin, L. Adler-Abramovich, E.
Gazit, Chem. Soc. Rev. 2016, 45, 3935-3953.
[5]
[6]
a) E. De Santis, M. G. Ryadnov, Chem. Soc. Rev. 2015, 44, 8288-
8300; b) K. Liu, R. Xing, Q. Zou, G. Ma, H. Mohwald, X. Yan, Angew.
Chem. Int. Ed. 2016, 55, 3036-3039; c) M. Abbas, Q. Zou, S. Li, X.
Yan, Adv. Mater. 2017, 29, 1605021.
Histopathology evaluation of tumor tissues. After treatment on day 20,
the mice were sacrificed and the tumors were resected. After photograph,
the tumors were frozen in optimal cutting temperature medium (OCT) and
cut into 10 μm thick slices using a vibrating-blade microtome. The
fluorescence images of the tumor tissue slices were then acquired on a
Leica TCS SP5 confocal laser scanning microscope. For H&E staining and
TUNEL staining, tumors were fixed in formalin. Then, the tumors were
a) J. F. Lovell, C. S. Jin, E. Huynh, H. Jin, C. Kim, J. L. Rubinstein,
W. C. Chan, W. Cao, L. V. Wang, G. Zheng, Nat. Mater. 2011, 10,
324-332; b) D. Ye, G. Liang, M. L. Ma, J. Rao, Angew. Chem. Int.
Ed. 2011, 50, 2275 -2279; c) J. Wang, F. Chen, S. J. Arconada-
Alvarez, J. Hartanto, L. P. Yap, R. Park, F. Wang, I. Vorobyova, G.
This article is protected by copyright. All rights reserved.

10.1002/chem.201801112
Chemistry - A European Journal
FULL PAPER
Dagliyan, P. S. Conti, J. V. Jokerst, Nano Lett. 2016, 16, 6265-6271;
b) L. Zhang, X. J. Miao, X. Wang, H. H. Pan, P. Li, H. Ren, Y. R. Jia,
C. Lu, H. B. Wang, L. Yuan, G. L. Zhang, Sci. Rep. 2016, 6, 28116.
[17] a) G. C. Pereira, A. F. Branco, J. A. Matos, S. L. Pereira, D. Parke,
E. L. Perkins, T. L. Serafim, V. A. Sardao, M. S. Santos, A. J.
Moreno, J. Holy, P. J. Oliveira, J. Pharmacol. Exp. Ther. 2007, 323,
636-649; b) K. N. Chidambara Murthy, G. K. Jayaprakasha, B. S.
Patil, Eur. J. Pharmacol. 2012, 688, 14-21.
d) M. Zheng, Y. Wang, H. Shi, Y. Hu, L. Feng, Z. Luo, M. Zhou, J.
He, Z. Zhou, Y. Zhang, D. Ye, ACS Nano 2016, 10, 10075-10085;
e) Y. Cai, P. Liang, Q. Tang, X. Yang, W. Si, W. Huang, Q. Zhang,
X. Dong, ACS Nano 2017, 11, 1054-1063; f) Q. Zou, M. Abbas, L.
Zhao, S. Li, G. Shen, X. Yan, J. Am. Chem. Soc. 2017, 139, 1921-
1927; g) J. Wang, C. Y. Lin, C. Moore, A. Jhunjhunwala, J. V.
Jokerst, Langmuir 2018, 34, 359-365.
[18] a) M. Yang, W. J. Brackenbury, Front. Physiol. 2013, 4, 185; b) B.
Chen, W. Le, Y. Wang, Z. Li, D. Wang, L. Lin, S. Cui, J. J. Hu, Y.
Hu, P. Yang, R. C. Ewing, D. Shi, Z. Cui, Theranostics 2016, 6,
1887-1898.
[7]
[8]
a) D. Ye, A. J. Shuhendler, P. Pandit, K. D. Brewer, S. S. Tee, L.
Cui, G. Tikhomirov, B. Rutt, J. Rao, Chem. Sci. 2014, 4, 3845-3852;
b) Y. Yuan, F. Wang, W. Tang, Z. Ding, L. Wang, L. Liang, Z. Zheng,
H. Zhang, G. Liang, ACS Nano 2016, 10, 7147-7153.
[19] M. Martínez-Diez, G. Santamaría, Á. D. Ortega, J. M. Cuezva, Plos
One 2006, 1, e107.
a) J. Li, Y. Kuang, J. Shi, J. Zhou, J. E. Medina, R. Zhou, D. Yuan,
C. Yang, H. Wang, Z. Yang, J. Liu, D. M. Dinulescu, B. Xu, Angew.
Chem. Int. Ed. 2015, 54, 13307-13311; b) H. Wang, Z. Feng, D. Wu,
K. J. Fritzsching, M. Rigney, J. Zhou, Y. Jiang, K. Schmidt-Rohr, B.
Xu, J. Am. Chem. Soc. 2016, 138, 10758-10761; c) P. K. Magut, S.
Das, V. E. Fernand, J. Losso, K. McDonough, B. M. Naylor, S.
Aggarwal, I. M. Warner, J. Am. Chem. Soc. 2013, 135, 15873-15879.
a) Z. Feng, H. Wang, R. Zhou, J. Li, B. Xu, J. Am. Chem. Soc. 2017,
139, 3950-3953; b) L. L. Li, S. L. Qiao, W. J. Liu, Y. Ma, D. Wan, J.
Pan, H. Wang, Nat. Commun. 2017, 8, 1276.
[20] C. Dalal, A. Saha, N. R. Jana, J. Phys. Chem. C 2016, 120, 6778-
6786.
[21] J. V. Georgieva, D. Kalicharan, P. O. Couraud, I. A. Romero, B.
Weksler, D. Hoekstra, I. S. Zuhorn, Mol. Ther. 2011, 19, 318-325.
[22] N. K. Y. Wong, R. A. Shenoi, S. Abbina, I. Chafeeva, J. N.
Kizhakkedathu, M. K. Khan, Biomacromolecules 2017, 18, 2427-
2438.
[9]
[23] a) N. Chuard, K. Fujisawa, P. Morelli, J. Saarbach, N. Winssinger,
P. Metrangolo, G. Resnati, N. Sakai, S. Matile, J. Am. Chem. Soc.
2016, 138, 11264-11271; b) D. Sobot, S. Mura, P. Couvreur, J.
Mater. Chem. B. 2016, 4, 5078-5100.
[10] a) A. Shao, Y. Xie, S. Zhu, Z. Guo, S. Zhu, J. Guo, P. Shi, T. D.
James, H. Tian, W. H. Zhu, Angew. Chem. Int. Ed. 2015, 54, 7275-
7280; b) S. R. MacEwan, A. Chilkoti, Angew.Chem. Int. Ed. 2017,
56, 6712-6733.
[24] a) E. Hondroulis, R. Zhang, C. Zhang, C. Chen, K. Ino, T. Matsue,
C. Z. Li, Theranostics 2014, 4, 919-930; b) C. Gui, E. Zhao, R. T. K.
Kwok, A. C. S. Leung, J. W. Y. Lam, M. Jiang, H. Deng, Y. Cai, W.
Zhang, H. Su, B. Z. Tang, Chem. Sci. 2017, 8, 1822-1830.
[25] a) V. R. Fantin, P. Leder, Cancer Res. 2004, 64, 329-336; b) S. Wu,
Q. Cao, X. Wang, K. Cheng, Z. Cheng, Chem. Commun. 2014, 50,
8919-8922.
[11] a) S. Karnewar, S. B. Vasamsetti, R. Gopoju, A. K. Kanugula, S. K.
Ganji, S. Prabhakar, N. Rangaraj, N. Tupperwar, J. M. Kumar, S.
Kotamraju, Sci. Rep. 2016, 6, 24108; b) C. J. Zhang, J. Wang, J.
Zhang, Y. M. Lee, G. Feng, T. K. Lim, H. M. Shen, Q. Lin, B. Liu,
Angew. Chem. Int. Ed. 2016, 55, 13770-13774.
[12] N. Yang, M. Weinfeld, H. Lemieux, B. Montpetit, I. S. Goping, Cell
Death Dis. 2017, 8, e2587.
[26] a) M. E. Fitch, C. M. Chang, T. G. Parslow, Cell Death Differ. 2000,
7, 338-349; b) M. A. McDonnell, D. Wang, S. M. Khan, M. G. Vander
Heiden, A. Kelekar, Cell Death Differ. 2003, 10, 1005-1015.
[27] a) J. Bellmunt, T. Powles, N. J. Vogelzang, Cancer Treat. Rev. 2017,
54, 58-67; b) H. Cai, Z.-H. Huang, L. Shi, Y.-F. Zhao, H. Kunz, Y.-
M. Li, Chem. Eur. J. 2011, 17, 6396-6406; c) C. H. Marzabadi, R.
W. Franck, Chem. Eur. J. 2016, 23, 1728-1742.
[13] J. Zielonka, J. Joseph, A. Sikora, M. Hardy, O. Ouari, J. Vasquez-
Vivar, G. Cheng, M. Lopez, B. Kalyanaraman, Chem. Rev. 2017,
117, 10043-10120.
[14] a) X. Zhang, Q. Ba, Z. Gu, D. Guo, Y. Zhou, Y. Xu, H. Wang, D. Ye,
H. Liu, Chem. Eur. J. 2015, 21, 17415-17421; b) A. Kurutos, I.
Orehovec, A. Tomašić Paić, I. Crnolatac, L. Horvat, N. Gadjev, I.
Piantanida, T. Deligeorgiev, Dyes and Pigments 2018, 148, 452-459.
[15] S. Luo, X. Tan, S. Fang, Y. Wang, T. Liu, X. Wang, Y. Yuan, H. Sun,
Q. Qi, C. Shi, Adv. Funct. Mater. 2016, 26, 2826-2835.
[16] a) N. Luiza Andreazza, C. Vevert-Bizet, G. Bourg-Heckly, F. Sureau,
M. Jose Salvador, S. Bonneau, Int. J. Pharm. 2016, 510, 240-249;
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Entry for the Table of Contents (Please choose one layout)
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Lipophilic anion-driven self-assembly
of fluorescent mitochondria-targeting
Ruibing An, Zhanni Gu, Haifeng Sun,
Yuxuan Hu, Runqi Yan, Deju Ye,* and
Hong Liu,*
dehydroberberine
into
organic
nanoparticles is demonstrated for
fluorescence imaging of tumor cells
and inhibition of tumor growth in mice.
Page No. – Page No.
Self-assembly
of
Fluorescent
Enhances
Antitumor
Dehydroberberine
Mitochondria-Dependent
Efficacy
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