Coordination-drIVen self-assembly of a Pt(IV) prodrug-conjugated supramolecular hexagon

Article abstract of DOI:10.1039/c7cc07622c

This article presents a new strategy to engage coordination-driven self-assembly for platinum drug delivery. The self-assembled supramolecular hexagon is conjugated with three equivalents of Pt(iv) prodrugs and displays a superior therapeutic index compared to cisplatin against a panel of human cancer cell lines.

Full text of DOI:10.1039/c7cc07622c

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Coordination-Driven Self-Assembly of a Pt(IV) Prodrug-Conjugated
Supramolecular Hexagon
Received 00th January 20xx,
Accepted 00th January 20xx
†
a
†a
b
c
c
a
a
Zhizhou Yueb, Han Wang,c Yiming Li, Yi Qin, Lin Xu, David J. Bowers, Mahinda Gangoda,
*a
Xiaopeng Li, Hai-Bo Yang, and Yao-Rong Zheng
DOI: 10.1039/x0xx00000x
www.rsc.org/
This article presents a new strategy to engage coordination-driven macromolecular/supramolecular inorganic structures with
1
2
self-assembly for platinum drug delivery. The self-assembled well-defined size and geometry.
Unlike many other
supramolecular hexagon is conjugated with three equivalents of macromolecular/supramolecular species, e.g. polymers or
Pt(IV) prodrugs and displays superior therapeutic index as liposomes, their structural features can be determined by
compared to cisplatin against a panel of human cancer cell lines.
NMR spectroscopy, mass spectrometry, and X-ray
1
3, 14
crystallography.
By virtue of their unique structural
Cisplatin, carboplatin, and oxaliplatin are the FDA-approved
platinum-based chemotherapeutics commonly used for cancer
features, these self-assembled structures have led to a variety
of innovative research in chemistry, material science, and
biology, such as the development of new catalysts, novel
energy storage platforms, etc. In recent years, studies about
applying coordination-driven self-assembly in the area of
1
therapy in clinics. Their anticancer activity arises from the
formation of Pt-DNA adducts through covalent bonds of the Pt
atoms with the purine nucleobases. These cross-links block
2
, 3
1
5-19
transcription, leading to apoptotic cell death.
Patients
cancer research has attracted increasing attention.
treated with Pt-based drugs usually suffer from a variety of
side effects including kidney damage, nerve damage, hearing
loss, and suppression of bone marrow activity. To overcome
theseundesired side effects, it is necessary to develop a
suitable drug delivery system for carrying anticancer agents to
Lippard, Therrien, Crowley, and Isaacs have respectively
published their works on designing self-assembled metal-
2
0-23
organic cages for anticancer drug delivery.
In all of these
reports, the therapeutic components were introduced via non-
covalent interactions.
4
, 5
the target location.
Pt(IV) prodrugs in combination with
much
In this report, we present the first example of using
covalent conjugation and coordination-driven self-assembly to
develop a delivery platform with well-defined size, geometry,
and Pt drug loading. As shown in Figure 1A, the Pt(IV) prodrug
nanodelivery is an approach that has received
attention, due to the versatility of Pt(IV) prodrugs to be
chemically modified and the superior therapeutic index of
nanodelivery. A large variety of delivery platforms have been
o
is covalently conjugated to a 120 dipyridyl building block.
6
investigated over the last decades, including polymeric
8
inorganic
Subsequently, the drug-conjugated building blocks (
o
assemble with the 120 organoplatinum linkers (
1
) self-
) and
7
nanoparticles, carbon-based nanomaterials,
2
9
-11
nanoparticles,
etc. However, a delivery system with well-
generate a supramolecular hexagon (
a payload of three Pt(IV) prodrug molecules. As a negative
control, the hexagon ( ) without Pt(IV) prodrugs was prepared
3), each of which carries
defined size and shape that demonstrates consistent drug
loading is still limited.
5
Coordination-driven self-assembly is a well-established
via the self-assembly of
2
and
4
(Scheme S1 in the
methodology
for
constructing
Supplementary Information).
o
The 120 Pt(IV) prodrug conjugated dipyridyl ligand (
1
) was
prepared via the amide-bond-formation reaction between
c,c,t-Pt(NH )Cl (ethyl carbamate)(succinate)] and the amino
[
3
2
dipyridyl linker with 45% yield (Scheme S2). The amino linker
was synthesized by the reaction between 3,5-di(2-(4-pyridyl
ethynyl)) phenol and 2-(Boc-amino)ethyl bromide and
subsequent
trifluoroacetic acid (v/v 2:1). Details of the synthesis can be
found in the Supplementary Information. Ligand is
deprotection
in
dichloromethane
and
1
anticipated to release cisplatin, ethylamine, CO , and a
2
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succinate dipyridyl ligand (9) upon reduction (Figure S1A). As with that observed in other supramolecular hexagons reported
1
4, 24
shown in Figure S1B, the HPLC peak (11.1 min) for
1
previously.
High-resolution ESI-MS was employed to
disappeared upon incubation with 20 eq. of ascorbate (10 further confirm the formation of the supramolecular hexagon.
o
mM) in aqueous solution for 15 h at 37 C, and a new peak (9.2 As shown in Figure 2B, the MS signals at m/z = 934.76, m/z =
min) corresponding to the reduction product (
9) was observed. 1151.47, m/z = 1476.60, and m/z = 2018.44 are corresponding
o
6+
5+
4+
3+
For the self-assembly, the Pt drug-conjugated 120
to [
) was reacted with the 120 organoplatinum respectively, and all the isotopically resolved patterns match
) in a 1:1 ratio in DMSO-d , and the mixture with the theoretical simulations (Figure S3). Combined
3 – 6OTf] , [3 – 5OTf] , [3 – 4OTf] , and [3 – 3OTf]
o
dipydriyl linker (
building block (
1
2
6
was stirred at R.T. for 2 h. As a result of coordination-driven evidence from NMR and MS support the coordination-driven
self-assembly (Figure 1A), the reaction generated the self-assembly of the supramolecular hexagons conjugated with
supramolecular hexagon (
Figure 2B), the hexagonal structure of
prodrug components. The structure of
3
). As depicted in the 3D model the three Pt(IV) prodrugs per ensemble.
bears three Pt(IV) The in vitro anticancer activity of the supramolecular
was characterized by hexagon was evaluated using the MTT assay (Figure 2A). Six
(
3
3
1
31
multinuclear ( H and P) NMR spectroscopy and electrospray human cancer cell lines were tested, including the non-small
1
ionization mass spectrometry (ESI-MS). In the H NMR spectra cell lung cancer cell line A549, ovarian cancer cell line A2780,
(
Figure 2C), the signals of the α- (peak a’) and β-pyridyl (peak and cisplatin-resistant ovarian cancer cell line A2780cis,
b’) protons of have been downfield shifted by 0.22 ppm and ovarian cancer cell line SKOV-3, colon cancer cell line HT29,
.32 ppm as compared to those (peak a and b) of , and is and triple-negative breast cancer cell line MDA-MB-231.
attributed to the coordination to the platinum centers of the Cancer cells were treated with cisplatin or the different test
3
0
1
3
1
1
acceptor (
2). Likewise, in the P{ H} spectrum (Figure S2 in SI), compounds for 72 h and cell viability was assessed.
95
1
a predominant singlet at δ = 13.61 ppm with two Pt Additionally, various control groups have been used in order to
1
satellites ( J = 2632 Hz) was observed corresponding to the clarify that the obtained cytotoxicity profiles represent the
Pt-P
formation of the hexagon (3). The NMR evidence is consistent intrinsic properties of the Pt prodrug-loaded hexagons, and
Figure 1. Coordination-driven self-assembly of the Pt(IV) prodrug-conjugated supramolecular hexagon: (A) illustration of the self-assembly
o
o
of the hexagon (3) using a 120 Pt(IV) prodrug-conjugated dipyridyl ligand (1) and 120 organoplatinum acceptor (2); (B). 3D computational
model of the self-assembled supramolecular hexagon (3), to which, three molecules of Pt(IV) prodrugs are conjugated (Carbon: grey;
2
| J. Name., 2012, 00, 1-3
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1
Oxygen: red; Nitrogen: blue; Phosphorous: purple; Chlorine: green; Platinum: pink); (C). H NMR spectra of 1 (top) and 3 (bottom) in
DMSO-d
6
; (D) ESI-MS spectra of 3. (Theoretical simulations in blue and experimental data in red).
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not from the individual components. These controls include with acetomethoxycalcein, or calcein AM (Figure 2C). Live cells
the individual building blocks (
1
and
2
), the mixture of
1
and
2
5
stain with calcein AM and yield a green fluorescence signal,
whereas dead cells exhibit no fluorescence or a red signal due
in cell culture media without self-assembly, and Hexagon
that does not carry Pt(IV) prodrugs (Scheme S1). Details of the to the ethidium homodimer-1. A549 lung cancer cells treated
synthesis and characterization of can be found in with ([Pt] = 7 μM) for 48 h were mostly dead, but those
5
3
Supplementary Information (Figure S4 and S5). The IC50 values, treated with cisplatin (7 µM) under the same conditions had
which represent the Pt concentration required to inhibit mostly survived. The observation in live/dead cell assay agrees
growth by 50%, are given in a table shown in Figure 2A. It is with the above-mentioned cytotoxicity profiles. Notably, as
clear that
lines and all test compounds. For example, in A549 lung cancer (IC50(A2780cis)/IC50(A2780) = 1.7) in ovarian cancer cell lines than
cell line (Figure 2B), IC50 ) = 0.87±0.13 μM is 18 times lower cisplatin (IC50(A2780cis)/IC50(A2780) = 7.0). To sum up, all these
than that of cisplatin (IC50 = 16.38±2.02 μM) and 100 times results suggest that the supramolecular hexagon shows
) itself (IC50 = 75.5±20.5 superior therapeutic index as compared to cisplatin in vitro.
was also confirmed by Further mechanistic studies were carried out to gain a
3 has the lowest IC50 values among all tested cell shown in Figure 2D, 3 displays a lower resistance factor
(
3
3
lower than that of the Pt(IV) prodrug (
1
μM). The in vitro anticancer activity of
3
using fluorescent microscopy and the LIVE/DEAD cell assay, a better insight into the anticancer activity of the
combination of the ethidium homodimer-1 assay and staining supramolecular hexagon . As shown in Figure 3A, the
cytotoxic effect of is hypothetically attributed to three key
3
3
steps, including cellular uptake, DNA damage triggered by
cisplatin released from reduction of the Pt(IV) prodrug, and
subsequent apoptosis. Cellular uptake of
3 and other
compounds has been determined using A549 cell line and
graphite furnace atomic absorption spectroscopy (GFAAS). As
a result (Figure 3B),
3
shows up to 20 times higher uptake as
. The high uptake of is
compared to cisplatin and ligand
1
3
believed to contribute to its high potency as discussed above.
To confirm DNA damage caused by cisplatin release, we
monitored changes in expression of biomarkers related to DNA
damage pathways. A549 cells incubated with
for 48 h showed a marked increase in phosphorylated H2AX
γH2AX) as determined by flow cytometry (Figure 3C), and
3 ([Pt] = 7 μM)
(
increases in p21 and p53 mRNA expression determined by real
time PCR (Figure S6). Consequently, the supramolecular
hexagon
programmed cell death (apoptosis). As shown in Figure S6, the
mRNA levels of BAX and Apaf-1 were upregulated in -treated
3 was able to kill cancer cells by triggering
3
A549 cells. By using a dual staining Annexin V/PI flow
cytometry assay, the occurrence of apoptosis was investigated
in A549 cells treated with
D clearly indicate that
A549 cells. Hexagon
undergo early (9.49%) and late (6.89%) stage apoptosis after
8 h of incubation, the populations of which were much higher
3
or cisplatin. The results in Figure
can efficiently induce apoptosis in
([Pt] = 7 µM) prompts A549 cells to
3
3
3
4
than those of cisplatin (Figure 3D). The evidence compiled
from the cell-based experiments well supports the conclusion
that
3 can readily enter cancer cells and release cisplatin to
damage DNA and trigger apoptosis to kill cancer cells.
Figure 2. Cytotoxicity profiles of the supramolecular hexagon (3):
A) A table of the IC50 values of cisplatin, 1, 2, and the mixture of 1
In conclusion, we demonstrate that coordination-driven
self-assembly can serve as a novel, precisely engineered drug
delivery platform and provide superior therapeutic index. For
the first time, through covalent conjugation of Pt(IV) prodrugs,
coordination-driven self-assembly allows for precise control of
drug loading, by which, three equivalents of the Pt payload are
incorporated onto each supramolecular hexagon. The drug-
loaded supramolecular complex has been well characterized
by multinuclear NMR spectroscopy and high-resolution mass
spectrometry. In cell-based experiments through MTT assay
and live/dead cell assay, we found that such supramolecular
(
and 2 in media without self-assembly, the hexagon conjugated
with Pt(IV) prodrugs (3), and the hexagon without Pt(IV) prodrugs
(5) determined by MTT assays; (B) bar graph of IC50 values obtained
using A549 cell line; (C) fluorescence microscopic images obtained
for the live/dead cell assay using A549 cells treated with 3 (7 μM
[Pt], 48 h) or cisplatin (7 μM [Pt], 48 h). Dead cells were labelled
with red and live cells were labelled with green; (D) representative
killing curves of cisplatin and 3 against A2780 and A2780cis ovarian
cancer cell lines.
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complexes have superior therapeutic properties, including sub-
μM potency against various human cancer cell lines and low
cross-resistance with cisplatin. According to our mechanistic
studies, such therapeutic properties mainly attribute to the
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Figure 3. Mechanistic studies of the anticancer activity of the Pt(IV)
prodrug-conjugated hexagon (3): (A) Schematic representation of
the key steps associated with the cytotoxic effect of 3; (B) Cellular
uptake determined by GFAAS. A549 cells were incubated with
different samples ([Pt] = 50 μM) for 4h; (C) Flow cytometric analysis
of phosphorylation of H2AX (γH2AX) level. A549 cells were
incubated with 3 ([Pt] = 7 μM) for 48 h; (D) Annexin V/PI flow
cytometric analysis of the apopotic events of A549 cells treated
with 3 or cisplatin ([Pt] = 7 μM, 48 h).
6
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Am. Chem. Soc., 2016, 138, 14488-14496.
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,
1
support by NSF (CHE-1506722). H.-B. Y. thanks the financial
support provided by the NSFC/China (Nos. 21625202 and
21672070) and STCSM (No. 16XD1401000).
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A supramolecular hexagon obtained from coordination-driven self-assembly can be used to
deliver platinum-based anticancer agents and promote their therapeutic index