Design, synthesis and evaluation of a novel π-π Stacking nano-intercalator as an anti-tumor agent

Article abstract of DOI:10.1039/c5md00507h

Based on the knowledge that cyclohexane-1,4-dione, piperazine and β-carboline are the essential building blocks of DNA intercalators, β-carboline-3-carboxylic acid is a π-π stack-like DNA intercalator, and β-carboline derivatives can form nanoparticles, this paper hypothesized that (2′S,5′S)-tetrahydropyrazino[1′,2′:1,6]- di{2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole}-1′,4′-dione (THPDTPI) would be a π-π stacking lead nano-intercalator. The docking investigation found that THPDTPI can intercalate into DNA in a π-π stacking manner. The simple condensation of 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid provided THPDTPI in good yield and high purity. The TEM, SEM and AFM imaging visualized that THPDTPI formed nanoparticles in ultrapure water, in the solid state and in rat plasma. The Faraday-Tyndall effect proved that THPDTPI exhibited nano-properties in pH 2.0 and pH 7.0 water. Spectrophotometric assays suggested that the interaction model of THPDTPI and CT DNA was π-π stacking intercalation. In vivo THPDTPI dose-dependently slowed the tumor growth of S180 mice with a minimal effective dose of 0.01 μmol kg^-1 per day. In vitro THPDTPI exhibited anti-proliferation activities on S180 and HeLa cells with IC₅₀ values of 0.39 and 3.5 μM, respectively. Even when the single dose was raised up to 10 000 fold of the minimal effective dose, i.e. 100 μmol kg^-1, THPDTPI still did not show liver, kidney and systemic toxicity in mice. These findings provide a strategy for designing THPDTPI-like π-π stacking nano-intercalators.

Full text of DOI:10.1039/c5md00507h

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Design, synthesis and evaluation of novel πꢀπ stacking
nanoꢀintercalator as antiꢀtumor agent†
Haimei Zhu,a‡ Yuanbo Song,c‡ Yuji Wang, a Ming Zhao,a,b* Yi Ren,a Yaonan Wang,a
Shurui Zhao,a Jianhui Wu,a Shiqi Penga*
aBeijing area major laboratory of peptide and small molecular drugs; Engineering
Research Center of Endogenous Prophylactic of Ministry of Education of China;
Beijing Laboratory of Biomedical Materials; College of Pharmaceutical Sciences, of
Capital Medical University, Beijing 100069, China; Eꢀmail: sqpeng@bjmu.edu.cn
bFaculty of Biomedical Science and Environmental Biology, Kaohsiung Medical
University, Kaohsiung, Taiwan
cGuangxi Pusen Biotechnology Co. ltd.
*To whom correspondence should be addressed. Eꢀmails: sqpeng@bjmu.edu.cn and
mingzhao@bjmu.edu.cn
.
‡ These authors contributed equally.
† The authors declare no competing interests.

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Abstract
Based on the knowledge that cyclohexaneꢀ1,4ꢀdione, piperazine and βꢀcarboline are
the essential building blocks of DNA intercalators, βꢀcarbolineꢀ3ꢀcarboxylic acid is a
πꢀπ stacking like DNA intercalator, and βꢀcarboline derivatives can form
nanoparticles, this paper hypothesized that (2’S,5’S)ꢀtetrahydropyrazino[1’,2’:1,6]ꢀ
di{2,3,4,9ꢀtetraꢀhydroꢀ1Hꢀpyrido[3,4ꢀb]indole}ꢀ1’,4’ꢀdione (THPDTPI) would be a
πꢀπ stacking lead nanoꢀintercalator. The docking investigation explored that
THPDTPI can intercalate into DNA with a πꢀπ stacking manner. The simple
condensation of 3Sꢀ1,2,3,4ꢀtetraꢀhydroꢀβꢀcarbolineꢀ3ꢀcarboxylic acid provided
THPDTPI in good yield and high purity. The TEM, SEM and AFM imaging
visualized that THPDTPI formed nanoparticles in ultrapure water, in solid state and in
rat plasma. FaradayꢀTyndall effect proved that THPDTPI exhibited nanoꢀproperties in
pH 2.0 and pH 7.0 water. Spectrophotometric assays suggested that the interaction
model of THPDTPI and CT DNA was πꢀπ stacking intercalation. In vivo THPDTPI
doseꢀdependently slowed the tumor growth of S180 mice with a minimal effective
dose of 0.01 ꢁmol/kg/day. In vitro THPDTPI exhibited antiꢀproliferation activities on
S180 and Hela cells with IC50 values of 0.39 and 3.5 ꢁM, respectively. Even when the
single dose was raised up to 10000 folds of the minimal effective dose, i.e.
100ꢁmol/kg, THPDTPI still did not show liver, kidney and systematic toxicity in mice.
These findings provide a strategy for designing THPDTPI like πꢀπ stacking
nanoꢀintercalators.
Keywords: πꢀπ stacking, intercalation, nanoparticles, antiꢀtumor, toxicity.

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Introduction
DNA is an inarguably indispensable target for developing antiꢀcancer drugs. The
constant struggle of developing potent anticancer drugs leads to the discovery of a
series of DNA binding lead compounds such as amino substituted
benzimidazoꢀ[1,2ꢀa]quinolines,1
3ꢀformylchromones,2
carbazoleꢀpyrrolo[2,1ꢀc][1,4]ꢀbenzodiazepine
acenaphtho[1,2ꢀb]pyrroles,6
ethidium
9ꢀaminoacridine
and
proflavine,3
conjugates,4
aaꢀTrpꢀTrpꢀOBzls,5
bromide
and
4ꢀmethylꢀ2,7ꢀdiaminoꢀ5,10ꢀdiphenylꢀ4,9ꢀdiazapyrenium hydrogensulfate,7 five and
six member ring dual DNA intercalators,8 triplexꢀselective indoloquinolines,9
conjugates of βꢀcarboline with chalcone,10 1,2,3ꢀtriazolꢀ1,8ꢀnaphthalimides,11 monoꢀ
and bisꢀphenothiaziniumepiperazinꢀexylene,12 nitroacridines, thienoquinolone,
cumoestrol, mitoxantrone, daidzein and genistein,13 bisꢀethidium and bisꢀacridine,14
acridineꢀ3ꢀcarboxamides
and
acridineꢀ4ꢀcarboxamides,15
and
Nꢀ(3ꢀbenzyloxycarbonylcarbolineꢀ1ꢀyl)ethylamino acid benzylesters.16 These lead
compounds are capable of targeting DNA either via groove binding or via
intercalation, of which piperazine, cyclohexaneꢀ1,4ꢀdione and βꢀcarboline are the
interesting building blocks. It is well documented that the groove binding determines
little perturbations of the Bꢀform DNA double helix, but via πꢀπ stacking the
intercalation can lead the paired DNA bases to an unwinding status and consequently
can overcome resistance phenomena common to other antiꢀcancer drugs.17,18,19 It is
also well documented that πꢀπ stacking is a key step to DNA intercalating mechanism
of βꢀcarbolines.20 These knowledge emphasize that the discovery of novel πꢀπ
stacking nanoꢀintercators is of chemotherapeutic importance.
In view of structural impression of piperazine, βꢀcarboline and cyclohexaneꢀ1,4ꢀdione,
a novel heterocycle, (2’S,5’S)ꢀtetrahydropyrazino[1’,2’:1,6]ꢀdi{2,3,4,9ꢀtetrahydroꢀ
1Hꢀpyrido[3,4ꢀb]indole}ꢀ1’,4’ꢀdione (THPDTPI) was constructed and the design is
graphically illustrated with Scheme 1. To the best of our knowledge, THPDTPI would
be a nanoꢀmedicine capable of targeting DNA.21,22 The present paper calculated the
docking energy of THPDTPI towards the double strand Bꢀform DNA to predict its
potential target, discussed its synthesis, visualized its nanoꢀstructure, evidenced

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physical and chemical mechanism of it intercalating DNA, evaluated its antiꢀtumor
activity in vitro and in vivo, and explored its liver and kidney toxicity.
Scheme 1 Design of novel DNA intercalator, heterocycle THPDTPI, wherein the blue arrowheads
represents the easy modifying positions.
Results and discussion
Docking of THPDTPI towards double strands Bꢀform DNA
The rationality of the design and the intercalation model were examined with docking
investigation. Fig. 1 shows that THPDTPI intercalates into the bases of double strands
Bꢀform DNA with a typical πꢀπ stacking manner. The carboline moieties of THPDTPI
properly approach the two base pairs of DNA. Here this proximity is illustrated with
the distances between 13 atoms of the carboline moieties and 23 atoms of two
purine/pyrimidine pairs, which ranged from 3.0 Å to 3.9 Å, and marked with the
green solid lines. This proximity also results in two hydrogen bond interactions
between the two hydrogens of the pyrroles of THPDTPI and the two oxygens of the
phosphate (1.9 Å) and the deoxyribose (2.2 Å) of DNA, and marked with green
dashed lines. Reasonably, the hydrogen bonds can stabilize the πꢀπ stacking. On the
other hand, the free energy of THPDTPI binding DNA is ꢀ9.32 kcal/mol.

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Fig. 1 Docking of THPDTPI towards the double strands Bꢀfrom DNA. THPDTPI is represented in
stick model, hydrogen bonds are represented with green dashed lines, while the πꢀπ stacking and
distances between THPDTPI and DNA bases are represented with green solid lines.
Synthetic route of preparing THPDTPI
THPDTPI was prepared according to a synthetic route depicted in Scheme 2, in which
two cyclizations were involved. Firstly the PictetꢀSpengler cyclization resulted in 3Sꢀ
1,2,3,4ꢀtetrahydroꢀβꢀcarbolineꢀ3ꢀcarboxylic acid, and secondly the intermolecular
cyclization provided THPDTPI. The detailed preparation was described in the
following synthetic procedure.
O
O
H
N
OH
N
i
ii
LꢀTrp + CH2O
NH
N
N
N
H
H
O
Scheme 2 Synthetic route of the preparation of THPDTPI. i) Sulfuric acid (1 M); ii) EDC,
Nꢀhydroxybenzotriazol, DMF, Nꢀmethylmorpholine.
Synthetic procedures of THPDTPI
Preparing 3Sꢀ1,2,3,4ꢀtetrahydroꢀβꢀcarbolineꢀ3ꢀcarboxylic acid
To a mixture of 5.0 g (24.5 mmol) of LꢀTrp, 25 mL of H2SO4 (1 M) and 80 mL of
water, 8 mL of formaldehyde (36ꢀ38%) were added. The reaction mixture was stirred
at room temperature for 2 h and adjusted to pH 7 with concentrated ammonia liquor.
The mixture was kept at 0 ºC for 12 h, and the formed precipitates were collected by
filtration. The recrystallization gave 3.97 g (75%) of the title compound as colorless
powders. Mp 280ꢀ282 ºC and the structure was confirmed with spectra. ESI/MS: 217
[M + H]+. IR (KBr): 3450, 3200, 3000, 2950, 2850, 1700, 1601, 1452, 1070, 900 cmꢀ1.

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1HNMR (BHSCꢀ500, DMSOꢀd6): δ=10.99 (s, 1H), 9.89 (s, 1 H), 7.30 (t,
J
=7.5 Hz,
1H), 7.22 (t,
J
=8.0 Hz, 1 H), 7.01 (d,
=4.8Hz, 1H), 3.75 (dd, =10.5 Hz,
=10.5 Hz, 2 H), 2.86 (s, 1 H).
J
=8.0 Hz, 1 H), 6.81 (d,
J
J
=7.5 Hz, 1 H), 4.01 (t,
=10.5 Hz, =2.4 Hz, 1
J
J
J
=5.0 Hz, 1 H), 3.64 (dd,
J
H), 2.91 (d,
J
Preparing THPDTPI
To a mixture of 648 mg (3 mmol) of 3Sꢀ1,2,3,4ꢀtetrahydroꢀβꢀcarbolineꢀ3ꢀcarboxylic
acid, a solution of 573 mg (3 mmol) of EDC and 405 mg (3 mmol) of
Nꢀhydroxybenzotriazol in 20 mL of anhydrous N,Nꢀdimethylformamide was added.
The reaction mixture was adjusted to pH 9 by adding 0.3 mL of Nꢀmethylmorpholine
and then was stirred at room temperature for 12 h. TLC (CH2Cl2/CH3OH, 15/1)
indicated the reaction was completed. At 45 °C the reaction mixture was evaporated
under vacuum. The residue was successively triturated with water and acetone, and
then purified on silica gel column (CH2Cl2/CH3OH, 30/1) to provide 535 mg (90%) of
the title compound as colorless powders, of which the structure was confirmed with
1
spectra. FTꢀMS 397.1586 [M + H]+. H NMR (800 MHz, CDCl3): δ=7.930 (s, 2H),
7.448 (d,
J
=8.0 Hz, 2H), 7.360 (d,
=8.0 Hz, 2H), 5.737 (d, =16.0 Hz, 2H), 4.468 (dd,
=16.0 Hz, 2H), 3.535 (dd, =14.4 Hz, =2.4 Hz, 2H), 2.927 (t, J
J
=8.0 Hz, 2H), 7.204 (t,
J
=8.0 Hz, 2H), 7.124 (t,
=4.0 Hz, 2H), 4.264
=13.6 Hz, 2H).
J
J
J
=12.0 Hz,
J
(d,
J
J
J
13C NMR (125 MHz, DMSOꢀd6): δ=169.98, 165.05, 136.71, 136.46, 130.31, 128.40,
126.75, 121.72, 121.60, 119.22, 118.24, 118.15, 111.63, 107.11, 106.01, 57.06, 56.46,
40.97, 28.06, 23.44.
HPLC purity of THPDTPI
By following the methods in ESI† the chromatogram was recorded, a sole peak of
THPDTPI occurred at 5.704 minutes and the relative area of the peak was 98.9%.
Therefore the retention time of THPDTPI is 5.704 minutes and the HPLC purity of
THPDTPI is 98.9%.
FTꢀMS spectrum and THPDTPI forming dimer
Mass spectrum was measured on a SolariX FTꢀICR mass spectrometer (Bruker
Daltonics) with ESI negative ion source and superconductive magnet of 9.4 T. Fig.
2A shows the spectrum of 1 nM solution of THPDTPI in ultrapure water, gives a

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negative ion peak at 827.28939, the mass of a dimer plus Cl, and gives a negative ion
peak at 431.12884, the mass of monomer plus Cl. Since Fig. 2A is a qCID spectrum
the monomer is the fragmentation product of the dimer, and the dimer is the unified
form of the existence of THPDTPI in ultrapure water. The full mass spectrum is given
in ESI.†
NOESY 2D NMR and four interesting crossꢀpeaks
To reveal the manner of the dimerization the NOESY 2D NMR was measured on 800
MHz in deuterated DMSO. Fig. 2B shows NOESY 2D 1HNMR spectrum of THPDTPI,
in which four interesting crossꢀpeaks are marked with red circles. Crossꢀpeak 1 mirrors
the interactions between the pyrrole H of the carboline moiety of one molecule with the
H at the 1ꢀposition of the carboline moiety of another molecule. Crossꢀpeak 2 mirrors
the interactions between the H at 7,8ꢀpositions of the carboline moiety of one molecule
with the pyrrole H of the carboline moiety of another molecule. Crossꢀpeaks 3 and 4
mirror the interactions between the H at 7 and 8ꢀpositions of the carboline moiety of
one molecule with the H at 4ꢀposition of the carboline moiety of another molecule. All
the NMR spectra are also given in the ESI.†
Energy minimized conformation based dimer
To visualize the dimer, THPDTPI was sketched in ChemDraw 10.0, converted to 3D
conformation in Chem3D 10.0, and then energy minimized in Discovery Studio 3.5
with a Merck molecular force field (Merck & Co.). Energyꢀminimized conformation
was utilized to form a dimer. Fig. 2C and 2D indicate that to match the requirements
of the 4 interesting crossꢀpeaks of Fig. 2B the two energyꢀminimized conformations
of THPDTPI have to approach in a typical πꢀπ stacking manner. NOESY 2D 1HNMR
experiment, therefore, implies that the πꢀπ stacking should be an essential manner of
THPDTPI intercalating DNA.

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Fig. 2 FTꢀMS and NOESY 2D 1HNMR spectra of THPDTPI, (A) qCID spectrum of 1 nM solution
of THPDTPI in ultrapure water; (B) NOESY 2D 1HNMR spectrum of THPDTPI and 4 interesting
crossꢀpeaks; (C) Top view of 2 molecules of THPDTPI approaching in πꢀπ stacking manner; (D)
Front view of 2 molecules of THPDTPI approaching in πꢀπ stacking manner.
FaradayꢀTyndall effect and nanoꢀproperties of THPDTPI in water
To explore the nanoꢀproperties of the aqueous solution of THPDTPI, the laser (650 nm)
induced FaradayꢀTyndall effect was tested and is shown in Fig. 3. As seen, when 1
nM solution of THPDTPI in pH 2.0 and pH 7.0 ultrapure water are irradiated with 650
nm laser beam the FaradayꢀTyndall effects clearly occur (Fig. 3D, E). In addition, the
zeta potential and size of THPDTPI in water were determined on a Malvern’s Zetasizer
(NanoꢀZS90; Malvern Instruments) with the DTS (Nano) Program. Fig. 3F indicates
that the zeta potential of THPDTPI in pH 7.0 ultrapure water (1 nM) is ꢀ20.60 mV with
4.42 mV of half width. Fig. 3G, H indicate that the mean size of THPDTPI in pH 2.0
and pH 7.0 ultrapure water are 164.2 ± 1.91 nm and 141.8 ± 1.91 nm, respectively.
Therefore, the pH 2.0 and pH 7.0 aqueous solution of THPDTPI possess
nanoꢀproperties.

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Fig. 3 Tyndall effect, zeta potential and size of THPDTPI in ultrapure water. (A) Ultrapure water
no radiation; (B) pH 2.0 ultrapure water with 650 nm laser radiation; (C) pH 7.0 ultrapure water
with 650 nm laser radiation; (D) 1 nM solution of THPDTPI in pH 2.0 ultrapure water with 650
nm laser radiation; (E) 1 nM solution of THPDTPI in pH 7.0 ultrapure water with 650 nm laser
radiation; (F) zeta potential of 1 nM solution of THPDTPI in pH 7.0 ultrapure water; (G) size of
THPDTPI in pH 2.0 ultrapure water; (H) size of THPDTPI in pH 7.0 ultrapure water.
Theoretically predicted nanoparticle size of THPDTPI
To theoretically predict the formation and the size of the nanoparticles, the mesoscale
simulation software was used to perform the calculation. The molecule of THPDTPI
was built and optimized in the Visualizer window. “Beads” were constructed from
atomistic simulations and placed at the centerꢀofꢀmass of groups of the atoms
corresponding to particular parts of a molecule. Fig. 4 indicates that in a nanoparticle
of 8.5 nm in diameter there are 608 molecules of THPDTPI.
Fig. 4 Proposed model of THPDTPI forming nanoparticles.
TEM image of THPDTPI nanoparticles
The shape and size of THPDTPI in aqueous solution were imaged with transmission
electron microscopy (TEM). The TEM images of THPDTPI in pH 7.0 ultrapure water
(Fig. 5Aꢀ5C) indicate that in 10.0, 1.0 and 0.1 nM of solutions THPDTPI forms nanoꢀ
particles of 8.5ꢀ146.5 nm, 30.2ꢀ46.5 nm and 46.5ꢀ116.3 nm in diameter, respectively.

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The comparison of the diameters of the nanoparticles in three concentration solutions
suggests that in 1.0 nM solution THPDTPI forms nanoparticles of the most suitable
size. On the other hand, in 10.0 nM solution THPDTPI forms the nanoparticle of 8.5
nm in diameter, the smallest particle. According to theoretical prediction, this particle
should contain 608 molecules of THPDTPI.
SEM image of THPDTPI nanoparticles
The shape and size of THPDTPI in solid state were imaged with scanning electron
microscopy (SEM). Fig. 5D indicates that in the solid state THPDTPI exists as sphere
of 2ꢀ14 µm in diameter. The amplified insert indicates that the spheres consist of the
nanoparticles of ~140 nm in diameter, which are marked with red circles. Thus it
could be hypothesized that during the evaporation of 10.0 nM aqueous solution of
THPDTPI the smaller nanoparticles gradually aggregate and finally form the
microspheres.
Fig. 5 TEM and SEM images of THPDTPI. (A) TEM image of THPDTPI in pH 7.0 ultrapure
water (10.0 nM); (B) TEM image of THPDTPI in pH 7.0 ultrapure water (1 nM); (C) TEM image
of THPDTPI in pH 7.0 ultrapure water (0.1 nM); (D) SEM image of the precipitates formed by
evaporating an aqueous solution of THPDTPI in pH 7.0 ultrapure water (10.0 nM).
AFM image of THPDTPI nanoparticles
The shape and size of THPDTPI in blood were imaged with atomic force microscopy
(AFM). Fig. 6A indicates that rat plasma alone gives no interesting nanoparticle. Fig.
6Bꢀ6D indicate that in 1 nM rat plasma THPDTPI forms the nanoparticles of 77.5 ꢀ
121.1 nm in height, suggesting in rat blood THPDTPI is still able to form the suitable
nanoparticles for delivery in the circulation.

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Fig. 6 AFM image of THPDTPI in rat plasma. (A) AFM image of rat plasma alone; (B) AFM
image of THPDTPI in rat plasma (0.1 nM), in which two nanoparticles been marked with a blue
ring; (C) scaled AFM image of the down particle inside the blue ring; (D) scaled AFM image of
the up particle inside the blue ring.
Antiꢀtumor activity of THPDTPI in vivo
To show the antiꢀtumor activity and the doseꢀdependent relationship, male S180 mice
were used and the assay was performed by following the standard procedure and the
activity was represented with tumor weight. Fig. 7 indicates that after 14ꢀday
treatment the tumor weights of the mice orally treated with THPDTPI (0.01, 0.1 and
1.0 ꢁmol/kg/day) are significantly lower than that of the mice treated with 0.5%
carmellose sodium (CMCNa, vehicle, 0.2 mL/mouse/day), suggesting THPDTPI is an
active antiꢀtumor agent. Beside, with the increase of the dose the tumor weight
gradually decreases, suggesting THPDTPI slows the tumor growth in a
doseꢀdependent manner. Furthermore, the tumor weight of the mice orally treated
with 1 ꢁmol/kg/day of THPDTPI is equal to that of the mice intraperitoneally treated
with 2 ꢁmol/kg/day of doxorubicin (Dox, positive control), suggesting the activity of
THPDTPI is 2 folds higher than that of Dox. Therefore, in respect of antiꢀtumor
activity and nanoꢀproperty, THPDTPI is an excellent lead antiꢀtumor agent possessing
nanoꢀproperties.

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Fig. 7 Tumor weights of the S180 mice treated with various doses of THPDTPI, n=12.
Spectrophotometric assays and intercalation of THPDTPI towards CT DNA
To confirm the intercalation model of THPDTPI binding DNA, the
spectrophotometric assays were performed. Fig. 8A gives increased absorption at 208
nm and 258 nm in the UV spectra of 0.9 µM THPDTPI with increased amount of CT
DNA. Fig. 8B gives increased absorption at 208 nm but decreased absorption at 258
nm in the UV spectra of 200 µM CT DNA with increased amount of THPDTPI.
Furthermore, Fig. 8B shows obvious red shift at 208 nm in the UV spectra while
increasing the amount of THPDTPI in 200 µM CT DNA. The increased absorption at
208 nm and 258 nm in Fig. 8A and the decreased absorption at 258 nm in Fig. 8B
demonstrate that CT DNA exhibits strong absorption at both 208 nm and 258 nm,
while THPDTPI exhibits strong absorption at 208 nm only. The obvious red shifts at
208 nm in Fig. 8B suggests THPDTPI intercalates into CT DNA and the maximal
absorption wave length of the complex drifts to 220 nm.23,24
Fig. 8C gives decreased fluorescence intensity at 345 nm in the fluorescence spectra
of 0.18 µM THPDTPI with increased amount of CT DNA, while Fig. 8D gives
increased fluorescence intensity at 345 nm in the fluorescence spectra of 80 µM CT
DNA with increased amount of THPDTPI. The decreased fluorescence intensity in
Fig. 8C and the increased fluorescence intensity in Fig. 8D demonstrate that
THPDTPI possesses much stronger fluorescence intensity than CT DNA does at 345
nm. The quenching of THPDTPI fluorescence intensity with increasing amount of CT
DNA in Fig. 8C suggests THPDTPI intercalates into CT DNA.25
According to a general understanding, the circular dichroism (CD) spectrum of right
handed Bꢀform DNA is characterized by its positive and negative bands, of which the
former is due to base stacking and the latter is due to helicity. The spectral changes of

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these two bands are usually the result of the intercalation of small molecules with CT
DNA.25 The circular dichroism (CD) spectra of 14 ꢁM CT DNA with increasing
amount of THPDTPI (Fig. 8E) exhibit decreased shift of the positive bands at 280 nm
and increased shift of negative bands at 255 nm, respectively. These results suggest
that the intercalation of THPDTPI into CT DNA induces constant conformational
changes of CT DNA.
Fig. 8F shows the effect of increasing amounts of THPDTPI on the relative viscosity
of CT DNA at 25 ± 0.1 ºC. The relative viscosity of 14 ꢁM of CT DNA gradually
increases while mixing with increased amount of THPDTPI, which suggests
THPDTPI intercalates into CT DNA and the viscosity of the formed complex
becomes larger than CT DNA alone.25
Fig. 8 Intercalation of THPDTPI towards CT DNA in vitro. (A) UV spectrum of 0.9 µM of
THPDTPI with increased amount of CT DNA; (B) UV spectra of 200 µM of CT DNA with
increased amount of THPDTPI; (C) fluorescence spectra of 0.18 µM of THPDTPI with increased

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amount of CT DNA; (D) fluorescence spectra of 80 µM of CT DNA with increased amount of
THPDTPI; (E) circular dichroism (CD) spectra of 14 ꢁM of CT DNA with increased amount of
THPDTPI; (F) the relative viscosity of 14 ꢁM of CT DNA with increased amount of THPDTPI.
Antiꢀproliferation activity of THPDTPI in vitro
The in vitro activity of THPDTPI (final concentrations: 0.01, 0.1, 1, 5, 10 and 50 ꢁM)
inhibiting cancer cell proliferation was assayed on S180 and HeLa cells using MTT
method, and represented with IC50 values. The concentration curves demonstrated that
the IC50 values of THPDTPI against S180 and HeLa cells are 0.39 ꢁM and 3.5 ꢁM,
respectively. Since THPDTPI has blue emission fluorescence, to visualize THPDTPI
inside cancer cells S180 and HeLa cells were incubated with DMEM medium or 3.5
ꢁM of THPDTPI for 24 h and the confocal images are shown in Fig. 9. As seen, no
blue fluorescence can be seen inside S180 and HeLa cells incubated with DMEM
medium (Fig. 9A and 9C), while obvious (Fig. 9B) and weak (Fig. 9D) blue
fluorescence can be seen inside the nucleus of S180 and HeLa cells incubated with
THPDTPI. These observations imply that THPDTPI inhibits the proliferation of
cancer cells by entering their nucleus. Both the IC50 values and confocal images show
that THPATPI possesses a stronger antiꢀproliferation activity on S180 cells than on
Hela cells.

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Fig. 9 Confocal images: (A) for S180 cells that incubated with DMEM medium; (B) for S180
cells that incubated with 3.5 ꢁM of THPDTPI; (C) for HeLa cells that incubated with DMEM
medium; (D) for HeLa cells that incubated with 3.5 ꢁM of THPDTPI.
Acute toxicity of THPDTPI
To estimate the acute toxicity the healthy ICR mice were orally treated with
THPDTPI at a single dose of 100 µmol/kg, a dose of 10000 times of the minimal
effective dose (0.01 µmol/kg), and the mice were monitored for 7 days. Four hours
after the administration the orbital blood of the mice were collected to determine the
serum level of alanine transaminase (ALT), aspartate transaminase (AST) and
creatinine (Cr). On 7th day the mice received ether anaesthesia and were sacrificed
to sample the blood and the organs to determine serum ALT, AST and Cr, as well as
to examine morphological change of the organs. Fig. 10Aꢀ10C indicates that
similar to CMCNa, a single oral dose of 100 µmol/kg of THPDTPI does not change
the serum ALT, AST and Cr of healthy mice, suggesting THPDTPI possesses no
liver and kidney toxicity. It is also found that during 7ꢀday observation the mice
exhibit normal behavior and their organs have no morphological change, suggesting
THPDTPI possesses no systematic toxicity. Besides, THPDTPI and CMCNa treated
S180 mice have similar organ weight which is shown in Fig. S6 of the ESI†. This
means that the administration THPDTPI induces no organ toxicity, and consequently
induces no Doxꢀlike heart toxicity in particular.
Fig. 10 Serum ALT (A), AST (B) and Cr (C) of the mice treated with 100 µmol/kg single dose
of THPDTPI
.
THPDTPI potentially targeting tumor tissue
To estimate the distribution of THPDTPI, the brain, heart, spleen, liver, kidney, lung,
blood and tumor of the S180 mice treated with CMCNa or 1.0 ꢁmol/kg/day of
THPDTPI were homogenized, ultrasonically extracted with dichloromethane,
centrifuged at 1000 g for 10 minutes to separate the supernatant for ESI(ꢀ/+)ꢀMS tests.

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It was found that ESI(ꢀ/+)ꢀMS spectra of the extracts of the brain, heart, spleen, liver,
kidney, lung and blood, but not the extracts of the tumor tissue, failed to give any ion
peak relate to THPDTPI. Fig. 11A and 11C are the ESI(ꢀ/+)ꢀMS spectra of the tumor
tissue of S180 mice treated with 1.0 ꢁmol/kg/day of THPDTPI for 7 days, while Fig.
11B and 11D are the ESI(ꢀ/+)ꢀMS spectra of the tumor tissue of S180 mice treated
with CMCNa. The ESI(+)ꢀMS and ESI(ꢀ)ꢀMS spectra of the extract of the tumor
tissue of S180 mice treated with THPDTPI give an ion peak at 419.10190 of [M +
Na]+ and an ion peak at 431.09567 of [M + Cl]ꢀ, respectively. The ESI(+)ꢀMS and
ESI(ꢀ)ꢀMS spectra of the extract of the tumor tissue of S180 mice treated with
CMCNa give no comparable ion peak at the corresponding area. ESI(ꢀ/+)ꢀMS
spectrum analysis suggests that THPDTPI would be a tumor targeting lead compound.
It is worthy to emphasize that ESI(ꢀ/+)ꢀMS spectra of the extract of the heart of 1.0
ꢁmol/kg/day of THPDTPI treated S180 mice failed to give any ion peak related to
THPDTPI. This means that the administration induces no accumulation of THPDTPI
in the heart of the treated mice, and consequently results in no Doxꢀlike heart toxicity.
Fig. 11 ESI(ꢀ/+)ꢀMS spectra of the extract of the tumor tissue. (A) ESI(+)ꢀMS spectrum of the
extract of the tumor tissue of S180 mice treated with 1.0 ꢁmol/kg/day of THPDTPI for 14 days,
the local amplified insert shows an ion peak of THPDTPI plus Na at 419.10190; (B) ESI(+)ꢀMS
spectrum of the extract of the tumor tissue of S180 mice treated with CMCNa for 14 days, the
local amplified insert shows no such an ion peak; (C) ESI(ꢀ)ꢀMS spectrum of the extract of the

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tumor tissue of S180 mice treated with 1.0 ꢁmol/kg/day of THPDTPI for 14 days, the local
amplified insert shows an ion peak of THPDTPI plus Cl at 431.09567; (D) ESI(ꢀ)ꢀMS spectrum of
the extract of the tumor tissue of S180 mice treated with CMCNa for 14 days, the local amplified
insert shows no such an ion peak.
Experimental section
The detailed methodologies for all experiments are given as ESI.†
Sprague Dawley rats and ICR mice were purchased from the Animal Center of Peking
University. Work performed was based on a protocol reviewed and approved by the
ethics committee of Capital Medical University. The committee assures that the
welfare of the animals was maintained in accordance to the requirements of the
Animal Welfare Act and in accordance to the NIH Guide for Care and Use of
Laboratory Animals.
Conclusions
Docking investigation and spectrophotometric experiments support that the
interaction between THPDTPI and CT DNA is πꢀπ stacking intercalation; FTꢀMS
spectrum and NOESY 2D 1HNMR spectrum together show that THPDTPI can form
πꢀπ stacking like dimer; FaradayꢀTyndall effect and the images of TEM, SEM and
AFM support that the dimers of THPDTPI can further selfꢀassemble to form
nanoꢀparticles, the in vivo antiꢀtumor assay, in vitro antiꢀproliferation assay and the
acute toxicity evaluation logically support the rationality of the present design, and
ensure the novel heptacyclic THPDTPI been a promising parent stock of
nanoꢀintercalotors. The present study also opens the gate to a promising area of
discovering diverse nanoꢀintercalation analogs of THPDTPI via constructing more
novel heptacyclic molecules with modified βꢀcarbolines.
Acknowledgements
The authors thank the BMSTC (Z141100002114049), TJSHG (201310025008), the
Project of Construction of Innovative Teams and Teacher Career Development for
Universities and Colleges Under Beijing Municipality, NSFC (81172930, 81273379,
81202412 and 81373264), Joint Research and Development of protective agents for
low dose radiation injury (2014DFR30930) and the Beijing NOVA Program
(XX2013039) for financial supports.

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Graphical Abstract
A strategy for designing safe and effective πꢀπ stacking nanoꢀintercalators as
antiꢀtumor agent was presented for the first time.