Modifying ICCA with trp-phe-phe to enhance in vivo activity and form nano-medicine

Article abstract of DOI:10.2147/IJN.S229856

Background: 1-(4-isopropylphenyl)-β-carboline-3-carboxylic acid (ICCA) was modified by Trp-Phe-Phe to form 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Trp-Phe-Phe (ICCA-WFF). Purpose: The object of preparing ICCA-WFF was to enhance the in vivo efficacy of ICCA, to explore the possible targeting action, and to visualize the nano-feature. Methods: The advantages of ICCA-WFF over ICCA were demonstrated by a series of in vivo assays, such as anti-tumor assay, anti-arterial thrombosis assay, anti-venous thrombosis assay, P-selectin expression assay, and GPIIb/IIIa expression assay. The nano-features of ICCA-WFF were visualized by TEM, SEM and AFM images. The thrombus targeting and tumor-targeting actions were evidenced by FT-MS spectrum analysis. Results: The minimal effective dose of ICCA-WFF slowing tumor growth and inhibiting thrombosis was 10-fold lower than that of ICCA. ICCA-WFF, but not ICCA, formed nano-particles capable of safe delivery in blood circulation. In vivo ICCA-WFF, but not ICCA, can target thrombus and tumor. In thrombus and tumor, ICCA-WFF released Trp-Phe-Phe and/or ICCA. Conclusion: Modifying ICCA with Trp-Phe-Phe successfully enhanced the anti-tumor activity, improved the anti-thrombotic action, formed nano-particles, targeted tumor tissue and thrombus, and provided an oligopeptide modification strategy for heterocyclic compounds.

Full text of DOI:10.2147/IJN.S229856

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O R I G I N A L R E S E A R C H
Modifying ICCA with Trp-Phe-Phe to Enhance
in vivo Activity and Form Nano-Medicine
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
Xiaoyi Zhang1,2
Background: 1-(4-isopropylphenyl)-β-carboline-3-carboxylic acid (ICCA) was modified by
Yixin Zhang1,2
Trp-Phe-Phe to form 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Trp-Phe-Phe (ICCA-WFF).
1,2
Purpose: The object of preparing ICCA-WFF was to enhance the in vivo efficacy of ICCA,
Yaonan Wang
1,2
to explore the possible targeting action, and to visualize the nano-feature.
Methods: The advantages of ICCA-WFF over ICCA were demonstrated by a series of
in vivo assays, such as anti-tumor assay, anti-arterial thrombosis assay, anti-venous throm-
bosis assay, P-selectin expression assay, and GPIIb/IIIa expression assay. The nano-features
of ICCA-WFF were visualized by TEM, SEM and AFM images. The thrombus targeting and
tumor-targeting actions were evidenced by FT-MS spectrum analysis.
Jianhui Wu
Haiyan Chen1,2
Ming Zhao1–3
1,2
Shiqi Peng
1Beijing Area Major Laboratory of Peptide
and Small Molecular Drugs, Department of
Medicinal Chemistry, School of
Pharmaceutical Sciences, Capital Medical
University, Beijing 100069, People’s
Republic of China; 2Engineering Research
Center of Endogenous Prophylactic of
Ministry of Education of China,
Department of Medicinal Chemistry,
School of Pharmaceutical Sciences, Capital
Medical University, Beijing 100069, People’s
Republic of China; 3Beijing Laboratory of
Biomedical Materials and Key Laboratory of
Biomedical Materials of Natural
Results: The minimal effective dose of ICCA-WFF slowing tumor growth and inhibiting throm-
bosis was 10-fold lower than that of ICCA. ICCA-WFF, but not ICCA, formed nano-particles
capable of safe delivery in blood circulation. In vivo ICCA-WFF, but not ICCA, can target thrombus
and tumor. In thrombus and tumor, ICCA-WFF released Trp-Phe-Phe and/or ICCA.
Conclusion: Modifying ICCA with Trp-Phe-Phe successfully enhanced the anti-tumor
activity, improved the anti-thrombotic action, formed nano-particles, targeted tumor tissue
and thrombus, and provided an oligopeptide modification strategy for heterocyclic
compounds.
Keywords: ICCA, modification, Trp-Phe-Phe, anti-tumor, thrombus targeting, release,
toxicity, nano-species
Macromolecules, Department of
Biomaterials, College of Materials Science
and Engineering, Beijing University of
Chemical Technology, Beijing 100026,
People’s Republic of China
Introduction
The malignant cancer is a heterogeneous disease and characterized by the different
phenotypes.1–3 Though early recognition, surgical extirpation and clinical therapy did
reduce the death rate,4 the diseases, such as deep venous and arterial embolisms can
induce bad outcomes of cancer patients, thereby the diseases made malignant cancer to
be a response for the elevated mortality of human being of the world.5–9 In fact, the
correlation between malignant cancer and thromboembolism was established for more
than 150 years. Later, it was reported that the risk of hospitalized patients with cancer to
develop venous thromboembolism was 4.1 times higher than that of the non-cancer
patients,10–16 and conversely venous thromboembolism was one of the leading causes
of the deaths of patients with some types of malignant cancer.17 In addition to venous
embolism, arterial embolism was correlated with specific malignant cancer18,19 and
cancer therapies.20,21 In this profile to discover lead compounds capable of simulta-
neously inhibiting arterial thrombosis, preventing venous thrombosis and slowing
tumor growth is of great clinical importance. As an effort, 1-(4-isopropylphenyl)-β-
carboline-3-carboxylic acid (ICCA) was recently reported as an agent capable of
Correspondence: Shiqi Peng; Ming Zhao
Department of Medicinal Chemistry,
School of Pharmaceutical Sciences,
Capital Medical University, No. 10,
Youanmenwaixitoutiao, Fengtai District,
Beijing 100069, People’s Republic of China
Tel +86 10 8391 1528; +86 10 8391 1535
Fax +86 10 8391 1528; +86 10 8391 1533
Email sqpeng@bjmu.edu.cn;
maozhao@126.com
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work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For
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http://doi.org/10.2147/IJN.S229856

Zhang et al
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simultaneously inhibiting GPIIb/IIIa, P-selectin, arterial cells (95D) and ascites tumor cells (S180) were purchased
thrombosis, venous thrombosis and tumor growth.22 from keyGEN BioTECH (Nanjing China).
However, its oral dose (5 μmol/kg) was a bit high. To lower
From the Laboratory Animal Center of Capital Medical
the dose, some oligopeptides were previously designed, pre- University male ICR mice (22–24 g) and male Sprague
pared and evaluated for modifying ICCA. Among them, Trp- Dawley rats (240–270 g) were purchased. Ethics
Phe-Phe was found having anti-tumor and anti-thrombotic Committee of Capital Medical University reviewed and
activities, as well as depressing serum P-selectin and GPIIb/ approved the protocol of the evaluations. In particular,
IIIa in particular. This led to a hypothesis that coupling ICCA the animal welfare fulfilled the requirements of the
with Trp-Phe-Phe should form the derivative capable of Animal Welfare Act and NIH Guide for Care and Use of
binding GPIIb/IIIa and P-selectin. In this case, 1-(4-iso Laboratory Animals.
propylphenyl)-β-carboline-3-carbonyl-Tyr-Phe-Phe (ICCA-
Biological data were statistically analyzed with ANOVA.
WFF) was designed and docked into the active sites of LSD for multiple group comparison. SPSS 19.0-program
P-selectin and GPIIb/IIIa.
was used to perform the analysis and a P-value less than
0.05 was considered statistically significant.
Materials and Methods
Synthesis
General
The preparations and the physical-chemical data of (1R,3S)-
1-(4-isopropylphenyl)-2,3,4,9-tetrahydro-β-carboline-3-car-
boxylic acid benzyl esters(1), benzyl 1-(4-isopropylphenyl)-
β-carboline-3-carboxylate (2) and ICCA were the same as
those of the literature.22
Trp and Phe purchased from Sigma were L-configuration. All
reactions were done in nitrogen (1 bar). On Bruker AMX-300
1
and AMX-800 spectrometer H (300 and 800 MHz) and
13C (75 and 200 MHz) NMR spectra were measured for the
intermediates or ICCA-WFF, for which dimethylsulfoxide
(DMSO-d6) was the solvent and tetramethylsilane was the
internal standard. Perkin-Elmer 983 instrument was used to
record IR spectra. ESI( )-ICR-FT-MS spectrum was recorded
on a 9.4 T solariX Fourier transform ion cyclotron resonance
mass spectrometer (Bruker Corp, Billerica, MA, USA) to get
the exact mass of ICCA-WFF. Chromatography was per-
formed by using Sephadex-LH20 or Qingdao silica gel GF254
or Qingdao silica gel H60. All solvents were distilled and dried
by the literature procedures for use. HPLC purity (Waters, C18
column 4.6×150 mm) of the intermediates and ICCA-WFF
were more than 96% and 99.63%, respectively. HPLC was
performed with Agilent Technologies 1200 Series HPLC sys-
tem (Agilent Technologies, Santa Clara, CA, USA) on Eclipse
XDB C18 column (5 μm, 4.6 mm × 150 mm). The column
temperature was 40°C, the mobile phase consisted of water
and methanol, and the gradient consisted of 60% methanol
(0–5 min), 70% methanol (5–10 min), 80% methanol (10–20
min) and 90% methanol (20–30 min), while the flow rate was
0.8 mL/min. Ultraviolet (UV) absorption spectrum was
recorded online. The UV detector was set to a scanning
range of 200–400 nm, and the chromatogram of ICCA-WFF
was monitored at a wavelength of 280 nm.
Preparation of Boc-Phe-Phe-OBzl
At 0ºC to a solution of 13.00 g (49.02 mmol) of Boc-Phe in
200mL of anhydrous THF 6.02 g (44.59 mmol) of HOBt
and 11.00 g (53.39 mmol) of DCC were added. This solu-
tion was at 0ºC stirred for 30 min and as solution A for the
coming use. A solution of 13.00 g (44.56 mmol) of
HCl·Phe-OBzl in 100 mL of anhydrous THF was adjusted
to pH 9 with NMM. This solution was firstly combined with
solution A to form a reaction mixture and then stirred at
room temperature, of which pH 9 was maintained by adding
NMM. Six hours after the stirring TLC (petroleum/acet-
one,2/1) indicated the complete disappearance of HCl·Phe-
OBzl. The reaction mixture was filtered, the filtrate was
evaporated in vacuum and the residue was dissolved in
150 mL of ethyl acetate. This solution was successively
washed with saturated aqueous NaHCO3 (50 mL×3), 5%
aqueous KHSO4 (50 mL×3), saturated aqueous NaCl
(50 mL×3), saturated aqueous NaHCO3 (50 mL×3) and
saturated aqueous NaCl (50 mL×3). Ethyl acetate phase
was isolated, dried with anhydrous Na2SO4 for 12 hrs,
filtered, the filtrate was evaporated in vacuum and the
residue was purified on silica gel column (petroleum/acet-
one,5/1) to give 20.2 g (90%) of title compound as colorless
powders. 1H NMR (300 MHz, DMSO-d6): δ/ppm =8.45 (d,
Animals and Cell Lines
Human myeloid leukemia cells (K562), human non-small J = 7.2 Hz, 1H), 7.24 (m, 15H), 6.88 (d, J = 8.7 Hz, 1H),
cell lung cancer cells (A549), non-small cell lung cancer 5.07 (m, 2H), 4.56 (m, 1H), 4.20 (m, 1H), 3.04 (m, 2H),
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Zhang et al
2.87 (m, 1H), 2.60 (m, 1H), 1.30 (s, 9H). ESI(+)-MS (m/z): DMSO-d6): δ/ppm =173.24, 172.78, 170.88, 138.38, 137.83,
503 [M+H]+.
136.78, 129.84, 129.71, 128.51, 128.39, 127.79, 126.67,
124.68, 121.42, 118.94, 118.75, 111.84, 109.85, 54.84,
54.47, 53.73, 38.18, 37.38, 30.04; HPLC purity: 99.17%;
ESI(-)-ICR-FT-MS: 497.2179 [M-H]− (calculated value:
497.2188).
Preparation of Phe-Phe-OBzl
At 0ºC to a solution of 10.0g (19.9 mmol) of Boc-Phe-Phe-
OBzl in 50 mL of anhydrous ethyl acetate, a solution of
hydrogen chloride in anhydrous ethyl acetate (50 mL, 4M)
was added. Four hours after stirring TLC (petroleum ether/
acetone, 2/1) indicated the complete disappearance of Boc-
Phe-Phe-OBzl. This reaction mixture was evaporated in
vacuum and the residue was dissolved in 50 mL of anhydrous
ethyl acetate for evaporation. This procedure was repeated
for at least 3 times to remove the access hydrogen chloride
thoroughly, which provided 7.72 g(96.5%) of the title com-
pound as colorless powders. ESI(+)-MS (m/z): 403 [M+H]+.
Preparation of 1-(4-isopropylphenyl)-β-
carboline-3-carbonyl-Tyr-Phe-Phe-OBzl (3)
By using the procedure of item 2 from 2.75 g (8.3 mmol) of
ICCA and 5.89 g (10.0 mmol) of Trp-Phe-Phe-OBzl 5.1
g (68%) of title compound was obtained as colorless pow-
ders. IR (KBr): 3396, 3295, 3064, 3029, 2960, 2921, 2862,
1725, 1651, 1523, 1495, 1454, 1392, 1356, 1212, 1173,
1100, 739, 698cm−1. 1H NMR (300 MHz, DMSO-d6): δ/
ppm = 11.82 (s, 1H), 10.84 (s,1H), 8.74 (s, 1H), 8.60 (s,
1H), 8.58 (s, 1H), 8.35 (d, J = 9 Hz, 1H), 8.30 (d, J = 9 Hz,
Preparation of Boc-Trp-Phe-Phe-OBzl
By using the procedure of item 2 from 12.39 g (40.7 1H), 7.85 (s, 1H), 7.83 (s, 1H), 7.67 (d, J = 9 Hz, 1H), 7.59
mmol) of Boc-Trp and 14.95 g (37.02 mmol) of Phe-Phe- (d, J = 7.8 Hz, 2H), 7.53 (s, 1H), 7.50 (s, 1H), 7.17 (m,
OBzl 10.02 g (40%) of the title compound was obtained as 19H), 6.85 (t, J = 6 Hz, 1H), 5.06 (t, J = 12 Hz, 2H), 4.64 (q,
1
colorless powders. H NMR (300 MHz, DMSO-d6): δ/ J = 6.6 Hz, 1H), 4.66 (m, 2H), 3.18 (m, 2H), 3.0 (m, 4H),
ppm = 10.76 (s, 1H), 8.62 (d, J = 7.5 Hz, 1H), 7.90 (d, 2.79 (m, 1H), 1.34 (d, J = 6.6 Hz, 6H). 13C NMR (75 MHz,
J = 8.4 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.27 (m, 16H), DMSO-d6): δ/ppm = 172.07, 171.58, 164.72, 149.79,
7.02 (m, 3H), 6.75 (d, J = 8.4 Hz, 1H), 5.07 (m, 2H), 4.64 142.02, 141.12, 139.55, 137.36, 136.60, 136.45, 136.15,
(m, 2H), 4.57 (q, J = 7.2 Hz, 1H), 4.14 (m, 1H), 2.90 (m, 135.50, 134.64, 130.14, 129.55, 128.93, 128.79, 128.73,
6H), 1.27 (s, 9H). ESI(+)-MS (m/z): 689 [M+H]+.
128.44, 128.33, 128.02, 127.84, 127.32, 126.99, 124.36,
123.93, 122.36, 121.65, 121.23, 120.63, 119.02, 118.82,
118.62, 113.16, 111.71, 110.44, 110.10, 66.45, 54.36,
53.83, 53.60, 38.71, 37.23, 33.90, 28.59, 28.25, 24.36,
24.30. ESI(+)-MS (m/z): 901[M+H]+.
Preparation of Trp-Phe-Phe-OBzl
By using the procedure of item 3 from 5.0 g (7.3 mmol) of
Boc-Trp-Phe-Phe-OBzl 4.06 g (95%) of the title com-
pound was obtained as colorless powders. ESI(+)-MS
(m/z): 589 [M+H]+.
Preparation of 1-(4-isopropylphenyl)-β-
carboline-3-carbonyl-Tyr-Phe-Phe (ICCA-WFF)
Preparation of Trp-Phe-Phe
By using the procedure of item 6 from 1.0 g (1.2
Into a suspension of 1.60 g (2.7 mmol) of Trp-Phe-Phe-OBzl mmol) of 1-(4-isopropylphenyl)-β-carboline-3-carbo-
and 160 mg of Pd/C in 30 mL of CH2Cl2 hydrogen was nyl-Tyr-Phe-Phe-OBzl (3) 802 mg (89%) of the title
introduced for 10 hrs, and TLC (CH2Cl2/CH3OH, 10/1) compound was obtained as powders with light pink
indicated the complete disappearance of Trp-Phe-Phe-OBzl. color. Mp 170–172 ºC; [α]25D=−53.27 (c=0.106, CH3
The reaction mixture was filtered, the filtrate was evaporated OH); IR (KBr): 3297, 3029, 2963, 1731, 1650, 1625,
under vacuum and the residue was grinded in anhydrous 1558, 1511, 1492, 1454, 1349, 841, 737, 698 cm−1;
ether to give 720 mg (53%) of the title compound as the 1H NMR (800 MHz, DMSO-d6): δ/ppm = 12.74 (s,
colorless powders. Mp 187–189ºC; [α]25D = −72.4 (c=0.106, 1H), 11.82 (s, 1H), 10.84 (s, 1H), 8.74 (s, 1H), 8.58 (d,
CH3OH); IR (KBr): 3290, 3060, 3029, 2926, 2853, 1656, J = 8.0 Hz, 1H), 8.36 (m, 2H), 8.26 (d, J = 8.0 Hz,
1639, 1519, 1495, 1454, 1435, 739, 697, cm−1; 1H NMR (300 1H), 7.84 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 8.0 Hz, 1H),
MHz, DMSO-d6): δ/ppm = 10.91 (s, 1 H), 8.33 (d, J = 7.5 7.58 (dd, J = 8.8 Hz, 7.2 Hz, 2H), 7.51 (d, J = 8.0 Hz,
Hz, 1H), 8.25 (d, J = 7.2 Hz, 1H), 7.59 (d, J = 7.5 Hz, 1H), 2H), 7.30 (dd, J = 17.6 Hz, 8.8 Hz, 2H), 7.25 (m, 4H),
7.34 (d, J = 7.8 Hz, 1H), 7.16 (m, 14H), 6.97 (t, J = 7.5 Hz, 7.19 (d, J = 7.2 Hz, 2H), 7.16 (m, 1H), 7.15 (m, 1H),
1H), 4.57 (m, 1H), 4.37 (m, 1H), 3.57 (m, 1H), 3.17 (s, 1H), 7.11 (t, J = 8.0 Hz, 2H), 7.04 (t, J = 7.2Hz, 1 H), 7.01
3.1 (m, 1H), 2.99 (m, 3H), 2.76 (m, 2H); 13C NMR (75 MHz, (t, J = 8.0 Hz, 1H), 6.84 (t, J = 8.0 Hz, 1H), 4.81
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(q, J = 7.2 Hz, 1H), 4.64 (m, 1H), 4.47 (q, J = 6.4 Hz, lung cancer cells (A549) and non-small cell lung cancer
1H), 3.38 (dd, J = 14.4 Hz, 7.2 Hz, 1H), 3.18 (m, 2H), cells (95D) were performed by using the method of
3.05 (m, 3H), 2.96 (m, 1H), 2.81 (m, 1H), 1.33 (s, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
6H).13C NMR (200 MHz, DMSO-d6): δ/ppm mide (MTT).23
=173.14, 171.45, 171.41, 164.72, 149.81, 142.01,
In vivo Anti-Tumor Assay
141.13, 139.49, 138.01, 137.82, 136.58, 135.48,
On S180 mouse model this assay was carried out, and the
134.64, 130.13, 129.71, 129.57, 129.02, 128.93,
mice were divided into 7 groups (each 12).24 The mice in
128.68, 128.35, 128.01, 127.33, 126.90, 126.58,
positive control group were intraperitoneally injected doxor-
124.31, 122.41, 121.65, 121.30, 120.66, 118.98,
ubicin (Dox, 2 μmol/kg/day) for 9 consecutive days, the mice
118.71, 113.20, 111.72, 110.06, 54.04, 53.96, 53.74,
in negative control group were orally given CMCNa (car-
99.63%;ESI(-)-ICR-FT-MS (m/z): 809.3448 [M-H]−
boxymethyl cellulose sodium, 0.5%) for 9 consecutive days,
38.00, 37.26, 33.91, 28.38, 24.37, 24.31. HPLC purity:
the mice in parent compound control group were orally given
(calculated value,809.3451).
ICCA (5 μmol/kg/day) for 9 consecutive days, the mice in
FT-ICR-MS and qCID Spectra
modification control group were orally given Trp-Phe-Phe (5
μmol/kg/day) for 9 consecutive days, the mice in object
compound group were orally given ICCA-WFF (5, 0.5 and
0.05 μmol/kg/day) for 9 consecutive days.
To reveal ICCA-WFF’s molecule assembly its FT-ICR-MS
and qCID spectra were measured on Bruker 9.4 T solariX
FT-ICR mass spectrometer. The measurement was per-
formed by following the procedure of the equipment.
Testing Serum ALT, AST and Cr
1
NOESY 2D H NMR Spectrum
The serum was prepared by centrifuging (1000 g, 10 min)
blood (0.5 mL) of S180 mice treated by CMCNa or ICCA-
WFF (5 µmol/kg/day). Serum glutamic pyruvictran-saminase
(ALT), glutamic oxaloacetictransaminase (AST) and creati-
nine (Cr) were tested by using the procedure of literature and
ALT/GPT Assay Kit (Nanjing Jiancheng Bioengineering
Institute, PR China).25
To reveal the manner of ICCA-WFF’s molecule assembly
its NOESY 2D NMR spectrum was measured on Bruker
800 MHz spectrometer. The measurement was performed
by following the procedure of the equipment.
SEM, TEM and AFM Feature
To visualize the nano-feature of ICCA-WFF in lyophilized
powders, in water and in rat plasma its scanning electron
microscopy (SEM) image, transmission electron micro-
scopy (TEM) image and atomic force microscopy (AFM)
image were recorded by following the procedure. In addi-
tion, the nano-property of aqueous ICCA-WFF was iden-
tified by Faraday-Tyndall effect.22
Testing Serum BUN
The serum was prepared by centrifuging (3000 g, 10 min) the
blood (0.5 mL) of S180 mice treated by CMCNa or ICCA-
WFF (5 µmol/kg/day). Blood urea nitrogen (BUN) was
tested by using the procedure of literature and the procedure
of the kit (Shanghai Lianshuo Biological Technology Co.,
Ltd, PR China).25
Mesoscale Simulation of Nano-Particle
The structure and conformation of ICCA-WFF were gen-
erated with ChemDraw Ultra 10.0 (Cambridge Soft) and
Materials Studio 3.2 (Accelrys, Inc. USA), and then the
conformation optimization, the molecular dynamic simula-
tion, the equilibration of the structure, the building of
a rigid coarse grain model of 4 connected spherical
beads, and the simulation of 15,000 ps was performed by
following the method.21
Arterial Thrombus Weight Assays
Arterial thrombus weight assay was performed on male ICR
mouse (22–24 g, each 12) model or on male Sprague Dawley
rat (240–270 g, each 12) model by following the procedure of
the literature.25,26 In mouse arterial thrombus weight assay
0.5% CMCNa (10 mL/kg, negative control) or a suspension
of aspirin in 0.5% CMCNa (240 µmol/kg, positive control)
or a suspension of ICCA in 0.5% CMCNa (5 µmol/kg) or
a suspension of WFF in 0.5% CMCNa (5 µmol/kg) or
a suspension of ICCA-WFF in 0.5% CMCNa (5, 0.5 and
0.05 µmol/kg) were orally given. In rat arterial thrombus
Bioassays
In vitro Anti-Proliferation Assay
The in vitro viability assays of human myeloid leukemia cells weight assay 0.5% CMCNa (3 mL/kg) or suspension of
(K562), ascites tumor cells (S180), human non-small cell aspirin in 0.5% CMCNa (167 µmol/kg) or a suspension of
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Zhang et al
ICCA in 0.5% CMCNa (5 µmol/kg) or a suspension of WFF
in 0.5% CMCNa (5 µmol/kg) or a suspension of ICCA-WFF
in 0.5% CMCNa (5, 0.5 and 0.05 µmol/kg) were orally
given.
Docking Toward the Active Sites of P-selectin and
GPIIb/IIIa
To show the superiority of ICCA-WFF to ICCA in fitting the
active site of both P-selectin and GPIIb/IIIa the CDOCKER
interaction energies were calculated. For this purpose, the
conformations of ICCA-WFF and ICCA were generated, and
10 energy optimized conformations of ICCA-WFF and ICCA
were docked into the active sites of P-selectin and GPIIb/IIIa
with CDOCKER protocol and using the procedure of the
literature.26,30
P-selectin and GPIIb/IIIa Expression Assays
The level of P-selectin expression was identified with an
enzyme-linked immune-sorbent assay (ELISA).The proce-
dure of the literature and the kit (rat P-selectin ELISA kit;
Wuhan Huamei Biotech Co., Ltd., Wuhan, Hubei Province,
PR China) was used.26 GPIIb/IIIa expression was similarly
identified with ELISA. The procedure of the literature and
the kit (rat GPIIb/IIIa ELISA kit; Wuhan Huamei Biotech
Co., Ltd., Wuhan, Hubei Province, PR China) was used.27 In
ELISA the serum was prepared from the blood of the rats
treated by ICCA (5 µmol/kg) or by aspirin (167 µmol/kg,
positive control) or by ICCA-WFF (5, 0.5 and 0.05 µmol/kg)
or by 0.5% CMCNa (3 mL/kg, negative control).
Molecular Assembly and Thrombus
Targeting Action
FT-ICR-MS and Corresponding Characterization
Molecular assembly of ICCA-WFF was identified with FT-
ICR-MS spectra, while the manner of the molecular assembly
was identified with the nuclear over hauser effect spectro-
scopy (NOESY) and energy optimization conformation.
These methods were in accordance with the literature.31
Rat Tail Bleeding Time Assay
FT-ICR-MS Analysis of Homogenate Extracts
This assay was performed by following the procedure of the
literature.28 In assay the male Sprague Dawley rats (240–270
g, each 12) were orally treated with 0.5% CMCNa (3 mL/kg,
negative control) or a suspension of aspirin in 0.5% CMCNa
(167 µmol/kg, positive control) or a suspension of ICCA in
0.5% CMCNa (5 µmol/kg) or a suspension of ICCA-WFF in
0.5% CMCNa (5, 0.5 and 0.05 µmol/kg).
To explore the targeting action the blood, brain, heart,
kidney, liver, spleen and thrombus of arterial thrombosis
rats treated with 0.5% CMCNa (3 mL/kg, negative control,
12 rats) or ICCA-WFF in 0.5% CMCNa (5 µmol/kg, 12
rats) were homogenized, the homogenates were extracted
with methanol and the extracts received FT-ICR-MS tests.
Besides, the tumor tissue, blood, brain, heart, kidney, liver
and spleen of S180 mice treated with 0.5% CMCNa
(3 mL/kg, negative control, 12 rats) or a suspension of
ICCA-WFF in 0.5% CMCNa (5 µmol/kg, 12 rats) were
homogenized, the homogenates were extracted with
methanol and the extracts received FT-ICR-MS analysis.
These methods were in accordance with literature.22,29,31
Venous Thrombus Weight Assay
This assay was performed by following the procedure of the
literature.26 In assay male Sprague Dawley rats (240–270 g,
each 12) were orally treated with 0.5% CMCNa (3 mL/kg,
negative control) or warfarin in 0.5% CMCNa (4.87 µmol/
kg, positive control) or ICCA in 0.5% CMCNa (5 µmol/kg)
or ICCA-WFF in 0.5% CMCNa (5, 0.5 and 0.05 µmol/kg).
Confocal Microscope Imaging
To show the cellular uptake of ICCA-WFF the confocal
microscope image was visualized. At 37°C 95D cells
(1×105 cells/mL) were incubated in complete RPMI 1640
medium containing 10% (v/v) fetal calf serum, penicillin
(60 μg/mL), and streptomycin (100 μg/mL), as well as
CO2 (a humidified atmosphere, 5%) for 24 hrs. After
removing the original medium, the complete RPMI 1640
medium or a solution of ICCA-WFF in the complete
RPMI 1640 medium (final concentration: 55 μM, 27.5
μM and 13.5 μM) was added (1 mL/well), and the cells
were incubated for another 12 h. After removing the
medium, the cells were washed with fresh medium
Testing PT, TT, APTT and FIB
To estimate the effect of ICCA-WFF on the prothrombin time
(PT), thrombin time (TT), activated partial thromboplastin
time (APTT) and plasma fibrinogen (FIB) the blood of 0.5%
CMCNa (3 mL/kg, negative control, 12 rats) or ICCA-WFF in
0.5% CMCNa (5 µmol/kg, 12 rats) treated rats in the venous
thrombosis assay was used to prepare the serum. Then, PT,
TT, APTT and FIB were recorded on the semi-automatic
blood coagulation instrument (Virtues Pacific Biological
Polytron Technologies Inc., Tianjin, People’s Republic of
China).22
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(1 mL×3) and resuspended in fresh medium (1 mL/well). consists of the synthesis of ICCA, the preparation of Trp-Phe-
The cells treated by the complete RPMI 1640 medium or
a solution of ICCA-WFF in the complete RPMI 1640
medium were analyzed by the use of TCS SP8 STED
confocal microscopy (Leica, Leica Microsystems,
Wetzlar, Germany). The excitation wavelength and the
emission wavelength were 405 nm and 575 nm,
respectively.
Phe-OBzl and the coupling of ICCA with Trp-Phe-Phe-OBzl.
It was found that the overall yields of the synthesis of ICCA,
the preparation of Trp-Phe-Phe-OBzl and the coupling of
ICCA with Trp-Phe-Phe-OBzl were 73%, 83% and 61%,
respectively, while the HPLC purity of ICCA-WFF was
99.63%. These data emphasize that this route is suitable for
preparing ICCA-WFF.
Results
ICCA-WFF Forming Trimer and Having
Z-Like Conformation
Trp-Phe-Phe Was Found to Be Suitable
for Modifying ICCA
To lower the dose of ICCA some oligopeptides were pre-
viously designed, prepared and evaluated. Among them Trp-
Phe-Phe was found to be capable of slowing tumor growth,
inhibiting thrombosis, and depressing serum P-selectin and
GPIIb/IIIa in particular (Figure 1).
FT-ICR-MS spectrum shows that the molecular assembly of
ICCA-WFF leads to the formation of a trimer.31 Figure 4A
shows that the FT-ICR-MS spectrum of ICCA-WFF gives an
anion peak at 809.34135 (the mass of monomer minus H,
calculated value: 809.34459), an anion peak at 1619.69296
(the mass of dimer minus H, calculated value: 1619.69296)
and an anion peak at 1214.52111 (the mass of trimer minus
2H, calculated value: 1214.52107). Figure 4B shows that the
qCID of the trimer minus 2H at 1214.52895 gives an anion
peak of monomer minus H at 809.34930 and an anion peak of
dimer minus H at 1619.71182. Therefore, the anion peaks of
the monomer minus H and the dimer minus H occur in
Figure 4A are the fragments of the trimer, and the trimer is
the sole form of ICCA-WFF to exist.
Thus, a derivative of ICCA coupling Trp-Phe-Phe could
bind GPIIb/IIIa and P-selectin was hypothesized, and
a docking investigation was performed. Figure 2 shows that
the CDOCKER interaction energies of ICCA and ICCA-WFF
with P-selectin are −20.5 kcal/mol and −40.3 kcal/mol,
respectively. Figure 2 also shows that the CDOCKER inter-
action energies of ICCA and ICCA-WFF with GPIIb/IIIa are
−23.9 kcal/mol and −41.6kcal/mol, respectively. For
P-selectin and GPIIb/IIIa, the CDOCKER interaction energies
of ICCA-WFF are significantly lower than those of ICCA.
The multiple activities of Trp-Phe-Phe in vivo with the
CDOCKER interaction energy of ICCA-WFF to be lower
than that of ICCA together suggested that Trp-Phe-Phe
was suitable for modifying ICCA.
NOESY 2D 1H NMR spectrum and energy optimization
conformation define the manner of molecular assembly of
ICCA-WFF to form the trimer.31 Figure 4C shows that there
is only one interesting cross-peak marked by a red circle.
According to the concept of NOESY, this cross-peak reflects
an interaction of the methine H of the isopropyl moiety of
one molecule and the amino H of the carboxyl terminal Phe
of Trp-Phe-Phe moiety of another molecule. This cross-peak
also means that the distance between these H is less than 4 Å.
Based on the distance between two H atoms, three energy
The Route Is Suitable to the Preparation
of ICCA-WFF
To process the forward investigations, ICCA-WFF was
synthesized by following the route of Figure 3. This route minimized monomers of ICCA-WFF were manually
Figure 1 In vivo activities of Trp-Phe-Phe (WFF), n=12: (A) anti-tumor activity of WFF against S180 mice; (B) anti-thrombotic activity of WFF on rat model; (C) inhibition of
WFF to serum P-selectin of arterial thrombosis rats; (D) inhibition of WFF to serum GPIIb/IIIa of arterial thrombosis rats.
Abbreviation: WFF, Trp-Phe-Phe.
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Figure 2 Docking feature of ICCA and ICCA-WFF in the active site of P-selectin and GPIIb/IIIa: (A) docking feature of ICCA and ICCA-WFF in the active site of P-selectin;
(B) docking feature of ICCA and ICCA-WFF in the active site of GPIIb/IIIa.
Abbreviations: ICCA, 1-(4-isopropylphenyl)-β-carboline-3-carboxylic acid; ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Trp-Phe-Phe.
accessed to form a trimer. This manual operation gives the J and N indicate that the radiation of 650 nm laser
trimer of ICCA-WFF a Z-like conformation (Figure 4D).
does not induce the ultrapure water of various pH to
occur Faraday-Tyndall effect. Figure 5C, G, K and
O indicate that 3.7 μM solutions of ICCA-WFF in
ultrapure water of various pH occur no Faraday-
Tyndall effect. Figure 5D, H, L and P indicate that the
radiation of 650 nm laser induces 3.7 μM solutions of
ICCA-WFF in ultrapure water of various pH to occur
Aqueous ICCA-WFF Is a Nano-Solution
To show the nano-property of aqueous ICCA-WFF the
Faraday-Tyndall effect was tested. Figure 5A, E,
I and M indicate that the ultrapure water of various
pH occurs no Faraday-Tyndall effect. Figure 5B, F,
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Figure 3 Synthetic route of ICCA-WFF: i) TFA, CH2Cl2; ii) SeO2, 1,4-dioxane and 70 °C; iii) aqueous NaOH (2M), 1,4-dioxane and 0°C; iv) anhydrous THF, HOBt, DCC; v)
hydrochloride in ethyl acetate (4M); vi) Pd/C and H2.
Abbreviations: ICCA, 1-(4-isopropylphenyl)-β-carboline-3-carboxylic acid; TFA, trifluoroacetic acid;CH2Cl2, dichloromethane;SeO2, selenium dioxide; NaOH, sodium
hydroxide; THF, tetrahydrofuran; HOBt, 1-Hydroxybenzotriazole; ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Tyr-Phe-Phe.
Faraday-Tyndall effect. These observations mean that various concentration and pH values, and the SEM images
the aqueous ICCA-WFF is nano-solution.
of the lyophilized powders tested. Figure 7 shows that the
lyophilized powders of ICCA-WFF consist of nano-
particles, the diameter of the nano-particles is less than
70 nm, and the diameter of the nano-particles is indepen-
dent of the concentration and the pH value of the solution
used for preparing the lyophilized powders.
In Aqueous Solution ICCA-WFF Exists as
Nano-Particles
To visualize the nano-feature of ICCA-WFF in the ultra-
pure water of pH 6.8 the TEM images were provided.
Figure 6 shows that in 1.23 nM, 123 nM and 12.3 μM
solutions of ICCA-WFF form nano-particles of 61.5–230.7
nm in diameter. Besides, the size of the nano-particles is
independent of the concentration of the solution.
In Rat Serum ICCA-WFF Exists as
Nano-Particles
To visualize the nano-feature of ICCA-WFF in rat plasma
the AFM images were recorded. Figure 8A shows that the
rat plasma alone has no interesting particles. Figure 8B–
D shows that in the rat plasma ICCA-WFF exists as nano-
particles of 12.3–59.2 nm in height, and the height is
In Solid State ICCA-WFF Exists as
Nano-Particles
To visualize the nano-feature of ICCA-WFF in solid state
the lyophilized powders were prepared from solution with independent of the concentration.
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Figure 4 FT-ICR-MS spectrum, qCID, NOESY and trimer conformation of ICCA-WFF: (A) FT-ICR-MS spectrum of ICCA-WFF gives an anion peak of the monomer at
809.34135, an anion peak of the dimer at 1619.69296 and an anion peak of the trimer at 1214.52111; (B) qCID of the trimer at 1214.52895 gives two anion peaks at
1619.71182 and 809.34930; (C and D) NOESY spectrum and the manner of three-molecule-assembly of ICCA-WFF.
Abbreviations: NOESY, nuclear over hauser effect spectroscopy; ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Tyr-Phe-Phe.
Figure 5 Tyndall effect of ICCA-WFFinvarious aqueous: (A)ultrapurewaterof pH2.0 withoutradiation; (B)ultrapurewaterof pH2.0 with 650 nm laser radiation; (C) 3.7μM solution
of ICCA-WFF in ultrapure water of pH 2.0 without radiation; (D) 3.7 μM solution of ICCA-WFF in ultrapure water of pH 2.0 with 650 nm laser radiation; (E) ultrapure water of pH 5.8
without radiation; (F) ultrapure water of pH 5.8 with 650 nm laser radiation; (G) 3.7 μM solution of ICCA-WFF in ultrapure water of pH 5.8 without radiation; (H) 3.7 μM solution of
ICCA-WFF in ultrapure waterof pH 5.8 with 650 nm laser radiation; (I) ultrapure water of pH 6.8 without radiation; (J) ultrapure waterof pH6.8 with 650 nm laser radiation; (K) 3.7 μM
solution of ICCA-WFF in ultrapure waterof pH 6.8 without radiation; (L) 3.7 μM solution of ICCA-WFF in ultrapure water of pH 6.8 with 650 nm laser radiation; (M) ultrapure waterof
pH 7.4 without radiation; (N) ultrapure water of pH 7.4 with 650 nm laser radiation; (O) 3.7 μM solution of ICCA-WFF in ultrapure water of pH 7.4 without radiation; (P) 3.7 μM
solution of ICCA-WFF in ultrapure water of pH 7.4 with 650 nm laser radiation.
Abbreviation: ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Tyr-Phe-Phe.
MTT method.22 Figure 9 shows that the IC50 values of ICCA-
ICCA-WFF Has Similar Cytotoxicity to
ICCA in vitro
To know if the modification of ICCA by Trp-Phe-Phe greatly
WFF against A549, K562, S180 and 95D cells are ~50–100 μM.
The IC50 of ICCA-WFF against 95D cells has the lowest value. In
was known that the IC50 values of ICCA against A549, K562,
and S180 cells treated by ICCA-WFF were measured by using S180 and 95D cells are also ~50–100 μM.22 Thus, for these cells,
alters the cytotoxicity of ICCA, the viabilities of A549, 95D, K562
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Figure 6 TEM images of ICCA-WFF in ultrapure water: (A) TEM image of 12.3 μM ICCA-WFF in ultrapure water of pH 6.8; (B) TEM image of 123 nM ICCA-WFF in
ultrapure water of pH 6.8; (C) TEM image of 1.23 nM ICCA-WFF in ultrapure water of pH 6.8.
Abbreviations: TEM, transmission electron microscopy; ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Tyr-Phe-Phe.
Figure 7 SEM images of lyophilized powders of ICCA-WFF: (A) SEM image of the lyophilized powders prepared from a solution of ICCA-WFF in ultrapure water of pH 2.0 (12.3 μM); (B)SEM
image of the lyophilized powders prepared from a solution of ICCA-WFF in ultrapure water of pH 2.0 (123 nM); (C) SEM image of the lyophilized powders prepared from a solution of ICCA-
WFF in ultrapure water of pH 2.0 (1.23 nM); (D) SEM image of the lyophilized powders prepared from a solution of ICCA-WFF in ultrapure water of pH 5.8 (12.3 μM); (E) SEM image of the
lyophilized powders prepared from a solution of ICCA-WFF in ultrapure water of pH 5.8 (123 nM); (F) SEM image of the lyophilized powders prepared from a solution of ICCA-WFF in
ultrapure water of pH 5.8 (1.23 nM); (G) SEM image of the lyophilized powders prepared from a solution of ICCA-WFF in ultrapure water of pH 5.8 (12.3 μM); (H) SEM image of the lyophilized
powders prepared from a solution of ICCA-WFF in ultrapure water of pH 6.8 (123 nM); (I) SEM image of the lyophilized powders prepared from a solution of ICCA-WFF in ultrapure water of
pH 6.8 (1.23 nM); (J) SEM image of the lyophilized powders prepared from a solution of ICCA-WFF in ultrapure water of pH 7.4 (12.3 μM); (K) SEM image of the lyophilized powders prepared
from a solution of ICCA-WFF in ultrapure water of pH 7.4 (123 nM); (L) SEM image of the lyophilized powders prepared froma solution of ICCA-WFF in ultrapure water of pH 7.4 (1.23 nM).
Abbreviations: SEM, scanning electron microscopy; ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Tyr-Phe-Phe.
ICCA-WFF and ICCA have similar IC50, and Trp-Phe-Phe mod- assay was performed on S180 mouse model.22 Figure 10A
ification does not greatly alter the cytotoxicity of ICCA.
shows that the tumor weight of S180 mice orally treated
with 5 μmol/kg/day of ICCA for 9 consecutive days is
significantly lower than that of S180 mice orally treated
with CMCNa. Figure 10A also shows that ICCA-WFF
dose-dependently slows tumor growth, and the tumor
Trp-Phe-Phe Modification Increases
Anti-Tumor Activity of ICCA in vivo
To evidence the enhanced the antitumor activity of Trp-
Phe-Phe modification of ICCA in vivo the tumor growth weight of S180 mice orally treated with 0.5 μmol/kg/day
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Figure 8 AFM images of ICCA-WFF in rat serum: (A) AFM image of rat serum alone; (B) AFM image of ICCA-WFF in rat serum (1.2×10−2 nM); (C) AFM image of ICCA-
WFF in rat serum (1.2×10−3 nM); (D) AFM image of ICCA-WFF in rat serum (1.2×10−4 nM).
Abbreviations: AFM, atomic force microscopy; ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Tyr-Phe-Phe.
orally treated by 5 μmol/kg of ICCA. Therefore, Trp-Phe-
Phe modification leads the anti-thrombotic activity of ICCA
for rats having a 100-fold increase.
Trp-Phe-Phe Modification Does Not
increase the Inhibition of ICCA to Venous
Thrombosis in vivo
To show the effect of Trp-Phe-Phe modification on the anti-
venous thrombotic activity of ICCA the venous thrombotic
assays were performed on rat models.22 Figure 10D shows
that ICCA-WFF dose-dependently decreases the venous
Figure 9 Effect of ICC-WFF on cell viability (n=5).
thrombus weight of the treated rats, and the minimal effective
dose is 0.5 μmol/kg. Besides, at 5 μmol/kg of dose ICCA-
of ICCA-WFF for 9 consecutive days is equal to that of
S180 mice orally treated with 5 μmol/kg/day of ICCA.
Therefore, Trp-Phe-Phe modification leads the anti-tumor
activity of ICCA to 10-fold increase.
WFF, ICCA and warfarin have equal activity. This means
that Trp-Phe-Phe modification does not enhance the inhibi-
tion of ICCA to venous thrombosis in vivo.
ICCA-WFF Down regulates P-selectin
and GPIIb/IIIa Expression in vivo
Trp-Phe-Phe Modification Increases the
To correlate P-selectin and GPIIb/IIIa with the inhibi-
tion of ICCA-WFF to arterial thrombosis in vivo the
expression levels of P-selectin and GPIIb/IIIa were
examined by using the reported methods.22 Figure
11A shows that P-selectin level in the serum of the
rats treated with 5 μmol/kg ICCA is significantly lower
than that of the rats treated with CMCNa, and is sig-
nificantly higher than that of the rats treated with 5
μmol/kg ICCA-WFF. Thus, by downregulating
P-selectin expression ICCA-WFF inhibits arterial
thrombosis. Figure 11B shows that GPIIb/IIIa level in
the serum of the rats treated with 5 μmol/kg ICCA is
significantly lower than that of the rats treated with
CMCNa, and is significantly higher than that of the
rats treated with 5 μmol/kg ICCA-WFF. Thus, by
downregulating GPIIb/IIIa expression ICCA-WFF
Inhibition of ICCA to Arterial
Thrombosis in vivo
To evidence the enhanced anti-thrombotic activity of Trp-
Phe-Phe modification of ICCA in vivo the arterial thrombotic
assays were performed on mouse and rat models.22
Figure 10B shows that ICCA-WFF dose-dependently
decreases the arterial thrombus weight of the mice, and the
arterial thrombus weight of the mice orally treated by 0.5
μmol/kg of ICCA-WFF is equal to that of the mice orally
treated by 5 μmol/kg of ICCA. Therefore, Trp-Phe-Phe mod-
ification leads the anti-thrombotic activity of ICCA for mice
to 10-fold increase. Figure 10C shows that ICCA-WFF dose-
dependently decreases the arterial thrombus weight of the
rats, and the arterial thrombus weight of the rats orally treated
by 0.05 μmol/kg of ICCA-WFF is equal to that of the rats
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A
B
C
D
Figure 10 Activity of ICCA-WFF in vivo assays (n=12): (A) anti-tumor activity of ICCA-WFF; (B) activity of ICCA-WFF in mouse arterial thrombotic assay; (C) activity of
ICCA-WFF in rat arterial thrombotic assay; (D) activity of ICCA-WFF in rat vein thrombotic assay.
Abbreviations: CMCNa, carboxymethylcellulose sodium; Dox, doxorubicin; ICCA, 1-(4-isopropylphenyl)-β-carboline-3-carboxylic acid; ICCA-WFF, 1-(4-isopropylphenyl)-
β-carboline-3-carbonyl-Tyr-Phe-Phe.
Figure 11 Expression of P-selectin and GPIIb/IIIa, as well as tail bleeding time of ICCA-WFF-treated rats in arterial thrombotic assays (n=12): (A) ICCA-WFF down-
regulates P-selectin expression; (B) ICCA-WFF down-regulates GPIIb/IIIa expression; (C) ICCA-WFF does not increase bleeding risk of ICCA.
Abbreviations: CMCNa, carboxymethyl cellulose sodium; ICCA, 1-(4-isopropylphenyl)-β-carboline-3-carboxylic acid; ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-car-
bonyl-Tyr-Phe-Phe.
effectively inhibits arterial thrombosis. Besides, the
ICCA-WFF Has No Bleeding Risk
efficacy of ICCA-WFF downregulating the expression
of P-selectin and GPIIb/IIIa is 33 folds higher than that
of aspirin.
To show the bleeding risk of ICCA-WFF the rats of
arterial thrombosis assay to be given tail bleeding time
assay by using the reported method.28 Figure 11C shows
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that at 5 μmol/kg of oral dose both ICCA and ICCA- that by adhering onto the surfaces of the rat platelets,
ICCA-WFF downregulates the expression of P-selectin
and GPIIb/IIIa.
WFF do not prolong tail bleeding time of the treated
rats. Thus, Trp-Phe-Phe modification does not increase
the bleeding risk of ICCA. Besides, the tail bleeding
time of the rats treated by 167 μmol/kg of aspirin is
significantly longer than that of the rats treated by 5
μmol/kg of ICCA-WFF.
Mesoscale Simulation of ICCA-WFF
Forming Nano-Particle
To dissect the process of the nano-particle formation
and to predict the molecular number of ICCA-WFF in
a nano-particle of definite diameter a mesoscale simu-
lation was performed. Figure 13 shows that the forma-
tion of the nano-particle consists of 3 steps, and
a nano-particle of 99.96 nm in diameter contains 1187
molecules of ICCA-WFF. This demonstrates that
mesoscale simulation not only reveals the course of
nano-particle formation but also estimates how much
molecules will be involved in a nano-particle of the
exact size.
By Adhering Rat Platelets ICCA-WFF
Inhibits P-selectin and GPIIb/IIIa
Expression
The in vitro action of ICCA-WFF on the resting rat
platelets and arachidonic acid (AA) activated rat plate-
lets was visualized by AFM. Figure 12A shows that the
resting rat platelets have a smooth surface and have no
pseudopodia. Figure 12B–D shows that in the presence
of ICCA-WFF (1.2×10−2 nM, 1.2×10−3 nM and
1.2×10−4 nM) the resting rat platelets neither have
smooth surface nor have pseudopodium. Figure 12E
ICCA-WFF Has No Liver and Renal
shows that the activation of AA induces rat platelets Toxicity
to extend pseudopodia and to form aggregation. Figure Serum ALT, AST, Cr and BUN are usually for estimating the
12F–H show that ICCA-WFF concentration (1.2×10−2 toxicity of the drug to kidney and liver.25 In this profile, the
nM, 1.2×10−3 nM and 1.2×10−4 nM) dependently inhi- ALT, AST, Cr and BUN of S180 mice treated with 5 μmol/
bits AA activated rat platelets to extend pseudopodia kg/day of ICCA-WFF or CMCNa for 9 consecutive days
and to form aggregation. These phenomena suggest were measured and are shown in Figure 14A. As seen, the
Figure 12 Imaging the interaction of ICCA-WFF and rat platelets by AFM: (A) AFM image of resting rat platelets; (B) AFM image of resting rat platelets with ICCA-WFF
(1.2×10−2 nM); (C) AFM image of resting rat platelets with ICCA-WFF (1.2×10−3 nM); (D) AFM image of resting rat platelets with ICCA-WFF (1.2×10−4 nM); (E) AFM image
of AA activated rat platelets; (F) AFM image of AA activated rat platelets with ICCA-WFF (1.2×10−2 nM); (G) AFM image of AA activated rat platelets with ICCA-WFF
(1.2×10−3 nM); (H) AFM image of AA activated rat platelets with ICCA-WFF (1.2×10−4 nM).
Abbreviations: AFM, atomic force microscopy; ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Tyr-Phe-Phe; AA, arachidonic acid.
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Figure 13 The course of forming nano-particle explored by mesoscale simulation and the molecular number of ICCA-WFF in a nano-particle of definite diameter.
Abbreviation: ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Tyr-Phe-Phe.
ALT, AST, Cr and BUN of S180 mice treated by ICCA-WFF arterial thrombus or venous thrombus of the rats treated with
are equal to those of S180 mice treated by CMCNa. These
data mean that, similarly to CMCNa, 5 μmol/kg of ICCA-
WFF does not injure the kidney and the liver of S180 mice.
CMCNa or ICCA-WFF (5 µmol/kg), as well as the extracts
of the homogenates of the blood, brain, heart, kidney, liver,
spleen and the tumor tissue of S180 mice treated with
CMCNa or ICCA-WFF (5 µmol/kg/day) received FT-ICR-
MS spectrum analysis. It was found that the FT-ICR-MS
spectra of the extract of the homogenate of the arterial
thrombus, but not the blood and the organs, of the arterial
thrombosis rats treated by ICCA-WFF gave the ion peas
related to ICCA-WFF. It was also found that the FT-ICR-
MS spectra of the extract of the homogenate of the venous
thrombus, but not the blood and the organs, of the venous
thrombosis rats treated by ICCA-WFF gave the ion peas
related to ICCA-WFF. It was again found that the FT-ICR-
MS spectra of the extract of the homogenate of the tumor
tissue, but not the blood and the organs, of the S180 mice
treated by ICCA-WFF gave the ion peas related to ICCA-
WFF. These observations are shown in Figure 15.
ICCA-WFF Affects No Coagulation
Function
To estimate the effect of ICCA-WFF on coagulation function
the PT, TT, APTTand FIB of the rats in the venous thrombosis
assay were measured. Figure 14B shows that the PT, TT, APTT
and FIB of the rats treated by ICCA-WFF (5 µmol/kg, 12 rats)
are equal to those of the rats treated by CMCNa (3 mL/kg,
negative control, 12 rats). The PT, TT, APTT and FIB suggest
that, similarly to CMCNa, 5 μmol/kg of ICCA-WFF does not
injure the coagulation system of the thrombosis rats.22
ICCA-WFF Targets Arterial Thrombus
and Tumor Tissue
To ensure the targeting action and release the extracts of the extracts of the homogenates of the arterial thrombus and
homogenates of the blood, brain, heart, kidney, liver, spleen, the venous thrombus, respectively. In both of the spectra,
Figure 15A and B are the FT-ICR-MS spectra of the
Figure 14 Effects of ICCA-WFF on the kidney and the liver of S180 mice (n=12) as well as the coagulation system of the venous thrombosis rats (n=12): (A) effect of ICCA-
WFF on the kidney and the liver of S180 mice; (B) effect of ICCA-WFF on the coagulation system of the venous thrombosis rats.
Abbreviations: ALT, glutamic pyruvictran-saminase; AST, glutamic oxaloacetic transaminase; Cr, creatinine; BUN, blood urea nitrogen; FIB, fibrinogen; PT, prothrombin
time; TT, thrombin time; APTT, activated partial thromboplastin time; CMCNa, carboxymethyl cellulose sodium; ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-
Tyr-Phe-Phe.
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Zhang et al
Figure 15 FT-ICR-MS spectra of the homogenate extracts of the arterial thrombus and venous thrombus of the rats treated with 5μmol/kg of ICCA-WFF: (A) FT-ICR-MS
spectrum of the homogenate extract of arterial thrombus of the rats treated with 5 μmol/kg of ICCA-WFF, in the local amplified inset an ion peak of ICCA plus H is at
331.14555 (calculated value: 331.14465) and an ion peak of Trp-Phe-Phe plus Na is at 521.21687 (calculated value: 521.21648); (B) FT-ICR-MS spectrum of the homogenate
extract of venous thrombus of the rats treated with 5 μmol/kg of ICCA-WFF, in the local amplified inset an ion peak of ICCA plus H is at 331.14665 (calculated value:
331.14465) and an ion peak of Trp-Phe-Phe plus Na is at 521.21550 (calculated value: 521.21648); (C) FT-ICR-MS spectrum of the homogenate extract of tumor tissue of
S180 mice treated with 5 μmol/kg of ICCA-WFF, in the local amplified inset an ion peak of ICCA plus Na is at 353.12644 (calculated value: 353.12660) and an ion peak of
ICCA plus K is at 369.10067 (calculated value: 369.10053).
Abbreviations: ICCA, 1-(4-isopropylphenyl)-β-carboline-3-carboxylic acid; ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Tyr-Phe-Phe.
there are the ion peaks of ICCA and Trp-Phe-Phe. These data and 575 nm of emission wavelength the nucleus of the cells
evidence that ICCA-WFF targets thrombus and releases anti- treated by the complete RPMI 1640 medium exhibits weak
thrombus pharmacophores ICCA and Trp-Phe-Phe. Figure red. Figure 16B–D show that at 405nm of excitation wave-
15C is FT-ICR-MS spectrum of the extract of the homoge-
nate of the tumor tissue. In the spectrum, there is the ion peak
of ICCA. This data evidences that ICCA-WFF targets tumor
tissue and releases anti-tumor pharmacophore ICCA.
length and 575 nm of emission wavelength the cytoplasm,
but not nucleus of the cells, treated by ICCA-WFF exhibits
strong red. This strong red is from the UV absorption of
ICCA-WFF at 254 nm, and the absorption strength depends
on the concentration of ICCA-WFF. Thus, confocal micro-
scope images reflect the uptake of ICCA-WF for 95D cells
in a concentration-dependent manner.
ICCA-WFF Enters Cytoplasm of 95D
Cells
To show the cellular uptake of ICCA-WFF the confocal
microscope images of 95D cells treated by complete RPMI Discussion
1640 medium and ICCA-WFF (55 μM, 27.5 μM and The synthetic route of Figure 2 provides ICCA-WFF in
13.5 μM) were recorded, and the images are visualized. proper yield and high HPLC purity. These results demon-
Figure 16A shows that at 405nm of excitation wavelength strate that the route is suitable for preparing ICCA-WFF. FT-
Figure 16 Confocal microscope images of 95D cells treated by complete RPMI 1640 medium or ICCA-WFF; (A) confocal microscope images of 95D cells treated by
complete RPMI 1640 medium; (B) confocal microscope images of 95D cells treated by 55 μM ICCA-WFF; (C) confocal microscope images of 95D cells treated by 27.5 μM
ICCA-WFF; (D) confocal microscope images of 95D cells treated by 13.5 μM ICCA-WFF; n=3.
Abbreviation: ICCA-WFF, 1-(4-isopropylphenyl)-β-carboline-3-carbonyl-Tyr-Phe-Phe.
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ICR-MS spectrum approves that due to molecular assembly
the trimer is the sole existence form of ICCA-WFF. To
explore whether the trimer can further assemble the aqueous
ICCA-WFF is radiated with 650 nm laser to test Faraday-
Tyndall effect. The appearance of Faraday-Tyndall effect
suggests that aqueous ICCA-WFF has nano-property and
this nano-property should contribute to the assembly of
ICCA-WFF’s trimer. The assembly of the trimer is further
Conclusions
Coupling 1-(4-isopropylphenyl)-β-carboline-3-carboxylic
acid (ICCA) and Trp-Phe-Phe smoothly provides 1-(4-iso-
propylphenyl)-β-carboline-3-carbonyl-Trp-Phe-Phe (ICCA-
WFF). The molecular assembly leads ICCA-WFF been
able to form nano-particles capable of safe delivery in the
blood circulation, adhering on the surfaces of platelets, tar-
geting tumor tissue and thrombus as well as releasing anti-
visualized with TEM, ESM and AFM images. The images tumor and anti-thrombotic pharmacophores wherein. ICCA-
show that even though in ultrapure water of pH 6.8 the WFF has no bleeding risk, no effect on the coagulation
function and no liver or kidney toxicity. ICCA-WFF is
a promising nano-medicine.
diameter of the nano-particles is 61.5–230.7 nm, in the
lyophilized powders, the diameter of the nano-particles is
less than 70 nm and in rat plasma the height of the nano-
particles is less than 60 nm in particular. The size of the
nanoparticles suggests that in the blood circulation ICCA-
WFF is able to avoid macrophage phagocytosis and is cap-
able of safe delivery. AFM images also visualize that the size
of the nano-particles benefits ICCA-WFF adhering onto the
surfaces of AA activated rat platelets. Logically, this kind of
adhering property benefits ICCA-WFF depressing the
expression of P-selectin and GPIIb/IIIa.
Acknowledgments
The authors thank the Special Project of China (2018
ZX097201003), NSFC (81673303 and 81703332), KZ201
610025029, BNSF (7172028), 2016000020124G096, and
2017000020124G264 for financial support.
Disclosure
The authors report no conflicts of interest in this work.
The minimal effective dose of ICCA-WFF to slow
tumor growth is 0.5 μmol/kg/day, and the minimal effec-
tive dose of ICCA-WFF to inhibit the arterial thrombosis
and the venous thrombosis is 0.5 μmol/kg. On the other
hand, however, Trp-Phe-Phe modification increases the
inhibition of ICCA to tumor growth and arterial thrombo-
sis, but does not increase the inhibition of ICCA to venous
thrombosis. In addition, the in vivo activities of ICCA-
WFF inhibiting the expression of P-selectin and GPIIb/IIIa
are significantly higher than those of ICCA. FT-ICR-MS
spectrum analysis ensures that after oral administration
ICCA-WFF selectively distributes to tumor tissue and
arterial thrombus, i.e. exhibits the so-called tumor and
arterial thrombus targeting actions. These targeting actions
should be responsible for the superiority of ICCA-WFF
over ICCA in anti-tumor action, anti-arterial thrombotic
action and down-regulation of P-selectin and GPIIb/IIIa.
The minimal effective dose of ICCA-WFF to inhibit
the arterial thrombosis and the venous thrombosis is 0.5
μmol/kg, both arterial thrombosis and venous thrombosis
are the multiple complications of malignant tumor
patients, and these emphasize that as an anti-thrombotic
agent ICCA-WFF is of clinical importance. This impor-
tance is further supported by ICCA-WFF has no bleeding
risk, no effect on the coagulation function and no liver or
kidney toxicity.
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