Design and synthesis of nanoscaled IQCA-TAVV as a delivery system capable of antiplatelet activation, targeting arterial thrombus and releasing IQCA

Article abstract of DOI:10.2147/IJN.S150205

Background: Arterial thrombosis has been associated with a series of pathological conditions, and the discovery of arterial thrombosis inhibitor is of clinical importance. Methods: By analyzing the pharmacophores of anti-platelet agents, thrombus targeting peptide and anti-thrombotic nano-systems 3S-1,2,3,4-tetrahydroisoquino- line-3-carbonyl-Thr-Ala- Arg-Gly-Asp(Val)-Val (IQCA-TAVV) was designed and prepared as a nano-scaled arterial thrombosis inhibitor. Results: In vitro the nanoparticles of IQCA-TAVV were able to adhere onto the surface of activated platelets, attenuate activated platelets to extend pseudopodia and inhibit activated platelets to form aggregators. In vivo IQCA-TAVV targeted arterial thrombus, dose depend- ently inhibited arterial thrombosis with a 1 nmol/kg of minimal effective dose, and the activity was ~1670 folds of that of aspirin. Conclusion: IQCA-TAVV represented the design, preparation and application of nanomedicine capable of adhering on the surface of activated platelets, attenuating platelet activation, targeting arterial thrombus and inhibiting arterial thrombosis.

Full text of DOI:10.2147/IJN.S150205

International Journal of Nanomedicine
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O r I g I N a l r e s e a r c h
Design and synthesis of nanoscaled IQca-TaVV as
a delivery system capable of antiplatelet activation,
targeting arterial thrombus and releasing IQca
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
Jianhui Wu1,2,*
haimei Zhu1,2,*
guodong Yang1
Jianhong he3
Yuji Wang1,2
Background: Arterial thrombosis has been associated with a series of pathological conditions,
and the discovery of arterial thrombosis inhibitor is of clinical importance.
Methods: By analyzing the pharmacophores of anti-platelet agents, thrombus targeting peptide
and anti-thrombotic nano-systems 3S-1,2,3,4-tetrahydroisoquino- line-3-carbonyl-Thr-Ala-
Arg-Gly-Asp(Val)-Val (IQCA-TAVV) was designed and prepared as a nano-scaled arterial
thrombosis inhibitor.
shurui Zhao1,2
Xiaoyi Zhang1,2
lin gui1,2
Results: In vitro the nanoparticles of IQCA-TAVV were able to adhere onto the surface of
activated platelets, attenuate activated platelets to extend pseudopodia and inhibit activated
platelets to form aggregators. In vivo IQCA-TAVV targeted arterial thrombus, dose depend-
ently inhibited arterial thrombosis with a 1 nmol/kg of minimal effective dose, and the activity
was ~1670 folds of that of aspirin.
Ming Zhao1,2,4
shiqi Peng1,2
1Beijing area Major laboratory of
Peptide and small Molecular Drugs,
engineering research center of
endogenous Prophylactic of Ministry
of education of china, Beijing, china;
2Beijing laboratory of Biomedical
Materials, college of Pharmaceutical
sciences, capital Medical University,
Beijing, china; 3Department of
Conclusion: IQCA-TAVV represented the design, preparation and application of nanomedicine
capable of adhering on the surface of activated platelets, attenuating platelet activation, targeting
arterial thrombus and inhibiting arterial thrombosis.
Keywords: arterial thrombosis, thrombus targeting, nanodelivery, antithrombosis
Introduction
Thrombus formation is responsible for elevated mortality worldwide. Arterial throm-
bosis has been associated with a series of pathologic conditions, such as acute coronary
syndromes,1–3 ischemic stroke,4,5 transient ischemic attack,5,6 tumor thrombosis,7,8
recurrence of thromboembolic events,9,10 metabolic syndrome,11–15 chronic pain and
swelling symptoms.16
Internal Medicine of TcM,The
First affiliated hospital of guanxi
University of chinese Medicine,
Nanning, china; 4Department of
Biomedical science and environmental
Biology, Kaohsiung Medical University,
Kaohsiung,Taiwan
Platelet activation plays an important role in the formation of arterial thrombosis.17,18
Thrombosis involves the complex interactions between coagulation, and fibrinolytic
and innate immune systems, while the interaction between the platelets, injured ves-
sel and a variety of specific membrane receptors plays an essential role.19 Through
activating the specific membrane receptors for adhesive proteins, the adhesion, acti-
vation and aggregation of platelets is triggered at the site of vascular injury to form
a plug, that is, thrombus.20 A series of drugs, such as heparin tissue plasminogen
activator,21,22 urokinase,23 warfarin24 and melagatran,25 have been used to dissolve the
formed thrombus or to inhibit the formation of thrombus. On the other hand, these
drugs have some clinical shortcomings. For instance, most of these drugs need to be
intravenously injected, can cause serious side effects and usually have short half-life
in the blood circulation.26–34 To solve these problems, a series of nanopreparations,
*These authors contributed equally
to this work
correspondence: shiqi Peng; Ming Zhao
Beijing laboratory of Biomedical
Materials, college of Pharmaceutical
sciences, capital Medical University,
Number 10 You an Men Wai Xi Tou
Tiao, Fengtai District, Beijing 100069,
china
Tel +86 10 8391 1535; +86 10 8391 1528
Fax +86 10 8391 1528; +86 10 8391 1535
email mingzhao@bjmu.edu.cn;
sqpeng@bjmu.edu.cn
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Figure 1 The combination of pharmacophores of the antiplatelet agents, thrombus-targeting peptide arg-gly-asp-Val and antithrombotic agents leads to the design of
IQca-TaVV as a thrombus-targeting nanodelivery system capable of antiplatelet activation, targeting arterial thrombus and releasing IQca.
Abbreviations: IQca, 3s-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; IQca-TaVV, 3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-ala-arg-gly-asp(Val)-Val.
such as hollow nanogels, magnetic nanoparticles and lipo- Beijing, China) and without correction. 1H nuclear magnetic
somes, were discovered.35–37 However, the oral nanodelivery resonance (NMR) spectra of all prepared compounds were
capable of inhibiting platelet activation, targeting arterial recorded at 500 MHz (1H NMR) and 125 MHz (13C NMR) on
thrombus and releasing pharmacophores remains unknown. a Bruker AVANCE II 500. Optical rotations of all prepared
Based on 3S-1,2,3,4-tetrahydroiso-quinoline-3-carbonyl- compounds were measured with a Jasco P-1020 Polarimeter.
Thr and 3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Ala Electrospray ionization mass spectrometry (ESI-MS) read-
being antiplatelet agents,38 Arg-Gly-Asp-Val in aspirin- ings of all prepared compounds were measured on a ZQ
Arg-Gly-Asp-Val being a thrombus-targeting peptide,39 2000 (Waters Corp) and 9.4 T solariX Fourier transform ion
3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-Ala-Arg- cyclotron resonance (FT-ICR) mass spectrometer (Bruker
Gly-Asp-(Phe)-Phe40 and 3S-1,2,3,4-tetrahydroisoquinoline- Corp, Billerica, MA, USA), with an ESI/matrix-assisted
3-carbonyl-Thr-Ala-Arg-Gly-Asp(Ser)-Ser41 being an oral laser desorption/ionization (MALDI) dual ion source. Male
antithrombotic nanodelivery system, this paper designed Sprague Dawley rats and ICR mice were purchased from
3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-Ala-Arg- the Animal Center of Capital Medical University. Animal
Gly-Asp(Val)-Val (IQCA-TAVV of Figure 1) as a nano- experiments were reviewed and approved by the ethics com-
scaled delivery system to inhibit the activation of platelets, mittee of Capital Medical University. The committee assures
to target the arterial thrombus and to release 3S-1,2,3,4- that the welfare of the animals is maintained in accordance
tetrahydroisoquinoline-3-carboxylic acid (IQCA).
with the requirements of the Animal Welfare Act and in
accordance with the NIH Guide for Care and Use of Labora-
tory Animals. Student’s t-test and analysis of variance were
used for statistical analyses of all data. P-values ,0.05 were
considered statistically significant.
Materials and methods
general
The chemicals were commercially available without further
purification. The chromatography was performed on silica
gel H (Qingdao Marine Chemical Factory, Qingdao, China).
Preparation of 3s-1,2,3,4-
The purity of IQCA-TAVV was analyzed with high-perfor- tetrahydroisoquinoline-3-carboxylic acid
mance liquid chromatography (Waters Corporation, Milford, Into a solution of 4.0 g (24.2 mmol) of L-Phe in 21.6 mL of
MA, USA; C18 column, 4.6×150 mm). Melting points (MPs) formaldehyde 36 mL of hydrochloric acid (35%) was added
of all prepared compounds were measured on an electrother- dropwise. This solution was stirred at 90°C for 12 h and TLC
mal XT5G apparatus (Beijing Keyi Electro-optic Instrument, (CHCl3/CH3OH, 5/1) indicated the complete disappearance
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Nanoscaled IQca-TaVV targeting arterial thrombus
of L-Phe. The reaction mixture was cooled to room tem- Preparation of 3s-N-Boc-1,2,3,4-
perature, and the formed precipitates were collected and
tetrahydroisoquinoline-3-carbonyl-
successively washed with water and acetone to give 4.17 g
Thr (3)
(98%) of the title compound as a colorless powder. ESI-MS
(m/z): 176 (M - H)-.
At 0°C, to a solution of 400 mg (1.0 mmol) of 3S-N-Boc-
1,2,3,4-tetrahydro-isoquinoline-3-carbonyl-Thr-OBzl (2)
in 4 mL of methanol, 10 mL of aqueous NaOH (2 M) was
Preparation of 3s-N-Boc-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid
added dropwise; this mixture was stirred at room tempera-
ture for 5 h, and TLC (acetone/petroleum, 1/3) indicated
(1)
the complete disappearance of 2. The reaction mixture was
At 0°C a solution of 10 g (56.5 mmol) of IQCA and 2.5 g
filtered and the filtrate was concentrated under reduced
(62.2 mmol) of NaOH in 63 mL of water and a solution of
pressure. The residue was dissolved in 30 mL of water and
14.8 g (67.8 mmol) of (Boc)2O in 40 mL of tetrahydrofuran
the solution was adjusted to pH 2 with hydrochloric acid
were mixed. This mixture was stirred at room temperature
(2 M). The formed precipitates were dissolved in 50 mL
for 24 h, TLC (CHCl3/CH3OH, 10/1) indicated the complete
of ethyl acetate, and the solution was washed successively
disappearance of IQCA, the solvent was removed under
with 40 mL of aqueous sodium bicarbonate (5%), 40 mL of
reduced pressure, and the residue was dissolved in 60 mL of
aqueous citric acid (5%) and 40 mL of aqueous saturated
ethyl acetate. The formed solution was washed successively
sodium chloride. The ethyl acetate phase was separated and
with 40 mL of hydrochloric acid (0.5 M), 40 mL of aqueous
dried over anhydrous sodium sulfate, filtered, and the filtrate
sodium bicarbonate (5%) and 40 mL of saturated aqueous
was concentrated under reduced pressure to give 305 mg
sodium chloride. The ethyl acetate phase was separated,
(96% yields) of the title compound as a colorless powder.
dried over anhydrous sodium sulfate, filtered, and the filtrate
ESI-MS (m/z): 377 [M - H]-.
was concentrated under reduced pressure to give 14.8 g
(95% yields) of the title compound as colorless powder.
ESI-MS (m/z): 276 [M - H]-.
Preparation of ala-arg(NO2)-gly-
asp(OBzl)-OBzl
The solution method and a stepwise strategy (from C ter-
Preparation of 3s-N-Boc-1,2,3,4-
tetrahydroisoquinoline-3-carbonyl-Thr-
minal to N terminal) were used to prepare Ala-Arg(NO2)-
Gly-Asp(OBzl)-OBzl as a colorless powder in 85% yield.
ESI-MS (m/z): 701 [M + H]+.
OBzl (2)
A solution of 149 mg (1.2 mmol) of N-hydroxy benzotriazole
and 290 mg (1.1 mmol) of 3S-N-Boc-1,2,3,4-tetrahydroiso-
Preparation of 3s-N-Boc-1,2,3,4-
quinoline-3-carboxylic acid (1) in 10 mL of anhydrous
tetrahydroisoquinoline-3-carbonyl-Thr-
tetrahydrofuran was at 0°C, stirred for 10 min, then 228 mg
(1.1 mmol) of dicyclohexylcarbodiimide was added to form ala- arg(NO2)-gly-asp(OBzl)-OBzl (4)
solution A. A solution of 381 mg (1.0 mmol) of Tos⋅Thr- By using the same procedure as for preparing 2, from
OBzl in 4 mL of anhydrous tetrahydrofuran was adjusted to 1.05 g (3 mmol) of 3 and 2.04 g (3 mmol) of Ala-
pH 9 with 1 mL of N-methyl-morpholine to form solution B. Arg(NO2)-Gly-Asp(OBzl)-OBzl, 926 mg (93% yields) of
Solution A and Solution B were mixed, at room temperature the title compound was obtained as a colorless powder.
stirred for 12 h, and TLC (ethyl acetate/petroleum, 1/3) MP 159°C–160°C. ESI-MS (m/z): 1,003 [M + H]+. [α]
25
D
indicated the complete disappearance of Tos⋅Thr-OBzl.
=-14 (c=1.1, CH3OH). 1H NMR (500 MHz, DMSO-d6)
This mixture was filtered, the filtrate was concentrated δ/ppm =8.42 (d, J=6.6 Hz, 1 H), 8.12 (s, 1 H), 7.99 (s, 1 H),
under reduced pressure and the residue was dissolved in 7.91 (d, J=7.5 Hz, 1 H), 7.74 (m, 1 H), 7.59 (m, 1 H), 7.34
ethyl acetate (50 mL). The formed solution was washed (s, 10 H), 7.17 (s, 4 H), 5.09 (d, J=6 Hz, 4 H), 4.97 (s, 1 H),
successively with 40 mL of aqueous sodium bicarbonate 4.78 (s, 1 H), 4.58 (m, 2 H), 4.49 (m, 1 H), 4.27 (m, 3 H), 3.94
(5%), 40 mL of aqueous citric acid (5%) and 40 mL of aque- (s, 1 H), 3.75 (s, 2 H), 3.14 (s, 4 H), 2.92 (dd, J=6.3 Hz, J=5.7
ous saturated sodium chloride. The ethyl acetate phase was Hz, 3 H), 2.01 (s, 1 H), 1.99 (s, 2 H), 1.30 (m, 13 H), 0.84
separated, dried over anhydrous sodium sulfate, filtered, and (m, 4 H). 13C NMR (125 MHz, DMSO-d6) (ppm) =172.7,
the filtrate was concentrated under reduced pressure to give 171.9, 170.8, 169.2, 169.1, 136.2, 136.1, 128.8, 128.5, 128.4,
453 mg (90% yields) of the title compound as a colorless 128.1, 127.3, 126.3, 66.8, 66.4, 52.7, 49.1, 48.9, 48.7, 42.1,
powder. ESI-MS (m/z): 469 [M + H]+.
36.3, 29.5, 28.4, 19.6.
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Preparation of 3s-N-Boc-1,2,3,4-
tetrahydroisoquinoline-3-carbonyl-
Thr-ala-arg(NO2)-gly-asp (5)
By using the same procedure as for preparing 3, from 1.0 g
(1.0 mmol) of 4, 756 mg (92%) of the title compound was
obtained as a colorless powder. ESI-MS (m/z): 821 [M - H]-.
δ/ppm =172.53, 172.46, 171.51, 170.89, 170.69, 168.80,
168.69, 167.98, 167.85, 156.15, 134.87, 131.15, 128.08,
126.37, 125.84, 125.62, 66.11, 64.30, 57.42, 56.99, 56.68,
54.21, 51.63, 47.73, 41.23, 36.55, 29.49, 29.37, 28.63, 19.02,
18.48, 17.49 (Figure S2).
energy-minimized conformation of
IQca-TaVV
Preparation of 3s-N-Boc-1,2,3,4-
tetrahydroisoquinoline-3-carbonyl-
Thr-ala-arg(NO2)-gly-asp(Val-OBzl)-
The structure of IQCA-TAVV was constructed, converted to
conformation using Chem Office 10.0, and energy minimiza-
tion was carried out in MM2 force field until the minimum
root mean squared (RMS) gradient reached 0.001. Systematic
search and BEST methods of Discovery Studio 3.5 sampled
the energy-optimized conformations of IQCA-TAVV. The
SMART minimizer was used for the conformation sampling.
The sampling parameters were set to CHARMm force field
such as 20 kcal/mol energy threshold, 300 K, maximum 200
minimization steps and minimization RMS gradient 0.1 Å.
In addition, maximum 255 conformations were generated with
an RMS deviation cutoff of 0.2 Å. The lowest energy confor-
mation of IQCA-TAVV was used for docking investigation.
Val-OBzl (6)
By using the same procedure as for preparing 3, from 822 mg
(1.0 mmol) of 5 and 909 mg (2.4 mmol) of Tos⋅Val-OBzl,
864 mg (72% yields) of the title compound was obtained
as a colorless powder. ESI-MS (m/e): 1,201 [M + H]+.
25
1
MP 128°C–129°C. [α]D =-17.86 (c=1.1, CH3OH), H
NMR (500 MHz, DMSO-d6) δ/ppm =8.51 (s, 1 H), 8.42
(d, J=8.0 Hz, 2 H), 8.14 (d, J=7.5 Hz, 1 H), 8.07 (s, 1 H),
7.97 (d, J=5.5 Hz, 1 H), 7.75 (d, J=8 Hz, 1 H), 7.34–7.26
(m, 10 H), 7.16 (m, 4 H), 5.19–5.10 (m, 4 H), 4.63 (m, 1 H),
4.51 (m, 1 H), 4.45 (q, J=8.0 Hz, 1 H), 4.39 (m, 2 H), 4.28
(m, 4 H), 4.12 (m, 4 H), 3.11 (m, 2 H), 3.04 (m, 2 H), Mesoscale simulation of nanoparticles of
3.15 (m, 2 H), 2.64 (dd, J=6.3 Hz, J=6.9 Hz, 1 H), 2.55
IQca-TaVV
(dd, J=6.3 Hz, J=6.9 Hz, 1 H), 2.01 (s, 2 H), 1.96 (s, 1 H),
1.77 (m, 2 H), 1.55 (m, 2 H), 1.49 (d, J=7.8 Hz, 3 H), 1.37
(s, 9 H), 1.02 (d, J=8.2 Hz, 12 H), 1.22 (d, J=8.2 Hz, 3 H).
The structure of IQCA-TAVV was sketched by use
of ChemDraw Ultra 10.0 (Cheminformatics company
CambridgeSoft, Cambridge, MA, USA), which was trans-
formed into conformation with Materials Studio 3.2 (Accelrys,
Inc., San Diego, CA, USA) and entirely optimized with MS
Forcite module. The geometry of the prepared model of
IQCA-TAVV was optimized until the maximum energy con-
Preparation of 3s-1,2,3,4-
tetrahydroisoquinoline-3-carbonyl-Thr-
ala-arg-gly-asp-(Val)-Val (IQca-TaVV)
At 0°C a solution of 50 mg (0.4 mmol) of 6 in 1 mL of vergence and the maximum force changed to 2×10-5 kcal/mol
dimethyl sulfide, 1 mL of phenyl methyl ether, 4 mL of and 0.001 kcal/mol/Å, respectively. The molecular dynamic
CF3CO2H and 1 mL of CF3SO3H (1 mL) was stirred for 1 h. simulation was performed at 500 K by use of NVT ensemble
Upon removal of CF3CO2H/CF3SO3H, the residue was tritu- till equilibration. The final structure of IQCA-TAVV was
rated with 150 mL of ether, and the residue was purified on further optimized with MS Dmol3 module, based on which a
Sephadex G10 to provide 28.4 mg (94% yields) as a colorless rigid coarse grain model of three connected spherical beads was
powder. ESI-MS (m/e): 874 [M - H]-. MP 164°C–165°C. built. In a cubic box of 200×200×200 Å3 three-spherical-bead
25
[α]D =-10.1 (c=1.4, CH3OH). IR (cm-1) 3,446, 2,963, 2,386, model was randomly distributed with a density of 0.15 g/cm3,
1,653, 1,542, 1,456, 1,390, 1,254, 1,172, 1,029, 762, 641, and a simulation of 15,000 ps was performed at 298 K by use
1
515, 437; H NMR (800 MHz, DMSO-d6) δ/ppm =8.36 of NVT ensemble.
(m, 1 H), 8.20 (m, 1 H), 8.08 (m, 1 H), 8.03 (m, 1 H), 7.98
recording FT-Ms spectrum of
(m,1H),7.94(m,1H),7.82(m,1H),7.71(m,1H),7.20(m,4H),
4.62 (m, 1 H), 4.31 (m, 4 H), 4.17 (m, 3 H), 4.14 (m, 2 H), 3.97 IQca-aTVV
(m,1H),3.72(m,2H),3.22(m,1H),3.08(s,2H),2.88(m,1H), On solariX FT-ICR mass spectrometer (Bruker Dalton-
2.58 (m, 1 H), 2.02 (m, 2 H), 1.71 (m, 1 H), 1.54 (m, 1 H), 1.50 ics, Billerica, MA, USA) consisting of an ESI/MALDI
(m, 3 H), 1.26 (t, J=7.2 Hz, 3 H), 1.09 (d, J=5.6 Hz, 3 H). dual ion source and a 9.4 T superconductive magnet, the
0.85 (m, 12 H) (Figure S1); 13C NMR (200 MHz, DMSO-d6) FT-MS spectrum of IQCA-ATVV was recorded in positive
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Nanoscaled IQca-TaVV targeting arterial thrombus
MALDI ion mode. The qualitative contingent influence dia- load IQCA-TAVV. The copper grid with IQCA-TAVV was
gram (qCID) spectrum of 1,773.58817 m/z was recorded, and put under the microscope, the randomly selected regions
the isolation window was 5 mass units. By use of software were viewed, and the morphology and size distribution of the
solariX and Bruker Daltonics Data Analysis the spectral data nanospecies were recorded over 100 species. Then 6,000–
were analyzed.
400,000× digitally enlarged nanospecies were recorded on the
imaging plate (Gatan Bioscan Camera Model 1792; Gatan,
Inc., Pleasanton, CA, USA) with 20 eV energy windows.
Each imaging was performed with triplicate grids.
recording NOesY 2D NMr spectrum of
IQca-aTVV
1H NMR tests were performed on Bruker AvanceIII 800 MHz
spectrometer operating at 18.8 T equipped a CPQCI cryo-
probe of 5 mm, and the signals were observed at 800.25 MHz
Imaging the nanofeatures of lyophilized
powders of IQca-TaVV with a scanning
and recorded at 303 K. DMSO-d6 and tetramethylsilane electron microscope (seM)
were used as the solvent and the internal standard, respec- The morphology and size of the lyophilized powders of
tively. The spectrum was acquired by use of spectral width IQCA-TAVV were imaged on an SEM (S-4800; Hitachi,
of 16,447 Hz, data points of 65,536, pulse width of 8.78 μs, Tokyo, Japan) at 15 kV. The SEM sample was prepared by
relaxation delay of 1.0 s, acquisition time of 2 s and 16 scans. fixing the lyophilized powders from a 10-8 M solution of
To enhance the sensitivity before Fourier transforms, the IQCA-TAVV in ultrapure water onto the stub with double-
spectrum was processed, accurately phased and baseline sided tape (Euromedex, Strasbourg, France), which was
adjusted by applying an exponential line broadening of then coated with 10-nm-thick gold–palladium by use of a
0.3 Hz. NOESY 2D NMR spectrum was recorded by a Joel JFC-1600 auto fine coater (JEOL, Tokyo, Japan). The
phase-sensitive mode with 2,048 points and 512 points in imaging conditions were 15 kV, 30 mA and 200 mTorr
the F2 and the F1 directions, respectively. NOESY 2D NMR (argon) for 60 s. The randomly selected regions of the coater
spectrum was acquired with 12 scans, a relaxation delay of were viewed, and the morphology and size distribution of
2 s and mixing time values of 0.30 s.
the lyophilized powders were recorded for over 100 species.
Then 100–10,000× digitally enlarged lyophilized powders
were recorded on the imaging plate (Gatan Bioscan Camera
Model 1792) with 20 eV energy windows. Each imaging was
Determining the particle size and zeta
potential of IQca-aTVV solution state
To characterize the nanoproperty of IQCA-TAVV in solution performed with triplicate samples.
state, the particle size of the compound in normal saline (NS,
atomic force microscopy (aFM) images
of the nanoparticles of IQca-TaVV
10 nM) was determined on a particle size analyzer (Nano-
ZS90; Malvern Instruments Ltd, Malvern, UK), while the
zeta potential of the compound in ultrapure water (10 nM, adhering on platelets
pH 6.7) was measured on a Zeta Potential Plus Analyzer Rat blood containing 3.8% sodium citrate (citrate/blood =1/9,
(Brookhaven Instruments Corp, Holtsville, NY, USA). To v/v) was centrifuged at 1,000 rpm for 10 min to get platelet-
explore time-induced change of the particle size, and thereby rich plasma (PRP). Then PRP was centrifuged at 3,000 rpm
to provide a basis for the application of the nanosolution, for an additional 10 min. The rat plasma and the platelets
these determinations were performed at 25°C for 7 days, were collected. The platelets were carefully washed with NS
and each experiment was repeated three times.
three times, and then diluted to 1×105 platelets per mL NS to
give the normal platelet solution. This solution (500 μL) was
activated at 37°C with arachidonic acid (AA: [Sigma-Aldrich
Co., St Louis, MO, USA] final concentration 350 mM)
or thrombin (TH, Sigma-Aldrich Co., final concentration
Imaging the nanospecies of IQca-TaVV
with transmission electron microscopy
(TeM)
The morphology and size of the nanospecies formed by 0.1 U/mL) for 5 min to give AA- or TH-activated platelet
IQCA-TAVV in water were imaged on a TEM (JSM- solution. At 37°C AA- and TH-activated platelet solutions
2100LV; JEOL, Tokyo, Japan). In brief, onto the surface of were incubated with 10 and 100 nM as well as 0.05, 0.5 and
a formvar-coated copper grid a solution of IQCA-TAVV in 5 nM of IQCA-TAVV for 30 min, respectively. The plate-
ultrapure water (pH 6.7, 100 μL) was dropped, which was let solutions were individually dropped onto a mica sheet,
followed by air-drying, and warming at 37°C for 7 days to fixed with glutaraldehyde (3%) for 10 min, carefully washed
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with ultrapure water, and dried overnight in air to record the 3,000 rpm for 15 min to precipitate the platelets. The precipi-
images of AFM. For this purpose, a gold-coated nonconduc- tated pellet was fixed with glutaraldehyde (3%) for 1 h, washed
tive silicon nitride (Si3N4) tip of 180–220 μm in length, a with PBS three times, at 0°C post-fixed with osmium tetroxide
cantilever with an spring constant of 0.15 N/m and scan rate (1%) for 1 h, and centrifuged and treated with 50% ethanol
of 0.5 Hz were used, while the pixel resolution was 512×512. two times (each 10 min). The platelets were successively
Three locations were imaged for each platelet solution.
dehydrated in 70%, 80%, 90% and 100% ethanol for 10 min.
To get insight into the nanoparticles of IQCA-TAVV The platelets were dried with critical point dryer (CPD300;
adhering on the surfaces of AA-activated platelets, AFM Leica Ltd., Wetzlar, Germany), mounted on an aluminum stub,
images were recorded on a Nanoscope 3D AFM or Multi- coated with platinum by the JFC-1600 Auto Fine Coater and
mode 8 instrument (Veeco Metrology, Santa Barbara, CA, imaged on an SEM (S-4800).
USA) by using the contact mode and the Nanoscope V531r1
In vivo assay of IQca-TaVV on rat
software. In addition, the AFM images of the resting rat
platelets, TH-activated rat platelets and TH-activated rat arterial thrombosis model
platelets treated with IQCA-TAVV (0.05, 0.5 and 5 nM) Platelets and fibrin are implicated in the arterial throm-
were recorded on the Multimode 8 instrument using the bus that can be cross-linked with thread thrombus of rat
Nanoscope analysis 1.7 software.
arteriovenous shunt-silk thread model. In this context the
in vivo anti-arterial thrombosis activity of IQCA-TAVV
was evaluated in this model. In brief, male Sprague
Dawley rats (200±20 g) were randomly divided into five
groups of 12 animals each to orally receive IQCA-TAVV
(0.1, 1 and 10 nmol/kg) or aspirin (167 μmol/kg) or NS
(3 mL/kg). Thirty minutes after the administration the rat
was anesthetized with sodium pentobarbital (80.0 mg/kg,
intraperitoneal injection [ip]), and then the right carotid
artery and left jugular vein were separated. A polyethylene
tube with an exact weighted thread of 6 cm in length was
filled with a solution of heparin sodium in NS (50 IU/mL).
One end of this polyethylene tube was inserted into the
left jugular vein, while the other end of this polyethylene
tube was implanted into the right carotid artery, prior to
which heparin sodium was injected as anticoagulant. After
conjugation of the left jugular vein and the right carotid
artery, blood was allowed to circulate for 15 min, and the
thread in the polyethylene tube was removed and weighed
to calculate the weight increase of the thread, which was
the weight of the thrombus which was used to represent the
antithrombotic activity.
In vitro platelet aggregation assay of
IQca-TaVV
Platelet aggregation was evaluated with two-channel
Chronolog aggregometer by following the manufacturer’s
instructions. The citrated pig blood was immediately centri-
fuged at 1,000 rpm for 15 min to get PRP and further cen-
trifuged at 3,000 rpm for another 10 min to get platelet-poor
plasma (PPP). With stirring in an optical glass cuvette, 500 μL
PRPwasdilutedwithPPPandtheconcentrationoftheplatelets
became ~2×108 platelets/mL, into which 5 μL NS or 5 μL solu-
tion of IQCA-TAVV in NS (final concentrations ranging from
0.01 to 10 nM) was added. After adjustment of the baseline,
5 μL solution of AA in NS (final concentration 350 μM) or
5 μL solution of TH in NS (final concentration 0.1 U/mL)
was added. At 37°C the change of the light transmission was
measured for 5 min, and the activity of IQCA-TAVV against
the aggregation induced by AA or TH was recorded. The
inhibition rate was calculated by the following: inhibition% =
[1 - (Am% of IQCA-TAVV)/(Am% of NS)] ×100%, wherein
Am% was the maximal rate of platelet aggregation and was rep-
resented by the peak height of aggregation curve. IC50 values
were obtained from the dose–response curves and calculated
with a logarithmic curve-fitting program.
In vivo assay of IQca-TaVV on mouse
arterial thrombosis model
Endothelial cell injury is one of the important mechanisms of
the arterial thrombosis and FeCl3 can induce endothelial cell
injury. To explore the efficacy of IQCA-TAVV preventing
seM image of IQca-TaVV-treated rat
platelets
To image the action of IQCA-TAVV on platelets, 10 μL solu- endothelial cells from injury, thereby preventing the arterial
tion of IQCA-TAVV in NS (final concentration, 200 nM) or vessel from thrombosis, the FeCl3-induced mouse thrombosis
10 μL of NS was added to 500 μL of PRP; 3 min after incuba- model was used. In brief, male ICR mice (25±2 g) were ran-
tion AA was added (final concentration, 15 μM); at 37°C the domly divided into three groups (each 12) for orally receiv-
resultant solution was incubated for 5 min and centrifuged at ing IQCA-TAVV (10 nmol/kg) or aspirin (240 μmol/kg)
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Nanoscaled IQca-TaVV targeting arterial thrombus
or NS (10 mL/kg). Thirty minutes after the administration while the qCID spectrum (Figure 2B) gives an ion peak at
the mice were intraperitoneally injected with chloral hydrate 876.45208, the mass of a monomer plus H. Thus FT-MS
(10 g/100 mL) for anesthesia. The segment abdominal aorta spectrum reveals that in water the intermolecular association
of the mouse was exposed and dissected, and then two strips drives IQCA-TAVV to carry out the dimerization and form
of filter paper (0.5×3 cm) saturated with 15% FeCl3 were the dimer as the existing form. In FT-MS condition the dimer
placed beneath and above the dissected carotid artery for undergoes fragmentation and forms the monomer.
15 min, while a paraffin strip of 4×8 mm was placed below
NOESY 2D NMR spectrum defines the
the filter paper to protect the surrounding tissue. An artery
segment of 0.5 cm with thrombus was excised, the blood manner of IQca-TaVV forming a dimer
was blotted with filter paper and the thrombus was weighed To explore the formation manner of the dimer, NOESY 2D
to represent the antithrombotic activity.
NMR spectrum of IQCA-TAVV was measured on Bruker
AvanceIII 800 MHz spectrometer. DMSO-d6 and tetrameth-
ylsilane were used as the solvent and the internal standard,
respectively. Figure 2C gives one interesting cross-peak
Results
synthesis of IQca-TaVV
3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr (3) was marked with a red circle. This cross-peak represents the
prepared with a four-step route. Via Pictet-Spengler conden- interaction between the NH of Val residue of one molecule
sation L-Phe was converted into IQCA (98% yield). Using and the β-CH of Thr residue of another molecule. Thus,
(Boc)2O, 2N of IQCA was blocked by Boc (95% yield) NOESY 2D NMR spectrum reveals that to form a dimer the
and formed 1. The coupling of 1 with Tos⋅Thr-OBzl gave aromatic ring of IQCA of one molecule should approach the
N-(3S-N-Boc-1,2,3,4-tetrahydroisoquino-line-3-carbonyl)- Asp residue of another molecule and the distance between
L-Thr-OBzl (2, 90% yield). The saponification of 2 provided the mentioned protons should be ,4 Å.
3 (96% yield). The protective Ala-Arg(NO2)-Gly-Asp(OBzl)-
energy minimization gives the dimer of
OBzl was prepared by use of the solution-phase method
and stepwise synthesis (from C-terminal to N-terminal) in IQca-TaVV aV-like conformation
85% yield. Coupling 3 with Ala-Arg(NO2)-Gly-Asp(OBzl)- To explore the conformation of the dimer the monomer of
OBzl yielded 3S-N-Boc-1,2,3,4-tetrahydroisoquinoline- IQCA-TAVV first received energy minimization. Figure 2D
3-carbonyl)-Thr-Ala-Arg(NO2)-Gly-Asp(OBzl)-OBzl (4, indicates that the energy minimization gives the monomer a
93% yield). Upon removing benzyl groups 4 was converted scorpion-like conformation. Based on the formation manner
to 3S-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-carbonyl- of the dimer defined by NOESY 2D NMR spectrum, that is,
Thr-Ala-Arg(NO2)-Gly-Asp (5). The conjugation of 5 with the aromatic ring of IQCA of one molecule approaching the
Tos·Val-OBzl gave 3S-N-Boc-1,2,3,4-tetrahydroiso quin- Asp residue of another molecule, two monomers of scorpion-
oline-3-carbonyl-Thr-Ala-Arg(NO2)-Gly-Asp(Val-OBzl)- like conformation were manually accessed to control the dis-
Val-OBzl (6, 72% yield). Upon removal of all protective tance between the mentioned protons within 4 Å. Figure 2E
groups of 6, IQCA-TAVV was obtained with 94% yield. indicates that this operation leads the energy-minimized
These data suggest that the reaction conditions are mild and dimer of IQCA-TAVV to the V-like conformation.
the yield of the individual reaction is acceptable. Thus, the
seM-,TeM- and mesoscale-simulation-
route depicted with Scheme 1 for the preparation of IQCA-
TAVV represents an acceptable strategy of a mild reaction predicted images of IQca-TaVV
condition, desirable yield and good purity.
The nanofeature of IQCA-TAVV in aqueous solution was
visualized with the TEM image. Figure 3A and B show that
in pH 6.7 ultrapure water the diameters of the nanoparticles
formed by 10 and 0.1 nM of IQCA-TAVV are 12–186 and
FT-Ms spectrum explores IQca-TaVV
forming a dimer
To explore the association profile the FT-MS spectrum was 48–197 nm, respectively, and the diameters of most particles
measured on a SolariX FT-ICR mass spectrometer (Bruker fall within a range of 75–125 nm (Figure 3C). Figure 3D
Daltonics) with an ESI positive ion source and a supercon- shows that the SEM images of lyophilized powders formed
ductive magnet of 9.4 T. Figure 2A is the FT-MS spectrum of from 10 nM aqueous IQCA-TAVV are nanoparticles of
1 nM solution of IQCA-TAVV in ultrapure water and gives 20–70 nm in diameter, and the diameters of most particles
an ion peak at 1,773.64447, the mass of a dimer plus Na, fall within a range of 50–80 nm (Figure 3E). Mesoscale
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Scheme 1 synthetic route to IQca-TaVV.
Notes: (a) hchO and hcl; (b) (Boc)2O and NaOh; (c) Dcc, hOBt, and NMM; (d) 2N NaOh and methanol; (e) hydrogen chloride in ethyl acetate (4N); (f) cF3cO2h/
cF3sO3h.
Abbreviations: IQca-TaVV, 3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-ala-arg-gly-asp(Val)-Val; Dcc, dicyclohexylcarbodiimide; hOBt, N-hydroxybenzo-
triazole; NMM, N-methylmorpholine.
simulation predicts that 824 molecules of IQCA-TAVV can and the solution is also clear. These observations suggest
form a particle of 12.1 nm in diameter (Figure 3F).
that the nanosolution formed by IQCA-TAVV in water has
no colloidal property. The zeta potential test indicates that
aqueous IQCA-TAVV (10 nM, pH 6.7) has a zeta potential
Nanoproperty of aqueous IQca-TaVV
The nanoproperty of aqueous IQCA-TAVV was characterized of -18.57 mV with a half-width of 2.32 mV (Figure 4G).
with 650 nm laser-induced Faraday–Tyndall effect, zeta poten- The mean size of 10 nM solution of IQCA-TAVV in NS
tial and 7-day mean size. Figure 4A indicates that ultrapure over 7 days ranges from ~160 to ~350 nm (Figure 4H). Thus,
water without radiation is a clear solution. Figure 4B indicates the 650-nm laser-induced Faraday–Tyndall effect, the zeta
that ultrapure water with 650 nm laser radiation shows no potential test and the 7-day size determination consistently
Faraday–Tyndall effect, and is a clear solution. Figure 4C evidence that aqueous IQCA-TAVV is a nanosolution.
and D indicate that the radiation of 650 nm laser induces the
Nanoparticles of IQca-TaVV adhere on
solution of IQCA-TAVV in ultrapure water (1 nM, pH 6.7 or
pH 1.2) to show Faraday–Tyndall effect, while the solution is platelet surface
clear. Figure 4E and F indicate that the radiation of 650 nm To clearly visualize the adhesion of the nanoparticles of
laser induces the solution of IQCA-TAVV in ultrapure water IQCA-TAVV on the surface of AA-activated platelets, the
(10 nM, pH 6.7 or pH 1.2) to show Faraday–Tyndall effect, AFM model of Veeco Nanoscope 3D was used. Figure 5A
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Figure 2 FT-Ms spectrum, NOesY 2D NMr spectrum and the dimer of IQca-TaVV.
Notes: (A and B) FT-Ms spectrum and qCID spectrum of 1 nM solution of IQCA-TAVV in ultrapure water, and the red box shows the amplified region in the MS spectrum;
(C) NOesY 2D NMr spectrum of IQca-TaVV and red ring–labeled interesting cross-peak; (D) energy-minimized conformation of IQca-TaVV’s monomer; (E) energy-
minimized conformation of IQca-TaVV’s dimer.
Abbreviations: FT-Ms, Fourier transform mass spectrometry; IQca-TaVV, 3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-ala-arg-gly-asp(Val)-Val; Intens, intensities;
NMR, nuclear magnetic resonance; qCID, qualitative contingent influence diagram.
shows the IC50 of IQCA-TAVV against AA-induced rat environment. Figure 5D is the AFM image of the resting rat
platelet aggregation. Figure 5B is the AFM image of rat platelets. The locally amplified platelet is marked with a blue
plasma without platelets, giving no nano particle. Figure 5C box, showing a smooth surface and no extended pseudopodia.
is the AFM image of IQCA-TAVV in rat plasma (10 nM) Figure 5E is the AFM image of AA-activated and -aggregated
to visualize the features of its particles in physiologic rat platelets without IQCA-TAVV. The locally amplified
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Figure 3 TeM and seM images, as well as mesoscale simulation software-predicted nanoparticle of IQca-TaVV.
Notes: (A) TeM image of 10 nM IQca-TaVV in ultrapure water; (B) TeM image of 0.1 nM IQca-TaVV in ultrapure water; (C) Particle size distribution of TeM image of
0.1 nM IQca-TaVV in ultrapure water; (D) seM image of lyophilized powders from 10 nM IQca-TaVV in ultrapure water; (E) Particle size distribution of TeM image of
lyophilized powders from 10 nM IQca-TaVV in ultrapure water; (F) Mesoscale simulation software-predicted nanoparticle of IQca-TaVV.
Abbreviations: IQca-TaVV, 3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-ala-arg-gly-asp(Val)-Val; TeM, transmission electron microscopy; seM, scanning electron
microscopy.
platelet aggregator is marked with a blue box, showing a shows the IC50 of IQCA-TAVV against TH-induced rat
smooth surface and extended pseudopodia. Figure 5F is the platelet aggregation. Figure 6B is the AFM image of the
AFM image of AA-activated rat platelets treated with 10 nM resting rat platelets, giving no aggregators and extended pseu-
of IQCA-TAVV. The locally amplified platelet with extended dopodia. Figure 6C is the AFM image of TH-activated rat
pseudopodia is marked with a blue box, showing some nano- platelets, showing a lot of aggregated platelets with extended
particles of 23.7–51.3 nm in height and some aggregators on pseudopodia. Figure 6D is the AFM image of TH-activated
its surface. Figure 5G is the AFM image of AA-activated rat rat platelets treated with 0.05 nM of IQCA-TAVV, where the
platelets treated with 100 nM of IQCA-TAVV. The locally extended pseudopodia and the aggregators are less than that
amplified platelet with extended pseudopodia is marked with of Figure 6C. Figure 6E is the AFM image of TH-activated
a blue box, showing some nanoparticles of 8.5–20.9 nm in rat platelets treated with 0.5 nM of IQCA-TAVV, where the
height and few aggregators on its surface. The features of extended pseudopodia and the aggregators are less than that
the AFM images show that the size of the nanoparticles of Figure 6D. Figure 6F is the AFM image of TH-activated
formed by IQCA-TAVV in rat plasma is smaller than that rat platelets treated with 5 nM of IQCA-TAVV, where the
of its nanoparticles in ultrapure water, which can adhere on extended pseudopodia and the aggregators are less than that
the surface of the activated platelets to inhibit their activation of Figure 6E. Thus, AFM images visualize that IQCA-TAVV
and aggregation.
concentration dependently inhibits platelet activation.
seM images and the effect of the
nanoparticles of IQca-TaVV on rat
To clearly visualize the effect of the nanoparticles of IQCA- platelets
TAVV on the platelet activation induced by TH, the AFM The effect of the nanoparticles of IQCA-TAVV on AA-
model of multimode 8 instruments was used. Figure 6A activated rat platelets was visualized with the SEM image.
effect of the nanoparticles of IQca-
TaVV on platelet activation
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Nanoscaled IQca-TaVV targeting arterial thrombus
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Figure 4 Tyndall effect, zeta potential and size of IQca-TaVV in ultrapure water.
Notes: (A) Ultrapure water without radiation; (B) ultrapure water with 650 nm laser radiation; (C) IQca-TaVV in ph 6.7 ultrapure water (1 nM) with 650 nm laser
radiation; (D) IQca-TaVV in ph 1.2 ultrapure water (1 nM) with 650 nm laser radiation; (E) IQca-TaVV in ph 6.7 ultrapure water (10 nM) with 650 nm laser radiation;
(F) IQca-TaVV in ph 1.2 ultrapure water (10 nM) with 650 nm laser radiation; (G) zeta potential of IQca-TaVV in ultrapure water (10 nM); (H) particle size of 10 nM
solution of IQca-TaVV in normal saline over 7 days (n=6).
Abbreviation: IQca-TaVV, N-(3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-ala-arg-gly-asp(Val)-Val.
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Figure 5 aFM images of the resting rat platelets and the interaction between nanoparticles of IQca-TaVV and aa-activated rat platelets.
Notes: (A) Ic50 of IQca-TaVV against aa-activated rat platelet aggregation; (B) aFM image of rat plasma without platelets; (C) aFM image of IQca-TaVV in rat plasma (10 nM)
to mirror the feature of the particles in physiologic environment; (D) aFM image of resting platelets without IQca-TaVV; (E) aFM image of aa-activated platelets without
IQca-TaVV; (F) AFM image of IQCA-TAVV (10 nM)-treated resting platelets; local amplified particle labeled by blue box; (G) aFM image of IQca-TaVV (100 nM)-treated aa-
activated platelets. Besides, the aFM images of normal saline and IQca-TaVV in normal saline (10 nM) are shown in Figure s5, the height of particle was labeled by red arrow.
Abbreviations: IQca-TaVV, N-(3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-ala-arg-gly-asp(Val)-Val; aa, arachidonic acid; aFM, atomic force microscopy.
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Figure 6 aFM images of the resting rat platelets and the effect of IQca-TaVV nanoparticles on Th-activated rat platelets.
Notes: (A) Ic50 of IQca-TaVV against Th-induced rat platelet aggregation; (B) aFM image of the resting rat platelets; (C) aFM image of Th-activated rat platelets without
IQca-TaVV; (D) aFM image of Th-activated platelets treated with 0.05 nM of IQca-TaVV; (E) aFM image of Th-activated platelets treated with 0.5 nM of IQca-TaVV;
(F) aFM image of Th-activated platelets treated with 5 nM of IQca-TaVV.
Abbreviations: IQca-TaVV, N-(3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-ala-arg-gly-asp(Val)-Val; Th, thrombin; aFM, atomic force microscopy.
Figure 7A shows the SEM images of AA-activated rat
IQca-TaVV effectively inhibits mouse
arterial thrombosis
Figure 8A indicates that 10 nmol/kg of oral IQCA-TAVV
significantly inhibits the mice to form an arterial thrombus,
and the thrombus weight is significantly lower than that of
the mice treated with NS and equal to that of the mice treated
with 240 μmol/kg aspirin. This means that the activity of
platelets treated with NS. These platelets have smooth sur-
faces and a series of extended pseudopodia. Figure 7B shows
the SEM images of AA-activated rat platelets treated with
200 nM IQCA-TAVV. In contrast, these platelets have no
extended pseudopodia. However, on the surfaces of these
platelets there are a lot of nanoparticles of IQCA-TAVV.
Figure 7 seM images of the action of the nanoparticles of IQca-TaVV on rat platelets.
Notes: (A) SEM images of AA-activated rat platelets treated with normal saline, in which an amplified platelet having a smooth surface is inserted and the extended
pseudopodia are labeled with red arrowheads; (B) SEM images of AA-activated rat platelets treated with 200 nM IQCA-TAVV, in which an amplified platelet without
pseudopodia and the nanoparticles of IQca-TaVV on the surface are labeled with blue arrowheads.
Abbreviations: seM, scanning electron microscopy; aa, arachidonic acid; IQca-TaVV, N-(3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-ala-arg-gly-asp(Val)-Val.
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Nanoscaled IQca-TaVV targeting arterial thrombus
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Figure 8 anti-arterial thrombosis activities of IQca-TaVV, n=12.
Notes: (A) at 10 nmol/kg dose IQca-TaVV effectively inhibits the mice forming an arterial thrombus; (B) IQca-TaVV dose-dependently inhibits the rats forming an
arterial thrombus.
Abbreviations: IQca-TaVV, N-(3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-ala-arg-gly-asp(Val)-Val; TaVV, Thr-ala-arg-gly-asp(Val)-Val; Ns, normal saline.
IQCA-TAVVis24,000-foldthatofaspirin. Themorphologyof the ESI(+)-FT-MS spectrum of the thrombus extract of the
arterial thrombus in the mouse model is shown in Figure S3.
treated rats. The local amplified inset of Figure 9B gives an
ion peak at 519.25641; the mass of TARGD (Thr-Ala-Arg-
Gly-Asp) plus H. Figure 9C is the ESI(-)-FT-MS spectrum of
the blood extract of the treated rats. The local amplified inset
of Figure 9C gives no IQCA-related ion peak. Figure 9D–I are
the ESI(-)-FT-MS spectra of the extracts of the brain, heart,
lung, liver, spleen and kidney of the treated rats. The local
amplified insets show no IQCA-related ion peak. Thus, the
ESI(-)-FT-MS spectrum of the thrombus extract is character-
ized by the ion peak of IQCA and is distinctly different from
the ESI(-)-FT-MS spectra of the blood, brain, heart, lung,
liver, spleen and kidney extracts that give no ion peak of
IQCA. The ESI(+)/FT-MS spectrum of the thrombus extract
is characterized by the ion peak of Thr-Ala-Arg-Gly-Asp, and
is distinctly different from the ESI(+)-FT-MS spectra of the
blood, brain, heart, lung, liver, spleen and kidney extracts that
give no ion peak of Thr-Ala-Arg-Gly-Asp. The ESI(-)-FT-MS
spectrum with the ESI(+)-FT-MS spectrum of the thrombus
extract together suggest that IQCA-TAVV targets arterial
thrombus, wherein it releases IQCA and TARGD(V)V.
IQca-TaVV effectively inhibits rat
arterial thrombosis
Figure 8B indicates that in the rat arterial thrombosis
model, IQCA-TAVV dose dependently inhibits rats form-
ing an arterial thrombus and the minimal effective dose is
1 nmol/kg. The thrombus weight of the rats orally receiving
1 nmol/kg IQCA-TAVV is significantly lower than those of
the rats orally receiving NS, 100 nmol/kg of Thr-Ala-Arg-
Gly-Asp(Val)-Val (TAVV) and 16.7 μmol/kg of aspirin. This
means that the activity of IQCA-TAVV is ~1,670-fold higher
than that of aspirin. The morphology of arterial thrombus in
the rat model is shown in Figure S4.
IQca-TaVV targets arterial thrombus to
release IQca
The benefit of the nanoparticles adhering on the surface
of rat platelets was reflected with IQCA-TAVV targeting
arterial thrombus to release IQCA and Thr-Ala-Arg-Gly-
Asp(Val)-Val (TAVV) in vivo. For this profile, the brain,
heart, lung, liver, spleen, kidney, blood and thrombus of the acute toxicity of IQca-TaVV in mice
thrombosis rats treated with 10 nmol/kg IQCA-TAVV were Acute toxic action of IQCA-TAVV on mice was examined. It
homogenized, ultrasonically extracted with methanol, cen- was found that even at a dose of 1 μmol/kg (100- or 1,000-fold
trifuged at 3,000 rpm for 10 min and the supernatants were of the minimal effective dose) IQCA-TAVV still induced no
used for ESI(±)-FT-MS analysis. Figure 8A is the ESI(-)- mouse death or neurotoxicity behavior, such as tremor, twitch,
FT-MS spectrum of the thrombus extract of the treated rats. jumping, tetanus and supination. This suggests that the LD50
The local amplified inset of Figure 9A gives an ion peak at value of IQCA-TAVV is more than 1 μmol/kg. Figure 10A–C
178.07821; the mass of IQCA subtracting H. Figure 9B is indicate that the serum levels of alanine transaminase,
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Nanoscaled IQca-TaVV targeting arterial thrombus
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Figure 10 effects of IQca-TaVV on mouse cr, alT and asT, n=5.
Notes: (A) serum cr of Icr mice treated with Ns and 1 μmol/kg of IQca-TaVV; (B) serum alT of Icr mice treated with Ns and 1 μmol/kg of IQca-TaVV; (C) serum
asT of Icr mice treated with Ns and 1 μmol/kg of IQca-TaVV.
Abbreviations: alT/gPT, alanine aminotransferase/glutamic pyruvic transaminase; asT/gOT, aspartate aminotransferase/glutamic oxaloacetic transaminase; cr, creatinine;
Ns, normal saline; Icr, institute of cancer research.
aspartate transaminase and creatinine of 1 μmol/kg IQCA- inhibit TH to activate rat platelets, thereby decreasing the
TAVV-treated mice are equal to those of NS-treated mice. amount of the extended pseudopodia and the aggregator of
All the observations together suggest that even at a high dose the platelets.
IQCA-TAVV still exhibits no neurotoxicity and does not
injure the kidneys, liver and heart.
Relying on inhibiting platelet activation, IQCA-TAVV
dose dependently inhibited the rats forming an arterial
thrombus, and at 1 nmol/kg of dose, its minimal effec-
tive dose, the activity is significantly higher than that of
16.7 μmol/kg aspirin. Relying on the inhibition of platelet
activation, IQCA-TAVV also effectively inhibited the mice
forming an arterial thrombus and at 10 nmol/kg of dose the
activity is equal to that of 240 μmol/kg aspirin. These data
suggest that in inhibiting arterial thrombosis the mice are
more sensitive to IQCA-TAVV therapy.
Discussion
The route to the preparation of 3S-1,2,3,4-tetrahydroiso-
quinoline-3-carbonyl-Thr-Ala-Arg-Gly-Asp(Val)-Val
(IQCA-TAVV) represents an acceptable strategy that uses
mild reaction condition, provides desirable yield and gives
good purity.
The FT-MS spectrum reveals that in water the intermo-
lecular association drives the dimerization of IQCA-TAVV
and the dimer is the unique form of it existing in water.
In FT-MS condition the dimer undergoes fragmentation to
form the monomer. Energy minimization gives the monomer
a scorpion-like conformation. The NOESY 2D NMR spectra
reveal that the intermolecular association is an aromatic
ring of IQCA of one molecule accessing the Asp residue of
another molecule. Energy minimization gives the dimer a
V-like conformation.
Relying on adhering onto the surface of the platelets,
IQCA-TAVV targets the arterial thrombus, wherein it
releases IQCA and Thr-Ala-Arg-Gly-Asp(Val)-Val. Both
of these compounds resulted from the thrombus-specific
metabolism of IQCA-TAVV and the course could be hypoth-
esized as in Figure 11.
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The intermolecular association–based self-assembly
drives IQCA-TAVV forming nanoparticles. TEM, SEM,
AFM and 650-nm laser-induced Faraday–Tyndall effect
visualize the nanoproperty of IQCA-TAVV. The particle
size of IQCA-TAVV in aqueous solution, in lyophilized
powders and in rat plasma is suitable to deliver and to avoid
phagocytosis of macrophages in blood circulation.
AFM images show that the nanoparticles of IQCA-
TAVV can adhere onto the surface of AA-activated rat
platelets to inhibit their activation and aggregation. AFM
images also show that the IQCA-TAVV can effectively
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Figure 11 Hypothesized thrombus-specific release of IQCA and Thr-Ala-Arg-Gly-
asp from IQca-TaVV.
Abbreviations: IQca-TaVV, 3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-
ala-arg-gly-asp(Val)-Val; IQca, 3s-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
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Conclusions
In aqueous solution, lyophilized powders and rat plasma
IQCA-TAVV form nanoparticles that not only could be
suitably delivered in blood circulation but also could adhere
onto the surface of activated platelets, and thereby inhibit
the activation of the platelets. This allows IQCA-TAVV to
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and leads to its minimal effective dose being as low as
1 nmol/kg. This high therapeutic efficacy is correlated with
IQCA-TAVV targeting arterial thrombus. Therefore, IQCA-
TAVV shows an example of the application of nanomedicine
capable of adhering on the surface of activated platelets,
attenuating platelet activation, targeting arterial thrombus
and consequently inhibiting arterial thrombosis.
Acknowledgments
ThisworkwassupportedbySpecialProject(2017ZX09201008)
of China, NSFC (81673303, 81373265 and 81373264),
BNSF (7162025 and 7172028), PHR (KZ201610025029
and KM201710025009), 863 program (2015AA020902) and
The Importation and Development of High-Caliber Talents
Project of Beijing Municipal Institutions. We are grateful to
Yaonan Wang of the Capital Medical University for helping
us with this project.
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Disclosure
The authors report no conflicts of interest in this work.
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Supplementary materials
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Figure S1 1h NMr spectrum of IQca-TaVV.
Abbreviations: IQca-TaVV, 3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-ala-arg-gly-asp(Val)-Val; NMr, nuclear magnetic resonance.
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Figure S2 13c NMr spectrum of IQca-TaVV.
Abbreviations: IQca-TaVV, 3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-ala-arg-gly-asp(Val)-Val; NMr, nuclear magnetic resonance.
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Nanoscaled IQca-TaVV targeting arterial thrombus
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Figure S3 The morphology of the arterial thrombus in mouse model.
Notes: (A) abdominal aorta of health mouse; (B) abdominal aorta of mouse treated with Ns; (C) abdominal aorta of mouse treated with 167 μmol/kg aspirin; (D) abdominal
aorta of mouse treated with 10 nmol/kg IQca-TaVV.
Abbreviations: IQca-TaVV, N-(3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-ala-arg- gly-asp(Val)-Val; Ns, normal saline.
Figure S4 The morphology of the arterial thrombus in rat model.
Notes: (A) Thread without thrombus; (B) thread with thrombus from the rat treated with Ns; (C) thread with thrombus from the rat treated with 16.7 μmol/kg aspirin;
(D) thread with thrombus from the rat treated with 167 μmol/kg aspirin; (E) thread with thrombus from the rat treated with 100 nmol/kg TaVV; (F) thread with thrombus
from the rat treated with 10 nmol/kg IQca-TaVV; (G) thread with thrombus from the rat treated with 1 nmol/kg IQca-TaVV; (H) Thread with thrombus from the rat
treated with 0.1 nmol/kg IQca-TaVV.
Abbreviations: IQca-TaVV, N-(3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-ala-arg- gly-asp(Val)-Val; Ns, normal saline.
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Figure S5 aFM image of IQca-TaVV in Ns (10 nM), showing a nanoparticle of IQca-TaVV of ~20 nm in height.
Notes: (A) aFM image of Ns; (B) aFM image of IQca-TaVV (10 nM).
Abbreviations: IQca-TaVV, N-(3s-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-ala-arg-gly-asp(Val)-Val; aFM, atomic force microscopy; Ns, normal saline.
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