Tetrahydro-β-carboline-3-carboxyl-thymopentin: A nano-conjugate for releasing pharmacophores to treat tumor and complications

Article abstract of DOI:10.1039/c5tb01930c

To improve the therapeutic efficacy of cancer patients a novel conjugate of thymopentin (TP5) and (1S,3S)-1-methyl-tetrahydro-β-carboline-3-carboxylic acid (MTC) was presented. In water and mouse plasma MTCTP5 forms the nanoparticles of 14-139 nm in diameter, the suitable size for delivery in blood circulation. In mouse plasma MTCTP5 releases MTC, while in the presence of trypsin MTCTP5 releases MTC and TP5. On mouse and rat models the MTCTP5 dose dependently slows down the tumor growth, inhibits inflammatory response and blocks thrombosis. The anti-tumor activity as well as the anti-inflammation activity and anti-thrombotic activity of MTCTP5 are 100 fold and 10 fold higher than those of MTC, respectively, which are attributed to the fact that it down-regulates the plasma levels of TNF-α and IL-8 of the treated animals. The immunology enhancing activities in vitro and in vivo of MTCTP5 are similar to those of TP5, which is attributed to the fact that MTCTP5 up-regulates the plasma levels of IL-2 and CD4 as well as down-regulates the plasma level of CD8 of the treated animals. The plasma alanine transaminase, aspartate transaminase and creatinine assays indicate that MTCTP5 therapy does not injure the liver and the kidney of the animals. The survival time of MTCTP5 treated mice is significantly longer than that of TP5 treated mice.

Full text of DOI:10.1039/c5tb01930c

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Tetrahydro-b-carboline-3-carboxyl-thymopentin:
a nano-conjugate for releasing pharmacophores
to treat tumor and complications†
Cite this:DOI: 10.1039/c5tb01930c
Xi Hu,a Ming Zhao,*ab Yuji Wang,a Yaonan Wang,a Shurui Zhao,a Jianhui Wu,a
Xiangmin Lic and Shiqi Peng*a
To improve the therapeutic efficacy of cancer patients a novel conjugate of thymopentin (TP5) and
(1S,3S)-1-methyl-tetrahydro-b-carboline-3-carboxylic acid (MTC) was presented. In water and mouse
plasma MTCTP5 forms the nanoparticles of 14–139 nm in diameter, the suitable size for delivery in
blood circulation. In mouse plasma MTCTP5 releases MTC, while in the presence of trypsin MTCTP5
releases MTC and TP5. On mouse and rat models the MTCTP5 dose dependently slows down the tumor
growth, inhibits inflammatory response and blocks thrombosis. The anti-tumor activity as well as the
anti-inflammation activity and anti-thrombotic activity of MTCTP5 are 100 fold and 10 fold higher than
those of MTC, respectively, which are attributed to the fact that it down-regulates the plasma levels of
TNF-a and IL-8 of the treated animals. The immunology enhancing activities in vitro and in vivo of
MTCTP5 are similar to those of TP5, which is attributed to the fact that MTCTP5 up-regulates the
plasma levels of IL-2 and CD4 as well as down-regulates the plasma level of CD8 of the treated animals.
The plasma alanine transaminase, aspartate transaminase and creatinine assays indicate that MTCTP5
therapy does not injure the liver and the kidney of the animals. The survival time of MTCTP5 treated
mice is significantly longer than that of TP5 treated mice.
Received 16th September 2015,
Accepted 17th December 2015
DOI: 10.1039/c5tb01930c
www.rsc.org/MaterialsB
TP5-thymosin a1 and TP5-iRGD peptides are for treating tumor,6,7
TP5-poly(butyl cyanoacrylate) is for enhancing immuno-
Introduction
Thymopentin (TP5, RKDVY), the Arg32–Tyr36 fragment of the modulatory activity,8 and TP5-PLGA-lectins are for improving
thymus hormone thymopoietin, is well known as an immuno- oral immune-modulatory activity.9 Of the formulations TP5
modulator, plays an important role in T-lymphocyte maturation formulated chitosan or bacitracin is for novel intranasal delivery,10
and differentiation, and is used in the clinic to treat some the co-sprayed dry powders of TP5 with lactose or mannitol are
immunodeficiencies, malignancies and infections.1–3 However, for improving the flow ability,11 the TP5 incorporated copoly-
TP5 has very short half-life in vivo and can be administered mer of poly-D-lactide and poly(ethylene glycol) is for slowing
either via intramuscular injection or via intravenous infusion.4 the release rate,12 the aerosol is for improving the systemic
Additionally TP5 is successfully used as a pharmacophore or an action of TP5,13 and the mixed polymer matrix of polylactide
activity enhancer in the modification of peptides and polymers and poly(lactic-co-glycolic) of TP5 is for preparing biodegradable
and formulations for various purposes. Of TP5 modified peptides microspheres.14
and polymers, the TP5-RR-6 peptide is for treating tuberculosis,5
b-Carbolines have diverse pharmacological functions, such
as inhibiting platelet activation,15 improving object recognition
memory,16 stimulating insulin secretion,17 interacting with
DNA,18,19 and blocking topoisomerases.20 Besides, b-carbolines
are the pharmacophores of a series of bioactive agents, such as
trypanocidal agents,21 anti-malarial agents,22,23 neuron-protective
and neuron-differentiating agents,24 antiviral agents,25 anti-
leishmanial agents,26 as well as antitumor agents,27–30 anti-
inflammatory agents,31 and anti-thrombotic agents in particular.32,33
It is well known that chronic inflammation can increase cancer
susceptibility, while anti-inflammatory drugs may retard the devel-
a Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering
Research Center of Endogenous Prophylactic of Ministry of Education of China,
Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences,
Capital Medical University, Beijing 100069, P. R. China.
E-mail: mingzhao@bjmu.edu.cn, sqpeng@bjmu.edu.cn; Fax: +86-10-8280-2482,
+86-10-8391-1528; Tel: +86-10-8280-2482, +86-10-8391-1528
b Faculty of Biomedical Science and Environmental Biology,
Kaohsiung Medical University, Kaohsiung, Taiwan
c Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047,
P. R. China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5tb01930c opment of cancers.34,35 Long-term and low-dose administration
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of aspirin is associated with lower cancer mortality, suggesting
that anti-thrombotic therapy is beneficial for the outcome of
cancer patients.36 Thrombotic microangiopathy (TMA) is now
recognized as a late complication of cancers.37 Several clinical
studies indicate that TMA can develop directly from certain
malignancies, but more often results from anticancer therapy.38
These findings emphasize the potential utility of a pharmaco-
phore capable of simultaneously inhibiting cancer, thrombosis
and inflammation. Our broad screening identified that (1S,3S)-
1-methyltetrahydro-b-carboline-3-carboxylic acid (MTC) is such a
pharmacophore.32,33 However, the antitumor, anti-inflammatory
and anti-thrombotic activities of MTC need to be enhanced. In
this context, the present paper used TP5 as an enhancer of MTC
to prepare nano-scale N-[(1S,3S)-1-methyltetrahydro-b-carboline-
3-carboxyl]-Arg-Lys-Asp-Val-Tyr (MTCTP5), and to evaluate the
bioactivities.
Materials and methods
General procedures
All L-amino acids were purchased from Sigma-Aldrich Co.
(St. Louis, MO, USA). The column chromatography was performed
using silica gel (200–300 mesh, Qingdao Haiyang Chemical Co.,
Qingdao, P. R. China). The purities of MTC, protective TP5 and
protective MTCTP5 (495%) were determined by TLC analysis
(Qingdao silica gel plates of GF254). The purity of MTCTP5
(496%) was determined by HPLC analysis (CHIRALPAK AH-H
column, 4.6 Â 250 mm, Daicel Chemical IND., Ltd).
Scheme 1 Synthetic route to MTCTP5. (i) Sulfuric acid; (ii) DMF, di-tert-
butyl dicarbonate (Boc2O) and triethylamine; (iii) dicyclohexylcarbodiimide
(DCC), N-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM) and
tetrahydrofuran (THF); (iv) hydrochloride in ethyl acetate (4 M); (v) aqueous
NaOH (4 M); (vi) H2, Pd/C and CH3OH.
1
The spectra of H NMR (800 MHz) and 13C NMR (200 MHz)
Preparing (1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-
carboxylic acid (1, MTC)
were recorded on a Bruker Avance II 800 MHz spectrometer with
DMSO-d6 as the solvent and tetramethylsilane as internal standard.
ESI/MS was tested on a ZQ 2000 (Waters, US) and solariX FT-ICR
mass spectrometer (Bruker Daltonik) consisting of an ESI/MALDI
dual ion source and a 9.4 T superconductive magnet.
K562, A549, HT-29 and HL-60 carcinoma cell lines were
purchased from ATCC. Bovine serum albumin (BSA) was pur-
chased from Sigma. Fetal bovine serum (FBS), basal DMEM
medium and 1640 medium were purchased from Gibco.
Male Wistar rats and ICR mice were purchased from the
Animal Center of Capital Medical University. The protocol was
reviewed and approved by the ethics committee of Capital
Medical University. The committee ensures that the welfare of
the animals was maintained in accordance with the requirements
of the Animal Welfare Act and in accordance with the NIH Guide
for Care and Use of Laboratory Animals.
To a solution of 10.0 g (49.0 mmol) of L-Trp, 4 mL of concen-
trated H2SO4 and 400 mL of water 10 mL of aldehyde (40%) were
added. The reaction mixture was stirred at room temperature for
6 h, adjusted to pH 7 with concentrated ammonia, and then
kept at 0 1C for 0.5 h. The formed precipitates were collected by
filtration to give 8.0 g (70%) of MTC. ESI-MS (m/z): 231 [M + H]+;
1H NMR (300 MHz, DMSO-d6): d/ppm = 11.144 (s, 1H), 7.449
(d, J = 7.8 Hz, 1H), 7.349 (d, J = 7.8 Hz, 1H), 7.090 (t, J = 7.2 Hz, 1H),
7.000 (t, J = 7.8 Hz, 1H), 4.536 (d, J = 6.6 Hz, 1H), 3.636 (dd,
J = 4.8 Hz, J = 12.0 Hz, 1H), 3.204 (dd, J = 4.8 Hz, J = 12.0 Hz, 1H),
2.792 (m, 1H), 1.630 (d, J = 6.6 Hz, 3H).
Preparing (1S,3S)-1-methyl-2-Boc-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (2)
All experimental data are presented as average Æ SD. Statis-
tical analyses of all the biological data were carried out by the
use of ANOVA by SPSS. P-values o0.05 were considered statis-
tically significant.
A solution of 8.0 g (34.8 mmol) of MTC and 8.0 g (37.0 mmol) of
Boc2O in 100 mL of DMF was stirred at 0 1C for 30 min and
at room temperature for additional 48 h, evaporated under
vacuum, the residue was triturated with ether, the residue was
dissolved in 100 mL of water and then was acidified with a
dilute hydrochloric acid to pH 2. The solution was extracted
Synthesis of MTCTP5
MTCTP5 was prepared by the use of the synthetic route with ethyl acetate (30 mL Â 3), the ethyl acetate phase was
depicted in Scheme 1; the preparations of the related inter- washed with water (30 mL Â 3) and dried with anhydrous
mediates and the chemical physical data thereof are given in sodium sulfate for 12 h. The ethyl acetate phase was filtered
the corresponding procedures.
and the filtrate was evaporated under vacuum to give 7.6 g
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(66%) of the title compound as colorless powders. ESI-MS (m/z): Preparing Boc-Arg-Lys(Z)-OBzl
331 [M + H]+.
At 0 1C a solution of 0.552 g (3.0 mmol) of Boc-Arg, 0.405 g
(3.0 mmol) of HOBt, 0.675 g (3.6 mmol) of DCC and 20 mL of
anhydrous THF was stirred for 30 min, to which a solution of
1.629 g (3.0 mmol) of TosÁLys(Z)-OBzl in 10 mL of anhydrous
THF was added, and adjusted to pH 9 with NMM. The reaction
mixture was stirred at room temperature for 8 h and TLC
(CH2Cl2 : CH3OH, 10 : 1) indicated the complete disappearance
of TosÁLys(Z)-OBzl. The formed DCU precipitates were removed
by filtration and the filtrate was evaporated under vacuum. The
residue was dissolved in 100 mL of ethyl acetate. The solution
was washed successively with 5% aqueous solution of sodium
bicarbonate, 5% aqueous solution of citric acid and saturated
aqueous solution of sodium chloride. The ethyl acetate phase
was dried with anhydrous sodium sulfate for 12 h. After filtra-
tion the filtrate was evaporated under vacuum to provide 1.428 g
(74%) of title compound as colorless powders. ESI+-MS (m/z):
627 [M + H]+.
Preparing Boc-Val-Tyr-OBzl
At 0 1C a solution of 0.434 g (2.0 mmol) of Boc-Val, 0.270 g
(2.0 mmol) of HOBt, 20 mL of anhydrous THF and 0.450 g
(2.2 mmol) of DCC was stirred for 30 min, to which a solution of
0.886 g (2.0 mmol) of TosÁTyr-OBzl in 10 mL of anhydrous THF
was added, and adjusted to pH 9 with NMM. The reaction
mixture was stirred at room temperature for 8 h and TLC
(CH2Cl2 : CH3OH, 40 : 1) indicated the complete disappearance
of TosÁTyr-OBzl. The formed precipitates of dicyclohexylurea
(DCU) were removed by filtration, the filtrate was evaporated
under vacuum and the residue was dissolved in 100 mL of ethyl
acetate. The solution was washed successively with 5% aqueous
solution of sodium bicarbonate, 5% aqueous solution of citric
acid and saturated aqueous solution of sodium chloride. The
ethyl acetate phase was dried with anhydrous sodium sulfate
for 12 h. After filtration the filtrate was evaporated under vacuum
to provide 0.688 g (88%) of the title compound as colorless
powders. ESI-MS (m/z): 471 [M + H]+.
Preparing HClÁArg-Lys(Z)-OBzl
Using the procedure of preparing HClÁVal-Tyr-OBzl from 1.290 g
(2.0 mmol) of Boc-Arg-Lys(Z)-OBzl, 1.021 g (92%) of the title
compound was obtained as colorless powders for next reaction
directly. ESI-MS (m/z): 527 [M + H]+.
Preparing HClÁVal-Tyr-OBzl
At 0 1C a solution of 0.874 g (1.8 mmol) of Boc-Val-Tyr-OBzl in
10 mL solution of hydrogen chloride in ethyl acetate was stirred
for 1 h and then evaporated under vacuum. The residue was
dissolved in 20 mL of ethyl acetate and the solution was
evaporated under vacuum to thoroughly remove hydrogen
chloride and provide 0.701 g (92%) of the title compound as
colorless powders for next reaction directly. ESI-MS (m/z):
371 [M + H]+.
Preparing (1S,3S)-1-methyl-2-Boc-1,2,3,4-tetrahydro-b-
carboline-3-carboxyl-Arg-Lys(Z)-OBzl (3)
At 0 1C a solution of 0.506 g (2.2 mmol) of (1S,3S)-1-methyl-
2-Boc-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid, 0.270 g
(2.0 mmol) of HOBt, 0.490 g (2.4 mmol) of DCC and 20 mL of
anhydrous THF was stirred for 30 min, to which a solution of
1.116 g (2.0 mmol) of HClÁArg-Lys(Z)-OBzl in 10 mL of anhy-
drous THF was added, and adjusted to pH 9 with NMM. The
reaction mixture was stirred at room temperature for 8 h and
TLC (CH2Cl2 : CH3OH, 20 : 1) indicated the complete disappear-
ance of HClÁArg-Lys(Z)-OBzl. The formed DCU precipitates were
removed by filtration, the filtrate was evaporated under vacuum
and the residue was dissolved in 100 mL of dichloromethane.
The solution was washed successively with 5% aqueous solution
of sodium bicarbonate, 5% aqueous solution of citric acid and
saturated aqueous solution of sodium chloride. The dichloro-
methane phase was dried with anhydrous sodium sulfate for
12 h. After filtration the filtrate was evaporated under vacuum to
provide 1.178 g (70%) of the title compound as colorless powders
for next reaction directly. ESI-MS (m/z): 839 [M + H]+.
Preparing Boc-Asp(OBzl)-Val-Tyr-OBzl
At 0 1C a solution of 0.578 g (1.8 mmol) of Boc-Asp(OBzl),
0.240 g (1.8 mmol) of HOBt, 20 mL of anhydrous THF and
0.401 g (2.0 mmol) of DCC was stirred for 30 min, to which a
solution of 0.758 g (1.8 mmol) of HClÁVal-Tyr-OBzl in 10 mL of
anhydrous THF was added, and adjusted to pH 9 with NMM.
The reaction mixture was stirred at room temperature for 8 h,
and TLC (CH2Cl2 : CH3OH, 40 : 1) indicated the complete dis-
appearance of HClÁVal-Tyr-OBzl. The formed DCU precipitates
were removed by filtration, the filtrate was evaporated under
vacuum, the residue was dissolved in 100 mL of ethyl acetate
and the solution was washed successively with 5% aqueous
solution of sodium bicarbonate, 5% aqueous solution of citric
acid and saturated aqueous solution of sodium chloride. The
ethyl acetate phase was dried with anhydrous sodium sulfate
for 12 h. After filtration the filtrate was evaporated under vacuum
to provide 1.004 g (82%) of the title compound as colorless
powders. ESI-MS (m/z): 676 [M + H]+.
Preparing (1S,3S)-1-methyl-2-Boc-1,2,3,4-tetrahydro-b-
carboline-3-carboxyl-Arg-Lys(Z) (4)
At 0 1C a solution of 1.178 g (1.4 mmol) of (1S,3S)-1-methyl-2-
Boc-1,2,3,4-tetrahydro-b-carboline-3-carboxyl-Arg-Lys(Z)-OBzl, 10 mL
of methanol and 2 mL of aqueous NaOH (2 M) was stirred for
Preparing HClÁAsp(OBzl)-Val-Tyr-OBzl
Using the procedure of preparing HClÁVal-Tyr-OBzl from 60 min, adjusted to pH 2 with hydrochloric acid (2 M) and
1.004 g (1.5 mmol) of Boc-Asp(OBzl)-Val-Tyr-OBzl, 0.687 g evaporated under vacuum. The residue was dissolved in 100 mL
(91%) of the title compound was obtained as colorless powders of ethyl acetate and the solution was dried with anhydrous
for the next reaction directly. ESI-MS (m/z): 576 [M + H]+.
sodium sulfate for 12 h. After filtration the filtrate was evaporated
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under vacuum to provide 1.046 g (99%) of the title compound 13C-NMR (200 MHz, DMSO-d6): d/ppm = 173.30, 171.95, 171.26,
as colorless powders for next reaction directly. ESI-MS (m/z): 171.06, 170.64, 157.53, 156.42, 136.85, 130.43, 128.55, 127.85,
749 [M + H]+.
126.20, 125.97, 125.95, 122.16, 119.70, 118.40, 115.48, 111.90,
105.29, 57.47, 56.37, 54.35, 52.99, 52.73, 50.09, 49.85, 49.06,
36.36, 31.53, 27.00, 25.43, 23.94, 22.54, 19.60, 18.04, 16.91; IR
(KBr): 3167.12, 3059.20, 2962.66, 2931.80, 2873.94, 1720.50,
Preparing (1S,3S)-1-methyl-2-Boc-1,2,3,4-tetrahydro-b-
carboline-3-carboxyl-Arg-Lys(Z)-Asp(OBzl)-Val-Tyr-OBzl (5)
At 0 1C a solution of 0.773 g (1.0 mmol) of (1S,3S)-1-methyl-2- 1654.92, 1543.05, 1516.05, 1450.47, 1396.46, 1319.31, 1226.73.
Boc-1,2,3,4-tetrahydro-b-carboline-3-carboxyl-Arg-Lys(Z), 0.135 g
Preparing Boc-Arg-Lys(Z)-Asp(OBzl)-Val-Tyr-OBzl
(1.0 mmol) of HOBt, 0.244 g (1.2 mmol) of DCC and 20 mL
of anhydrous THF was stirred for 30 min, to which a solution of At 0 1C a solution of 0.627 g (1.0 mmol) of Boc-Arg-Lys(Z)-OBzl,
0.610 g (1.0 mmol) of HClÁAsp(OBzl)-Val-Tyr-OBzl in 10 mL of 10 mL of methanol and 2 mL of aqueous NaOH (2 M) was
anhydrous THF was added, and adjusted to pH 8 with NMM. stirred for 60 min, adjusted to pH 7 with hydrochloric acid
The reaction mixture was stirred at room temperature for 48 h (2 M), and evaporated under vacuum. The residue was dissolved
and TLC (CH2Cl2 : CH3OH, 10 : 1) indicated the complete dis- in 100 mL of THF, mixed with 0.135 g (1.0 mmol) of HOBt and
appearance of HClÁAsp(OBzl)-Val-Tyr-OBzl. The formed DCU 0.244 g (1.2 mmol) of DCC, stirred at 0 1C for 30 min, to which a
precipitates were removed by filtration, the filtrate was evapo- solution of 0.550 g (0.9 mmol) of HClÁAsp(OBzl)-Val-Tyr-OBzl in
rated under vacuum and the residue was dissolved in 100 mL of 10 mL of anhydrous THF was added, and adjusted to pH 8 with
dichloromethane. The solution was washed successively with 5% NMM. The reaction mixture was stirred at room temperature
aqueous solution of sodium bicarbonate, 5% aqueous solution of for 48 h and TLC (CH2Cl2 : CH3OH, 10 : 1) indicated the disap-
citric acid and saturated aqueous solution of sodium chloride. The pearance of HClÁAsp(OBzl)-Val-Tyr-OBzl. The formed DCU pre-
dichloromethane phase was dried with anhydrous sodium sulfate cipitates were removed by filtration, the filtrate was evaporated
for 12 h. After filtration the filtrate was evaporated under vacuum under vacuum, the residue was dissolved in 100 mL of CH2Cl2,
to provide 0.824 g (68%) of the title compound as colorless and the solution was successively washed with 5% aqueous
powders. ESI-MS(m/z): 1307 [M + H]+; 1H-NMR (300 MHz, solution of sodium bicarbonate, 5% aqueous solution of citric
DMSO-d6): d/ppm = 10.910 (s, 1H), 9.292 (s, 1H), 8.455 (m, 2H), acid and saturated aqueous solution of sodium chloride. The
8.166 (m, 1H), 7.531 (m, 2H), 7.312 (m, 15H), 7.223 (m, 4H), CH2Cl2 phase was dried with anhydrous sodium sulfate for 12 h.
6.976 (d, J = 2.7 Hz, 2H), 6.652 (d, J = 2.7 Hz, 2H), 5.001 (m, 7H), After filtration the filtrate was evaporated under vacuum to
4.683 (m, 1H), 4.438 (m, 1H), 4.306 (m, 3H), 3.233 (m, 1H), 3.168 provide 0.564 g (57%) of the title compound as colorless powders.
(m, 2H), 2.949–2.785 (m, 6H), 2.665 (m, 1H), 1.929 (m, 1H), ESI-MS (m/z): 1095 [M + H]+; 1H-NMR (300 MHz, DMSO-d6):
1.540–1.247 (m, 21H), 0.729 (dd, J = 7.5 Hz, J = 15.0 Hz, 6H).
d/ppm = 8.441 (m, 2H), 7.803 (m, 2H), 7.597–7.434 (m, 4H),
7.321–7.223 (m, 15H), 7.140 (d, J = 8.4 Hz, 2H), 6.938 (d, J = 8.4 Hz,
2H), 5.031 (m, 6H), 4.666 (m, 1H), 4.502 (m, 1H), 4.260 (m, 2H),
3.918 (m, 1H), 3.165 (m, 2H), 3.012–2.778 (m, 6H), 1.923 (m, 1H),
Preparing (1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl-Arg-Lys-Asp-Val-Tyr (MTCTP5, 6)
To a suspension of 0.100 g (0.077 mmol) of (1S,3S)-1-methyl-2- 1.722–1.485 (m, 6H), 1.369–1.238 (m, 13H), 0.743 (m, 6H).
Boc-1,2,3,4-tetrahydro-b-carboline-3-carboxyl-Arg-Lys(Z)-Val-Tyr-
Preparing Arg-Lys-Asp-Val-Tyr (TP5)
OBzl, 20 mL of methanol, 0.2 mL of formic acid and 20 mg of
Pd/C (10%) hydrogen gas were bubbled for 20 h and TLC To a suspension of 0.100 g (0.091 mmol) of Boc-Arg-Lys(Z)-Val-
indicates the complete disappearance of (1S,3S)-1-methyl- Tyr-OBzl, 20 mL of methanol, 0.2 mL of formic acid and 20 mg of
2-Boc-tetrahydro-b-carboline-3-carboxyl-Arg-Lys(Z)-Val-Tyr-OBzl. Pd/C (10%) hydrogen gas were bubbled for 20 h and TLC indicates
The reaction mixture was filtered and the filtrate was evapo- the complete disappearance of Boc-Arg-Lys(Z)-Asp(OBzl)-Val-Tyr-
rated under vacuum. The residue was treated with 10 mL of OBzl. The reaction mixture was filtered, the filtrate was evaporated
ethyl acetate containing hydrogen chloride (4 M) to remove Boc. under vacuum and the residue was treated with 10 mL of ethyl
After removal of hydrogen chloride 0.056 g (63%) of the title acetate containing hydrogen chloride (4 M) to remove Boc. After
compound was obtained as colorless powders. Mp 123–124 1C; removal of hydrogen chloride 0.036 g (58%) of the title com-
[a]2D5 = À89.0 (c 0.10, CH3OH); FT-MS (m/z): 892.47541 [M + H]+; pound was obtained as colorless powders. ESI-MS (m/z): 680
HPLC purity (CH3OH : H2O = 95% : 5%, C18, 1.0 mL minÀ1): [M + H]+; 1H-NMR (300 MHz, D2O): d/ppm = 7.039 (d, J = 8.4 Hz,
96%. 1H-NMR (800 MHz, DMSO-d6): d/ppm = 11.287 (s, 1H), 2H), 6.628 (d, J = 8.4 Hz, 2H), 4.606–4.525 (m, 2H), 4.323
9.246 (s, 1H), 8.946 (s, 1H), 8.532 (s, 1H), 8.257 (s, 1H), 8.194 (d, (m, 1H), 3.984–3.871 (m, 2H), 3.151–3.040 (m, 3H), 2.922–2.819
J = 8.0 Hz, 1H), 8.005 (s, 3H), 7.485 (d, J = 8.0 Hz, 1H), 7.472 (m, (m, 3H), 2.802–2.603 (m, 2H), 1.925–1.502 (m, 9H), 1.419–1.312
1H), 7.432 (d, J = 8.0 Hz, 1H), 7.383 (m, 1H), 7.130 (t, J = 8.0 Hz, (m, 2H), 0.738 (m, 6H).
1H), 7.040 (t, J = 8.0 Hz, 1H), 6.974 (t, J = 8.0 Hz, 2H), 6.657
(d, J = 8.0 Hz, 2H), 4.722 (m, 1H), 4.567 (m, 1H), 4.433 (m, 1H),
Trypsin promoted release of MTCTP5
4.315 (m, 2H), 4.225 (m, 1H), 3.122 (m, 3H), 2.896 (m, 2H), To a solution of MTCTP5 (1 mM) in ultrapure water 200 mL of
2.898 (m, 2H), 2.785 (m, 1H), 2.753 (m, 2H), 2.672 (m, 1H), trypsin (800 UI) was added, at 37 1C incubated for 10, 20, 30, and
2.562 (m, 1H), 1.984 (m, 1H), 1.859 (m, 1H), 1.694 (d, J = 6.4 Hz, 60 min, and 10 mL of the supernatant was used to carry out ESI-MS
3H), 1.645–1.241 (m, 8H), 0.799 (dd, J = 7.2 Hz, J = 24.0 Hz, 6H); experiments using positive and negative MALDI ion modes.
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Mouse plasma stability of MTCTP5
an imaging plate (Gatan Bioscan Camera Model 1792, Pleasanton,
CA, USA) with 20 eV energy windows at 6000–400 000Â and were
digitally enlarged.
Mouse blood was collected in 1 mg mLÀ1 of aqueous solution
of heparin sodium (1 : 9, v/v) and immediately centrifuged at
3000 rpm for 15 min to collect the plasma. To 200 mL of plasma
200 mL solution of MTCTP5 (2 mM) in ultrapure water was added
and incubated at 37 1C for 10, 20, 30, 60 and 90 min. To the
plasma 800 mL of methanol was added and the sample was
centrifuged at 3000 rpm for 10 min, and then 10 mL of the
supernatant was used to carry out the ESI-MS experiment.
Measuring SEM images of MTCTP5
The shape and the size of the nanospecies of MTCTP5 in the
solid state were measured using scanning electron microscopy
(SEM, JEM-1230, JEOL, Tokyo, Japan) at 50 kV. In brief, the
lyophilized powders from 10À7 nM solution of MTCTP5 in
ultrapure water were attached to a copper plate with double-
sided tape (Euromedex, Strasbourg, France). The specimens were
coated with 20 nm gold–palladium using a Joel JFC-1600 Auto Fine
Coater. The coater was operated at 15 kV, 30 mA, and 200 mTorr
(argon) for 60 s. The shape and size distribution of the nanospecies
were measured by examining 4100 species in randomly selected
regions on the SEM alloy. Each measurement was performed with
triplicate grids. Images were recorded on an imaging plate
(Gatan Bioscan Camera Model 1792) with 20 eV energy windows
at 100–10 000Â, and were digitally enlarged.
Measuring the FT-MS spectra of MTCTP5
The positive MALDI FT-MS spectrum of aqueous solution of
MTCTP5 (10À6 nM) was measured on a solariX FT-ICR mass
spectrometer (Bruker Daltonics, Billerica, MA, USA) with an ESI/
MALDI dual ion source and a 9.4 T superconductive magnet. The
ion source was a Smart-beam-II laser (wavelength, 355 nm; focus
setting, ‘medium’; repetition rate, 1000 Hz). The QCID spectrum
was set to 2000 m/z, and the isolation window was 5 m/z. Data
were collected using solariX-control software. Spectral data were
processed using data analysis software (Bruker Daltonics).
Measuring AFM images of MTCTP5
Atomic force microscopy (AFM) experiments were performed in
the contact mode using a Nanoscope 3D AFM (Veeco Metrology,
Santa Barbara, CA, USA) under ambient conditions. AFM images
of mouse plasma alone or MTCTP5 in mouse plasma (10À7 nM)
were recorded using a standard procedure.
Measuring the NOESY 2D NMR spectra of MTCTP5
One-dimensional 1H NMR spectra of 12 mg of MTCTP5 in
0.5 mL of deuteron dimethyl sulfoxide (DMSO-d6) were measured
on a Bruker 800 MHz spectrometer. The probe temperature was
regulated to 298 K. Using a simple pulse-acquire sequence zg30
the spectra were recorded. To ensure the full relaxation of
1H resonance typical acquisition parameters consisting of 64 K
points covering a sweep width of 16447 Hz, a pulse width (pw90)
of 8.63 ms and a total repetition time of 24 s were used. Before FT
the digital zero filling to 64 K and a 0.3 Hz exponential function
were applied to the FID. The resonance at 2.5 ppm presented
CD2HSOCD2H (the impurity in the residual solvents) and tetra-
methylsilane (TMS) was used as internal reference. Standard
absorptive 2D 1H–1H COSY was measured using the same
spectrometer. Each spectrum consisted of a matrix of 2 K (F2)
by 0.5 K (F1) covering a sweep width of 9615.4 Hz. Before FT the
matrix was zero filled to 1 K by 1 K and the standard sinebell
apodization functions were applied in both dimensions. 2D
NOESY tests were carried out in the phase-sensitive mode by
using the same spectrometer. Spectra were obtained using spin-
lock mixing periods of 200 ms.
Measuring the particle size of MTCTP5
The particle size of MTCTP5 in ultrapure water was measured
on a Particle Size Analyzer (Zeta Plus S/N 21394, Brookhaven
Instruments Corporation) using a dynamic light scattering
(DLS) model. The concentration of the solution of MTCTP5 in
ultrapure water was 10À7 nM and the testing temperature was
25 1C. The size measurement was repeated for 3 runs per sample
every day, and totally tested for 7 consecutive days.
Measuring zeta potential of the nanospecies of MTCTP5
The surface zeta potential of the nanospecies of MTCTP5 in
ultrapure water or in mouse plasma was measured on a ZetaPlus
Potential Analyzer (ZetaPlus S/N 21394, Brookhaven Instruments
Corporation) with a BIC Zeta Potential Analyzer. The concen-
tration of the solution of MTCTP5 in ultrapure water or in mouse
plasma was 10À7 nM and the testing temperature was 25 1C. The
zeta potential measurement was repeated for 3 runs per sample,
and the data were calculated automatically using the software
Measuring the TEM image of MTCTP5
The shape and the size of the nanospecies of MTCTP5 in water from the electrophoretic mobility based on Smoluchowski’s
were measured using transmission electron microscopy (TEM, formula.
JSM-6360 LV, JEOL, Tokyo, Japan). In brief, an aqueous solution
of MTCTP5 (pH 6.7, 10À7 nM) was dripped onto a formvar-
Energy-minimization conformations of MTCTP5
coated copper grid. The grid was allowed to dry thoroughly in MTCTP5 was sketched in ChemDraw 10.0, converted to 3D
air and then was heated at 35 1C for 14 days. The copper grids conformation in Chem3D 10.0 and then energy was minimized
were viewed under a TEM. The shape and size distribution of in Discovery Studio 3.5 with a MMFF force field. The energy-
the nanospecies were determined by counting 4100 species in minimized conformation was utilized as the starting one for
randomly selected regions on the copper grid. Each determina- conformation generation. The energy-minimized conformations
tion was carried out with triplicate grids and at 80 kV (the were sampled in the whole conformational space via systematic
electron beam accelerating voltage). Images were recorded on search and BEST methods in Discovery Studio 4.0. Both of the
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systematic search and BEST methods were practiced using a inserted into the middle of a polyethylene tube. The poly-
SMART minimizer with a CHARMm force field. The energy ethylene tube was filled with NS solution of heparin sodium
threshold was set to 20 kcal molÀ1 at 300 K. The maximum (50 IU mLÀ1) and one end was inserted into the left jugular vein.
minimization steps and the RMS gradient were set to 200 Å and From the other end of the polyethylene tube heparin sodium was
0.1 Å, respectively. The maximum generated conformations injected as an anticoagulant and this end was inserted into the
were set to 255 with a RMSD cut-off of 0.2 Å.
right carotid artery. Blood was allowed to flow from the right
carotid artery to the left jugular vein through the polyethylene
tube for 15 min. The thread was removed to obtain the weight of
the wet thrombus.
Molecular docking of MTCTP5 towards the active site of bovine
trypsin
The 3D structure of MTCTP5 was built using a 3D-sketcher
module and energy minimized in Discovery Studio 4.0 with a
In vivo anti-tumor assay
SMART minimizer using the CHARMm force field. The crystal Male ICR mice were 10–12 weeks old at the beginning of
structure of bovine trypsin (PDB ID: 4MTB) was from the experiments. S180 cells for initiation of subcutaneous tumors
Protein Data Bank, and the binding sphere (9.0 Å) of the (2R)- were obtained in ascitic form of the mouse tumors, which were
2-amino-N-(2S)-1-[(4-carbamimi-doylbenzyl)amino]-1-oxopro-pan- serially transplanted once a week. Subcutaneous tumors were
2-yl-4-(4-hydroxyphenyl)-butanamide domain was defined. The implanted by injecting 0.2 mL of NS containing 1.8 Â 107 viable
water molecules were removed, and hydrogen atoms were added tumor cells under the skin on right oxter. Twenty four hours after
under the CHARMm force field. Docking calculations were implantation the tumor bearing mice were randomized into
performed using the LibDock module implemented in Accelrys 6 experimental groups (12 per group). All mice were given a daily
Discovery studio 4.0, which is a high-through put docking algo- intraperitoneal injection of doxorubicin (2 mmol kgÀ1 per day
rithm that positions catalyst generated ligand conformations in in NS) or MTC (2 mmol kgÀ1 per day in NS) or NS (10 mL kgÀ1
the protein active site based on polar and apolar interaction sites per day) or MTCTP5 (0.002, 0.02, 0.2 and 2 mmol kgÀ1 per day
(hotspots). The results could be displayed by analyzing and in NS) for 12 consecutive days. Twenty four hours after the last
scoring docked ligand poses. To find a top rank pose and administration, all mice were sacrificed by ether anesthesia and
measure the goodness of a docking study, the LibDock Score the tumors were dissected and weighed.
was used as the criteria. The binding site ‘‘hotspots’’ were set to
100. The conformation generation of the ligands was carried
In vivo carbon clearance assay
out using the BEST method. The energy threshold was set to In vivo carbon clearance assay was performed using a standard
20 kcal molÀ1, the maximum minimization steps were set procedure. In brief, ICR mice were divided into 3 groups of
to 1000, the minimization RMS gradient was set to 0.001 Å, 10 animals each and given a daily intraperitoneal injection of
and the maximum generated conformations were set to 255 with MTCTP5 (2 mmol kgÀ1 per day in NS) or TP5 (positive control,
a RMSD cut-off of 1.0 Å.
2 mmol kgÀ1 per day in NS) or NS (blank control, 10 mL kgÀ1 per
day) or for 7 consecutive days. On the 8th day the mice were
intraperitoneally injected with a suspension of 0.1 mL of indian
In vivo xylene-induced ear edema assay
Male ICR mice were 10 weeks old at the beginning of the ink and NS solution of gelatine (1%, w/v). One and ten minutes
experiment, randomly divided into 6 groups (12 per group) after the administration the blood was sampled, mixed with
and intraperitoneally administered with MTCTP5 (2, 0.2 and aqueous Na2CO3 (0.1%, 4 mL) and the absorbance was
0.02 mmol kgÀ1) or MTC (2 mmol kgÀ1) or aspirin (1100 mmol kgÀ1
)
measured at 660 nm to calculate carbon clearance based on
or NS (10 mL kgÀ1). Thirty minutes after the administration mbody/(mliver + mspleen) Â [(Log OD1 À Log OD2)/9]1/3, wherein
0.03 mL of xylene was applied to the anterior and posterior OD1 refers to the absorbance at 10 min and OD2 refers to the
surfaces of right ears of the mice, and left ears without xylene absorbance at 1 min, mbody, mliver and mspleen refer to the
application were used as the controls. Two hours after xylene bodyweight, liver weight and spleen weight of the tested mice,
application the mice were anesthetized with ether and sacrificed respectively.
for the removal of both ears. Using a cork borer of 7 mm in
diameter the ears were punched to take circular pieces for
In vivo IL-2, CD4 and CD8 assays
weighing. Ear edema was represented with the weight difference 0.9 mL of blood of the mice receiving carbon clearance assay
between the circular pieces of right and left ears.
was collected into a syringe containing 0.1 mL of 3.8% sodium
citrate and immediately centrifuged at 3000 rpm for 10 min.
The separated plasma was used to measure plasma levels of
In vivo anti-thrombotic assay
Male Wistar rats (250–300 g) were randomly divided into IL-2, CD4 and CD8 by ELISA according to the manufacturer’s
6 groups (12 per group), and MTCTP5 (2, 0.2 and 0.02 mmol kgÀ1
or MTC (2 mmol kgÀ1) or aspirin (167 mmol kgÀ1) or NS (10 mL kgÀ1
)
)
protocols. In brief 100 mL of plasma or standard solution was
added into the wells of anti-body coated 96 microtiter plates,
was intraperitoneally administered. Thirty minutes after the incubated at 37 1C for 90 min, and washed with 100 mL of wash
administration the rats were anesthetized with intraperitoneally buffer 5 times; 100 mL of biotinylated antibody was added,
pentobarbital sodium (80.0 mg kgÀ1), the right carotid artery and incubated at 37 1C for 60 min, and washed with 100 mL of wash
the left jugular vein were separated. A weighed 6 cm thread was buffer 5 times; 100 mL of HRP-conjugate reagent was added,
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incubated at 37 1C for 30 min, and washed with 100 mL of wash solution B were added, gently mixed and at 37 1C and protec-
buffer 5 times; 50 mL of chromogen solution A and 50 mL of tion from light incubated for 10 min. To each well 50 mL of stop
chromogen solution B were added, and incubated at 37 1C for solution was added, and the color in the well changed from
15 min; 100 mL of stop solution was added, and the absorbance blue to yellow. The plate was read at 450 nm using a microtiter
of the yellow solutions was recorded at 450 nm.
plate reader within 15 min to record the O.D. value. According
to the standard curve the concentrations of TNF-a in plasma
were calculated.
In vitro spleen lymphocyte proliferation assay
The effects of MTCTP5 on Babl/c spleen lymphocyte proliferation
were measured using MTT assay. The spleen lymphocytes were
Plasma IL-8 assay on S180 mice
prepared according to a standard procedure. In brief under sterile In the preparation of the plasma sample 0.9 mL of blood was
conditions after rapid collection spleens were placed in cold collected from healthy mice or S180 mice receiving MTCTP5
RPMI-1640 containing 10% FCS supplemented with antibiotics (2, 0.2, 0.02 mmol kgÀ1) or NS into a syringe containing 0.1 mL
(100 UI mLÀ1 of penicillin and 100 mg mLÀ1 of streptomycin). of 3.8% sodium citrate. The sample was centrifuged at 4 1C and
Cells were teased apart and gently passed through a 40 gm nylon 3000 rpm for 10 min to prepare the plasma sample. To each of
mesh to remove clumps of cells and connective tissue. After two the three blank wells nothing was added. To each of the six
time wash of PBS the cell suspension was adjusted to a final standard wells 100 mL of standard solution was added. To each
concentration of 5 Â 105 cells per mL RPMI-1640 with 10% FCS in of the three testing wells 100 mL of the plasma sample from
96-well microtiter plates, and the blastogenic assays were carried healthy mice or the mice receiving NS or MTCTP5 (2, 0.2,
out with 0.2 mL of suspensions. Cell viability was tested by the use 0.02 mmol kgÀ1) was added. Then the plate was closed with a
of fluorescein diacetate. The cultures of splenocyte cells were at closure plate membrane and at 37 1C incubated for 40 min.
37 1C incubated with mitogen concanavalin A (Con-A, 5 Â 10À5 M) Upon uncovering the closure plate membrane the liquid was
and 5% CO2 for 48 h, and the cells received MTT assay to calculate discarded from the well and dried by swinging, to the residue in
the relative spleen lymphocyte proliferation (%) according to the well sufficient washing buffer, which was prepared by diluting
proliferation% = (ODT À ODC)/ODC Â 100%, wherein ODT the wash solution with 30 fold distilled water, was added, main-
refers to the absorbance of MTCTP5 and ODC refers to the tained for 30 s, and then drained. After 5 repeats of the procedure,
absorbance of RPMI-1640 (blank control).
50 mL of biotinylated antibody solution and 50 mL of distilled
water were added, then the plate was closed with a closure plate
membrane and at 37 1C incubated for 30 min. Upon uncovering
Plasma TNF-a assay on S180 mice
In the preparation of the plasma sample 0.9 mL of blood was the closure plate membrane the liquid was discarded from the
collected from healthy mice or S180 mice receiving MTCTP5 well and washed by sufficient washing buffer 5 times. After
(2, 0.2, 0.02 mmol kgÀ1) or NS into a syringe containing 0.1 mL 5 repeats of the procedure, 100 mL of biotinylated antibody
of 3.8% sodium citrate. The sample was centrifuged at 4 1C and solution was added and the plate was closed with a closure
3000 rpm for 10 min to prepare the plasma sample. To each of plate membrane and at 37 1C incubated for 30 min. Upon
the three blank wells nothing was added. To each of the six uncovering the closure plate membrane the liquid was dis-
standard wells 100 mL of standard solution was added. To each carded from the well and washed by sufficient washing buffer
of the three testing wells 100 mL of the plasma sample from 5 times and dried by patting. To the residue in the well 50 mL of
healthy mice or the mice receiving NS or MTCTP5 (2, 0.2, chromogen solution A and 50 mL of chromogen solution B were
0.02 mmol kgÀ1) was added. Then the plate was closed with a added, gently mixed and at 37 1C and protection from light
closure plate membrane and at 37 1C incubated for 40 min. incubated for 10 min. To each well 50 mL of stop solution was
Upon uncovering the closure plate membrane the liquid was added, and the colour in the well changed from blue to yellow.
discarded from the well and dried by swinging, to the residue in The plate was read at 450 nm using a microtiter plate reader
the well sufficient washing buffer, which was prepared by within 15 min to record the O.D. value. According to the standard
diluting the wash solution with 30 fold distilled water, was curve the concentrations of IL-8 in plasma were calculated.
added, maintained for 30 s, and then drained. After 5 repeats of
Lifespan assay
the procedure, 50 mL of biotinylated antibody solution and
50 mL of distilled water were added, then the plate was closed Lifespan assay was performed by using a standard procedure.
with a closure plate membrane and at 37 1C incubated for In brief, male ICR mice were 10–12 weeks old at the beginning
30 min. Upon uncovering the closure plate membrane the of experiments. S180 cells for the initiation of subcutaneous
liquid was discarded from the well and washed by sufficient tumors were obtained from the ascitic form of the tumors in mice,
washing buffer 5 times. After 5 repeats of the procedure, 100 mL which were serially transplanted once a week. Subcutaneous
of biotinylated antibody solution was added and the plate was tumors were implanted by injecting 0.2 mL of NS containing
closed with a closure plate membrane and at 37 1C incubated 1 Â 107 viable tumor cells under the skin on right oxter. Twenty
for 30 min. Upon uncovering the closure plate membrane the four hours after implantation the tumor bearing mice were
liquid was discarded from the well and washed by sufficient randomized into 4 experimental groups (12 per group). All
washing buffer 5 times and dried by patting. To the residue in mice were given a daily intraperitoneal injection of TP5
the well 50 mL of chromogen solution A and 50 mL of chromogen (2 mmol kgÀ1 per day in NS) or MTC (2 mmol kgÀ1 per day in NS)
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or NS (10 mL kgÀ1 per day) or MTCTP5 (2 mmol À1kg per day in at 546 nm to calculate vitality based on (ODtest À ODcontrol) Â
NS) for 33 consecutive days. Mouse death due to tumor burden Kcurve, wherein ODtest refers to the absorbance of the test well,
was noted and the lifespan was calculated as [(T À C)/C]%, ODcontrol refers to the absorbance of the control well and
wherein T and C represent the survival days of treated and Kcurve refers to the standard curve.
untreated mice, respectively.
Results and discussion
Plasma ALT and AST assays
1. Synthesis and structural characterization
0.9 mL of blood of the mice receiving anti-tumor assay was
collected into a syringe containing 0.1 mL of 3.8% sodium
citrate and immediately centrifuged at 3000 rpm for 10 minutes
to get the plasma samples. The separated plasma was used
to measure plasma levels of alanine transaminase (ALT) and
aspartate transaminase (AST) according to the guidance of the
kits (AST/GOT testing kit, ALT/GPT testing kit; JCBIO Co.,
Nanjing, People’s Republic of China). In brief 5 mL of plasma
was added into the test wells of 96 microtiter plates, then 20 mL
of Matrix liquid was added and incubated at 37 1C for 30 min,
5 mL of plasma was added into the control wells of 96 microtiter
plates, 20 mL of 2,4-dinitrophenylhydrazine was added to all
wells, and incubated at 37 1C for 20 min, 200 mL of 0.4 mol LÀ1
NaOH was added, and incubated at 37 1C for 15 min, and the
absorbance of the yellow solutions was recorded at 510 nm to
calculate vitality based on (ODtest À ODcontrol) Â Kcurve, wherein
ODtest refers to the absorbance of the test well, ODcontrol refers
to the absorbance of the control well, and Kcurve refers to the
standard curve which is calculated using the absorbance of the
standard well following the manufacturer’s protocols.
1.1. Preparation and chemical structure of MTCTP5. MTC
(2) and the intermediates (3–5) were prepared by following the
previous procedure.39,40 TP5 and the intermediates were pre-
pared by following the general route.41–43
As depicted in Scheme 1 MTCTP5 was prepared via 12-step
reactions, the yields ranged from 63% to 99% and the total
1
yield was 5.6%. The H NMR, 13C NMR, FT-MS and IR spectra
are given as Fig. S1–S8 of the ESI,† which supports MTCTP5
having a correct chemical structure.
1.2. Nanofeature and size of MTCTP5
The nanofeature and the size of MTCTP5 in water, at the solid
state and in mouse plasma were described using TEM, SEM and
AMF images, respectively.
TEM images (Fig. 1A) indicate that when the powders of MTCTP5
are dissolved in ultrapure water to form a solution of 10À7 nM
MTCTP5 the nanoparticles of 30–110 nm in diameter can be formed.
The inset indicates that in the amplified nanoparticle 7 small
particles are included and on the surface of the amplified nano-
particle numerous holes of B7 nm in diameter are observed.
SEM images (Fig. 1B) indicate that the solids resulted from
Plasma Cr assay
0.9 mL of blood of the mice receiving anti-tumor assay was 10À7 nM solution of MTCTP5 in ultrapure water are equally
collected into a syringe containing 0.1 mL of 3.8% sodium distributed nanoparticles of 10–92 nm in diameter, for most
citrate and immediately centrifuged at 3000 rpm for 10 minutes nanoparticles the diameter is less than 50 nm.
to get the plasma samples. The separated plasma was used to
AFM images (Fig. 1C) indicate that in mouse plasma MTCTP5
measure plasma levels of creatinine (Cr) according to the (final concentration, 10À7 nM) forms nanoparticles of 14–139 nm
guidance of the kits (Cr testing kit; JCBIO Co., Nanjing, People’s in diameter, for most nanoparticles the diameter is less than
Republic of China). In brief 6 mL of plasma or standard solution 100 nm, and some nanoparticles can form aggregates. On the
was added into the wells of 96 microtiter plates, 180 mL of the other hand no any comparable nanoparticle is found in the AFM
enzyme solution A was added to incubate at 37 1C for 5 min, image of mouse plasma alone (Fig. 1D).
60 mL of the enzyme solution B was added to incubate at 37 1C for
The DLS determinations of 7 consecutive days explored that
5 min, and the absorbance of the purple solutions was recorded in ultrapure water the nano-size of MTCTP5 ranged from 186 nm
Fig. 1 Nanofeature and size of MTCTP5. (A) TEM image of MTCTP5 in ultrapure water (10À7 nM); (B) SEM image of the solids from a solution of MTCTP5
in ultrapure water (10À7 nM); (C) AFM image of MTCTP5 in mouse plasma (10À7 nM); (D) AFM image of mouse plasma alone; (E) the change of the nano-
size of MTCTP5 in ultrapure water within 7 days.
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to 320 nm in diameter. These data imply that the nanoparticles’ 3. Bioactivity related to the TP5 moiety of MTCTP5
diameter of MTCTP5 in water increases slightly in 7 days
(Fig. 1E).
3.1. Proliferation of splenocyte cells treated with MTCTP5.
The immunology enhancing activity related to the TP5 moiety
was tested with the in vitro proliferation of splenocyte cells
The zeta-potential determinations explored that in ultrapure
water and in mouse plasma the zeta-potentials of MTCTP5 were
treated with MTCTP5. Fig. 3A indicates that the proliferation of
À8.56 Æ 0.89 mV and À25.13 Æ 0.98 mV, respectively. These
splenocyte cells treated with 50 mM of ConA plus 100 mM of TP5
is equal to that of splenocyte cells treated with 50 mM of ConA
plus 100 mM of MTCTP5, and is significantly higher than that of
splenocyte cells treated with 50 mM of ConA alone.
data imply that the nanoparticles’ diameter of MTCTP5 in
ultrapure water is larger than that of MTCTP5 in mouse plasma,
and this should benefit the delivery of the nanoparticles in
blood circulation.
3.2. In vivo phagocytosis index of MTCTP5 treated ICR
mice. The immunologic enhancing activity related to the TP5
2. Bioactivity related to the MTC moiety of MTCTP5
moiety was further tested using carbon clearance assay and is
represented with the phagocytosis index of MTCTP5 treated
ICR mice. Fig. 3B indicates that the phagocytosis index of ICR
mice treated with 2 mmol kgÀ1 of MTCTP5 is equal to that of
ICR mice treated with 2 mmol kgÀ1 of TP5, and is significantly
higher than that of ICR mice treated with NS.
2.1. In vivo anti-tumor activity of MTCTP5. The anti-tumor
activity of MTCTP5 was examined using S180 tumor bearing
mouse assay and represented with tumor weight. Fig. 2A
indicates that the tumor weights of the mice intraperitoneally
receiving 2 mmol kgÀ1 of MTC and 0.02 mmol kgÀ1 of MTCTP5
are significantly lower than those of the mice intraperitoneally
receiving NS, while the tumor weight of the mice intraperito-
neally receiving 0.2 mmol kgÀ1 of MTCTP5 is equal to that of the
mice intraperitoneally receiving 2 mmol kgÀ1 of Dox.
4. Effect of MTCTP5 on TNF-a, IL-8, IL-2, CD4 and CD8 of the
treated mice
4.1. Effect of MTCTP5 on plasma TNF-a of the treated
2.2. In vivo anti-thrombotic activity of MTCTP5. The mice. To explore the action mechanism of MTCTP5 inhibiting
anti-thrombotic activity of MTCTP5 was examined using rat thrombosis, inflammation and tumor growth the plasma TNF-a
thrombosis assay and represented with thrombus weight. of MTCTP5 treated S180 mice was tested. The data of Fig. 4A
Fig. 2B indicates that the thrombus weights of the rats receiving indicate that the plasma TNF-a of 2 and 0.2 mmol kgÀ1 of
2 mmol kgÀ1 of MTC are significantly lower than those of the MTCTP5 treated S180 mice is significantly lower than that of NS
rats receiving NS, suggesting that at 2 mmol kgÀ1 of dose MTC treated S180 mice, while the plasma TNF-a of 0.02 mmol kgÀ1
can inhibit the rats to form thrombus. Fig. 2B also indicates of MTCTP5 treated S180 mice is equal to that of NS treated
that the thrombus weight of the rats receiving 2 mmol kgÀ1 of S180 mice.
MTC is significantly higher than that of the rats receiving
4.2. Effect of MTCTP5 on IL-8 of the treated mice. To
0.2 mmol kgÀ1 of MTCTP5, while the thrombus weight of the explore the action mechanism of MTCTP5 inhibiting thrombosis,
rats receiving 2 mmol kgÀ1 of MTCTP5 is equal to that of the rats inflammation and tumor growth the plasma IL-8 of MTCTP5
receiving 167 mmol kgÀ1 of aspirin.
treated S180 mice was also tested. The data of Fig. 4B indicate that
2.3. In vivo anti-inflammation activity of MTCTP5. The the plasma IL-8 of 2 and 0.2 mmol kgÀ1 of MTCTP5 treated S180
anti-inflammation activity of MTCTP5 was examined using mice is significantly lower than that of NS treated S180 mice,
xylene-induced ear edema assay. Fig. 2C indicates that the while the plasma IL-8 of 0.02 mmol kgÀ1 of MTCTP5 treated S180
anti-inflammation activity of 2 mmol kgÀ1 of MTC is signi- mice is equal to that of NS treated S180 mice.
ficantly higher than that of NS and is equal to that of
4.3. Plasma IL-2, CD4 and CD8 of MTCTP5 treated ICR
0.02 mmol kgÀ1 of MTCTP5. Besides, the anti-inflammation activity mice. The immunology enhancing activities related to the TP5
of 0.2 mmol kgÀ1 of MTCTP5 is equal to that of 1100 mmol kgÀ1 moiety of MTCTP5 were tested using plasma IL-2, CD4 and CD8
of aspirin.
assays in vivo. Fig. 4C–E indicate that the plasma IL-2 and CD4
Fig. 2 In vivo anti-tumor, anti-thrombotic and anti-inflammatory activities of MTCTP5. (A) At 0.002 mmol kgÀ1 of dose MTCTP5 effectively allows the
tumor growth of S180 mice; (B) at 0.2 mmol kgÀ1 of dose MTCTP5 effectively inhibits the rats to form thrombus; (C) at 0.2 mmol kgÀ1 of dose MTCTP5
effectively inhibits xylene inducing the mice to develop ear edema; n = 12.
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Cr was measured according to the guidance of the kit, and the
data are shown in Fig. 5C. The plasma Cr of sham mice, of
NS treated mice and of 2 mmol kgÀ1 of MTCTP5 treated mice
exhibits no significant difference.
5.4. Survival time of MTCTP5 treated S180 mice. The
systemic toxic action of MTCTP5 was firstly examined with
the vital stage of S180 tumor bearing mouse assay and repre-
sented with the survival time. Fig. 5D indicates that the survival
time of NS treated mice is equal to that of 2 mmol kgÀ1 of TP5
treated mice. Fig. 5D also indicates that the survival time of
2 mmol kgÀ1 of MCTP5 treated mice is significantly longer than
those of NS and 2 mmol kgÀ1 TP5 treated mice.
Fig. 3 The immunology enhancing activities of MTCTP5. (A) Effect of
MTCTP5 on the in vitro proliferation of splenocyte cells, n = 6; (B) effect of
MTCTP5 on the phagocytosis index of MTCTP5 treated ICR mice, n = 10.
6. MTCTP5 releasing MTC and/or TP5
6.1. MTCTP5 releasing MTC in mouse plasma. To explore
the release profile 1 mg of MTCTP5 in 1 mL of mouse plasma at
of 2 mmol kgÀ1 of MTCTP5 treated ICR mice are equal to those 37 1C was incubated for 10, 20, 30, 60 and 90 min, and the
of 2 mmol kgÀ1 of TP5 treated ICR mice and are significantly plasma samples were extracted with ultrapure water for ESI-MS
higher than those of NS treated ICR mice, meanwhile CD8 of analysis. Fig. 6A indicates that 60 min after the incubation the
2 mmol kgÀ1 of MTCTP5 treated ICR mice is equal to that of ESI(+)-MS spectrum of the extract gives an ion peak at 892.39 of
2 mmol kgÀ1 of TP5 treated ICR mice and is significantly lower the molecule of MTCTP5 plus H, [M + H]+ (exact mass: 892.46).
than that of NS treated ICR mice.
Fig. 6B shows the total ion current chromatograms of 1 mg of
MTCTP5 in 1 mL of mouse plasma at 37 1C incubated for 1, 5,
10, 30 and 60 min, and suggests that at 37 1C mouse plasma
5. Toxicity of MTCTP5
5.1. Plasma ALT of MTCTP5 treated S180 mice. The hepa- MTCTP5 can stably exist for more than 60 min. Fig. 6C plots the
totoxicity of MTCTP5 was examined using plasma alanine incubation time vs. the area of total ion current chromatogram,
transaminase (ALT) assay. In brief, the blood from the mice and suggests that at 37 1C mouse plasma MTCTP5 has a half-
receiving in vivo antitumor assay was collected to obtain the life of 9.98 min. Fig. 6D shows the ESI(À)-MS spectrum of
plasma. The plasma ALT was measured according to the guidance MTCTP5 in mouse plasma at 37 1C incubated for 30 min, and
of the kit, and the data are shown in Fig. 5A. Plasma ALT of the gives the ion peak at 264.98 of the molecule of MTC plus Cl,
sham mice, the NS treated mice and 2 mmol kgÀ1 of MTCTP5 [M + Cl]À (exact mass: 265.07). Fig. 6E shows the ESI(À)-MS
treated mice exhibits no significant difference.
spectrum of mouse plasma alone at 37 1C incubated for 30 min,
5.2. Plasma AST of MTCTP5 treated S180 mice. The hepa- and gives no such peak.
totoxicity of MTCTP5 was also examined using plasma aspartate
6.2. MTCTP5 releasing MTC and TP5 in trypsin. To esti-
transaminase (AST) assay. In brief, the blood of the mice receiving mate the release profile of MTCTP5 in the presence of trypsin a
in vivo antitumor assay was collected to obtain the plasma. The solution of 1 mg of MTCTP5 and 800 IU of trypsin in 1 mL of
plasma AST was measured according to the guidance of the kit, ultrapure water was at 37 1C incubated for 30 min, of which the
and the data are shown in Fig. 5B. Plasma AST of sham mice, of ESI(+/À)-MS spectra are shown in Fig. 7. The locally amplified
NS treated mice and of 2 mmol kgÀ1 of MTCTP5 treated mice ESI(+)-MS spectrum between 800 and 900 gives a peak of the
exhibits no significant difference.
molecular ion of MTCTP5 plus H at 892.21, [M + H]+ (exact
5.3. Plasma Cr of MTCTP5 treated S180 mice. The renal mass: 892.46, Fig. 7A). The locally amplified ESI(À)-MS spec-
toxicity of MTCTP5 was examined using plasma creatinine trum between 200 and 850 gives a peak of the molecular ion of
(Cr) assay. In brief, the blood from the mice receiving in vivo MTC plus Cl at 264.75, [M + Cl]+ (exact mass: 265.07), and a
antitumor assay was collected to obtain the plasma. The plasma peak of the molecular ion of TP5 minus H at 678.63, [M À H]À
Fig. 4 Effect of MTCTP5 on TNF-a and IL-8. (A) Plasma TNF-a of MTCTP5 treated S180 mice, n = 12; (B) plasma IL-8 of MTCTP5 treated S180 mice,
n = 12; (C) peripheral blood IL-2 of MTCTP5 treated ICR mice, n = 10; (D) peripheral blood CD4 of MTCTP5 treated ICR mice, n = 10; (E) peripheral blood
CD8 of MTCTP5 treated ICR mice, n = 10.
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Fig. 5 Plasma ALT, AST and Cr, as well as survival time of the mice treated with MTCTP5. (A) Plasma ALT of the mice treated with MTCTP5, n = 12;
(B) plasma AST of the mice treated with MTCTP5, n = 12; (C) plasma Cr of the mice treated with MTCTP5, n = 12; (D) survival time of the mice treated with
MTCTP5, n = 12.
Fig. 6 ESI-MS spectra, total ion current chromatogram/time course of MTCTP5 in mouse plasma. (A) ESI(+)-MS spectrum of MTCTP5 in mouse plasma
incubated for 60 min; (B) total ion current chromatograms of MTCTP5 in mouse plasma incubated for 1, 5, 10, 30 and 60 min; (C) area of the total ion
current chromatogram of MTCTP5 in mouse plasma incubated for 1, 5, 10, 30 and 60 min; (D) ESI(À)-MS spectrum of MTCTP5 in mouse plasma
incubated for 30 min and the ion peak of MTC; (E) ESI(À)-MS spectrum of mouse plasma alone incubated for 30 min.
(exact mass: 678.36, Fig. 7B). The total ESI(+)-MS spectrum of a in forming the trimer. Due to the distances between two H atoms
solution of 1 mg of MTCTP5 and 800 IU of trypsin in 1 mL of of the cross-peak are less than 4 Å, thus to form a trimer three
ultrapure water incubated at 37 1C for 30 min is shown in molecules of MTCTP5 have to approach in the manner shown in
Fig. 7C. Fig. 7D shows the locally amplified ESI(À)-MS spectrum Fig. S10A and B (ESI†) and should take flyer-like conformation of
between 200 and 850 of 800 IU of trypsin in 1 mL of ultrapure Fig. S10C (ESI†).
water incubated at 37 1C for 30 min, and gives no comparable
ion peaks of MTC plus Cl and TP5 minus H.
The images of TEM, SEM and AFM explore that the self-
assembly of the trimers leads MTCTP5 to spontaneously form
To explain the molecular mechanism of trypsin promoting nanoparticles. The appropriate size of 14–139 nm in diameter is
MTCTP5 to release MTC and TP5, MTCTP5 was docked into the beneficial for the delivery of the nanoparticles in blood circula-
active site of bovine trypsin (Fig. 7E). It was found that MTCTP5 tion. The zeta potentials of the nanoparticles of MTCTP5 in water
well fitted the active site, and well interacted with the amino and mouse plasma are À8.56 Æ 0.89 mV and À25.13 Æ 0.98 mV,
acid residues of the active site.
respectively, and support the stability of the nanoparticles.
The anti-tumor assay indicates that the tumor weight of the
mice treated with 0.02 mmol kgÀ1 of MTCTP5 is significantly
lower than those of the mice treated with both 2 mmol kgÀ1 of
MTC and NS, suggesting that the minimal effective dose of
Discussion
Using the synthetic route MTCTP5 can be successfully prepared MTCTP5 in treating tumor is 0.02 mmol kgÀ1, and the conjuga-
in acceptable yield and desirable purity. In water the molecular tion of TP5 with MTC leads the anti-tumor activity of MTC to
association leads to MTCTP5 to form trimers. The qCID spec- an increase of 100 fold. Besides, the tumor weight of the mice
trum suggests that under the conditions of FT-MS fragmenta- treated with 0.2 mmol kgÀ1 of MTCTP5 is equal to that of the mice
tion the trimer directly forms the monomer, and no any dimer treated with 2 mmol kgÀ1 of Dox, meaning that the activity of
could be observed. Three interesting cross-peaks of the NOESY MTCTP5 is 10 fold higher than that of Dox. It is worth indicating
2D NMR spectrum define the association manner of 3 molecules that in the in vitro MTT assay the IC50 of MTCTP5 against K562,
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Fig. 7 ESI-MS spectra of MTCTP5 in the presence of trypsin. (A) ESI(+)-MS spectrum of MTCTP5 in the presence of trypsin at 37 1C incubated for 30 min;
(B) locally amplified ESI(À)-MS spectrum of MTCTP5 in the presence of trypsin at 37 1C incubated for 30 min; (C) ESI(+)-MS spectrum of MTCTP5 in the
presence of trypsin at 37 1C incubated for 30 min; (D) locally amplified ESI(À)-MS spectrum of trypsin alone at 37 1C incubated for 30 min; (E) the
interaction of MTCTP5 in the active site of bovine trypsin was hypothesized to be responsible for the release of TP5 and MTC.
A549, HT-29 and HL-60 cells is higher than 150 mM, suggesting encountered implications of cancer patients.45,46 The facts that at
that the anti-tumor action of MTCTP5 in vivo is independent of 0.2 mmol kgÀ1 of dose MTCTP5 simultaneously slows tumor
the cytotoxic action.
growth, inhibits thrombosis and depresses inflammation demon-
The anti-thrombotic assay indicates that the thrombus weight strate that MTCTP5 is a promising lead compound for treating
of the rats receiving 0.2 mmol kgÀ1 of MTCTP5 is significantly cancer patients without complicating chronic inflammation and
lower than those of the rats receiving 2 mmol kgÀ1 of MTC and NS, thromboembolism.
meaning that the minimal effective dose of MTCTP5 in treating
Splenocyte cell proliferation assay indicates that for ConA
thrombus is 0.2 mmol kgÀ1 as well as the conjugation of TP5 and treated splenocyte cells the enhanced proliferations of 100 mM
MTC leads the anti-thrombotic activity of MTC to an increase of of TP5 and 100 mM of MTCTP5 are the same, suggesting that
410 fold. Besides, the thrombus weight of the rats receiving the conjugation of MTC with TP5 does not weaken the in vitro
2 mmol kgÀ1 of MTCTP5 is equal to that of the rats receiving immunology enhancing activity of TP5. Carbon clearance assay
167 mmol kgÀ1 of aspirin, suggesting that the anti-thrombotic indicates that at 2 mmol kgÀ1 of dose the phagocytosis index of
activity of MTCTP5 is 83.5 fold higher than that of aspirin.
TP5 and MTCTP5 treated mice is the same and significantly
The anti-inflammatory assay indicates that xylene-induced higher than that of NS treated mice, suggesting that the conju-
ear edema of the mice treated with 0.2 mmol kgÀ1 of MTCTP5 is gation of MTC with TP5 does not weaken the in vivo immunology
significantly lower than that of the mice treated with NS, enhancing activity of TP5.
suggesting that the minimal effective dose of MTCTP5 in
The plasma TNF-a and IL-8 levels of 2, 0.2 and 0.02 mmol kgÀ1
treating inflammation is 0.2 mmol kgÀ1. Xylene-induced ear edema of MTCTP5 treated S180 mice indicate that MTCTP5 down
of the mice treated with 0.2 mmol kgÀ1 of MTCTP5 is equal to that regulates the plasma levels of TNF-a and IL-8 of S180 mice in
of the mice treated with 2 mmol kgÀ1 of MTC, suggesting that the a dose-dependent manner. The significantly different plasma
conjugation of TP5 and MTC leads the anti-inflammatory activity levels of TNF-a and IL-8 of 0.2 mmol kgÀ1 of MTCTP5 and NS
of MTC to an increase of 10 fold. Besides, xylene-induced ear treated S180 mice indicate that the minimal effective dose of
edema of the mice treated with of 2 mmol kgÀ1 of MTCTP5 is equal MTCTP5 down regulating plasma levels of TNF-a and IL-8 of
to that of the mice treated with 1100 mmol kgÀ1 of aspirin, i.e. S180 mice is 0.2 mmol kgÀ1. The plasma levels of TNF-a and IL-8
the anti-inflammatory activity of MTCTP5 is 550 fold higher of 2 mmol kgÀ1 of MTCTP5 treated S180 mice and the sham mice
than that of aspirin.
are the same indicating that at 2 mmol kgÀ1 of dose MTCTP5 can
Chronic inflammation has been recently correlated with resume the plasma TNF-a and IL-8 of S180 mice to the normal
thromboembolism,44 which is well documented as the commonly level. Due to the fact that plasma TNF-a and IL-8 are considered
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as the biomarker of tumor, thrombus and inflammation, Notes and references
MTCTP5 dose-dependently down-regulates the plasma levels of
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Even though ESI(+/À)-MS spectra of the aqueous extracts fail to
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should be a nanomedicine capable of releasing MTC and TP5.
Conclusions
The nano-scale conjugate of TP5 and MTC (MTCTP5) is a novel
delivery system for TP5 and MTC. At 37 1C plasma MTCTP5 can
exist for at least 60 min and can release MTC and TP5.
Compared to MTC, MTCTP5 possesses significantly higher
anti-inflammatory, anti-thrombotic and anti-tumor activities.
Compared to TP5, MTCTP5 possesses similar immunology
enhancing activity. Besides, MTCTP5 minimally injures the
liver and kidney. The appropriate nano-structure, the potent
efficacy, the minimal toxic action, and the correlations of the
bioactivities with IL-8, THF-a, IL-2, CD4 and CD8 demonstrate
that MTCTP5 would be a promising nanomedicine.
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Acknowledgements
This work was supported by Beijing Municipal Science & Technol-
ogy Commission (Z141100002114049), PXM2013014226000002,
TJSHG (201310025008), IHLB (KZ201210025021), 863 program
(AA2015020902), Beijing Nova programme XX2013039, National
Natural Science Foundation (81172930, 81273379, 81373264,
81373265 and 81270046) and Beijing Natural Science Founda-
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