Novel amphiphilic PEG-hydroxycamptothecin conjugates as glutathione-responsive prodrug nanocapsules for cancer chemotherapy

Article abstract of DOI:10.1007/s11051-017-3897-4

A series of novel hydroxycamptothecin (HCPT) conjugates (13a–14d), which contained a polyethylene glycol moiety and disulfide bond, were designed and synthesized in five to six steps, with overall yields of 20–39%. The anticancer activities and toxicities of these new conjugates were evaluated using an in vitro MTT assay in K562, HepG2, and HT-29 cell lines and HUVECs. The conjugates displayed enhanced antitumor activity and reduced toxicity in comparison with their parent molecule, HCPT. Among these conjugates, compound 13a exhibited 100-fold better selectivity to the tumor cells than to HUVECs. TEM and DLS experiments demonstrated that 13a formed nanosized micelles with a diameter of approximately 200?nm in aqueous solution and that the conjugate could undergo glutathione-responsive degradation to release HCPT at the tumor site. The improved potency and reduced toxicity of these conjugates may be caused by the enhanced permeation and retention (EPR) effect of nanoparticles.

Full text of DOI:10.1007/s11051-017-3897-4

J Nanopart Res (2017) 19:205
DOI 10.1007/s11051-017-3897-4
RESEARCH PAPER
Novel amphiphilic PEG-hydroxycamptothecin conjugates
as glutathione-responsive prodrug nanocapsules for cancer
chemotherapy
Na Guo & Tiantian Hao & Xiuzhuan Shang & Tianle Zhang &
Huan Liu & Qian Zhang & Jing Wang & Du Jiang & Yao Rong &
Yuou Teng & Peng Yu
Received: 5 December 2016 /Accepted: 19 May 2017
#
Springer Science+Business Media Dordrecht 2017
Abstract A series of novel hydroxycamptothecin
(HCPT) conjugates (13a–14d), which contained a poly-
ethylene glycol moiety and disulfide bond, were designed
and synthesized in five to six steps, with overall yields of
20–39%. The anticancer activities and toxicities of these
new conjugates were evaluated using an in vitro MTT
assay in K562, HepG2, and HT-29 cell lines and
HUVECs. The conjugates displayed enhanced antitumor
activity and reduced toxicity in comparison with their
parent molecule, HCPT. Among these conjugates, com-
pound 13a exhibited 100-fold better selectivity to the
tumor cells than to HUVECs. TEM and DLS experiments
demonstrated that 13a formed nanosized micelles with a
diameter of approximately 200 nm in aqueous solution
and that the conjugate could undergo glutathione-
responsive degradation to release HCPT at the tumor site.
The improved potency and reduced toxicity of these
conjugates may be caused by the enhanced permeation
and retention (EPR) effect of nanoparticles.
.
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Keywords PEG EPR Cytotoxicity Nanoparticles
.
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Self-assembly Nanomedicine Water solubility
Introduction
Nanocarriers with an enhanced permeation and retention
(EPR) effect on the tumor site have been widely used to
improve the properties and potency of anticancer drugs
(Kamaly et al. 2012; Blanco et al. 2011; Maeda and
Matsumura 2011). Nanocarrier systems composed of mi-
celles and vesicles formed from the self-assembly of
amphiphilic polymers have been extensively studied; em-
bedded drugs or conjugated drugs have demonstrated
significant therapeutic advantages over free drugs
(Matsumura 2011; Nalwa 2011; Wang et al. 2013; Xu
et al. 2014). However, the most efficient methods of
achieving free drug release at the tumor site remain a
challenge. Therefore, scientists from both academia and
industry have invested much time and effort into the
design of novel nanocarrier-drug conjugates with the abil-
ity to release the free drug in the tumor microenvironment;
this is generally considered a critical requirement for a
high therapeutic index (Denison and Bae 2012; Gao et al.
2011; Mullen and Banaszak Holl 2011).
Electronic supplementary material The online version of this
article (doi:10.1007/s11051-017-3897-4) contains supplementary
material, which is available to authorized users.
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N. Guo T. Hao X. Shang T. Zhang H. Liu
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Q. Zhang J. Wang D. Jiang Y. Rong Y. Teng
P. Yu (*)
Key Laboratory of Industrial Fermentation Microbiology of
Ministry of Education, Tianjin University of Science and
Technology, Tianjin 300457, China
e-mail: yupeng@tust.edu.cn
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N. Guo T. Hao X. Shang T. Zhang H. Liu
Polyethylene glycol (PEG) has been widely used in
medicinal fields. PEG is a good choice for grafting
anticancer drugs; the advantages include excellent solu-
bility in many solvents, good biocompatibility,
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Q. Zhang J. Wang D. Jiang Y. Rong Y. Teng P. Yu
China International Science and Technology Cooperation Base of
Food Nutrition/Safety and Medicinal Chemistry, Tianjin
University of Science and Technology, Tianjin 300457, China

205 Page 2 of 13
J Nanopart Res (2017) 19:205
nontoxicity, and approval by the USFDA (Veronese and
Pasut 2005; Zhou et al. 2011).
and of analytical or chemical purity. Thin layer chroma-
tography (TLC) was performed using E. Merck silica gel
60 GF254-precoated plates (Darmstadt, Germany) and
There are many methods for the conjugation of the
drug molecule and the nanocarrier. Among them, the
linker containing a disulfide bond which could be cleaved
by glutathione (GSH) (Xu et al. 2014) has been used
frequently in prodrug research. Tumor cells differ from
normal cells in many ways. For example, cancer cells may
exist in reducing conditions as a result of their relatively
higher intracellular GSH levels (2–8 mM), which is much
higher than that of plasma and normal cells (1–2 μM)
(Ryu et al. 2010; Zheng et al. 2005). The introduction of a
disulfide bond into small prodrugs or polymer-drug con-
jugates has been a popular and powerful method to pre-
pare redox-sensitive prodrugs (Cheetham et al. 2013;
Chen et al. 2012; Jackson and Fulton 2012; Li et al.
2011; Wei et al. 2012; Xing et al. 2012).
visualized using a combination of UV254 and UV365
.
Silica gel (particle size 200–400 mesh, Marine Chemical
Group Co., Qingdao, China) was used for flash chroma-
tography. NMR spectra were recorded on Bruker
AVANCE III (400 MHz) instruments. Chemical shifts
(δ) were reported in parts per million downfield from
TMS, the internal standard; J values were given in Hertz.
The molecular weights of the compounds were confirmed
by a Bruker ultrafleXtreme MALDI-TOF/TOF mass
spectrometer (Bruker Daltonics, Bremen, Germany).
Synthesis of compounds 13a–14d
Synthesis of compound 16
To demonstrate our continued research into the discov-
ery of novel anticancer molecules with high potency and
low toxicity, here we would like to report the synthesis and
characterization of a series of novel amphiphilic PEG-
hydroxycamptothecin (HCPT) conjugates with the ability
to form core-shell nanoparticles that displayed passive tu-
mor targeting by the EPR effect. In the tumor microenvi-
ronment, the HCPT molecule fragment is released through
a mechanism induced by GSH, which is present at a much
higher level in tumor cells than in normal cells (Fig. 1).
The amphiphilic conjugates (Fig. 2) characterized
herein possessed significant advantages and displayed
promise as HCPT prodrugs for cancer treatment.
To a stirred solution of 1-benzothiophen-2-ylboronic acid
(2.0 g, 11.2 mmol) in ethanol (20 mL) at 0 °C was added
30% H2O2 dropwise (3.7 mL). The reaction mixture was
stirred at room temperature overnight. When the reaction
was completed monitored by TLC, saturated Na2SO3 was
added to the mixture at 0 °C, then extracted with CH2Cl2.
The organic layers were combined and dried with anhy-
drous sodium sulfate. Removal of solvent under vacuum
1
gave compound 16 (92% yield). H NMR (400 MHz,
DMSO-d6, δ): 7.51–7.25 (m, 4H), 4.21 (s, 2H).
Synthesis of compound 17
Compound 16 (1.5 g, 10.0 mmol) was dissolved in THF
(40 mL), then 40 mL aqueous solution of LiOH·H2O
was added dropwise at 0 °C. The mixture was stirred
overnight under argon at 60 °C until it completed mon-
itored by TLC. Then, the mixture was diluted with ethyl
acetate and water, the pH adjusted to 3 with 2 N HCl,
and the organic layer collected, dried with anhydrous
Experimental
Materials
HPLC-grade acetonitrile was purchased from Tedia
(USA). The other reagents were commercially available
Fig. 1 Release mechanism of
HCPT induced by GSH

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Fig. 2 Structures of PEG-10 hydroxycamptothecin conjugates 13a–14d
sodium sulfate, concentrated under vacuum, and puri-
fied by column chromatography (petroleum ether/ethyl
acetate, 10/1, v/v) to give compound 17 (1.4 g, 80%
then di-tert-butyl dicarbonate (13.5 g, 62.4 mmol) was
added. After overnight stirring at room temperature,
0.1 N HCl aqueous solution was added to dilute the
mixture, then extracted with ethyl acetate, and the or-
ganic layer was collected, dried with anhydrous sodium
sulfate, concentrated under vacuum, and recrystallized
with n-hexane to give compound 19 (6.0 g, 65% yield).
1H NMR (400 MHz, DMSO-d6, δ): 12.83 (s, 2H), 7.21
(d, J = 8.4 Hz, 2H), 4.20–4.14 (m, 2H), 3.12–3.08 (m,
2H), 2.91–2.85 (m, 2H), 1.38 (s, 18H).
1
yield). H NMR (400 MHz, DMSO-d6, δ): 12.36 (s,
1H), 7.54–7.10 (m, 4H), 3.37 (s, 2H).
Synthesis of compound 19
To a solution of L-cystine (5.0 g, 20.8 mmol) in H2O
(50 mL) was added Et3N (8.7 mL, 62.4 mmol) at 0 °C,

205 Page 4 of 13
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Synthesis of compound 20
Compound 22b:1H NMR (400 MHz, DMSO-d6, δ):
8.48 (d, J = 4.0 Hz, 1H), 7.83–7.74 (m, 2H), 7.55 (d,
J = 8.0 Hz, 1H), 7.27 (t, J = 4.0 Hz, 1H), 4.29–4.10 (m,
3H), 3.70–3.42 (m, MPEG), 3.25 (s, 3H), 3.21–3.09 (m,
2H),1.40 (s, 9H).
Compound 19 (1.0 g, 2.3 mmol) was dissolved in THF
(18 mL), then Ph3P (0.9 g, 3.4 mmol) and H2O (2 mL)
were added. The mixture was stirred overnight at 50 °C,
after which saturated NaHCO3 was added at 0 °C to
adjust pH to 8–9, the solution was extracted with ethyl
acetate, the water layer was collected, pH was adjusted
to 2–3 with 1 N HCl at 0 °C, extracted with ethyl acetate,
and the organic layer was dried with anhydrous sodium
sulfate. Removal of solvents under vacuum gives com-
Compound 22c:1H NMR (400 MHz, DMSO-d6, δ):
8.46 (d, J = 4.4 Hz, 1H), 7.81–7.72 (m, 2H), 7.54 (d,
J = 8.0 Hz, 1H), 7.25 (t, J = 5.8 Hz, 1H), 4.25–4.08 (m,
3H), 3.68–3.50 (m, MPEG), 3.23 (s, 3H), 3.19–3.07 (m,
2H), 1.38 (s, 9H).
Compound 22d:1H NMR (400 MHz, DMSO-d6, δ):
8.48 (d, J = 4.0 Hz, 2H), 7.83–7.75 (m, 4H), 7.57 (d,
J = 8.0 Hz, 2H), 7.27 (t, J = 5.8 Hz, 2H), 4.29–4.11 (m,
6H), 3.57–3.48 (m, PEG), 3.21–3.09 (m, 4H), 1.40 (s,
18H).
1
pound 20 (1.0 g, 99% yield). H NMR (400 MHz,
DMSO-d6, δ): 12.84 (s, 1H), 7.04 (d, J = 8.4 Hz, 1H),
4.02–4.06 (m, 1H), 2.81–2.86 (m, 1H), 2.67–2.72 (m,
1H), 1.39(s, 9H).
Synthesis of compound 21
Synthesis of compounds 23a–23d
Compound 20 (1.3 g, 6.0 mmol) was dissolved in
CH2Cl2 (100 mL), then the solution was slowly dropped
to the 2, 2′-dipyridyl disulfide (4.0 g, 18 mmol) solution
in CH2Cl2 (15 mL) at −10 °C under argon. After the
reaction completed, washed with water, the organic
layer was dried with anhydrous sodium sulfate, concen-
trated under vacuum, and purified by column chroma-
tography (petroleum ether/ethyl acetate, 3/1, v/v) to give
compound 21 (1.3 g, 67% yield). 1H NMR (400 MHz,
DMSO-d6, δ): 12.86 (s, 1H), 8.47 (d, J = 4.0 Hz, 1H),
7.82–7.75 (m, 2H), 7.40 (d, J = 8.0 Hz, 1H), 7.26 (t,
J = 5.6 Hz, 1H), 4.19–4.15 (m, 1H),3.21–3.07 (m,
2H),1.40 (s, 9H).
2-Mercaptophenylacetic acid (17) (94.5 mg, 0.6 mmol)
was dissolved in CH2Cl2 (20 mL), and 22a–22c
(0.4 mmol) and 22d (0.2 mmol) were respectively dis-
solved in CH2Cl2 (5 mL); the 2-mercaptophenylacetic
acid solution was dropped separately to the 22a–22d
solution at −10 °C under argon. Removal of CH2Cl2
under vacuum and recrystallization with ether give com-
pounds 23a–23d (89–95% yield).
Compound 23a:1H NMR (400 MHz, DMSO-d6,
δ):12.46 (s, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.46–7.31
(m, 4H), 4.32–4.01 (m, 3H), 3.87 (s, 2H), 3.51–3.50 (m,
MPEG), 3.25 (s, 3H), 3.06–2.94 (m, 2H), 1.40 (s, 9H).
Compound 23b:1H NMR (400 MHz, DMSO-d6,
δ):12.44 (s, 1H),7.71 (d, J = 7.2 Hz, 1H), 7.43–7.30
(m, 4H), 4.31–4.11 (m, 3H), 3.78 (s, 2H), 3.70–3.41 (m,
MPEG), 3.24 (s, 3H), 2.05–2.93 (m, 2H), 1.38 (s, 9H).
Compound 23c:1H NMR (400 MHz, DMSO-d6, δ):
12.45 (s, 1H), 7.70 (d, J = 7.2 Hz, 1H), 7.43–7.30 (m,
4H), 4.28–4.18 (m, 3H), 3.77 (s, 2H), 3.68–3.50 (m,
MPEG), 3.23 (s, 3H), 3.03–2.92 (m, 2H), 1.38 (s, 9H).
Compound 23d:1H NMR (400 MHz, DMSO-d6, δ):
12.56 (s, 2H), 7.77 (d, J = 7.2 Hz, 2H), 7.51–7.35 (m,
8H), 4.37–4.12 (m, 6H), 3.84 (s, 4H), 3.65–3.53 (m,
PEG), 3.09–2.99 (m, 4H), 1.40 (s, 18H).
Synthesis of compounds 22a-22d
Compound 21 (1.0 g, 3.0 mmol) was dissolved in dry
CH2Cl2 (40 mL), then different molecular weight
mPEG (1.4 mmol) or PEG (0.7 mmol) was added,
respectively; DMAP (0.1 g, 0.8 mmol) and CH2Cl2
solution (20 mL) of EDCI (0.8 g, 4.1 mmol) were added.
The mixture were stirred at 40 °C for 2 h, diluted with
CH2Cl2, washed with 0.1 N HCl, saturated NaHCO3,
brine, dried with anhydrous sodium sulfate, concentrat-
ed under vacuum, recrystallized with ether to give com-
pounds 22a–22d (85–96% yield).
Synthesis of compounds 13a–13d
Compound 22a:1H NMR (400 MHz, DMSO-d6, δ):
8.48 (d, J = 4.4 Hz, 1H), 7.84–7.75 (m, 2H), 7.57 (d,
J = 8.0 Hz,1H), 7.27 (t, J = 5.8 Hz, 1H), 4.29–4.10 (m,
3H,), 3.57–3.42 (m, MPEG), 3.24 (s, 3H), 3.20–3.09
(m, 2H), 1.40 (s, 9H).
10-Hydroxycamptothecin (0.4 g, 1.0 mmol) was dis-
solved in dry DMF (10 mL), 23a–23c (0.7 mmol) and
23d (0.4 mmol) were added, respectively, then pyridine
(1.7 mL), DMAP (85 mg, 0.7 mmol), and CH2Cl2

J Nanopart Res (2017) 19:205
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solution (10 mL) of EDCI (0.5 g, 2.8 mmol) were added
sequentially. The mixture was stirred at room tempera-
ture for 3 h and diluted with CH2Cl2; brine was washed
with 0.1 N HCl, dried with anhydrous sodium sulfate,
concentrated under vacuum, and purified by column
chromatography (CH2Cl2/MeOH, 25/1, v/v) and recrys-
tallization with ether to give compounds 13a–13d (75–
81% yield).
dropped to the 2,2′-dipyridyldisulfide (5.8 g, 26.4 mmol)
solution in CH2Cl2 (10 mL) at −10 °C under argon.
After that, the solvents were removed under vacuum,
dry acetonitrile (120 mL) was added, then Et3N
(1.8 mL) and succinic anhydride (1.1 g, 10.6 mmol)
were added at 0 °C and stirred at room temperature for
2 h, acetic acid (0.4 mL) was added, solvents were
removed under vacuum, and the crude residue was
purified by column chromatography (CH2Cl2/MeOH,
60/1, v/v) and recrystallized with ether to give com-
MPEG350-Cys(Boc)-HCPT (13a): 1H NMR
(400 MHz, DMSO-d6, δ): 8.72(s, 1H), 8.22(d,
J = 9.2 Hz, 1H), 7.94 (s, 1H), 7.80 (d, J = 8.4 Hz,
1H), 7.70–7.67 (dd, J = 9.2, 2.0 Hz, 1H), 7.52–7.47
(m, 2H), 7.43–7.41 (m, 2H), 7.36 (s, 1H), 6.53 (s, 1H),
5.44 (s, 2H), 5.30 (s, 2H), 4.41–4.34 (m, 1H), 4.30 (s,
2H), 4.23–4.11 (m, 2H), 3.58–3.42 (m, MPEG), 3.23 (s,
3H), 3.10–3.03 (m, 2H), 1.92–1.85 (m, 2H), 1.41 (s,
9H), 0.90 (t, J = 7.2 Hz, 3H). Purity: 95%.
1
pound 25 (1.3 g, 50% yield). H NMR (400 MHz,
DMSO-d6, δ): 12.06 (s, 1H), 8.46 (d, J = 4.4 Hz, 1H),
8.08 (t, J = 5.2 Hz,1H), 7.85–7.81 (m, 1H), 7.71 (d,
J = 8.0 Hz, 1H), 7.25 (t, J = 5.6 Hz, 1H), 3.33 (t,
J = 6.4 Hz, 2H), 2.89 (t, J = 6.8 Hz, 2H), 2.42 (t,
J = 6.6 Hz, 2H), 2.32 (t, J = 6.8 Hz, 2H).
MPEG2000-Cys(Boc)-HCPT (13b): 1H NMR
(400 MHz, DMSO-d6, δ): 8.69 (s, 1H), 8.21 (d,
J = 9.2 Hz, 1H), 7.93 (s, 1H), 7.78 (d, J = 6.8 Hz,
1H), 7.67 (d, J = 6.8 Hz, 1H), 7.51–7.47 (m, 2H),
7.40–7.39 (m, 2H), 7.34 (s, 1H), 6.53 (s, 1H), 5.42 (s,
2H), 5.29 (s, 2H), 4.37–4.33 (m, 1H), 4.28 (s, 2H),
4.20–4.09 (m, 2H), 3.67–3.50 (m, MPEG), 3.23 (s,
3H), 3.11–2.98 (m, 2H), 1.91–1.81 (m, 2H), 1.39 (s,
9H), 0.88 (t, J = 6.8 Hz, 3H). Purity: 96%.
Synthesis of compounds 26a–26d
Compound 25 (0.3 g, 1.1 mmol) was dissolved in dry
CH2Cl2 (15 mL), then different molecular weight
mPEG (0.5 mmol) and PEG (0.2 mmol) were added
respectively; DMAP (33.6 mg, 0.3 mmol) and CH2Cl2
solution (6 mL) of EDCI (0.3 g, 1.5 mmol) were added.
The mixture was stirred at 40 °C for 2 h, diluted with
CH2Cl2, and washed with 0.1 N HCl; brine was saturat-
ed with NaHCO3, dried with anhydrous sodium sulfate,
concentrated under vacuum, and recrystallized with
ether to give compounds 26a–26d (89–95% yield).
Compound 26a: 1H NMR (400 MHz, DMSO-d6, δ):
8.46 (d, J = 4.4 Hz, 1H), 8.11 (t, J = 5.2 Hz,1H), 7.83 (t,
J = 7.6 Hz,1H), 7.77 (d, J = 7.6 Hz, 1H), 7.25 (t,
J = 6.0 Hz,1H), 4.10 (t, J = 4.6 Hz, 2H), 3.69–3.41
(m, MPEG), 3.33 (m, 2H, overlapped with water peak),
3.24 (s, 3H), 2.88 (t, J = 6.8 Hz,2H), 2.35 (t,
J = 6.8 Hz,2H).
MPEG5000-Cys(Boc)-HCPT (13c): 1H NMR
(400 MHz, DMSO-d6, δ): 8.72 (s, 1H), 8.24 (d,
J = 9.2 Hz, 1H), 7.95 (s, 1H), 7.80 (d, J = 7.6 Hz,
1H), 7.70 (d, J = 8.8 Hz, 1H), 7.54–7.45 (m, 2H),
7.43–7.42 (m, 2H), 7.37 (s, 1H), 6.55 (s, 1H), 5.44 (s,
2H), 5.31 (s, 2H), 4.39–4.37 (m, 1H), 4.30 (s, 2H),
4.20–4.14 (m, 2H), 3.69–3.52 (m, MPEG), 3.25 (s,
3H), 3.13–3.00 (m, 2H), 1.94–1.83 (m, 2H), 1.42 (s,
9H), 0.90 (t, J = 7.2 Hz, 3H). Purity: 97%.
HCPT-Cys(Boc)-PEG300-Cys(Boc)-HCPT (13d): 1H
NMR (400 MHz, DMSO-d6, δ): 8.68 (s, 2H), 8.20 (d,
J = 8.4 Hz, 2H), 7.91 (s, 2H), 7.78 (d, J = 5.6 Hz, 2H),
7.66 (d, J = 7.6 Hz, 2H), 7.51–7.49 (m, 4H), 7.40 (s,
4H), 7.33 (s, 2H), 6.55 (s, 2H), 5.43 (s, 4H), 5.27 (s,
4H), 4.37 (s, 2H), 4.28 (s, 4H), 4.17–4.12 (m, 2H),
3.56–3.44 (m, PEG), 3.10–2.98 (m, 4H), 1.89–1.85
(m, 4H), 1.40 (s, 18H), 0.88 (t, J = 7.0 Hz, 6H). Purity:
97%.
Compound 26b: 1H NMR (400 MHz, DMSO-d6, δ):
8.48 (d, J = 4.4 Hz, 1H), 8.12 (t, J = 5.2 Hz, 1H), 7.85 (t,
J = 7.6 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.26 (t,
J = 6.0 Hz, 1H), 4.11 (t, J = 4.8 Hz, 2H), 3.70–3.43
(m, MPEG), 3.33 (m, 2H, overlapped with water peak),
3.25 (s, 3H), 2.90 (t, J = 6.6 Hz, 2H), 2.36 (t, J = 6.8 Hz,
2H).
Compound 26c: 1H NMR (400 MHz, DMSO-d6, δ):
8.47 (d, J = 4.4 Hz, 1H), 8.12 (t, J = 5.0 Hz,1H), 7.85 (t,
J = 7.6 Hz,1H), 7.78 (d, J = 8.0 Hz, 1H), 7.26 (t,
J = 6.0 Hz,1H), 4.11 (t, J = 4.6 Hz, 2H), 3.71–3.44
(m, MPEG), 3.33 (m, 2H, overlapped with water peak),
Synthesis of compound 25
Cysteamine hydrochloride (1.0 g, 8.8 mmol) was dis-
solved in MeOH (100 mL), then the solution was slowly

205 Page 6 of 13
J Nanopart Res (2017) 19:205
3.25 (s, 3H), 2.90 (t, J = 6.8 Hz,2H), 2.36 (t, J = 6.8 Hz,
2H).
(0.6 mL), DMAP (31.5 mg, 0.3 mmol), and CH2Cl2
solution (7.5 mL) of EDCI (0.2 g, 1.0 mmol) were added
sequentially. The mixture was stirred at room tempera-
ture for 3 h, diluted with CH2Cl2, and washed with 0.1 N
HCl; brine was dried with anhydrous sodium sulfate,
concentrated under vacuum, and purified by column
chromatography (CH2Cl2/MeOH, 20/1, v/v) and recrys-
tallization with ether to give compounds 14a–14d (61–
91% yield).
Compound 26d: 1H NMR (400 MHz, DMSO-d6, δ):
8.45 (d, J = 4.4 Hz, 2H), 8.10 (t, J = 5.0 Hz,2H), 7.82 (t,
J = 7.6 Hz,2H), 7.76 (d, J = 8.0 Hz, 2H), 7.24 (t,
J = 6.0 Hz,2H), 4.09 (t, J = 4.2 Hz, 4H), 3.57–3.50
(m, PEG), 3.33 (m, 4H, overlapped with water peak),
2.87 (t, J = 6.8 Hz,4H), 2.35 (t, J = 6.8 Hz, 4H).
Synthesis of compounds 27a–27d
MPEG350-cysteamine-HCPT (14a): 1H NMR
(400 MHz, DMSO-d6, δ): 8.71 (s, 1H), 8.23 (d,
J = 9.2 Hz, 1H), 8.11 (t, J = 5.2 Hz, 1H), 7.94 (s, 1H),
7.81 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 9.2 Hz, 1H), 7.50
(d, J = 7.2 Hz, 1H), 7.45–7.35 (m, 3H), 6.53 (s, 1H),
5.43 (s, 2H), 5.30 (s, 2H), 4.28 (s, 2H), 4.09 (t,
J = 4.4 Hz, 2H), 3.58–3.41 (m, MPEG), 3.33 (m, 2H,
overlapped with water peak), 3.23 (s, 3H), 2.83 (t,
J = 6.8 Hz, 2H), 2.36 (t, J = 6.8 Hz, 2H),1.92–1.82
(m, 2H), 0.88 (t, J = 7.2 Hz, 3H). Purity: 93%.
2-Mercaptophenylacetic acid (72.3 mg, 0.4 mmol) was
dissolved in CH2Cl2 (10 mL), and 26a–26c (0.3 mmol)
and 26d (0.2 mmol) were respectively dissolved in
CH2Cl2 (5 mL); the 2-mercaptophenylacetic acid solu-
tion was dropped separately to the 26a–26d solution at
−10 °C under argon. Removal of CH2Cl2 under vacuum
and recrystallization with ether give compounds 27a–
27d (73%–91% yield).
Compound 27a: 1H NMR (400 MHz, DMSO-d6, δ):
12.50 (s, 1H), 8.13 (t, J = 5.2 Hz, 1H), 7.80 (d,
J = 8.0 Hz, 1H), 7.43–7.34 (m, 3H), 4.16 (t,
J = 4.4 Hz, 2H), 3.83 (s, 2H), 3.66–3.48 (m, MPEG),
3.33 (m, 2H, overlapped with water peak), 3.30 (s, 3H),
2.82 (t, J = 6.8 Hz, 2H), 2.42 (t, J = 6.6 Hz, 2H).
Compound 27b: 1H NMR (400 MHz, DMSO-d6, δ):
12.43 (s, 1H), 8.07 (t, J = 5.4 Hz, 1H), 7.73 (d,
J = 8.0 Hz, 1H), 7.36–7.27 (m, 3H), 4.10 (t,
J = 4.6 Hz, 2H), 3.76 (s, 2H), 3.69–3.41 (m, MPEG),
3.33 (m, 2H, overlapped with water peak), 3.24 (s, 3H),
2.76 (t, J = 6.6 Hz, 2H), 2.34 (t, J = 6.8 Hz, 2H).
Compound 27c: 1H NMR (400 MHz, DMSO-d6, δ):
12.41 (s, 1H), 8.08 (t, J = 5.4 Hz, 1H), 7.75 (d,
J = 7.6 Hz,1H), 7.38–7.29 (m, 3H), 4.11 (t,
J = 4.4 Hz, 2H), 3.78 (s, 2H), 3.74–3.52 (m, MPEG),
3.33 (m, 2H, overlapped with water peak), 3.25 (s, 3H),
2.78 (t, J = 6.8 Hz, 2H), 2.35 (t, J = 6.6 Hz, 2H).
Compound 27d: 1H NMR (400 MHz, DMSO-d6, δ):
12.54 (s, 2H), 8.06 (t, J = 5.4 Hz, 2H), 7.72 (d,
J = 7.6 Hz, 2H), 7.34–7.27 (m, 6H), 4.09 (s, 4H), 3.76
(s, 4H), 3.57–3.50 (m, PEG), 3.33 (m, 4H, overlapped
with water peak), 2.75 (t, J = 6.6 Hz, 4H), 2.33 (t,
J = 6.8 Hz, 4H).
MPEG2000-cysteamine-HCPT (14b): 1H NMR
(400 MHz, DMSO-d6, δ): 8.70 (s, 1H), 8.21 (d,
J = 8.8 Hz, 1H), 8.09 (s, 1H), 7.93 (s, 1H), 7.80 (d,
J = 7.6 Hz, 1H), 7.66 (d, J = 9.2 Hz, 1H), 7.48 (d,
J = 6.8 Hz, 1H), 7.44–7.34 (m, 3H), 6.52 (s, 1H), 5.42
(s, 2H), 5.29 (s, 2H), 4.27 (s, 2H), 4.09 (t, J = 4.4 Hz,
2H), 3.66–3.41 (m, MPEG), 3.33 (m, 2H, overlapped
with water peak), 3.22 (s, 3H), 2.81 (t, J = 6.4 Hz, 2H),
2.34 (t, J = 6.6 Hz,2H), 1.88–1.84 (m, 2H), 0.87 (t,
J = 6.8 Hz, 3H). Purity: 94%.
MPEG5000-cysteamine-HCPT (14c): 1H NMR
(400 MHz, DMSO-d6, δ): 8.72 (s, 1H), 8.23 (d,
J = 9.2 Hz, 1H), 8.10 (t, J = 5.4 Hz, 1H), 7.94 (s, 1H),
7.81 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.50
(d, J = 7.2, 1H), 7.45–7.35 (m, 3H), 6.53 (s, 1H), 5.43 (s,
2H), 5.30 (s, 2H), 4.28 (s, 2H), 4.10 (t, J = 4.4 Hz, 2H),
3.69–3.43 (m, MPEG), 3.33 (m, 2H, overlapped with
water peak), 3.24 (s, 3H), 2.83 (t, J = 6.6 Hz, 2H), 2.36
(t, J = 6.8 Hz, 2H), 1.91–1.82 (m, 2H), 0.89 (t,
J = 7.2 Hz, 3H). Purity: 96%.
HCPT-cysteamine-PEG300-cysteamine-HCPT (14d):
1H NMR (400 MHz, DMSO-d6, δ): 8.69 (s, 2H), 8.21
(d, J = 9.2 Hz, 2H), 8.11 (t, J = 5.0 Hz, 2H), 7.92 (s, 2H),
7.80 (d, J = 7.6 Hz, 2H), 7.66 (d, J = 8.0, 2H), 7.49 (d,
J = 6.8 Hz, 2H), 7.44–7.34 (m, 6H), 6.53 (s, 2H), 5.43
(s, 4H), 5.28 (s, 4H), 4.27 (s, 4H), 4.09 (t, J = 4.4 Hz,
4H), 3.56–3.47 (m, PEG), 3.33 (m, 4H, overlapped with
water peak), 2.82 (t, J = 6.6 Hz, 4H), 2.35 (t, J = 6.6 Hz,
4H), 1.90–1.83 (m, 4H), 0.88 (t, J = 7.2 Hz, 6H). Purity:
93%.
Synthesis of compounds 14a–14d
10-Hydroxycamptothecin (0.1 g, 0.4 mmol) was dis-
solved in dry DMF (5 mL), 27a–27c (0.3 mmol) and
27d (0.1 mmol) were added respectively, then pyridine

J Nanopart Res (2017) 19:205
Page 7 of 13 205
Preparation of 13a nanoparticles
and samples for TEM measurements were made by
casting one drop of the sample’s water solution on
carbon-coated copper grids. The size distribution of
13a nanoparticles was determined by dynamic light
scattering (DLS) with a BI-APD instrument
(Brookhaven, USA) with angle detection at 90°. The
molecular weight was measured by MALDI-TOF mass
spectrometry (Bruker Daltonics, Inc., USA).
Compound 13a (10.0 mg) was dissolved in THF
(2 mL). The solution was dropped into 10 mL water
with stirring. The THF was removed under vacuum, and
the solution was lyophilized. The required vesicle solu-
tion was prepared by adding the lyophilized powder to
PBS at the desired concentration.
Cell viability assay
Results and discussion
The cytotoxicity of the target compounds was evaluated
by the MTT assay. Cells were seeded at a density of
5 × 104 cells/mL in 96-well microplates (100 μL/well).
After 2 h for K562 cells and 24 h for HepG2 and HT-29
cells, media containing tested compounds were added in
triplicate. After a 48-h incubation, the media were re-
placed with PBS medium containing 0.5 mg/mL MTT
and the cells were incubated for another 4 h. Next, the
medium was removed and 100 μL DMSO was added to
each well to dissolve the formazan. The absorbance at
570/630 nm was measured for K562 cells and the ab-
sorbance at 490/630 nm was measured for HepG2 and
HT-29 cells using a Thermo (Waltham, MA, USA)
microplate reader. The untreated controls were consid-
ered as having a cell viability value of 100%. The half-
maximal inhibiting concentration (IC50) values were
obtained by nonlinear regression using GraphPad Prism
5.0. IC50 was measured three times for each compound.
The synthesis routes of compounds 13a–13d are shown
in Scheme 1. First, 2-(2-mercaptophenyl) acetic acid
(17) was prepared from compound 15 by oxidation
and hydrolysis. The linker intermediate 21 was synthe-
sized from cysteine (18), which was sequentially
protected by di-tert-butyl dicarbonate, reduced by
PPh3, and reacted with 2,2′-dithiodipyridine. Com-
pound 21 was conjugated with the PEG molecule via
an ester bond to produce 22a–22d, and compounds
23a–23d, which contained the final disulfide bond, were
prepared via condensation between 22a–22d and 17.
Finally, the target conjugates 13a–13d were prepared
by coupling 23a–23d with HCPT.
The synthesis of compounds 14a–14d (Scheme 2)
was achieved using a strategy similar to the synthesis of
13a–13d, but used 25 as the linker instead of 21.
The in vitro cytotoxicities of 13a–14d toward the
cancer cells K562, HepG2, and HT-29 and the normal
human cells, HUVECs, were evaluated and compared
with the free drug HCPT by using the MTT assays. As
shown in Table 1, most conjugates resulted in un-
changed or improved potency against the three tumor
cell lines and exhibited much lower toxicity against
normal cells (HUVECs) in comparison with their parent
molecule, HCPT. This selectivity to tumor cells may
result from the unique GSH-induced release mechanism
as the GSH level is much higher in tumor cells than in
normal cells. Surprisingly, 13a exhibited greater inhib-
itory activity against all three tested tumor cells, but was
also significantly more selective for tumor cells than
HCPT and other conjugates. The toxicity of 13a against
HUVECs was at least 100 times lower than that against
tumor cells. Therefore, 13a was chosen for further
investigation.
HCPT release test
The HCPT release of all the compounds in PBS was
examined by HPLC using a C18 analytical column. The
mobile phase was composed of water and acetonitrile at
a grade ratio from 5–95% acetonitrile. The amount of
the compound and its metabolite HCPTwere flowed at a
rate of 1.0 mL min−1 and detected by a UV wavelength
of 360 nm. The HCPT release test in PBS was as
follows: each compound was dissolved in PBS
(pH 7.4 and pH 5.4), incubated at 37 °C, and analyzed
at different times.
Characterization
The 1H NMR spectra of the samples were recorded with
a Bruker AVANCE 400 MHz spectrometer (Germany).
The morphology of the samples was recorded with a
JEM-2100 transmission electron microscope (Japan),
As was consistent with recent reports (Li et al. 2011;
Wang et al. 2013; Xu et al. 2014), the amphiphilic
structures self-assembled to form nanoparticles in

205 Page 8 of 13
J Nanopart Res (2017) 19:205
Scheme 1 Synthetic route of conjugates 13a–13d

J Nanopart Res (2017) 19:205
Page 9 of 13 205
Scheme 2 Synthetic route of
conjugates 14a–14d

205 Page 10 of 13
J Nanopart Res (2017) 19:205
Table 1 Antiproliferative activi-
ty of the conjugates in vitro
Compd.
IC50 (μM)
HUVEC
K562
HepG2
HT-29
HCPT
13a
13b
13c
0.63 0.24
11.80 2.32
0.31 0.15
0.28 0.21
0.42 0.28
0.40 0.19
0.55 0.23
0.46 0.18
0.30 0.15
0.15 0.05
0.09 0.04
0.04 0.00
0.03 0.01
0.03 0.02
0.04 0.02
0.13 0.02
0.12 0.03
0.05 0.02
0.18 0.01
0.10 0.01
0.05 0.01
0.05 0.01
0.04 0.01
0.05 0.03
0.18 0.03
0.26 0.14
0.11 0.08
0.51 0.33
0.29 0.09
0.15 0.05
0.19 0.00
0.20 0.10
0.14 0.03
0.31 0.12
0.37 0.09
0.14 0.04
13d
14a
14b
14c
14d
aqueous solution. The 1H-NMR spectra, DLS, and TEM
results of 13a indicated the formation of nanoparticles.
As shown in Fig. 3, the protons of the HCPT, linker, and
PEG segments of 13a were all clearly detectable in
deuterated dimethyl sulfoxide (DMSO-d6). In contrast,
only signals associated with PEG were detected in
Fig. 3 1H NMR spectra of conjugate 13a in DMSO-d6 (top) and D2O (bottom)

J Nanopart Res (2017) 19:205
Page 11 of 13 205
Fig. 4 TEM image of conjugate
13a
deuterated water (D2O), while all signals from the hy-
drophobic HCPT and the linkage disappeared, which
suggested that the conjugates formed a core-shell struc-
ture in water (Lu et al. 2015; Tai et al. 2014). The TEM
images (Fig. 4) confirmed the formation of nanoparti-
cles of 13a, which showed that they existed in aqueous
solution as a spherical shape. The dynamic light scatter-
ing (DLS) (Fig. 5) illustrated that this conjugate could be
distributed in water as nanoparticles with a diameter of
approximately 200 nm. Such nanoparticles could pro-
long residence time in the bloodstream, which would
lead to more effective targeting of diseased tissues by
the EPR effect.
To evaluate the feasibility of the HCPT conjugates as
an anticancer drug delivery system, the HCPT release
behavior of conjugate 13a was studied in vitro under
simulated physiological conditions (pH 7.4 in PBS
buffer). As expected, the disulfide linker exhibited a
unique GSH-induced release of HCPT from the nano-
particles. As shown in Fig. 6, the rapid release of HCPT
was observed in the presence of GSH (10 mM), as
expected.
Fig. 5 Size distribution of
conjugate 13a by DLS

205 Page 12 of 13
J Nanopart Res (2017) 19:205
Fig. 6 In vitro release of HCPT
from the 13a nanoparticles in
PBS with or without GSH
treatment
Chen W, Shi Y, Feng H, Du M, Zhang JZ, Hu J, Yang D (2012)
Preparation of copolymer paclitaxel covalently linked via a
disulfide bond and its application on controlled drug delivery.
J Phys Chem B 116:9231–9237. doi:10.1021/jp303260f
Denison TA, Bae YH (2012) Tumor heterogeneity and its impli-
cation for drug delivery. J Control Release 164:187–191.
doi:10.1016/j.jconrel.2012.04.014
Gao Y et al (2011) Controlled intracellular release of doxorubicin
in multidrug-resistant cancer cells by tuning the shell-pore
sizes of mesoporous silica nanoparticles. ACS Nano 5:9788–
9798. doi:10.1021/nn2033105
Conclusions
In summary, a series of unique disulfide-containing
HCPT molecules were successfully synthesized with
the objective to increase the water solubility of the drug
and to improve targeting to the tumor site by EPR effect.
Their amphiphilic structure formed nanoparticles in
aqueous solution, which were degraded in response to
the increased levels of GSH of the environment and
rapidly released HCPT under tumor-relevant GSH
concentrations.
Jackson AW, Fulton DA (2012) Triggering polymeric nanoparticle
disassembly through the simultaneous. Application of Two
Different Stimuli Macromolecules 45:2699–2708
Kamaly N, Xiao Z, Valencia PM, Radovic-Moreno AF, Farokhzad
OC (2012) Targeted polymeric therapeutic nanoparticles:
design, development and clinical translation. Chem Soc
Rev 41:2971–3010. doi:10.1039/c2cs15344k
Li XQ et al (2011) Self-assembling nanomicelles of a novel
camptothecin prodrug engineered with a redox-responsive
release mechanism. Chem Commun (Camb) 47:8647–8649.
doi:10.1039/c1cc12495a
Acknowledgements The authors are thankful to the Research
Centre of Modern Analytical Technology, Tianjin University of
Science and Technology, for NMR measurements and MALDI-
TOF analysis.
Compliance with ethical standards
Lu J et al (2015) The self-assembling camptothecin-tocopherol
prodrug: an effective approach for formulating camptothecin.
B i o m a t e r i a l s 6 2 : 1 7 6 – 1 8 7 . d o i : 1 0 . 1 0 1 6 / j .
biomaterials.2015.05.046
Maeda H, Matsumura Y (2011) EPR effect based drug design and
clinical outlook for enhanced cancer chemotherapy. Adv Drug
Deliv Rev 63:129–130. doi:10.1016/j.addr.2010.05.001
Matsumura Y (2011) Preclinical and clinical studies of NK012, an
SN-38-incorporating polymeric micelles, which is designed
based on EPR effect. Adv Drug Deliv Rev 63:184–192.
doi:10.1016/j.addr.2010.05.008
Conflict of interest The authors declare that they have no con-
flict of interest.
Funding This work was supported by the National Natural
Science Foundation of China (No. 81302649) and Natural Science
Foundation of Tianjin City (14JCQNJC13200 and
16JCTPJC46000).
Mullen DG, Banaszak Holl MM (2011) Heterogeneous ligand-
nanoparticle distributions: a major obstacle to scientific un-
derstanding and commercial translation. Acc Chem Res 44:
1135–1145. doi:10.1021/ar1001389
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