Novel cathepsin B-sensitive paclitaxel conjugate: Higher water solubility, better efficacy and lower toxicity

Article abstract of DOI:10.1016/j.jconrel.2012.02.020

In order to realize the targeted delivery of paclitaxel (PTX) to tumor through an environment-sensitive mechanism, increase its solubility in water and reformulate without toxic excipients, a novel PTX conjugate, PEG-VC-PABC-PTX was designed and synthesiz

Full text of DOI:10.1016/j.jconrel.2012.02.020

Journal of Controlled Release 160 (2012) 618–629
Contents lists available at SciVerse ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Novel cathepsin B-sensitive paclitaxel conjugate: Higher water solubility, better
efficacy and lower toxicity
1
1
Liang Liang , Song-Wen Lin , Wenbing Dai, Jing-Kai Lu, Ting-Yuan Yang, Yu Xiang, Yang Zhang,
Run-Tao Li ⁎, Qiang Zhang ⁎⁎
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to realize the targeted delivery of paclitaxel (PTX) to tumor through an environment-sensitive
mechanism, increase its solubility in water and reformulate without toxic excipients, a novel PTX conjugate,
PEG–VC–PABC–PTX was designed and synthesized in this study, using p-aminobenzylcarbonyl (PABC), a
Received 5 September 2011
Accepted 26 February 2012
Available online 3 March 2012
B
spacer, and valine–citrulline (VC), a substrate of cathepsin B (C ), to link polyethylene glycol (PEG) and
PTX. Pegylated PTX (PEG-PTX) which was synthesized and Taxol formulation were prepared as controls.
The conjugates were purified and characterized by melting points, 1H-NMR, ESI-MS or MALDI-TOF-MS. The
two conjugates were similar in particle size, water solubility and their effects on MCF-7 cell line in vitro,
and both of them induced no obvious toxicity in vivo. The release of PTX from PEG-PTX was faster due to
Keywords:
Paclitaxel
Pegylated
Cathepsin B-sensitive
Conjugate
B
its ester bond, while PEG–VC–PABC–PTX was proved to be C -sensitive in terms of PTX release and its effect
Anti-tumor effect
on cell cycle. Additionally, PEG–VC–PABC–PTX exhibited significant effects of antitumor, anti-angiogenesis
and anti-proliferation in vivo, while the control conjugate was almost inefficacious at the same in vivo test.
On the other hand, PTX conjugates demonstrated a thousand-time or more improvement in water solubility
compared to PTX, suggesting a very easy way in the preparation and use of its injection. Without involvement
of Cremophore EL and ethanol, the PTX conjugate will guarantee less adverse effects as frequently reported
for Taxol formulation. Taxol formulation had a higher cytoxicity in vitro than PEG–VC–PABC–PTX likely
B
because of toxic additives. Importantly, the C -sensitive conjugate indicated a similar in vivo efficacy with
the Taxol control, but much lower in vivo toxicity at the same doses evidenced by body weight, animal status,
liver toxicity and blood count. Moreover, at the tolerant dose, this novel conjugate exhibited significantly
better antitumor effect than that of Taxol formulation. In general, the PEG–VC–PABC–PTX conjugate designed
in this study demonstrated significant advantages in terms of high water solubility, no toxic surfactant or
organic solvent, tumor environment-sensitivity and high therapeutic index.
©
2012 Elsevier B.V. All rights reserved.
1
. Introduction
PTX, isolated from the trunk bark of the Pacific Yew tree, Taxus
brevifolia [3], is a potent inhibitor of cell replication which was used
in the treatment of various cancers including breast and ovarian
cancer [4]. However, PTX suffers from significant drawbacks such as
aqueous insolubility and dose-limiting toxicity at clinically adminis-
tered doses [5]. Therefore, in Taxol formulation presently available
in the market, PTX needs to be formulated with surfactant Cremophor
EL and ethanol. However, Cremophor EL was reported to induce
severe toxicities to patients such as hypersensitivity after systemic
administration [6,7].
The idea of covalently attaching chemotherapeutic agents to a
water-soluble polymer was first proposed by Ringsdorf in the mid-
1970s [8]. Since then, accompanied by the development of synthetic
and natural polymers, it is becoming a fast-growing field. Such conju-
gates were developed pre-clinically in 1980s and started to enter the
clinical pipeline in the 1990s [9]. For instance, the poly(L-glutamic
acid)-paclitaxel conjugate (PG-PTX, coded as CT-2103), showed satis-
factory safety and efficacy through the Phases I, II and III studies
Cancer is a leading cause of death worldwide: it accounted for
.9 million deaths (around 13% of all deaths) in 2007. Still, deaths
7
from tumors worldwide are supposed to rise continuously, with an
estimated 12 million deaths in 2030 [1]. However, the lack of tumor-
specific agents is a long-standing problem faced in cancer treatment.
Chemotherapeutics kill cancer cells, and cause a number of undesir-
able severe side effects due to their non-specific distribution in vivo.
Additionally, there is a great challenge in terms of solubilizing the
water-insoluble anticancer agent, like paclitaxel (PTX) [2]. Therefore,
it is imperative to find a new kind of agent which is water-soluble
and specifically active at tumor site.
⁎
Corresponding author.
⁎⁎ Corresponding author. Tel./fax: +86 10 82802791.
E-mail addresses: lirt@bjmu.edu.cn (R.-T. Li), zqdodo@bjmu.edu.cn (Q. Zhang).
These authors contributed equally to this work.
1
0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2012.02.020

L. Liang et al. / Journal of Controlled Release 160 (2012) 618–629
619
[
10–13]. Other polymer-drug conjugates under development in-
and fetal bovine serum were from GIBCO. CD31 and Ki67 were pur-
chased from Abcam. All other chemicals were of analytical or HPLC
grade.
clude hydroxypropyl methacrylate (HPMA) copolymer-doxorubicin
(
(
coded as PK1, PK2), HPMA copolymer-diamino-cyclohexane
DACH)-platinate (coded as AP5346) and PEG-camptothecin etc.
[
14]. Polyethelene glycol (PEG) is a hydrophilic polymer approved
2.2. Synthesis of MPEG-COCH
2
CH
2
CO-2′-PTX conjugate (PEG-PTX)
by FDA and used in pharmaceutical industry for many years as
an excipient which shows little toxicity and immunogenicity [15].
In the past forty years, several antitumor agents, such as proteins,
peptides and low molecular weight drugs, have been considered
for PEGylation [16,17].
However, the concept of linking chemotherapeutic agents to
water-soluble polymer is challenged greatly by the fact that polymer
like PEG may not be cleavable from the conjugate, even in tumor
tissue, led to obvious decrease of the antitumor effect [16]. Whereas,
tumor environment-sensitive drug delivery system, which is cur-
rently in rapid development, may provide a novel strategy to over-
come the obstacles for both drug solubility and site-specific delivery
of chemo-drugs [18,19]. In such circumstances, a special linker, sensi-
tive to the pH condition or enzyme system of tumor tissue, may be
used to connect the chemotherapeutic agent and the water-soluble
polymer, ensuring the drug solubility in water and also the drug
release in time from the conjugate at the tumor site [20–23].
1
Melting points were determined on X4 microscope. H NMR spec-
III
tra were recorded on a Bruker AVANCE 400 (400 MHz) instrument
at room temperature, with CDCl as solvent and TMS as internal ref-
3
erence [6]. ESI-MS spectra were performed on a MDS SCIEX QSTAR
spectrometer. On an AXIMA-CFR Plus spectrometer with a nitrogen
laser (337.1 nm), MALDI-TOF-MS spectra were obtained in positive
ion and linear mode with an acceleration voltage of 20 kV and aver-
aged over 100 laser shots. Each spectrum was externally calibrated
with insulin at m/z 2867.80, 5534.59 and 11468.17 respectively pro-
viding mass measurement accuracies of approximately 50 ppm across
the 1 kDa ~12 kDa mass range.
2 2
2.2.1. MPEG-COCH CH COOH
MPEG5000 (20 g, 4 mmol) was added to toluene (100 mL) and
refluxed for 1 h to remove water. After cooling, toluene was evaporat-
ed and the residue was dissolved in CHCl
dride (2.00 g, 20 mmol) and pyridine (1 mL) were added and the
mixture was refluxed for 48 h. CHCl was evaporated and distilled
water (150 mL) was added. The mixture was filtered, and the filtrate
was extracted with CHCl (100 mL×3). The combined organic layers
4
were washed with brine (100 mL×2), dried over anhydrous Na SO ,
3
(100 mL). Succinic anhy-
B
Cathepsin B (C ) is a 30 KDa lysosomal cysteine protease from a
family of 11 cysteine proteases [24,25]. In many kinds of cancer,
cathepsin B acts as an important proteinase of matrix materials so
that tumor cells can actively invade and metastasize [26,27]. It is
never found extracellularly, except in pathological conditions such
as tumors or in the areas of tissue destruction in rheumatoid arthritis
3
3
2
filtered and concentrated under reduced pressure. Ether was added,
and the resulting white solid was collected by filtration, washed
with ether, and dried in vacuo (15.10 g, 74%). The product was char-
acterized by 1H NMR and MALDI-TOF-MS.
[28]. The general substrates of cathepsin B include valine–citrulline
and phenylalanine–arginine [29]. It was reported that some antibody-
drug conjugates using the protease-sensitive dipeptide as well as
some kind of spacer as the linker showed efficient release of drug in
tumor tissues [30].
The purpose of this study is to obtain a water-soluble and tumor
environment-sensitive delivery system of PTX through PEGylation
2.2.2. MPEG-COCH
MPEG-COCH CH
(10 mL) and the mixture was refluxed for 1 h to remove water. Tolu-
ene was then evaporated and the residue was dissolved in CH Cl
2
CH
2
CO-2′-PTX
2
2
COOH (0.56 g, 0.11 mmol) was added to toluene
as well as the introduction of C
B
-sensitive linker with additional spac-
2
2
er, in the hope of that it will exhibit significant advantages over PTX
or Taxol formulation in terms of higher water solubility, no toxic
surfactant or organic solvent involvement, tumor environment-
sensitivity and higher therapeutic index. So, a novel paclitaxel
conjugate was synthesized here by using valine–citrulline dipeptide
(10 mL). EDC·HCl (0.038 g, 0.20 mmol) was added and the mixture
was stirred at 0 °C for 30 min. PTX (0.086 g, 0.10 mmol), DIEA
(33 μL, 0.20 mmol) and DMAP (1.2 mg, 0.01 mmol) was added, and
the mixture was warmed to room temperature and stirred for anoth-
er 24 h. Then the mixture was washed with brine (10 mL), and the
(
(
(
Val–Cit, or VC), a substrate of C
PABC), a spacer, to link PEG and paclitaxel. The PEGylated PTX
PEG-PTX) and the Taxol formulation were used as the controls. For
B
, and p-aminobenzylcarbonyl
aqueous layer was extracted with CH
organic layers were dried over anhydrous Na
orated. The residue was purified by column chromatography
(CHCl :CH OH=40:1 then 10:1) to afford the product as a white
2
Cl
2
(10 mL). The combined
2
SO , filtered and evap-
4
the proof of concept, PEG–VC–PABC–PTX conjugate was synthesized,
3
3
characterized and evaluated in vitro and in vivo.
foamed solid (0.54 g, 91%). The product was characterized by
MALDI-TOF-MS.
2
. Material and methods
2 2
2.3. Synthesis of MPEG-COCH CH CO-Val-Cit-PABC-2′-PTX conjugate
2
.1. Materials
(PEG–VC–PABC–PTX)
Diisopropylethylamine (DIEA), 2-ethoxy-1-ethoxycarbonyl-1, 2-
2.3.1. Fmoc-Val-Cit [29]
dihydroquinoline (EEDQ), N-hydroxysuccinimide (HOSu), poly-
ethylene glycol monomethyl ether 5000 (MPEG5000), p-aminobenzyl
alcohol (PABOH) and p-nitrophenyl (PNP) chloroformate were
purchased from Alfa Aesar (Tianjin, China). L-citrulline (L-Cit), 9-
fluorenylmethyloxycarbonyl-L-valine (Fmoc-Val), 4-dimethylaminopyridine
Fmoc-Val (5.06 g, 14.91 mmol), HOSu (1.72 g, 14.91 mmol) and
DCC (3.08 g, 14.91 mmol) were added to THF (50 mL) at 0 °C and
the mixture was stirred at room temperature for 16 h, and then fil-
tered and washed with THF. The solvent was removed under reduced
pressure, and the resulting glassy solid (Fmoc-Val-OSu) was used
without purification in the next step.
(
DMAP), 1,2-dimethoxyethylene (DME), tetrahydro furan (THF), 1-
Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC·
HCl), Succinic anhydride, 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU)
and dicyclohexylcarbodiimide (DCC) were obtained from Bo-Mai-Jie
Fmoc-Val-OSu (14.91 mmol) in DME (40 mL) was added to a solu-
tion of Cit (2.74 g, 1.05 equiv) and NaHCO (1.32 g, 1.05 equiv) in
3
water (40 mL). THF (20 mL) was added to aid solubility, and the mix-
ture was stirred at room temperature for 16 h. Aqueous citric acid
(15%, 75 mL) was added, and the mixture was extracted with 10% iso-
propanol/ethyl acetate (100 mL×3). The combined organic layers
were washed with water (150 mL×3), and the solvents were evapo-
rated. The resulting white glassy solid was added to ether (80 mL).
B
Co., Ltd. (Beijing, China). Cathepsin B (C ) and sulforhodamine B
(
SRB) were from Sigma Aldrich (St. Louis, MO, USA). Paclitaxel (PTX)
was the product of Mei-Lian Co., Ltd. (Chongqing, China). Cremophor
EL was purchased from BASF Corporation of Germany (Local Agent in
Shanghai, China). Cell culture media DMEM, penicillin streptomycin,

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L. Liang et al. / Journal of Controlled Release 160 (2012) 618–629
After sonication and trituration, the white solid product was collected
by filtration, washed with ether, and dried in vacuo (4.64 g, 63%).
The product was characterized by 1H NMR.
Malvern Zetasizer Nano ZS (Malvern, UK) at 25 °C, and the shape
and surface morphology of conjugates were investigated by transmis-
sion electron microscope (TEM, JEM-1230, JEOL, JAPAN) after nega-
tive staining with uranyl acetate solution (1%, w/v).
2
.3.2. Fmoc-Val-Cit-PABOH [29]
Fmoc-Val-Cit (1.04 g, 2.10 mmol) and PABOH (0.52 mg,
2.6. Drug release in vitro
4
2 2 3
.20 mmol) in 2:1 CH Cl /CH OH (24 mL/12 mL) were treated with
EEDQ (1.04 g, 4.20 mmol). The mixture was stirred in the dark at
room temperature for 36 h. The solvents were removed under re-
duced pressure at 40 °C, and ether (75 mL) was added. The mixture
was triturated and sonicated for 5 min and then left to stand for
The conjugate prepared in this study was incubated in 1 mL
DMEM with 10% FBS at 37 °C with shaking (100 rpm). Aliquots of
100 μL of incubation medium were removed at predetermined time
points (1, 2, 4, 6, 8, 12, 24 h), and mixed with 2.5 mL methyl-butyl
ether in a vortex mixer for 2 min. Then 2 mL supernate was collected
and dried in nitrogen at 40 °C. The residue was dissolved in 100μL
mobile phase. The supernatant (20 μL) was injected into the HPLC
system for drug analysis.
3
0 min. The solid was collected by filtration, washed with ether, and
1
dried in vacuo (1.10 g, 82%). The product was characterized by
NMR.
H
2
.3.3. Fmoc-Val-Cit-PABC-PNP
A mixture of Fmoc-Val-Cit-PABOH (0.30 g, 0.5 mmol), PNP chloro-
formate (0.20 g, 1.0 mmol) and pyridine (80 μL, 1.0 mmol) in THF
25 mL) was stirred at room temperature over night. The solvent
was evaporated under vacuum and the residue was purified by col-
umn chromatography (CHCl :CH OH=30:1 then 20:1) to afford the
B
2.7. C sensitivity of conjugates
(
C
B
was preincubated in the activation buffer containing 50 mmol/L
sodium acetate (pH5.0), 2 mmol/L dithiothreitol (DTT), and 25% (v/v)
glycerol for 15 min at 37 °C. The activated C solution then was dilut-
ed with 2 mL of 25 mM acetate/1 mM EDTA buffer (pH5.0, preincu-
bated at 37 °C) [29]. The final concentration of C in this solution
was 10 unit/mL. Then conjugate was added to C solution. The
3
3
B
product as a pink foamed solid (0.20 g, 52%). The product was charac-
terized by H NMR and ESI-MS.
1
B
B
2
.3.4. Fmoc-Val-Cit-PABC-2′-PTX
A mixture of Fmoc-Val-Cit-PABC-PNP (0.16 mg, 0.21 mmol), PTX
whole system was incubated at 37 °C for 24 h. The free PTX was
detected by HPLC [31]. The HPLC condition was mentioned above.
(
0.22 mg, 0.25 mmol) and DMAP (0.031 g, 0.25 mmol) in CH
2 2
Cl
(
20 mL) was stirred at room temperature for 48 h. The solvent was
2.8. Cell culture
evaporated under vacuum and the residue was purified by column
chromatography (CHCl :CH OH=30:1 then 20:1) to afford the prod-
3
3
Human breast cancer cells MCF-7 (Institute of Material Medical,
Chinese Academy of Medical Sciences and Peking Union Medical Col-
lege, Beijing, China) were employed as an in vitro cell model. The
MCF-7 cells were maintained in the DMEM medium with 10% FBS
and 1% penicillin–streptomycin solution. The cells were incubated at
uct as a pink foamed solid (0.18 g, 59%). The product was character-
ized by 1H NMR and ESI-MS.
2
.3.5. MPEG-COCH
MPEG-COCH CH
10 mL) and the mixture was refluxed for 1 h to remove water. Tolu-
ene was then evaporated and the residue was dissolved in CH Cl
10 mL). The resulting solution was cooled to 0 °C, and EDC·HCl
0.047 g, 0.25 mmol) was added. The mixture was stirred for 30 min
2
CH
2
CO-Val-Cit-PABC-2′-PTX
2
2
COOH (0.69 g, 0.14 mmol) was added to toluene
2
37 °C in the humidified environment of 5% CO . The medium was
(
replenished every two days until confluence was achieved. The cells
were then washed with PBS and harvested with 0.25% trypsin solu-
tion. PTX solution (dissolved in DMSO then diluted by DMEM) and
conjugate solution (dissolved in water then diluted by DMEM) were
used in all the cell tests.
2
2
(
(
at 0 °C, and then transferred to a solution of Fmoc-Val-Cit-PABC-2′-
PTX (0.18 g, 0.12 mmol) and DBU (37 μL, 0.25 mmol) in THF (8 mL)
which was previously stirred for 10 min to remove the Fmoc protec-
tive group. After reacting at room temperature for 24 h, the mixture
was washed with brine (10 mL), and the aqueous layer was extracted
2.9. In vitro cytotoxicity
MCF-7 cells were seeded at a density of 2×103 cells/well in a 96-
well transparent plate and incubated for 24 h. Then various concen-
trations of PTX or conjugate solution were added to the medium.
After 72 h, the cell viability was determined by the SRB assay [32].
Briefly, the medium was removed, and then cells were fixed with tri-
chloroacetic acid, washed and stained with SRB. The absorbance was
measured at 540 nm using a 96-well plate reader (Bio-rad, 680,
America). The survival percentages were calculated using the follow-
ing formula: Survival%=(A540 nm for the treated cells/A540 nm for
the control cells)×100%, where A540 nm is the absorbance value.
Each assay was repeated for triplicates. Finally, dose–effect curves
were made and IC50 values were calculated.
with CH
anhydrous Na
by chromatography (CHCl
2
Cl
2
(10 mL). The combined organic layers were dried over
SO , filtered and evaporated. The residue was purified
:CH OH=40:1 then 12:1) to afford the
2
4
3
3
product as a white foamed solid (0.23 g, 30%). The product was char-
acterized by MALDI-TOF-MS.
2
.4. Solubility test
Excess PTX powder and PTX conjugate prepared in this study were
put into three different kinds of medium, pure water, 5% glucose and
physiological saline, respectively. Then the test system was shaken
at room temperature for 48 h, followed by the centrifugation at
1
0000 rpm for 10 min. Finally, each supernate was diluted and deter-
2.10. Cell cycle analysis
mined by HPLC [31]. The HPLC assay was conducted using a Phenom-
enex® C18 column (5 μm, 250×4.6 mm) kept at 35 °C and an UV
detector set at 232 nm. Acetonitrile and water (53:47, V/V) were
used as mobile phase at a flow-rate of 1.0 mL/min.
Cell cycle perturbations induced by PTX or conjugates were ana-
lyzed by propidium iodide (PI) DNA staining. Briefly, exponentially
growing MCF-7 cells were treated with 20 nM PTX, 20 nM PEG-PTX
and 20 nM PEG–VC–PABC–PTX conjugate for 12 h, with or without
2
.5. Particle size and morphology
B
C , respectively. The MCF-7 cells incubated with DMEM were used
as the control. At the end of each treatment, cells were collected
after a gentle centrifugation at 1200 rpm for 5 min and then fixed in
70% ethanol for 12 h at 4 °C. Ethanol-suspended cells were diluted
The apparent particle sizes of the nanoparticles formed from con-
jugates were determined by dynamic light scattering (DLS) using

L. Liang et al. / Journal of Controlled Release 160 (2012) 618–629
621
with PBS and then centrifuged at 1200 rpm for 5 min to remove resid-
ual ethanol. For cell cycle analysis, the pellets were suspended in
nude mice were randomly divided into three groups of 6 mice: 5%
glucose solution (control), PEG-PTX and PEG–VC–PABC–PTX. The
conjugates in 5% glucose solution were given at a dosage of
10 mg PTX/kg. Administration via tail vein injection was started at
day 6 post tumor inoculation when the average tumor size reached
80 mm3 and continued at days 8, 10, 12 and 14. The tumor size was
recorded at days 7, 9, 11, 13 and 15 accordingly. The tumor volume
was measured with digital caliper in two dimensions and calculated
0
.5 mL of PBS, and incubated with 1 mg/mL of DNase-free RNase A
at 37 °C for 30 min. Then 0.1 ml of 0.1% of Triton X-100 containing
.02 mg/mL of PI was added. Cell cycle profiles were studied using a
0
FACScan flow cytometer (Becton Dickinson FACSCalibur, Mountain
View, CA, USA). The data were analyzed through FCS Express V 3 soft-
ware. Each assay was repeated for triplicates.
2
as follows: (length)×(width) /2 [33]. At the end of the test, tumors
2
.11. In vivo anti-tumor efficacy
were harvested and weighted.
Then PEG–VC–PABC–PTX conjugate at various doses was com-
pared with Taxol formulation (prepared according to the formulation
of Taxol), MCF-7 tumor-bearing nude mice were randomly divided
into five groups of 6 mice: 5% glucose, Taxol formulation (10 mg/
kg), PEG–VC–PABC–PTX (equimolar with 10 mg/kg PTX), PEG–VC–
PABC–PTX (equimolar with 15 mg/kg PTX) and PEG–VC–PABC–PTX
The 6–8 week old female BALB/c nude mice inoculated with MCF-
cells (10 ) in the left armpit were used in the studies. All care and
handling of animals were performed with the approval of Institution-
al Authority for Laboratory Animal Care of Peking University. In the
comparison test among different PTX conjugates, tumor-bearing
6
7
Fig. 1. MALDI-TOF spectra of MPEG-COCH
2
CH
2
COOH (a), PEG-PTX (b), and PEG–VC–PABC–PTX (c).

622
L. Liang et al. / Journal of Controlled Release 160 (2012) 618–629
(
equimolar with 20 mg/kg PTX). The manipulation was the same as
vasculature, CD31 antibody (Abcam, 1:200) was used for microvessel
staining. For cell proliferation analysis, sections were incubated with
Ki-67 antibody (Abcam, 1:200). The method of immunohisto-chemistry
used here was basically according to reference [34].
mentioned above, except that tumor size was recorded until day 17.
2
.12. Effect on the angiogenesis, cell proliferation and toxicity in vivo
At end point, 20 μl blood sample was collected from retro-orbital
sinus for each mouse. The blood count was determined by a MEK-
6318K Hematology Analyzer (Nihon Kohden, Japan) to preliminarily
The tumors of different treatment groups were embedded and pre-
pared into serial sections (4 μm thick). For analysis of tumor
Scheme 1. Synthetic routes. Reagents and conditions: (a) pyridine, CHCl
3
, reflux; (b) PTX, EDC·HCl, DIEA, DMAP, CH
2
Cl
2
; (c) i) HOSu, DCC, THF; ii) Cit, NaHCO
3
, H
2
O, DME, THF
Cl
(
d) PABOH, EEDQ, CH Cl , CH OH; (e) PNP chloroformate, pyridine, THF; (f) PTX, DMAP, CH
2
2
3
2
Cl ; (g) i) DBU, THF; ii)MPEG-COCH
2
2
CH COOH, EDC·HCl, DIEA, DMAP, CH
2
2
2
.

L. Liang et al. / Journal of Controlled Release 160 (2012) 618–629
623
assess the blood toxicity of PTX and PTX conjugate. Additionally, H&E
Table 2
Apparent particle size of the two conjugates (n=3).
staining of the liver tissue sections was conducted for the histological
study of liver toxicity.
Group
Particle size (nm)
PDI
PEG-PTX
PEG–VC–PABC–PTX
159.4± 5.0
122.5± 0.4
0.223± 0.017
0.61± 0.15
2
.13. Data analysis
Data are shown as means± SD unless particularly outlined. Stu-
dent's t-test or one-way analyses of variance (ANOVA) were
performed in statistical evaluation. A p value less than 0.05 was con-
sidered to be significant.
[M+Na]+, respectively. All these results supported that Fmoc-Val-
1
Cit-PABC-PNP had been obtained. H NMR (400 MHz, DMSO-d
6
): δ
0.86 (d, J=6.8 Hz, 3H), 0.88 (d, J=6.8 Hz, 3H), 1.39–1.69 (m, 4H),
.97–2.02 (m, 1H), 2.97–3.06 (m, 2H), 3.94 (t, J=7.6 Hz, 1H), 4.23–
4.43 (m, 4H), 5.25 (s, 2H), 5.41 (br s, 2H), 5.98 (t, J=6.0 Hz, 1H),
.31–7.77 (m, 13H), 7.89 (d, J=7.6 Hz, 2H), 8.14 (d, J=7.6 Hz, 1H),
1
3
3
3
. Results
7
+
.1. Characterization of PEG-PTX
8.23–8.33 (m, 2H), 10.14 (s, 1H). MS (ESI+): m/z 766.14 [M+H] ,
+
7
89.10 [M+Na]
.
2 2
.1.1. MPEG-COCH CH COOH
The characteristic peaks for MPEG-COCH
2
CH
COOH. As shown in Fig. 1a,
The MALDI-TOF-MS spectrum exhibited the center of the peak at m/z
100. All these results supported that MPEG had been conjugated
2
COOH were found in
3.2.4. Fmoc-Val-Cit-PABC-2′-PTX
1
the H NMR spectrum of MPEG-COCH
2
CH
2
The characteristic peaks of Fmoc-Val-Cit-PABC-2′-PTX were
1
observed in the H NMR spectrum. The ESI-MS spectrum exhibited
+
5
the peak at m/z 1503.81, which corresponds to the [M+Na] . All
1
with succinic anhydride. H NMR (400 MHz, CDCl
.38 (s, 3H), 3.46 (t, J=5.2 Hz, 2H), 3.55 (t, J=5.2 Hz, 2H), 3.64
m), 3.82 (t, J=4.8 Hz, 4H), 4.26 (t, J=4.8 Hz, 2H).
3
): δ 2.60 (m, 4H),
these results supported that Fmoc-Val-Cit-PABC-2′-PTX had been
obtained. 1H NMR (400 MHz, CDCl
): δ 0.85–0.89 (m, 6H), 1.13
(s, 3H), 1.17 (s, 3H), 1.42–1.92 (m, 12H), 2.04–2.59 (m, 11H), 3.11–
.30 (m, 2H), 3.67 (d, J=6.8 Hz, 1H), 3.69 (d, J=5.6 Hz, 1H), 4.09–
3
(
3
3
3
.1.2. MPEG-COCH
The synthetic route and structure of MPEG-COCH
2
CH
2
CO-2′-PTX
4.76 (m, 10H), 4.96–5.31 (m, 3H), 5.47 (d, J=3.6 Hz, 1H), 5.64–5.73
(m, 2H), 5.95 (dd, J=3.6, 8.8 Hz, 1H), 6.14 (t, J=8.4 Hz, 1H), 6.27
2 2
CH CO-2′-PTX
are shown in Scheme 1. The MALDI-TOF-MS spectrum exhibited the
center of the peak at m/z 5800. All these results supported that
(s, 1H), 7.11–7.74 (m, 27H), 8.12 (d, J=7.2 Hz, 2H), 9.25 (br s, 1H).
+
MS (ESI+): m/z 1503.81 [M+Na]
.
MPEG-COCH
2 2
CH COOH had been conjugated with PTX successfully.
MS (MALDI-TOF): m/z peak at 5700 (Fig. 1b).
3.2.5. MPEG-COCH
2
CH
2
CO-Val-Cit-PABC-2′-PTX
CH CO-Val-Cit-PABC-2′-PTX
The synthetic route of MPEG-COCH
2
2
3
.2. Characterization of MPEG–VC–PABC–PTX
is presented in Scheme 1. The MALDI-TOF-MS spectrum exhibited
3
.2.1. Fmoc-Val-Cit
The structure of Fmoc-Val-Cit was confirmed by the characteristic
peaks which were in accordance with that reported in reference
1
[
(
(
29]. H NMR (400 MHz, DMSO-d
6
): δ 0.86 (d, J=6.8 Hz, 3H), 0.89
d, J=6.4 Hz, 3H), 1.36–1.73 (m, 4H), 1.94–2.00 (m, 1H), 2.92–2.97
m, 2H), 3.92 (t, J=8.0 Hz, 1H), 4.12–4.31 (m, 4H), 5.36 (br s, 2H),
5
7
.92 (t, J=4.8 Hz, 1H), 7.31–7.44 (m, 5H), 7.75 (t, J=7.2 Hz, 2H),
.90 (d, J=7.6 Hz, 2H), 8.16 (d, J=7.2 Hz, 1H), 12.51 (br s, 1H).
3
.2.2. Fmoc-Val-Cit-PABOH
The structure of Fmoc-Val-Cit-PABOH was approved by the char-
acteristic peaks which were in accordance with that reported in refer-
1
ence [29]. H NMR (400 MHz, DMSO-d
0
3
6
): δ 0.86 (d, J=6.8 Hz, 3H),
.88 (d, J=6.8 Hz, 3H), 1.37–1.69 (m, 4H), 1.94–2.03 (m, 1H), 2.92–
.05 (m, 2H), 3.93 (t, J=7.6 Hz, 1H), 4.23–4.44 (m, 6H), 5.09
(t, J=5.6 Hz, 1H), 5.40 (br s, 2H), 5.97 (t, J=5.6 Hz, 1H), 7.23
(d, J=8.4 Hz, 2H), 7.31–7.44 (m, 5H), 7.54 (d, J=8.4 Hz, 2H), 7.74
(t, J=7.2 Hz, 2H), 7.89 (d, J=7.6 Hz, 2H), 8.09 (d, J=7.2 Hz, 1H),
9
.97 (s, 1H).
3
.2.3. Fmoc-Val-Cit-PABC-PNP
The characteristic peaks of Fmoc-Val-Cit-PABC-PNP were all ob-
1
served in the H NMR spectrum. The ESI-MS spectrum exhibited the
+
peak at m/z 766.14, 789.10, which corresponds to the [M+H] and
Table 1
Solubility of PTX and PTX conjugates in different medium (PTX: mg/mL).
PTX
PEG-PTX
PEG–VC–PABC–PTX
3
3
Water
Saline
4.03×10−
4.26×10
4.33
3.05
2.43
5.36
4.13
4.32
−
5.83×10−3
Fig. 2. Transmission electron microscopy image of conjugates suspend in PBS at a con-
centration of 0.1 mg/mL. A: PEG–VC–PABC–PTX; B: PEG-PTX.
5% glucose

624
L. Liang et al. / Journal of Controlled Release 160 (2012) 618–629
one molecule of PTX. So, the amount of PTX (MW 853) bound to
the conjugate was 13.98%. The total synthesis yield of PEG–VC–
PABC–PTX was 17.7% based on PTX and calculated from the reaction
yield in each of the two steps related.
3.3. Solubility
The solubility of free PTX and two PTX conjugates is shown in
Table 1. As been seen, the PTX had a very poor solubility in three
commonly-used media. After connection to PEG with or without link-
er, the solubility of PTX conjugates increased enormously, from the
μg/mL to the mg/mL order of magnitude, suggesting a thousand-
time or more improvement in solubility. The solubility of two PTX
conjugates in different medium was in the same order of magnitude.
3.4. Particle size and morphology
As shown in Table 2, each kind of conjugate possessed an apparent
mean particle size in DLS analysis between 100 and 200 nm, with a
polydispersity index (PDI) between 0.2 and 0.7. In TEM graph
(
Fig. 2), the diameter of PEG–VC–PABC–PTX on average was larger
than 100 nm also with a rather big fluctuation in particle size. Addi-
tionally, the conjugate seemed to form the “nano-aggregate”.
3.5. Drug release in vitro
In vitro release profiles of PTX from the two conjugates are shown
in Fig. 3A. There was obvious difference in the drug release pattern
between the two conjugates. In PEG-PTX group, more than 50% of
free PTX was detected in 24 h. While only about 10% of free drug
was released from PEG–VC–PABC–PTX conjugate after 24 h test.
B
3.6. C sensitivity of conjugates
The sensitivity of conjugates to C
the drug released from this conjugate in the presence of C
B
was tested here by monitoring
, and the
B
data is shown in Fig. 3B and C. In comparison with 8.03% of PTX re-
leased from PEG–VC–PABC–PTX conjugate after incubated without
C at 37 °C for 24 h, 24.06% of PTX was released when C was added
B B
to the same system. There was a significant difference between the
two groups (pb0.01), revealing the obvious sensitivity of PEG–VC–
B
PABC–PTX conjugate to C . However, PEG-PTX conjugate changed lit-
tle (p>0.1) after C was added.
B
3.7. In vitro cytotoxicity
The cytotoxicities of two PTX conjugates and PTX solution are
shown in Table 3. The free drug (PTX) exhibited the highest cytotox-
icity as expected. The cytotoxicity of PEG-PTX or PEG–VC–PABC–PTX
was much lower than PTX group, indicating the negative effect of
chemical linkage at 2′-OH of PTX on their cytotoxicity. There is no
significant difference between PEG-PTX group and PEG–VC–PABC–
PTX group.
Fig. 3. In vitro release of PTX from each conjugate in DMEM with 10% FBS at 37 °C. A:
release curve without C ; B: C -sensitivity test for two conjugates at 24 h; C: C -sensi-
tivity test for PEG–VC–PABC–PTX at different time; a: pb0.01 versus that without C
B
B
B
B
.
the center of the peak at m/z 6100. All these results supported
that the conjugate had been obtained successfully. MS (MALDI-
TOF): m/z peak at 6100 (Fig. 1c).
3.8. Cell cycle analysis
From the conjugate structure as well as its molecular weight of
6
100, it was clear that one molecule of the conjugate contains only
A proliferating culture of MCF-7 cells was incubated with 20 nM of
PTX or PTX conjugates for 12 h with or without the existence of adsci-
B
titious C . The results are shown in Fig. 4 and Table 4. Based on the
antitumor mechanism of PTX, the fraction of cells in G
the most important parameters [35,36].
2
-M phase is
Table 3
IC50 values of PTX conjugates and PTX after incubation with MCF-7 cell for 72 h (Unit:
nM, n=3).
As shown in Table 4, there was a significant difference between
cell group (negative control) and PTX group (positive control)
PTX
PEG-PTX
PEG–VC–PABC–PTX
(
pb0.001), suggesting the feasibility of the methodology. However,
6.38± 1.06
40.32± 3.39a
34.28± 3.38a
the effect of PEG–VC–PABC–PTX and PEG-PTX conjugate on cell
cycle was significantly lower than PTX groups. PEG–VC–PABC–PTX
a
pb0.0005 versus PTX.

L. Liang et al. / Journal of Controlled Release 160 (2012) 618–629
625
B B
Fig. 4. Effects of different treatments on the cell cycle of MCF-7 cells after incubation for 12 h. A: without additional C ; B: with additional C .
and PEG-PTX was similar to each other, and there was no significant
difference among each of them and the negative control (p>0.1).
without C
significance), indicating that PEG–VC–PABC–PTX is significantly
sensitive to C . For PEG-PTX, the cells in G -M phase still remained
a relatively low level even in the presence of C
B
(pb0.05), while all other groups changed little (without
After C
-M phase than the cell control (PEG-PTX: pb0.05, PEG–VC–PABC–
PTX: pb0.0005). However, only the PEG–VC–PABC–PTX group with
showed a significant higher cell number of G -M phase than that
B
was added, all groups showed significantly more cells in
B
2
G
2
B
.
C
B
2
3.9. In vivo anti-tumor efficacy
Table 4
The anti-tumor efficacy of two conjugates, PEG-PTX and PEG–VC–
2
Effect of different treatments on the G -M phase (%) in cell cycle of MCF-7 cells incu-
bated with or without C
B
for 12 h (n=3).
PABC–PTX was compared in animal experiment here, while the toxic-
ities of these prodrugs were also evaluated by recording the changes
in animal weight, as well as the animal status. As shown in Fig. 5,
there was no anti-tumor efficacy observed in PEG-PTX group. Howev-
er, compared to the 5% Glu and PEG-PTX group, the PEG–VC–PABC–
PTX conjugate (equimolar with 10 mg/kg PTX) significantly inhibited
the tumor growth (pb0.05). All treatments showed no significant dif-
ference in body weight changes of animals (data not show), and the
animal status in each test group was similar with the control group.
Control
%)
PTX
(%)
PEG-PTX
(%)
PEG–VC–
PABC–PTX (%)
(
a
c
G
G
2
-M(without CB) 8.75± 1.60 41.91± 5.86
-M (with C 7.82± 3.88 46.76± 2.88
21.15± 9.15
22.80± 9.26
42.17± 1.10
28.14± 7.61b
c,d
2
B
)
a
b
c
pb0.001 vs control.
pb0.05 vs control.
pb0.0005 vs control.
d
B
pb0.05 vs itself without C .

626
L. Liang et al. / Journal of Controlled Release 160 (2012) 618–629
Fig. 5. Anti-tumor activity of different treatments in MCF-7 cells baring nude mice.
A) Tumor volume. (B) Tumor weight at the end of test. Treatment started when the
(
3
average tumor size reached 80 mm via tail vein every other day (n=6). Animals
were sacrificed on day 15 post-tumor inoculation. a, pb0.05 versus control; *, pb0.05
versus PEG-PTX.
Furthermore, various dosage of PEG–VC–PABC–PTX conjugate was
compared with Taxol formulation, which is displayed in Fig. 6. Dra-
matic growth of tumor was observed in the 5% glucose group. Both
Taxol formulation and PEG–VC–PABC–PTX (equimolar with 10 mg/
kg PTX) conjugate showed a significant tumor inhibition in compari-
son with the control (pb0.001). Nevertheless, there was no signifi-
cant difference in terms of therapeutic effect between Taxol
formulation and PEG–VC–PABC–PTX conjugate (p>0.5) at the same
dose of 10 mg/kg PTX. However, the Taxol formulation showed signif-
icant toxicity at this dose, evidenced by the most body weight loss
and worst animal status, such as anorexia, lack of vitality, etc., pre-
venting the further increase of drug dose. In contrast, the animals
treated with PEG–VC–PABC–PTX (10 mg/kg PTX) demonstrated
much less loss of body weight and much better status.
With the increase of dosage, the anti-tumor activity of the PEG–
VC–PABC–PTX conjugate further improved. When the dose of PTX
reached 20 mg/kg, this group exhibited significantly better antitumor
effect than that of 10 mg/kg PTX group (pb0.05). It was observed that
the body weight loss in PEG–VC–PABC–PTX (20 mg/kg PTX) group
was similar to that of Taxol group (10 mg/kg PTX), while the animals
in conjugate group were found with better vitality than that of Taxol
group.
Fig. 6. Anti-tumor activity and body weight change of different treatments in MCF-7
cells baring nude mice. (A) Tumor volume. (B) Tumor weight at the end of test.
(
8
C) Body weight changed. Treatment started when the average tumor size reached
0 mm via tail vein every other day (n=6). Animals were sacrificed on day 17
3
post-tumor inoculation. (1) 5% glucose, (2) Taxol formulation (10 mg/kg), (3) PEG–
VC–PABC–PTX (10 mg PTX/kg), (4) PEG–VC–PABC–PTX (15 mg PTX/kg), (5) PEG–
VC–PABC–PTX (20 mg PTX/kg). a, pb0.005 versus control; b, pb0.05 versus Taxol
formulation.
difference between Taxol and PEG–VC–PABC–PTX group, but the lat-
ter is slightly better than the former.
Fig. 7B presents the anti-proliferation effects of different treat-
ments. Compared with the negative group, both Taxol formulation
and PEG–VC–PABC–PTX could significantly inhibit tumor cell prolifer-
ation. However, no cell proliferation effect was observed in the PEG-
PTX group.
3
.10. Effect on the angiogenesis, cell proliferation and toxicity in vivo
As shown in Fig. 7A, PEG–VC–PABC–PTX exhibited obvious antian-
giogenesis effect, indicated by the lower density of tumor neovascula-
ture compared with the negative control (5% Glucose), while PEG-PTX
had no such effect at all. At the same dosage, there's no apparently
Liver toxicity of different formulations is illustrated in Fig. 7C.
After 5-time administration at the dosage of 10 mg PTX/kg, the two
conjugates showed no apparent toxicity to mice liver. In contrast,

L. Liang et al. / Journal of Controlled Release 160 (2012) 618–629
627
Fig. 7. Effect of various treatments on the angiogenesis, proliferation of tumors and in vivo toxicity. At the end of antitumor test in vivo, MCF-7 tumors were removed from mice
with different treatments and sectioned. Tumor microvessel was stained by CD31 (Rabbit polyclonal antibody, Abcam, 1:200) and its proliferating cells were stained by Ki67 (Rabbit
polyclonal antibody, Abcam, 1:200). The blood count was determined by a MEK-6318 K Hematology Analyzer (Nihon Kohden, Japan), and H&E staining of the liver tissue sections
was conducted. (A) CD31; (B) Ki67; (C) Liver toxicity; (D) Hematotoxicity; (1) 5% glucose (negative control); (2) Taxol formulation (10 mg/kg); (3) PEG-PTX (equimolar to 10 mg/
kg PTX); (4) PEG–VC–PABC–PTX (equimolar to 10 mg/kg PTX); (a) Hemoglobin concentration; (b) Platelet count; * pb0.05 vs 5% glucose group.

628
L. Liang et al. / Journal of Controlled Release 160 (2012) 618–629
pyknosis can be observed in the Taxol group, indicating its effect on
liver.
and the enzyme-sensitive linker. In vivo, the concentration of PEG–
VC–PABC–PTX may not be high because of the dilution of blood.
As shown in Table 3, in vitro cytotoxicity study shows that the
efficacy of PEG–VC–PABC–PTX was significantly lower than PTX
(pb0.0005), while in vivo PEG–VC–PABC–PTX was found to be signif-
icantly effective than PTX (Taxol) at most of test points (Fig. 6). In
vitro, free PTX could directly act on tumor cells while PEG–VC–
PABC–PTX had to release PTX first then interact with tumor cells.
Since conjugate release PTX slowly (Fig. 3), it was expected that
free PTX showed better effect in vitro. However, the selectivity in
drug release from the conjugate in tumor tissue in vivo was guaran-
Moreover, platelet count and hemoglobin concentration are
shown in Fig. 7D-a and D-b, respectively. None of the conjugates in-
duced significant changes compared to negative control. However,
Taxol formulation showed the lowest values among all treatment
and also a significant inhibition on hemoglobin (pb0.05 vs negative
control). The higher toxicity of Taxol formulation was likely due to
the additives in its formulation.
4
. Discussion
teed by both EPR effect and C
B
-sensitivity, while free PTX in vivo
suffered from quick elimination, resulting in better efficacy of the
It is well known that the presence of Cremophore EL and ethanol
C
B
-sensitive conjugate.
(
50/50, V/V) causes an array of severe adverse effects, which ex-
It was noticed that the behaviors of the two conjugates were al-
tremely narrow the use of PTX [37,38]. Without the use of Cremo-
phore EL and ethanol, the two PTX conjugates prepared in this
study will definitely have advantages over the present formulation
of PTX injection (Taxol) in terms of less adverse effects if used in clin-
ic, especially the hypersensitivity. Studies reveal that each PEG subu-
nit is tightly associated with two or three water molecules [15], which
make the conjugates water-soluble. PEG5000 was used here to provide
enough PEG subunit, resulted in completed solubilization of highly
insoluble PTX and a thousand time increase in the solubility. So, the
second advantage of the two PTX conjugates here is their high solu-
bility in water, saline or 5% glucose as seen in Table 1, which makes
the preparation and use of PTX injection a very easy process.
most the same in cell tests in vitro, cytotoxicity or cell cycle test with-
out C , though they released PTX differently in vitro. Firstly, this may
B
be partially related to the different test conditions between drug re-
lease and cell tests, such as the shaking (100 rpm) in drug release
study. In other words, PTX release from PEG-PTX may slow down in
the condition of cell tests, narrowing the difference in drug release
between these two conjugates. Secondly, in the tumor cell tests, the
C sensitivity of PEG–VC–PABC–PTX is favorable for its effect on
B
cells. All these facts may result in the similar effect of these two con-
jugates in cell tests in vitro.
As we know, PTX molecules exert their anti-tumor activity through
the stabilization of microtubule assemblies, increase the fraction of
In the design of our prodrugs, Val–Cit dipeptide was chosen as the
linker because it was reported to be the most stable one under blood
cells in G
[35,36]. When MCF-7 cells were incubated with PEG–VC–PABC–PTX,
cell number in G -M phase dramatically increased after C was
added, while other groups changed little (Table 4 and Fig. 4). This
study indicated that PEG–VC–PABC–PTX is C -sensitive and it works
through the release of free PTX molecules. The C -sensitivity of
2
-M phase, and thus interrupt mitosis and cellular division
circulation (without C
B
in blood) among several substrates of C
B
[30].
2
B
PABC was also used as the spacer according to the previous report
that drug will not release if dipeptide is directly connected to a
B
drug, due to the lack of enough space for C
B
to recognize [29]. It is
B
supposed that PEG–VC–PABC–PTX conjugate may hydrolyze to
PABC–PTX which is unstable and then quickly releases free PTX
through spontaneous 1, 6-benzyl elimination and decarboxylation
based on the chemical point of view [40,41].
PEG–VC–PABC–PTX had been proved again in sensitivity experiment
as seen in Fig. 3B and C.
As seen in Fig. 4, it is obvious that PEG–VC–PABC–PTX conjugate
showed the optimal anti-tumor efficacy in the two conjugates. This
The apparent particle size of each kind of conjugate was between
B
could be explained by their release properties and C sensitivity. In
1
00 nm and 200 nm. Different from micelles with a mean particle size
vivo, thanks to the PEGylation, both conjugates will not eliminate
very faster in blood circulation, however, this is not the case for free
drug. For PEG-PTX, it release free PTX faster than PEG–VC–PABC–
PTX (Fig. 3), may leading to faster drug elimination in vivo and less
lower than 100 nm, conjugates were more likely to form the “nano-
aggregation” when dissolved in aqueous phase, with a bigger inner
core and a relatively large diameter (larger than 100 nm in average),
which was previously reported [39]. At a given concentration of con-
jugates, some of them may aggregate while some of them may not
aggregate, which was observed in TEM photograph of conjugates
drug accumulation into tumor tissue. Additionally, without the C
sensitive linkage, PEG–PTX demonstrated no C sensitivity (Table 4),
B
B
suggesting a non-specific drug distribution in vivo. On the other
hand, the slow release of PTX from PEG–VC–PABC–PTX (Fig. 3) sug-
gests a long circulation time of drug in vivo and more drug distribu-
(
Fig. 2). Here, the uniformity of particle size might be affected by
the conjugate structure, molecular weight, concentration, preparation
condition, and so on. The concentration of conjugates in DLS study
B
tion in tumor. Furthermore, the C -sensitive conjugate may release
(
Table 2) was much high than that in TEM observation, which may
more PTX than other groups in tumor tissue. As a result, a better anti-
tumor efficacy was observed in the group of PEG–VC–PABC–PTX. On
the other hand, based on the efficacy as well as toxicity (Fig. 6 and
so on), it was clear that PEG–VC–PABC–PTX provides advantage
over the current Taxol formulation with respect to therapeutic index.
As for the tumor model, typically, MCF-7 cells are estrogen-
dependent and require ovariectomy and estrogen supplementation
for predictable growth in vivo [42], although the biological properties
of different MCF-7 cell lines may be different when derived from
different sources [43]. By the way, Cremophor/ethanol was well un-
derstood for causing severe adverse effects but not for efficacy im-
provement, so they were not used as controls in the efficacy study.
For the body weight loss (Fig. 6C), there was no significant differ-
ence among all groups. The biggest difference in the average weight
loss among all groups was about 5%. The reasons for this fact was
not very clear, however, likely it was resulted from the large fluctua-
tion frequently seen in animal test due to complicated factors. For in-
stance, both tumor growth and drug treatment will cause body
cause greater fluctuation in particle size. These might be the reasons
for the higher polydispersity.
As indicated in Fig. 3A, PEG-PTX released PTX faster than PEG–VC–
PABC–PTX. This could be elucidated by the different structures
between these two conjugates, ester bond for PEG-PTX and amide
linkage for PEG–VC–PABC–PTX, namely, amide bond is more stable
than ester bond.
B
It was also observed that even with C , the drug release from PEG–
VC–PABC–PTX was not very fast (Fig. 3), possibly due to the steric
hindrance of PEG at the condition of drug release. Because of the de-
tectability of PTX by HPLC, we have to test the drug release at a rather
high concentration of PEG–VC–PABC–PTX (800 nm/mL). At such high
condition, the intense and long chains of PEG may interfere with the
interaction of C
concentration of PEG–VC–PABC–PTX such as that in cell cycle test
20 nm/L), PEG–VC–PABC–PTX took effect within 12 h in the pres-
ence of C (Table 4), suggesting much easier interaction between C
B
with enzyme-sensitive linker. In the case of lower
(
B
B

L. Liang et al. / Journal of Controlled Release 160 (2012) 618–629
629
[
[
[
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exhibited no antitumor efficacy in vivo (Fig. 5). At the same dosage,
effect of Taxol group and PEG–VC–PABC–PTX group was basically
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5
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fect on cell cycle. Besides, this novel conjugate exhibited significant
effects of antitumor, anti-angiogenesis and anti-proliferation in vivo,
while the control conjugate was almost inefficacious at the same
test. In comparison with PTX, the conjugates were very water soluble.
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B
with less adverse effects. Moreover, the C -sensitive conjugate had a
similar in vivo efficacy with the Taxol formulation, but much lower
in vivo toxicity at the same doses. At the tolerant dose, however,
PEG–VC–PABC–PTX showed significantly better antitumor efficacy
than that of Taxol formulation. Therefore, design and application of
a water-soluble and C -sensitive conjugate, like PEG–VC–PABC–PTX
B
in this study, might be a potential strategy for the therapy with PTX.
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Acknowledgment
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30] S.O. Doronina, B.E. Toki, M.Y. Torgov, B.A. Mendelsohn, C.G. Gerveny, D.F. Chace,
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This study was supported by projects from Ministry of Science
and Technology, PR China (No. 2009CB930300 and No. 2009ZX09310-
0
01) and National Science Foundation (No. 81130059).
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