Fabrication of reduction-degradable micelle based on disulfide-linked graft copolymer-camptothecin conjugate for enhancing solubility and stability of camptothecin

Article abstract of DOI:10.1016/j.polymer.2010.09.004

This research is aimed to develop a reduction-degradable micelle delivery system based on polymer-camptothecin (CPT) conjugate in order to enhance the solubility and stability of CPT in aqueous media. Firstly, disulfide-linked poly(amido amine) (SS-PAA) containing alkyne groups was synthesized by Michael addition polymerization between propargyl amine and N,. N'-bis(acryloyl) cystamine (BAC). And then, azide-functionalized CPT derivatives were conjugated with SS-PAA by click cycloaddition. Further grafting of residual alkyne groups in SS-PAA with azide-terminated poly(ethylene glycol) methyl ether (mPEG) gave mPEG-. g-SS-PAA-CPT conjugate. At last, micelles with size of ca. 88 nm were fabricated from mPEG-. g-SS-PAA-CPT conjugate, suggesting their passive targeting potential to tumor tissue. It was worthy of note that the drug-loaded system of mPEG-. g-SS-PAA-CPT micelles improved the solubility and stability of CPT in aqueous media. Owing to the reduction degradability of disulfide linker in main chain of mPEG-. g-SS-PAA-CPT, the CPT sustainably release from micelles together with the gradual cleavage of polymer in the presence of dithiothreitol (DTT) at the concentration of simulating the intracellular condition while almost no change occurred at the level of DTT corresponding to extracellular condition. Furthermore, the cell viability results showed the essential decrease of cytotoxicity to L929 cell line. These results present a strategy in designing anti-tumor CPT-polymer conjugates for highly selective delivery to tumor cells.

Full text of DOI:10.1016/j.polymer.2010.09.004

Polymer 51 (2010) 5107e5114
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Fabrication of reduction-degradable micelle based on disulfide-linked graft
copolymer-camptothecin conjugate for enhancing solubility and stability
of camptothecin
a
a
,
b
,
, Yaping Li , Jiahui Yu ,**, Jinghua Chen d
c
b
*
Honglei Fan , Jin Huang
a
College of Chemical Engineering, Wuhan University of Technology, Wuhan 430070, PR China
b
c
Institutes for Advanced Interdisciplinary Research, East China Normal University, Shanghai 200062, PR China
Center for Drug Delivery System, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, PR China
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 June 2010
Received in revised form
This research is aimed to develop a reduction-degradable micelle delivery system based on polymer-
camptothecin (CPT) conjugate in order to enhance the solubility and stability of CPT in aqueous media.
Firstly, disulfide-linked poly(amido amine) (SS-PAA) containing alkyne groups was synthesized by
Michael addition polymerization between propargyl amine and N,N -bis(acryloyl) cystamine (BAC). And
then, azide-functionalized CPT derivatives were conjugated with SS-PAA by click cycloaddition. Further
grafting of residual alkyne groups in SS-PAA with azide-terminated poly(ethylene glycol) methyl ether
17 August 2010
0
Accepted 1 September 2010
Available online 15 September 2010
(
mPEG) gave mPEG-g-SS-PAA-CPT conjugate. At last, micelles with size of ca. 88 nm were fabricated from
Keywords:
mPEG-g-SS-PAA-CPT conjugate, suggesting their passive targeting potential to tumor tissue. It was
worthy of note that the drug-loaded system of mPEG-g-SS-PAA-CPT micelles improved the solubility and
stability of CPT in aqueous media. Owing to the reduction degradability of disulfide linker in main chain
of mPEG-g-SS-PAA-CPT, the CPT sustainably release from micelles together with the gradual cleavage of
polymer in the presence of dithiothreitol (DTT) at the concentration of simulating the intracellular
condition while almost no change occurred at the level of DTT corresponding to extracellular condition.
Furthermore, the cell viability results showed the essential decrease of cytotoxicity to L929 cell line.
These results present a strategy in designing anti-tumor CPT-polymer conjugates for highly selective
delivery to tumor cells.
Reduction-degradable
Camptothecin
Micelle
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
polymers, and usually locates in main chain, side chain or cross-
linker. A notable fact has been found that the cleavage of disulfide
Besides the traditional intelligent drug carriers based on the
temperature- and pH-sensitive micelles and polymersomes [1e3],
the reduction-response biodegradable polymers have emerged as
a fascinating class of drug-loading materials [4], in the forms of
drug-conjugate [5,6], nano/micro-gel [7,8] and micelle [9,10], for
intracellular triggered delivery and release of protein and low-
molecular-weight drug. In the view of molecular level, the disulfide
linkage is the typical character of these reduction-sensitive
linkage, due to the thiol-disulfide exchange reaction, is sensitive to
the reduction conditions in human body. In the body circulation
and the extracellular milieus, the disulfide bonds showed enough
stability to a low concentration of glutathione tripepetide (GSH) as
ca. 2e20 mM. However, the intracellular high concentration of GSH
(0.5e10 mM) can lead to the quick degradation of disulfide-linked
polymers. As a result, the drugs conjugated or encapsulated with
these reducible polymers are destined for intracellular delivery and
release. In addition, these reduction-response polymers are espe-
cially suitable for the triggered delivery of tumor-specific drug
because of the higher concentration of GSH in tumor tissue at least
4
-fold over normal tissues [4].
*
Corresponding author. College of Chemical Engineering, Wuhan University of
2
0(S)-Camptothecin (CPT), a pentacyclic alkaloid isolated from
Technology, Wuhan 430070, PR China. Tel.: þ86 27 63373510; fax: þ86 27
the Chinese tree Camptotheca acuminate [11], has been verified
as a broad range of anti-cancer activity in animal models
[12]. However, both low solubility in aqueous medium and ring-
8
7859019.
** Corresponding author.
E-mail addresses: huangjin@iccas.ac.cn (J. Huang), jhyu@sist.ecnu.edu.cn (J. Yu).
0
032-3861/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymer.2010.09.004

5108
H. Fan et al. / Polymer 51 (2010) 5107e5114
opening of active lactone at physiological pH, as well as high
toxicity, greatly limit its clinical application [13,14]. It is known
that a pH-dependent equilibrium between lactone form and
carboxylate form in aqueous solution for CPT (Fig. 1) results in the
inactive carboxylate form of most CPT molecules at physiological
pH, and hence produces bone marrow and nonhematologic
toxicities. On the other hand, the closed lactone ring (E-ring) of
CPT is essential for its anti-tumor activity [15,16]. The 20-OH
group of the E-ring of CPT is necessary for ring opening, and can
participate into the enhanced rate of lactone hydrolysis at phys-
iological pH. At the same time, serum albumin preferentially
binds the carboxylate form of CPT to shift the lactone/carboxylate
equilibrium in favor of the carboxylate form [17,18]. As a result,
acylation of the 20-OH group has been verified to inhibit the
participation of lactone hydrolysis, and hence significantly
enhanced the stability of the E-ring of CPT. Meanwhile, 20-O-
acylated CPT possesses no intrinsic topoisomerase I inhibitory
activity [19e21]. Currently, it become popular that the 20-OH of
CPT is covalently conjugated with water-soluble polymer, such as
2. Experimental part
2.1. Materials
Cystamine hydrochloride, propargyl amine, acryloyl chloride,
poly (ethylene) methyl ether (M
n
¼ 1900) and p-Toluesulfonyl
chloride were purchased from Alfa Aesar and used as received.
Dithiothreitol (DTT), Sodium azide, CuBr was obtained fromAladdin.
6-Bromohexanoic acid (98%), N,N’-dicyclohexylcarbodiimide (DCC)
(99%), N,N-Diisopropylethylamine (DIEA), N,N-dimethylformamide
(DMF), triethylamine, and dimethylamino pyridine (DMAP) were
purchased from Sinopharm Chemical Reagent Co., Ltd. DMF was
dried with 4 Å molecular sieve and redistilled before use. CPT,
5-diphenyltetrazolium bromide, MTT were obtained from
SigmaeAldrich.
2.2. Cell line and culture
Mouse muscular cell line L929 was supplied from Institute of
Biochemistry & Cell Biology, Chinese Academy of Sciences. Cells
were cultured in RPMI 1640 (Gibco BRL, Paris, France), supple-
mented with 10% fetal bovine serum (FBS, HyClone, Logan, Utah),
polyethylene glycol (PEG) [21], poly(
-cyclodextrin based polymers [23], poly(1-hydroxymethyl-
ethylene hydroxyl-methyl formal) [24], poly[N-(2-hydroxypropyl)
methacrylamide] (HPMA) [25], -poly[(N-carboxybutyl)-
L-glutamic acid) (PGA) [22],
b
a
,
b
L
-
streptomycin at 100
mg/mL, and penicillin at 100 U/mL. All cells
ꢀ
aspartamide] (PBAsp) [26], and so on. This not only enhanced the
stability of CPT in the active lactone form, but also improved
its water-solubility. In this case, the release of CPT was under
control by the hydrolysis of ester bonds between polymer and
CPT. It has been found that the PGA-CPT conjugate contributed to
superior in vivo activity to the HT-29 colon and NCI-H460 lung
carcinoma models, in contrast to free CPT [22]. The further study
revealed that the conjugation of CPT with PGA altered drug
pharmacokinetics, and increased drug accumulation in tumors
based on the “enhanced permeability of tumor vessels and
retention of macromolecules (EPR)” effects [27]. It is worth of
noting that the combination between hydrophobic CPT and
hydrophilic polymer contributes to the possibility of self-
assembly. The formed micelles generally exhibited the decreased
toxicity, and showed the potential of passive targeting to tumor
tissue [23e25].
In this study, we synthesized the reduction-sensitive polymer-
CPT conjugate by successively reacting azide-functionalized CPT
and poly (ethylene glycol) methyl ether (mPEG) with alkyne focal
groups in disulfide-linked poly (amido amine) (SS-PAA), and then
fabricated the micelles to expectedly achieve the passive targeting
to tumor tissue. Moreover, the effects of nanoencapsulation on the
stability of its active form in physiological pH level and its cyto-
toxicity were evaluated. At last, the dithiothreitol (DTT) was used to
simulate the different reduction conditions in body, and hence the
drug release profiles together with the reduction degradation of the
micelle were investigated.
were incubated at 37 C in humidified 5% CO atmosphere. Cells
were splited by using trypsin/EDTA solution when almost
confluent.
2
0
2.3. Synthesis of N,N -bis(acryloyl) cystamine (BAC)
BAC was synthesized according to the literature [28]. Cystamine
dihydrochloride (10 mmol) was dissolved in a mixture of 15 mL of
3.5 M NaOH and 10 mL chloroform. This solution was heated up to
ꢀ
50 C, and 5 mL of chloroform containing 20 mmol of acryloyl
chloride was added dropwise under constant stirring over 15 min
while the reaction temperature was maintained at 50 C. After
ꢀ
separating the phases while still warm, the aqueous phase was
discarded. The remaining organic phase was cooled to room
temperature, and the product precipitated directly from the solu-
tion. The white crystal product was recovered by filtration and
recrystallized from chloroform to give a yield 50%.
2.4. Synthesis of disulfide-linked poly(amido amine) (SS-PAA)
As shown in Fig. 2, the SS-PAA was synthesized by the Michael
addition polymerization between propargyl amine and BAC [29].
Firstly, BAC (2.60 g, 10 mmol) and propargyl amine (550.8 mg,
10 mmol) were added into a flask and dissolved in 20 mL MeOH.
The solution was stirred at room temperature in the dark under
nitrogen atmosphere for 7 days. Subsequently, 10 mol% excess
propargyl amine was added into the reaction solution to consume
O
O
OH
N
O
N
pH>7
N
O
N
COO
OH
OH
Lactone form
Carboxylate form
Fig. 1. Chemical structure of the two forms of CPT.

H. Fan et al. / Polymer 51 (2010) 5107e5114
5109
Fig. 2. Synthesis scheme of mPEG-g-SS-PAA-CPT conjugate.
any unreacted acrylamide groups, and the reaction was performed
2.7. Characterizations
at room temperature for at least additional one week. At last, the
reaction solution was evaporated, and then the residue was dried in
vacuum at 40 C to give the SS-PAA with alkyne focal groups.
1
1
H nuclear magnetic resonance ( H NMR) spectra were recorded
ꢀ
TM
with a Bruker Avarice
measurement, the sample concentration was 35 mg mL in CDCl
500 NMR spectrometer. For NMR
ꢂ1
3
6
or DMSO-d .
2.5. Synthesis of mPEG-g-SS-PAA-CPT conjugate
FT-IR spectra were recorded on Nicolet Nexus 670 spectrometer.
The samples were pressed into pellets with KBr. UVeVis experi-
ments were conducted on a UV-1900PC spectroscope (Shanghai,
China).
mPEG-g-SS-PAA-CPT conjugate was synthesized by click reac-
tion between alkyne focal groups in SS-PAA and azide-functional-
ized mPEG (mPEG-N ) and CPT (CPT-N ). Herein, the CPT-N was
synthesized from the CPT using a protocol same as that described
by Emrick and Parrish [30]. while mPEG-N was prepared from
3
3
3
The molecular weights of mPEG-g-SS-PAA-CPT were measured
with Agilent 1200 gel permeation chromatography (GPC) (Agilent
Technologies Inc. Shanghai Branch). Agilent 1200 refractive index
detector and aqueous SEC start-up kit were used. Chromatography
columns (PL aquagel-OH MIXED columns, Polymer Laboratories
Ltd. Amherst, MA, USA) were calibrated with poly (ethylene glycol)
3
mPEG according to the previous literature [31]. A representative
procedure for the click conjugation is as follows: SS-PAA
(
0
315.46 mg, 1 mmol acetylene) and camptothecin azide (97.4 mg,
.20 mmol) were dissolved in DMF in a small reaction vessel to
target 20% camptothecin grafting. N,N-Diisopropylethylamine
DIEA) (51.7 mg, 0.4 mmol) and CuBr (57.4 mg, 0.4 mmol), were
then added sequentially, and the reaction mixture was stirred in
the dark at room temperature for 48 h. Then mPEG-N (1.86 g,
.96 mmol) was added into the reaction solution to consume the
ꢀ
kit. The column temperature was maintained at 35 C. Mobile phase
was PBS buffer(0.137 M NaCl, 2.68 mM KCl, 3.2 mM Na
.5 mM KH PO , pH 7.4), and the flow rate was 1.0 mL/min.
The particle sizes and size distributions of mPEG-g-SS-PAA-CPT
2 4
HPO and
(
1
2
4
3
micelles were measured by dynamic light scattering (DLS) (Zeta-
sizer Nano ZS, Malvern Instruments, UK).
0
unreacted acetylene groups and the reaction was performed at
room temperature for additional two days. After removal of solvent,
the residue was dispersed in water, and thoroughly dialyzed against
distilled water for 7 days using a dialysis membrane (MWCO:
000 Da). The product was lyophilized to yield a slightly yellow
solid of 1.81 g (90%), and labeled as mPEG-g-SS-PAA-CPT.
Morphological observation of mPEG-g-SS-PAA-CPT micelles was
performed using transmission electron microscopy (TEM). A copper
grid with a carbon film was used. The copper grid was immersed in
a drop of polymer solution for 60 s, and then dried at room
temperature.
7
2.6. Micelle formation and critical micelle concentration
2.8. Degradation of mPEG-g-SS-PAA-CPT micelle
One milligram of mPEG-g-SS-PAA-CPT was dissolved in 8 mL of
DMSO, and stirred at room temperature for 2 h, then dialyzed
against de-ionized water for 48 h. The critical micelle concentration
mPEG-g-SS-PAA-CPT micelle was degraded in the presence of
water-soluble reducing agents, dithiothreitol (DTT) in PBS buffer
[9]. The concentrations of DTT in mixture solution were set at
40 mM, 20 mM and 10 mM, respectively. In a typical procedure,
mPEG-g-SS-PAA-CPT micelle (20 mg, 0.011 mmol disulfide) was
(
CMC) was determined using pyrene as a fluorescence probe. The
concentration of mPEG-g-SS-PAA-CPT conjugate was varied from
ꢂ6
ꢂ7
ꢀ
8
6
ꢁ 10 to 1 mg/mL and the concentration of pyrene was fixed at
treated with DTT in 27.5 mL PBS buffer under nitrogen at 37 C.
ꢁ 10 mol/L. The fluorescence spectrum was recorded on F-4500
Aliquots were periodically withdrawn, diluted with PBS buffer at
ambient temperature, and analyzed by GPC immediately to deter-
mine the extent of cleavage of the disulfide bonds in the copolymer.
The size change of micelles was monitored by DLS measurement at
different time intervals. Finally, the solution was dialyzed against
Fluorescence Spectrophotometer (Hitchi F-4500) with the excita-
tion wavelength of 390 nm. The CMC was estimated as the cross-
point when extrapolating the intensity ratio of I339/I337 from low to
high concentration regions.

5110
H. Fan et al. / Polymer 51 (2010) 5107e5114
Fig. 3. 1H NMR spectra of (A) BAC in CDCl
, (B) SS-PAA in DMSO-d , (C) mPEG-g-SS-PAA-CPT conjugate in DMSO-d and (D) mPEG-g-PAA-SH in CDCl .
3 6 6 3
distilled water for 7 days using a dialysis membrane (MWCO:
000 Da) to remove the excess DTT. The lyophilized sample was
characterized by H NMR.
carried out 2 h after preparing the standard solution to ensure the
complete conversion of CPT-lactone to CPT-carboxylate. Standard
calibration samples were prepared at concentrations ranging from
1
1
0.1 to 50 mg/mL. The calibration curves exhibited linear behavior
over the concentration range of about 3 orders of magnitude. The
detection limits were evaluated on the basis of a signal-to-noise
2.9. Stability of CPT in mPEG-g-SS-PAA-CPT micelle
ratio of 3 and were 0.02 mg/mL for CPT.
To elucidate the stability of CPT lactone in mPEG-g-SS-PAA-CPT
micelle over time at physiologic pH (7.4), the micelle was incubated
ꢀ
in PBS buffer at 37 C at a concentration of 0.5 mg/mL. 1 mL aliquots
2
.11. Cell viability assays
were taken from the medium for monitoring by using a UV/vis
spectrophotometry according to the method described by Zhao
et al. [20]. The ratios (A324/A345) were calculated from the intensity
of the characteristic UV absorption of CPT lactone form (
24 nm) and carboxylate form ( max: 345 nm), and used to evaluate
the stability of CPT lactone in mPEG-g-SS-PAA-CPT micelle.
In vitro cytotoxicity of mPEG-g-SS-PAA-CPT conjugate and CPT
to inhibit cells growth was determined by evaluation of the viability
of mouse muscular cell line (L929) by MTT method. Cells were
max
l :
3
l
3
seeded in 96 well plates at an initial density of 6 ꢁ 10 cells/well in
100 mL growth medium and incubated for 24 h to reach 80% con-
fluency at the time of treatment. Growth medium was replaced
with 100 L fresh serum-free media containing various amounts of
mPEG-g-SS-PAA-CPT conjugate or CPT. Cells were incubated for
48 h. And then, the culture medium was replaced by 100 L of MTT
solution (0.5 mg/mL). After further incubation for 4 h in incubator,
100 L of dimethyl sulfoxide (DMSO) was added to each well to
replace the culture medium and dissolve the insoluble formazan-
2
.10. Release profiles of CPT from micelle
m
Release of CPT from polymeric micelle was performed by the
method described by Opanasopit et al. with minor modification
m
[17]. The release profiles of CPT from mPEG-g-SS-PAA-CPT micelle
m
ꢀ
were studied at 37 ꢃ 0.5 C in three different media, i.e. 30 mL PBS
buffer (7.4) with 10 mM and 40 Mm DTT and neat PBS buffer (7.4)
under constant stirring. 5 mg of free CPT or 10 mg of mPEG-g-SS-
PAA-CPT were dissolved in a mixture of PBS (pH ¼ 7.4) and DMSO
(
10:1). Five milliliter of each sample solution was charged into
a dialysis tube (MWCO: 3500 Da). Then the dialysis tube was
immersed in the dialysis medium. At certain time intervals, 0.6 mL
aliquot of the dialysis medium was withdrawn, and the same
volume of fresh media was added, respectively. The sample solution
was analyzed by reverse-phase HPLC. Each experiment was carried
out in triplicate. Means and corresponding standard deviations
(
mean ꢃ SD) were shown as results.
The concentrations of the drugs were assayed by reverse-phase
HPLC (Agilent Technologies Inc. Shanghai Branch). Chromato-
graphic separations were achieved using a Zorbax Eclipse XDB-C18
ꢀ
column at 35 C. The mobile phase was a mixture of triethylamine
acetate (TEAA) buffer (0.1% v/v triethylamine, adjusted with glacial
acetic acid to pH ¼ 5.5)/acetonitrile with a ratio of 73:27 (v/v) for
the assay of CPT. Mobile phase was delivered at a flow rate of
1.0 mL/min. Agilent 1200 UV/vis detector was set at 368 nm for CPT.
The standard solution of CPT lactone and CPT-carboxylate were
made by dilution of CPT stock solution in DMSO with pH ¼ 3.5 and
1
2 PBS buffer, respectively. The analysis of CPT-carboxylate was
3
Fig. 4. FT-IR spectra (a) BAC, (b) mPEG-N , and (c) mPEG-g-SS-PAA-CPT conjugate.

H. Fan et al. / Polymer 51 (2010) 5107e5114
5111
Fig. 5. I339/I337 band intensity ratio of pyrene as a function of logarithm concentration
of micelle.
containing crystals. The optical density (OD) was measured at
570 nm using an automatic BIO-TEK microplate reader (Powerwave
XS, USA), and the cell viability was calculated from following
equation:
ꢀ
ꢁ
Cell viability ð%Þ ¼ ODsample=ODcontrol ꢁ 100
(1)
where ODsample represents an OD value from a well treated with
samples and ODcontrol from a well treated with PBS buffer only. Each
experiment was carried out in sextuplet. Mean and corresponding
standard deviations (mean ꢃ SD) were shown as results.
3
. Results and discussion
3.1. Synthesis of mPEG-g-SS-PAA-CPT conjugate
Fig. 7. (A) UV absorption curves of (1) CPT in pH 7.4 PBS, (2) mPEG-g-SS-PAA-CPT
conjugate in pH 3.5 PBS, (3) mPEG-g-SS-PAA-CPT conjugate in pH 12 PBS, (4) mPEG-g-
SS-PAA-CPT conjugate before incubation, and (5) mPEG-g-SS-PAA-CPT conjugate after
In this study, we report a water-soluble polymer-drug conjugate
ꢀ
3
0-day incubation in pH 7.4 PBS at 37 C; (B) A324/A345 ratios of mPEG-g-SS-PAA-CPT
from functionalized mPEG, camptothecin (CPT) and reduction-
degradable disulfide-linked poly(amido amine) (SS-PAA) by click
chemistry as shown in Fig. 2. Firstly, the BAC monomer was
ꢀ
micelle against incubation time in pH 7.4 PBS at 37 C.
synthesized according to a classic reaction pathway with acryloyl
1
chloride under conventional SchotteneBaumann conditions.
H
Fig. 8. Cell viability of mPEG-g-SS-PAA-CPT micelle at various CPT concentrations
against L929 cell line for 48 h (mean ꢃ SD, n ¼ 6).
Fig. 6. (A) Size distributions, and (B) TEM photographs of mPEG-g-SS-PAA-CPT micelle.

5112
H. Fan et al. / Polymer 51 (2010) 5107e5114
Fig. 9. Scheme of the reduction of mPEG-g-SS-PAA-CPT conjugate by DTT.
ꢂ1
typical of triazole ring
NMR confirmed the structure of BAC, and the detailed data of
chemical shifts were shown in Fig. 3 A. The SS-PAA was synthesized
by Michael addition of molar equivalents of the propargyl amine to
BAC. The reaction was continued until the solution became viscous
for magnetic stirring 7e10 days. Then, ca. 20 mol% excess of
propargyl amine was added and the mixture was allowed to react
for another 7 days to terminate the polymerization with amino end
groups. The final structure of SS-PAA was confirmed by 1H NMR
absorption at about 1600e1640 cm
appeared. In addition, the mPEG-g-SS-PAA-CPT conjugate showed
ꢂ1
ꢂ1
ꢂ1
the intense stretching bands at 2883 cm and 840 cm for mPEG
block, and a broad band eNH at about 3200e3600 cm for the
SS-PAA.
3.2. Micelle formation
(
Fig. 3B). The signal at 2.23 ppm (eC^CeH) indicated the presence
The mPEG-g-SS-PAA-CPT conjugate was found to be water-
soluble and to form micelle in aqueous solution. The critical micelle
concentration (CMC) was determined using pyrene as a probe. The
plot of intensity ratio I339/I337 of the pyrene excitation spectra
against the logarithm of the micelle concentration is shown in
Fig. 5. The CMC value can be determined at a micelle concentration
of onset of I339/I337 ratio increase. The mPEG-g-SS-PAA-CPT conju-
gate showed a CMC of ca. 0.003 mg/mL.
Dynamic light scattering (DLS) measurement shows that mPEG-
g-SS-PAA-CPT conjugate formed micelle with sizes of ca. 88 nm
(Fig. 6 A), suggestion of their efficient passive target potential to
tumor tissue. TEM micrograph revealed spherical shapes and good
dispersity with nanoscale sizes of 50 nm (Fig. 6B). The smaller size
observed by TEM as compared to that measured by DLS, which
might attribute to evaporation shrinkage of the PEG shell.
of alkyne focal group. The molecular weight of SS-PAA measured by
GPC (mobile phase: THF; standard sample: polystyrene) was about
M
4
4
n
¼ 2.1 ꢁ 10 , M
w
¼ 9.8 ꢁ 10 . The inherent nature of Michael
addition resulted in relatively high PDI of 4.67.
Coupling of acetylene-functionalized SS-PAA with azide-deri-
vated camptothecin was carried out using N,N-diisopropylethyl-
amine (DIEA)/CuBr as catalyst in DMF at room temperature by
targeting 20 mol% CPT incorporation (or one CPT per five monomer
repeat units). Further grafting with mPEG is necessary to improve
the water solubility of SS-PAA-CPT conjugate. Click cycloaddition of
the residual acrtylene groups with azide-terminated mPEG was
performed by adding mPEG-N into the reaction solution. The
3
molecular weight of mPEG-g-SS-PAA-CPT conjugate measured by
GPC was about M
high PDI of SS-PAA, grafting degree of mPEG and conjugation
probability of CPT resulted in high weight distribution of the mPEG-
4
5
n
¼ 5.9 ꢁ 10 , M
w
¼ 3.8 ꢁ 10 , and PDI ¼ 6.44. The
3.3. Stability of CPT in mPEG-g-SS-PAA-CPT micelle
1
g-SS-PAA-CPT. H NMR confirmed the structure of mPEG-g-SS-PAA-
CPT conjugate, and the detailed data of chemical shifts were shown
in Fig. 3C. The click reaction was confirmed from the appearance of
new proton signals at 7.51 ppm (i, singlet) and 7.27 ppm (q, singlet)
typical of methine proton of the triazole ring from the mPEG and CPT
grafts, as well as the methylene protons at 4.53 (j, singlet) and 4.28
As well known, the carboxylate conversion limits the bioavail-
ability and the in vivo therapeutic efficacy of CPT, and thus, main-
tenance of the lactone structure is a prerequisite for an improved
stability of CPT in mPEG-g-SS-PAA-CPT micelles. The characteristic
UV absorption of mPEG-g-SS-PAA-CPT micelles was monitored in
PBS (pH 7.4). As shown in Fig. 7 A, the maximum UV wavelength
(
C1) ppm adjacent to the triazole, respectively. The integral ratios of
protons at 4.53 and 4.28 ppm were considered to suggest the
conjugation probability of CPTand grafting extent of mPEG, in terms
of percent monomers containing the conjugation with ca. 20% CPT
and ca. 72% mPEG. The FT-IR spectra (Fig. 4) show a complete
absorption (
l
) of mPEG-g-SS-PAA-CPT in acidic media (pH ¼ 3.5)
max
and in basic media (pH ¼ 12) located at 324 and 345 nm respec-
tively, which represented the lactone and carboxylate form of CPT
in mPEG-g-SS-PAA-CPT micelle [20]. However, the maximum UV
absorptions of native CPT lactone and carboxylate forms were at
ꢂ1
disappearance of the azide vibrational peak at 2106 cm , and a new
ꢀ
Fig. 10. (A) The molecular weight distribution of mPEG-g-SS-PAA-CPT conjugate in the incubation with 40 mM DTT in PBS buffer (pH ¼ 7.4) at 37 C; (B) The degradation rate of the
copolymer in the incubation in the incubation with various DTT in PBS buffer (pH ¼ 7.4) at 37 ꢀC.

H. Fan et al. / Polymer 51 (2010) 5107e5114
5113
of the copolymers are shown in Fig. 10A. The peak intensity of small
molecules around 9 min of elution time increased together with the
decrease of peak intensity of polymer fraction with high molecular
weight, indicating that the copolymer degraded as smaller
segments. At a longer incubation time (i.e. up to 7 d), most of the
copolymers were broken into oligomers and small complexes. The
GPC trace indicated that the degraded segments had
3
3
M
n
¼ 2.35 ꢁ 10 , M
w
¼ 2.47 ꢁ 10 and PDI ¼ 1.05. The degradation
rate of the copolymer increased with an increase in the amount of
DTT as shown in Fig. 10B. When the concentration of DTT was
40 mM, over 85% copolymer was degraded in 7 d. The degradation
rate of the copolymer was slower than those of previous reports [4],
which might be ascribed to the steric hindrance of grafted mPEG
and conjugated CPT.
The size change of micelle in response to DTT in PBS buffer
(pH ¼ 7.4) was traced by DLS measurement as shown in Fig. 11.
Notably, aggregation of micelle occurred in present of DTT and the
size increased from 87 nm to 350 nm after 7 d when the concen-
tration of DTT was 40 mM. Furthermore, the micelles size increased
with an increase of the DTT concentration. The aggregation of
micelle might be ascribed to the reductive cleavage of the internal
disulfide bonds, which changed the hydrophilicityehydrophobicity
equilibrium for the original assembly of the copolymer.
Fig. 11. The size change of mPEG-g-SS-PAA-CPT micelle in respone to DTT in PBS buffer
pH ¼ 7.4) determined by DLS measurement.
(
3
55 and 368 nm. That is to say, the conjugation of CPT with the
SS-PAA resulted in a blue shift of the maximum UV absorption of
lactone and carboxylate forms to 324 and 345 nm, respectively. So
A
324/A345 ratios were calculated from the intensity of the maximum
3
.6. Release of CPT from micelle
UV absorption of lactone and carboxylate forms of CPT, and used to
evaluate the stability of CPT in mPEG-g-SS-PAA-CPT micelle as
shown in Fig. 7B. The A324/A345 ratios of mPEG-g-SS-PAA-CPT
micelle surrounded an analogical value in agreement with experi-
mental error in the whole incubation time (even in one month
incubation). On the other hand, the lactone form of native CPT in
PBS disappeared quickly. It was thought that the esterification of
the 20-OH group of CPT was able to improve its stability. In addi-
tion, the micellization of mPEG-g-SS-PAA-CPT conjugate in aqueous
medium was also beneficial for the enhancement of CPT stability
Since the CPT was conjugated to the reduction-degradable
polymer via hydrolysable ester bonds, it was believed that the CPT
could be released from mPEG-g-SS-PAA-CPT prodrug by the
hydrolysis of ester bonds. As shown in Fig. 12, the free CPT drug was
nearly completely released during 24 h. Compared with free CPT,
the accumulative drug release behaviors of mPEG-g-SS-PAA-CPT
micelle in the absence of DTT showed slow gradual profiles,18.6% of
CPT was released in 14 days due to the hydrolysis of ester bonds
between CPT and polymer backbone, suggesting its sustained drug
release behaviors. Interestingly, CPT was rapidly released from the
mPEG-g-SS-PAA-CPT micelle in the presence of DTT. For example,
ca. 66% CPT was released in 14 days. It was thought that the
structure of mPEG-g-SS-PAA-CPT micelle was destroyed due to the
degradation of disulfide bonds in polymer backbone under
a reduction environment, which resulted in the enhanced hydro-
lysis of ester bonds between CPT and polymer backbone. This was
in line with the results that the mPEG-g-SS-PAA-CPT conjugate was
degraded and the micelle was destabilizad in the presence of DTT.
[32].
3.4. Cytotoxicity of mPEG-g-SS-PAA-CPT micelle
The in vitro cytotoxicity of mPEG-g-SS-PAA-CPT micelle was
evaluated with mouse muscular cell line (L929) by MTT assay.
Representative concentration-growth inhibition curves showing
the effects of treatment with mPEG-g-SS-PAA-CPT micelle on the
growth of L929 after 48 h are shown in Fig. 8. Although both mPEG-
g-SS-PAA-CPT micelle and CPT inhibited cell growth in a dose-
dependent manner, the former was consistently less toxic than that
of CPT. It was thought that the decreased cytotoxicity of mPEG-g-
SS-PAA-CPT conjugate resulted from the slow release of CPT from
the mPEG-g-SS-PAA-CPT micelle.
3.5. In vitro degradation of mPEG-g-SS-PAA-CPT Conjugate
The degradability of the mPEG-g-SS-PAA-CPT conjugate was
investigated by the incubation of copolymers with DTT under
physiological conditions. The disulfide bond was cleaved by DTT,
which has been explained by the formation of more stable six-
membered cyclic disulfide after the oxidation of such dithiol as
1
shown in Fig. 9 [33]. H NMR and GPC measurements confirmed the
cleavage of disulfide bonds to yield mPEG-g-PAA-SH. The new
proton signal at 1.5 ppm (r, eSH) included the presence of thiol
end-group and new format peaks at 2.75 ppm of ethylene protons
next to the thiol end-group (eCH
the signal at 2.92 ppm attributable to the methylene protons
neighboring to disulfide bond (eCH eSSeCH e) completely dis-
appeared. In addition, the changes of molecular weight distribution
2
eSH) are shown in Fig. 3D while
2
2
Fig. 12. In vitro release profiles of CPT from free drug and mPEG-g-SS-PAA-CPT micelle
in pH 7.4 PBS at 37 C (means ꢃ SD, n ¼ 3).
ꢀ

5114
H. Fan et al. / Polymer 51 (2010) 5107e5114
Although the enhanced hydrolysis of ester bonds between CPT and
polymer backbone could produce faster drug release, the hydrolysis
of ester bonds was still much lower than the cleavage of disulfide
bonds in the presence of DTT. At the same time, the AA-CPT
monomer also released when the depolymerization extent of main
chains is very high. As a result, the release of CPT was lower than
the polymer degradation rate.
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The research work was supported by the 973 Projects of Chinese
Ministry of Science and Technology (2009CB930300), National
Natural Science Foundation of China (50873080), Shanghai Munici-
pality Commission for Special Project of Nanometer Science and
Technology (0952nm05300), Shanghai Municipality Commission for
Non-governmentalInternationalCorporationProject(09540709000)
and International Corporation Project (10410710000), Foundation for
New Century Excellent Talents in University of Ministry of Education
of China (NCET-10-0435), and Fundamental Research Funds for the
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