Preparation of a camptothecin prodrug with glutathione-responsive disulfide linker for anticancer drug delivery

Article abstract of DOI:10.1002/asia.201301030

We present here a novel camptothecin (CPT) prodrug based on polyethylene glycol monomethyl ether-block-poly(2-methacryl ester hydroxyethyl disulfide-graft-CPT) (MPEG-SS-PCPT). It formed biocompatible nanoparticles (NPs) with diameters of approximately 122 nm with a CPT loading content as high as approximately 25 wt % in aqueous solution. In in vitro release studies, these MPEG-SS-PCPT NPs could undergo triggered disassembly and much faster release of CPT under glutathione (GSH) stimulus than in the absence of GSH. The CPT prodrug had high antitumor activity, and another anticancer drug, doxorubicin hydrochloride (DOX×HCl), could also be introduced into the prodrug with a high loading amount. The DOX×HCl-loaded CPT prodrug could deliver two anticancer drugs at the same time to produce a collaborative cytotoxicity toward cancer cells, which suggested that this GSH-responsive NP system might become a promising carrier to improve drug-delivery efficacy.

Full text of DOI:10.1002/asia.201301030

FULL PAPER
DOI: 10.1002/asia.201301030
Preparation of a Camptothecin Prodrug with Glutathione-Responsive
Disulfide Linker for Anticancer Drug Delivery
Zhigang Xu,[a, b] Dongdong Wang,[a] Shuang Xu,[a] Xiaoyan Liu,[a] Xiaoyu Zhang,[c] and
Haixia Zhang*[a]
Abstract: We present here a novel
camptothecin (CPT) prodrug based on
polyethylene glycol monomethyl ether-
block-poly(2-methacryl ester hydrox-
yethyl disulfide-graft-CPT) (MPEG-SS-
PCPT). It formed biocompatible nano-
particles (NPs) with diameters of ap-
proximately 122 nm with a CPT load-
ing content as high as approximately
25 wt% in aqueous solution. In in vitro
release studies, these MPEG-SS-PCPT
NPs could undergo triggered disassem-
bly and much faster release of CPT
under glutathione (GSH) stimulus than
in the absence of GSH. The CPT pro-
drug had high antitumor activity, and
another anticancer drug, doxorubicin
hydrochloride (DOX·HCl), could also
be introduced into the prodrug with
a high loading amount. The DOX·HCl-
loaded CPT prodrug could deliver two
anticancer drugs at the same time to
produce
a collaborative cytotoxicity
toward cancer cells, which suggested
that this GSH-responsive NP system
might become a promising carrier to
improve drug-delivery efficacy.
Keywords: cancer · cytotoxicity ·
disulfides · drug delivery · drug
design
Introduction
could not only improve in vivo drug efficacy but also signifi-
cantly reduce drug-associated side effects.
Since the concept of polymeric drug-delivery systems
(PDDSs) was first proposed in 1975 by Helmut Ring-
sdorf,[1,2] compelling achievements have been made in the
preparation of intelligent PDDSs owing to their unique
properties and structures such as nanoscale size, core–shell
structure, relatively high stability, and prolonged circulation
in the blood.[3–11] As we expected, drugs embedded in or
conjugated with inert nanocarriers showed therapeutic ad-
vantages over free drugs, but for most PDDSs, the carrier
materials were the major component (generally more than
90%), thereby resulting in low drug-loading contents and
thus excessive use of parenteral excipients.[12] Recently, to
improve the medical efficiency of drugs, many studies have
explored the attributes of 1) good stability in blood circula-
tion, 2) higher drug loading, 3) increasing the water solubili-
ty of lipophilic drugs, and 4) higher cytotoxicity toward
tumor tissues and cells.[13–15] Therefore, polymeric prodrugs
In the family of biocompatible water-soluble polymers in
polymeric prodrugs, poly(ethylene glycol) (PEG) and its de-
rivatives are most often used for grafting anticancer drugs
and therapeutic proteins on account of their advantages,
which include excellent solubility in many solvents, good
biocompatibility and natural nontoxicity, high flexibility, and
approval by the USFDA (United States Food and Drug Ad-
ministration).[13,16] Glutathione (GSH) is a thiol-containing
tripeptide, and it has been demonstrated that the concentra-
tion of GSH (2–8 mm) in tumor cells is significantly higher
than that in plasma (1–2 mm).[17,18] Introduction of a disulfide
bond into PDDSs has been widely reported.[19–22] Notably, in
the case of polymeric prodrugs, there have been only a few
examples of this reported.[23,24] One recent study also report-
ed the introduction of disulfide bridges into a camptothecin
(CPT) prodrug with a high drug-loading amount. Unfortu-
nately, limited by the chemical structure, it was difficult to
directly obtain the native CPT molecule during the release
process.[23]
[a] Dr. Z. Xu, D. Wang, S. Xu, Dr. X. Liu, Prof. Dr. H. Zhang
State Key Laboratory of Applied Organic Chemistry
Lanzhou University, Lanzhou 730000 (P.R. China)
Fax : (+86)931-8912582
In this study, we introduced a unique, GSH-responsive
prodrug design of CPT covalently linked to the polymer of
poly(ethylene glycol) monomethyl ether-block-poly(2-meth-
acryl ester hydroxyethyl disulfide) (MPEG-b-PMABHD)
and engineered with a GSH-induced drug-release mecha-
nism. The aim of our research was to synthesize a CPT pro-
drug with an amphiphilic structure that contained a hydro-
philic MPEG and hydrophobic CPT that could form core–
shell nanoparticles suitable for passive tumor targeting by
the enhanced permeability and retention (EPR) effect.[25–27]
Multiple CPT molecules could be grafted onto the polymer
by means of a disulfide linker,[28,29] which could induce faster
E-mail: zhanghx@lzu.edu.cn
[b] Dr. Z. Xu
School of Chemical and Biomedical Engineering
Nanyang Technological University
70 Nanyang Drive, Singapore 637457 (Singapore)
[c] Prof. Dr. X. Zhang
School of Basic Medical Science, Lanzhou University
Lanzhou 730000, (P.R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201301030.
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Haixia Zhang et al.
release of CPT in an endo-lyso-
some environment. As a result
of the low molecular weight of
PEG (1.9 kDa) and the well-de-
fined structure of polyethylene
glycol monomethyl ether-block-
poly(2-methacryl ester hydrox-
yethyl
disulfide-graft-CPT)
(MPEG-SS-PCPT), the prodrug
nanoparticles had a high CPT
loading amount. Meanwhile, we
were able to obtain the native
CPT molecules during the re-
lease process owing to a special
release mechanism. Further-
more, another anticancer drug,
doxorubicin
hydrochloride
(DOX·HCl), could also be
loaded into the prodrug nano-
particles. The resulting prodrug
was carefully characterized and
its biological effects were also
evaluated. The obtained results
indicated that the MPEG-SS-
PCPT prodrug had enough ad-
vantages to become a promising
CPT drug carrier for cancer
treatment.
Scheme 1. Synthesis of MPEG-SS-PCPT prodrug and the proposed release mechanism.
to achieve controlled conjugation of CPT, 2-methacryl ester
hydroxyethyl disulfide (MABHD) monomer that contained
a disulfide bond was designed and prepared. The H NMR
1
Results and Discussion
spectrum (Figure 1A) showed signals at d=5.60, 6.13, and
1.95 ppm that were attributable to the methacryl ester moi-
eties; resonances at d=4.42, 3.90, 2.96, and 2.87 ppm were
assignable to bis(2-hydroxyethyl) disulfide (BHD) moieties.
Furthermore, it was noteworthy that signals at d=5.60/
6.13 ppm (methylene protons of methacryl ester moieties)
and d=4.42 or 2.87 ppm (methylene protons of BHD moiet-
ies) had an intensity ratio of close to 1:1, which indicated
equivalent coupling of methacryloyl chloride and BHD.
Secondly, MPEG-b-PMABHD copolymers were prepared
by RAFT copolymerization of MABHD by using MPEG–
CPADB as a macro-RAFT agent and azodiisobutyronitrile
(AIBN) as the radical agent. The 1H NMR spectra displayed
clear characteristic peaks of MPEG moieties (d=3.38 and
3.65 ppm) and MABHD units (d=4.42, 3.90, 2.96, and
2.87 ppm) (Figure 1B). On the basis of the integral ratio of
peak k in Figure 1B, the average repeating unit number of
MABHD was determined to be 6. In this paper, the block
copolymer was referred to as PEG43-PMABHD6. Further-
more, the results of gel permeation chromatography (GPC)
showed that the resulting copolymer had a molecular weight
The synthesis of the MPEG-SS-PCPT prodrug is shown in
Scheme 1. Firstly, the macro radical addition–fragmentation
chain transfer (RAFT) agent MPEG-CPADB (CPADB: 4-
cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid) was prepared
by an esterification reaction; 1H NMR spectroscopic and
FTIR results indicated that MPEG-CPADB was prepared
successfully (see the Supporting Information). Furthermore,
Abstract in Chinese:
1
close to those determined from H NMR spectroscopic anal-
yses and low polydispersities of 1.12 (see the Supporting In-
formation), thus indicating the controlled synthesis of
PEG43–PMABHD6 copolymer.
Finally, the CPT drug molecules were grafted onto the
PEG–PMABHD block copolymer chains by means of cou-
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Haixia Zhang et al.
pling the hydroxy groups between CPT drug molecules and
PMABHD block copolymers in the presence of triphosgene.
The conjugation efficiency of CPT is about 55.8%, which
could be determined from the result of the drug-loading
amount of CPT. The resulting CPT prodrug was confirmed
1
by H NMR spectroscopy (see the Supporting Information).
The signals at d=8.70–8.72, 8.23, 8.20–8.12, 7.85-7.90, 7.71–
7.73, 7.35–7.37, 6.95–7.03, and 5.29–5.55 ppm corresponded
to CPT aromatic group protons, thus indicating the success-
ful grafting of CPT molecules.
The obtained MPEG-SS-PCPT could self-assemble to
form stable nanoparticles in phosphate buffer solution
(PBS; pH 7.4) because of its amphiphilic structure. The ag-
gregation behavior of aqueous MPEG-SS-PCPT solutions
was determined by fluorescence spectroscopy using Nile red
as a probe. From these results, an apparent critical micelle
concentration (CMC) of 41 mgmLÀ1 was obtained (see the
Supporting Information). Thus, we determined that micellar
assemblies of this novel CPT prodrug were formed at con-
centrations that exceeded CMC. Scheme 2 illustrates sponta-
neous formation of these nanoparticles. The TEM images
(Figure 2a) showed that the MPEG-SS-PCPT nanoparticles
Figure 1. 1H NMR spectra of A) MABHD and B) PMEG-b-PMABHD.
Scheme 2. Illustration of how GSH-activatable CPT prodrug nanoparti-
cles selectively release CPT in endosomal compartments.
Figure 2. A) TEM image and B) size distribution of MPEG-SS-PCPT nanoparticles.
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exhibited a spherical shape and was approximately 80 nm in
diameter. The size distribution of the nanoparticles was fur-
ther illustrated by dynamic light scattering (DLS) (Fig-
ure 2B), and its size distribution in water ranged from 100 to
148 nm with an average size of approximately 122 nm.
DOX·HCl was further loaded into the nanoparticles, and
the DOX·HCl-loaded MPEG-SS-PCPT nanoparticles had
a larger diameter of approximately 105 nm (see the Support-
ing Information). Such nanosized nanoparticles could easily
avoid renal filtration, thereby leading to prolonged resi-
dence time in the bloodstream, which would allow more ef-
fective targeting of diseased tissues.[30–33]
To assess the feasibility of the resulting CPT prodrug as
an anticancer drug delivery system, we studied the release
behavior in vitro under simulated physiological conditions
(pH 7.4). As reported previously,[29] the disulfide linker had
a characteristic GSH-induced release behavior, which might
result in a favorable GSH-induced release behavior of CPT
from MPEG-SS-PCPT nanoparticles. As shown in Fig-
ure 4A, an initial burst release of CPT from MPEG-SS-
Relative to the reported water-soluble CPT prodrugs or
conjugates, the resulting CPT prodrug had several special
features. For example, a low-molecular-weight hydrophilic
PEG polymer was used to increase the drug-loading amount
and simultaneously ensured the water solubility of prodrugs.
In addition, the release of CPT from the prodrug was also
different from that of other reports,[23,24] in which an addi-
tional ester bond cleavage was needed, whereas for MPEG-
SS-PCPT, the native CPT molecule could be obtained
during the release process owing to a special GSH-induced
mechanism.[28] Moreover, the PEG polymer might effective-
ly reduce the nonspecific uptake by the reticuloendothelial
system, prolong circulation time, and result in specific tumor
targeting by EPR effects.[34,35]
The UV-visible spectrum was used to further demonstrate
the loading of DOX·HCl into MPEG-SS-PCPT nanoparti-
cles. As shown in Figure 3, the aqueous solution of MPEG-
Figure 4. A) In vitro release of CPT from the MPEG-SS-PCPT nanopar-
ticles and b) DOX·HCl release from the DOX·HCl-loaded nanoparticles
in PBS (pH 7.4) at 378C with or without GSH treatment.
PCPT nanoparticles was obtained, which might be directly
caused by the physically trapped unreacted CPT in MPEG-
SS-PCPT nanoparticles. Although free CPT was removed as
much as possible in the synthesis and release process, it was
still difficult to remove unreacted CPT thoroughly. Further-
more, the burst release might also be due to the different
hydrolysis rates of MPEG-SS-PCPT nanoparticles caused by
the different conjugation efficiency of CPT, followed by
a very slow release. However, in the presence of GSH,
MPEG-SS-PCPT had a faster degradation and more CPT
was released. Based on the chemistry involved, the scission
of the disulfide bonds was expected to be followed by intra-
molecular cyclization and cleavage of the neighboring carba-
mate bond.[28] This, in turn, would serve to release native
CPT molecules from the MPEG-SS-PCPT nanoparticles.
The release of another drug, DOX·HCl, using MPEG-SS-
Figure 3. A) UV-visible spectra of a) MPEG-SS-PCPT, b) DOX·HCl, and
c) DOX·HCl-loaded MPEG-SS-PCPT dissolved in water.
SS-PCPT nanoparticles had a characteristic peak at around
365 nm, and for DOX·HCl it was at about 485 nm. Mean-
while, the water solution of DOX·HCl-loaded MPEG-SS-
PCPT nanoparticles had two characteristic peaks at around
365 and 485 nm. The DOX·HCl loading efficiency was ap-
proximately 60%, and the loading content was 10.7 wt%. In
addition, the color of free DOX·HCl in water was red,
whereas the color of DOX·HCl-loaded MPEG-SS-PCPT
was light purple, a phenomenon that might further demon-
strate the successful introduction of DOX·HCl.[3,12]
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PCPT was also studied (Figure 4B); the results demonstrat-
ed that the release of DOX·HCl was also a controlling pro-
cess and indicated that the drug DOX·HCl had a slow diffu-
sion process owing to the hydrophobic interaction between
the drug molecules and hydrophobic parts of the nanoparti-
cles.[12]
The overall in vitro cytotoxicity of MPEG-SS-PCPT
toward TCA8113 and PC12 cancer cells was evaluated and
compared with free drug CPT by using sulforhodamine B
(SRB) assays, as was the DOX·HCl-loaded MPEG-SS-
PCPT under the same conditions. As shown in Figure 5,
As shown in Figure 5, MPEG-SS-PCPT/DOX·HCl
showed a comparable level of cytotoxicity toward TCA8113
and PC12 cancer cells to that of MPEG-SS-PCPT in all the
concentrations tested. For example, the IC50 values of
MPEG-SS-PCPT/DOX·HCl toward TCA8113 and PC12
cancer cells were 5.7 and 8.7 mgmLÀ1, respectively. The re-
sults indicated that MPEG-SS-PCPT successfully served as
a platform to carry DOX·HCl into cells, thereby resulting in
expected synergistic anticancer effects.
In vitro studies were performed to elucidate the cell
uptake of MPEG-SS-PCPT/DOX·HCl. The results were ob-
served by laser-scanning confocal microscopy (LSCM) with
DOX·HCl as a fluorescent dye to label the nanoparticles.
Meanwhile, 4’,6-diamidino-2-phenylindole (DAPI) was used
to label the cell nuclei (shown in blue). As revealed by
LSCM studies (Figure 6), MPEG-SS-PCPT/DOX·HCl was
Figure 6. LSCM images of TCA8113 cells incubated with MPEG-SS-
PCPT/DOX·HCl nanoparticles and free DOX·HCl (10 mgequivmLÀ1).
For each panel, the images from left to right show DOX·HCl fluores-
cence in cell nuclei stained by DAPI (blue), cells (red), and overlays of
the two images. A) MPEG-SS-PCPT/DOX·HCl nanoparticles, 3 h incu-
bation; B) MPEG-SS-PCPT/DOX·HCl nanoparticles, 24 h incubation;
C) free DOX·HCl, 3 h incubation. Scale bars, 20 mm.
Figure 5. Relative cell viabilities of A) PC12 cells and B) TCA8113 cells
incubated with different concentrations of CPT, MPEG-SS-PCPT,
DOX·HCl, and MPEG-SS-PCPT/DOX·HCl for 24 h.
MPEG-SS-PCPT showed similar cytotoxicity toward both
TCA8113 and PC12 cancer cells when compared with free
CPT. For instance, the half-maximal inhibitory concentration
(IC50) values for MPEG-SS-PCPT against TCA8113 and
PC12 cancer cells were 6.9 and 9.4 mgmLÀ1, respectively,
whereas those for free CPT were 8.2 and 5.5 mgmLÀ1, re-
spectively. In conjunction with the above results, MPEG-SS-
PCPT nanoparticles may enter tumor cells by endocytosis,
followed by endo-lysosomal escape and subsequent drug dis-
tribution in the cytosol and nucleus.[35] These release pro-
cesses were much slower than the passive diffusion process
of free CPT and would result in slow release of CPT from
MPEG-SS-PCPT nanoparticles.
quickly taken up and found to be associated with or near
the nuclear membrane, and some was even located in the
nuclei (pink spots in Figure 6A and B). Notably, free
DOX·HCl was distributed to the perinuclei and nuclei of
TCA8113 cells in 3 h (Figure 6C). Thus, the punctuated dis-
tribution of the fluorescence of MPEG-SS-PCPT/DOX·HCl
in cells indicated that DOX·HCl entered TCA8113 cancer
cells by the delivery of MPEG-SS-PCPT nanoparticles
rather than by means of a diffusion process of the free
DOX·HCl.
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Preparation of MPEG-SS-PCPT and DOX·HCl-Loaded Nanoparticles
Conclusion
The nanoparticles were prepared by a dialysis method. Typically, a prede-
termined amount of MPEG-SS-PCPT (5.0 mg) was dissolved in dimethyl
sulfoxide (DMSO; 1 mL) and added dropwise to buffer (5 mL, pH 7.4);
after that, the solution was transferred to a dialysis bag (MWCO 3500)
and dialyzed against deionized water (2 L) for 24 h to remove the organic
solvents. The DOX-loaded nanoparticles were prepared by a similar
method. Typically, MPEG-SS-PCPT (5.0 mg) and DOX·HCl (1.0 mg)
were dissolved in DMSO (1 mL) and added dropwise to buffer (5 mL,
pH 7.4) in the dark. After that, the solution was transferred to a dialysis
bag (MWCO 3500) and dialyzed against deionized water (2 L) for 24 h to
remove the organic solvents and free DOX·HCl.
In summary, a novel, disulfide-containing CPT prodrug
(MPEG-SS-PCPT) was successfully synthesized with the ob-
jective to increase the drug-loading amount and reduce the
proportion of inactive materials. The MPEG-SS-PCPT
nanoparticles formed in aqueous solution; they could under-
go triggered disassembly in response to the GSH of the en-
vironment and release CPT quickly under tumor-relevant
GSH concentrations. The CPT prodrug could further adsorb
another anticancer drug, DOX·HCl, with a high loading
amount to achieve collaborative cytotoxicity toward cancer
cells.
Cell Viability Assay
The human tongue squamous cell carcinoma TCA8113 cell lines and rat
adrenal pheochromocytoma PC12 cell lines were provided by the Biology
Preservation Center of the Shanghai Institute of Materia Medica and
maintained with RPMI 1640 medium that contained 10% fetal bovine
serum (FBS), penicillin (100 UmLÀ1), and streptomycin (100 mgmLÀ1) at
378C in a humidified atmosphere with 5% CO2. The in vitro cytotoxicity
of MPEG-SS-PCPT and MPEG-SS-PCPT/DOX·HCl, free CPT, and
DOX·HCl were evaluated by sulforhodamine B (SRB) assays. TCA 8113
and PC12 cells were all seeded in a 96-well plate (Costar, USA) (3.5ꢁ
104 cellsmLÀ1) overnight. Then culture medium was replaced with fresh
medium that contained the above four samples with varied concentra-
tions that ranged from 0.125 to 10 mgmLÀ1 for 24 h, respectively. Then
cells were fixed by the addition of cold 10% trichloroacetic acid (TCA;
100 mL at 48C) for 1 h in each well. The wells were gently washed five
times with deionized water and then stained with 0.4% SRB solution
(150 mL per well) for 30 min at room temperature. At the end of the
staining period, unbound SRB was rinsed out with 1% acetic acid. As
the plate air-dried again, aqueous tris base (tris(hydroxymethyl)amino-
methane) (150 mL, 10 mm) was added into each well to solubilize the
SRB dye. When the crystals were dissolved, the absorbance values were
read on a microplate reader (Huake, Shanghai) at 570 nm. This proce-
dure was repeated three times.
Experimental Section
Synthesis of 2-Methacrylester Hydroxyethyl Disulfide (MABHD)
Monomer
A solution of methacryloyl chloride (0.89 mL, 10 mm) in THF (10 mL)
was added dropwise at once to an anhydrous solution of bis(2-hydrox-
yethyl) disulfide (7.7 g, 50 mmol) in THF (60 mL) and triethylamine
(1.47 mL, 10.0 mmol) at 08C. The reaction was stirred at room tempera-
ture for 24 h after completion of the addition. The solution was then fil-
tered to remove the salt, and the crude product was recovered by evapo-
ration of THF. The product was purified by passing it through silica
column chromatography (eluent: petroleum ether/CH2Cl2/methanol
70:10:1 v/v/v). Then the product was concentrated and dried under
vacuum. Yield: 62%; 1H NMR (600 MHz, CDCl3): d=5.60, 6.13 (s, 2H),
4.42 (t, 2H), 3.90 (t, 2H), 2.96 (t, 2H), 2.87 (t, 2H), 1.95 ppm (s, 3H).
Synthesis of MPEG-b-PMABHD by RAFT Polymerization
The diblock copolymer was synthesized by RAFT of MABHD using
MPEG-CPADB as a macro-RAFT agent. In a typical example, under
a nitrogen atmosphere, MPEG-CPADN (90.0 mg, 0.04 mmol), MABHD
Cellular Uptake Studies
(444.0 mg,
2.0 mmol),
azodiisobutyronitrile
(AIBN)
(2.4 mg,
The ability of MPEG-SS-PCPT/DOX·HCl nanoparticles to enter cancer
cells was observed by using a laser scanning confocal microscope (LSCM,
ZEISS, LSM 510 Meta, Germany). In a typical procedure, the TCA8113
cells (3.5ꢁ104 cells per well) were seeded on a six-well plate (Costar,
USA) at 378C overnight. After that, free DOX·HCl, MPEG-SS-PCPT)/
DOX·HCl (at 5 mg DOX·HCl per mL) were added, respectively. The cell
nucleus was stained by 4’,6-diamidino-2-phenylindole (DAPI) (Sigma Al-
drich). After a further 3 or 24 h of incubation, the cells were washed by
phosphate buffered saline (PBS) three times to remove the dead cells
and the drugs adsorbed on the outer surface of the cell membrane, and
the MPEG-SS-PCPT/DOX·HCl prodrug uptake was visualized at an ex-
citation wavelength of 488 nm.
0.0133 mmol), and THF (1.5 mL) were added into a 10 mL Schlenk flask.
The flask was sealed and placed in an oil bath thermostatted at 608C.
The polymerization proceeded with stirring for 24 h. The resulting copo-
lymer was isolated by precipitation in cold diethyl ether, filtration, and
drying under vacuum. Yield: 71%. On the basis of the GPC results (see
the Supporting Information), the number-average molecular weight and
the molecular-weight distribution of the MPEG-b-PMABHD block copo-
lymer were 4.2 K and 1.12, respectively. H NMR (600 MHz, CDCl3): d=
3.38 (s, CH3O ), 3.65 (s, MPEG, CH2CH2O ), 4.22 (m, MPEG–
CH2OCO), 2.43–2.76 (m, OCOCH2CH2 ), 1.96 (s, CH3), 4.42, 3.90,
2.96, 2.87 ppm (s, CH2CH2SSCH2CH2 , MABHD).
1
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Synthesis of MPEG-SS-PCPT Prodrug
Characterization
In
a
typical example, under
a
nitrogen atmosphere, CPT (50 mg,
The 1H NMR spectra of the samples were recorded with a Bruker
AVANCE 600 MHz spectrometer (Rheinstetten, Germany) using tetra-
methylsilane as an internal standard at 258C. The Fourier transform in-
frared (FTIR) spectra were acquired with a Nicolet 20 NEXUS 670
FTIR spectrophotometer (Ramsey, MA, USA) using KBr pellets. The
number-average molecular weight (Mw) and molecular-weight distribu-
tion (Mw/Mn) were measured by gel permeation chromatography (GPC)
system (Waters 1515, USA) equipped with a Waters 1515 pump and
a Waters 2414 refractive index detector and a Styragel HT column. THF
was used as the eluent (1 mLminÀ1), and polystyrene was used as the
standard for calibration. The morphology of the samples was recorded
with a JEM-1230EX transmission electron microscope (Japan), and sam-
ples for TEM measurements were made by casting one drop of the sam-
pleꢂs water solution on carbon-coated copper grids. The size distribution
of MPEG-SS-PCPT nanoparticles was determined by dynamic light scat-
tering (DLS) with a BI-200SM instrument (Brookhaven, USA) with
0.15 mmol) and dimethylaminopyridine (DMAP; 49 mg, 0.4 mmol) were
dissolved in anhydrous dichloromethane (CH2Cl2; 5 mL); the reaction
mixture was stirred at room temperature for 10 min. Then triphosgene
(18 mg, 0.06 mmol, 2 mL) in a solution of CH2Cl2 was added and the mix-
ture was stirred for 30 min at room temperature. MPEG-b-PMABHD
(51 mg, 0.01 mmol in anhydrous THF (2 mL)) was added dropwise using
a gastight syringe. The reaction was stirred for 24 h. The free CPT was re-
moved by filtration using a 0.45 mm filter. The resulting copolymers were
isolated by precipitation in cold diethyl ether (50 mL), filtration, and
1
drying under vacuum. Yield: 62%. The H NMR spectrum of MPEG-SS-
PCPT is shown in the Supporting Information. 1H NMR (600 MHz,
[D6]DMSO): d=8.70–8.72 (m), 8.23 (s), 8.20–8.12 (m), 7.85–7.90 (m),
7.71–7.73 (m), 7.35–7.37 (m), 6.95–7.03 (m), 5.29–5.55 (m), 3.95–4.19 (m),
4.24–4.45 (m), 3.51 (br), 2.75–3.0 (m), 2.09–2.21 (m), 1.81–1.90 (m), 1.22–
1.24 (m), 1.04–1.07 (m), 0.87–0.90 ppm (m).
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Haixia Zhang et al.
angle detection at 908. Absorbance spectra were carried out with a Puxi
UV-1810 visible spectrophotometer (Beijing, China). Fluorescence spec-
tra were recorded with a RF-5301PC fluorescence spectrometer (Shimad-
zu, Japan). The cellular images were acquired with a laser scanning con-
focal microscope (ZEISS, LSM 510 Meta, Germany).
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