A multi-stimuli responsive nanoparticulate SN38 prodrug for cancer chemotherapy

Article abstract of DOI:10.1039/c6tb02262f

Modification of drug delivery systems (DDSs) with stimuli-responsive elements could significantly increase the tumor-specific delivery of anticancer drugs. Herein we synthesized a novel multiple stimuli-responsive SN38 prodrug, named PEG-S-S-SN38, by conjugating PEG (M_W: 2000) and SN38 with disulfide bonds and carbonic ester linkages as linkers for efficient delivery of SN38. The amphiphilic PEG-S-S-SN38, with a high SN38 loading content, could self-assemble into nanoparticles (NPs) with a stable diameter of ～73 nm. PEG-S-S-SN38 NPs release SN38 very slowly at physiological pH, while they quickly release SN38 in the presence of GSH, esterase and H₂O₂, all of which are abundant in the cytoplasm of cancer cells. PEG-S-S-SN38 NPs could be quickly internalized into tumor cells, achieve vesicular escape and nuclear localization, and exhibit remarkable in vitro anticancer activity similar to SN38. Encouragingly, PEG-S-S-SN38 NPs exhibit the same effects on cell cycle regulations as SN38 in vitro. Most importantly, the inhibition rate of tumor growth induced by PEG-S-S-SN38 NPs in a xenograft tumor model reached 72.49% ± 6.26%, which was nearly double that of the corresponding clinical drug CPT-11 (38.64% ± 13.04%) at a dosage equivalent to 10 mg kg^?1 SN38. Our data suggest that the multi-stimuli responsiveness of PEG-S-S-SN38 NPs remarkably enhances their therapeutic activity against heterogeneous or mixed cell population in tumors, making this new DDS a promising alternative to CPT-11 for cancer treatment.

Full text of DOI:10.1039/c6tb02262f

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January 2016 Pages 1–178
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PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
rsc.li/materials-b

Page 1 of 22
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Multi-stimuli Responsive Nanoparticulate SN38 Prodrug for Cancer
Chemotherapy
a,b
※
a,b
※
a,b,c
a,b
a,b
a
Xun Liu,
Qian Huang,
Caixia Yang,
Qianzhi Zhang, Wan Chen, Youqing Shen, Mei-
b,a,*
hua Sui
a
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China 310027;
,
b
Center for Cancer Biology and Innovative Therapeutics, Key Laboratory of Tumor Molecular Diagnosis and
Individualized Medicine of Zhejiang Province, Clinical Research Institute, Zhejiang Provincial People’s Hospital,
Hangzhou, China, 310014;
c
ACEA BIO(Hangzhou) Co. LTD, Hangzhou, China, 310030
*
Corresponding author at the Center for Cancer Biology and Innovative Therapeutics, Key Laboraꢀ
tory of Tumor Molecular Diagnosis and Individualized Medicine of Zhejiang Province, Clinical Reꢀ
search Institute, Zhejiang Provincial People’s Hospital, Hangzhou, 310014, China.
Eꢀmail address: suim@zju.edu.cn (M. Sui)
※
equal contribution for the first two authors.
Keywords: nanocarrier; stimuliꢀsensitive; PEG; SN38; CPTꢀ11
1

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Abstract:
Modification of drug delivery system (DDS) with stimuliꢀresponsive elements could signifiꢀ
cantly increase the tumorꢀspecific delivery of anticancer drugs. Herein we synthesized a novel mulꢀ
tiple stimuliꢀresponsive SN38 prodrug, named as PEGꢀSꢀSꢀSN38, through conjugating PEG (MW:
2
000) and SN38 with disulfide bond and carbonic ester linkage as the linkers for efficient delivery
of SN38. The amphiphilic PEGꢀSꢀSꢀSN38, with a high SN38 loading content, could selfꢀassemble
into nanoparticles (NPs) with a stable diameter of ~73 nm. PEGꢀSꢀSꢀSN38 NPs release SN38 very
2 2
slowly in physiological pH, while quickly release SN38 in the presence of GSH, esterase and H O ,
all of which are abundant in the cytoplasm of cancer cells. PEGꢀSꢀSꢀSN38 NPs could be quickly
internalized into tumor cells, achieve vesicular escape and nuclear localization, and exhibit remarkꢀ
able in vitro anticancer activity similar to SN38. Encouragingly, PEGꢀSꢀSꢀSN38 NPs exhibit the
same effects on cell cycle regulations as SN38 in vitro. Most importantly, the inhibition rate of tuꢀ
mor growth induced by PEGꢀSꢀSꢀSN38 NPs in a xenograft tumor model reached 72.49% ± 6.26%,
which was nearly double that of the corresponding clinical drug CPTꢀ11 (38.64% ± 13.04%) at a
ꢀ1
dosage equivalent to 10 mg kg SN38. Our data suggest that the multiꢀstimuli responsiveness of
PEGꢀSꢀSꢀSN38 NPs remarkably enhances their therapeutic activity against heterogeneous or mixed
cell population in tumors, making this new DDS a promising alternative of CPTꢀ11 for cancer
treatment.
2

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Introduction
The clinical application of anticancer drugs is often restricted by multiple factors, for instance,
low solubility or stability, lack of selectivity to tumor tissues or tumor cells, which result in deꢀ
creased therapeutic activity and severe side effects. Formulation strategies such as nanoparticleꢀ
based drug delivery systems (DDS) have emerged as the most promising technology to increase
drug solubility or stability, improve pharmacokinetics, increase the passive targeting ability of carꢀ
1
ꢀ3
ried drugs by the enhanced permeability and retention effect (EPR). Moreover, modification of
DDS with specific stimuliꢀresponsive elements or with active targeting ligands (e.g., folic acid) furꢀ
4
ther increases the tumorꢀspecific drug delivery and retention.
CPTꢀ11 (irinotecan) is a widely used chemotherapeutic drug to treat a range of cancers. Howꢀ
ever, it needs to be converted to 7ꢀethylꢀ10ꢀhydroxyꢀcamptothecin (SN38), which has 100ꢀ1000
5
ꢀ7
fold higher cellꢀkilling potency than CPTꢀ11, before exhibiting significant antiꢀcancer effect. Unꢀ
fortunately, the conversion rate of CPTꢀ11 to SN38 in cancer patients is only around 2ꢀ8% after
CPTꢀ11 infusion, with great interindividual difference, leading to significant interpatient variability
8
,9
in clinical responses. CPTꢀ11 may also cause severe gastrointestinal toxicity and myelosuppresꢀ
10
sion. Therefore, direct administration of SN38 would bring more clinical advantages and may
prevent the side effects of CPTꢀ11. As SN38 has very low solubility in all kinds of pharmaceutically
acceptable solutions, considerable interests have been focused on the development of DDS for tarꢀ
1
1ꢀ14
geted delivery of SN38.
delivering DDS reported in the literature, e.g. low drug loading and unfavorable release profile.
For example, polymer conjugate of SN38 EZNꢀ2208 has a payload of only 3.7% even though four
However, some significant limitations are related with the SN38ꢀ
11, 15
15
SN38 moleculars were conjugated to the PEG backbone.
Stimuliꢀsensitive anticancer DDS, through responses to either an internal physiopathologic
changes (pH, redox potential, etc.) or external stimulus (temperature, magnetic field, etc.), could
1
6ꢀ19
achieve targeted delivery, controlled release and accumulation of drug at tumor sites.
For inꢀ
stance, the intracellular redox potential contains 100ꢀ1000 times higher concentration of reducing
3

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glutathione (GSH) tripeptide than extracellular environment. Moreover, the levels of GSH in tumor
2
0ꢀ22
tissues are at least 4ꢀfold higher than those in normal tissues.
As disulfide bonds are stable in
extracellular space with low concentration of GSH, while could be quickly degraded in a highly reꢀ
ductive intracellular environment, redoxꢀresponsive delivery systems containing disulfide bonds
have attracted much attention for targeted delivery of bioactive molecules for both therapeutics and
23, 24
diagnosis.
Disulfide bonds may be incorporated in the backbones, side chains, crossꢀlinkers of
polymeric carriers, or directly used for conjugation of anticancer drug (e.g. doxurobucin, cisplatin
2
5ꢀ29
and camptothecin) with the polymer backbone.
Another strategy to design stimuliꢀresponsive
DDS is based on enzymatic responsiveness that has multiple advantages including high selectivity,
substrate specificity and mild operating condition, e.g. ester bonds for esteraseꢀcatalyzed bioconverꢀ
30, 31
sion.
Encouragingly, novel dual and multiꢀstimuli responsive delivery systems that respond to a
combination of two or more stimulus have recently been developed and further improved the perꢀ
3
2
formances of DDS.
Through using a hydrophobic drug as the hydrophobic part of the block copolymer, we recentꢀ
ly established a selfꢀassembling prodrug strategy to prepare nanoꢀvehicles for delivery of several
33ꢀ35
anticancer drugs, which has shown additional advantages over traditional DDS.
Herein, for the
first time, we successfully synthesized a novel multiple stimuliꢀresponsive nanocarrier, named as
PEGꢀSꢀSꢀSN38, through conjugating PEG and SN38 with both disulfide bonds and carbonic ester
linkage as the linkers (Scheme 1). PEG (MW: 2000) was selected as the hydrophilic part to improve
36
the biocompatibility of the nanoparticles and prolong their circulation in blood. This novel system
was carefully characterized and its biological effects were investigated using a series of assays. Our
data demonstrated that P EGꢀSꢀSꢀSN38 nanoparticles possess significant superiority and may beꢀ
come a promising alternative of CPTꢀ11 for cancer treatment.
4

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Scheme 1. Illustration of the formation, cellular internalization, distribution and multiꢀstimuli responsive drug
release of PEGꢀSꢀSꢀSN38 nanoparticles. The amphiphilic PEGꢀSꢀSꢀSN38 prodrug could selfꢀassemble into nanoꢀ
particles. The obtained PEGꢀSꢀSꢀSN38 nanoparticles invaginate and pinch off of the plasma membrane to form
endocytic vesicles, followed by a fraction of vesicles containing nanoparticles fusing with lysosomes. After vesicꢀ
ular escape (e.g. endosomal and lysosomal escape), the internalized nanoparticles could be quickly degraded in
2 2
the presence of GSH, esterase and H O , leading to controlled release of active SN38 to target nucleus.
Experimental
Chemicals, cell cultures and mice
Chloromethyl methyl ether (98%) (MOMꢀCl) was purchased from Xiya Reagent (Chengdu,
China). N,NꢀDiisopropylethylamine (DIPEA), 4ꢀdimethylaminopyridine (DMAP) and
bis(trichloromethyl)carbonate (BTC) were purchased from Energy Chemical (Shanghai, China). 7ꢀ
ethyꢀ10ꢀhydroxylꢀcamptothecin (SN38) was purchased from Sendflld Science and Technology
(
Xi’an, China). Irinotecan hydrochloride was purchased from Chengdu Yuancheng Biotechnology
Chengdu, China). Bis(2ꢀhydroxyethyl) disulfide (ca. 50% in water) was purchased from Tokyo
(
Chemical Industry (Tokyo, Japan).
BCap37 (human breast cancer), MCFꢀ7 (human breast cancer) and its multidrug resistant
(MDR) subline MCFꢀ7/ADR, KB (human epidermoid cancer) and its MDR subline KBv200,
5

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SKOV3 (human ovarian cancer), LoVo (human colon cancer) and Vero (monkey fibroblastꢀlike
kidney cell) cell lines are maintained in our laboratory and have been used in our previous studies.
Cancer cell lines were incubated in either RPMI1640 or DMEM supplemented with 10% heatꢀ
ꢀ1
ꢀ1
inactivated fetal bovine serum (FBS), penicillin (100 units mL ) and streptomycin (100 ꢁg mL ) in
a humidified atmosphere of 5% CO at 37°C. Both culture medium and fetal serum were purchased
2
from Genom Biological Technology (Hangzhou, China). The 6ꢀ8ꢀweekꢀold female BALB/c homoꢀ
zygous athymic nude mice were purchased from the Animal Center of Zhejiang Academy of Mediꢀ
cal Sciences, and maintained under standard conditions. All animal studies were approved by the
Animal Care and Use Committee of Zhejiang University and Zhejiang Academy of Medical Sciꢀ
ences.
Synthesis of MOM-SN38
2 2
SN38 (2 g) was dissolved in 20 mL CH Cl and 4.46 mL DIPEA, then 1.93 mL MOMꢀCl was
added into the mixture with stirring. After stirring for 24 h at room temperature, the mixture was
filtered. The solid was purified by passing through a column chromatography packed with silica gel
1
(
eluent MeOH/CH
2
Cl
2
=3/97), and pure MOMꢀSN38 was obtained and characterized by H NMR
spectroscopy and High Performance Liquid Chromatography (HPLC).
Synthesis of MOM-SN38-S-S-OH
2 2
MOMꢀSN38 (0.7 g) was mixed with DMAP (0.63 g) in 70 mL dry CH Cl , and BTC (0.18 g)
was added into the solution under argon atmosphere. The reaction was allowed to proceed at room
temperature for 30 mins, then 15 mL THF solution containing dry bis(2ꢀhydroxyethyl) disulfide
(
2.47 g) was injected under argon atmosphere. After stirring for 24 h at room temperature, the soluꢀ
tion was washed with HCl aqueous solution (pH 3.0) and saturated NaHCO aqueous solution sucꢀ
cessively. After evaporation of the solvent, the product was purified by passing through a column
3
2 2
chromatography packed with silica gel (eluent MeOH/ CH Cl =2/98), and the pure MOMꢀSN38ꢀSꢀ
1
SꢀOH obtained was characterized by H NMR spectroscopy and HPLC.
Synthesis of PEG-S-S-SN38
6

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MOMꢀSN38ꢀSꢀSꢀOH (0.45 g) was mixed with DMAP (0.29 g) in 70 mL dry CH Cl , and BTC
2
2
(74 mg) was added into the solution under argon atmosphere. The reaction was allowed to proceed
at room temperature for 30 mins, then the solution of 10 mL dry CH Cl containing mPEG2000 (0.7 g)
2
2
was added dropwise into the solution with stirring in the ice bath. After stirring for 48 h at room
temperature, the solvent was evaporated and the residue was dialyzed for 5 days against DMSO reꢀ
freshed every 24 h. After further dialysis against water to remove DMSO, the purified product was
1
obtained after freeze drying and characterized by H NMR spectroscopy and HPLC.
Preparation of PEG-S-S-SN38 nanoparticles (NPs) and stability measurement
10 mg PEGꢀSꢀSꢀSN38 was dissolved in 2 mL DMSO. The solution was added dropwise into
1
0 mL deionized water with stirring, followed by dialysis against deionized water and concentrated
ꢀ1
to a final SN38 concentration of 1 mg mL . The size and size distribution of the nanoparticles were
measured using dynamic light scattering (DLS, Malvern Zen3600, UK) and the morphology of the
nanoparticles was observed by transmission electron microscopy (TEM). Furthermore, a certain
amount of FBS was added into 1 mL of the above solution at the final concentration of 40% to inꢀ
vestigate the stability of PEGꢀSꢀSꢀSN38 NPs. The samples were tested by DLS at 2.5 h, 6 h and 32
h respectively.
Determination of the critical micelle concentration (CMC)
A known amount of nile red solution in CH
2
2
Cl was added to a series of vials to give a final
ꢀ6
concentration of 1×10 M. After CH Cl was evaporated, 10 mL of a series of measured amount of
2 2
PEGꢀSꢀSꢀSN38 solution was added to each vial, and the solution was stirred overnight in darkness.
The fluorescence emission intensity of each solution was determined at the wavelength of 650 nm
(exited at 540 nm) using a microplate reader. CMC was obtained as the intersection of the tangents
to the two linear portions of the graph of the fluorescence intensity as a function of PEGꢀSꢀSꢀSN38
concentration.
Measurement of SN38 release
7

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ꢀ1
The PEGꢀSꢀSꢀSN38 NPs (equivalent to 200 ꢁg mL SN38) were prepared in PBS and incuꢀ
bated at 37°C. At timed intervals, 100 ꢁL solution was withdrawn for HPLC test. The MOMꢀSN38
concentration was calculated using a relative method from the total peak area of the absorbance at
3
2 2
60 nm. The MOMꢀSN38 release in the presence of GSH (or esterase, H O ) was carried out in the
same method except adding each agent to the solution to give the final concentration stated.
Cellular uptake and subcellular distribution
The cellular uptake, accumulation and internalization of different formulations of SN38 were
monitored by confocal laser scanning microscopy (CLSM, NikonꢀA1, Japan) and flow cytometric
analysis (FACS, Becton Dickinson FACS Calibur, USA). To visualize cells using CLSM, cells were
cultured on glassꢀbottomed petri dishes for 24 h before treatment. 3,3′ꢀdioctadecyloxacarbocyanine
perchlorate (DIO) staining solution at a final concentration of 5 ꢁM was then added into the dishes
for 20 mins to label cell membrane. Afterwards, the solution was replaced with fresh medium conꢀ
ꢀ1
taining PEGꢀSꢀSꢀSN38 NPs (equivalent to 10 ꢁg mL SN38) for designated time points. In another
two experiments, after the treatment of PEGꢀSꢀSꢀSN38 NPs, tumor cells cultured in petri dishes
were incubated with either LysoTracker green (Molecular Probes, USA) at a concentration of 200
nM for 1.5 h to label lysosomes, or DRAQ5 at a concentration of 5 ꢁM for 20 mins to label nucleus.
DIO for cell membrane and LysoTracker green for lysosomes were observed using a 488 nm laser,
and the emission wavelength was read from 500 to 550 nm and shown as green. PEGꢀSꢀSꢀSN38
was observed using a 405 nm laser, and the emission wavelength was read from 425 to 475 nm and
shown as red. DRAQ5 for nucleus was observed using a 640 nm laser, and the emission wavelength
was read from 662 to 737 nm and shown as blue.
For flow cytometric analysis, tumor cells plated in 6ꢀwell plates were treated with nile redꢀ
ꢀ1
loaded PEGꢀSꢀSꢀSN38 NPs (equivalent to 0.04 ꢁg mL SN38) for 1 h, 2 h and 6 h at 37° C, respecꢀ
tively. Then the cells were collected and rinsed twice with cold PBS. Finally, the cells were disꢀ
persed in cold PBS again and subjected to flow cytometric analysis with the excitation set to be FL2
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(488 nm).
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and xCELLigence
system real time cellular analysis (RTCA)
The in vitro anticancer activity of CPTꢀ11, SN38 and PEGꢀSꢀSꢀSN38 NPs was determined by
4
8 hꢀMTT assay in multiple cell lines including MCFꢀ7, MCFꢀ7/ADR, KB, KBv200, BCap37 and
32, 34
SKOV3, according to the methods described previously.
The absorbance in each individual well
was determined at 562 nm with a microplate spectrophotometer (SpectraMaxM2E, Molecular Deꢀ
vices, USA). Each drug concentration was tested in triplicate and in three independent experiments.
Moreover, as a complementary method of MTT assay, xCELLigence RTCA MP system (ACEA Biꢀ
osciences, USA) was used to evaluat the cytotoxicity of CPTꢀ11, SN38 and PEGꢀSꢀSꢀSN38 NPs in
6
LoVo and Vero cells. In brief, LoVo and Vero cells were seeded into the Eꢀplates 96 at 1.5×10 cells
6
per well and 3.0×10 cells per well, respectively, and incubated for 24 h. Then cells were adminisꢀ
tered with designated concentrations of CPTꢀ11, SN38 and PEGꢀSꢀSꢀSN38 NPs respectively and
monitored for at least 72 h. The normalized cell index were calculated according to RTCAꢀ
integrated software (ACEA Biosciences, USA).
Analysis of cell cycle and apoptosis
After the treatment of CPTꢀ11, SN38 and PEGꢀSꢀSꢀSN38 NPs, at an equivalent SN38 concenꢀ
ꢀ1
tration of 0.04 ꢁg mL for 24 h and 48 h, respectively, cell samples were prepared according to the
Cell Cycle and Apoptosis Analysis Kit (Beyotime Biotechnology, China). In brief, at the end of
each time point, tumor cells were collected and fixed in 70% cold ethanol diluted in PBS. After
rinsed with cold PBS, cell samples were incubated in water bath at 37°C in darkness, with each
sample containing 0.5 mL buffer solution, 25 ꢁL propidium iodide solution and 10 ꢁL RNase. Fiꢀ
nally, the samples were examined using FACScan and the obtained data were processed with Modiꢀ
fit LT 3.2 Software.
In vivo antitumor studies
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In vitro cultured BCap37 cells were collected and washed with PBS, then implanted into the
6
3
right flanks of nude mice (1×10 cells). When the tumor volumes were around 90 mm , mice were
randomly divided into three groups (n=6 per group): (a) CTL (PBS), (b) irinotecan (equivalent to 10
ꢀ1
ꢀ1
mg kg SN38) and (c) PEGꢀSꢀSꢀSN38 (equivalent to 10 mg kg SN38). The treatment regimens
were initiated on day 1 (i.v.), repeated every 3 days for a total of 6 cycles. The body weight of each
2
animal and the tumor size ([major axis]×[minor axis] ×1/2) were measured before each intravenous
injection. Three days after the last treatment, animals were sacrificed following institutional guideꢀ
3
lines. The curve of tumor growth was drawn based on tumor volume (mm ) and corresponding time
(days) after the first treatment. Inhibition rates (IR) of tumor growth were calculated using the folꢀ
lowing formula: IR= (mean tumor weight of control group – mean tumor weight of experimental
group)/mean tumor weight of control group×100%. Tumors were resected, weighed, and fixed in
formalin for paraffin embedding, followed by preparation of tissue sections and staining with hemaꢀ
toxylin and eosin for histological examinations. The in vivo proliferation of tumor cells was studied
by immunohistochemistry staining for proliferation marker Kiꢀ67 (Beijing Zhongshan Golden
Bridge Biotechnology, China) in BCap37 xenograft tumors and counterstained with hematoxylin.
Statistical analysis
Data are presented as means±standard errors. Tumor volumes over time were analyzed by oneꢀ
way ANOVA (analysis of variance) and subsequently by Student's
tꢀtest. All other statistical analꢀ
yses were performed using Student's
level of P<0.05.
tꢀtest. Differences were considered statistically significant at a
Results and discussion
Synthesis and characterization of SN38 prodrug and PEG-S-S-SN38 NPs
The design and synthesis route for a new SN38 prodrug, named as PEGꢀSꢀSꢀSN38, is shown in
Figure 1A. SN38 has a C20 alcoholic hydroxyl group and a C10 phenolic hydroxyl group. The C20
10

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alcoholic hydroxyl group, along with the lactone ring and pyridone group, are essential for the
topoisomerase inhibitory activity of SN38, while the presence of the αꢀOH group at the C20 chiral
centre facilitates the hydrolysis of the lactone ring. Herein, we first protected the C10 phenolic hyꢀ
droxyl group with chloromethyl methyl ether using N,NꢀDiisopropylethylamine as a catalyst in
2 2
CH Cl to obtain MOMꢀSN38. Next, the reaction of C20 alcoholic hydroxyl group of MOMꢀSN38
with BTC and bis(2ꢀhydroxyethyl) disulfide in the presence of DMAP afforded MOMꢀSN38ꢀSꢀSꢀ
OH. Finally, the prodrug PEGꢀSꢀSꢀSN38 was obtained through the reaction of MOMꢀSN38ꢀSꢀSꢀ
OH with mPEG2000 using BTC in the presence of DMAP as the catalyst. The structures and purity
of MOMꢀSN38, MOMꢀSN38ꢀSꢀSꢀOH and PEGꢀSꢀSꢀSN38 were confirmed by NMR (see Supportꢀ
ing Information, Figure S1ꢀS3) and HPLC (Figure S4). As determined with DLS and TEM, the amꢀ
phiphilic PEGꢀSꢀSꢀSN38, with a SN38 loading content as high as 15 wt%, could selfꢀassemble into
nanoparticles with a diameter of about 73 nm in PBS (Figure 1B). Further studies indicated that
PEGꢀSꢀSꢀSN38 NPs have good stability, with the size only slightly increased even after 32 h incuꢀ
bation with 40% FBS (Figure S5). The appropriate size of PEGꢀSꢀSꢀSN38 NPs and their favorable
stability may increase the targeting ability of nanocarriers through EPR effect, and prevent sequesꢀ
tration by reticuloendothelial system (RES), both of which leading to the effective accumulation of
nanoparticles into tumor tissues. Moreover, the CMC of PEGꢀSꢀSꢀSN38 in aqueous solution was 53
ꢀ1
µ
g mL (Figure S6).
Multi-stimuli responsive release of SN38 from PEG-S-S-SN38 NPs
The release of SN38 from PEGꢀSꢀSꢀSN38 NPs was detected by HPLC. As shown in Figure 1C,
PEGꢀSꢀSꢀSN38 NPs released SN38 very slowly in PBS at pH 7.4, without the phenomenon of iniꢀ
tial burst release and with only 11.3% of SN38 released after 48 h of incubation. However, in the
presence of 10 mM GSH, these NPs quickly hydrolyzed and up to 91.3% of SN38 was released
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Figure 1. Synthesis and characterization of SN38 prodrug PEGꢀSꢀSꢀSN38. (A) Synthesis route of PEGꢀSꢀSꢀSN38
prodrug. (B) The size distribution of PEGꢀSꢀSꢀSN38 nanoparticles determined by DLS and the morphology of
PEGꢀSꢀSꢀSN38 nanoparticles observed by TEM (insert, scale bar=100 nm). (C) SN38 release kinetics from PEGꢀ
2 2
SꢀSꢀSN38 nanoparticles in PBS at pH 7.4 and in the presence of 10 mM GSH, 10 U esterase and 1 mM H O .
after 48 h of incubation, indicating that the fast SN38 release could be triggered by reduction with
GSH. Meanwhile, in the presence of esterase, which is abundant in cytoplasm, up to 85% of SN38
was released from PEGꢀSꢀSꢀSN38 NPs after 26 h of incubation. This data coincides with our anticꢀ
ipation that the carbonic ester linkage between PEG and SN38 could be easily cleaved when exꢀ
posed to intracellular esterase. In some ways, this esteraseꢀresponsive property of PEGꢀSꢀSꢀSN38 is
12

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superior to the carboxylesteraseꢀresponsiveness of CPTꢀ11 because of the remarkable interspecies
and individual variability of carboxylesterase. Interestingly, we further found that in the presence of
1
2 2
mM H O , one of the intracellular prevailing reactive oxygen species (ROS) overproduced in canꢀ
cer cells compared with normal cells, PEGꢀSꢀSꢀSN38 could also be quickly hydrolyzed and effecꢀ
tively release SN38. This accelerated drug release might be caused by the oxidation of the disulfide
bonds in the presence of high level of ROS. These data demonstrates that for the first time, through
conjugating PEG and SN38 with both disulfide bond and carbonic ester linkages, we have successꢀ
fully obtained a tripleꢀstimuli responsive nanoparticulate SN38 prodrug to achieve better controlled
release of SN38.
Cellular uptake and subcellular distribution of PEG-S-S-SN38 NPs
When observed with CLSM (Figure 2AꢀJ), PEGꢀSꢀSꢀSN38 NPs exhibited red fluorescence exꢀ
cited at 405 nm. It is worth mentioning that the absorption spectrum of free SN38 is 360 nm and the
emission wave is 530 nm. Therefore, no red fluorescence would be observed with 405 nm excitaꢀ
tion if free SN38 was released from PEGꢀSꢀSꢀSN38 NPs. Herein, lipophilic carbocyanine dyes DIO,
LysoTracker green and DRAQ5 were used to label cell membrane (green in Figure 2AꢀD), lysoꢀ
somes (green in Figure 2EꢀH) and nucleus (blue in Figure 2IꢀJ), respectively.
As shown in Figure 2AꢀD, exposure to PEGꢀSꢀSꢀSN38 NPs resulted in a significant timeꢀ
dependent accumulation of SN38 in BCap37 cells via endocytosis, as indicated by endocytic interꢀ
nalization of the plasma membrane and the coꢀlocalization of drug and membrane (shown as yelꢀ
low). After incubation for 30 mins, a large amount of PEGꢀSꢀSꢀSN38 NPs had been trapped in enꢀ
docytic vesicles (shown as yellow, Figure 2C), followed by most of the nanoparticles escaped from
the vesicles at 2 h (Figure 2D, shown as the reduced yellow fluorescence). Interestingly, data preꢀ
sented in Figure 2EꢀH revealed that most PEGꢀSꢀSꢀSN38 NPs were not coꢀlocalized with lysoꢀ
somes at these time points. This phenomena might be attributed to the occurrence of fast vesicular
escape of PEGꢀSꢀSꢀSN38 NPs into cytosol, which preventing the further transportation of nanoparꢀ
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ticles into the lysosomes. Encouragingly, after 6 h of incubation, some PEGꢀSꢀSꢀSN38 NPs entered
the cell nuclei (shown as pink in Figure 2IꢀJ). These findings demonstrate that PEGꢀSꢀSꢀSN38 NPs
could be quickly internalized into tumor cells, achieve vesicular escape and nuclear localization.
Moreover, the cellular uptake of PEGꢀSꢀSꢀSN38 NPs was also monitored by flow cytometry. As
summarized in Figure 2K, these nanoparticles quickly entered into BCap37 cells, with rates as high
as 50.4%, 70.1% and 89.6%, respectively, after 1 h, 2 h, and 6 h of incubation, which was consistent
with the data obtained with CLSM.
Figure 2. Cellular uptake and subcellular localization of PEGꢀSꢀSꢀSN38 nanoparticles in BCap37 cells as obꢀ
served with CLSM. AꢀD, images were taken after incubation with PEGꢀSꢀSꢀSN38 for 0 min (A), 10 mins (B), 30
mins (C) and 120 mins (D), respectively. Cell membrane was labeled with lipophilic carbocyanine dyes DIO
(
shown as green), and PEGꢀSꢀSꢀSN38 were shown as red. EꢀH, images were taken after incubation with PEGꢀSꢀ
SꢀSN38 for 10 mins (E), 30 mins (F), 60 mins (G) and 120 mins (H), respectively. Lysosomes were labeled with
LysoTracker green (shown as green), and PEGꢀSꢀSꢀSN38 were shown as red. IꢀJ, observation of the nuclear localꢀ
ization after incubation with PEGꢀSꢀSꢀSN38 for 2 h (I) and 6 h (J), respectively. Cell nucleus was labeled with
DRAQ5 (shown as blue), and PEGꢀSꢀSꢀSN38 was shown as red. K, cellular uptake of PEGꢀSꢀSꢀSN38 nanopartiꢀ
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cles into BCap37 cells after incubation for 0 h, 1 h, 2 h and 6 h, respectively, as determined by flow cytometric
ꢀ1
analysis. AꢀJ, scale bar=10 ꢁm; PEGꢀSꢀSꢀSN38 at a concentration equivalent to 10 ꢁg mL SN38.
Figure 3 In vitro cytotoxicity of CPTꢀ11, SN38 and PEGꢀSꢀSꢀSN38 NPs in BCap37, SKOV3 and other two pairs
of parental sensitive and MDR cancer cell lines (KB and KBv200; MCFꢀ7 and MCFꢀ7/ADR) after 48 h incubaꢀ
tion with equivalent CPTꢀ11, SN38 and PEGꢀSꢀSꢀSN38 NPs.
In vitro cell-killing activity of CPT-11, SN38 and PEG-S-S-SN38 NPs
The in vitro cellꢀkilling activity of PEGꢀSꢀSꢀSN38 NPs, in comparison with CPTꢀ11 and free
SN38, was evaluated with MTT assays (Figure 3) and RTCA (Figure S7). Our data showed that
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CPTꢀ11 had little cytotoxicity against all these cell lines, while PEGꢀSꢀSꢀSN38 NPs exhibited simiꢀ
lar doseꢀdependent and comparable anticancer activity as SN38 in most tested cancer cell lines
(Figure 3). These data indicates that the multiꢀstimuli responsiveness enables PEGꢀSꢀSꢀSN38 NPs
efficiently release active SN38 after taken up by cancer cells, which is critical for their cellꢀkilling
activity. In RTCA assays, PEGꢀSꢀSꢀSN38 NPs also produced significant in vitro cellꢀkilling activity
comparable to free SN38, which was much higher than that of CPTꢀ11 in both LoVo and Vero cells
(Figure S7).
Cell cycle regulations induced by CPT-11, SN38 and PEG-S-S-SN38 NPs
Cell cycle distribution of BCap37 cells exposed to CPTꢀ11, SN38 and PEGꢀSꢀSꢀSN38 NPs, at
ꢀ1
a concentration equivalent to 0.04 ꢁg mL SN38, were monitored by flow cytometry (Figure 4). It
is known that SN38, a topoisomerase I inhibitor, can induce irreversible doubleꢀchain DNA damꢀ
age, thereby resulting in cell cycle arrest at S and G2/M phases or even inducing cell death. Indeed,
our data showed that BCap37 cells were arrested at S phase after 24 h of incubation with 0.04 ꢁg
ꢀ1
mL SN38 (percent of S phase: 90.35%), which progressed to G2 phase arrest at 48 h (percent of
G2 phase: 99.70%). Encouragingly, PEGꢀSꢀSꢀSN38 NPs at the equivalent SN38 concentration exꢀ
hibited exactly the same effects on cell cycle changes as SN38 (with 90.58% Sꢀphase cells at 24 h
and 96.14% G2ꢀphase cells at 48 h, respectively), indicating that PEGꢀSꢀSꢀSN38 NPs could quickly
enter into cells and release active SN38 effectively. It is worth mentioning that in our previous reꢀ
port, the regulatory effect of OEGꢀSN38, a prodrug prepared through attaching oligo (ethylene glyꢀ
col) (OEG) to SN38 via ester bond at the C20 position, on the cell cycle arrest of cultured cancer
34
cells was significantly less than SN38. These findings suggest that PEGꢀSꢀSꢀSN38 NPs possess
better in vitro anticancer activity than OEGꢀSN38 NPs, which might be attributed to the promoted
release of SN38 from PEGꢀSꢀSꢀSN38 NPs due to the multiꢀstimuli responsiveness. Compared with
the control groups, CPTꢀ11 did not cause obvious in the cell cycle distribution at both time points.
These findings coincide with the fact that CPTꢀ11 has little or no anticancer activity in vitro, and
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needs to be converted into active metabolite SN38 to exhibit therapeutic activity.
Figure 4. Cell cycle distribution and regulation. BCap37 cells treated with CPTꢀ11, SN38 and PEGꢀSꢀSꢀSN38 at a
ꢀ1
concentration equivalent to 0.04 ꢁg mL SN38 for 24 h and 48 h, respectively, were monitored by flow cytometric
analysis.
In vivo antitumor activity of CPT-11 and PEG-S-S-SN38 NPs
All animal studies were performed in strict accordance with Chinese legislation on the use and
the care of laboratory animals, and with the guidelines established by the Institute for Experimental
Animals. To evaluate their in vivo antitumor activity, PEGꢀSꢀSꢀSN38 NPs and CPTꢀ11 were adminꢀ
ꢀ1
istered intravenously via tail vein at a dosage equivalent to 10 mg kg SN38 in nude mice bearing
BCap37 xenograft tumors for a total of 6 cycles (Figure 5). Our data showed that both treatment
significantly repressed the tumor growth (P<0.0005 for CPTꢀ11 vs PBS; P<0.00005 for PEGꢀSꢀSꢀ
SN38 vs PBS). Meanwhile, the IR (inhibition rate of tumor growth) induced by PEGꢀSꢀSꢀSN38
NPs reached 72.49% ± 6.26%, which is nearly double that of CPTꢀ11 (IR=38.64% ±
1
3.04%)(Figure 5A, 5C and 5D, P<0.0005 for PEGꢀSꢀSꢀSN38 vs CPTꢀ11). In addition, neither
CPTꢀ11 nor PEGꢀSꢀSꢀSN38 NPs induced significant changes on body weight in comparison with
the control group (Figure 5B). These data indicate that PEGꢀSꢀSꢀSN38 NPs possess not only excelꢀ
lent in vivo safety, but much better therapeutic efficacy than CPTꢀ11 at equivalent dosage. Moreoꢀ
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ver, in a preliminary study examining the drug distribution in different organs/tissues of mice bearꢀ
ing BCap37 xenograft tumors, we found that similar to many other nanoꢀDDSs, PEGꢀSꢀSꢀSN38
NPs were mainly cleared by liver and spleen (Figure S8).
Figure 5. In vivo therapeutic effects of CPTꢀ11 and PEGꢀSꢀSꢀSN38 against BCap37 human breast xenograft tuꢀ
mors. (A) Changes in tumor volume. (B) Body weight changes of the nude mice. (C) The average tumor weight of
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each group at the end of experiment; IR, the inhibition rate of tumor growth. (D) Photos of xenograft tumors at the
termination of animal experiment. (E) and (F) Representative histological and Kiꢀ67 immunohistochemical feaꢀ
tures of tumor tissues; a) PBS group, b) CPTꢀ11 group, c) PEGꢀSꢀSꢀSN38 group. **P<0.005, and ***P<0.0005.
Scale bars: 100 µm for E; 50 µm for F.
Histological studies and immunohistochemical staining of proliferation marker Kiꢀ67 with
xenograft tumors further confirmed the therapeutic superiority of PEGꢀSꢀSꢀSN38 NPs over CPTꢀ11.
In H&E staining, the tumor tissues were composed of tightly packed tumor cells and stroma in the
control group, while apoptotic cell death were observed in a small portion of tumor cells in tissue
sections obtained from CPTꢀ11 group, which was characterized by membraneꢀbound, small nuclear
fragments surrounded with a rim of cytoplasm. However, in xenograft tumors treated with PEGꢀSꢀ
SꢀSN38 NPs, nearly all the tumor cells exhibited typical apoptotic features and even obvious vacuꢀ
olization was observed (Figure 5E). Moreover, administration of CPTꢀ11 and PEGꢀSꢀSꢀSN38 NPs
significantly reduced both the percentage of Kiꢀ67 positiveꢀcells and the expression level of Kiꢀ67,
especially in PEGꢀSꢀSꢀSN38ꢀtreated tumors (Figure 5F). These data also demonstrated that conꢀ
sistent with their better activity in inducing cell cycle arrest in vitro (see above section), PEGꢀSꢀSꢀ
SN38 NPs exhibit significantly increased in vivo anticancer activity compared with OEGꢀSN38
NPs, as the latter only achieved anticancer activity comparable to CPTꢀ11 against BCap37 xenoꢀ
34
graft tumors.
Conclusions
Dual and multiꢀstimuli responsive drug delivery systems hold the promise to further improve
the performances of DDS. In the present study, we successfully synthesized a novel SN38 prodrug
PEGꢀSꢀSꢀSN38, for the first time, through conjugating PEG and SN38 with both disulfide bond and
carbonic ester linkage as the linkers for efficient delivery of SN38. The amphiphilic PEGꢀSꢀSꢀSN38
has a high SN38 loading content and could selfꢀassemble into nanoparticles with a stable diameter
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of about 73 nm. These nanoparticles have no initial burst release, but exhibit favorable stimuliꢀ
responsiveness to GSH, esterase and H for the controlled release of SN38. Most importantly,
2
O
2
PEGꢀSꢀSꢀSN38 NPs possess excellent in vitro anticancer activity comparable to free SN38, and
show dramatically increased in vivo anticancer activity compared with both the corresponding cliniꢀ
cal chemotherapy drug CPTꢀ11 and OEGꢀSN38 NPs previously reported. Our data suggest that the
responsiveness of PEGꢀSꢀSꢀSN38 NPs to a combination of three stimulus remarkably enhances
their therapeutic activity against heterogeneous or mixed cell population in tumors, making this new
DDS a promising alternative of CPTꢀ11 for cancer treatment.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (NSFC 21104065,
2
1274125 and 21574118) and the Zhejiang Provincial Natural Science Foundation for Distinꢀ
guished Young Scientists (No. LR16H160002).
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