Mucoadhesive nanoparticles based on ROS activated gambogic acid prodrug for safe and efficient intravesical instillation chemotherapy of bladder cancer

Article abstract of DOI:10.1016/j.jconrel.2020.03.028

Chemotherapy is the standard of care for bladder cancer after transurethral resection of the tumor. However, the rapid excretion of clinically used formulations of anticancer drugs make the common intravesical instillation chemotherapy far from efficient. Therefore, improving the muco-adhesion and penetrability of chemotherapeutic drugs became the key factors in the post-surgery treatment of superficial bladder cancers. Here, a reduction sensitive vehicle was developed to deliver the reactive oxygen species activated prodrug of gambogic acid for treatment of orthotopic bladder cancer. The positively charged chitosan can significantly enhance the adhesion and permeability of prodrug within the bladder wall. Moreover, by utilizing the different glutathione and ROS level between cancer cells and normal cells, the dual responsive nanoparticle can selectively and rapidly deliver drug in bladder cancer cells, and thus can significantly inhibit the proliferation of bladder cancer cells in an orthotopic superficial bladder cancer model without causing damage to normal cells. This work demonstrates that the smart prodrug nanomedicine may act as a promising drug-delivery system for local chemotherapy of bladder cancer with unprecedented clinical benefits.

Full text of DOI:10.1016/j.jconrel.2020.03.028

Journal Pre-proof
Mucoadhesive nanoparticles based on ROS activated gambogic
acid prodrug for safe and efficient intravesical instillation
chemotherapy of bladder cancer
Xin Xu, Kunpeng Liu, Binbin Jiao, Kejun Luo, Jian Ren, Guan
Zhang, Qingsong Yu, Zhihua Gan
PII:
S0168-3659(20)30180-2
DOI:
https://doi.org/10.1016/j.jconrel.2020.03.028
COREL 10230
Reference:
To appear in:
Journal of Controlled Release
Received date:
Revised date:
Accepted date:
11 November 2019
10 March 2020
19 March 2020
Please cite this article as: X. Xu, K. Liu, B. Jiao, et al., Mucoadhesive nanoparticles based
on ROS activated gambogic acid prodrug for safe and efficient intravesical instillation
chemotherapy of bladder cancer, Journal of Controlled Release (2020), https://doi.org/
1
0.1016/j.jconrel.2020.03.028
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2020 Published by Elsevier.

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Mucoadhesive nanoparticles based on ROS activated
gambogic acid prodrug for safe and efficient intravesical
instillation chemotherapy of bladder cancer
a,#
b,c,e,#
a,h
b,c,e
a
Xin Xu, Kunpeng Liu,
Binbin Jiao, Kejun Luo,
Jian Ren, Guan
a,g,h,
b,c,d,e,f,
b,c,d,e,f,
Zhang, * Qingsong Yu,
* Zhihua Gan
*
a
Department of Urology, China-Japan Friendship Hospital, Beijing, China, 100029;
b
The State Key Laboratory of Organic−inorganic Composites, Beijing University of
Chemical Technology, Beijing, China, 100029;
c
Beijing Laboratory of Biomedical Materials, Beijing University of Chemical
Technology, Beijing, China, 100029;
d
Beijing Advanced Innovation Center for Soft Matter Science and Engineering,
Beijing University of Chemical Technology, Beijing, China, 100029;
e
College of Life Science and Technology, Beijing University of Chemical Technology,
Beijing, China, 100029;
f
Key Laboratory of Biomedical Materials of Natural Macromolecules (Beijing
University of Chemical Technology), Ministry of Education, Beijing, China, 100029;
g
Graduate School of Peking Union Medical College and Chinese Academy of Medical
Sciences, China-Japan Friendship Hospital, Beijing, China, 100029;
h
Peking University China-Japan Friendship School Clinical Medicine, Beijing, China,
1
#
*
00029.
These authors contributed equally to this work.
Corresponding author. E-mail address: gzhang40@hotmail.com (Guan Zhang);
yuqs@mail.buct.edu.cn (Qingsong Yu), zhgan@mail.buct.edu.cn (Zhihua Gan).
Abstract
Chemotherapy is the standard of care for bladder cancer after transurethral
resection of the tumor. However, the rapid excretion of clinically used formulations of
anticancer drugs make the common intravesical instillation chemotherapy far from
efficient. Therefore, improving the muco-adhesion and penetrability of
chemotherapeutic drugs became the key factors in the post-surgery treatment of

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superficial bladder cancers. Here, a reduction sensitive vehicle was developed to
deliver the reactive oxygen species activated prodrug of gambogic acid for treatment
of orthotopic bladder cancer. The positively charged chitosan can significantly
enhance the adhesion and permeability of prodrug within the bladder wall. Moreover,
by utilizing the different glutathione and ROS level between cancer cells and normal
cells, the dual responsive nanoparticle can selectively and rapidly deliver drug in
bladder cancer cells, and thus can significantly inhibit the proliferation of bladder
cancer cells in an orthotopic superficial bladder cancer model without causing damage
to normal cells. This work demonstrates that the smart prodrug nanomedicine may act
as a promising drug-delivery system for local chemotherapy of bladder cancer with
unprecedented clinical benefits.
Keywords: Bladder cancer, intravesical instillation chemotherapy, mucoadhesive,
stimuli-responsive, prodrug
1
. Introduction
Bladder cancer (BC), approximately 75% of which was diagnosed as
non-muscle-invasive type (NMIBC),[1, 2] is one of the most common malignancies
worldwide.[3, 4] The mainstay standard of care for NMIBC management is
transurethral resection of bladder tumor (TURBT), with an 80% early success rate.[5]
However, after TURBT, many patients will experience a 50% risk of recurrence and
2
0% chance of progression within 5 years.[6] Because of the special physiological
structure of bladder cavity, post-surgery intravesical instillation chemotherapy (IIC)
can be directly administrated through the urethra to attain high local concentrations
and minimal systemic toxicity.[7, 8] This could reduce the recurrence rate of NMIBC
by 15-20%, and reduce the long-term recurrence rate by about 5%.[9, 10]
Although chemotherapy plays an important role in the treatment of BC, some
characteristics of bladder present challenges for IIC. Firstly, the bladder permeability
barrier (BPB) lining the luminal surface of the bladder prevents the efficient
penetration of drugs into tumors.[7] Secondly, the residence of free drugs inside

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bladder is quite short due to the voiding of urine,[11, 12] so that continuous infusion
is required to maintain high drug concentration, which may result in increased
expense, elevated side effects and occurrence of drug-resistance. To address these
issues, various nanoscopic drug delivery vehicles have been applied, such as
polymer-drug conjugates,[13] carbon nanotubes,[14] liposomes,[15, 16] polymeric
nanoparticles,[17-19] nanogel,[20, 21] and so forth. Two major strategies have been
applied in the development of nano-vehicles aiming at intravesical delivery of
anticancer drugs. One is the introduction of active targeting moieties, for example,
RGD peptide[22] and folic acid,[23, 24] which have been proved to selectively target
BC cells. The other is the application of mucoadhesive materials in the construction of
drug carriers, such as chitosan and its derivatives,[25, 26] polyethylene glycol,[27]
hyaluronic acid,[28] alginate,[29] gelatin,[30] cationic polypeptides[20, 31] and
fluorinated polyethylenimine,[32] thiolated particles,[33, 34] etc.
With these advanced nanotechnologies, the therapeutic efficacy of IIC has been
greatly improved. However, the payloads of most current nanomedicines are general
cytotoxic drugs like doxorubicin,[22] paclitaxel,[35] cisplatin,[36] mitomycin[19] or
gemcitabine.[26] Their limitations in selectivity will inevitably be passed on to the
formulated nanomedicine, which may cause the damage of normal urothelium and
urethra, and further increase the risk of infection and urethral obstruction[37].
Therefore, it is essential to develop a new drug delivery system with high selectivity.
Even though BC-selective peptide based nanomedicine, which resulted in great
therapeutic efficacies including the prolonged lifetime and the delayed tumor growth,
has been reported recently,[32] tumor-selective nanomedicine based on small
molecule chemotherapeutics has merely been mentioned. In response to this demand,
the increased numbers of reactive oxygen species (ROS), one characteristic of cancer
cells that distinguishes themselves from normal cells,[38, 39] underlie the design
criteria of efficient tumor-specific nanomedicines. Moreover, Given the different
metabolic pathways and rates of cancer cells in comparison with normal ones, the
high intracellular glutathione (GSH) concentration of about 10.0 mM[40, 41] in
malignant cells also provide an interesting target.

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Herein, based on the above considerations, a new GSH responsive delivery
system based on positively charged chitosan, a well-established mucoadhesive
biomolecule used as a drug carrier for enhancing the contact between drugs and
bladder for better tissue permeability and longer residence time,[42] was developed to
deliver ROS activated gambogic acid prodrug (noted as GB). Gambogic acid (GA), a
naturally occurring xanthone-based moiety, extracted from Garcinia hanburyi tree, is
known to be an efficient anticancer drug through numerous intracellular and
extracellular actions, including programmed cell death, autophagy, cell cycle arrest,
anti-angiogenesis, anti-metastatic, and anti-inflammatory activities.[43] In this way,
drug release can only be triggered by the higher concentration of GSH and ROS level
inside BC cells, which will then exempt normal urothelium. To verify our hypothesis,
the solution properties, drug release profiles, in vitro activity and selectivity has been
investigated. Meanwhile, an orthotopic BC xenograft model was established to
evaluate the anticancer efficacy and safety profiles.
Scheme 1. The schematic illustration of intravesical instillation of tumor cells or
various drugs and the synthetic routes of vehicle and prodrug.

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2
. Materials and methods
2
.1 Materials
Gambogic acid was purchased from Sichuan Weikeqi Biological Technology Co.
Ltd. 4-Dimethylaminopyridine (DMAP), N-hydroxy succinimide (NHS),
dicyclohexyl carbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDC), 4-hydroxybenzylboronic acid pinacol ester, chitosan
(Mw=5kDa), 4,4’-dithiodipropionic acid, adipic acid, benzyl alcohol,
dihydroethidium, anhydrous dimethylsulfoxide (DMSO) were purchased from
Aladdin and used without further purification. Mouse BC cells (MB49) and mouse
fibroblasts (NIH-3T3) were purchased from cell bank of typical culture preservation
committee of Chinese Academy of Sciences. Dulbecco’s modified eagle’s medium
(DMEM), fetal bovine serum (FBS) and penicillin/streptomycin solution were
obtained from Wisent (Canada). The other chemicals (reagent grade) in this work
were purchased from Sigma Aldrich and used directly.
2
.2 Synthesis and characterization of ROS responsive Gambogic acid prodrug (GB)
25 mg of gambogic acid (0.2 mmol), 140 mg 4-hydroxybenzylboronic acid
1
pinacol ester (0.6 mmol) were dissolved in 10 mL dichloromethane (DCM). 61.8 mg
DCC and 10 mg DMAP were added and reacted under room temperature for 12 h.
The precipitate was then filtered, the filtrate was washed with 1M HCl, saturated
sodium bicarbonate, saturated sodium chloride. The organic phase was collected and
dried with anhydrous sodium sulphate followed by rotary evaporation. The product
was further purified though column chromatography using ethyl acetate and n-hexane
(1:5) as elution phase. The final product was obtained after vacuum drying. The
1
chemical structure of the GB was analyzed by H NMR (Bruker AVANCE 400MHz).
2
.3 Synthesis and characterization of hydrophobically modified chitosan
The functionalized chitosan was synthesized by two-step reaction. At the first
step, dithiodipropionic acid or adipic acid was reacted with benzyl alcohol to yield
dithiodipropionic acid mono benzyl ester or adipic acid mono benzyl ester. Taking
dithiodipropionic acid as an example, 4.205 g dithiodipropionic acid (20 mmol) and
0
.432 g benzyl alcohol (4 mmol) were firstly dissolved in 40 mL dimethylformamide.

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Then 0.62 g EDC (4 mmol) and 0.049 g DMAP were added and reacted under room
temperature for 12 h. DMF was removed by rotary evaporation and DCM was added
to precipitate the unreacted dithiodipropionic acid. The filtrate was washed with
saturated sodium chloride and dried with anhydrous sodium sulphate. The product
was purified with column chromatography. The column was firstly washed with
DCM:methanol (15:1) and then with DCM:methanol (10:1) to yield final product.
The second step was the amidation of chitosan with mono benzyl ester. Briefly,
1
.08 g chitosan (6.7 mmol amino group) and 0.244 g dithiodipropionic acid mono
benzyl ester (0.8 mmol) were dissolved in 50 mL DMF. 0.23 g EDC (1.2 mmol) and
.138 g NHS (1.2 mmol) were then added and reacted under room temperature for 24
0
h. The product was purified by repeated precipitation in acetone to remove the
unreacted dithiodipropionic acid mono benzyl ester. Then the precipitate was
re-dissolved in DMF and dialyzed in water. The pure product dithiodipropionic acid
SS
mono benzyl ester modified chitosan ( CB) could be obtained after freeze drying.
2
.4 Preparation and characterization of mucoadhesive nanoparticles
The mucoadhesive nanoparticles based on chitosan polymer were prepared by
SS
SS
nanoprecipitation method. Briefly, taking CB as an example, 10 mg CB and 5
GB
mg GB was dissolved in 1 mL of DMF and slowly injected into 10 mL water with
syringe pump (Langer Pump Co. Ltd.) at a flow rate of 2 mL/h. After injection, the
solution was further stirred for 1 h and then dialyzed in water (MWCO = 8,000 ~
1
4,000 Da).
The size and zeta potential of as-prepared nanoparticles were characterized by
dynamic light scattering using Malvern Zeta sizer (ZS90, Malvern, Germany). The
morphology of nanoparticles was further detected using transmission electron
microscope (TEM, JEM-2100, JEOL, Japan).
2
.5 Cell culture
The MB49 and NIH-3T3 cells were grown in DMEM medium supplemented
with 10% FBS. Cells were maintained at 37 °C in a humidified atmosphere of 5%
carbon dioxide and 95% air.
2
.6 In vitro cytotoxicity assay

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MB49 cells were plated in 96-well plates and treated by different amount of GA,
CC
SS
CC
SS
GB, CB , CB , CBGB and CB for 24 h. The cytotoxicity was evaluated
GA
GA
GB
using the MTT [3-(4, 5-dimethyltiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] cell
viability assay as previously described.[44] The plates were read
spectrophotometrically at 570nm using a microplate spectrophotometer (Multiskan
GO, Thermo Scientific). The IC50 values were calculated by GraphPad Prism
software.
2
.7 Detection of apoptosis and cell cycle arrest in vitro
To measure apoptosis and cell cycle arrest caused by various drugs, MB49 cells
5
were seeded on six-well plates at a density of 3×10 cells/well and cultured for 24
CC
SS
CC
SS
hours. Then GA, GB, CB , CB , CBGB and CB with 2μM GA equivalent
GA
GA
GB
was added and cultured for 24 hours. Cells were collected, washed twice with PBS
and treated with apoptosis detection Kit and cell cycle Kit separately. Apoptotic cells
and cell cycle of treated cells were then analyzed with Flow Cytometer (FCM, BD
Biosciences).
2
.8 Cellular uptake of responsive and non-responsive nanoparticles
To detect the cellular uptake of nanoparticles, 2% weight of DiI was added
during nanoparticle fabrication. MB49 cells were seeded onto 35 mm glass bottom
5
petri dish (NEST) at a density of 2×10 cells/well and cultured for 24 hours. Then
-1
SS
CC
medium containing 0.1 mg·mL DiI labeled CBGB and CBGB was added and
cultured for 3 hours or 9 hours. The medium containing the drugs was discarded and
the cell nucleus was stained with Hoechst 33342 for 15 min. Cells were observed
under a confocal laser scanning microscope (CLSM, TCS SP8, Leica) after washed
with PBS.
2
.9 Endocytic mechanism study
Analysis of endocytic pathway of nanoparticles was conducted by flow
5
cytometry. Briefly, MB49 cells were seeded into 6-well plates (3×10 cells/well) at 24
h before the experiment. The cells were pretreated for 30 min with 37°C, 4 °C, with
chlorpromazine (10 μM) to inhibit the formation of clathrin vesicles and filipin III (1
μg/mL) to inhibit caveolae, and amiloride (50 μM) to inhibit macropinocytosis, and

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then treated with DiI labeled nanoparticles for an additional 1 h. Next, the cells were
washed twice with ice cold PBS (pH 7.4) and observed with ARVOX3
multifunctional microplate reader (Perkin Elmer, Waltham, MA, USA).
2
.10 Co-localization of nanoparticles with lysosomes
MB49 cells were seeded onto 35 mm glass bottom petri dish at a density of
5
2
×10 cells/well and cultured for 24 hours. Then medium containing 0.1 mg/mL DiI
CC
SS
labeled CBGB and CB nanoparticles was added and cultured for 3 hours. The
GB
cells were then washed with PBS and the nuclei and lysosome were stained with
Hoechst 33342 dye and Lysotracker Green DND-26 for 15 min and 30 min,
respectively. After washing with PBS, the colocalization was investigated with
CLSM.
2
.11 Establishment of orthotopic BC xenograft model
Female C57BL/6J mice (5-6 weeks old) were purchased from Beijing Huafukang
biological Co. Ltd. All animal experiments were performed in accordance with
guidelines of Peking University Health Science Center Animal Care and Use
Committee under the protocols approved by the Institutional Animal Care and Use
Committee at Peking University (Beijing, China). During the experiment, the animals
were under isoflurane anesthesia. After the anesthesia was stable, urethral intubation
(flexible tube of the 24G BD intravenous catheters) was performed on the mice. Clean
the bladders with PBS buffer solution for 3 times. The mouse bladders were then
infused with 0.1mol/L HCl for 25 seconds, and then instilled with 0.1 mol/L NaOH
for 5 seconds. After washing with PBS for 3 times, the bladders were infused with
0
.025% trypsin solution for 10 min followed by 3 times PBS washing. To enhance the
adhesive ability of MB49 cells, the bladders were instilled with 0.1 mg/mL
Poly-L-lysine solution for 10 min. Finally, the mouse bladders were infused with
MB49 cells suspension, and to avoid spontaneous micturition, the animals remained
anesthesia for approximately 60 min after intravesical instillation.
To prove the successful establishment of orthotopic BC model, MB49 cells
labeled with a membrane dye, DiR, was intravesical instilled. The in situ fluorescent
signal at five days post-instillation was monitored using IVIS Spectrum in vivo living

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imaging system (PerkinElmer, U.S.A). Meanwhile, the bladders were also excised and
subjected to hemoxylin & eosin (H&E) staining to confirm the formation of tumor
mass.
2
.12 The ROS level in normal bladder and cancerous bladder
Dihydroethidium (DHE), a lipophilic probe which readily diffuses across cell
membranes and be rapidly oxidized to ethidium (a red fluorescent compound) by
superoxide and hydrogen peroxide (H O ) (in the presence of peroxidase), was used
2
2
for the measurement of intracellular ROS content. The normal and tumor bearing
mice were anesthetized and sacrificed. Then the bladders were excised and stored
o
immediately under -80 C for frozen section. The bladder slices were then stained cells
with 5μM DHE and DAPI and observed by CLSM.
2
.13 Retention of drug delivery system in the bladder of mice (In vivo fluorescence
imaging)
days after intravesical instillation of MB49 cells, mice were randomized into 3
5
SS
groups (3 mice in each group). Then 50 μL free Cy7.5, Cy7.5 labeled CBGB and
CC
CBGB at 0.1 mg/mL Cy7.5 concentration were infused via urethra (after anesthesia).
Then the mice were placed supine on an IVIS light-tight chamber. Optical imaging of
the Cy7.5 was conducted on an IVIS Spectrum in vivo imaging system using 745/800
filter combination. The exposure time were set as 10 s. The fluorescence intensity at
the bladder region was quantified by Living Image version 4.4 In Vivo Imaging
Software (PerkinElmer, U.S.A).
2
.14 Permeability of nanoparticle in urothelium
To investigate the penetration of drug delivery system in urothelium, normal
C57BL/6J mice were divided into 3 groups (3 mice in each group), and were
CC
intravesical instilled with 50 μL of 0.1 mg/mL of free DiI or DiI labeled CBGB and
SS
CBGB nanoparticles. After staying for two hours under anesthesia, the flexible tube
was extracted, and the fluid in mice bladder was expelled by gently pressing the
abdomen of the mice. To analyze the penetration, the mice were sacrificed, then the
bladders were excised and were spread out on cover slides. The penetration of
fluorescence in the mucosa of bladder was observed by confocal microscope.

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Furthermore, sections were also cut transversely through the midportion of the
excised bladder. The fluorescence distribution in the bladder wall was investigated
with CLSM.
2
.15 Anticancer efficacy and in vivo safety
To investigate the tumor suppression effect of various drugs, tumor bearing mice
CC
were divided into 5 groups with 5 mice in each group. 50 μL of GA, GB, CB ,
GB
SS
CBGB and Saline at 5mg/kg GA equivalent were instilled every three days. Mice
were weighted after every instillation. After 5 times instillation, the mice were
sacrificed. The bladders were excised, weighed, embedded in paraffin after fixed in 4%
paraformaldehyde for 24 h, and submitted for histopathological staining. Sections
were also cut transversely through the midportion of the bladder and subjected to
Ki-67 and Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling
(TUNEL) staining. The heart, liver, spleen, lung and kidney were excised from one
mouse in each group for H&E staining to evaluate the toxicity of drug administration
on normal organs.
2
.16 Statistical analysis
All data were reported as averages plus/minus the standard deviation (mean ±
SD). Statistical significance was assessed by one-way ANOVA with Bonferroni
post-tests using GraphPad Prism 5.0 software.
3
. Results and discussion
3
.1 Preparation and characterization of gambogic acid prodrug and chitosan
nanoparticles

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1
Figure 1. The H NMR spectra of (A) dithiodipropionic acid mono benzyl ester, (B)
SS
CC
adipic acid mono benzyl ester, (C) CB and (D) CB.
As a naturally originated positively charged polysaccharide, the intrinsic
mucoadhesive property of chitosan make itself an ideal intravesical delivery
vehicle.[45] As shown in Scheme 1, in order to efficiently encapsulate hydrophobic
payloads, part of the amino groups of low molecular weight chitosan (Mw = 5kDa)
was modified with hydrophobic moieties containing benzyl group. Benzyl alcohol
was firstly reacted with 3, 3'-dithiodipropionic acid and adipic acid, respectively, to
yield the GSH responsive and non-responsive intermediates. The structure of these
1
intermediates could be confirmed by H NMR spectra (Figure 1A&B). As shown in
Figure 1A, the resonance at 7.39 ppm (a) could be attributed to the benzene ring. The
signals at 2.96 ppm (c) and 2.87 ppm (d) could be ascribed to the protons near
carbonyl group and disulfide group, respectively. The integration ratio between peak
(a) and peak (c) was found to be 5:3.8, indicating the hetero-functionalization of the
two acid groups. Similarly, the structure of mono-modified adipic acid could also be
confirmed by the integration ratio of 5:3.8:3.9:3.9 between the signals at 7.39 ppm (a),

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1
2
.38 ppm (c), 1.76 ppm (d) and 1.55 ppm (e) in the H NMR spectrum (Figure 1B).
The responsive and non-responsive intermediates were then linked to chitosan to
SS
CC
obtain responsive ( CB) and non-responsive carriers ( CB), respectively. The
successful amidation could be proved by the presence of benzene signals in the
1
respective H NMR spectra after repeated precipitation with acetone (Figure 1C&D).
The substitution degree calculated according to the integration ratio between (a) and
SS
CC
(
b) was 5.9% and 6.3% for CB and CB, respectively.
SS
CC
Figure 2. (A) Rh and (B) Typical TEM images of CB and CBGB nanoparticles,
GB
SS
CC
scale bar represents 100 nm. (C) Zeta potential of CB and CBGB in 10 mM PBS
GB
1
(
pH 7.4). (D) The speculative ROS activate mechanism of GB prodrug. (E) The H
NMR spectra of GA and GB. (F) The HPLC elution curves of GA, GB and GB plus
SS
H O . (G) Release profiles of GA from CBGB nanoparticle under different
2
2

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conditions (***p<0.001 for GSH + H O group compared with the other groups. All
2
2
the statistical data are represented as mean ± SD (n=3). (H) ROS level in normal
bladder and cancerous bladder. Cell nucleus were stained by DAPI (in blue), ROS
was stained by DHE (in red).
−
Intracellular ROS signaling including H O , superoxide anions (O ) and
2
2
2
hydroxyl radicals are known to be elevated in cancerous cells[46] and capable of
oxidizing benzylboronic acid/esters[47, 48] to trigger drug release[49] or activation of
prodrugs.[50] Therefore, in order to achieve selective inhibition of cancer cells, a
ROS responsive prodrug GB was synthesized by the esterification between GA and
4
-(hydroxymethyl) benzylboronic acid pinacol ester. As shown in Figure 2E, the
chemical structure of GB could be confirmed by the presence of 4-(hydroxymethyl)
benzene boronic acid pinacol ester signals and the integration ratio of 2:1.08 between
1
peak (b) and peak (c) in the H NMR spectrum. GB prodrug were then loaded by
SS
CC
SS
CC
CB or CB to form CBGB and CBGB nanoparticles through nanoprecipitation
method. The DLC and DLE reached relatively high levels, that is, 36.7% and 73.5%
SS
CC
SS
CC
for CBGB and 35.8% and 71.8% for CB . The resulted CBGB and CBGB
GB
nanoparticles exhibited spherical morphology under TEM (Figure 2B). The average
SS
CC
diameters of CBGB and
CBGB were 83.4 nm (PDI=0.183) and 78.8 nm
(PDI=0.207), respectively, as determined by DLS (Figure 2A), which were slightly
larger than that obtained from TEM, possibly due to the dehydration. Because the
nanoparticles were administered after voiding of bladder, the stability of nanoparticle
was evaluated in PBS only, in which stable size could be obtained during 7 days
SS
incubation (Figure S1). Notably, due to the positive nature of chitosan, both CB
GB
CC
and CBGB nanoparticles were positively charged in PBS at pH 7.4 with zeta
potential of 12.3 ± 1.1 mV and 13.1 ± 1.2 mV (Figure 2C), which may facilitate the
interaction between nanoparticles and biological membranes.
To evaluate whether GB prodrug could be transformed into GA by ROS
activation. GB prodrug was co-incubated with 1 mM H O at pH 7.4 for 1 h, then the
2
2
products were analyzed with HPLC. As shown in Figure 2F, compared with GB, a

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new peak corresponding to GA appeared after treatment, indicating the successful
SS
CC
transformation of GB. The in vitro release behaviors of CBGB and
CBGB
nanoparticles were investigated in PBS (pH 7.4) with or without 10mM GSH or 1mM
H O . As shown in Figure 2G, GA could be efficiently released in the presence of
2
2
both 10mM GSH and 1mM H O , with a total 78.4% of GA detected at 72h by HPLC.
2
2
In contrast, without GSH, 27.8% of GA could be released, while without H O , only
2
2
trace amount of free GA could be detected, indicating that the rapid GA release could
only be triggered by the presence of both high level GSH and H O inside cells.
2
2
SS
Moreover, the dual-sensitivity of CB could also be proved by the release profiles
GB
CC
of CBGB nanoparticles under the same conditions (Figure S2), in which much
slower release rate were observed even in the presence of both GSH and H O .
2
2
Considering the higher ROS level in cancerous tissues, as showed by the stronger red
fluorescence in cancerous bladder compared with the normal bladder (Figure 2H), the
ROS- and GSH- dual responsiveness of as-prepared prodrug nanomedicine may
predict higher toxicity in cancer cells.
3
.2 Cellular uptake, intracellular transportation and cytotoxicity of prodrug
nanomedicine

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SS
CC
Figure 3. (A) The cellular uptake of DiI labeled CBGB and CBGB nanoparticles
(red) at 3 h and 9 h after co-incubation, the nuclei were stained with Hoechst 33342
SS
CC
(
blue). (B) The colocalization between DiI labeled CBGB and CBGB nanoparticles
CC
SS
(
red) with lysosome (green). The in vitro cytotoxicity of GA, GB, CB , CB ,
GA GA
CC
SS
CBGB and CB after co-incubation with (C) MB49 cells and (D) NIH-3T3 cells
GB
for 24 h. Data are presented as mean ± SD (n=6). (E) The IC50 values of GA, GB,
CC
SS
CC
SS
CB , CB , CBGB and CB against MB49 and NIH-3T3 cells (***P<0.001
GA
GA
GB
for MB49 cells compared with NIH-3T3 cells). (F) The pro-apoptotic ability and (G)
CC
SS
CC
SS
cell cycle arrest capacity of Saline, GA, GB, CB , CB , CBGB and CB
GB
GA
GA
(***p<0.001 for saline group compared with the other groups.
SS
CC
The internalization of CBGB and CBGB nanoparticles by MB49 cells were

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monitored with CLSM. For CLSM observation, MB49 cells were incubated with 100
SS
CC
μg/mL DiI labeled CBGB and CBGB nanoparticles for 3 or 9 h. As depicted in
Figure 3A, after 3 h co-incubation, red fluorescence of DiI was observed both on the
membrane and inside cells. Markedly enhanced fluorescence was observed at 9 h,
indicating the time-dependent uptake kinetics. The membrane bounded fluorescence
at both 3 and 9 h might be attributed to the electrostatic interaction between positively
charged chitosan and the membrane. The endocytic mechanism study was carried out
by pre-incubating MB49 cells under different conditions (4 ℃, 37 ℃) or in the
presence of various endocytosis inhibitors (such as chlorpromazine (CPZ), filipin III
(Fil), and amiloride (Amil)).[51] The cellular fluorescence was evaluated using
multifunctional microplate reader (Figure S4). It could be found that the endocytosis
of nanoparticles by MB49 cells was energy dependent and mediated by multiple
SS
CC
mechanisms. The colocalization study of CBGB and CBGB nanoparticles with
lysosome was carried out after 6 h co-incubation. As shown in Figure 3B, both
SS
CC
CBGB and CBGB nanoparticles were found to partially colocalized with Lysosome,
corroborating that multiple endocytic pathways were involved in the uptake of
prodrug nanoparticles. It should be noted that the fluorescence outside lysosome was
much stronger than that inside the lysosome. This may facilitate the release of loaded
drugs since GSH was reported to mainly distribute in the cytoplasm.[52]
SS
CC
The cytotoxicity of GA, GB, CB and CBGB against MB49 and NIH-3T3
GB
cells was estimated following a standard MTT procedure. The cell growth inhibition
SS
CC
capability of CBGA and CBGA in these two cell lines was also characterized to
reveal the tumor selectivity of prodrug nanomedicine. As shown in Figure 3C, MB49
SS
SS
CC
CC
cells treated with GA, GB, CB , CB , CBGB and CBGA exhibited a
GB
GA
concentration-dependent proliferation inhibition. GA (0.52 ± 0.31μM), GB (0.99 ±
SS
SS
0
.68μM), CB (1.56 ± 0.61μM), CB (1.87 ± 0.53μM) showed similar toxicity
GA GB
CC
against MB49 cells. In comparison, the toxicity of CBGA (IC = 3.69 ± 0.71μM)
50
CC
and CBGB (IC50 = 5.98 ± 0.87μM) was slightly lower (P<0.001), revealing the
important role of disulfide bond. The similar toxicity between GA and GB or between
SS
SS
CBGA and CBGB indicated that the prodrug could be efficiently transformed into

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the parent GA inside cancer cells. Compared with cancer cells, the toxicity of GB,
SS
CC
CBGB and CBGB against NIH-3T3 was found to be much lower than that of GA
SS
and CB (Figure 3D) (P<0.001), which might be ascribed to the higher ROS level
GA
in tumor cells than that in normal cells.[38]
To evaluate the apoptosis of BC cells induced by the prodrug nanomedicine,
CC
SS
SS
CC
MB49 cells were exposed to GA, GB, CB , CB , CB , and CB , at an
GA
GA
GB
GB
equivalent GA dosage of 2 μM for 24 h. The cells were then double stained with PI
and Annexin-V FITC for viability and apoptosis assay, and analyzed by FCM. As
shown in Figure 3F, the viability of MB49 cells was significantly reduced after
treatment with different GA or GB formulations. Free GA contributed to 34.7% early
apoptotic cells and 33.3% late apoptotic cells. While for free GB, the level was up to
3
3.6% and 33.7%, respectively. Slightly weaker pro-apoptotic ability was observed
for the nano-formulations of both GA and GB. The results were consistent with the
MTT assay in which GA and GB showed the highest toxicity. Furthermore, the
influence of free drugs and nanomedicines on the cell cycle of MB49 cells was also
investigated. As shown in Figure 3G, GA treatment caused a significant increase of
G1 phase and decrease of S phase compared with normal cells, suggesting that the
cells were arrested in G0/G1 phase by GA. Similar G1 phase increase and S phase
decrease could also be observed in the rest GA or GB containing groups, further
confirming that both free GB and GB nanomedicine might present the same
mechanism of action as free GA and further implying the efficient transformation of
GB prodrug inside cells.
3
.3 Muco-adhesiveness, retention and permeation of prodrug nanomedicine in bladder
The muco-adhesiveness and permeability of free drugs and nanomedicines in
bladder were confirmed by small animal living imaging system and CLSM. The
SS
CC
tumor-bearing mice were anesthetized and then Cy7.5 labeled CBGB and CBGB
solution at an equivalent GA dosage of 5.0 mg per kg body weight was intravesically
instilled into the bladders through the urethra and stay inside bladder for 2 h. Same
dosage of free Cy7.5 was used as control. At different time points after instillation

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(i.e., 0, 6, 12, 24, 36, 48, and 60 hours), the mice were imaged with IVIS Spectrum
living imaging system. As depicted in Figure 4A, at all time points, there was an
SS
CC
obviously stronger fluorescence in the bladders of CB and CB -treated mice
GB
GB
than that in the bladders of free Cy7.5-treated mice. The strongest Cy7.5 fluorescence
was recorded at 0 h for all groups. The fluorescence intensity of free Cy7.5 decreased
SS
CC
rapidly and became undetectable after 24 h, while the CBGB and CB -treated
GB
bladders continually showed strong fluorescence until 60 h, indicating the better
SS
CC
muco-adhesiveness of CB and CBGB than free drugs.
GB
Figure 4. (A) The in vivo living imaging photographs at different time points after
SS
CC
intravesical instillation of free Cy7.5, Cy7.5 labeled CB and CBGB for 2 h. (B)
GB
The time related fluorescent intensity changes at the bladder region of free Cy7.5,
SS
CC
SS
CC
Cy7.5 labeled CB and CBGB treated mice (***P<0.001 for CB and CBGB
GB
GB
compared with free Cy7.5, n=3). (C) The three-dimensional fluorescence distribution
SS
CC
in the urothelium of free DiI, DiI labeled CB and CBGB nanoparticles (red) at 2
GB
h after instillation. (D) The fluorescence distribution of DiI or DiI-labeled
nanoparticles in the cross-section of bladder urothelium.

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The quantitative analysis of fluorescence intensity was showed in Figure 4B. The
initial fluorescence intensity of bladder at 0 h was defined as “100%”. It could be
found that the fluorescence intensity of free Cy7.5 decreased rapidly after instillation,
and less than 10% residual fluorescence could be observed in the bladder region at 24
h. In contrast, the excretion of nanomedicine exhibited much slower velocity.
SS
Approximately 50% residual fluorescence could still be observed for both CBGB and
CC
CBGB after 60 hours, which was comparable with the literature reported polypeptide
nanogels.[20] Therefore, the quantitative data more convincingly confirmed the
excellent muco-adhesiveness of chitosan-based nanomedicine.
To determine the permeability of free drug and positively charged nanomedicine,
SS
CC
the penetration depth of free DiI and DiI labeled
nano-formulations was detected after instillation by CLSM and showed in Figure 4C
D. As observed from the 3D fluorescence distribution in the bladder wall (Figure
C), free DiI was mainly confined near the mucous membrane in almost the entire
CBGB and
CBGB
&
4
bladder, and the largest penetration depth was found to be c.a. 40 μm. The relatively
weak and punctate distributed fluorescence might be attributed to the weak
muco-adhesiveness of free dye. Compared with free DiI, the nano-formulations of DiI
SS
CC
exhibited much stronger mucous penetrating ability. Both CBGB and
nanoparticles showed strong fluorescence with largest penetration depth reaching c.a.
00 μm, which is comparable with the reported nanogel with similar size.[20, 31] The
CBGB
2
dispersed fluorescence in the whole bladder wall could be due to the strong
muco-adhesiveness of positively charged chitosan nanoparticles. However, due to the
limitation of tissue transparency and laser penetration, the fluorescence distribution in
deeper tissue cannot be visualized by this method. Therefore, we rechecked the
mucous penetration of free dye and nano-formulations by observing the fluorescence
distribution in the cross-section of bladder wall. As shown in Figure 4D, it was
consistent with the 3D imaging, punctate fluorescence could be observed near the
mucous membrane of free DiI treated bladder. In contrast, strong and dispersive
fluorescence which extended straightly from mucous membrane to serosa could be

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SS
CC
observed in the bladder wall of CBGB and CBGB groups, further revealing the
adhesiveness and permeability of as-prepared prodrug nanomedicine. Overall, these
SS
CC
phenomena indicated that the exposure to CB and CBGB could maintain a high
GB
level of drug in bladder wall, which was necessary for BC chemotherapy.
3
.4 In vivo antitumor efficacy
Figure 5. In vivo tumor inhibition. (A) The appearance and (B) the average weight of
CC
SS
normal bladder and bladder treated with saline, GA, GB, CBGB and CBGB
CC
SS
(
***P<0.001 for CBGB and CBGB treated bladder compared with GA, GB and
SS
saline groups; n.s. no significance for CBGB compared with normal bladder). (C)

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Body weight changes of tumor bearing mice during treatment with saline, GA, GB,
CC
SS
CC
CBGB and CB (***P<0.001 for GA and saline compared with GB, CBGB and
GB
SS
CB ). (D) Histopathological (i.e., H&E) analyses of normal bladder and tumor
GB
CC
tissue cross-sections after treatment with saline (as a control), GA, GB, CBGB and
SS
CB . The scale bar represents 2.5 mm. (E) The enlarged H&E micrographs of
GB
normal bladder and tumor region and urothelial region of tumor tissue cross-sections
CC
SS
after treatment with saline (as a control), GA, GB, CBGB and CB . (F) The
GB
immunohistochemical (i.e., TUNEL) analyses of normal bladder and tumor region
and urothelial region of tumor tissue cross-sections after treatment with saline (as a
CC
SS
control), GA, GB, CBGB and CB . The scale bar in both (E) and (F) represent
GB
1
00 μm.
To investigate the antitumor efficacy of various drugs in vivo, orthotopic BC
xenograft models in female C57BL/6J mice were established by intravesical injection
of MB49 cells after acid induced urothelium damage. To confirm whether the BC was
successfully induced or not, near-infrared dye DiR labeled MB49 were injected and
monitored with living imaging system at 5 days after instillation (Figure S3A).
Meanwhile, the average bladder weight (Figure S3B) and histological analysis of
tumor slices (Figure S3C) were investigated at 5, 10 and 15 days after tumor
inoculation. The tumor bearing mice were then randomly divided into five groups (n =
CC
SS
5
5
for each group) and treated with saline, free GA, free GB, CBGB or CB at a
GB
.0 mg/kg dosage of GA equivalent by intravesical instillation every three day for a
total of five treatments. Over the entire treatment period, the body weight was
measured as an indicator of systemic toxicity. At the end of treatment, mice were
anaesthetized and sacrificed, and the bladder was excised, photographed and weighed,
followed by hematoxylin and eosin (H&E) staining along with major organs like heart,
liver, spleen, lung and kidney. As shown in Figure 5A&B, the average weight of
CC
SS
normal bladder as well as GA, GB, CB , CB and saline treated bladder was
GB
GB
2
6.5, 83.5, 113, 46.4, 31.3 and 243 mg, respectively. It was approved that both
CC
SS
CBGB and CB could dramatically inhibit the growth of BC as compared to free
GB

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GA or GB (P<0.001), which was mostly benefited from the improved
muco-adhesiveness and permeability of nanoparticles. Despite the efficient in vitro
anticancer activity, the suboptimal antitumor activity of free GA and GB was
attributed to their quick excretion with urine. It should be noted that, both the average
SS
weight and appearance of CB treated bladder are similar as the healthy mice
GB
bladder, which showed general weight and smooth surface. Overall, similar anticancer
efficacy as the previously chitosan formulations,[25, 26] mucoadhesive[20, 31] or
fluorinated formulations[32] could be obtained with as-prepared prodrug
nanomedicine.
At the end of the intravesical instillation of various drugs, the bladders were
excised and sectioned for histopathological examination to further verify the
antitumor efficacy. For H&E staining, cancer cells were characterized by the spherical
or spindle nuclei with obvious atypia compared to normal cells. However, the
apoptotic cells had an ill-defined morphology, and the nuclei became darker, pyknotic,
and even disappeared.[51] As shown in Figure 5D, the cross-sections of bladder
CC
treated with saline, GA and GB were filled with dense tumor mass, while CBGB and
SS
CBGB treated bladders exhibited similar appearance as normal bladder as revealed
by intact urothelium and few cancer cells. The enlarged images of these sections
could be found in Figure 5E. Large amounts of tumor cells and few tumor necrosis
areas were observed in the H&E-stained tumor tissue section in the saline group,
indicating the active tumor growth. Higher degrees of tumor necrosis were observed
for the tumor region in GA treated bladder than that for GB, which is in accordance
with the different bladder weight. Meanwhile, tumor cell infiltration into the muscular
layer could also be observed in the urothelium region of GA and GB treated mice,
indicating the disease progression. Encouragingly, very few tumor cell clusters could
CC
be observed in CBGB treated bladder, while the tumor cells could hardly be
SS
distinguished by naked eyes in CBGB treated bladder, suggesting that the
muco-adhesiveness and permeability played important roles in treating BC. Except
for the efficient tumor inhibition, the urothelium maintained intact after treatment
CC
SS
with both CBGB and CB . This could be explained by their “double insurance”
GB

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drug release mechanism, which led to efficient tumor cell selectivity.
Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL)
assay has been designed to detect apoptotic cells that undergo extensive DNA
degradation during the late stages of apoptosis.[53] This method is based on the
ability of TdT to label blunt ends of double-stranded DNA breaks independent of a
template. As shown in Figure 5F, more intense positive signal of TUNEL was
observed in the GA group than in GB group, which suggested the existence of more
apoptotic cells, especially in urothelial region. Since the tumor mass was almost
CC
SS
completely eradiated by CBGB and CB , the rest tissue was mainly urothelium.
GB
Therefore, the lack of apoptotic cells indicated the remarkable safety profiles of
prodrug nanomedicine rather than the lack of anticancer efficacy. Moreover,
immunohistochemical staining for Ki-67, which is expressed from G1 phase to
anaphase and thus is suitable for estimating the fraction of a given cell population
with cell-cycle activity,[54] was performed to analyze the cell proliferation status,
which was shown in Figure S5. The largest number of Ki-67 positive cells was
observed in the saline group, while less Ki-67 positive cells was found in the GB
groups. Compare with GB, fewer Ki-67 positive cells could be observed after GA
treatment, and only a few clustered proliferated tumor cells could be found for
CC
CBGB treated bladder, indicating the better tumor growth inhibition. Because the
SS
rest tissue in CB treated bladder was mainly the highly differentiated urothelial
GB
cells, no obvious cell proliferation could be observed, confirming the remarkable
SS
capability of CB nanomedicine for BC chemotherapy. Considering the selective
GB
killing ability of the prodrug nanomedicine, the remarkable anticancer efficacy may
also be attributed to the exempt of normal cells whose damage may cause the
activation of a series of senescence-associated secretory phenotype (SASP) so as to
promote drug resistance and tumor outgrowth.[55, 56]

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Figure 6. Histopathologic analysis of major organs including heart, lung, liver, spleen
CC
and kidney excised from normal mice or mice treated with saline, GA, GB, CBGB
SS
and CB . The scale bar represents 100 μm.
GB
In order to assess the toxicity of free drug and prodrug nanomedicine in vivo, the
change in body weight was monitored after every injection. As shown in Figure 5C,
during the entire process of chemotherapy, free GA and saline treated group exhibited
significant weight loss. The weight loss in saline group might be due to the malignant
proliferation of tumor mass which will inevitably cause the obstruction of urethra and
bladder. In contrast, the weight loss after GA treatment might be explained by the
sustained damage of urothelium caused by GA as indicated by the TUNEL staining of
GA treated bladder (Figure 5F). The mice treated with free GB or GB nanomedicine
did not show any weight loss, suggesting the negligible toxicity of prodrug strategy.
Moreover, the safety of prodrug strategy can also be proved by the histopathologic

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analysis of major organs like heart, liver, lung, spleen and kidney (Figure 6), in which
no obvious organ toxicity could be observed for all groups. These data strongly
SS
suggested that CB prodrug nanomedicine could efficiently adhere and penetrate
GB
bladder urothelium and selectively release GA in tumor cells, which enhanced the
tumor growth inhibition effect and decreased the side effects.
4
. Conclusions
In summary, a reduction- and ROS- dual responsive prodrug nanomedicine was
developed for the potential intravesical chemotherapy of BC. The cationic chitosan
promoted the muco-adhesiveness and penetrability of GB prodrug within the bladder
SS
wall. In addition, the reduction- and ROS-sensitive properties gave CB the ability
GB
to accurately release GA in BC cells when compared with free drugs. Consequently,
SS
the intravesical instillation of CBGB significantly inhibited tumor growth in an
orthotopic BC model while caused no toxicity to normal urothelium. The safety
profiles and the efficient tumor suppression in vivo demonstrate the great potential of
as-prepared prodrug nanomedicine in intravesical chemotherapy.
Data availability statement
The raw/processed data required to reproduce these findings cannot be shared at this
time due to technical or time limitations.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of
China (Grant Nos. 51503013, 51390481, 21774008, and 81472412) and by the
long-term subsidy mechanism from the Ministry of Finance and the Ministry of
Education of PRC for BUCT. This work was also supported by the Fundamental
Research Funds for the Central Universities of China (Grant Nos. XK1701,

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XK1802-8) and Research Projects on Biomedical Translation of China-Japan
Friendship Hospital (Nos. PYBZ1823, PYBZ1838).
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