Smart Responsive Quercetin-Conjugated Glycol Chitosan Prodrug Micelles for Treatment of Inflammatory Bowel Diseases

Article abstract of DOI:10.1021/acs.molpharmaceut.0c01245

The incidence and progression of inflammatory bowel disease are closely related to oxidative stress caused by excessive production of reactive oxygen species (ROS). To develop an efficacious and safe nanotherapy against inflammatory bowel diseases (IBD),

Full text of DOI:10.1021/acs.molpharmaceut.0c01245

pubs.acs.org/molecularpharmaceutics
Article
Smart Responsive Quercetin-Conjugated Glycol Chitosan Prodrug
Micelles for Treatment of Inflammatory Bowel Diseases
Cuiyun Shen,∥ Luqing Zhao,∥ Xueying Du, Jiaxin Tian, Yi Yuan, Mengdi Jia, Ye He, Rong Zeng,*
Renzhong Qiao,* and Chao Li*
Cite This: Mol. Pharmaceutics 2021, 18, 1419−1430
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ABSTRACT: The incidence and progression of inflammatory bowel disease are closely related to oxidative stress caused by
excessive production of reactive oxygen species (ROS). To develop an efficacious and safe nanotherapy against inflammatory bowel
diseases (IBD), we designed a novel pH/ROS dual-responsive prodrug micelle GC-B-Que as an inflammatory-targeted drug, which
was comprised by active quercetin (Que) covalently linked to biocompatible glycol chitosan (GC) by aryl boronic ester as a
responsive linker. The optimized micelles exhibited well-controlled physiochemical properties and stability in a physiological
environment. Time-dependent NMR spectra traced the changes in the polymer structure in the presence of H2O2, confirming the
release of the drug. The in vitro drug release studies indicated a low release rate (<20 wt %) in physiological conditions, but nearly
complete release (>95 wt % after 72 h incubation) in a pH 5.8 medium containing 10 μM H2O2, exhibiting a pH/ROS dual-
responsive property and sustained release behavior. Importantly, the negligible drug release in a simulated gastric environment in 1 h
allowed us to perform intragastric administration, which has potential to achieve the oral delivery by mature enteric-coating
modification in future. Further in vivo activities and biodistribution experiments found that the GC-B-Que micelles tended to
accumulate in intestinal inflammation sites and showed better therapeutic efficacy than the free drugs (quercetin and mesalazine) in
a colitis mice model. Typical inflammatory cytokines including TNF-α, IL-6, and iNOS were significantly suppressed by GC-B-Que
micelle treatment. Our work promoted inflammatory-targeted delivery and intestinal drug accumulation for active single drug
quercetin and improved the therapeutic effect of IBD. The current study also provided an alternative strategy for designing a smart
responsive nanocarrier for a catechol-based drug to better achieve the target drug delivery.
KEYWORDS: glycol chitosan, quercetin, nanomicelles, dual responsive, drug delivery, IBD
prevent or treat IBD. It has been determined that the
pathophysiology of IBD is closely related with the over-
production of reactive nitrogen species (RNS) and reactive
oxygen species (ROS)6−9 and this may cause an imbalance
between these reactive species (RS) and the antioxidant activity.
1. INTRODUCTION
Inflammatory bowel diseases (IBD) are the idiopathic chronic
multifactorial inflammatory diseases of the gastrointestinal tract,
mainly including ulcerative colitis (UC) and Crohn’s disease
(CD).1,2 Nowadays, corticosteroids, immunosuppressants, anti-
biotics, and biological agents are used as the first-line of
treatment for patients with mild to moderate symptoms of IBD.
However, taking these drugs for a long time may cause serious
side effects, and IBD is difficult to cure with them.3−5 When
pharmacotherapy becomes inadequate to control inflammation,
the IBD patients were only treated through surgical approaches,
as colectomy. Therefore, it is an urgent clinical need to develop
new and safer drugs as complementary and alternative therapy to
Received: December 23, 2020
Revised: January 18, 2021
Accepted: January 20, 2021
Published: February 1, 2021
https://dx.doi.org/10.1021/acs.molpharmaceut.0c01245
© 2021 American Chemical Society
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Scheme 1. Schematic Presentation of the GC-B-Que Prodrug Micelle Structure and its Inflamed-Selective Anti-Inflammatory
Drug Delivery by a pH/ROS-Triggered Mechanism
Thus, oxidative damage is the main cause of ulcerative colitis.
Within this context, flavonoids are attracting attention as
effective anti-inflammatory and antioxidant agents.10−12
advantageous properties of its parent molecule. As a hydrophilic
polymer, GC grafted with hydrophobic fragments can generate
an amphiphilic copolymer, which can self-assemble into
nanoparticles in an aqueous solution.26−28 Therefore, we here
proposed a functional prodrug micelle as an inflammation-
targeted drug, which was comprised by quercetin (Que)
covalently linked to biocompatible glycol chitosan by aryl
boronic ester as a responsive linker. This nanosystem expected
that glycol chitosan-based prodrug micelles effectively delivered
quercetin to the inflamed tissue by a pH/ROS dual-responsive
mechanism, improving its bioavailability in the specific site
(Scheme 1). The synthesis, characterization, self-assembly, and
H2O2-triggered hydrolysis (under different pH media) of
polymeric GC-B-Que were explored to optimize the micelle
structure. We also studied the ability of the system to release
quercetin in physiological, gastric, and simulated intestinal pH.
Further, the anti-inflammatory effect and biodistribution of this
formulation were evaluated in a mice model of dextran sulfate
sodium (DSS)-induced colitis, which showed the same
characteristics as human ulcerative colitis. Although the current
system was evaluated by intragastric administration, resulting in
the early release of a small amount of drugs, the substantial
therapeutic effect in the mice model indicated that most drugs
can be released and accumulated in the inflamed tissue due to
short stay in a gastric environment. It will inspire us to improve
the current system by mature enteric-coating modification to
really achieve oral delivery in future.
The main representatives of flavonols are quercetin and its
glycosylated derivatives, such as rutin and quercetin (quercetin
3-rhamnoside), which have been shown to weaken the
pharmacological model of colitis significantly.13−16 Lots of
studies in vitro have provided evidence that a quercetin
monomer could better reduce the inflammatory response
compared with its derivatives but the same efficacy was not
observed in in vivo studies. Poor absorption in the stomach and
intestines of these derivatives and colon microflora degradation
are main reasons for the low concentration of derivatives in the
colon.17−20 Quercetin’s availability and absorption rate are
pivotal to exert its claimed biological activities at its site of
action.21 Therefore, colon-specific drug delivery systems are a
necessary strategy, which can keep quercetin from absorption
and degradation through the gastrointestinal tract and to
increase its availability. Castangia and co-workers reported that
quercetin-loaded poly(ethylene glycol)-containing vesicles were
coated with chitosan, which represented a suitable method to
increase quercetin concentration in the colon.22 Very recently,
Soliman et al. also prepared quercetin-encapsulated chitosan
microcapsules and evaluated anti-inflammatory and antioxidant
effects on pharmacological rabbit models of colitis.23 Compared
with these physical encapsulation, covalent modification may
allow for intelligent systems with certain desired response
characteristics (such as pH and reactive oxygen species (ROS)
response) for specific drug delivery at the site of inflammation.24
The high level of ROS and a mild acidic environment in
inflamed tissues can be considered as a trigger for the rational
design of drug delivery systems at specific sites. Given the unique
structure of catechol-based quercetin, boronate would be
appropriate for ROS-responsive trigger because it allows
quercetin to directly conjugate to phenylboronic acid under
mild conditions. Meanwhile, boronic ester is an inherently dual-
responsive group: in addition to oxidizability, it can be easily
hydrolyzed into diols and boronic acids at moderately acidic
pH.25 Glycol chitosan (GC) has good water solubility with the
2. MATERIALS AND METHODS
2.1. Materials. Glycol chitosan (GC, Mw = 8.4 kDa, degree
of deacetylation = 82.7%) was purchased from Japan Wako
Industrial Corporation. Quercetin (Que) was purchased from
Tianjin Xiensi Biochemical Technology Co., Ltd. The Spectra/
Por 1 dialysis membrane (molecular weight cutoff (MWCO):
3500 Da) was purchased from Spectrum Laboratories. Pyrene
was purchased from Macklin. 4-Nitrophenyl chloroformate and
L-serine were purchased from Beijing Coupling Co., Ltd.
Dulbecco’s modified eagle medium (DMEM), fetal bovine
serum (FBS), and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-
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Figure 1. (A) Synthesis route of a GC-B-Que polymer and (B) 1H NMR spectra of GC (black line), GC-B (red line), and GC-B-Que (blue line). The
insets are the proton signals in the aromatic region.
H-tetrazolium bromide (MTT) were purchased from Sigma-
Aldrich Co., Ltd. All of the other reagents and solvents were
purchased from Beijing Chemical Reagent Co., Ltd. and used
without further purification. A Bruker AV-400 nuclear magnetic
resonance spectroscope was used to record all NMR spectra at
400 MHz in CDCl3 (unless otherwise specified). An Agilent
6540 UHD Q-TOF MS (analyzed ions up to m/z 6000)
equipped with a gas nebulizer probe was used to record data of
high-resolution mass spectra (HRMS).
2.2. Preparation of Prodrug GC-B-Que Micelles.
2.2.1. Synthesis of 4-(Hydroxymethyl)phenylboronic Acid
Pinacol Ester (2). 4-Hydroxymethylphenylboronic acid 1 (1.0 g,
6.58 mmol) and pinacol (816 mg, 6.91 mmol) were dissolved in
toluene (40 mL) and the mixture was stirred at 100 °C for 2 h.
After the reaction, the solution was allowed to cool to room
temperature and extracted three times with water, and the
organic phases were evaporated in vacuum. The residue was
separated by silica gel column chromatography (CH2Cl2/EA =
25:1) to give the product as a white solid 2 (0.92 g, yield 60.8%),
1H NMR (400 MHz, CDCl3): δ/ppm = 1.34 (s, 12H, CH3),
4.70 (S, 2H, −CH2−), 7.35 (d, J = 8 Hz, 2H, Ph-H), 7.79 (d, J =
8.4 Hz, 2H, Ph-H). 13C NMR (100 MHz, CDCl3) δ/ppm
144.01, 135.06, 126.08, 83.83, 65.25, 24.87. ESI-HRMS (m/z):
calculated for C13H18BO3: 233.0349 [M − H]−; found:
233.0357 [M − H]−.
2.2.2. Synthesis of 4-Nitrophenyl 4-(4,4,5,5-Tetramethyl-
1,3,2-dioxaborolan-2-yl)benzyl carbonate (3). 4-Hydroxyme-
thylphenylborate (2, 0.500 g, 2.1 mmol) and triethylamine (445
μL, 3.2 mmol) were dissolved in 20 mL of tetrahydrofuran. 4-
Nitrophenyl chloroformate (0.470 g, 2.3 mmol) was then added
and stirred at 25 °C for 24 h. Ethyl acetate (20 mL) was added to
terminate the reaction, and the mixture was extracted four times
with saturated NaHCO3. The combined organic phases were
removed in vacuum and the residue was separated by silica gel
column chromatography (n-hexane/EA = 5:1) to give the
product as a white solid 3 (0.60 g, yield 71.6%). 1H NMR (400
MHz, DMSO-d6): δ/ppm = 1.31 (s, 12H, CH3), 5.35 (s, 2H,
−CH2−), 7.48 (d, J = 8 Hz, 2H, Ph-H), 7.58 (d, J = 8.8 Hz, 2H,
Ph-H), 7.73 (d, J = 8 Hz, 2H, Ph-H), 8.33 (d, J = 9.2 Hz, 2H, Ph-
H). 13C NMR (100 MHz, DMSO-d6) δ/ppm 155.53, 152.44,
145.43, 137.07, 135.21, 127.64, 125.31, 121.79, 84.01, 70.82,
24.88. ESI-HRMS (m/z): calculated for C20H23BNO7:
400.2668 [M + H]+; found: 400.2679 [M + H]+.
2.2.3. Synthesis of GC-B. Glycol chitosan (0.96 g, 1.0 mmol)
and 0.3 mL of triethylamine were dissolved in 15 mL of
deionized water and the mixture was stirred for 30 min.
Compound 3 (320 mg, 0.8 mmol) and N-hydroxysuccinimide
(NHS, 115 mg, 1.0 mmol) were dissolved in 15 mL of
tetrahydrofuran (THF), which was added dropwise to the above
mixture at 25 °C. After the reaction with stirring for 9 h, the
mixture was washed twice with methanol. The solution was
dialyzed against methanol for 1 day and then distilled water for 3
days. The product GC-B (295 mg) was obtained by
1
lyophilization. See Figure 1B for the H NMR spectrum. The
preparation of GC-B with various feed ratios was similar to the
above general procedure.
2.2.4. Preparation of GC-B-Que Micelles. The micelles were
prepared using the dialysis method. In brief, 60 mg of GC-B was
dissolved in 25 mL of deionized water and then 5 mL of THF
was added. After stirring at room temperature for 10 min, Que
(20 mg) dissolved in THF (10 mL) was slowly added and the
reaction was allowed to proceed for 10 h. The GC-B-Que
conjugate with a feed ratio of 1:0.33 was obtained, and the
preparation of GC-B-Que with various feed ratios was similar to
1
the above general procedure. See Figure 1B for the H NMR
spectrum and assignment.
The abovementioned resulting mixture was dialyzed against
water/methanol (100:2, v/v) for 2 days (molecular weight
cutoff: 3500 Da) to remove the byproducts and then lyophilized
to obtain a dry yellow flocculent product of GC-B-Que micelles
(60 mg).
2.3. Characterization of Physiochemical Properties.
Gel permeation chromatography (GPC) (TDK 302, Viscotek)
was used to determine the molecular weight distribution of the
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analytes and detected using a DAWNEOS laser light scattering
spectrometer (Wyatt Technology Corporation). A column set
consisting of two PL gel 5 μm MIXED-D columns (7.5 mm ×
300 mm) was used with dimethylformamide (DMF) as an
eluent. The instrument conditions of GPC were as follows: flow
rate, 0.5 mL·min−1; injection volume, 0.2 mL; and detection
wavelength, 690 nm. A Zetasizer Nano instrument (Zetaplus,
Brookhaven) and a HeeNe laser (633 nm) were used to measure
the particle size of micelles by dynamic light scattering (DLS) to
collect optical measurements. All analytes were suspended in a
pH 7.4 phosphate-buffered solution (PBS) at a concentration of
1 mg·mL−1 in DLS measurement. Positive staining was also
performed using 2% uranyl acetate; the morphology was
observed by transmission electron microscopy (TEM) and
inspection was performed using JEM-3010F (JEOL, Tokyo,
Japan) operated at an acceleration voltage of 200 kV. For ζ-
potential measurement, a Nano-ZS ZEN3600 particle sizer
(Malvern Instruments) was used.
We determined the critical micelle concentration (CMC) of
the micelles in water through fluorescence spectroscopy. In
short, a certain amount of pyrene in an acetone solution (1.0 ×
10−4 mol·L−1) was added to a series of test tubes and evaporated
under nitrogen to remove acetone, and then the micelle
solutions at different concentrations varying from 0.0001 to 1.0
mg·mL−1 were added into the above tubes, making the final
concentration of pyrene 2.0 × 10−6 mol·L−1. The pyrene and
micelle mixtures were equilibrated overnight under gentle
shaking (80 rpm) at 37 °C, and then the mixtures were sonicated
in an ultrasonic bath (KQ-800KDE, China) for 10 min at 100 W.
The fluorescence emission spectra of samples were recorded
using a fluorescence spectrophotometer (LS55, PerkinElmer) at
a range from 360 to 500 nm with an exciting wavelength of 343
nm. To determine the CMC value of the micelles, the peak
intensity ratios (I3/I1) of the third band (384 nm, I3) to the first
band (373 nm, I1) were analyzed.
kDa), sealed, and placed in 200 mL of the release medium under
continuous shaking at 100 rpm at 37 °C. At various time
intervals, 1 mL of the buffer solution was collected and
replenished with an equal volume of a fresh medium. The
cumulative amounts of Que were determined by high-
performance liquid chromatography (HPLC) (UV-detector, λ
= 374 nm, C-18 column, eluent: 0.4% formic acid in water/
methanol/acetonitrile/trifluoroacetate (20:50:25:5, v/v), flow
rate: 0.8 mL·min−1).
2.6. Cytotoxicity. Based on the reduction of MTT by
mitochondria in viable cells to water-insoluble formazan, the
cytotoxicity of small-molecule drugs and the GC-B-Que micelle
was determined by MTT assay. Different cells were seeded into
96-well plates at a density of 8000 cells/well, including L929
cells, human colon carcinoma Caco-2 clone C2BBe1, and
human colon adenocarcinoma HT29-MTX. After incubating for
12 h, we aspirated the cell culture medium and replaced it with
200 μL of the sample solution with different concentrations (1,
10, 50, 100, and 200 μg·mL−1) in the Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 0.5% fetal bovine
serum (FBS). Then, the cells were incubated at 37 °C for 24 h,
respectively. Afterward, 200 μL of a 5 mg·mL−1 MTT solution
was added to each well. After further incubation in the dark at 37
°C for 4 h, the medium was aspirated and formazan crystals were
dissolved in 200 μL of dimethylsulfoxide (DMSO) per well. A
microplate reader (PT3502G) was used to perform measure-
ment at a wavelength of 570 nm.
2.7. In Vivo Assessment of Anti-Inflammatory Activity.
A total of 56 male C57BL/6 mice (7-week-old, 20−22 g) were
purchased from SPF (Beijing) Biotechnology Co., Ltd. The
mice were housed in a standardized environment with a 12 h
light/dark cycle, at a temperature of 20−26 °C (humidity, 50
5%) and acclimated for 3 days prior to the experiment. All of the
mice were weighed and confirmed to be healthy before
induction of colitis. In each treated group, colitis was induced
by oral administration of DSS (160110, Mp Biomedicals,
Canada) at 3% (w/v) in tap water ad libitum for 6 days. All
animal experiment protocols were in compliance with the
guidelines for ethical conduct in the care and use of animals in
research by the Chinese Society of Toxicology.
The animals were then grouped into seven subgroups, each
group consisting of eight animals: untreated control group
(saline group), PBS-treated DSS group (model group),
mesalazine-treated DSS group (100 mg of mesalazine/kg
equivalent), Que-treated DSS group (100 mg of Que/kg
equivalent), high-dose GC-B-Que (H)-treated DSS group
(150 mg of GC-B-Que/kg equivalent), medium-dose GC-B-
Que (M)-treated DSS group (100 mg of GC-B-Que/kg
equivalent), and low-dose GC-B-Que (L)-treated DSS group
(50 mg of GC-B-Que/kg equivalent).
The encapsulation efficiency (EE) and loading capacity (LC)
of the GC-B-Que micelle are depicted as eqs 1 and 2,
respectively. The drug solution was filtered, and the concen-
tration of quercetin was measured with a ultraviolet−visible
(UV−vis) spectrophotometer (Genway, England) at a wave-
length of 374 nm.
weight of Que in micelle
weight of Que feed
EE(%) =
LC (%) =
× 100%
(1)
weight of encapsulated Que
total weight of micelle
× 100%
(2)
2.4. H2O2-Responsive Behaviors of GC-B-Que Micelles.
GC-B-Que (6 mg) was fully dissolved in 600 μL of
dimethylsulfoxide (DMSO)-d6 in an NMR tube. H2O2 (30 wt
%) (0.5 μL) was added into the tube for the responsiveness test
at 25 °C. 1H NMR signals at 0, 15, 45, 75, 105, 135, 165, 210, and
240 min were collected, respectively. Using the same protocols
mentioned above, DLS and ζ-potentials were measured.
2.5. In Vitro Drug Release of GC-B-Que Micelles. The
release of GC-B-Que micelles was evaluated using a dialysis
membrane under sink conditions. Various release media
consisting of a phosphate-buffered solution (PBS, pH 7.4, 6.8
and 5.8) or a diluted HCl solution (pH = 2) were used in the
release study. For pH/ROS dual-responsive cases, 10 μM H2O2
was added into the release media. Briefly, 2 mg of GC-B-Que
micelles (DS = 6.76%) was kept in a dialysis bag (MWCO = 3.5
The micelles were administrated to mice by intragastric
administration once a day for seven consecutive days. The body
weights of mice were recorded daily before starting intragastric
administration. The mice were sacrificed on the 8th day. The
blood samples were collected into centrifuge tubes for serum
biochemistry and blood hematological parameter analysis. After
receiving an intraperitoneal injection of sodium pentobarbital
for euthanasia, the colon was divided into two parts, one for
H&E staining, and the other section and other tissues samples
(heart, liver, spleen, lung, kidney, and colon) were snap frozen in
liquid nitrogen and stored at −80 °C for the (RT-q) polymerase
chain reaction (PCR) and/or the determination of drug tissue
distribution.
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Table 1. Physicochemical Properties of GC-B and GC-B-Que with Different Feed Ratios
a
b
c
d
polymer
GC-B
feed ratio
DS (%)
Mn (Da)
Mw (Da)
particle size (nm)
PDI
EE (%)
LC (%)
1:0.33
1:0.5
1:1
1:0.25
1:0.33
1:0.5
8.08
11.24
16.02
6.32
6.65
6.76
16530 949
15420 1015
34171 2333
24535 1873
32308 2140
44531 1278
33540 2726
30480 2791
76194 4725
36720 2066
51417 2927
61245 2621
344.2 18.5
382.1 23.2
355.3 27.2
222.1 10.1
216.5 12.3
215.8 14.5
2.07 0.26
2.00 0.31
2.25 0.29
1.51 0.20
1.60 0.18
GC-B-Que
45.3 0.7
30.2 1.3
23.5 2.3
8.2 0.2
7.9 0.1
6.7 0.1
1.38 0.10
a
b
Ratio of glycol chitosan to phenylboronic ester for GC-B, and GC-B to quercetin for GC-B-Que, respectively. Polydispersity index, PDI = Mw/
c
d
Mn. Encapsulation efficiency, see Section 2.3 for calculation. Loading capacity, see Section 2.3 for calculation.
Colon tissues were fixed with a 4% paraformaldehyde solution
and embedded in a paraffin block for hematoxylin and eosin
(H&E) staining. The blocks were sliced (5 mm in thickness) and
stained by H&E. The optical microscopy (BX 51, Olympus) was
used for observation of the stained slides.
spontaneously react with catechol in water to form stable borate
ester (GC-B-Que). The amphiphilic GC-B-Que conjugates
readily formed self-assembled micelles in an aqueous environ-
ment. In a mild acidic or excess ROS environment, the stimuli-
responsive borate ester was broken to release quercetin
molecules.
Total RNA was extracted from the colon tissue using a TRIzol
reagent (Thermo Fisher Scientific, Inc.). The gene expression
levels of TNF-a, iL-6, and iNOS were tested using the SYBR
Master Mix (Promega Corporation) and the CFX 96 Real-time
(RT) PCR System (Bio-Rad Laboratories, Inc.). The
thermocycling conditions were as follows: an initial step at 95
°C for 2 min, 40 cycles of denaturation at 95 °C for 15 s, and
annealing/extension at 60 °C for 1 min. The primer sequences
used were: TNF-α forward, 5′-GGTCCCCAAAGGGATGA-
GAA-3′, and reverse, 5′-TGAGGGTCTGGGCCATAGAA-3′;
IL-6 forward, 5′-ATGGATGCTACCAAACTGGAT-3′, and
reverse, 5′-TGAAGGACTCTGGCTTTGTCT-3′; iNOS for-
ward, 5′-CAGCTGGGCTGTACAAACCTT-3′, and reverse,
5′-CATTGGAAGTGAAGCGTTTCG-3′.
The successful synthesis of a GC-B-Que nanocarrier was
confirmed by 1H NMR spectra. The characterization of GC and
GC-B was also presented as controls to better assign the proton
signals of quercetin in GC-B-Que (Figure 1B). The character-
istic signals of glycol chitosan can be clearly assigned in the high
field of all spectra.31,32 The broad peak from 3.0 to 3.8 ppm was
assigned to chitosan H3−H6 in D2O. The two peaks at 2.4 and
4.4 ppm were assigned to H2 and H1, respectively. The proton
signals on a glycol side chain (−OCH2CH2O−) were probably
covered by a H3−H6 signal. In GC-B spectra (red line), Ha and
Hb in phenylboronic acid appeared at 7.4 and 7.7 ppm with the
integration of 1:1, respectively. The peaks c−f at 6.8−7.2 ppm
were assigned to aromatic protons of quercetin in GC-B-Que
(blue line), indicating successful conjugation of quercetin to
phenylboronic acid. We calculated the relative peak area ratio of
quercetin protons (e, f) to a glycol chitosan H2 proton to
determine the degree of substitution (DS). The effect of
different feed ratios on the degree of substitution was
investigated, and results are shown in Table 1.
3.2. Physicochemical Properties of Micelles. The
physical and chemical properties of a nanocarrier should be
carefully considered to achieve target special delivery of the
particles. The primary concerns including the degree of
substitution (DS), particle size, surface charge, encapsulation
efficiency (EE), and loading capacity (LC) were determined, as
shown in Table 1. The feed ratio of the reaction could be
changed to adjust the DS of phenylboronic acid and quercetin,
which can greatly affect their self-assembly behaviors and
hydrophilic−lipophilic balance of the micelles. Phenylboronic
ester was first grafted to GC with different DSs ranging from 8.08
to 16.02%, and the mean diameters of the micelles, measured by
DLS, were in the range of 340−380 nm. Subsequently, quercetin
through catechol covalently linked to the optimized GC-B (DS
= 16.02%), and GC-B-Que prepared from different feed ratios
were almost the same in their DSs and sizes (220 nm), with PDI
ranging from 0.17 0.05 to 0.28 0.12. The poor solubility of
quercetin and the reactivity of phenolic hydroxyl (alcoholic
hydroxyl was more likely to react with phenylboric acid), to a
large extent, limited the increase of DS. The optimized GC-B-
Que (DS = 6.76%) was used to evaluate the subsequent drug
release and in vivo anti-inflammatory efficacy.
2.8. In Vivo Biodistribution. Various tissues including
heart, liver, spleen, lung, kidney, and colon were excised and
lightly rinsed to remove any excess blood, weighed, and stored at
−80 °C until assay. The organs were homogenized with 1 mL of
lysis buffer (50 mM Tris−HCl, 0.2 mM NaCl, 5 mM CaCl2, 1%
Triton X-100). Next, 1 mL of acidified isopropanol was added to
the homogenate. The mixture was then incubated overnight at 4
°C. We clarified the tissue extracts by centrifugation at 15 000
rpm for 15 min. The concentration of quercetin in homogenized
tissues was determined by HPLC (UV-detector, λ = 374 nm,
Agilent ODS C-18 column, 4.6 mm × 250 mm, 5 μm particle
size, eluent/methanol: 0.2% phosphoric acid (53:47, v/v), flow
rate: 1.0 mL·min−1, 25 °C).
2.9. Statistic Analysis. One-way analysis of variance
(ANOVA) was used for analyzing the statistical significance of
differences between the groups. Significance of the results was
indicated according to p values (* p < 0.05; **p < 0.01).
3. RESULTS AND DISCUSSION
3.1. Design and Synthesis of GC-B-Que. To improve the
solubility and biocompatibility of a nanocarrier for drug delivery,
we chose glycol chitosan as a carrier skeleton. Stimuli-responsive
phenylborate ester as a linker conjugated quercetin to glycol
chitosan by an effective aminolysis reaction. The synthesis route
of the nanocarrier GC-B-Que is depicted in Figure 1A. Initially,
compound 3 was prepared according to previous reports,29,30 as
a key intermediate for linking glycol chitosan and quercetin. A
phenyl boronate moiety was linked to 2-NH2 on glycol chitosan
by aminolysis, and subsequently, borate ester was then easily
hydrolyzed under alkaline conditions to give GC-B. A simple
strategy was proposed to conjugate quercetin by its unique
catechol structure. The exposed boronic acid group can
The optimized GC-B-Que conjugate (DS = 6.76%) as an
amphiphilic macromolecule can self-assemble to form a
nanomicelle in an aqueous solution. The histogram in Figure
2A clearly presented the size distribution of GC-B-Que, and the
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Figure 2. (A) Size distribution of GC-B-Que micelles. The inset is the TEM image of the micelle. The scale bar is 200 nm. (B) Variations in the
fluorescence intensity ratio I3/I1 with logarithm concentrations of GC-B (red) and GC-B-Que (blue). (C) The particle size (black line) and ζ-
potential (red line) of micelles with different incubation times at pH = 1.2 and 7.4 media.
Figure 3. (A) Probable H2O2-responsive path for the GC-B-Que complex. (B) 1H NMR spectra of GC-B-Que micelles recorded from 0 to 4 h in the
presence of 10 mM H2O2 at 25 °C. (C) The particle size (black line) and ζ-potential (blue line) of micelles under different H2O2 incubation (10 mM)
times.
inset shows the TEM image of corresponding micelles. The
average diameter was around 100 nm, while the DLS results
were slightly larger than the TEM results. This was mainly
because the structure was in a hydrated state during DLS
measurement, which made the size of the structure larger. For
GC-B, however, no desirable result of size distribution and the
TEM image was obtained, which was mainly because of weak
hydrophobicity of boronic acid terminal. Pyrene was used as a
hydrophobic fluorescence probe to study the CMC value to
demonstrate the formation of micelles, and results are shown in
Figure 2B. The CMC value of GC-B-Que was calculated to be
7.1 × 10−3 mg·mL−1, which was obtained by plotting I384/I373
against the logarithm of the micelle concentration. The low
CMC value indicates that micelles have good stability under
highly diluted conditions. In contrast, the case of GC-B showed
a higher CMC of ∼0.1778 mg·mL−1, implying poor micelle-
forming performance. Later, time- and pH-dependent stabilities
of GC-B-Que micelles were monitored on the basis of variation
in sizes and surface charges. In a pH = 7.4 medium, the micelles
showed insignificant changes in size and a negligible decrease in
the ζ-potential at 37 °C after 24 h (Figure 2C), suggesting the
good stability of the micelles that was essential for a prolonged
blood half-life in vivo.33 The remarkable changes in particle size
and surface charge were observed at pH = 1.2 that simulated the
gastric environment. After incubation for 1.5 h, the surface
charge of micelles began to decrease and approached to 0 mV
rapidly and the size also sharply increased. Although these
changes indicated the micelle dissociation, the occurrence was
well over the gastric emptying time.34 Thus, the micelle was
probably intended for intragastric administration but was strictly
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Figure 4. In vitro drug release profiles of quercetin. (A) The GC-B-Que micelle was incubated in a pH = 1.2 HCl solution without and with H2O2 (10
μM); (B) pH = 5.8, 6.8, and 7.4 PBS buffer without H2O2; (C) pH = 5.8, 6.8, and 7.4 PBS buffer with H2O2 (10 μM), as measured through HPLC (see
SI).
Figure 5. Viability of (A) L929 cells, (B) HT29-MTX cells, and (C) C2BBe1 cells treated with glycol chitosan, GC-B, mesalazine, quercetin, and GC-
B-Que with different concentrations at 37 °C for 24 h. Data represent mean standard deviation (SD), (n = 3). The level of significance was set at the
probabilities of *P < 0.05 and **P < 0.01 compared to the control (GC).
based on the following evaluation of drug release under a gastric
medium.
phenyl signals. The characteristic signals of free aryl compounds
moved to a high field and gradually increased. These results
structurally proved the supposed degradation. Additionally, the
particle size and surface charge were also monitored during the
H2O2 response of micelles, as shown in Figure 3C. The size
sharply increased after 2 h of incubation with H2O2, suggesting
H2O2-induced dissociation of B-Que and macromolecule
breakage. The unstable particles featured decreasing surface
charges, affording ζ-potentials from 17.2 to −0.3 mV. These
provided credible evidence for H2O2-induced stimuli-responsive
GC-B-Que micelles.
3.4. In Vitro Drug Release Profile. The stability of GC-B-
Que micelles and the drug release behavior under highly acidic
pH were the major concerns if gavage administration was
considered. Thus, the drug release profile in a simulated gastric
environment was first investigated, and the results were detected
by HPLC, as shown in Figure 4A. Only 12% quercetin was
collected when drug micelles were incubated in a pH = 1.2 HCl
solution without H2O2 for 1 h. It demonstrated that the
nanomicelles could rarely be dissociated under a highly acidic
medium in a short time (<1 h), which effectively prevented the
premature release of the drug in the stomach. After 1 h of
incubation, the quercetin was dramatically released from drug
micelles, and near 95% quercetin was obtained at 2 h. The burst
release of drugs may be related to some dynamic factors.
Although the total quercetin in the micelle was almost released
within 2 h, we can assume that the short periods of drug micelles
in the stomach just caused a negligible premature release. The
3.3. H2O2-Responsive Mechanism. The responsive
behavior of GC-B-Que micelles in the presence of H2O2 was
investigated, and structural changes are shown in Figure 3A. The
reaction first proceeded by temporary quaternarization of the
boron atom and then an oxygen atom was inserted between the
carbon and boron atom. The resulting borate was then
hydrolyzed in the second step of the reaction. The phenol
group underwent a rearrangement based on benzyl 1,6-
elimination,35,36 thus forming p-quinone methide, free querce-
tin, and boronic acid. Due to the presence of a carbonate linker,
the elimination in the formed quinone methide was accom-
panied by the release of CO2.37 The p-quinone methide was
expected to readily convert to p-hydroxybenzyl alcohol (HBA)
after the reaction with water, so the potential toxicity of these
compounds was generally considered not to be an issue.38
Moreover, HBA was the main pharmaceutical ingredient of the
herb Gastrodia elata and was known for its antioxidant
properties.39 Subsequently, time-dependent H NMR signals
1
of the GC-B-Que complex incubated with 10 mM H2O2 were
recorded to confirm the target degradation products (Figure
3B). The signals that appeared at 7.70 and 7.36 ppm
corresponded to the phenyl protons in an aryl boronate linker,
while two weak signals at 6.81 and 7.18 ppm were assigned to
aromatic protons in grafted quercetin. When the micelles were
incubated with H2O2 over time, the responsive side chain was
degraded as the approach mentioned above, showing decreasing
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Figure 6. In vivo anti-inflammatory activity. (A) The body weight variation of DSS-induced inflammatory male C57BL/6 mice during treatment. (B)
Changes in DAI. (C) Average lengths of the whole colon treated by different drugs. (D) The spleen index in each group, calculated by the spleen
weight/body weight. (E) The representative macroscopic view of colon tissue of mice, healthy (saline) and after the induction of colitis with DSS and
treated with different drugs (quercetin, mesalazine, GC-B-Que (L), GC-B-Que (M), and GC-B-Que (H)). (F) Histological study of inflammatory
tissues after treatment by hematoxylin and eosin staining. The scale bar is 200 μm. (G) RT-PCR analysis of the expression of proinflammatory
cytokines IL-6, (H) TNF-α, and (I) iNOS. Statistical analysis was performed using one-way ANOVA followed by Dunnet’s post hoc test. All data were
represented as mean standard error of the mean (SEM) (eight mice in each group). *P < 0.05 and **P < 0.01 compared to the DSS group.
presence of H2O2 has no significant effect on the drug release
profile in a pH = 1.2 medium. The result provided a vital basis for
intragastric administration. Subsequently, the drug release
profiles in physiological and weak acidic environments were
explored. As shown in Figure 4B without H2O2, less than 20%
quercetin was released at pH 7.4 and 6.8 in 72 h of incubation,
indicating the satisfying stability of the micelles around
physiological conditions. At pH 5.8, however, 50% of quercetin
was collected in 15 h of incubation, suggesting the pH sensitivity
of GC-B-Que micelles. At last, 60% of drugs were released over
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the tested time. The same assays were performed again with 10
μM H2O2 incubation, and results are shown in Figure 4C. The
rate of quercetin release sharply increased in the presence of
H2O2 for all cases. More than 50% drugs can be released in 24 h
of incubation at pH 7.4 and 6.8, showing the sensitivity to H2O2.
In the presence of 10 μM H2O2 at pH 5.8 to mimic an
inflammatory environment,40,41 almost all of the quercetin was
released over 48 h of incubation. The sustained and thorough
release indicated that the nanomicelles have a favorable response
ability in inflamed tissues with a high level of ROS and a mild
acidic environment, which promoted drug delivery efficacy.
3.5. Cell Viability. The cytotoxicity of the nanoparticles was
evaluated by MTT assay using L929, human colon carcinoma
Caco-2 clone (C2BBe1), and human colon adenocarcinoma
(HT29-MTX) cell lines, representing the fibroblast, the
enterocytes, and the mucus-producing goblet cells of the
intestine, respectively.6,42 The viability levels of cells after 24 h
of incubation with small-molecule drugs, the micelle, and the
blank vehicles are shown in Figure 5A−C. The cytotoxicity of
the blank vehicles GC and GC-B was first evaluated to obtain
higher than 80% viabilities for the three cell lines at all test
concentrations up to 200 μg·mL−1 after 24 h of incubation,
showing their safety and favorable compatibility to the cells. The
cell viability treated with small-molecule drugs (free quercetin
and mesalazine) had no obvious difference in the three cell lines;
however, a significant difference in the case of GC-B-Que
micelles was observed. Compared with L929 cells, the
noticeable toxicities in the C2BBe1 and HT29-MTX cells
treated by GC-B-Que micelles were observed with increasing
doses. The cell viability decreased to 50% for both cell lines
when the concentration of GC-B-Que micelles (DS of quercetin
was 6.76%) was 200 μg·mL−1. The possible explanation could be
that the amount of quercetin released due to the phenylborate
linker was triggered by relatively high levels of ROS, indicating
the effective response to the special microenvironment.
mucosal structure, crypt abscess formation, and intense cell
infiltration (Figure 6F). Treatment with the three doses of GC-
B-Que micelles significantly restored the colon structure and
goblet cells, improved the mucosal structures, and mildly
infiltrated inflammatory cells compared to the model group,
which were also superior to that in free quercetin and mesalazine
groups. Notably, GC-B-Que micelles contained only about 7%
quercetin (DS = 6.76%), which was far less than the content of
free quercetin under the same tested dose (100 mg·kg−1). The
results indicated that quercetin in the nanomicelles could be
effectively utilized through environmental response in inflam-
matory sites.
The levels of typical inflammatory cytokines including TNF-
α, IL-6, and iNOS significantly increased in mice with DSS-
induced acute colitis (Figure 6G−I). Although free quercetin
and mesalazine as single drugs also showed different degrees of
reduction, therapy with GC-B-Que micelles effectively reduced
the mRNA levels of these inflammatory cytokines. The best
result was obtained from the case of GC-B-Que (M) (100 mg·
kg−1). Considering only 7% quercetin conjugated to the
nanoparticles, the GC-B-Que micelles showed remarkably
suppressed expression of these typical inflammatory cytokines.
Notably, the medium-dose (100 mg·kg−1) micelles showed the
best effect on the DAI, spleen index, and the levels of
inflammatory cytokines. For the similar or decreased efficacy
of the high-dose (150 mg·kg−1) group, we suspected that it was
probably not dose-dependent in the range (over 100 mg·kg−1).
On the contrary, the high concentration polymers may have a
negative effect on the drug release and absorption in vivo.
3.7. In Vivo Biodistribution. In vivo biodistribution of
various doses of GC-B-Que micelles was also evaluated by
quantitatively detecting the quercetin amounts in different
tissues (Figure 7). Quercetin and mesalazine were used as
3.6. In Vivo Anti-Inflammatory Efficacy. The anti-
inflammatory activity of GC-B-Que micelles was evaluated by
recording the body weights, colon lengths, spleen index, and
disease activity index (DAI) of male C57BL/6 mice for 9 days
after treatment. The GC-B-Que drug micelle (DS = 6.76%) was
administered orally at three different doses, 50 mg·kg−1 (GC-B-
Que (L)), 100 mg·kg−1 (GC-B-Que (M)), and 150 mg·kg−1
(GC-B-Que (H)). A 100 mg·kg−1 dosage of free quercetin and
mesalazine was used as a control, respectively, to compare the
therapeutics effect with GC-B-Que micelles. Except for the
saline group as the negative control, the other tested groups
showed similar growth trends in the body weight and DAI within
4 days. Beginning from the 5th day, DSS-treated mice exhibited
significantly increased body weight loss and higher DAI (Figure
6A,B). Significant improvement in weight loss and DAI showed
that treatment of mice with test drugs including mesalazine,
quercetin, and three doses of GC-B-Que micelles significantly
delayed the development of chronic colitis. Figure 6E depicts the
representative macroscopic view of colon tissue treated by
different drugs after 9 days. The saline group had no
macroscopic damage in the colon, while the DSS group showed
obvious inflammation. Compared with other groups, treatment
with quercetin and GC-B-Que (M) showed considerable
recovery in the colon length and appearance. The results of
the colon length and spleen index were quantified, as shown in
Figure 6C,D. H&E-stained histologic images showed that the
normal saline group had a typical colonic mucosal structure,
while the DSS group showed goblet cell depletion, the loss of the
Figure 7. Biodistribution profiles of drug accumulation in the heart,
liver, spleen, lung, colon, and kidney of DSS-induced inflammatory
male C57BL/6 mice following intragastric administration. 100 mg·kg−1
for quercetin, mesalazine, and GC-B-Que (M); 50 mg·kg−1 for GC-B-
Que (L); and 150 mg·kg−1 for GC-B-Que (H).
controls to compare the distribution behavior with GC-B-Que
micelles. Compared with small-molecule drug accumulation in
the colon (54.6 μg·g−1 for quercetin and 69.5 μg·g−1 for
mesalazine), the quercetin amount released from the dose of
GC-B-Que (M) was up to 39.2 μg·g−1, but the DS of quercetin
in the micelle was only about 6.76%. The GC-B-Que micelle
exhibited the effective drug accumulation in inflamed tissue, and
the quercetin amount in the colon was 2−4-fold higher than the
other organs. It was proved that the micelle can reduce the loss
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Authors
of quercetin in the delivery and improve the accumulation in the
Cuiyun Shen − State Key Laboratory of Chemical Resource
Engineering, Beijing University of Chemical Technology,
Beijing 100029, P. R. China
Luqing Zhao − Beijing Hospital of Traditional Chinese
Medicine, Capital Medical University, Beijing 100010, P. R.
China
Xueying Du − State Key Laboratory of Chemical Resource
Engineering, Beijing University of Chemical Technology,
Beijing 100029, P. R. China
Jiaxin Tian − State Key Laboratory of Chemical Resource
Engineering, Beijing University of Chemical Technology,
Beijing 100029, P. R. China
Yi Yuan − State Key Laboratory of Chemical Resource
Engineering, Beijing University of Chemical Technology,
Beijing 100029, P. R. China
Mengdi Jia − Beijing Hospital of Traditional Chinese Medicine,
Capital Medical University, Beijing 100010, P. R. China
Ye He − Department of Materials Science and Engineering,
College of Chemistry and Materials, Jinan University,
Guangzhou 510632, P. R. China
diseased region through inflamed-responsive drug release.
4. CONCLUSIONS
In summary, GC-B-Que as glycol chitosan-modified nano-
micelles were prepared and characterized. Anti-inflammatory
single drug quercetin was conjugated to glycol chitosan by aryl
boronic ester, giving potential pH/ROS dual responsiveness for
effective delivery of the drug to the inflamed colon. Desirable
particle size and stability in physiological conditions were
obtained, supporting the effective delivery in the system
circulation. In vitro drug release assays, GC-B-Que micelles
presented significant responsive drug release in a pH 5.8
medium containing 10 μM H2O2. More than 80% quercetin can
be released with 24 h of incubation in a simulated inflammatory
environment, showing a favorable response ability to excess
ROS and weak acidic pH. The release profiles of quercetin in
gastric pH showed that 12% drugs were collected in 1 h of
incubation, suggesting relative stability of the micelle in gastric
acid for a short time. This result allowed us to attempt to
evaluate in vivo anti-inflammatory activity by intragastric
administration. The following in vivo assays showed that GC-
B-Que micelles can effectively treat DSS-induced colitis and
their efficacy was significantly better than that of free quercetin
and a control drug mesalazine, and the DSS-stimulated
overproduction of TNF-α, IL-6, and iNOS was significantly
inhibited. In vivo biodistribution experiments indicated that the
drug can effectively be accumulated in the inflamed colon
compared to other organs. For active single drug quercetin, the
current work provided a practical strategy to give the inflamed
intestinal tissue targeting and improve the therapeutic index, as
well as a promising drug delivery approach in the treatment of
inflammatory bowel diseases.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.molpharmaceut.0c01245
Author Contributions
∥C.S. and L.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was financially supported by the National Natural
Science Foundation of China (Nos. 21977012, 21672021,
21572018, 81973764, and 31370974), the Joint Project of BRC-
BC (Biomedical Translational Engineering Research Center of
BUCT-CJFH) (XK2020-06), and the Beijing Municipal Science
and Technology Project (No. Z181100001718218)
ASSOCIATED CONTENT
■
sı
* Supporting Information
REFERENCES
■
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.molpharma-
ceut.0c01245.
(1) Dulai, P. S.; Peyrin-Biroulet, L.; Danese, S.; Sands, B. E.; Dignass,
A.; Turner, D.; Mantzaris, G.; Scholmerich, J.; Mary, J.-Y.; Reinisch, W.;
Sandborn, W. J. Approaches to Integrating Biomarkers Into Clinical
Trials and Care Pathways as Targets for the Treatment of Inflammatory
Bowel Diseases. Gastroenterology 2019, 157, 1032−1043.
(2) Leuti, A.; Fazio, D.; Fava, M.; Piccoli, A.; Oddi, S.; Maccarrone, M.
Bioactive Lipids, Inflammation and Chronic Diseases. Adv. Drug
Delivery Rev. 2020, 159, 133−169.
1H, 13C NMR, and HRMS spectra of compounds 2 and 3;
the HPLC analysis of the drug release; and the HPLC
analysis of the drug in vivo biodistribution (PDF)
(3) Jess, T.; Jensen, B. W.; Andersson, M.; Villumsen, M.; Allin, K. H.
Inflammatory Bowel Diseases Increase Risk of Type 2 Diabetes in a
Nationwide Cohort Study. Clin. Gastroenterol. Hepatol. 2020, 18, 881−
888.
(4) Meserve, J.; Aniwan, S.; Koliani-Pace, J. L.; Shashi, P.; Weiss, A.;
Faleck, D.; Winters, A.; Chablaney, S.; Kochhar, G.; Boland, B. S.;
Singh, S.; Hirten, R.; Shmidt, E.; Hartke, J. G.; Chilukuri, P.; Bohm, M.;
Sagi, S. V.; Fischer, M.; Lukin, D.; Hudesman, D.; Chang, S.; Gao, Y.;
Sultan, K.; Swaminath, A.; Gupta, N.; Kane, S.; Loftus, E. V.; Shen, B.;
Sands, B. E.; Colombel, J.-F.; Siegel, C. A.; Sandborn, W. J.; Dulai, P. S.
Retrospective Analysis of Safety of Vedolizumab in Patients With
Inflammatory Bowel Diseases. Clin. Gastroenterol. Hepatol. 2019, 17,
1533−1540.
(5) Kochar, B.; Cai, W.; Cagan, A.; Ananthakrishnan, A. N.
Pretreatment Frailty is Independently Associated with Increased Risk
of Infections after Immunosuppression in Patients with Inflammatory
Bowel Diseases. Gastroenterology 2020, 158, 2104−2111.
(6) Bertoni, S.; Liu, Z.; Correia, A.; Martins, J. P.; Rahikkala, A.;
Fontana, F.; Kemell, M.; Liu, D.; Albertini, B.; Passerini, N.; Li, W.;
AUTHOR INFORMATION
■
Corresponding Authors
Rong Zeng − Department of Materials Science and Engineering,
College of Chemistry and Materials, Jinan University,
Guangzhou 510632, P. R. China; Email: tzengronga@
jnu.edu.cn
Renzhong Qiao − State Key Laboratory of Chemical Resource
Engineering, Beijing University of Chemical Technology,
Beijing 100029, P. R. China; orcid.org/0000-0002-6672-
9609; Phone: +86-10-64413899; Email: qiao_group@
163.com; Fax: +86-10-64416428
Chao Li − State Key Laboratory of Chemical Resource
Engineering, Beijing University of Chemical Technology,
Beijing 100029, P. R. China; orcid.org/0000-0002-0320-
5509; Phone: +86-10-64413899; Email: lichao@
mail.buct.edu.cn; Fax: +86-10-64416428
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Article
Santos, H. A. pH and Reactive Oxygen Species-Sequential Responsive
Nano-in-Micro Composite for Targeted Therapy of Inflammatory
Bowel Disease. Adv. Funct. Mater. 2018, 28, No. 1806175.
(7) Li, S.; Xie, A.; Li, H.; Zou, X.; Zhang, Q. A Self-Assembled, ROS-
Responsive Janus-Prodrug for Targeted Therapy of Inflammatory
Bowel Disease. J. Controlled Release 2019, 316, 66−78.
(8) Yao, Y.; Zhang, H.; Wang, Z.; Ding, J.; Wang, S.; Huang, B.; Ke, S.;
Gao, C. Reactive Oxygen Species (ROS)-Responsive Biomaterials
Mediate Tissue Microenvironments and Tissue Regeneration. J. Mater.
Chem. B 2019, 7, 5019−5037.
(24) Maji, T.; Banerjee, S.; Biswas, Y.; Mandal, T. K. Dual-Stimuli-
Responsive l-Serine Based Zwitterionic Ucst-Type Polymer with
Tunable Thermosensitivity. Macromolecules 2015, 48, 4957−4966.
(25) Gennari, A.; Gujral, C.; Hohn, E.; Lallana, E.; Cellesi, F.; Tirelli,
N. Revisiting Boronate/Diol Complexation as a Double Stimulus-
Responsive Bioconjugation. Bioconjugate Chem. 2017, 28, 1391−1402.
(26) Yang, H.; Tang, C.; Yin, C. Estrone-Modified pH-Sensitive
Glycol Chitosan Nanoparticles for Drug Delivery in Breast Cancer. Acta
Biomater. 2018, 73, 400−411.
́
(27) Ariza-Saenz, M.; Espina, M.; Calpena, A.; Gómara, M. J.; Pérez-
Pomeda, I.; Haro, I.; García, M. L. Design, Characterization, and
Biopharmaceutical Behavior of Nanoparticles Loaded with an HIV-1
Fusion Inhibitor Peptide. Mol. Pharmaceutics 2018, 15, 5005−5018.
(28) Lee, S. H.; Back, S.-Y.; Song, J. G.; Han, H.-K. Enhanced Oral
Delivery of Insulin via the Colon-Targeted Nanocomposite System of
Organoclay/Glycol Chitosan/EudragitS100. J. Nanobiotechnol. 2020,
18, No. 104.
(29) Yik-Sham Chung, C.; Timblin, G. A.; Saijo, K.; Chang, C. J.
Versatile Histochemical Approach to Detection of Hydrogen Peroxide
in Cells and Tissues Based on Puromycin Staining. J. Am. Chem. Soc.
2018, 140, 6109−6121.
(30) Major Jourden, J. L.; Cohen, S. M. Hydrogen Peroxide Activated
Matrix Metalloproteinase Inhibitors: a Prodrug Approach. Angew.
Chem., Int. Ed. 2010, 49, 6795−6797.
(9) Balmus, I. M.; Ciobica, A.; Trifan, A.; Stanciu, C. The Implications
of Oxidative Stress and Antioxidant Therapies in Inflammatory Bowel
Disease: Clinical Aspects and Animal Models. Saudi J. Gastroenterol.
2016, 22, 3−17.
(10) Ribeiro, D.; Proenca, C.; Rocha, S.; Lima, J. L. F. C.; Carvalho, F.;
Fernandes, E.; Freitas, M. Immunomodulatory Effects of Flavonoids in
the Prophylaxis and Treatment of Inflammatory Bowel Diseases: A
Comprehensive Review. Curr. Med. Chem. 2018, 25, 3374−3412.
(11) Ribeiro, D.; Freitas, M.; Lima, J. L. F. C.; Fernandes, E.
Proinflammatory Pathways: The Modulation by Flavonoids. Med. Res.
Rev. 2015, 35, 877−936.
(12) Vezza, T.; Rodríguez-Nogales, A.; Algieri, F.; Utrilla, M. P.;
Rodriguez-Cabezas, M. E.; Galvez, J. Flavonoids in Inflammatory Bowel
Disease: A Review. Nutrients 2016, 8, 211.
(31) Na, J. H.; Koo, H.; Lee, S.; Han, S. J.; Lee, K. E.; Kim, S.; Lee, H.;
Lee, S.; Choi, K.; Kwon, I. C.; Kim, K. Precise Targeting of Liver Tumor
Using Glycol Chitosan Nanoparticles: Mechanisms, Key Factors, and
Their Implications. Mol. Pharmaceutics 2016, 13, 3700−3711.
(32) Lee, D.-E.; Na, J. H.; Lee, S.; Kang, C. M.; Kim, H. N.; Han, S. J.;
Kim, H.; Choe, Y. S.; Jung, K.-H.; Lee, K. C.; Choi, K.; Kwon, I. C.;
Jeong, S. Y.; Lee, K.-H.; Kim, K. Facile Method To Radiolabel Glycol
Chitosan Nanoparticles with 64Cu via Copper-Free Click Chemistry
for MicroPET Imaging. Mol. Pharmaceutics 2013, 10, 2190−2198.
(33) Yhee, J. Y.; Son, S.; Kim, S. H.; Park, K.; Choi, K.; Kwon, I. C.
Self-Assembled Glycol Chitosan Nanoparticles for Disease-Specific
Theranostics. J. Controlled Release 2014, 193, 202−213.
(13) Lau, A. J.; Chang, T. K. H. 3-Hydroxyflavone and Structural
Analogues Differentially Activate Pregnane X Receptor: Implication for
Inflammatory Bowel Disease. Pharmacol. Res. 2015, 100, 64−72.
(14) Guazelli, C. F. S.; Fattori, V.; Colombo, B. B.; Georgetti, S. R.;
Vicentini, F. T. M. C.; Casagrande, R.; Baracat, M. M.; Verri, W. A., Jr.
Quercetin-Loaded Microcapsules Ameliorate Experimental Colitis in
Mice by Antiinflammatory and Antioxidant Mechanisms. J. Nat. Prod.
2013, 76, 200−208.
(15) Habtemariam, S.; Belai, A. Natural Therapies of the
Inflammatory Bowel Disease: The Case of Rutin and its Aglycone,
Quercetin. Mini-Rev. Med. Chem. 2018, 18, 234−243.
(16) Dicarlo, M.; Teti, G.; Verna, G.; Liso, M.; Cavalcanti, E.; Sila, A.;
Raveenthiraraj, S.; Mastronardi, M.; Santino, A.; Serino, G.; Lippolis,
A.; Sobolewski, A.; Falconi, M.; Chieppa, M. Quercetin Exposure
Suppresses the Inflammatory Pathway in Intestinal Organoids from
Winnie Mice. Int. J. Mol. Sci. 2019, 20, No. 5771.
(17) Mukhopadhyay, P.; Prajapati, A. K. Quercetin in Anti-Diabetic
Research and Strategies for Improved Quercetin Bioavailability Using
Polymer-Based Carriers - A Review. RSC Adv. 2015, 5, 97547−97562.
(18) Guo, Y.; Mah, E.; Bruno, R. S. Quercetin Bioavailability is
Associated with Inadequate Plasma Vitamin C Status and Greater
Plasma Endotoxin in Adults. Nutrition 2014, 30, 1279−1286.
(19) Tamura, M.; Nakagawa, H.; Hori, S.; Suzuki, T.; Hirayama, K.
Plasma Quercetin Metabolites are Affected by Intestinal Microbiota of
Human Microbiota-Associated Mice Fed with a Quercetin-Containing
Diet. J. Clin. Biochem. Nutr. 2019, 65, 232−239.
(20) Orrego-Lagarón, N.; Martinez-Huélamo, M.; Quifer-Rada, P.;
Lamuela-Raventos, R. M.; Escribano-Ferrer, E. Absorption and
Disposition of Naringenin and Quercetin after Simultaneous
Administration via Intestinal Perfusion in Mice. Food Funct. 2016, 7,
3880−3889.
(21) Thilakarathna, S. H.; Rupasinghe, H. P. V. Flavonoid
Bioavailability and Attempts for Bioavailability Enhancement. Nutrients
2013, 5, 3367−3387.
(34) Ferreira, C. H. F.; Shifrin, Y.; Pan, J.; Ivanovska, J.; McNamara, P.
J.; Belik, J. The Newborn Rat Gastric Emptying Rate is Volume and not
Developmentally Dependent. Neurogastroenterol. Motil. 2018, 30,
No. e13233.
(35) Iturmendi, A.; Monkowius, U.; Teasdale, I. Oxidation
Responsive Polymers with a Triggered Degradation via Arylboronate
Self-Immolative Motifs on a Polyphosphazene Backbone. ACS. Macro.
Lett. 2017, 6, 150−154.
̈
(36) Jager, E.; Hocherl, A.; Janouskova, O.; Jager, A.; Hruby, M.;
Konefal, R.; Netopilik, M.; Panek, J.; Slouf, M.; Ulbrich, K.; Stepanek, P.
Fluorescent Boronate-Based Polymer Nanoparticles with Reactive
Oxygen Species (ROS)-Triggered Cargo Release for Drug-Delivery
Applications. Nanoscale 2016, 8, 6958−6963.
(37) Jourden, J. L.; Daniela, K. B.; Cohen, S. M. Investigation of Self-
Immolative Linkers in the Design of Hydrogen Peroxide Aactivated
Metalloprotein Inhibitors. Chem. Commun. 2011, 47, 7968−7970.
(38) Weinert, E. E.; Dondi, R.; Colloredo-Melz, S.; Frankenfield, K.
N.; Mitchell, C. H.; Freccero, M.; Rokita, S. E. Substituents on Quinone
Methides Strongly Modulate Formation and Stability of Their
Nucleophilic Adducts. J. Am. Chem. Soc. 2006, 128, 11940−11947.
(39) Yu, S. J.; Kim, J. R.; Lee, C. K.; Han, J. E.; Lee, J. H.; Kim, H. S.;
Hong, J. H.; Kang, S. G. Gastrodia Elata Blume and an Active
Component, p-Hydroxybenzyl Alcohol Reduce Focal Ischemic Brain
Injury through Antioxidant Related Gene Expressions. Biol. Pharm.
Bull. 2005, 28, 1016−1020.
(22) Castangia, I.; Nacher, A.; Caddeo, C.; Merino, V.; Diez-Sales, O.;
Catalan-Latorre, A.; Fernandez-Busquets, X.; Fadda, A. M.; Manconi,
M. Therapeutic Efficacy of Quercetin Enzyme-Responsive Nano-
vesicles for the Treatment of Experimental Colitis in Rats. Acta
Biomater. 2015, 13, 216−227.
(40) Nunes, R.; Neves, J. D.; Sarmento, B. Nanoparticles for the
Regulation of Intestinal Inflammation: Opportunities and Challenges.
Nanomedicine 2019, 14, 2631−2644.
(23) Helmy, A. M.; Elsabahy, M.; Abd-Elkareem, M.; Ibrahim, E. A.;
Soliman, G. M. High-Payload Chitosan Microparticles for the Colonic
Delivery of Quercetin: Development and In-Vivo Evaluation in a
Rabbit Colitis Model. J. Drug Delivery Sci. Technol. 2020, 58,
No. 101832.
(41) Hua, S.; Marks, E.; Schneider, J. J.; Keely, S. Advances in Oral
Nano-Delivery Systems for Colon Targeted Drug Delivery in
Inflammatory Bowel Disease: Selective Targeting to Diseased versus
Healthy Tissue. Nanomed. Nanotechnol. Biol. Med. 2015, 11, 1117−
1132.
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Mol. Pharmaceutics 2021, 18, 1419−1430

Molecular Pharmaceutics
pubs.acs.org/molecularpharmaceutics
Article
́
(42) Araujo, F.; Sarmento, B. Towards the Characterization of An In
Vitro Triple Co-culture Intestine Cell Model for Permeability Studies.
Int. J. Pharmaceut. 2013, 458, 128−134.
1430
https://dx.doi.org/10.1021/acs.molpharmaceut.0c01245
Mol. Pharmaceutics 2021, 18, 1419−1430