Treating acute cystitis with biodegradable micelle-encapsulated quercetin.

Abstract

Intravesical application of an anti-inflammatory drug is an efficient strategy for acute cystitis therapy. Quercetin (QU) is a potent anti-inflammatory agent; however, its poor water solubility restricts its clinical application. In an attempt to improve water solubility of QU, biodegradable monomethoxy poly(ethylene glycol)-poly(ɛ-caprolactone) (MPEG-PCL) micelles were used to encapsulate QU by self-assembly methods, creating QU/MPEG-PCL micelles. These QU/MPEG-PCL micelles with DL of 7% had a mean particle size of <34 nm, and could release QU for an extended period in vitro. The in vivo study indicated that intravesical application of MPEG-PCL micelles did not induce any toxicity to the bladder, and could efficiently deliver cargo to the bladder. Moreover, the therapeutic efficiency of intravesical administration of QU/MPEG-PCL micelles on acute cystitis was evaluated in vivo. Results indicated that QU/MPEG-PCL micelle treatment efficiently reduced the edema and inflammatory cell infiltration of the bladder in an Escherichia coli-induced acute cystitis model. These data suggested that MPEG-PCL micelle was a candidate intravesical drug carrier, and QU/MPEG-PCL micelles may have potential application in acute cystitis therapy.

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International Journal of Nanomedicine 2012:7 2239–2247
International Journal of Nanomedicine
Treating acute cystitis with biodegradable
micelle-encapsulated quercetin
Bi Lan Wang1
Xiang Gao1,2
Ke Men1
Jinfeng Qiu1
Bowen Yang3
Ma Ling Gou1
Mei Juan Huang1
Ning Huang2
Zhi Yong Qian1
Xia Zhao1
Yu Quan Wei1
1State Key Laboratory of Biotherapy
and Cancer Center, West China
Hospital, West China Medical School,
2Department of Pathophysiology,
College of Preclinical and Forensic
Medical Sciences, 3College of Life
Science, Sichuan University, Chengdu,
People’s Republic of China
Correspondence: Ma Ling Gou
State Key Laboratory of Biotherapy
and Cancer Center, West China
Hospital, West China Medical School,
Sichuan University, Chengdu 610041,
People’s Republic of China
Tel +86 28 85164063
Fax +86 28 85164060
Email basad@163.com
Abstract: Intravesical application of an anti-inflammatory drug is an efficient strategy for
acute cystitis therapy. Quercetin (QU) is a potent anti-inflammatory agent; however, its poor
water solubility restricts its clinical application. In an attempt to improve water solubility of QU,
biodegradable monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) micelles
were used to encapsulate QU by self-assembly methods, creating QU/MPEG-PCL micelles.
These QU/MPEG-PCL micelles with DL of 7% had a mean particle size of ∼34 nm, and could
release QU for an extended period in vitro. The in vivo study indicated that intravesical appli-
cation of MPEG-PCL micelles did not induce any toxicity to the bladder, and could efficiently
deliver cargo to the bladder. Moreover, the therapeutic efficiency of intravesical administration
of QU/MPEG-PCL micelles on acute cystitis was evaluated in vivo. Results indicated that QU/
MPEG-PCL micelle treatment efficiently reduced the edema and inflammatory cell infiltration
of the bladder in an Escherichia coli-induced acute cystitis model. These data suggested that
MPEG-PCL micelle was a candidate intravesical drug carrier, and QU/MPEG-PCL micelles
may have potential application in acute cystitis therapy.
Keywords: nanomedicine, MPEG-PCL, self-assembly
Introduction
Urinary tract infections (UTIs) are one of the most common bacterial infections, and
Escherichia coli bacteria are the main cause of UTI.1–4 Bacterial infection of the blad-
der always leads to acute cystitis. Patients with acute cystitis account for 95% of all
visits to physicians for UTIs. Commonly, patients with acute cystitis have symptoms
of dysuria and increased frequency and urgency of urination, which greatly affect their
quality of life.5 In the past decades, great efforts have been made to develop efficient
therapeutics to treat acute cystitis. Despite many patients with acute cystitis having
benefited from these efforts,6 acute cystitis therapy still poses great challenges.
Currently, acute cystitis is commonly treated by systemic application of antibi-
otics and anti-inflammation agents. However, only a small amount of systemically
administered drugs can reach the bladder. Intravesical administration means directly
instilling the drug solution into the bladder through a urethral catheter, ensuring maxi-
mum delivery of active ingredients to the bladder.7–10 In addition, intravesical drug
administration has other potential benefits such as avoiding the first-pass metabolism.
Compared with systemic drug administration, intravesical drug administration, as a
local drug delivery method, can increase drug utilization and reduce systemic toxicity
and side effects. Thus, intravesical application of therapeutics attracts some attention
for cystitis therapy.
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International Journal of Nanomedicine 2012:7
Quercetin (QU; 3,3,4,5,7-pentahydroxyflavone) is one of
the most abundant flavonoids found in plants with biological
effects; its molecular structure is presented in Figure 1A.11,12
To the authors’ knowledge, QU has a wide range of bio-
logical properties, including anti-viral,13 anti-inflammatory,14
anti-oxidative,15 and anti-tumor activities.16,17 In recent
years, the anti-inflammatory effect of QU has been well
recognized,18,19 suggesting that QU as an anti-inflammatory
drug has promising clinical application. Recently, it was
found that QU can be used to prevent interstitial cystitis.20
The authors presumed that QU might have potential appli-
cation in intravesical therapy of acute cystitis, and set out
to prove this hypothesis in this work. However, QU is water
insoluble, and developing an aqueous formulation for QU
remains difficult, greatly restricting the clinical application
of QU. Therefore, it is necessary to develop an aqueous
QU formulation as well as study the application of QU in
depth in vivo.
Nanotechnology provides a novel platform for delivery of
lipophilic drugs.21,22 Encapsulation of hydrophobic drugs into
nanoparticles can render the drug completely dispersible in
solution. Biodegradable polymeric nanoparticles are regarded
as excellent candidates for drug-delivery systems.23–25 Poly(ε-
caprolactone) (PCL)/poly(ethylene glycol) (PEG) copoly-
mers are biodegradable, amphiphilic, and easy to produce,
showing promising applications in drug-delivery systems.26,27
Monomethoxy PEG (MPEG)-PCL is a diblock PCL/PEG
copolymer; its molecular structure is shown in Figure 1B.
The authors have previously used MPEG-PCL micelles to
deliver curcumin, creating a novel curcumin formulation with
potential application in colon cancer therapy.28 In an attempt
to develop an aqueous formulation for QU, MPEG-PCL
micelles were used to encapsulate QU, creating QU/MPEG-
PCL micelles. The effect of intravesical application of QU/
MPEG-PCL micelles on acute cystitis was then evaluated.
These results suggest that QU/MPEG-PCL micelles are a new
formulation of QU, with potential applications in bacterial
cystitis therapy.
Materials and methods
Materials
Escherichia coli DH5α was saved in the laboratory and was
cultured at 37°C on solid Luria-Bertani (LB) medium or in
liquid LB medium. QU was purchased from Sigma-Aldrich
(St Louis, MO). MPEG-PCL diblock copolymer with a
designed molecular weight of 4000 was synthesized accord-
ing to previous reports.27,28
Female BALB/c mice (8–10 weeks old) were purchased
from the Laboratory Animal Center of Sichuan University
(Chengdu, China). All studies involving mice were approved
by the Institute’s Animal Care and Use Committee.
Preparation of QU/MPEG-PCL
micelles
QU/MPEG-PCL micelles were prepared by a self-assembly
method. Briefly, QU (7 mg) and MPEG-PCL diblock
c opolymer (93 mg) were co-dissolved in 6 mL of organic
solvent (dichloromethane [DCM] : methanol = 2:1), fol-
lowed by evaporation under reduced pressure in a rotary
evaporator at 55°C. Normal saline (NS) (5 mL) was then
added into the polymer and drug mixture, allowing the
self-assembly of MPEG-PCL and QU, creating core-shell
structured QU/MPEG-PCL micelles with core-encapsulated
QU. The QU/MPEGPCL micelle solution was filtered using
a syringe filter (pore size: 220 nm) (Millex-LG, Millipore Co,
Billerica, MA) to remove the impurity and bacteria. Finally,
the prepared QU/MPEG-PCL micelles were lyophilized and
stored at 4°C.
Characterization of QU/MPEG-PCL
micelles
The particle size and zeta potential of QU/MPEG-PCL
micelles were determined by dynamic light scattering (DLS)
(Malvern Nano-ZS 90; Malvern Instruments, Malvern, UK).
The temperature was kept at 25°C during the measuring
process. All results were the mean of three test runs.
HO
OH
OH
OH
OH
O
O
O
O HOO
C
CH3
H2
C
H2
C
H2
C
5
y
x
AB
Figure 1 Molecular structure of (A) quercetin and (B) monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) copolymer.
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The morphology of QU/MPEG-PCL micelles was
observed under a transmission electron microscope
(H-6009IV; Hitachi, Tokyo, Japan): micelles were diluted
with distilled water and placed on a copper grid covered
with nitrocellulose. Samples were negatively stained with
phosphotungstic acid and dried at room temperature.
The concentration of QU was determined by high-
performance liquid chromatography (HPLC) (Waters Alliance
2695; Waters, Milford, MA). The solvent delivery system
was equipped with a column heater and a plus autosampler.
Detection was carried out on a Waters 2996 detector. Chro-
matographic separations were performed on a reversed phase
C18 column (4.6 × 150 mm-5µm, Sunfire Analysis column),
with the column temperature kept at 35°C. Methanol–water
(70/30, v/v) was used as eluent at a flow rate of 1 mL⋅min−1.
Drug loading (DL) and encapsulation efficiency (EE)
of QU/MPEG-PCL micelles were determined as follows:
Briefly, 10 mg of lyophilized QU/MPEG-PCL micelles were
dissolved in 0.1 mL DCM and diluted with methanol. The
amount of QU in the solution was determined by HPLC.
Finally, the DL and EE of QU/MPEG-PCL micelles were
calculated according to equations (1) and (2):

DL =
+
Drug
polymerDrug×100% (1)

EE
Experimental drug loading
Theoretical drug loading
=×100% (2)
In vitro drug release
To determine the release kinetics of QU from QU/MPEG-PCL
micelles, 0.5 mL of QU/MPEG-PCL micelle solution was
placed in a dialysis bag (molecular weight cutoff, 3.5 kDa).
Dialysis bags were incubated in 30 mL of phosphate buffer
solution (pH 7.4) containing Tween® 80 (Sigma-Aldrich)
(0.5% w/w) at 37°C with gentle shaking. At predetermined
time points, the incubation medium was replaced with fresh
incubation medium. The amount of released drug in the
incubation medium was quantified by determining absor-
bance at 370 nm using HPLC. This study was repeated three
times, and the result was expressed as mean value ± standard
deviation (SD).
Analysis of the acute toxicity
of MPEG-PCL micelles
Fifteen female BALB/c mice (8 weeks old) were used to
evaluate the acute toxicity of MPEG-PCL micelles after
intravesical application. These mice were divided into three
groups (five mice per group): one group as the control did not
receive any treatment, while the other two groups were treated
with NS or MPEG-PCL micelles (50 mg/mL × 0.1 mL),
respectively. Twenty-four hours after perfusion, all mice
were killed by cervical vertebra dislocation. Bladders were
immediately removed, weighted, and fixed in neutral buffer
formalin (10%). The bladders were then paraffin-embedded,
and sections were stained with hematoxylin and eosin (H&E).
Moreover, the heart, liver, spleen, kidney, and lung of each
mouse were also examined by H&E staining.
Distribution in vivo
Thirty-three female BALB/c mice (8 weeks old) were used to
evaluate the distribution of MPEG-PCL micelles. These mice
were divided into eleven groups (three mice per group).
Due to its specific fluorescence spectrum, coumarin-6
was used as a model drug to evaluate the capacity of MPEG-
PCL micelles to deliver cargo to bladder. Coumarin-6/
MPEG-PCL micelles were prepared by a rotary evapora-
tion method. Briefly, coumarin-6 (0.5 mg) and MPEG-PCL
diblock copolymer (99.5 mg) were co-dissolved in 6 mL of
DCM and evaporated to dryness under reduced pressure in
a rotary evaporator. The dried films were hydrated in 5 mL
of 0.9% NS.
The effect of the administration method on the bladder
drug deposition of MPEG-PCL micelle-encapsulated drugs
was studied in vivo. Coumarin-6/MPEG-PCL micelles
(0.1 mL) were intravenously or intravesically administered
into 8-week-old female BALB/c mice. At different time points
(0.5, 1, 2, 4, and 6 hours) after administration, mice were killed
by cervical vertebra dislocation, and their bladders were imme-
diately harvested and examined by the curmarin-6-associated
green fluorescence under a fluorescence light box (Lightools
Research, Encinitas, CA). Moreover, to study the effect of
administration method on the distribution of MPEG-PCL
micelle-encapsulated drugs in vivo, the heart, liver, spleen,
lung, and kidney of the mice were also harvested 2 hours after
treatment and examined by the curmarin-6-associated green
fluorescence under live image analysis instrument (Lightools
Research). These experiments were repeated three times, and
representative images were obtained.
Establishment of acute cystitis model
The acute cystitis model was established by intravesical
application of bacterial strain E. coli DH5α into 8-week-
old female BALB/c mice by transurethral catheterization as
previously described.29 E. coli DH5α was cultured at 37°C
in 5 mL LB for 18 hours, then centrifuged for 1 minute at
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10,000 rpm, washed with phosphate-buffered saline (PBS)
three times and diluted to 2 × 108 CFU/mL. 50 µL of this
suspension was used to infect each mouse with inoculums
of ∼1 × 107 CFU.
Treatment of acute cystitis
by QU/MPEG-PCL micelles
Twenty 8-week-old female BALB/c mice were infected with
E. coli DH5α (∼1 × 107 CFU per mouse). Twenty-four hours
later, the mice were divided into four groups. One group
as the control did not receive any treatment, and the other
groups were intravesically treated with NS (0.9%), MPEG-
PCL micelle saline solution (700 µg/mL × 100 uL) and QU/
MPEG-PCL micelle saline solution (QU: 50 µg/mL × 100 uL),
respectively. After 24 hours of treatment, the mice were killed
by cervical vertebra dislocation. The bladder of each mouse
was harvested and fixed with 10% buffered formaldehyde.
The bladders were then embedded in paraffin, and at least
four cross sections (5 µm thick) were taken from each blad-
der, followed by H&E staining.
Statistical analysis
Data were expressed as the mean ± SD. Statistical analysis
was performed with one-way analysis of variance using SPSS
Statistics (version 19.0.1; IBM Corporation, Somers, NY)
software. P values less than 0.05 were considered to be sta-
tistically significant.
Results
Preparation and characterization
of Qu/MPEG-PCL micelles
In an attempt to develop an aqueous formulation for QU,
biodegradable MPEG-PCL micelles were used to encapsu-
late QU. QU/MPEG-PCL micelles were prepared through
a self-assembly method, as schematically presented in
Figure 2. First, MPEG-PCL copolymer and QU were co-
dissolved in a DCM and methanol mixture. Then, the organic
solvent was evaporated under reduced pressure in a rotary
evaporator, forming an MPEG-PCL and QU mixture. Finally,
NS was added to the mixture, allowing the self-assembly of
MPEG-PCL and QU in water. In the structure of MPEG-PCL,
PEG is the hydrophilic segment, and PCL is the hydrophobic
segment; thus, MPEG-PCL micelles always have a core-shell
structure, with a PCL core and a PEG shell. The self-assembly
of MPEG-PCL and QU created core-shell QU/MPEG-PCL
micelles with core-encapsulated QU.
The QU/MPEG-PCL micelles were characterized in detail.
The QU/MPEG-PCL micelles had DL and EE of 6.9% and
98.1%, respectively. In Figure 3A, the particle size distribu-
tion spectrum of freshly prepared QU/MPEG-PCL micelles
are presented. It was indicated that QU/MPEG-PCL micelles
had a very narrow particle size distribution (polydispersity
index [PDI] = 0.12), with a mean particle size of 34.1 ± 1.2 nm
(determined by DLS). The zeta potential spectrum of QU/
MPEG-PCL micelles is presented in Figure 3B; QU/MPEG-
PCL micelles had a zeta potential of −0.269 mV . Moreover,
the morphology of QU/MPEG-PCL micelles was studied by
transmission electron microscopy (TEM), and the result is
shown in Figure 3C. According to the TEM image, QU/MPEG-
PCL micelles were spherical with a mean diameter of ∼23 nm.
TEM determined the size of dry particles, while the DLS
determined the hydrodynamic diameter of particles in water.
Because amphiphilic block polymeric micelles always have a
loose structure in water, the particle size determined by DLS
was always slightly larger than that determined by TEM.
One of the major purposes of the encapsulation
of QU in MPEG-PCL micelles was to make QU
completely d ispersible in aqueous media. The appearance
PCL
PEG
Self-assembly
QU + MPEG-PCL QU/MPEG-PCL micelle
QU
Figure 2 Preparation of QU-loaded MPEG-PCL nanoparticles. MPEG-PCL and QU were co-dissolved in organic solvent, followed by evaporation to dryness under reduced
pressure in a rotary evaporator, creating QU and MPEG-PCL mixture (QU + MPEG-PCL).
Notes: The QU and MPEG-PCL mixture was then hydrated in 0.9% normal saline, allowing QU and MPEG-PCL to self-assemble into QU/MPEG-PCL micelles. This micelle
has a core-shell structure (hydrophilic PEG shell and hydrophobic PCL core) with core-encapsulated QU.
Abbreviations: MPEG, monomethoxy poly(ethylene glycol); PCL, poly(ε-caprolactone); QU, quercetin.
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of QU/MPEG-PCL micelle aqueous solution is shown
in Figure 3D. QU cannot be dissolved in pure water, as
confirmed by the observation of a turbid yellow slurry.
In contrast, QU/MPEG-PCL micelle solution with an
equivalent quantity of QU was transparent, indicating full
dispersibility of QU in water.
The release profile of QU/MPEG-PCL micelles in vitro
was studied using a dialysis method. As shown in Figure 4,
QU was released from QU/MPEG-PCL micelles over an
extended period.
Analysis of toxicity
of MPEG-PCL micelles
In this work, the authors attempted to treat acute cystitis by
intravesical application of MPEG-PCL micelle-encapsulated
QU. Thus, the toxicity of intravesically applied MPEG-PCL
micelles was evaluated before treatment experiments. As
shown in Figure 5A, intravesical application of MPEG-
PCL micelles (50 mg/mL × 0.1 mL) did not significantly
induce bladder weight change (control: 16.604 ± 0.739 mg;
NS: 17.032 ± 1.846 mg; MPEG-PCL: 16.874 ± 0.780 mg),
which implied that the intravesical application of MPEG-
PCL micelles did not significantly induce bladder edema.
Moreover, pathological section of bladder in each treat-
ment group is shown in Figure 5B. We found that intravesi-
cal application of MPEG-PCL micelles did not induce any
AB
CD
16
14
12
10
8
6
4
2
0
1
100 nm
10100
Size (nm)
Intensity (%)
Total counts
1000
−200
−100
Zeta potential (mV)
0
100200
0
200000
400000
600000
800000
1000000
a
b
c
Figure 3 Characterization of QU/MPEG-PCL micelles: (A) size distribution spectrum of QU/MPEG-PCL micelles; (B) zeta potential spectrum of MPEG-PCL micelles;
(C) transmission electron microscopy image of QU/MPEG-PCL micelles; (D) the encapsulation of QU in MPEG-PCL/QU nanoparticles renders QU completely dispersible
in aqueous media (a, water (pH = 7.0); b, QU in water (pH = 7.0, 2 mg/mL); c, QU/MPEG-PCL micelles in water (pH = 7.0, 2 mg/mL).
Abbreviations: MPEG, monomethoxy poly(ethylene glycol); PCL, poly(ε-caprolactone); QU, quercetin.
100
90
80
70
60
50
40
30
20
10
Released drug (%)
0
020 4060
Time (h)
80 100120
Figure 4 In vitro release study.
Note: The in vitro release profile of QU/MPEG-PCL micelles was examined using
a dialysis method.
Abbreviations: MPEG, monomethoxy poly(ethylene glycol); PCL, poly
(ε-caprolactone); QU, quercetin.
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Biodegradable micelle-encapsulated quercetin and cystitis treatment