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Floating Hydrogel with Self-Generating
Micro-Bubbles for Intravesical Instillation
Tingsheng Lin 1,† , Xiaozhi Zhao 1,† , Yifan Zhang 2 , Huibo Lian 1 , Junlong Zhuang 1 ,
Qing Zhang 1 , Wei Chen 1 , Wei Wang 1 , Guangxiang Liu 1 , Suhan Guo 1 , Jinhui Wu 2, *,
Yiqiao Hu 2, * and Hongqian Guo 2, *
1
2
*
†
Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology,
Nanjing University, Nanjing 210008, China; [email protected] (T.L.); [email protected] (X.Z.);
[email protected] (H.L.); [email protected] (J.Z.); [email protected] (Q.Z.);
[email protected] (W.C.); [email protected] (W.W.); [email protected] (G.L.);
[email protected] (S.G.)
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China;
[email protected]
Correspondence: [email protected] (J.W.); [email protected] (Y.H.); [email protected] (H.G.);
Tel.: +86-139-1302-6062 (J.W.); +86-136-0140-2829 (Y.H.); +86-136-0517-1690 (H.G.);
Fax: +86-25-83596143 (J.W. & Y.H.)
These authors contributed equally to the work.
Academic Editor: Franz E. Weber
Received: 7 November 2016; Accepted: 5 December 2016; Published: 12 December 2016
Abstract: Intravesical instillation is the main therapy for bladder cancer and interstitial cystitis.
However, most drug solutions are eliminated from bladder after the first voiding of urine. To solve
this problem, we proposed a floating hydrogel with self-generating micro-bubbles as a new delivery
system. It floated in urine, avoiding the urinary obstruction and bladder irritation that ordinary
hydrogels caused. In this study, we abandoned traditional gas-producing method like chemical
decomposition of NaHCO3 , and used the foamability of Poloxamer 407 (P407) instead. Through
simple shaking (just like shaking SonoVue for contrast-enhanced ultrasound in clinical), the P407
solution will “lock” many micro-bubbles and float in urine as quickly and steadily as other gas
producing materials. In vivo release experiments showed that drug was released continually from
hydrogel for 10 h during the erosion process. Thus, the residence time of drug in bladder was
prolonged and drug efficacy was improved. In vivo efficacy study using rabbit acute bladder
injury model showed that prolonged drug residence time in bladder increased the efficiency of
heparin in the protection of bladder mucosal permeability. Therefore, our floating hydrogel system
with self-generating micro-bubbles was single-component, simply prepared and efficacy enhancing,
successfully exempting users from worries on safety and clinical efficiency from bench to bedside.
Keywords: floating hydrogel; shaking; thermo-sensitive hydrogel; intravesical instillation; acute
bladder injury
1. Introduction
Diseases of the urinary bladder account for a large number of severe medical conditions affecting
patients of all ages. The major diseases affecting the urinary bladder are bladder cancer and intractable
interstitial cystitis. Intravesical instillation is the most common treatment for the bladder cancer and
intractable interstitial cystitis [1]. It is performed by instilling chemical drugs to bladder using a
urethral catheter. The efficacy of the intravesical treatment depends on the residence time of drug
inside the bladder, which relates to the adhesive capability of the drug onto the urinary bladder wall [2].
However, most drug solutions were eliminated during the first voiding of urine after intravesical
Materials 2016, 9, 1005; doi:10.3390/ma9121005
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Materials 2016, 9, 1005
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instillation. The efficacy of intravesical instillation relies on the drug residence time in bladder, so it is
imperative to develop a controlled drug delivery system.
Recently, hydrogel had been used as drug reservoir to extend the drug residence time in
bladder [3–5]. However, due to the high viscosity of hydrogel, one serious problem is urinary
obstruction. Gels adhering to the bladder may block the urinary tract, such as internal urethral orifice
and ureteric orifice with narrow opening. As hydrogels attach to the bladder wall, another problem
is serious bladder irritation symptoms, such as blood clots in bladder [6]. The two floating hydrogel
drug delivery systems we developed before were able to overcome these two shortcomings and
prolonged the resident time of drug in the bladder [7,8]. The first floating hydrogel system consisted of
thermo-sensitive polymer (Poloxamer 407) and NaHCO3 , which was liquid at low temperature while
forming gel at body temperature (37 ◦ C). In the presence of H+ , NaHCO3 decomposed and produced
CO2 , which attached to the surface of hydrogel and float the hydrogel in urine. Hence, the urinary
tract will not be blocked, and the encapsulated drug released in a controlled manner. However, we
need to acidify the urine to provide enough acid environment (pH < 5.6) for hydrogel floating in urine.
To realize urine acidification, patients have to take oral drugs, like ammonium chloride and vitamin C.
Extra drugs increase the risk of side effects including acid–base imbalance and electrolyte disturbances.
Thus, the practical application of this floating hydrogel system was limited. In the second floating
hydrogel system, NaHCO3 was substituted by Ammonium bicarbonate (NH4 HCO3 ) as micro-bubbles
producers. NH4 HCO3 decomposed at body temperature without acidifying the urine and generated
micro-bubbles inside hydrogel, which also float the hydrogel in urine. However, NH4 HCO3 is unstable
and easily decomposed, especially in the liquid solution, so this floating hydrogel system could not be
stored for a long time. In addition, both NaHCO3 and NH4 HCO3 are inorganic compounds, which
might react with drugs in floating hydrogel and lead to chemical change of drugs. Therefore, more
ideal floating scheme is required for the floating hydrogel system in practical clinical application.
In this study, we developed a floating hydrogel drug delivery system merely with Poloxamer
407, with no additive for producing micro-bubbles. In fact, P407 solution could produce many
micro-bubbles through shaking, stirring, or homogenizing, as P407 is a non-ionic surfactant [9].
In this system, no extra conditions, such as specific temperature and pH, were required; hence, safety
and efficiency problems from bench to bedside were solved radically. By just shaking the hydrogel
solution (like shaking SonoVue for contrast-enhanced ultrasound in clinical), micro-bubbles can be
viewed as gas-filled micelles formed by surfactant molecules, whose hydrophobic tail groups face the
hydrophobic gas and hydrophilic head groups face the aqueous phase [10,11]. Due to the viscosity
of P407 solution, micro-bubbles were suspended for a certain amount of time and did not escape
easily. Moreover, the micro-bubbles could be “locked” in hydrogel when the P407 solution converted
to gel state at higher temperature due to its thermo-sensitive property (Figure 1). The P407 solution
after shaking converted to gel and float in urine immediately after injected into bladder, and the
encapsulated drug was released in a controlled manner. Our study showed that this floating hydrogel
drug delivery system was easily prepared and safe. Because the production of micro-bubbles was
milder and slower, the residence time of drug was extended and efficacy of intravesical drug delivery
was improved.
Materials 2016, 9, 1005
Materials 2016, 9, 1005
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Figure1.1. The
The schematic
schematicdiagram
diagramof
offloating
floatinghydrogel
hydrogelpreparation
preparationby
byshaking.
shaking.
Figure
2. Materials and Methods
2. Materials and Methods
2.1. Materials
Materials
2.1.
Poloxamer407
407(P407)
(P407) was
was purchased
purchased from
from Meilun
Meilun Biology
Biology Technology
Technology Company,
Company,Ltd.
Ltd. (Dalian,
(Dalian,
Poloxamer
China).Rhodamine
Rhodamine
B (RhB),
Protamine
sulfate
and
heparin
were
purchased
from
China).
B (RhB),
Protamine
sulfate (PS)
and (PS)
heparin
were
purchased
from
Sigma-Aldrich
Sigma-Aldrich
(St.
Louis,
MO,
USA).
Thirty
New
Zealand
white
male
rabbits,
weighting
on
average
(St. Louis, MO, USA). Thirty New Zealand white male rabbits, weighting on average 2000 g (range,
2000 g (range,
1800–2500
g),from
were
from the
Experimental
Animal Center,
University
of
1800–2500
g), were
obtained
theobtained
Experimental
Animal
Center, University
of Yangzhou,
China.
Yangzhou,
China.
All
animal
protocols
were
approved
by
Institutional
Animal
Care
and
Use
All animal protocols were approved by Institutional Animal Care and Use Committee (IACUC) of
Committee
(IACUC) of Nanjing University.
Nanjing
University.
2.2. Preparation
Preparationand
andOptimization
OptimizationofofFloating
FloatingHydrogel
Hydrogel
2.2.
Thenon-floating
non-floating
hydrogel
is mixture
the mixture
andwater,
distilled
water,
the P407
The
hydrogel
is the
of P407of
andP407
distilled
and the
P407and
concentration
concentration
45% (w/v).
To prove thatcould
micro-bubbles
could
be produced
solution
and
was
45% (w/v).was
To prove
that micro-bubbles
be produced
by P407
solution by
andP407
reserved
in P407
reserved
in
P407
solution,
the
P407
solutions
was
shaken
by
hand
up
and
down
at
a
speed
of
≥2
solution, the P407 solutions was shaken by hand up and down at a speed of ≥2 round/s (Figure 1),
◦
◦
round/s
(Figure
and37stored
at 0 °C and 37 °C, respectively.
and
stored
at 0 C1),and
C, respectively.
Todetermine
determine the
the volume
volume change of hydrogel
1010
mL
of
To
hydrogel in
inrespect
respectto
tothe
thedensity
densityofofmicro-bubble,
micro-bubble,
mL
P407
solution
was
shaken
by
hand
for
0,
10,
20,
30,
40
times
respectively.
The
volume
changes
of
of P407 solution was shaken by hand for 0, 10, 20, 30, 40 times respectively. The volume changes
hydrogels
after
effect of
of environmental
environmental
of
hydrogels
afterdifferent
differentshaking
shakingtimes
timeswere
wererecorded.
recorded. To
To determine
determine the effect
factors and
andshaking
shaking force
forceon
onmicro-bubble
micro-bubble generation,
generation, the
the volume
volume change
change of
of hydrogel
hydrogel in
inrespect
respectto
to
factors
thedensity
densityof
ofmicro-bubble
micro-bubblewas
wasmeasured.
measured. Ten
Tenmilliliters
millilitersof
ofP407
P407solution
solutionwas
wasshaken
shaken by
byhand
handfor
for
the
20times
timesat
atdifferent
differentenvironmental
environmentalfactors
factorsand
andthe
thevolume
volumechanges
changesof
ofhydrogels
hydrogelsafter
aftershaking
shakingwere
were
20
recorded.ToTo
observe
environmental
gel solutions
were atshaking
the local
different
recorded.
observe
environmental
effect,effect,
gel solutions
were shaking
the localatdifferent
temperature
◦
◦
◦
◦
temperature
(3.525.3
°C, 11.5
°C,31.9
25.3 C),
°C and
°C), local
different
humidity
46.8%,
69.4%local
and
(3.5
C, 11.5 C,
C and
local31.9
different
humidity
(22.6%,
46.8%,(22.6%,
69.4% and
88.2%),
88.2%), local
different
atmosphere
pressure
(1005/100Pa,
Pa,1013/100
1009/100 Pa
Pa,and
1013/100
Pa and
Pa).
different
atmosphere
pressure
(1005/100
Pa, 1009/100
1015/100
Pa).1015/100
To evaluate
To
evaluate
shaking
force
on
micro-bubble
generation,
shaking
speed
in
respect
to
the
shaking
force
shaking force on micro-bubble generation, shaking speed in respect to the shaking force was measured.
was measured.
Shaking
defined
asmove
the time
of hand
and(hand
downmove
in one
(hand
Shaking
speed defined
as speed
the time
of hand
up and
downmove
in oneup
cycle
upcycle
and down
move
upcycle,
and down
was one
theor
distance
of hand
up or down was 50 cm).
was
one
the distance
of cycle,
hand up
down was
50 cm).
Toevaluate
evaluatethe
theeffect
effectof
ofshaking
shakingtime
timeon
onmicro-bubbles
micro-bubblesamount
amountin
inhydrogel,
hydrogel,P407
P407solution
solutionwas
was
To
shakenby
byhand
handfor
for0,
0,10,
10,20,
20,30,
30,40
40times
timesrespectively.
respectively.Micro-bubbles
Micro-bubblesproduced
producedin
inhydrogel
hydrogelsolution
solution
shaken
after
different
shaking
time
were
recorded
with
photos
and
detected
by
ultrasound
(B/K,
after different shaking time were recorded with photos and detected by ultrasound (B/K, Copenhagen,
Copenhagen,
To count
number of micro-bubbles,
hydrogel
was slide
put on
a glass
slide
Denmark).
To Denmark).
count the number
of the
micro-bubbles,
hydrogel was put
on a glass
and
observed
anda observed
via microscope
a Zeiss M2Bio
microscope
(Zeiss,Germany).
Oberkochen,
The zoom was
of microscope
via
Zeiss M2Bio
(Zeiss,
Oberkochen,
TheGermany).
zoom of microscope
40×, and
was 40×,
andcaptured.
images were
P407 shaking
solutionwas
afterput
shaking
put in
°C water
to observe
its
images
were
P407captured.
solution after
in 37 ◦was
C water
to37
observe
its floating
state.
floating state.
Materials 2016, 9, 1005
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2.3. Characterizations of Floating Hydrogel
To obtain information on the pore structure of hydrogels, the floating hydrogel shaken for 20 times
was observed using Scanning electron microscopy. The samples were plunged in liquid nitrogen and
freeze-dried to maintain the porous structure without any collapse. The samples were mounted on the
base plate and coated with gold. The morphology was investigated using a Hitachi (Tokyo, Japan)
S-570 Scanning Electron Microscope.
The bubble size distribution of the floating hydrogel was investigated using electron microscope
(Nikon, Tokyo, Japan). The floating hydrogel after 20-times shaking was put on a glass slide, the zoom
of microscope was 40×. Three fields were randomly selected to calculate the bubble size distribution.
The apparent viscosity was determined by a NDJ-1 viscometer (Shanghai Balance Instrument
Factory, Shanghai, China). Twenty milliliters of hydrogel solution after 20-times shaking was put in a
25 mL beaker and placed in water bath. Then the solution was heated at a speed of 1 ◦ C/min and the
viscosity was recorded.
The erosion time was the time it takes for gel to dissolve completely in water. The volume of
5 mL, 10 mL, 15 mL, 20 mL floating hydrogels was injected into 37 ◦ C water, and the erosion time of
floating hydrogels were recorded.
The storage stability of the hydrogel of 45% P407 was also recorded. A digital timer (Thermo
Fisher Scientific, Boston, MA, USA) was used for recording.
2.4. Incorporation of Drug in Floating Hydrogel
To study the release of floating hydrogel in vitro and in vivo, Rhodamine B was incorporated into
hydrogel. A calculated amount of P407 (45%) was successively dissolved in RhB solution at 4 ◦ C to
form a RhB-loaded hydrogel solution. The concentration of RhB was 0.005% (w/v).
To study the efficacy of floating hydrogel in acute bladder injury model, Heparin was incorporated
into hydrogel. A calculated amount of P407 (45%) was successively dissolved in Heparin solution
at 4 ◦ C to form a Heparin-loaded hydrogel solution. The concentration of Heparin was 1500 IU/mL.
2.5. Release Study In Vitro
The release study was performed in the dissolution tester (ZRS-8G, Instruments of Tianjin
University, Tianjin, China). The PBS (pH = 7, 200 mL) in the beaker of dissolution tester was heated
to 37 ◦ C. RhB solution (50 ug/mL, 5 mL), RhB-loaded floating hydrogel (5 mL) and RhB-loaded
non-floating hydrogel (5 mL) was injected into solution, respectively. At predetermined time points,
3 mL solution was collected and substituted with the same volume of fresh solution. The amount of
RhB was determined by UV spectrophotometry (UV-2450, Shimadzu, Kyoto, Japan) at 555 nm.
2.6. Verification of Hydrogel Floating In Vivo
Rabbit was anesthetized with pentobarbital (30 mg/kg, intravenous injection). Floating hydrogel
solution was intravesically instilled into bladder using a catheter. The floating hydrogel in bladder
was detected by B ultrasound.
2.7. Release Study In Vivo
The rabbits were randomly chosen. They were maintained in a controlled atmosphere of 12 h
dark/light cycle, 22 ± 2 ◦ C temperature and 50%–70% humidity, with free access to pellet feed and
fresh tap water. The animals were supplied with dry food pellets commercially available.
In fact, the gel solution was stored at 0 ◦ C condition before injected to the bladder and the viscosity
of 45% P407 was about 200 mPa·s at this temperature. The viscosity was low. Therefore, it is easy to
inject the gel solution from the catheter to bladder. Rabbits were intravesically instilled with 3 mL
normal saline, free RhB solution, RhB-loaded floating hydrogel and RhB-loaded non-floating hydrogel
solution respectively. The RhB -loaded non-floating hydrogel solution did not undergo shaking.
Materials 2016, 9, 1005
5 of 14
All animals were fastened on a desk. Intravesical administration was accomplished using a catheter
(9F) inserted into the bladder through the urethra. Urine samples were collected. The concentration
of RhB was determined by a fluorescence microplate reader (Safire, TECAN or Molecular Devices
M3, Männedorf, Switzerland). The frozen section was immediately prepared after isolating rabbit
bladder tissues. The fluorescence in the bladder section was observed using a Zeiss M2Bio fluorescence
microscope. The thickness of frozen biopsy was 10 µm, and the exposure time of fluorescence
microscope was 1 s.
2.8. The Efficacy of Floating Hydrogel in Acute Bladder Injury Model
Fifteen rabbits were randomly divided into five groups, with three rabbits for each group.
All rabbits were fastened on the desk for intravesical instillation. For the control group, the bladders
of rabbits were pretreated with 10 mL normal saline for 60 min, followed by 3 mL normal saline.
For the PS + Saline group, the bladders of rabbits were pretreated with 10 mL Protamine sulfate (PS)
(20 mg/mL) for 60 min, followed by 3 mL saline. For the PS + Gel group, the bladders of rabbits were
pretreated with 10 mL PS (20 mg/mL) for 60 min, followed by 3 mL non-floating hydrogel (45% P407
hydrogel solution without shaking). For the PS + Heparin solution group, the bladders of rabbits were
pretreated with 10 mL PS (20 mg/mL) for 60 min, followed by 3 mL heparin solution (1500 IU/mL).
For the PS + Heparin loaded floating hydrogel group, the bladders of rabbits were pretreated with
10 mL PS (20 mg/mL) for 60 min, followed by 3 mL heparin loaded floating hydrogel (1500 IU/mL).
After the treatments, all rabbits bladders were intravesically instilled 20 mL saline to trigger the first
voiding of urine. After 10 h, all rabbits were intravesical instilled RhB solution (5 ug/mL) for 30 min.
Then all rats were executed and bladder tissues were obtained. The frozen section was immediately
prepared. The fluorescence in the bladder section was observed using a Zeiss M2Bio fluorescence
microscope. The thickness of frozen biopsy was 5 µm, the exposure time of fluorescence microscope
was 83 ms. The fluorescence depth of bladder tissues were calculated by the software Image-pro 6.0
(Media Cybernetics, Bethesda, MD, USA).
3. Results
3.1. Preparation and Optimization of Floating Hydrogel
P407 was chosen as the matrix of floating hydrogel, and 45% (w/v) P407 was selected as optimal
concentration according to our previous work [8]. As P407 was a kind of surfactant, the surfactant
molecules could coat micro-bubbles by shaking, stirring, or homogenizing [9]. Enough micro-bubbles
enlarge the volume of hydrogel and hence enhance the buoyancy, which eventually float the hydrogel.
A large amount of micro-bubbles were produced in hydrogel solution after shaking for 40 times, while
the hydrogel became opaque (Figure 2). To confirm that micro-bubbles were blocked in hydrogel at
different temperature, hydrogel solutions after shaking were placed at 0 ◦ C and 37 ◦ C respectively.
Results showed that the hydrogel could hold micro-bubbles in gel during the whole experiment at
37 ◦ C (Figure 2a), while micro-bubbles dissipated gradually at 0 ◦ C (Figure 2b). Because P407 is a
thermo-sensitive material, it was gel state at 37 ◦ C, but liquid state at 0 ◦ C. Once the gel network
formed, micro-bubbles was “locked” in hydrogel, and the hydrogel volume increased at the same time,
so the buoyancy of hydrogel was increased and ensure the hydrogel stably float in liquid. We then
investigate the effects of shaking time on generation of micro-bubbles. Hydrogel solution was shaken
for 0, 10, 20, 30, 40 times respectively. Micro-bubbles produced in hydrogel solution were recorded with
camera (Figure 3a). The density of micro-bubbles in gel increased with times of shaking. However, the
hydrogel solution before shaking was transparent, with no micro-bubbles observed. To further observe
the micro-bubbles produced in hydrogel solution, they were detected by ultrasound and images were
captured (Figure 3b). As micro-bubbles in hydrogel was an ultrasound contrast agents, significant
enhancement will be observed when a lot of micro-bubbles produced. The enhancement of hydrogel
ultrasound images was also proportional to the shaking time. Similarly, the microscope images
Materials 2016, 9, 1005
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showed that the number of micro-bubbles in hydrogel increased with times of shaking (Figure 3c).
According to these images, the mean number of micro-bubbles in hydrogel shaken for 10, 20, 30 and
Materials 2016, 9, 1005
6 of 14
40 times
were 49 ± 5,107 ± 15, 165 ± 8 and 250 ± 16, respectively (Figure S2). To determine
the
volume change of hydrogel in respect to the density of micro-bubble, 10 mL of P407 solution was
of micro-bubbles in hydrogel shaken for 10, 20, 30 and 40 times were 49 ± 5,107 ± 15, 165 ± 8 and 250 ±
shaken
hand 0, 10,
20, 30,
40 times. the
Thevolume
volume
changes
of hydrogels
after
16,by
respectively
(Figure
S2).and
To determine
change
of hydrogel
in respect
to different
the densityshaking
of
timesmicro-bubble,
were recorded.
Theofmean
change
of hydrogel
for and
10, 20,
30 and
40volume
times were
10 mL
P407 volume
solution was
shaken
by hand 0,shaken
10, 20, 30,
40 times.
The
0.47 ±changes
0.15, 1.23
0.25, 1.9 after
± 0.20,
and 2.23
± 0.25,
respectively
(FigureThe
S6),mean
hydrogel
volume
increased
of ±
hydrogels
different
shaking
times
were recorded.
volume
change
of
hydrogel
shaken
for 10, 20, 30
andhydrogel
40 times were
0.47 ± 0.15,
1.23 ± 0.25, 1.9To
± 0.20,
and 2.23the
± 0.25,
indicated
air gas
incorporated
into
and formed
micro-bubbles.
determine
effect of
respectivelyfactors
(Figure
S6),shaking
hydrogel
volume
increased indicated
air gasthe
incorporated
into hydrogel
environmental
and
force
on micro-bubble
generation,
volume change
of hydrogel
and formed
To determine
themeasured.
effect of environmental
factors
and
shaking force on
in respect
to the micro-bubbles.
density of micro-bubble
was
Results showed
that
environmental
factors
micro-bubble
generation,
the
volume
change
of
hydrogel
in
respect
to
the
density
of
micro-bubble
(temperature, humidity, and atmosphere pressure) had no significant effect on micro-bubble generation
was measured. Results showed that environmental factors (temperature, humidity, and atmosphere
(Figure S7a–c). However, the hand shaking speed had great impact on micro-bubble generation of
pressure) had no significant effect on micro-bubble generation (Figure S7a–c). However, the hand
gel solution,
shaking time greater than 1 s led to inadequate micro-bubbles generated for floating
shaking speed had great impact on micro-bubble generation of gel solution, shaking time greater
◦ C water, these hydrogel solutions
hydrogel
To test micro-bubbles
the floating state
of hydrogel
in 37hydrogel
than (Figure
1 s led toS7d).
inadequate
generated
for floating
(Figure S7d). To test the
◦
were floating
put intostate
37 of
C hydrogel
water. Results
that hydrogel
the groups
of hydrogels
20 times
floated
in 37 °Cshowed
water, these
solutions
were putshaken
into 37over
°C water.
Results
stablyshowed
in water,
groups
hydrogel
(not over
shaken
or shaken
times)
float
(Figure
thatother
the groups
of of
hydrogels
shaken
20 times
floated10stably
in failed
water, to
other
groups
of 3d).
hydrogel
(notand
shaken
or shaken
10 times)of
failed
to float were
(Figure
3d).
The erosion
and The
gelation
The erosion
time
gelation
temperature
hydrogels
also
tested
(Figuretime
S1a,b).
gelation
◦ C, 11.5
◦ C, 11.7of
◦ C,
temperature
of hydrogels
were 10,
also20,
tested
(Figure
S1a,b).was
The11.5
gelation
temperature
hydrogels
temperature
of hydrogels
shaken
30 and
40 times
11.9 ◦ C and
10, 20, 30 The
and 40
times time
was 11.5
°C, 11.5 °C, 11.7
°C, 11.9(not
°C and
11.9 or
°C,shaken
respectively.
The and
11.9 ◦shaken
C, respectively.
erosion
of non-floating
hydrogel
shaken
10 times)
erosion time of non-floating hydrogel (not shaken or shaken 10 times) and floating hydrogel (shaken
floating hydrogel (shaken 20–40 times) were about 6.5 h and 6 h, respectively. The gelation temperature
20–40 times) were about 6.5 h and 6 h, respectively. The gelation temperature and erosion time of
and erosion time of hydrogels showed no differences before and after shaking. These parameters
hydrogels showed no differences before and after shaking. These parameters indicated that the
indicated
that the shaking did not affect the inherent property of hydrogel, but only floated it.
shaking did not affect the inherent property of hydrogel, but only floated it.
Figure 2. Bubbles in hydrogel solutions after shaking at 0 °C and 37 °C. To confirm that
Figure 2. Bubbles in hydrogel solutions after shaking at 0 ◦ C and 37 ◦ C. To confirm that micro-bubbles
micro-bubbles were blocked in hydrogel at different temperature, hydrogel solutions after shaking
were blocked
in hydrogel
at37
different
temperature,
hydrogel
solutions
after shakinginwere
placedthe
at 0 ◦ C
were placed
at 0 °C and
°C, respectively.
The hydrogel
could
hold micro-bubbles
gel during
◦
and 37
C, respectively.
hydrogel
could hold
micro-bubbles
in at
gel0 during
whole
experiment atThe
37 °C
(a). Micro-bubbles
dissipated
gradually
°C (b). the whole experiment at
37 ◦ C (a). Micro-bubbles dissipated gradually at 0 ◦ C (b).
3.2. Characterization of Floating Hydrogel
3.2. Characterization
Floatingmorphology
Hydrogel was investigated by SEM (Figure 4a). The floating hydrogel
The floatingofhydrogel
was floating
porous, with
clear polymer
strands.
Micro-bubbles
produced
floating4a).
hydrogel
could be hydrogel
seen
The
hydrogel
morphology
was
investigated
by SEMin(Figure
The floating
in gel networks. The size of micro-bubbles in hydrogel ranged from 20 μm to 400 μm, most were
was porous, with clear polymer strands. Micro-bubbles produced in floating hydrogel could be seen
80~140 μm (Figure 4b). The viscosity of hydrogel increased drastically at 10.5 °C (its sol-gel
in gel networks. The size of micro-bubbles in hydrogel ranged from 20 µm to 400 µm, most were
transition temperature) and reached maximum at 12 °C (Figure 4c). To evaluate the effect of gel
◦ C (its sol-gel transition
80~140
µm (Figure
4b). The
increased
drastically
10.5
volume
on hydrogels,
weviscosity
recorded of
thehydrogel
erosion time
in aqueous
solution.at
The
erosion
time of floating
◦ C (Figure 4c). To evaluate the effect of gel volume on
temperature)
reached
at 12
hydrogel and
increased
withmaximum
the hydrogel
volume,
ranging from 6 to 24 h as the hydrogel volume
hydrogels,
wefrom
recorded
the20erosion
time4d).
in aqueous
solution.
erosion
time
floating
increased
5 mL to
mL (Figure
The storage
stability The
of the
hydrogel
wasofalso
tested.hydrogel
The
Materials 2016, 9, 1005
7 of 14
increased with the hydrogel volume, ranging from 6 to 24 h as the hydrogel volume increased from
2016,
9, 1005 4d). The storage stability of the hydrogel was also tested. The hydrogel
7 of
14
5 mLMaterials
to 20 mL
(Figure
could
be
stored up to nine months without significant change of the gelation. The drug entrapment efficiency
hydrogel could be stored up to nine months without significant change of the gelation. The drug
Materials
2016,
1005gelation of hydrogel.
7 of 14
was 100%
due
to9,the
entrapment efficiency was 100% due to the gelation of hydrogel.
hydrogel could be stored up to nine months without significant change of the gelation. The drug
entrapment efficiency was 100% due to the gelation of hydrogel.
Figure 3. Effects of shaking time on bubbles in hydrogel: (a) bubbles in hydrogel solution recorded
Figure
3. photos;
Effects (b)
of shaking
on bubbles
hydrogel:
(a) bubbles
hydrogel
solutionunder
recorded
with
bubbles intime
hydrogel
solutionin
detected
by ultrasound;
(c)in
bubbles
in hydrogels
with microscope;
photos; (b) and
bubbles
in
hydrogel
solution
detected
by
ultrasound;
(c)
bubbles
in
hydrogels
under
the floating
of hydrogels.
Figure 3. Effects(d)
of shaking
timestate
on bubbles
in hydrogel: (a) bubbles in hydrogel solution recorded
microscope; and (d) the floating state of hydrogels.
with photos; (b) bubbles in hydrogel solution detected by ultrasound; (c) bubbles in hydrogels under
microscope; and (d) the floating state of hydrogels.
Figure 4. Characterizations of floating hydrogel: (a) the floating hydrogel morphology under SEM;
(b) bubble size distribution of the floating hydrogel; (c) viscosity of the floating hydrogel; and (d)
erosion
time
of the floating hydrogel
with
different(a)
volume.
Figure 4.
Characterizations
of floating
hydrogel:
the floating hydrogel morphology under SEM;
Figure 4. Characterizations of floating hydrogel: (a) the floating hydrogel morphology under SEM;
(b) bubble size distribution of the floating hydrogel; (c) viscosity of the floating hydrogel; and (d)
(b)
size
distribution
3.3.bubble
Release
Study
In Vitro of the floating hydrogel; (c) viscosity of the floating hydrogel; and (d) erosion
erosion time of the floating hydrogel with different volume.
time of the floating hydrogel with different volume.
The release profiles of free RhB solution, RhB-loaded non-floating hydrogel and RhB-loaded
3.3. Release
Study In
floating
hydrogel
inVitro
vitro were investigated (Figure 5). RhB served as fluorescence indicator. The
The release profiles of free RhB solution, RhB-loaded non-floating hydrogel and RhB-loaded
floating hydrogel in vitro were investigated (Figure 5). RhB served as fluorescence indicator. The
Materials 2016, 9, 1005
8 of 14
3.3. Release Study In Vitro
The release profiles of free RhB solution, RhB-loaded non-floating hydrogel and RhB-loaded
floating
hydrogel in vitro were investigated (Figure 5). RhB served as fluorescence indicator. The
curves
Materials 2016, 9, 1005
8 of 14
depicted the
cumulative release amount of RhB as a function of time. The control (free RhB)
dispersed
Materials 2016, 9, 1005
8 of 14
curves depicted
thehomogenous
cumulative release
amount
of RhBand
as a the
function
of time. The
control
(free RhB)
immediately
to form
solution
in water,
cumulative
release
reached
96.3% in
curves
depictedwhile,
the to
cumulative
release amount
of RhBin
as
a function
time.
The control
(free RhB)
dispersed
immediately
form homogenous
solution
water,
andofthe
cumulative
release
reached
10 min
after
injection,
after
injecting
RhB-loaded
non-floating
hydrogel
or RhB-loaded
floating
dispersed
immediately
to form homogenous
solution
in water,
and the cumulative
release hydrogel
reached
96.3%
in
10
min
after
injection,
while,
after
injecting
RhB-loaded
non-floating
or for
hydrogel into water, floating hydrogel could float on the surface of water immediately. The time
96.3% in
10 minhydrogel
after injection,
while, floating
after injecting
RhB-loaded
non-floating
hydrogeloforwater
RhB-loaded
floating
into
water,
hydrogel
could
float
on
the
surface
RhB to beRhB-loaded
completely
released
from
gelwater,
in the
non-floating
hydrogel
group
and floating
floating
hydrogel
into
floating
hydrogel could
float on
the surface
of water hydrogel
√
immediately. The time for RhB to be completely released from gel in the non-floating
hydrogel
group were
6.5
and
6
h,
respectively.
We
also
found
that
the
Higuchi
model
(Qt
=
K
t) could predict
immediately. The time for RhB to be completely released from gel in the non-floatingHhydrogel
group and floating hydrogel group were 6.5 and 6 h, respectively. We also found that the Higuchi
group and
floating
hydrogel
group were
6.5 and in,
6 h,which
respectively.
We also constant
found that (K
the Higuchi
the drug release
from
floating
hydrogel
perfectly
the release
39.9 and the
H ) is
modelmodel
(Qt =(Qt
KH=√t)
predict
the
from floating
floatinghydrogel
hydrogel
perfectly
in,
which
KHcould
predict
thedrug
drugrelease
release from
perfectly
in, which
the the
√t) could
correlation
coefficient
(R)
is
0.99
[12],
which
indicated
that
drug
release
from
floating
hydrogel
releaserelease
constant
(KH)(K
isH39.9
and
thethe
correlation
(R)isis0.99
0.99
[12],
which
indicated
that drugwas a
constant
) is 39.9
and
correlationcoefficient
coefficient (R)
[12],
which
indicated
that drug
controlled-release
process
and
square
time dependent.
releaserelease
from from
floating
hydrogel
was
a root
controlled-release
processand
and
square
root
dependent.
floating
hydrogel
was
a controlled-release
process
square
root
timetime
dependent.
Figure 5. Cumulative release of free-RhB solution, RhB-loaded non-floating hydrogel and RhB-loaded
Figure 5. Cumulative
release of free-RhB solution, RhB-loaded non-floating hydrogel and RhB-loaded
floating hydrogel (Mean ± SD, n = 3).
floating
hydrogel
(Mean release
± SD, of
n =free-RhB
3).
Figure
5. Cumulative
solution, RhB-loaded non-floating hydrogel and RhB-loaded
3.4. Release
Study (Mean
In Vivo± SD, n = 3).
floating
hydrogel
3.4. Release Study
In the
Vivo
Firstly,
process of intravesical injection of floating hydrogel into rabbit bladder was detected
3.4. Release
Study In Vivo
by ultrasound
(Figure 6). Catheter was inserted into rabbit bladder via urethra, then the
Firstly,
the process of
intravesical
of floating
hydrogel
into rabbit
bladder
was detected by
thermo-sensitive
hydrogel
solutioninjection
was injected
into bladder
using catheter.
After that,
the hydrogel
Firstly,
the
process
of
intravesical
injection
of
floating
hydrogel
into
rabbit
bladder
was
detected
and6).
floated
in bladder
More
information
about
floating process
canbe
seen
ultrasoundformed
(Figure
Catheter
wasimmediately.
inserted into
rabbit
bladder
viathe
urethra,
then the
thermo-sensitive
by ultrasound
(Figure
6). Catheter
was The
inserted into image
rabbit non-floating
bladder via
urethra,
then the
in the Video
S1 injected
(Supplementary
Materials).
hydrogel
in rabbit
hydrogel solution
was
into bladder
using ultrasound
catheter. After of
that, the hydrogel
formed
and floated
thermo-sensitive
solution
was
injected
into
bladder
catheter.
After was
that,unable
the hydrogel
bladder was hydrogel
also captured
(Figure
S3).
From the
image,
the using
non-floating
hydrogel
to
in bladder
immediately.
More
information
about
the
floating
process
canbe
seen
in
the
Video S1
formed
and
inattached
bladdertoimmediately.
More information about the floating process canbe seen
float
in floated
urine and
the bladder wall.
(Supplementary
Materials).
The ultrasound
image
of non-floating
hydrogel in
rabbit in
bladder
in the Video S1
(Supplementary
Materials). The
ultrasound
image of non-floating
hydrogel
rabbit was
also captured
(Figure
S3).
From
the
image,
the
non-floating
hydrogel
was
unable
to
float
in
urine
bladder was also captured (Figure S3). From the image, the non-floating hydrogel was unable
to and
attached
bladder
wall. to the bladder wall.
float to
in the
urine
and attached
Figure 6. Process of intravesically injection of floating hydrogel into rabbit bladder (detected by
ultrasound). The white dashed curves in the pictures represented the rabbit bladder wall. The white
dashed circles represented gels in rabbit bladder. The catheters are indicated with white arrows.
Then, we measured RhB release rate from non-floating and floating hydrogels, compared with
Figure 6. Process of intravesically injection of floating hydrogel into rabbit bladder (detected by
in vivo
7). The first
voiding of
was triggered
filling
the rabbit
bladder
2~10
Figurefree
6. RhB
Process
of(Figure
intravesically
injection
ofurine
floating
hydrogelbyinto
rabbit
bladder
(detected
by
ultrasound). The white dashed curves in the pictures represented the rabbit bladder wall. The white
min
after
intravesical
instillation
of
saline,
free
RhB
solution,
non-floating
hydrogel
or
floating
ultrasound).
The
white
dashed
curves
in
the
pictures
represented
the
rabbit
bladder
wall.
The
white
dashed
circles
represented
gels
in rabbit bladder.
Theofcatheters
are indicated
with white arrows.
hydrogel
(Figure
S4). After
intravesical
instillation
RhB solution,
RhB concentration
in bladder
dashed circles represented gels in rabbit bladder. The catheters are indicated with white arrows.
reached the peak immediately, but dropped sharply after the first voiding of urine. As to the
Then,
we measured
release
rate
non-floating
and
hydrogels,
compared
non-floating
hydrogelRhB
group,
due to
thefrom
incapacity
of floating
in floating
urine, it attached
on the
bladder with
free
RhB
in
vivo
(Figure
7).
The
first
voiding
of
urine
was
triggered
by
filling
the
rabbit
bladder
Then, we measured RhB release rate from non-floating and floating hydrogels, compared2~10
with free
min after intravesical instillation of saline, free RhB solution, non-floating hydrogel or floating
RhB in vivo (Figure 7). The first voiding of urine was triggered by filling the rabbit bladder 2~10 min
hydrogel (Figure S4). After intravesical instillation of RhB solution, RhB concentration in bladder
after intravesical instillation of saline, free RhB solution, non-floating hydrogel or floating hydrogel
reached the peak immediately, but dropped sharply after the first voiding of urine. As to the
non-floating hydrogel group, due to the incapacity of floating in urine, it attached on the bladder
Materials 2016, 9, 1005
9 of 14
(Figure S4). After intravesical instillation of RhB solution, RhB concentration in bladder reached the
peak immediately, but dropped sharply after the first voiding of urine. As to the non-floating hydrogel
group, due
to the
incapacity
of floating in urine, it attached on the bladder wall, so it 9caused
severe
Materials
2016,
9, 1005
of 14
bladder irritation. Consequently, most gels were expelled from bladder during the first voiding of
so it caused severe bladder irritation. Consequently, most gels were expelled from bladder
urine, andwall,
only
little hydrogel remained. In contrast, the intravesical delivery of floating hydrogel
during the first voiding of urine, and only little hydrogel remained. In contrast, the intravesical
showed sustained
of
RhB from
gel.
The concentration
of RhB
from floating
hydrogel
delivery2016,
ofrelease
floating
hydrogel
showed
sustained
release of RhB from
gel.released
The concentration
of9 RhB
Materials
9, 1005
of 14
reached 35.6%
before
the second
voiding.
hydrogel
releasing drug,
released
from floating
hydrogel
reachedAfter
35.6%voiding,
before thefloating
second voiding.
Afterremained
voiding, floating
wall,
so itremained
caused reached
severe
bladder
most gels
werebefore
expelled
from voiding,
bladder
hydrogel
releasing
drug,irritation.
and
RhB Consequently,
concentration
reached
23.2%
the
third
and RhB concentration
23.2%
before
the third voiding,
and
12.5%
before
the last voiding.
during
the first
voiding
of urine,
andCompared
only littletohydrogel
remained.
In contrast,
the intravesical
and 12.5%
before
the last
voiding.
free RhB
and non-floating
hydrogel,
floating
Compared to free RhB and non-floating hydrogel, floating hydrogel could significantly extend the
delivery
floating
hydrogel extend
showedthe
sustained
release
gel. The
concentration
of RhB
hydrogelofcould
significantly
residence
time of
ofRhB
RhBfrom
in bladder
and
hence increase
the
residence released
time offrom
RhBfloating
in bladder
andreached
hence 35.6%
increase
the
hydrogel
before
thebioavailability.
second voiding. After voiding, floating
bioavailability.
hydrogel remained releasing drug, and RhB concentration reached 23.2% before the third voiding,
and 12.5% before the last voiding. Compared to free RhB and non-floating hydrogel, floating
hydrogel could significantly extend the residence time of RhB in bladder and hence increase the
bioavailability.
Figure 7. Drug release of floating hydrogel, non-floating hydrogel and free RhB solution in vivo
Figure 7. (Mean
Drug± release
SD, n = 3).of floating hydrogel, non-floating hydrogel and free RhB solution in vivo
(Mean ± SD, n = 3).
Finally, the residual amount of RhB in rabbit bladder of normal saline, free RhB solution,
RhB-loaded
hydrogel
RhB-loaded
floating
hydrogel
groups
were investigated
Figure 7.non-floating
Drug release of
floating and
hydrogel,
non-floating
hydrogel
and free
RhB solution
in vivo
Finally,
the8).residual
amount of RhB in rabbit bladder of normal saline, free RhB solution,
(Figure
(Mean ±The
SD,results
n = 3). showed that the fluorescence intensity of floating hydrogel group was much
RhB-loaded
non-floating
and
RhB-loaded
floating
hydrogel
groups
higher
than free RhBhydrogel
solution and
non-floating
hydrogel
groups. No
fluorescence
of RhBwere
could investigated
be
Finally,
residual
amount
offluorescence
RhB
in residual
rabbit bladder
saline,
free RhBtissue
solution,
in the
theshowed
normal saline
group.
The
amountofofnormal
in
the hydrogel
bladder
waswas much
(Figure 8).observed
The
results
that the
intensity
ofRhB
floating
group
RhB-loaded
non-floating
hydrogel
and so
RhB-loaded
floating
hydrogel
were investigated
proportional
to the
fluorescent
the residual
amount
of RhB ingroups
rabbit
bladder
of floating
higher than
free RhB
solution
andintensity,
non-floating
hydrogel
groups.
No
fluorescence
of RhB could
(Figure
8). group
The results
that thethan
fluorescence
intensity
of floating
group was
much
hydrogel
was showed
much higher
other groups.
Besides,
therehydrogel
is no targeted
delivery
be observed
in
the
normal
saline
group.
The
residual
amount
of
RhB
in
the
bladder
higher
than on
freethe
RhB
solution
and non-floating
hydrogel
No fluorescence
RhB
could
betissue was
mechanism
floating
hydrogel
drug delivery
systemgroups.
selectively
to the cancer of
cells
rather
than
proportional
to thein fluorescent
intensity,
soThe
the
residual
of drug
RhB
inbladder
rabbit
bladder
observed
the normal
saline
residual
amount
of RhB
in the
was
the healthy
cells.
Therefore,
thegroup.
floating
hydrogel
was
aamount
promising
carrier
for tissue
raising
theof floating
proportional
to
the fluorescent
intensity,
the residual
amountthere
of RhBisinno
rabbit
bladderdelivery
of floatingmechanism
efficiency
of intravesical
administration.
hydrogel group
was
much
higher
than
othersogroups.
Besides,
targeted
hydrogel group was much higher than other groups. Besides, there is no targeted delivery
on the floating
hydrogel drug delivery system selectively to the cancer cells rather than the healthy
mechanism on the floating hydrogel drug delivery system selectively to the cancer cells rather than
cells. Therefore,
the
floating
hydrogel
washydrogel
a promising
drug carrier
for raising
the the
efficiency of
the healthy
cells.
Therefore,
the floating
was a promising
drug carrier
for raising
intravesical
administration.
efficiency
of intravesical administration.
Figure 8. Phase and fluorescence picture of frozen sections of bladder tissue to evaluate residual of
RhB in the bladder. (a) Bright-field of frozen sections of bladder tissue; (b) Fluorescence images of
frozen sections of bladder tissue, the fluorescence intensity of floating hydrogel group was much
higher than free RhB solution and non-floating hydrogel groups.
Figure 8. Phase and fluorescence picture of frozen sections of bladder tissue to evaluate residual of
Figure 8. Phase
fluorescence
pictureofoffrozen
frozen
sections
of bladder
to evaluate
residual
of RhB
RhB in and
the bladder.
(a) Bright-field
sections
of bladder
tissue; tissue
(b) Fluorescence
images
of
frozen (a)
sections
of bladderof
tissue,
the sections
fluorescence
floating(b)
hydrogel
group was
much of frozen
in the bladder.
Bright-field
frozen
of intensity
bladderoftissue;
Fluorescence
images
than tissue,
free RhBthe
solution
and non-floating
hydrogel
groups. hydrogel group was much higher than
sections ofhigher
bladder
fluorescence
intensity
of floating
free RhB solution and non-floating hydrogel groups.
Materials 2016, 9, 1005
10 of 14
Materials 2016, 9, 1005
10 of 14
3.5. The Efficacy of Floating Hydrogel in Acute Bladder Injury Model
3.5. The Efficacy of Floating Hydrogel in Acute Bladder Injury Model
Interstitial cystitis (IC) patients have typical symptoms such as pain, frequency, urgency and
Interstitial
(IC) to
patients
have typical symptoms
such of
asthe
pain,
frequency,
urgency
and
nocturia,
owing cystitis
to damages
the glycosaminoglycan
(GAG) layer
bladder
[13]. Acute
bladder
nocturia,
owing
to damages
to the
glycosaminoglycan
(GAG)
layer interstitial
of the bladder
Acute
injury model
induced
by protamine
sulfate
was commonly used
to study
cystitis.[13].
Protamine
bladder
injury
model
induced
by
protamine
sulfate
was
commonly
used
to
study
interstitial
cystitis.
sulfate destroyed the GAG layer of bladder mucosa, thus increasing the permeability of bladder
Protamine
sulfate
destroyed
the
GAG
layerbeofattenuated
bladder mucosa,
thus increasing
the permeability
of
mucosa. The
protamine
sulfate
effect
might
by exogenous
sulfated polysaccharide,
such
bladder
mucosa. The protamine sulfate effect might be attenuated by exogenous sulfated
as heparin.
polysaccharide,
such as
We investigated
theheparin.
efficacy of saline, floating hydrogel, heparin solution and heparin-loaded
We
investigated
theinefficacy
of saline,
floating hydrogel,
solution
heparin-loaded
floating hydrogel groups
preventing
the permeation
of RhB inheparin
protamine
sulfateand
pretreated
bladders.
floating
hydrogel
groups
in
preventing
the
permeation
of
RhB
in
protamine
sulfate
pretreated
The higher permeability of RhB indicated poorer efficacy of drugs. In our experiment, the permeability
bladders.
higherwas
permeability
RhB
poorer
efficacy
of drugs.
In our experiment,
of bladderThe
mucosa
evaluatedofby
theindicated
fluorescence
depth
of RhB
in bladder
wall (Figurethe
9).
permeability
of
bladder
mucosa
was
evaluated
by
the
fluorescence
depth
of
RhB
in
bladder
wall
The fluorescence depth was indicated by the width of fluorescence zone in the bladder wall, and
(Figure
9). The fluorescence
was indicated
the width
of fluorescence
in the bladder
the fluorescence
distributiondepth
with width
was alsoby
plotted
(Figure
9b,c). Figure zone
9d showed
that the
wall,
and
the
fluorescence
distribution
with
width
was
also
plotted
(Figure
9b,c).
Figure
9d than
showed
fluorescence depth of RhB in the PS + saline group and PS + Gel group were much larger
the
that
the
fluorescence
depth
of
RhB
in
the
PS
+
saline
group
and
PS
+
Gel
group
were
much
larger
other two groups (p < 0.05), proving the capacity of heparin to reverse the wall leakage that PS caused.
than
the other
two+groups
(psolution
< 0.05), proving
the +capacity
of heparingel
to group
reverseshowed
the wallmuch
leakage
that
Compared
to PS
heparin
group, PS
heparin-loaded
poorer
PS
caused.
Compared
to
PS
+
heparin
solution
group,
PS
+
heparin-loaded
gel
group
showed
much
permeation of RhB (p < 0.05), with width similar to control group, indicating better efficiency of
poorer
permeation
of RhB
0.05),voiding
with width
towere
control
group,
better efficiency
heparin.
Because the
time(pof< first
of allsimilar
groups
2~10
min,indicating
and the heparin
solution
of
heparin.
Because
the
time
of
first
voiding
of
all
groups
were
2~10
min,
and
the
heparin
solution
was eliminated during that time. However, floating hydrogel could stay in bladder and continued
was
eliminated
during
thatimproving
time. However,
floating
hydrogelTherefore,
could staythe
in bladder
continued
to
to release
heparin,
largely
the efficacy
of heparin.
floating and
hydrogel
system
release
heparin,
largely
improving
the
efficacy
of
heparin.
Therefore,
the
floating
hydrogel
system
could successfully extend the residence time of drug and hence enhance drug efficacy.
could successfully extend the residence time of drug and hence enhance drug efficacy.
Figure 9. The efficacy of different treatments in acute bladder injury model: (a) phase picture of
Figure 9. The efficacy of different treatments in acute bladder injury model: (a) phase picture of frozen
frozen sections of bladder tissue; (b) fluorescence picture of frozen sections of bladder tissue; (c)
sections of bladder tissue; (b) fluorescence picture of frozen sections of bladder tissue; (c) fluorescence
fluorescence intensity–distance profiles of different bladder frozen sections; and (d) penetration
intensity–distance profiles of different bladder frozen sections; and (d) penetration depth of different
depth of different bladder frozen sections. (Mean ± SD, n = 3).
bladder frozen sections. (Mean ± SD, n = 3).
4. Discussion
4. Discussion
The safety problem of medical material is a tough obstacle lying in its application from bench to
The safety problem of medical material is a tough obstacle lying in its application from bench
bedside. In this study, we chose Poloxamer 407 as the only ingredient of our delivery system. The
to bedside. In this study, we chose Poloxamer 407 as the only ingredient of our delivery system.
safety of P407 hydrogel in vivo has been widely studied by previous reports [14]. In terms of
The safety of P407 hydrogel in vivo has been widely studied by previous reports [14]. In terms of
biocompatibility studies in vivo, there was no damage observed in muscle cells after injection of 20%
P407 [15]. Moreover, after intravesical instillation, the concentration of P407 in urine was much
lower than 20%. Furthermore, preparation of this system was just shaking the P407 aqueous solution
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biocompatibility studies in vivo, there was no damage observed in muscle cells after injection of 20%
P407 [15]. Moreover, after intravesical instillation, the concentration of P407 in urine was much lower
than 20%. Furthermore, preparation of this system was just shaking the P407 aqueous solution for
20 s, which is extremely simple. Therefore, there is no safety problem in the clinical application of
our system. The efficacy of our system is inspiring according to in vivo drug release and efficacy
experiments, much better than non-floating hydrogels and drug solutions.
P407 is polyoxyethylene-polyoxypropylene-polyoxyethylene (PEOn-PPOn-PEOn) tri-block
copolymer, non-ionic surfactants, possessing biocompatible, thermo-sensitive, low toxicity, foamability
and weak immunogenic properties [14,16]. In this study, its foamability enabled P407 to act as
micro-bubbles source of the floating hydrogel system. By shaking the P407 solution, air was
incorporated in the solution, a bubble rising in a surfactant solution accumulates surfactant molecules
on its surface (the surfactant adsorption at the air/water interface) [17]. The number or growth rate of
micro-bubbles increased with the concentration of surfactant (P407) in solution, as well as air amount
incorporated in the solution (i.e., more shaking times) [18]. The stability of micro-bubbles in solution
was positively correlated with the concentration or viscosity of surfactant [19]. Thus, micro-bubbles
would suspend in solution for a period because of the viscosity of the P407 solution. When P407
solution converted to gel state (at 37 ◦ C), so the air micro-bubbles in gel could not move any more thus
the micro-bubbles were “locked” in gel. When the gel changed to the solution state (at 0 ◦ C), the air
micro-bubbles in solution would gradually dissipate away because of the stochastic nature of bubble
coalescence in viscous liquids. We also have tested the gelation properties of the solution at 25 ◦ C,
the gelation temperature of the hydrogel solution was about 11.5 ◦ C, so the hydrogel converted to
the gel state from the solution state at 25 ◦ C. Since in clinical operation, the injection will be operated
under room temperature, so we stored the gel solution at 0 ◦ C condition before injected to the bladder,
the viscosity of 45% P407 was low (about 200 mPa·s) at this temperature. When take the gel solution
under room temperature from 0 ◦ C condition, the gelation time of the gel solution was about 5 min
after shaking quickly. Therefore, it has enough time to inject the gel solution into bladder. As the
shear force have impact on the stability of the micro-bubbles during injection, we injected the hydrogel
solution (3 mL) into the bladder quickly (about 10 s). The short injection time may reduce the impact
of shear force. The floating state of hydrogel was confirmed by ultrasound after injection. We also used
homogenizer to produce micro-bubbles. Hydrogel solution stirred for 10 s at the speed of 5000 rpm
could float in water stably (Figure S5). Considering the convenience in clinical application, we used
shaking by hand to replace machine.
The time to float of hydrogel should be as short as possible since the short time can decrease the
risk of urinary obstruction or bladder irritation. In this floating hydrogel system, the hydrogel could
float immediately due to the whole hydrogel was full of micro-bubbles at the beginning by shaking the
P407 solution. However, the time to float of hydrogels we previously developed were several minutes,
because chemical reaction took time [7,8].
P407 formed micelles to entrap drugs, so drugs was released throughout the diffusion process
of gel matrix. Drugs released with the process of P407 dissolution and ended once P407 dissolved
completely [20]. The erosion time of hydrogel should be longer to extend the release time of drug.
Increasing concentration and hydrogel volume of P407 will lengthen its erosion time and hence extend
the release time of drug. Moreover, the viscosity of gels also increased with the concentration of the
P407 [20]. Therefore, 45% P407 was chosen to obtain optimal erosion time and viscosity. The results
showed that the erosion time of this floating hydrogel system (about 6 h/5 mL) was much longer
than our previous floating hydrogel containing NH4 HCO3 (about 3 h/5 mL) [8]. These are due to the
chemical reaction inside hydrogel accelerated the gel erosion.
In this study, the drug release time in vivo was not consistent with that in vitro. The erosion time
of the 3 mL floating hydrogel was about 3.5~4 h in vitro. However, in vivo release study showed that
drug was released for about 10 h. The time discrepancy of drug release might result from the volume
difference between bladder (5~40 mL) and beaker (200 mL). Another reason was the urine residues in
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the bladder [21]. In addition, the autofluorescence of rabbit urine might increase the measurement of
RhB release amount. Moreover, the contraction of bladder during injection and gelation process will
reduce the amount of micro-bubble generated and thus result in the reduction in erosion time in vivo.
Bladder irritation is often induced by urinary infection or foreign bodies in the bladder (such as
bladder stone or blood clots) [22]. In our experiments, most non-floating hydrogels were expelled
from rabbit bladder after intravesical instillation. It indicated that non-floating hydrogel caused severe
bladder irritation as it attached to the bladder wall, and caused the rabbit bladder detrusor forcefully
contracted to expel it out. Compared to the non-floating hydrogel, the floating hydrogel was floating
in urine and was not directly in contact with the bladder wall, so the floating hydrogel could avoid
cause the bladder irritation.
Previous reports have shown that patients of interstitial cystitis endure damage of the
glycosaminoglycan (GAG) layer of urothelium [23]. The surface GAG layer has been proposed
as a protective barrier that coats the transitional cell surface. Parsons has demonstrated that the
increasing bladder mucosal permeability to solu-urea in patients with interstitial cystitis could be
imitated by intravesical administration of protamine sulfate [24]. The protamine effect appeared to be
reversed by treatments with exogenous sulfated polysaccharide (i.e., intravesical administration of
heparin, hyaluronic acid, or pentosan polysulfate) [25]. Heparin was initially reported to relieve the
symptoms of IC in the 1960s, and subsequent studies also proved its efficacy [26]. Its exact mechanism
of action was not understood at that time. One hypothesis was that heparin provided exogenous
sulfated polysaccharide which coated the epithelium to maintain the bladder mucosa integrity. In this
study, rabbit acute bladder injury model was constructed by intravesical administration of protamine
sulfate, and the efficacy of heparin loaded floating hydrogel was evaluated by fluorescence depth in
the bladder wall. Our results showed that the heparin-loaded floating hydrogel was more effective
than free heparin solution in alleviating the damage effects of protamine sulfate. Because the floating
hydrogel released RhB persistently and extended the residence time of heparin in bladder, while the
free heparin solution was eliminated of bladder along with the first voiding of urine. Our results
also showed that the P407 hydrogel did not affect the bladder mucosal permeability to RhB. As the
heparin served as the exogenous sulfated polysaccharide, the prolonged drug resident time in bladder
increased the efficiency of heparin in the protection of bladder mucosal permeability. The floating
hydrogel system was a promising drug delivery system in the treatment of IC patients whose bladder
GAGs layer was damaged.
5. Conclusions
This paper presents a new self-generating micro-bubbles floating hydrogel drug delivery system
for intravesical instillation. The Matrix of floating hydrogel is P407 without any additives, addressing
safety concerns for clinical use. The floating property of floating hydrogel avoids urinary obstruction
and bladder irritation that conventional hydrogels cause, and improves drug efficacy by prolonging
the residence time. In other words, this new drug delivery system is safe, simply prepared and efficient
for future clinical use.
Supplementary Materials: The following are available online at www.mdpi.com/1996-1944/9/12/1005/s1.
Figure S1: Effects of shaking time on erosion time (a) and gelation temperature (b) in hydrogel, Figure S2: Effects
of shaking time on micro-bubbles number in hydrogel, Figure S3: Non-floating hydrogel in rabbit bladder,
Figure S4: The time of first voiding after intravesical instillation, Figure S5: Hydrogel solution was homogenized,
then hydrogel floats stably in water, Figure S6: Volume change of hydrogel in respect to the density of micro-bubble,
Figure S7: Effect of environmental factors (temperature (a), humidity (b), and atmosphere pressure (c)) and the
shaking force (d) on micro-bubble generation, Video S1: Floating hydrogel in vitro and vivo.
Acknowledgments: This study was supported by National Natural Science Foundation (No. 81202474, 81273464
and 81473146), Changzhou Special Project of Biotechnology and Biopharmacy (No. CE20105006). Science Bridges
China-Changzhou Biotechnology and Pharmaceutical Technology Special Project (Grant Number: CE20105006).
J. Wu is sponsored by Qing Lan Project.
Author Contributions: Hongqian Guo, Jinhui Wu and Yiqiao Hu conceived and designed the experiments;
Tingsheng Lin and Xiaozhi Zhao performed the experiments; Tingsheng Lin analyzed the data; Huibo Lian,
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Junlong Zhuang, Qing Zhang, Wei Chen, Wei Wang, Guangxiang Liu and Suhan Guo contributed
reagents/materials/analysis tools; Tingsheng Lin and Yifang Zhang wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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