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Zhang, J. Miao, Z. Zhao, X. Jin, L. Liu, L. Yu, C. Shen and J. Ding, J. Mater. Chem. B, 2020, DOI:
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Volume 6
Number 3
21 January 2018
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ARTICLE

Journal of Materials Chemistry B Accepted Manuscript

An injectable thermosensitive hydrogel loaded with an
Cite this: DOI: 10.1039/x0xx00000x
ancient natural drug colchicine for myocardial repair
after infarction
Yu Chena,‡, Jiayue Shib,‡, Yaping Zhanga, Jiajun Miaob, Zhe Zhaoa, Xian Jina,
Published on 31 December 2019. Downloaded on 1/3/2020 1:26:29 AM.

Received 00th January 20xx, Liang Liua, Lin Yub,c,*, Chengxing Shena,*, Jiandong Dingb,c
Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x Localized administration of anti-inflammatory agents benefits patients after myocardial infarction (MI) by
repressing/modulating inflammatory response of the MI region and thus accelerating repair of the impaired
www.rsc.org/
tissues. Colchicine (Col), an ancient natural drug, has excellent anti-inflammatory effects; however, its
utilization is strictly limited due to its severe systemic toxicity and narrow therapeutic window. In this
study, we developed an intramyocardial delivery system of Col using an injectable, thermosensitive
poly(lactide-co-glycolide)-poly(ethylene glycol)-poly(lactide-co-glycolide) (PLGA-PEG-PLGA)
polymer hydrogel as the vehicle for the treatment of MI while minimizing its systemic toxicity. The
aqueous PLGA-PEG-PLGA solution loaded with Col (Col@Gel) underwent a sol-gel transition at 35 °C
and maintained a gel state at body temperature. Col was released from the Col@Gel in an initial burst
followed by a sustained release manner for over 8 days. The in vitro cell tests showed that the Col@Gel
system significantly inhibited macrophage proliferation and migration. In a mouse model of MI, a single
intramyocardial administration of the Col@Gel effectively alleviated cardiac inflammation, inhibited
myocardial apoptosis and fibrosis, improved cardiac function and structure, and increased mouse survival
without inducing severe systemic toxicity, which was observed following intraperitoneal administration
of Col solution. These results suggested that the Col@Gel system is a reliable drug delivery system for the
sustained local release of Col and has great potential as an anti-inflammatory therapy for the treat of MI.

Introduction promotes cardiomyocyte apoptosis and accelerates extracellular

Acute myocardial infarction (MI) and follow-up induced heart matrix synthesis, leading to myocardial fibrosis, adverse ventricular

failure are one of the leading causes of death in many countries remodeling, and cardiac dysfunction.2-4 In view of the important roles

and remain a therapeutic challenge worldwide.1, 2 In addition to of inflammation in myocardial injury, increasing attention has been

ischemia and hypoxia-induced myocardial damage, post-MI paid to the potential benefits of anti-inflammatory therapies to

injury is exacerbated by excessive local inflammation, which repress/modulate local inflammation post-MI, thereby accelerating
myocardial repair, inhibiting left ventricular remodeling, and
improving patient prognosis.2, 5, 6
aDepartment of Cardiology, Shanghai Jiao Tong University Affiliated Sixth
People’s Hospital, Shanghai 200233, China. Colchicine (Col), an ancient natural drug extracted from Colchicum
E-mail: shencx@sjtu.edu.cn
bState Key Laboratory of Molecular Engineering of Polymers, Department of autumnale, was first described and utilized in Egypt more than 3000
Macromolecular Science, Fudan University, Shanghai 200438, China.
E-mail: yu_lin@fudan.edu.cn
years ago.7 Col prevents mitotic spindle formation, arrests
cZhuhai Fudan Innovation Institute, Zhuhai, Guangdong 51900, China
microtubule polymerization and inhibits crucial inflammatory

This journal is © The Royal Society of Chemistry 2019 J. Mater. Chem. B, 2019, 00, 1-3 | 1
 Journal of Materials Chemistry B Page 2 of 15
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Paper Journal of Materials Chemistry B

signaling pathways, such as inflammasomes, proinflammatory convenient synthesis, good reproducibility and wide recognition of
cytokines, as well as expression of adhesion molecules.7, 8 Therefore, safety.38, 39 It has been developed into a platform for the delivery of
Col has potent anti-inflammatory effects. Nowadays, Col is various kinds of drugs to treat systemic or regional diseases, including
recognized as a wonder drug for the treatment of refractory gout and anti-cancer treatments,40-42 anti-diabetic treatments,43-45 anti-
familial Mediterranean fever.7, 9 Col has also been tried to prevent osteopenia treatments,39 and others.46-50 For example, the local

Journal of Materials Chemistry B Accepted Manuscript

cardiovascular disease in clinic.6 However, the therapeutic window of administration of ropivacaine hydrochloride-loaded PLGA-PEG-
Col is narrow. Col with excessive dose can cause severe, even lethal, PLGA hydrogel was able to prolong its analgesic efficacy and thus
systemic toxicity including gastrointestinal disturbance and alleviated postoperative pain.49 Zhu et al. found that the
multiorgan failure,7, 10, 11 which significantly limited its clinic use, subconjunctival co-delivery of metformin and levofloxacin
even if it has a very long application history and well-characterized hydrochloride using PLGA-PEG-PLGA hydrogel as the depot
effects. effectively inhibited corneal neovascularization.50 Also, Ding and
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Several studies found that the gout patients treated with Col exhibited Chen developed a tumor microenvironment-sensitive PLGA-PEG-
a reduced prevalence of MI.12, 13 Two recent clinic trials revealed that PLGA-doxorubicin conjugate hydrogel and a synergistic anticancer
the patients with acute MI receiving the systemic administration of effect was achieved after intratumoral injection of the conjugate
Col showed a decrease in left ventricular remodeling or infarct size.14, hydrogel containing docetaxel.51 Recently, the Hotta’s group revealed
15 More recently, in a mouse model of MI, oral ingestion or that the introduction of laponite clay nanoparticles to an aqueous
intraperitoneal injection of Col had beneficial effects on myocardial solution of PLGA-PEG-PLGA could facilely modulate the sol-gel
repair.8, 16 These findings opened up a new avenue for the old drug in transition temperature from 25 °C to 37 °C, while the degradation rate
new use and provided a promising strategy for the treatment of acute of composite hydrogel was mainly dependent on the LA/GA ratio.52
MI. Of note, the risk of systemic toxicity remains a considerable 53 It is worth pointing out that the application of PLGA-PEG-PLGA
hurdle for the application of Col. One valid alternative to avoid the hydrogel for MI treatment is rarely reported.
systemic toxicity of Col is to realize the regional sustained delivery of In this study, we developed a sustained delivery of Col based on an
the drug. Meanwhile, advances in echocardiography and injectable thermosensitive PLGA-PEG-PLGA hydrogel for
thoracoscopy technologies have made intramyocardial injection of myocardial repair after MI. First, a PLGA-PEG-PLGA triblock
drugs feasible. copolymer with rational molecular composition was designed and
Injectable hydrogels formed by in situ gelation have received synthesized to ensure its temperature-responsive sol-gel transition in
increasing attention as vehicles for drug delivery and as scaffolds for water below body temperature. The delivery system of Col (labeled
tissue repair due to the unique superiority of minimally invasive Col@Gel) was then fabricated using the PLGA-PEG-PLGA hydrogel
application of these systems.17-26 In particular, temperature-induced as the vehicle, as shown in Fig. 1A. Next, in vitro release behavior and
gelling materials composed of poly(ethylene glycol) effects of the Col@Gel on macrophage proliferation and migration
(PEG)/polyester,27-30 PEG/polypeptide,31-33 poly(N- were evaluated. Finally, a mouse model of MI was constructed and
isopropylacrylamide-co-2- hydroxyethyl methacrylate-co- the safety and efficacy of intramyocardial injection of Col@Gel for
methacrylate-polylactide) (poly(NIPAAm-co-HEMA- co- the post-MI treatment was confirmed by analysis of mouse weight,
MAPLA),34, 35 poly(phosphazene)s copolymers36, 37 are low-viscous survival, liver function, cardiac structure and function, and
liquids at ambient temperature, but can rapidly convert into standing myocardial inflammation, apoptosis, and fibrosis. Fig. 1B
in situ gels in response to physiological temperature. Therefore, schematically presents the creation of mouse model of MI and the use
various therapeutic agents can readily be loaded into these materials of Col@Gel after MI.
by blending them in the sol state without drug loss. Upon in vivo
injection, the in situ forming hydrogels can offer sustained release of Materials and Methods
the encapsulated drugs.
Materials
So far, the thermosensitive and biodegradable poly(lactide-co-
Poly(ethylene glycol) (PEG) with molecular weight (MW) 1500 and
glycolide)-poly(ethylene glycol)-poly(lactide-co-glycolide) (PLGA-
stannous octoate (Sn(Oct)2) were purchased from Sigma-Aldrich (St.
PEG-PLGA) hydrogel enjoys great popularity because of its

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Journal of Materials Chemistry B Accepted Manuscript

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Fig. 1 A) The fabrication of Col@Gel system. The Col@Gel system exhibited a free-flowing sol at ambient temperature (25 °C) and
spontaneously converted into a semi-solid hydrogel at body temperature. This sol-gel transition was reversible. B) The establishment of
mouse MI model and the subsequent administration of Col@Gel system into three sites around the infarct region after MI. MI was
induced by ligating the left anterior descending coronary artery (LAD).
Louis, MO, USA). DL-Lactide (LA) and glycolide (GA) were supplied 0.01 mol) was placed in a three-necked flask and dried under vacuum
by Hangzhou Mingzhong Biotechnology Co. Ltd. (China) and used as with stirring at 125 °C for 3 h to remove moisture. LA and GA at a
received. given amount were added to the flask and the mixture was incubated
All animal experimental procedures were approved by the at 80 °C for 30 min. After LA and GA had melted, 0.08 g of Sn(Oct)2
Institutional Authority for Laboratory Animal Care of Shanghai Jiao was added, and the reaction mixture was heated with stirring at 150
Tong University Affiliated Sixth People’s Hospital and conformed to °C for 12 h under an argon atmosphere. The temperature was then
the Guide for the Care and Use of Laboratory Animals (National cooled to 120 °C with stirring for 3 h under vacuum to eliminate the
Research Council, Eighth Edition, 2010) and the AVMA Guidelines unreacted monomers. Subsequently, the crude polymers were poured
for the Euthanasia of Animals (2013 Edition). Male C57BL/6 mice (6 into 80 °C water to remove water-soluble oligomers and unreacted
weeks of age) were supplied by SLAC Laboratory Animal Co. Ltd. monomers. This washing procedure was carried out for at least three
(Shanghai, China). Mice were housed under appropriate humidity and times. Afterwards, residual water was removed by freeze drying. The
temperature conditions and received fed standard chow. Tap water purified products were harvested and stored at −20 °C until use and
was available ad libitum. the yield was approximately 80%.
The structure and composition of PLGA-PEG-PLGA was analyzed
Synthesis and characterization of PLGA-PEG-PLGA via 1H-nuclear magnetic resonance (NMR) on a 500 MHz proton

A ring-opening copolymerization approach using LA and GA spectrometer (DMX500 spectrometer, Bruker). Deuterated

monomers was used to prepare PLGA-PEG-PLGA triblock chloroform with the internal standard tetramethylsilane was selected

copolymers and PEG 1500 and Sn(Oct)2 were selected as macro- as the solvent. The value of molar mass dispersity (ĐM) of the polymer
initiator and catalyst, respectively.39, 47 Briefly, PEG 1500 (15.0 g, was analyzed by gel permeation chromatography (GPC; Agilent

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 Journal of Materials Chemistry B
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1260). Tetrahydrofuran was selected as the eluent, the flow rate was
set at 1 mL/min and the measurement temperature was fixed at 35 °C.
Monodisperse polystyrene microspheres were used as standards.
Measurement of sol-gel transition temperature
The tube-inverting method39 was used to determine the sol-gel
transition point of aqueous polymer solution. 0.5 mL of polymeric
aqueous solution was added into a 2-mL vial and equilibrated at 4 °C
for 12 h. Subsequently, the capped vial was submerged in a water bath
and the temperature of water bath was increased from 20 to 50 °C at
a rate of 1 °C/step. At each temperature, the vial was incubated for 10
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min and then inverted by 180°. If the sample showed no evidence of
flow within 30 s, it was considered to be a gel.
The temperature-induced gelation behaviors of the aqueous polymer
solutions in the presence or absence of Col were further measured
using a dynamic rotational rheometer (Kinexus PRO, Malvern) with
a cone plate (1° steel cone, 60 mm diameter). The time sweep program
was set at an oscillatory frequency of 10 rad/s and the temperature was
increased from 15 °C to 50 °C at a heating rate of 1 °C/min.
In vitro Col release
The aqueous polymer solutions containing Col (0.25, 0.5 or 1 mg/mL)
were readily prepared by blending the aqueous polymer solution (25
wt%) with Col at 4 °C to form a homogenous solution. The Col-loaded
aqueous polymer solution maintained a free-flowing sol state at room
temperature while a gel state at body temperature, which was labeled
as Col@Gel. The in vitro release of Col from Col@Gel was
investigated and the method was implemented following the previous
protocols.18, 45 Briefly, the samples (0.5 mL) were injected into 10 mL
vials, equilibrated at 4 °C for at least 12 h, and then incubated in a
shaking water bath (50 rpm) at 37 °C for 10 min to form gels.
Phosphate-buffered solution (PBS, pH 7.4) adding 0.025 wt % NaN3
was introduced at 5 mL/vial. At various time points, 3 mL of PBS was
withdrawn from the vials and replaced with 3 mL of prewarmed (37
°C) fresh PBS. The Col contents in the PBS samples were detected
using a double beam UV-vis spectrophotometer (TU-1950). The UV
absorbance differences at 354 nm between the Col-containing
hydrogel samples and the Col-free blank hydrogel samples were
converted into the amounts of Col released at different time points by
comparison with the standard curve.
In vivo hydrogel degradation
Changes in visual appearance, mass, and MW of the PLGA-PEG-
PLGA hydrogel after subcutaneous injection into mice were
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monitored for up to 20 days. Male C57BL/6 mice were anesthetized
with chloral hydrate and then injected subcutaneously with 0.1 mL of
aqueous polymer solution (25 wt %) into the dorsal region. 3 mice
were euthanized at each specified time point and the remaining
hydrogels were dissected. The appearance of the injected gels was
Journal of Materials Chemistry B Accepted Manuscript
recorded with a digital camera, mass was measured using an analytical
balance, and MW was analyzed by GPC after freeze drying. The
tissues around the administration sites were collected and subjected to
hematoxylin-eosin (HE) staining to examine the tissue responses.
Cellular experiments
Cell culture. The mouse macrophage cell line Raw264.7 was
supplied by the Cell Bank of the Chinese Academy of Sciences
(Shanghai, China). The cells were incubated in DMEM medium
(Gibco) containing fetal bovine serum (FBS, 10%) and penicillin-
streptomycin (100 U/mL) under a 5% CO2 atmosphere at body
temperature.
In vitro cytotoxicity assay. The cytotoxicity of blank hydrogel and
Col@Gel system against Raw264.7 cells was evaluated by measuring
cell viability with a Cell Counting Kit-8 (CCK-8, Dojondo) assay.
Transwell chambers and a cell culture plate are shown in Fig. S1A.
As presented in Fig. S1B, aqueous polymer solutions (25 wt%) with
or without Col (1.0 or 0 mg/mL) were added to Transwell chambers
at 100 μL/chamber and incubated at 37 C for 10 min to form
hydrogels in situ. Raw264.7 cells were seeded into 24-well culture
plates at a density of 1×104 cells/well in DMEM growth medium and
incubated for 24 h. The cells were then washed for three times and
incubated in fresh DMEM. Next, the Transwell chambers containing
the gel samples were placed into the culture wells and the DMEM
could get touch with the gel through the permeable membrane of
Transwell chamber. After co-culture for 48 h, the cells were washed
with PBS, and CCK-8 reagent was then added to the wells in
accordance with the instructions provided by the manufacturer.
Control cells were incubated in the same manner in the absence of gel-
containing Transwell inserts, and their viability was set at 100% for
the purpose of calculating relative cell viability. Samples were
analyzed in triplicate.
In vitro cell migration assay. In vitro migration of Raw264.7
macrophages was measured using a Transwell assay. As presented in
Fig. S1C, aqueous polymer solutions (25 wt%) with or without Col
(1.0 or 0 mg/mL) were added to cell culture plates and then kept at 37
℃ to form gels in situ. Each well with 1 mL of hydrogel was incubated
in DMEM (1 mL) at 37 C for 24 h and subsequently the DMEM was
This journal is © The Royal Society of Chemistry 2019
 Page 5 of 15 Journal of Materials Chemistry B
Journal Name
collected. Raw264.7 cells (1×104 cells in the 100 μL of DMEM
collected) were added to the Transwell chambers, the chambers were
placed in new cell culture plates, and an additional 600 μL of fresh
DMEM was added per well. After incubation of 48 h, the chambers
were taken out, washed using PBS, and the cells on the membrane of
chamber were fixed with 4% phosphate-buffered paraformaldehyde
for 1 h at ambient temperature and then crystal violet was used to stain
the cells. Cells on the upper surface of the membrane were eliminated
with a cotton swab, and cells on the lower surface (migrated) were
observed under an OLYMPUS microscope (Japan).
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Animal experiments
In vivo application of the Col@Gel in a mouse model of MI.
Male C57BL/6 mice were randomized into four groups (15 per
group): Sham, MI control, MI + Col@Gel, and MI + Col (i.p.). MI
was induced by ligating the LAD of mouse. The Sham group
underwent surgery but the LAD was not ligated. The MI control group
underwent MI surgery but did not receive any treatment. The
Col@Gel group received intramyocardial injections of 1 mg/mL Col-
loaded polymer solution (10 μL) into three sites around the infarct
region immediately after LAD ligation. The Col (i.p.) group received
a single intraperitoneal injection of Col solution at the same dose.
Electrocardiography was performed using a Biological Signal
Acquisition System (Techman, Chengdu, China) during LAD
ligation.
The ST-segment elevation of electrocardiogram suggested that MI
was successfully modeled, as illustrated in Fig. S2. The mice were
monitored three times per day during the 4-week follow-up period.
The weights per week and the death of mice post-MI were recorded.
Echocardiography measurement. At day 28 after MI, the survival
mice were shaved in the anterior and left lateral thoracic regions and
then placed in the supine position. Ultrasound transonic gel was
smeared on the thoracic regions to optimize visibility. Two-
dimensional and m-mode echocardiographic images were acquired
using echocardiography. Left ventricular internal dimensions were
recorded during systole (LVIDs) and diastole (LVIDd). The ejection
fraction (EF) and fractional shortening (FS) were calculated from the
measured parameters.
Brain natriuretic peptide (BNP) and liver function tests. To
assess serum levels of BNP, a biomarker of heart failure,54, 55
alanine transaminase (ALT) and aspartate aminotransferase (AST),
two biomarkers of liver function,56 the mice were anesthetized using
isoflurane at day 28 post-treatment and blood samples were sampled
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ARTICLE
retro-orbitally and centrifuged to obtain serums. Serum
concentrations of BNP, ALT, and AST were measured using a BNP
ELISA Assay Kit (FineTest, Wuhan, China) or a Catalyst One®
Chemistry Analyzer (IDEXX, Westbrook, USA) according to the
manufacturers’ instructions.
Journal of Materials Chemistry B Accepted Manuscript
Myocardial tissue collection. Following collection of blood
samples, the anesthetized mice were euthanized and the hearts were
collected. Samples of ventricular myocardium were excised, fixed in
formalin, embedded in paraffin, cut into 5-μm-thick sections, and
mounted on slides for analysis of collagen deposition, inflammation,
and apoptosis by Masson’s trichrome, F4/80 immunofluorescence,
and terminal deoxynucleotidyl transferase dUTP nick end labeling
(TUNEL) staining, respectively.
Analysis of tissue fibrosis. Myocardial tissue sections were stained
with Masson’s trichrome. Sections were observed using an
OLYMPUS microscope (Japan). The fibrotic area within each tissue
section was measured and analyzed via ImageJ software 1.48
(National Institutes of Health, Bethesda, MD, USA).
TUNEL assay. A TUNEL assay was employed to detect apoptotic
cells in myocardial tissues. The sections were probed using an
TUNEL detection kit (Beyotime Biotechnology, Shanghai, China)
according to the manufacturer’s recommendations. Three images of
the infarct zone in each section were acquired using an OLYMPUS
microscope (Japan). The number of TUNEL-positive and total
cardiomyocyte nuclei in each zone were counted using ImageJ
software, and the percentage of apoptotic cardiomyocytes was
calculated.
Immunofluorescence staining of macrophages. Myocardial
inflammation was assessed by counting the number of F4/80-positive
macrophages in tissue sections. After deparaffinization, antigen
unmasking, and permeabilization, the myocardial sections were
incubated overnight at 4 °C with anti-F4/80 antibody (1:700 dilution,
CST, Danvers, USA), washed, and incubated with a goat anti-mouse
Alexa Fluor 488-labeled antibody (1:400 dilution, Jackson, West
Grove, USA) for 1 h at 37 C in the dark. Cell nuclei were
counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 10 min
at room temperature. Three images of the infarct zone in each section
were acquired using an OLYMPUS microscope and the number of
F4/80-positive cells per mm2 were counted using ImageJ software.
and Tumor necrosis factor-α (TNF-α) assay. The myocardial content
of TNF-α was evaluated using a Mouse TNF-α ELISA kit (Sigma-
Aldrich). Another gang of male C57BL/6 mice that underwent surgery
and then received different treatments were likewise divided into four
J. Name., 2019, 00, 1-3 | 5
 Journal of Materials Chemistry B
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groups: Sham, MI control, MI + Col@Gel, and MI+Col (i.p.). 3 mice
per group were euthanized each week, the hearts were removed, and
the myocardial tissues in the infarct zone were excised and stored at -
80°C until analysis. Then, the stored myocardial tissues were
homogenized five times in ice-cold lysis buffer for 1 min each time
and centrifuged at 10000 g for 20 min at 4 °C. The supernatants were
collected and TNF-α content was measured using the ELISA kit
according to the manufacturer’s instruction. The protein
concentrations in the supernatants were also measured, and the data
are presented as the TNF-α content in pg per 200 μg of protein.
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Statistical analysis
Data are presented as the mean ± standard deviation (SD). Group
means were compared via one-way analysis of variance or unpaired t-
test. A p value of <0.05 was considered statistically significant.
Results
Synthesis and characterization of the PLGA-PEG-PLGA triblock
copolymers
The PLGA-PEG-PLGA triblock copolymers were obtained through
ring-opening polymerization of LA and GA in the presence of PEG
1500, which contained two hydroxyl end groups, with Sn (Oct)2 as the
catalyst. The structure and composition of PLGA-PEG-PLGA were
assayed by 1H-NMR (Fig. S3). This analysis gave a number-average
molecular weight (Mn) of up to PLGA1850-PEG1500-PLGA1850 and a
molar ratio of LA:GA of 1:1. The ĐM value of PLGA-PEG-PLGA
was determined to be 1.30 by GPC analysis using monodisperse
polystyrene microspheres as standards. These characterizations
confirmed the successful preparation of expected sample.
Sol-gel transition of the PLGA-PEG-PLGA aqueous solution
The temperature-induced sol-gel transition of the PLGA-PEG-PLGA
aqueous solution was first investigated by the tube-inverting method
and the phase-transition diagram was obtained, as shown in Fig. S4.
Above a critical gel concentration (approximately 12 wt%), the
aqueous polymer solution underwent three physical states, namely,
sol, gel, and sol (suspension), with an increase in temperature.
The thermogelling properties of the 25 wt% PLGA-PEG-PLGA
aqueous solutions in the presence or absence of Col were further
assessed using dynamic rheological analysis. As shown in Fig. 2, the
changes in the elastic modulus (G′) and viscous modulus (G″) of the
aqueous polymer solutions upon heating were monitored. Generally,
G′ represents the elastic behavior and energy storage when a sample
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undergoes deformation, while G″ denotes the viscous behavior. At
low temperatures, G″ was higher than G′, suggesting a free-flowing
liquid state and good injectability. As the temperature increased, both
of them increased, but G′ increased more rapidly. The sol-gel
transition point was observed at 35 °C, when G′ was equal to G″.
Journal of Materials Chemistry B Accepted Manuscript
Meanwhile, the addition of 1.0 mg/mL Col had virtually no effect on
the rheological curves of the polymer/water system as a function of
temperature.
Fig. 2 Elastic modulus (G′) and viscous modulus (G″) of the
aqueous polymer solutions in the presence or absence of Col. The
concentration of the polymer solution was 25 wt % and the drug
loading amount was 1.0 mg/mL.
In vitro Col release
The in vitro release rate of Col was tested using three Col@Gel
systems loaded with various amounts of Col. The results are shown in
Fig. 3. All of the three Col@Gel systems exhibited a burst in the first
12 h and about 21 wt% of loaded Col was released, suggesting that
the loaded drug could be rapidly released and then delivered to the
adjacent lesions after local injection in vivo. Thereafter, Col continued
to be released over the next week, and more than 90 wt % of the loaded
drug was released within 8 days. The Col release profiles of the three
Col@Gel systems were similar, indicating a concentration-
independent of release dynamics.
In vivo degradation of the PLGA-PEG-PLGA hydrogel
To determine how the in vivo environment affected hydrogel
degradation, the 25 wt% PLGA-PEG-PLGA aqueous solution was
injected into the subcutaneous layer of back of C57BL/6 mouse,
which rapidly converted into a semi-solid hydrogel within 30 s at the
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administration site in response to the body heat. As displayed in Fig.

4A, the in situ-forming hydrogel showed an oval or flat shape and was
maintained for over 20 days, but its volume gradual decreased over
time. At predetermined time points, the remaining hydrogels were

Journal of Materials Chemistry B Accepted Manuscript

excised from the mice after euthanasia, and the mass was then
weighed. The mass percentage of remaining gel is shown in Fig. 4C.
This analysis showed a consistent decrease in mass as the time
prolonged, and only 20 % of the initial mass of the hydrogel was left
after 20 days of in vivo degradation. Meanwhile, the excised gels were
freeze-dried and then analyzed by GPC (Fig. 4D). The results revealed
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that the MWs of the recovered PLGA-PEG-PLGA polymers gradually

Fig. 3 Cumulative drug release curves of Col@Gel systems with
different amounts of Col. The results were presented as mean ± SD
(n=3). The photographs of the Col@Gel system containing 1
mg/mL Col were taken at preinstalled time points.
reduced, while the ĐMs continued to increase, confirming the in vivo
degradation feature of PLGA-PEG-PLGA polymers.
Moreover, the tissues around the hydrogels were collected and
subjected to HE staining to examine the tissue responses (Fig. 4B).
No severe inflammatory response was observed at the various time

Fig. 4 In vivo degradation and biocompatibility of the thermosensitive PLGA-PEG-PLGA hydrogel. A) In vivo hydrogel maintenance.
The pictures were shoot at various time points after dorsal subcutaneous injection. B) Optical micrographs of HE-stained slices of
surrounding tissues at designated time points after subcutaneous administration of hydrogel. M: muscle; G: hydrogel; S: skin; F: fibrous
capsule. C) The mass percentage of remaining gel excised from C57BL/6 mice measured by the gravimetric method. D) GPC traces of
residual PLGA-PEG-PLGA polymers collected from the excised gels during the in vivo degradation.

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DOI: 10.1039/C9TB02523E
Paper Journal of Materials Chemistry B

Journal of Materials Chemistry B Accepted Manuscript

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Fig. 5 A) The effect of Col@Gel on the proliferation of Raw264.7 macrophages in vitro after 48 h of incubation (n=3). The viability of
Raw264.7 cells that did not receive any treatment was defined as 100%. B) The effect of Col@Gel on the migration of Raw264.7 cells after 48
h of incubation (n=3). ***p<0.001. C) Representative micrographs of migrated Raw264.7 cells. Raw264.7 cells were stained purple using
crystal violet. The cells that received different treatments migrated from the upper surface of membrane of chamber to the lower surface of
membrane and then the lower surface of membrane was photographed after 48 h of incubation.
points. Apparently, the in vivo biodegradability and biocompatibility
of the PLGA-PEG-PLGA hydrogel supports the feasibility and safety
of clinical application as a long-acting drug release system.

Effects of the Col@Gel on macrophage proliferation and

migration in vitro
Macrophage is one of the most vital inflammatory cells involved in
MI.57, 58 To determine the cytotoxicity of blank hydrogel and
Col@Gel against macrophages, we first measured their effects on the
proliferation of Raw264.7 macrophages in vitro using the CCK-8
assay. As shown in Fig. 5A, the viability of Raw264.7 cells that
received the treatment of the blank hydrogel for 48 h did not suffer
from any influence, indicating that the hydrogel itself was
biocompatible, which did not interfere with the proliferation of
Raw264.7 cells. However, incubation with the Col@Gel containing
1.0 mg/mL Col for 48 h led to a remarkable decrease in the viability
of Raw264.7 cells.
Furthermore, the effect of the Col@Gel treatment on macrophage
migration was assessed using the Transwell assay. As presented in
Fig. 5B and 5C, the migration of Raw264.7 cells treated with the
Col@Gel system for 48 h was significantly inhibited compared with
the blank hydrogel-treated group. These outcomes affirmed that the
Col released from the gel depot could remarkably suppress the
proliferation and migration of Raw264.7 macrophages.
Fig. 6 The cumulative survival rate of mice that received the
different treatments post-MI.
Effects of the Col@Gel on a mouse model of MI
Post-operative survival
A mouse model of MI was constructed and then the mice received the
different treatments. Post-operative survival of mice was observed for
4 weeks and the results are shown in Fig. 6. None of the mice in the
Sham group died, whereas 7 mice in the MI group died (days 3, 9, 16,
20, 23, 23, and 26) and the overall survival rate was only 53.3%. The
survival rate of the Col@Gel group was markedly higher than that of
the MI group (86.7% vs 53.3%, p<0.05), and just 2 mice died at the
late stage of the experiment (days 20 and 26). The number of mice
that survived to day 28 in the Col (i.p.) group (73.3%) was also lower

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Fig. 7 The systemic toxicity of Col. A) Weight changes of mice receiving the indicated treatments at 4 weeks post-MI. B) Serum ALT
and AST levels of mice at 4 weeks post-MI. n=the survival mice number per group at 4 weeks post-MI, specially, nSham=15, nMI=8,
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nCol@Gel=13, nCol (i.p.)=11. *p<0.05, ***p<0.001.
Fig. 8 Myocardial inflammation of mice receiving the indicated
treatments at 4 weeks post-MI. A) Immunofluorescence-stained
myocardial tissues. F4/80-positive macrophages were stained
green with Alexa Fluor 488-labeled antibody, and cell nuclei
were counterstained blue with DAPI. B) Statistical analysis of the
abundance of F4/80-positive macrophages in the myocardium of
each group. n=the survival mice number per group at 4 weeks
post-MI, specially, nSham=15, nMI=8, nCol@Gel=13, nCol (i.p.)=11.
***p<0.001.
This journal is © The Royal Society of Chemistry 2019
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DOI: 10.1039/C9TB02523E
ARTICLE
Journal of Materials Chemistry B Accepted Manuscript
than that in the Col@Gel group, although the difference between them
did not reach statistical significance.
In vivo toxicity
The systemic toxicity of Col in vivo was assessed by recording the
changes in body weights and serum ALT and AST levels of mice. As
shown in Fig. 7A, the mice in the Col@Gel group gained a mean
weight increase of 3.42±0.5 g after MI for 4 weeks, which was not
significantly different from the gain in either the Sham group or the
MI group. The Col (i.p.) group gained significantly less weight than
the other three groups (2.95±0.4, p<0.05), illustrating the
intraperitoneal administration of Col solution caused the remarkably
systemic toxicity compared with the intramyocardially injected
Col@Gel. In a similar way, serum AST and ALT levels in the Col
(i.p.) group were significantly higher than those in the other three
groups (p<0.001), suggesting that the systemic exposure to Col
induced severe liver injury (Fig. 7B). It is worth noting that the slight
increases in AST and ALT levels in the MI and Col@Gel groups
compared with the Sham group may be attributed to the MI surgery
itself.
Myocardial inflammation
As macrophages are key players in MI-associated inflammation,
F4/80, a macrophage marker in myocardial tissue, was stained with
immunofluorescence. As shown in Fig. 8A, lots of macrophages were
detected in the myocardium of mice with MI compared with the Sham
group; however, the abundance of macrophages in mice treated with
Col@Gel after MI was significantly reduced. Fig. 8B further presents
the statistical results. The number of F4/80-positive cells per mm2 was
9±3 in the Sham group, 1031±133 in the MI group (p<0.001 vs Sham),
249±33 in the Col@Gel group (p<0.001 vs MI), and 448±59 in the
J. Name., 2019, 00, 1-3 | 9
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DOI: 10.1039/C9TB02523E
Paper Journal of Materials Chemistry B

Col (i.p.) group (p<0.001 vs MI). More importantly, the number of

macrophages in the myocardial sections of the Col@Gel-treated mice
was significantly lower than that in the Col (i.p.)-treated mice
(p<0.001).
To further confirm the aforementioned findings, we performed an

Journal of Materials Chemistry B Accepted Manuscript

additional experiment to measure the TNF-α content in myocardial
tissue extracts. TNF-α is produced by a variety of cells, including
neutrophils and macrophages, and it plays an important role not only
in MI-related inflammation response but also in myocardial apoptosis
and fibrosis.59, 60 As shown in Fig. 9, the TNF-α level in the MI group

Fig. 9 Changes of myocardial TNF-α levels of mice that contributed to the timely suppression of the overactive acute
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received different treatments after MI (n = 3 per time point). inflammatory response as well as the subsequent chronic
inflammation in the infarct region. In contrast, the anti-inflammation
effect was much weaker after a single intraperitoneal injection of Col
solution.

Myocardial fibrosis
peaked at 1 week post-MI and gradually declined thereafter. Both the
Col@Gel and Col (i.p.) treatments significantly reduced the
myocardial TNF-α content at each time point examined, and the use
of Col@Gel was far more effective in suppressing TNF-α production
than that of Col (i.p.) (p<0.05). Combined with the results presented
in Fig. 3, we believed that the sustained release of Col for up to 1 week
To detect MI-induced myocardial fibrosis, myocardial tissues were
stained with Masson’s trichrome to reveal collagen fibers. As
displayed in Fig. 10A, the fibrosis degree of myocardial tissue in the
infarction zone of mice in the MI group was significantly higher than
that in the Sham group, while that was significantly reduced in the
mice treated with Col@Gel. Specifically, the proportion of total tissue
area with evidence of fibrosis was 1.03±0.07% in the Sham group,
24.83±3.55% in the MI group (p<0.001 vs Sham), 11.53±2.50% in the
Col@Gel group (p<0.001 vs MI), and 19.47±2.93% in the Col (i.p.)
group (p<0.001 vs MI group) (Fig. 10B). Also, the fibrosis area of
myocardial tissue in the Col@Gel group was significantly lower than
that in the Col (i.p.) group, indicating that the intramyocardial
injection of Col@Gel and the subsequent sustained release of Col was
more effective in reducing myocardial fibrosis post-MI than the

Fig. 10 The myocardial fibrosis degree of mice that received intraperitoneal injection of Col solution.

different treatments at 4 weeks post-MI. A) Masson’s trichrome-

stained myocardial tissues. B) Statistical analysis of the
Myocardial apoptosis
myocardial fibrosis area of each group. n=the survival mice
Myocardial apoptosis caused by LAD ligation was assessed by the
number per group at 4 weeks post-MI, specially, nSham=15,
TUNEL assay. This analysis revealed that the MI operation
nMI=8, nCol@Gel=13, nCol (i.p.)=11. ***p <0.001.
significantly caused myocardial apoptosis in the MI group compared

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Fig. 11 The myocardial apoptosis of mice that received different
treatments at 4 weeks post-MI. A) TUNEL-stained myocardial
tissues. Apoptotic cell nuclei in the myocardium were stained
green (TUNEL-positive). The total cardiomyocyte nuclei were
stained blue with DAPI. B) Statistical analysis of the percentage of
apoptotic cardiomyocytes (TUNEL-positive cells) of each group.
n=the survival mice number per group at 4 weeks post-MI,
specially, nSham=15, nMI=8, nCol@Gel=13, nCol (i.p.)=11. ***p <0.001
with the Sham group, but here we observed that the Col@Gel
treatment significantly alleviated apoptosis of cardiomyocytes (Fig.
11A). As shown in Fig. 11B, the percentage of apoptotic
cardiomyocytes was 0.25±0.06% in the Sham group, 12.93±2.00% in
the MI group (p<0.001 vs Sham), 4.44±1.08% in the Col@Gel group
(p<0.001 vs MI), and 9.04±1.44% in the Col (i.p.) group (p<0.001 vs
MI and Col@Gel). These findings mirrored the analysis of tissue
fibrosis, as presented in Fig. 10, and revealed that treatment with
Col@Gel provided more benefits in inhibiting apoptosis of
cardiomyocytes after MI than Col by intraperitoneal administration.
Cardiac structure and function
Changes in cardiac structure and function following MI induction
were assessed by echocardiography. Fig. 12A shows representative
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DOI: 10.1039/C9TB02523E
ARTICLE
Journal of Materials Chemistry B Accepted Manuscript
Fig. 12 The cardiac structure and function of mice that received
different treatments at 4 weeks post-MI. A) Representative
echocardiographic images. B) Statistical analysis of the
echocardiographic parameters of each group. n=the survival mice
number per group at 4 weeks post-MI, specially, nSham=15, nMI=8,
nCol@Gel=13, nCol (i.p.)=11. *p<0.05, ***p<0.001.
echocardiographic images and Fig. 12B displays the statistical
outcomes. In the MI group, EF and FS were significantly decreased
(p<0.001), while LVIDs and LVIDd were significantly increased
(p<0.001) compared with the Sham group, indicating severe MI-
induced structural and functional damage to the myocardium. In the
Col@Gel group, all of these echocardiographic parameters were
significantly improved compared with the MI group (p<0.05 or
p<0.001), whereas only EF and FS were improved in the Col (i.p.)
group (p<0.05 or p<0.001). Apparently, the Col@Gel treatment post-
MI had a strikingly beneficial effect on myocardial structure and
function, whereas the response to Col (i.p.) treatment was much
weaker.
Heart failure biomarker
J. Name., 2019, 00, 1-3 | 11
 Journal of Materials Chemistry B
Paper
Finally, serum concentrations of BNP, a biomarker of congestive
heart failure, were measured at 4 weeks post-MI (Fig. 13). Compared
with the Sham group, the MI group showed a significantly higher BNP
level (p<0.001), as expected. However, the serum BNP levels were
significantly lower in both the Col@Gel- and Col (i.p.)-treated groups
compared with the MI group (p<0.001). It was exciting that the BNP
level in the Col@Gel group was much lower than that in the Col (i.p.)
group (p<0.001). This finding further confirmed the superiority of a
locally deposited Col@Gel system in preventing heart failure
compared with a systemically administered Col solution.
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Fig. 13 The serum BNP concentrations of mice that received
different treatments at 4 weeks post-operation. n=the survival mice
number per group at 4 weeks post-MI, specially, nSham=15, nMI=8,
nCol@Gel=13, nCol (i.p.)=11. ***p <0.001.
Discussion
Overactive, persistent tissue inflammatory response post-MI, which
not only disturbs tissue repair but also induces myocardial apoptosis,
necrosis, fibrosis and leads to ventricular remodeling and cardiac
insufficiency, is a major cause of myocardial injury.2-4 Therefore, the
timely suppression of excess early myocardial inflammatory response
is in favor of promoting and accelerating myocardial repair of patients
after MI.2, 3, 61, 62 For example, some clinic studies have demonstrated
that administration of anti-inflammatory drugs, such as canakinumab,
an anti-IL-1β monoclonal antibody, has beneficial effects on the
prognosis of MI patients.2, 63 However, the studies on the anti-
inflammatory remedy for patients after MI are still limited compared
with the other therapies, such as antiplatelet and lipid-lowering
treatments.
Col is a mitotic spindle poison utilized for more than 3000 years and
has pleiotropic anti-inflammatory properties, but the widespread
clinical use is precluded because of its severe toxicity profile and
narrow therapeutic window.7, 10, 11 Recently, Fujisue and Akodad
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Journal of Materials Chemistry B
confirmed that oral or intraperitoneal administration of Col had anti-
inflammatory and anti-fibrotic effects on experimentally induced MI
in mice, but their findings on improvement of cardiac function were
inconsistent.8, 16 Moreover, they were unable to draw a clear
conclusion about the systemic toxicity of Col in their studies.
Journal of Materials Chemistry B Accepted Manuscript
Considering that its systemic toxicity is the greatest obstacle for Col
use in clinic, we speculated that localized administration of Col maybe
provide better therapeutic efficacy while significantly alleviating the
side effects. With the development of echocardiographic and
thoracoscopic technologies, intramyocardial administration of anti-
inflammatory drugs has become a potentially feasible strategy for
post-MI treatment.
In the current work, we constructed an injectable drug delivery
system, enabling the localized, sustained release of Col in the
myocardium. First, we prepared a thermosensitive hydrogel
composed of PLGA-PEG-PLGA polymers that was an injectable
liquid at room temperature and converted into a non-flowing gel
within 30 s when exposed to body temperature. Col was readily,
homogenously dispersed in the aqueous polymer solution by blending
them at low or ambient temperature. The loading of Col did not
influence the injectability and temperature-induced gelation of
polymer/water system (Fig. 2). The acute stage in the first 7 days after
an MI is the crucial period for myocardial repair as well as the peak
period of myocardial necrosis, apoptosis, and inflammation
response.64, 65 The in vitro release of Col from the Col@Gel system
exhibited an initial burst followed by a sustained release pattern for
over 8 days (Fig. 3), suggesting that the liberated Col could rapidly
exert the anti-inflammatory effect upon injection as well as
throughout the follow-up crucial acute stage post-MI. This feature was
well confirmed by sustained, effective inhibition of the expression of
the inflammatory cytokine TNF-α following the intramyocardial
administration of the Col@Gel system post-MI (Fig. 9).
The in vitro cell tests showed that the Col released from the Col@Gel
effectively inhibited the proliferation and migration of macrophages
(Fig. 5), which make vital negative contributions to cardiac
remodeling post-MI.57, 58 On the basis of demonstrating the in vivo
biodegradability and biocompatibility of PLGA-PEG-PLGA hydrogel
itself (Fig. 4), the effect of Col@Gel system on myocardial repair of
mice after infarction was evaluated. We found that a single
intramyocardial administration of the Col@Gel post-MI and the
follow-up sustained release of Col significantly reduced the
accumulation of macrophages and inhibited myocardial apoptosis and
fibrosis in the infarct region, lowered the expression of the heart
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Journal Name
failure biomarker BNP, improved the cardiac structure and function,
and prolonged the survival of mice (Figs. 6, 8, 10-13). Apparently,
intramyocardial injection of the Col@Gel imparted multiple benefits
at the most critical stage of treatment after MI.
Furthermore, we compared the safety and efficacy of Col
administered by intramyocardial injection and intraperitoneal
injection. On the one hand, we found that a single intraperitoneal
injection of the Col solution has some beneficial effects on anti-
inflammation and improvement of cardiac function, which was
consistent with the Fujisue’s study.16 However, the effects were less
striking than those obtained from intramyocardial injection of the
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Col@Gel, possibly because of poor local uptake and accumulation of
Col at the infarct region after a single systemic administration. On the
other hand, a single intraperitoneal injection of the Col solution still
caused systemic toxicity, such as liver damage and reduced weight
gain (Fig. 7), that were negligible in the mice treated with Col@Gel.
In addition, the survival rate of mice in the Col (i.p.) group after MI
was worse compared with the Col@Gel group, which also reflected
either inferior myocardial efficacy or systemic toxicity. In contrast, a
single intramyocardial administration of the Col@Gel and the follow-
up continuous liberation of Col ensured the high local drug
concentration in the myocardium while minimizing the systemic
toxicity, both of which are beneficial to the patients post-MI.
Consequently, the intramyocardial injection of Col@Gel has great
potential for myocardial repair after MI.
It is worth noting that some synthetic or natural derived hydrogels,
such as poly(NIPAAm-co-HEMA-co-MAPLA) hydrogel or
hyaluronic acid-based hydrogel, without drug loading could also
improve the heart function post-MI by intramyocardial injection.34, 35,
66
These hydrogels presented high gel moduli, and the researchers
believed that their intramyocardial administration enhanced the
mechanical stiffness of infarct zone, thereby attenuating left
ventricular remodeling and improving cardiac function. Nevertheless,
our PLGA-PEG-PLGA hydrogel is much softer, whether such a soft
hydrogel alone could enhance the mechanical stiffness of ventricular
wall remains to be explored in future.
Conclusions
Herein, we successfully developed an injectable PLGA-PEG-PLGA
hydrogel for the intramyocardial sustained delivery of Col to treat MI.
The prepared Col@Gel system maintained the temperature-
responsive gelling feature, and the release of Col from the Col@Gel
exhibited a sustained manner over an 8-day period. In vitro and in vivo
This journal is © The Royal Society of Chemistry 2019
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DOI: 10.1039/C9TB02523E
ARTICLE
tests confirmed the safety and efficacy of Col@Gel for the treatment
of MI after intramyocardial injection were significantly superior to
those of intraperitoneal injection of Col solution, including lower
myocardial inflammation, apoptosis, and fibrosis, better heart
function and structure, higher animal survival and negligible systemic
Journal of Materials Chemistry B Accepted Manuscript
side effects. Overall, the local sustained delivery of Col offers a new
perspective on the old drug in new use and the Col@Gel system has
great potential for promoting tissue repair after MI.
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgements
This work was supported by the Natural Science Foundation of China
(grant nos. 81603327, 81670321, 51773043 and 51533002), China
Postdoctoral Science Foundation (grant no. 2018M632144), the
Research Fund of Shanghai Jiao Tong University Affiliated Sixth
People’s Hospital (No. 0004) and National Key R&D Program of
China (grant no. 2016YFC1100300).
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 Page 15 of 15 Journal of Materials Chemistry B

For Table of Contents use only View Article Online

DOI: 10.1039/C9TB02523E

Graphical and textual abstract for TOC

Journal of Materials Chemistry B Accepted Manuscript

An injectable thermosensitive hydrogel loaded with an

ancient natural drug colchicine for myocardial repair

after infarction

Yu Chen‡, Jiayue Shi‡, Yaping Zhang, Jiajun Miao, Zhe Zhao, Xian Jin, Liang Liu, Lin
Published on 31 December 2019. Downloaded on 1/3/2020 1:26:29 AM.

Yu*, Chengxing Shen*, Jiandong Ding

The intramyocardial injection of colchicine-loaded hydrogel system effectively

promoted myocardial repair after infarction while minimizing the systemic toxicity of

colchicine.