A reactive oxygen species (ROS)-responsive low molecular weight gel co-loaded with doxorubicin and Zn(II) phthalocyanine tetrasulfonic acid for combined chemo-photodynamic therapy

Article abstract of DOI:10.1039/c7tb02359f

Low molecular weight gels (LMWGs) have significant advantages in drug delivery such as high drug loading capacity, in situ delivery of drug to the lesion site, sustaining drug release with excellent bioavailability, and minimized side effects. Here, we sy

Full text of DOI:10.1039/c7tb02359f

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ISSN 2050-750X
PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
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A reactive oxygen species (ROS)-responsive low molecular weight
gel co-loaded with doxorubicin and Zn(II) phthalocyanine
tetrasulfonic acid for combined chemo-photodynamic therapy
Received 00th January 20xx,
Accepted 00th January 20xx
Long Xua,b, Mingying Zhaoc, Yidi Yanga, Yan Liangd, Changzhen Suna, Wenxia Gao*b, Sai Lic, Bin Hea,
and Yuji Pu*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Low molecular weight gels (LMWGs) have significant advantages in drug delivery such as high drug loading capacity, in situ
delivery of drug to the lesion site, sustaining drug release with excellent bioavailability, and minimized side effects. Here,
we synthesized a reactive oxygen species (ROS)-responsive gelator to prepare an injectable gel. Anticancer drug
doxorubicin hydrochloride (DOX) and photosensitizer Zn(II) phthalocyanine tetrasulfonic acid (ZnPCS4) were loaded in the
gel for combined chemo-photodynamic therapy. The ROS-responsive gelator was characterized by proton nuclear
magnetic resonance (1H NMR) and the morphology of gels were investigated by scanning electron microscopy (SEM). The
rheological properties and destroy-recovery capability of both blank and drug-loaded gels were studied. The cytotoxicity of
LMWGs against 3T3 fibroblasts and 4T1 breast cancer cells were tested. The in vitro drug release of both drugs were
studied and the in vivo anticancer activities of DOX-ZnPCS4-coloaded LMWGs were investigated in tumor-bearing mice. The
results revealed that the injectable DOX-ZnPCS4-coloaded LMWGs had excellent anti-tumor efficacy and played a
coordinated treatment efficacy.
gels to realize localized drug delivery by intratumor
injection.10,12−14 Specifically, low molecular weight gels
(LMWGs) have aroused extensive interest in various
Introduction
Chemotherapy, a very important cancer therapy mode, has
been widely used in the clinical treatment of various cancers.1,2
Small-molecule chemotherapeutics has obvious limits, such as
systemic toxicity due to lack of specific selectivity to
distinguish healthy and cancer cell sand short blood circulation
because of rapid clearance by endothelial reticular system
(RES).3 Nanoparticulate drug delivery systems (NDDSs) have
been developed to prolong the blood circulation and reduce
side effects by affecting the bio-distribution and tuning the
drug release behaviours.4−7 NDDS is generally administrated by
intravenous injection that may lead to drug leakage during
long blood circulation and toxicity to normal organs.8,9 The
localized drug delivery strategy provides an alternative way for
cancer therapy, which directly delivers the chemotherapeutics
to the lesion site and thus minimized toxicity to other
organs.10,11 One approach is to exploit drug-loaded injectable
biomedical applications such as drug delivery, 3D-cell culture,
enzyme immobilization, and induction of mesenchymal
stromal cells (MSC) differentiation.15-20 LMWGs, as an
anticancer drug delivery system, have great advantages and
application prospects: (1) They are biocompatible, non-toxic,
and biodegradable due to low toxicity of low molecular weight
gelators; (2) They display high drug loading contents; (3) Drug-
loaded LMWGs could be administrated by intratumor
injection, which directly delivers chemotherapeutic drugs to
tumor sites and thus lowers the toxicity to normal organs; (4)
Most importantly, the drug-loaded gels, not limited to LMWGs,
can sustain drug release by stimuli-responsive degradation and
maintain an effective therapeutic concentration.17,21−23 Many
research groups had employed LMWGs for efficient tumor
inhibition. For example, Barthélémy et al. investigated the
influence of cation on hydrogel and provided a paradigm for in
vivo sustained drug delivery.24 Our group developed
a
pheneylboronic acid-based LMWG for efficient local
chemotherapy by loading anticancer drug doxorubicin.25,26
Different from chemotherapy, photodynamic therapy (PDT),
an externally available treatment modality for tumor
treatment, employs singlet oxygen (SO) or reactive oxygen
species (ROS) generated by photosensitizers (PS) under light
irradiation to kill cancer cells.27,28 The lesion is selectively
illuminated with light after administration of PS and cytotoxic
ROS are generated, leading to cell death and tissue
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destruction. Porphyrin and its derivates have been widely used morphology of gel. The rheological property was studied on a
as PS for PDT.29,30 Specifically, nanoparticle-based PDT has rheometer (Discovery DHR-2, TA, American).
been developed and shown encouraging results in vitro and in
vivo.31 Compared with normal tissues, tumor tissue has a more
Synthesis of the ROS-responsive gelator
acidic microenvironment and higher concentration of ROS.32−34
The synthetic scheme of ROS-responsive gelator is shown in
Scheme 1.
Many ROS-responsive drug delivery systems, mainly containing
Synthesis of ROS-cleavable neotype thioketal (TK). A mixture
ROS-cleavable thioketal and thioether bonds, have shown
good anticancer effect both in vitro and in vivo.35,36
of 3-mercaptopropionic acid (11.44 g, 108.02 mmol) and p-
hydroxybenzaldehyde (6.0 g, 49.1 mmol) were dissolved in
In this study, a LWMG with ROS-responsive property was
ethyl acetate. Catalytic amount of trifluoroacetic acid were
designed for combined local chemo-photodynamic therapy. A
added and the solution was stirred at room temperature (RT).
low molecular weight gelator containing thioketal was
The reaction was monitored by thin layer chromatography.
designed and synthesized (Scheme 1), and the resulting gel
When p-hydroxybenzaldehyde was exhausted, the solvents
was used for loading chemotherapeutics doxorubicin
were removed and the crude product was washed by CH2Cl2
hydrochloride (DOX) and photosensitizer Zn(II) phthalocyanine
and cold water for several times. White product was obtained
tetrasulfonic acid (ZnPCS4) to form DOX-ZnPCS4-coloaded
after drying.
LWMGs. A control gelator without thioketal bond was also
Synthesis of Phe-TK-Phe. L-Phenylalanine methyl ester
designed and its blank gel and drug-loaded gels were used as
hydrochloride (4.48 g, 22 mmol) was dissolved in dry CH2Cl2
control (Scheme 1). In our hypothesis, after intratumor
(50 mL). DIEA (2.84 g, 22 mmol) was added dropwise to the
injection of the DOX-ZnPCS4-coloaded LWMG, ROS generated
solution at an ice bath and stirred for 10 min. In a separate
by ZnPCS4 under light irradiation would accelerate the
flask,
3,3'-(((4-
acid
degradation of ROS-responsive LWMG, releasing DOX and
ZnPCS4 for combined chemotherapy and photodynamic
therapy (Scheme 2). This dual modal therapy is expected to
display synergistic effect for cancer inhibition.37 The in vitro
ROS-responsive, rheological property, and drug release of
these gels were investigated. The in vitro cytotoxicity of gels
against mouse fibroblasts NIH/3T3 and 4T1 breast cancer cells
was tested. The in vivo antitumor efficiencies of these gels
were studied.
hydroxyphenyl)methylene)bis(sulfanediyl))dipropanoic
(3.16 g, 10 mmol) and CDI (3.57 g, 22 mmol) were dissolved in
30 mL dry CH2Cl2 and the solution was stirred at RT for 30 min.
The activated acid solution was added dropwise to the mixture
of -phenylalanine methyl ester hydrochloride at an ice bath
L
followed by stirring under N2 atmosphere for 24 h. The mixed
solution was washed with 1 M HCl solution, saturated NaHCO3
solution and NaCl solution, and dried with anhydrous sodium
sulfate. The solvents were removed by rotary evaporation and
purified by column chromatography (petroleum ether
and ethyl acetate, v/v, 1:2) to obtain the product.
Experimental
Synthesis of ROS-responsive and control gelator. A mixture of
Phe-TK-Phe (3.19 g, 5 mmol) and hydrazine hydrate (2 mL) was
dissolved in CH2Cl2 and methanol (40mL, v/v, 1:1). The mixture
was then stirred under N2 atmosphere for 24 h. The mixture
was filtrated and washed with dichloromethane for several
times. The gelator was obtained after vacuum drying. The
control gelator was synthesized similarly (Scheme 1).
Materials
All chemical reagents used in this study were commercially
available and chemically pure. L-Phenylalanine methyl ester
hydrochloride, 1,1'-carbonyldiimidazole (CDI), sebacoyl
chloride, and thiazolyl blue (MTT) were purchased from the
Saen chemical technology (Shanghai) Co. Ltd. 3-
Mercaptopropionic acid was purchased from Aladdin Bio-
Gel preparation
Chem
technology
(Shanghai)
Co.
Ltd.
N,
N-
Diisopropylethylamine (DIEA) was purchased from Asta Tech
(Chengdu) Biopharm. Co. Ltd. Zn(II) Phthalocyanine
tetrasulfonic acid (ZnPCS4) was purchased from J&K Scientific
Ltd. Doxorubicin hydrochloride (DOX) was purchased from
Zhejiang Hisun Pharmaceutical. Poly(ethylene glycol) (PEG, Mw
= 200 g/mol), p-hydroxybenzaldehyde, hydrazine hydrate
(80%), and all solvents were purchased from Chengdu Kelong
Chemical Co. (China) without further purification prior to use.
Dulbecco's modified Eagle's medium (DMEM), 100 × mycillin,
and fetal bovine serum (FBS) were purchased from Gibco Inc.
Characterizations
A given mass of gelator in 1 mL of PEG200 and H2O (1:1, v/v)
was placed in a flask fitted with a reflux condenser and heated
until complete dissolution of the gelator. When cooled to RT,
the solution was transferred into a closed glass vial. The gel
was obtained by cooling to RT to gelate the solvent. The critical
gelation concentration (CGC) was determined by measuring
the minimum amount of gelator required for the formation of
a stable gel at 37 ºC. The SEM images were obtained from gels
prepared in ethanol, which were dried under reduced pressure
by oil pump at RT.
Drug-loaded gel was prepared by dissolving the payload
(DOX and/or ZnPCS4) and gelator in PEG200 and H2O (1:1, v/v)
under heat and then cooling the solution to RT to form DOX-
loaded, ZnPCS4-loaded, and DOX-ZnPCS4-coloaded gels. The
concentration of gelator and payload was 30 and 2 mg/mL,
respectively. The blank, DOX-loaded, ZnPCS4-loaded, and DOX-
All the compounds were characterized by proton nuclear magnetic
resonance (1H NMR), which were performed on a Bruker Avance II
NMR spectrometer at 400 MHz using tetramethylsilane as the
internal standard. Scanning electron microscope (SEM, S4800,
Hitachi Ltd, Tokyo, Japan) was employed to observe the
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ZnPCS4-coloaded gels were abbreviated as G1, G2, G3, and G4. 315 nm and emission wavelength at 560 and 355 nm,
The drug-loaded control gel was prepared similarly, except the respectively. The release experiments were conducted in three
concentration of control gelator was 10 mg/mL. The blank, parallel experiments.
DOX-loaded, ZnPCS4-loaded, and DOX-ZnPCS4-coloaded control In vivo antitumor activity
gels were abbreviated as G1*, G2*, G3*, and G4*.
All animal experiments were performed in compliance with
I
n vitro degradation of gelator under ROS condition
the Animal Management Rules of the Ministry of Health of the
The degradation of gelator under ROS condition was People's Republic of China (Document no. 55, 2001) and the
1
investigated by H NMR. Gelator (10 mg) was incubated in 4 institutional guidelines. The experiments were approved by
mL of fresh aqueous solution with 400 mM H2O2 and 3.2 μM Sichuan University Animal Care and Use Committee. Male
CuCl2 and 2 mL acetone for 48 h. The solution was lyophilized BALB/c mice (body weight: 18−22 g) were purchased from
and the 1H NMR spectrum of the obtained mixture was Dashuo (Chengdu) Biotechnology Co. Ltd. The tumor-bearing
studied.
mice model was established by inoculated the 4T1 breast
cancer cells into right flank subcutaneously of BALB/c mice.
After the inoculated tumor volume reached 100−200 mm3, the
mice were randomly divided into 11 groups. Six groups were
treated with the formulations of G1, G3, G4, G1*, G3*, and G4*.
All these groups were irradiated for 5 min with a 660 nm (100
Rheological measurements
In order to investigate the viscoelastic properties of the blank
and drug-loaded gels, the storage modulus (G′), loss modulus
(G″), and dynamic complex viscosity (η*) of gels were
measured.18,38−40 The angular frequency (
ω) applied to the gel
and drug-loaded gel samples increased from 0.1 to 100 rad/s
with a strain of 1.0%. In order to obtain more information of
the gel network structure, the dynamic strain sweep and
dynamic time sweep was carried out. The dynamic strain
sweep was used to destroy the gel network structure, then the
dynamic time sweep was used to study the recovery
properties of the gel. In details, after the gel was entirely
formed, the strain was applied to the gel with an increase from
w) laser after 30 min of intratumor injection (L
groups were treated with the formulations of G2, G3, G2*, G3*,
and saline without irradiation (L ). All groups were injected for
+). The other
−
four times at a 3-day interval with a drug dose of 5 mg/kg. The
body weights of mice and the tumor volumes were measured
at a 3-day interval. The behaviours of mice were determined
by the animal healthcare technicians. Tumor volumes were
calculated using the formula V= (a
× (b × b))/2, with a the
largest and b the smallest diameters. All the dates of tumor
volumes and body weight were expressed as relative values,
compared to the tumor volume and body weight determined
on the first day of therapy. After the experiment, all of the
animals were euthanized and sacrificed, and the heart, liver,
spleen, lung, kidney, and tumor were separated and washed
with PBS, and fixed by 4% formaldehyde. Subsequently, all the
organs and tumor were dealed with the histopathological
procedures. Hematoxylin and eosin (H&E) staining and the
terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling (TUNEL) assay were taken to evaluate the acute
toxicity and the apoptosis of cancer cells.
0.01 to 50% (10 rad/s and 25 ºC), then the dynamic time sweep
was carried out with the strain 1% (1 rad/s and 25 ºC).
Cytotoxicity test
Mouse fibroblasts NIH/3T3 and 4T1 breast cancer cells were
used to evaluate the biocompatibility and toxicity of the gels.
The cytotoxicity of gel was carried out by testing the
cytotoxicity of gelator according to International Organization
for Standardization (ISO) tests (ISO 10993-5).25 The gelators
with different concentration (0.5, 1, 5, 10, 20, and 30 mg/mL)
were added to complete culture medium at 37 ºC and
incubated for 24 h, then extracts were filtered and added to
NIH/3T3 cells and 4T1 breast cancer cells for additional 48 h.
The culture medium was removed and the wells were washed
with PBS (pH7.4). MTT in blank culture medium was added to
each well. Subsequently, the cells were incubated for
additional 3 h. The culture medium was removed and DMSO
(100 μL) was added into each well. The cell viability was
determined by measuring the absorption at 490 nm using a
microplate reader (Thermo Scientific MK3).
Results and discussion
Synthesis and characterization of gelator
In vitro drug release
The DOX-ZnPCS4-coloaded gel and control gel were prepared
according to the aforementioned method. Subsequently, 1 mL
drug-coloaded gel in 4 mL threaded bottle was transferred into
a threaded bottle with 25 mL PBS buffer solutions. Hydrogen
peroxide (H2O2, 500 mM) was used to provide ROS.41,42 The
threaded bottle was incubated in a constant temperature
water bath oscillator at 37 ºC immediately. At a predetermined
time interval, 1 mL PBS was taken out and 1 mL fresh PBS was
added. The released DOX and ZnPCS4 were determined by a
fluorescence detector with excitation wavelength at 480 and
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the disappearance of the characteristic peak of −OCH3 (δ 3.70
ppm, Fig. S1C). The control gelator was similarly synthesized
and characterized by 1H NMR in Fig. S2.
OH
ROS
(A)
SH
b'
S
S
a
a
(B)
b'
10
9
8
7
6
5
4
3
2
1
0
Chemical shift (ppm)
Fig. 1. 1H NMR spectra of gelator before (A) and after (B)
treatment of ROS stimulus where Fenton reagent was used to
mimic ROS conditions in CDCl3 and DMSO-d6, respectively. The
inset showed the chemical change of thioketal to thiol in the
presence of ROS.
Scheme 1. The synthetic scheme of TK (A), thioketal containing
and control gelators (B).
In order to verify the ROS-responsiveness of the thioketal
containing gelator, the gelator was incubated with fresh
Fenton reagent that mimics the ROS condition. The obtained
1
product was characterized by H NMR.43 After the incubation
in ROS condition, the thioketal characteristic peak (δ = 5.03
ppm, peak a in Fig. 1A) of gelator disappeared and a new peak
at 1.38 ppm (peak b in Fig. 1B) emerged, which was ascribed to
the proton of thiol group, suggesting the ROS-responsiveness
of thioketal containing gelator. However, no change was
observed in the 1H NMR spectra of control gel before and after
treatment of ROS (Fig. S3), suggesting no response to ROS.
Gel characterizations
Scheme 2. (A) Chemical structures of thioketal containing
gelator, DOX, and ZnPCS4. (B) The illustration of ROS-
responsive degradation of DOX-ZnPCS4-coloaded gel and the
release of DOX and ZnPCS4 for combined chemo-
photodynamic therapy.
Fig. 2. The SEM images of xerogel prepared in ethanol with a
gelator concentration of 15 and 20 mg/mL; the bar was 500
nm.
The thioketal containing and control gelators were synthesized
(Scheme 1) and characterized by 1H NMR. For the ROS-
responsive
gelator,
the
3,3'-(p-hydroxybenzylidene)
The critical gel concentrations (CGCs) of the two gelators were
tested in different solvents at 37 ºC; the results were shown in
Table S1. Both ROS-responsive gelator and control gelator can
facilely form gels in ethanol or methanol with a CGC of 10
mg/mL. The microtopography of ROS-responsive xerogel
prepared in ethanol was characterized by SEM and typical
dithiodipropionic acid (TK) was successfully obtained through
the aldolization, which has a methyne characteristic peak
appeared at δ 5.15 = ppm (Fig. S1A). The L-phenylalanine
methyl ester hydrochloride was conjugated to TK through
amide condensation reaction, followed by a reaction with
hydrazine hydrate to obtain thioketal containing gelator. The network structure was observed (Fig. 2).
successful synthesis of ROS-responsive gelator was proved by
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function of angular frequency of the gels; (C) dynamic complex
viscosity as a function of angular frequency for the gels; (D) destroy
and recovery of the gels.
The viscoelastic properties of these gels (G1−G4) were
investigated. All the samples used for rheological test were real
gels, which were confirmed by the fact that the storage modulus
(G′) of gels was higher than their corresponding loss modulus (G″)
(Fig. 4A and 4B). The dynamic modulus decreased with the order of
G3 > G1 > G2 > G4, implying that G3 (ZnPCS4-loaded gel) and G4
(DOX-ZnPCS4-coloaded gel) had the biggest and smallest mechanical
strength, respectively. The enhanced mechanical strength of G3
might be ascribed to the supramolecular interactions of ZnPCS4 and
gelator, where aromatic structures and opposite charges (-NHNH2
and -SO3H) might induce π-π interactions and electrostatic
interactions, respectively. Although DOX has an aromatic structure,
the amine group in DOX might result in electrostatic repulsion
between DOX and gelator, reducing the mechanical strength. The
G4, however, showed the lowest mechanical strength, which might
be due to its higher drug loading content (the total drug loading
contents for G2, G3, and G4 were 6.25%, 6.25%, and 12.5%,
respectively) that somehow disrupt the tacticity and lead to more
disorder of gel network, resulting lower mechanical strength. The
frequency dependence of dynamic complex viscosity (η*) indicated
that all the gels had a shear-thinning behaviour (Fig. 4C). As the
dynamic strain and time sweep results (Fig. 4D) showed, the
network structure of gel was destroyed after increasing strain and
recovered after the removal of the strain. This result was owing to
the intrinsic dynamic and reversible supramolecular interactions
between gelators, which could respond to the external strain.44,45
The shear-thinning property and excellent destroy-recover ability
make supramolecular drug-loaded gels potential candidates for
intratumor injection.46 The rheological data of control gel and its
drug-loaded gel were also tested and similar rheological results
were observed (Fig. S4).
Fig. 3. The images of various ROS-responsive gels (top row)
and tumors (down row) treated with these gels. Gels: blank gel
(A, a); DOX-loaded gel (B, b); ZnPCS4-loaded gel (C, c); and
DOX-ZnPCS4-coloaded gel (D, d).
Besides, both gelators can form gels in PEG200-H2O
(5:1−1:1, v/v) mixed solution (Table S1). The biocompatible
and nontoxic PEG200 was added to deionized water to
improve the solubility of gelator and facilitate gel formation.18,
25
The thiolketal containing gelator showed a higher CGC in
PEG200-H2O of 30 mg/mL than that of control gelator (5
mg/mL). Moreover, the gel was demonstrated to have an
excellent capability to load anticancer drug doxorubicin
hydrochloride (DOX) and photosensitizer Zn(II) phthalocyanine
tetrasulfonic acid (ZnPCS4) alone or both (structures of DOX
and ZnPCS4 were shown in Scheme 2A). The drug loading
contents of both DOX and ZnPCS4 were 6.25% in these gels.
The typical blank gel (G1), DOX-loaded (G2), ZnPCS4-loaded
(G3), and DOX-ZnPCS4-coloaded gels (G4) formed by thiolketal
containing gelator were shown in Fig. 3A−3D. For the
dispersion form of drug molecules, when the drug molecules
were dissolved in aqueous solution, the crystal structure was
destroyed to be soluble in the solvent, and no crystallization
was observed in the process of drug-loaded gel formation or
during the storage period. Therefore, DOX and ZnPCS4 were
moleculalry dispersed in the gel network and crystal was hard
to form because of dispersion in the gel network. Control gels
were prepared similarly and the drug loading contents of DOX
and ZnPCS4 were 9.09%.
Cytotoxicity
Fig. 5. The cytotoxicity of ROS-responsive and control gelators
incubated with NIH/3T3 fibroblasts (A) and 4T1 breast cancer cells
(B).
The biocompatibility of ROS-responsive gels and control gels was
evaluated by MTT assay using NIH/3T3 fibroblasts and 4T1 breast
cancer cells. As shown in Fig. 5B, the cell viabilities of 4T1 cells were
higher than 90% for both gelator extracts even at a high gelator
concentration of 30 mg/mL. Neglectable cytotoxicity against
NIH/3T3 fibroblasts cells was observed when gelator concentration
Fig. 4. The rheological properties of ROS-responsive blank gel and
drug-loaded gels: storage modulus (A) and loss modulus (B) as a
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was no more than 20 mg/mL (Fig. 5A). These results showed that
the gels showed low cytotoxicity and good biocompatibility.
In vitro drug release
The in vitro release of DOX-ZnPCS4-coloaded gel and DOX-ZnPCS4-
coloaded control gel in PBS (pH 7.4), PBS with 500 mM H2O2, and
PBS with 500 mM H2O2 and irradiation was studied. Hydrogen
peroxide (H2O2) was used to provide ROS. We found that the
fluorescence intensity of ZnPCS4 was not proportional to the
concentration of the ZnPCS4 (Fig. S5) even though the concentration
was as low as 1.22 × 10−4 ꢀg/mL, and fluorescence quenches at a
high concentration of ZnPCS4. Therefore, we cannot detect the
amount of released ZnPCS4 by fluorescence spectroscopy. Although
the quantitive release was hard to obtain, we can qualitively
evaluate the release of the ZnPCS4 by its color (Fig. S6). The green
color of ZnPCS4 in the release medium of DOX-ZnPCS4-coloaded gel
treated with ROS condition (Fig. S6e) was obviously darker than that
of DOX-ZnPCS4-coloaded control gel (no obvious green color was
observed, Fig. S6b), demonstrating its ROS-responsive release
behavior. Moreover, the greenest color of the release medium was
observed in the DOX-ZnPCS4-coloaded gel treated with ROS
condition and light irradiation (Fig. S6f), demonstrating the most
release of ZnPCS4 in all groups. This result was ascribed to its light-
activated ROS generation in situ, which simultaneously triggered
the cleavage of thioketal bond and gel degradation, and the
acceleration of drug release.
Fig. 6. The in vivo antitumor activity of drug-loaded gels: tumor
volumes (A and B), body weights (C and D), and survival rates (E and
F) of mice treated with saline, and drug-loaded gels. (L+: light
irradiation for 5 min; L−: without light irradiation)
Unexpectedly, we did not observe the typical emission peak of
DOX (560 nm) under ROS condition by fluorescence spectroscopy.
We hypothesize that the H2O2 could oxidize the phenolic hydroxyl
groups on the anthraquinone ring of DOX, then the conjugated
structure is destroyed and leads to fluorescence quenching of DOX.
We then tested the fluorescence emission of DOX in the presence
of H2O2.The PBS (pH 7.4) solution of DOX was incubated with
different concentrations of H2O2 (0, 50, 150, and 250 mM) for
different times (1, 3, and 6 h). The more fluorescence quenching
was observed when the solution was treated with higher
concentration of H2O2 or longer incubation time (Fig. S7). Therefore,
it is inacurate to quantify the release of DOX by fluorescence
spectroscopy in the presence of H2O2. Taking together, light-
activated rapid drug release was qualitively demonstrated although
the quantitive calculation of drug release was not obtained in our
testing condition.
The in vivo antitumor efficacy of ROS-responsive gels and control
gels was then studied. The typical images of tumor after intratumor
injection of various gels were shown in Fig. 3a−3d. Gel formation on
the surface of tumors was observed. The gels were then
administrated into tumors by intratumor injection and the changes
of tumor volume were showed in Fig. 6A. The tumor volume of
saline group increased rapidly, suggesting that the saline had no
antitumor effect. The groups of G1 and G1*showed little inhibition
effect, which was probably due to oppression of gel to tumor tissue
and thus leading to limited cell apoptosis. The groups of G2 and G2*
showed obvious tumor suppression effect, demonstrating the
efficiency of chemotherapy; the group of G2 shows slightly better
antitumor effect than G2*, which was due to its ROS-response in
ROS-rich tumor tissue environment, resulting in faster gel
degradation and more DOX release. The groups of G3(L+) and
G3*(L+) had obvious antitumor effect, yet G3(L−) and G3*(L−)
showed little inhibition, indicating the efficiency of photodynamic
therapy. More importantly, the group of G4(L+) showed the best
antitumor effect and significantly superior to the groups of G2(L−),
and G3(L+), suggesting the synergistic effect of chemotherapy and
photodynamic therapy. Meanwhile, the groups of G2*(L−), G3*(L+),
and G4*(L+) showed comparable antitumor effects, demonstrating
that the thioketal containing gelator was the key for the localized
chemo-photodynamic therapy. Without thioketal in the backbone
of control gels, DOX and ZnPCS4 cannot efficiently diffuse from the
gel network and no synergistic effect was obtained. Taking together,
these results demonstrated our hypothesis that the light-triggered
ROS-responsive degradation of DOX-ZnPCS4-coloaded gel, resulting
In vivo antitumor assay
6 | J. Name., 2012, 00, 1-3
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ARTICLE
into faster release of anticancer drug DOX and photosensitizer
ZnPCS4, which was also verified in the in vitro drug release, and
realizing the synergistic effect of localized combined chemo-
photodynamic therapy.
Conclusions
A ROS-responsive injectable low molecular weight gel (LMWG)
was fabricated in the mixture solvent of PEG200 and deionized
water and showed excellent capacity as a reservoir for
anticancer drug and photosensitizer. The ROS-responsive
property of thioketal containing gelator was demonstrated by
1H NMR. The cytotoxicity test and in vivo histological analysis
showed that the gels had low cytotoxicity and good
biocompatibility. The shear thinning and rapidly recovery
capability ensured that the gel formation after intratumor
injection. The in vitro drug release showed that light irradiation
triggered the acceleration of drug release in the thioketal
containing LWMGs. The ROS-responsive DOX-ZnPCS4-coloaded
gel showed the best tumor inhibition and reduced side effects
under light irradiation due to the light-triggered cleavage of
thioketal bond that resulted in the degradation of gel and
release of DOX and ZnPCS4 for combined chemo-
photodynamic therapy. Our injectable gel with combined
therapy function may provide a platform of engineering
localized stimuli-responsive drug delivery system for
multimodal therapy.
The systemic toxicity of these gels was also investigated. As
shown in Fig. 6C and 6D, negligible weight loss was observed in all
groups, except the group of G2* that showed a maximum 5% weight
loss, indicating the low systemic toxicity because of in situ gel
injection. The survival rates of tumor bearing mice treated with
ROS-responsive gels and control gels were supervised and the
results were shown in Fig. 6E and 6F, respectively. The groups of
saline and G1*showed no survival after 45 d. The survival rate of
group of G1 was 16.7% for 45 d. The mice treated with G2 and G4
with light irradiation showed the best survival rate (80%) for 45 d.
The survival rate of group of G4* was relatively low, indicating the
enhanced antitumor effect of ROS-responsive thioketal containing
gels. In summary, the combination of chemotherapy and
photodynamic therapy effectively improved the antitumor effect
and minimized systemic toxicity.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors thank for the financial support of National Natural
Science Foundation of China (No. 51573111, 51773130,
21672164), Program for Changjiang Scholars and Innovative
Research Team in University (No. IRT-15R48).
Fig.7. Histological analysis and TUNEL staining of tumors
treated with different formulations. A, a: saline(L−); B, b:
G1*(L+); C, c: G2*(L−); D, d: G3* (L+); E, e: G3* (L−); F, f: G4* (L+);
G, g: G1(L+); H, h: G2 (L−); I, i: G3 (L+); J, j: G3 (L−); K, k: G4 (L+).
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Table of Content
A ROS-responsive low molecular weight hydrogel was fabricated and loaded with
anticancer drug and photosensitizer for efficient chemo-photodynamic therapy.