Lenvatinib-zinc phthalocyanine conjugates as potential agents for enhancing synergistic therapy of multidrug-resistant cancer by glutathione depletion

Article abstract of DOI:10.1016/j.ejmech.2019.02.065

The therapeutic efficacy of targeted therapy is dramatically hindered by multidrug resistance (MDR) because of elevated GSH levels. Thus, depletion of intracellular GSH level is highly desirable for targeted-therapeutic agents to reverse tumor drug resist

Full text of DOI:10.1016/j.ejmech.2019.02.065

Accepted Manuscript
Lenvatinib-zinc phthalocyanine conjugates as potential agents for enhancing
synergistic therapy of multidrug-resistant cancer by glutathione depletion
Gaofei Wei, Liangfeng Huang, Yali Jiang, Yifeng Shen, Zeqian Huang, Yanjuan
Huang, Xiaoqi Sun, Chunshun Zhao
PII:
S0223-5234(19)30189-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.065
EJMECH 11157
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 January 2019
Revised Date: 23 February 2019
Accepted Date: 23 February 2019
Please cite this article as: G. Wei, L. Huang, Y. Jiang, Y. Shen, Z. Huang, Y. Huang, X. Sun, C. Zhao,
Lenvatinib-zinc phthalocyanine conjugates as potential agents for enhancing synergistic therapy of
multidrug-resistant cancer by glutathione depletion, European Journal of Medicinal Chemistry (2019),
doi: https://doi.org/10.1016/j.ejmech.2019.02.065.
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ACCEPTED MANUSCRIPT
Lenvatinib-Zinc Phthalocyanine Conjugates as Potential Agents for
Enhancing Synergistic Therapy of Multidrug-Resistant Cancer by
Glutathione Depletion
Gaofei Wei‡*, Liangfeng Huang‡, Yali Jiang‡, Yifeng Shen, Zeqian Huang, Yanjuan Huang,
Xiaoqi Sun, Chunshun Zhao*
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
Corresponding Author
* Chunshun Zhao E-mail: zhaocs@mail.sysu.edu.cn
* Gaofei Wei E-mail: weigaof@mail.sysu.edu.cn
Author Contributions
‡ These authors contributed equally.
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Highlights
1. A photosensitive multifunctional conjugate ZnPc-C8-Len was synthesized to realize
photodynamic therapy to reverse multidrug resistance and enhanced antitumor therapy.
2. Upon the irradiation, ZnPc-C8-Len could generate ROS to deplete intracellular GSH.
3. The decreased GSH would enhance apoptotic cell death by Bcl-2/caspase 3 pathway and
reduce expression of P-gp to reverse lenvatinib resistance.
4. ZnPc-C8-Len displayed significantly enhanced tumor accumulation and excellent in vivo
antitumor activity.
Keywords
photodynamic therapy; targeted therapy; multidrug resistance; glutathione depletion; reactive
oxygen species
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ABSTRACT
The therapeutic efficacy of targeted therapy is dramatically hindered by multidrug resistance
(MDR) because of elevated GSH levels. Thus, depletion of intracellular GSH level is highly
desirable for targeted-therapeutic agents to reverse tumor drug resistance. In this study, a
photosensitive multifunctional conjugate ZnPc-C8-Len, in which lenvatinib (a VEGFR inhibitor)
is linked to a photosensitizer ZnPc through an alkyl chains, was synthesized to realize
photodynamic therapy to reverse multidrug resistance and enhanced antitumor therapy. Upon the
irradiation, ZnPc-C8-Len could generate ROS to deplete intracellular GSH. The decreased GSH
would enhance apoptotic cell death by Bcl-2/caspase 3 pathway and reduce expression of P-gp to
reverse lenvatinib resistance. Moreover, through PEG2000-PLA2000 encapsulation, ZnPc-C8-Len
NPs displayed significantly enhanced tumor accumulation and excellent in vivo antitumor
activity. And the fluorescence characteristics of ZnPc-C8-Len could monitor the changes of
nanoparticles in vivo in real time to guide when and where to conduct the subsequent therapy. As
a result, conjugate ZnPc-C8-Len had an outstanding capability to enhance synergistic therapy of
multidrug-resistant cancer by glutathione depletion. And the approach reported here provide a
promising strategy in development of conjugate integrated targeted therapy with photodynamic
therapy to reverse targeted drug multidrug resistance and enhance synergistic therapy.
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1. Introduction
Over the last few years, there is increasing evidence to demonstrate the prominent role of
tyrosine kinases (TKs) in tumor initiation and progression.1 Targeted therapies against the TKs
(known as tyrosine kinase inhibitors or TKIs) have revolutionized the practice of oncology and a
variety of TKIs have been developed, evaluated in clinical trials, and approved for cancer.2
However, despite significant responses to TKIs, the majority of patients will develop multidrug
resistant (MDR) to treatment within two years.3 Therefore, effectively solving the problem of
MDR becomes one of the biggest challenges in clinical applications.
MDR has been known to be associated with multiple factors, such as the alterations of drug
target, the repair of DNA damage and blockade of drug-induced apoptosis, among which, high
levels of glutathione (GSH) is correlated with a lot.4-7 As a well-known intracellular antioxidant,
GSH is widely expressed in normal tissues and cells as an important antioxidant that
significantly attenuates oxidative stress level caused by toxic xenobiotics and reactive oxygen
species (ROS).8 Conceivably, increased GSH levels are a universal phenomenon in various types
of cancer cells and solid tumors, and this tends to make these cells and tissues more resistant.9, 10
The main reason for drug resistance is that GSH can conjugate therapeutic agents to facilitate
their efflux by P-glycoprotein (P-gp), which is the one of most important proteins in MDR, and
overexpressed in many tumors.11, 12 GSH also is able to influence the apoptotic process by
affecting Bcl-2 family anti-apoptotic proteins and caspase activity. Thus, depletion of
intracellular GSH level is highly desirable for therapeutic agents to reverse tumor drug
resistance.
Recently, photodynamic therapy (PDT) has been developed to reverse tumor resistance.13-17
PDT is a noninvasive medical technology that employs ROS generated from photosensitizers
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(PS) by light activation, triggering an apoptotic or necrotic response.18 Excessive ROS can react
rapidly with biological substrates GSH to disrupt the redox balance in cancer cells.19, 20 PDT has
also been demonstrated to cause the photo-destruction of P-gp by superabundant ROS. The
reduction of P-gp expression facilitates the cytosolic retention delivery of therapeutic agents,
resulting in a synergistic effect.14, 16, 21-23
Accordingly, an ideal scenario that integrates targeted therapy with photodynamic therapy can
substantially overcome the MDR to enhanced antitumor therapy. Lenvatinib (Len) is first
approved in 2015 as a vascular endothelial growth factor receptor (VEGFR) inhibitor, which is
the majority of approved TKIs.24 Unfortunately, Len remains severely multidrug resistant that is
a substrate for the MDR transport protein P-glycoprotein (P-gp) (Eisai data on file).25 For
overcoming the drug resistance, PDT was recommended. Zinc phthalocyanine has strong tissue
penetration and ROS production capacity under red-light.26 In the present study, the preparation
and anti-tumor activities of a conjugate (ZnPc-Cn-Len), in which Len is linked to a tetra (4-
carboxyphenoxy)-zinc phthalocyanine (ZnPc) core through an alkyl chains with different lengths
was reported. As illustrated in Scheme 1, upon the irradiation of red light, the conjugate ZnPc-
Cn-Len generate ROS, which depletes intracellular GSH, thus resulting in down-regulation of P-
gp and enhanced apoptotic cell death to reverse Len resistance. Also, the fluorescence
bioimaging properties of conjugate can provide us with real-time information about when and
where to apply the light illumination. The synergistic effect of Len and ZnPc can enhance anti-
tumor effects in vitro and in vivo. Herein, this report demonstrates that the rationally designed
conjugate ZnPc-Cn-Len has ‘all in one’ mode of anticancer actions to reverse multidrug
resistance with synergistic effects in the proof of concept studies in vitro as well as in vivo.
2. Results and Discussion
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2.1 Synthesis of ZnPc-Cn-Len.
The ZnPc and Len were covalently linked by a flexible carbon chain, which can improve the
biocompatibility of the conjugate while maintaining the maximum targeting capacity of Len. The
synthesis of ZnPc was prepared according to our reported procedures.27 The synthesis methods of
the conjugates ZnPc-C0-Len, ZnPc-C8-Len and ZnPc-C12-Len were demonstrated in Scheme 2.
The detailed structure of ZnPc-C0-Len, ZnPc-C8-Len and ZnPc-C12-Len were confirmed by
FTIR spectrum (Figure 1A) and Matrix-Assisted Laser Desorption/Ionization Time of Flight
Mass Spectrometry (MALDA-TOF-MS) (Figure S16–S18, Supporting Information). As showed
in Figure 1A, the vibration bands centered at 2920 cm-1 (-CH) and 1369 cm-1 (-CH) are
corresponding to the alkane belong to ZnPc-C8-Len and ZnPc-C12-Len. Moreover, the stretching
vibrations of -NH-, -C=O- and -C-O-C- are around 3338, 3177, 1655 and 1229 cm-1 respectively
in the spectra of ZnPc-Cn-Len, which were the representative peaks of Len segment. These
results of the above FTIR data confirmed that Len has been successfully covalently conjugated
with zinc phthalocyanine. And ZnPc-Cn-Len demonstrated the similar UV/vis absorbance to
ZnPc (Figure 1B). Moreover, the maximum excitation wavelength and emission wavelength of
ZnPc-C8-Len were at 681 nm and 687 nm respectively (Figure 1C). The fluorescence
spectroscopy of ZnPc, ZnPc-C0-Len and ZnPc-C12-Len were showed at Figure S1-S3. The
fluorescence spectroscopy of ZnPc-Cn-Len was similar to ZnPc and suggested that ZnPc-Cn-Len
may have a parallel photosensitivity of ZnPc.
2.2 In Vitro ROS Generation
The ROS production capacity of three conjugates ZnPc-Cn-Len upon light irradiation were first
investigated. The generation of ROS was indicated by DBPF as a ROS sensitive probe. DPBF
could rapidly capture and react with ROS, then reduce its absorbance at 416 nm. As shown in
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Figure 1D, upon the irradiation, DPBF was degraded rapidly in the present of ZnPc or ZnPc-Cn-
Len, whereas the group of purely DBPF declared no significant degradation upon the same
irradiation, which confirmed the capacity of ROS generation of ZnPc-Cn-Len with light
irradiation. Then the photobleaching of ZnPc-Cn-Len were evaluated in Figure S4, which
confirmed that ZnPc-Cn-Len possessed a great photostability during the ROS generation.
2.3 Anticancer efficiency of ZnPc-Cn-Len.
First, to evaluate the anticancer efficiency of three derivatives, the MTT assay was used to
detect cell viability compared with free Len, free ZnPc and ZnPc/Len (equimolar mixture) and
ZnPc-Cn-Len against MCF7 cells (20 µM), MCF7/ADR cells (100 µM) and 4T1 cells (20 µM)
respectively. As shown in Figure 2, in the absence of irradiation, free Len and ZnPc/Len
(equimolar mixture) exhibited cell cytotoxicity rates of 30.16% and 28.71% against MCF7 cells.
But conjugates ZnPc-C0-Len, ZnPc-C8-Len and ZnPc-C12-Len showed lower cell cytotoxicity
rates of 9.52%, 27.23% and 19.27%, respectively. From the above dark cytotoxicity data, it was
verified that the modification of a bulky group on the amino group of Len reduced slight activity
of Len. Among the dark cytotoxicity of three conjugates, ZnPc-C8-Len conjugate showed the
least decrease compared to the prototype drug Len, which might be possible that the alkyl chains
of eight carbons have the least effect on the binding of Len and its target protein. The above
same phenomenon was also observed in drug-resistant MCF7/ADR cells and 4T1 cells.
However, under a relatively low light dose (660-670 nm, 50 mW cm-2, 10 min), all three
conjugates show significantly higher cytotoxicity than Len in all of three cancer cells, among
which ZnPc-C8-Len conjugate showed the strongest cytotoxicity and MDR reversal efficacy with
74.71% for MCF7 cells, 74.55% for MCF7/ADR cells and 49.71% for 4T1 cells respectively.
More interestingly, the cytotoxicity of Len in drug-resistant MCF7/ADR cells was almost
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negligible (approximately 18%), but the activity of Len modified photosensitizer (ZnPc-C8-Len)
was significantly increased with irradiation, which suggested that the conjugated form can
increase the activity of Len in drug-resistant cells. Thus, from above the cell viability, it was
reasonable to infer that the covalent combination of photosensitizer and Len can effectively
improve cytotoxic activity even in drug-resistant cells.
2.4 Correlation between GSH and Len-resistance.
With the above desirable observation of significant cytotoxicity and MDR reversal efficacy of
ZnPc-Cn-Len and the previous reports about GSH participates in the development of various
multidrug-resistance cells,28, 29 it is suspicious that whether Len is cross-resistant to MCF7/ADR
cells and whether GSH was involved in Len-cross-resistant. To elucidate the first concern, both
MCF7 and MCF7/ADR cells were investigated for their sensitivity towards Len. MCF7 and
MCF7/ADR cells were seeded in 96-well plates and exposed to different concentrations of Len
for 24 h. Cell viability tested by MTT assay indicated that MCF7/ADR cells were less sensitive
towards Len (Figure 3A and 3B). Noteworthy, high dose (42.9 µM) of Len that sufficed to
reduce MCF7 viability to 50% only reduced MCF7/ADR cells viability to 95%. These results
agreed well with the surmise that Len was indeed resistant to MCF7/ADR cells.
Then whether GSH contributed to Len cross-resistance observed in MCF7/ADR was
investigated. MCF7 and MCF7/ADR cells were pretreated with BSO (buthionine sulfoximine, a
GSH synthetase inhibitor) or exogenous GSH, then tested for their sensitivity towards various
concentrations of Len. Firstly, GSH and BSO itself were confirmed to have no toxicity on cell
growth after 72 h exposure (Figure S5 and S6). Then cells were incubated with BSO (500 µM) or
GSH (2.5 mM) for 24 h to disrupt GSH balance, followed by being exposed to Len to test the
sensitivity. As shown in Figure 3, the results suggested that the combined application of BSO
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could increase the cytotoxicity of Len. Compared with MCF7 cells (~2 -fold, from 73.1 µM to
42.9 µM), a significant decrease of the IC50 values was found in MCF7/ADR cells (~5 -fold,
291.5 µM to 58.8 µM). On the contrary, the concomitant application of exogenous GSH reduced
the sensitivity of Len, with 1.4-fold improvement of the IC50 values in MCF7/ADR cells and 1.2-
fold in MCF7. Combine the above results, it can be clearly obtained the correlation between
GSH and Len-resistance in MCF7/ADR cells.
2.5 Monitoring GSH change
To shed light on the mechanism of Len-resistance, ZnPc-C8-Len was chosen as a representative
compound to investigate the subsequent studies because it had the strongest cytotoxicity and
MDR reversal efficacy. First, the level of GSH in MCF7 and MCF7/ADR cells impacted by
ZnPc-C8-Len was investigated. Cells were treated with ZnPc-C8-Len, BSO, Len and ZnPc for 24
h follow by exposing to irradiation for 10 min and then the level of GSH was quantitatively
measured. As shown in Figure 4, Free Len could slightly increase GSH level in MCF7/ADR
cells. This observation can be explained by the rationales that MDR cells can launch robust GSH
biosynthetic system to produce GSH to detoxify therapeutic agents through phase II
metabolism.4, 30, 31 However, treatment of BSO, ZnPc or ZnPc-C8-Len resulted in significant
reduction of the GSH level. Compared with MCF7 cells, a more significant decrease of GSH
levels by ZnPc-C8-Len was found in MCF7/ADR cells. Through the above experiments, it can be
reasonably concluded that conjugate ZnPc-C8-Len could efficiently deplete GSH in MCF7 and
MCF7/ADR cells.
2.6 Mechanism of MDR Reversal
Resistant cells increase the capacity of tolerating therapy drugs by high GSH levels mediated
escaping apoptosis.4 Nevertheless, ZnPc-C8-Len was convincingly verified to effectively deplete
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GSH in MCF7/ADR cells. Naturally, it can be conjectured that ZnPc-C8-Len could alleviate
apoptotic silence to reverse MDR. To test this hypothesis, the apoptosis induced by ZnPc-C8-Len
in MCF7 and MCF7/ADR cells was measured by Annexin V-FITC/PI double staining assay.
After incubation with free Len, the apoptosis rate of the MCF7 cells was 44.1%, respectively, but
only 16.3% in the MCF7/ADR cells (Figure 5A). This result was consistent with accepted
knowledge that apoptosis can be silenced in drug-resistant cells. Compared with free drug
formulation, it was discovered that ZnPc-C8-Len promoted much higher level of apoptosis in
both MCF7 and MCF7/ADR cells with 82.4% and 75.0%. Thus, the outstanding performance of
apoptosis activation by ZnPc-C8-Len in MCF7/ADR may lead to a better therapeutic outcome.
After obtaining the above results, the probable mechanism of apoptosis caused by ZnPc-C8-Len
was investigated. The previous studies have showed that drug resistance mediated from high
GSH concentration is associated with overexpression of anti-apoptotic protein Bcl-2 which is
known as an important gatekeeper to the apoptotic response.32, 33 Next, western blot was used to
analyze the level of Bcl-2 expression in both MCF7 and MCF7/ADR cells with ZnPc-C8-Len. As
shown in Figure 5B and 5D, after exposure of MCF7 or MCF7/ADR cells to ZnPc-C8-Len with
irradiation, the expression of Bcl-2 protein was significantly decrease compared to free Len.
Furthermore, Caspase 3, the downstream protein of Bcl-2 and the essential executioner caspase
required for apoptosis signaling were also investigated. 34 It was obviously that ZnPc-C8-Len
upregulated the expression of apoptosis related cleaved-caspase 3 in MCF7/ADR cells (Figure
5B and 5E). All these results indicated that ZnPc-C8-Len is more efficiently decrease the
expression of Bcl-2, thus activating caspase 3 to promote MCF7/ADR cell apoptosis compared
with the single free drug Len.
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However, there may be another reason that ZnPc-C8-Len could induce greater apoptosis in
MCF7/ADR cells, which is probably due to the enhanced intracellular drug accumulation by
reducing the efflux of the drug. As well-known, P-gp, an efflux pump transporter, was able to
increase Len efflux and cause therapy failure.35 Thus, the expression level and activity of P-gp
will make a great contribution to the MDR effect. In order to confirm this hypothesis, the
expression of P-gp after treating with ZnPc-C8-Len in MCF7/ADR cells was investigated. As
shown in Figure 5C and 5F, the expression of P-gp was downregulated significantly by ZnPc-C8-
Len upon the irradiation in the MCF7/ADR cells. In brief, it could be inferred that the
mechanism of MDR reversal might lie on the induction of apoptosis through down-expression of
Bcl-2 and up-expression of caspase 3 accompanied with the reduction of P-gp expression.
2.7 In vitro cytotoxicity evaluation
First, intracellular drug accumulation efficiency of ZnPc-C8-Len in MCF7 and MCF7/ADR
cells were evaluated by confocal laser scanning microscopy (CLSM). Cells were incubated with
ZnPc-C8-Len for 24 h to arrive a same absorbance (Figure S7) before the different treatment.
Then cells were rinsed with PBS and incubated with fresh culture medium to investigate the drug
retention. As shown in Figure 6A, MCF7 cells presented higher fluorescence intensity than
MCF7/ADR cells under no light irradiation. This can be explained that MCF7/ADR increased
efflux of ZnPc-C8-Len assisted by overexpressed P-gp, resulting in limited drug accumulation in
cells. In contrast, upon light irradiation, the intracellular fluorescence intensity became obviously
stronger in MCF7/ ADR cells which may be due to the decrease of the expression of P-gp. Such
improvement of intracellular drug retention of ZnPc-C8-Len was also observed in MCF7/ADR
cells with the treatment of verapamil, which is a P-gp inhibitor. Taken together, the findings
confirmed that ZnPc-C8-Len could indeed accumulate effectively inside MCF7/ADR cells.
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Subsequently, its cytotoxicity against MCF7 cells, 4T1 cells and MCF7/ADR cells were tested
(Figure 6B, 6C & 6D), and IC50 values were calculated and summarized in Table S1. The IC50
values of ZnPc-C8-Len against 4T1 cells was 19.4 µM compared with 67.7 µM for Len, a 3.5-
fold decrease, and a 4.5-fold decrease against MCF7 cells correspondingly. More importantly,
ZnPc-C8-Len showed a significantly improved cytotoxicity against MCF7 /ADR cells, with an
IC50 values 47.32 µM while 329.2 µM for Len, a 6.9-fold decrease, indicating that ZnPc-C8-Len
could indeed reverse MDR in cancer cells.
To evaluate the synergistic effect between ZnPc and Len, the combination index (CI) of ZnPc-
C8-Len was calculated according to Equation (1).36 And the CI index indicates antagonism (CI
>1), additivity (CI = 1), or synergism (CI <1) between the drugs. ZnPc and Len conjugated in
ZnPc-C8-Len were synergistic against all three cell lines, with CI values of 0.82, 0.80 and 0.42 in
the 4T1, MCF7, and MCF7/ADR cell lines, respectively. In the MCF7/ADR cells, the CI values
was much lower than that in other cancer cells 4T1 and MCF7, indicating an excellent
synergistic effect in the drug resistant strain. The synergy in MCF7 ADR suggested that ZnPc-
C8-Len might minimize drug resistance in cancer cells.
2.8 In vivo antitumor efficacy
The in vivo therapeutic effects and potent toxicity were eventually verified on the tumor bearing
mice. Therapeutic nanoparticles (NPs) have been widely investigated to enhance cancer
treatment. Because of the low solubility of ZnPc-C8-Len, it was loaded into biodegradable
PEG2000-PLA2000 nanoparticles to improve solubility. In addition, ZnPc-C8-Len nanoparticles can
be accumulated in tumor tissues after tail vein injection due to enhanced permeability and
retention (EPR) effects. At the same time, the fluorescence characteristics of ZnPc-C8-Len can
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be utilized to monitor the changes of nanoparticles in vivo in real time so as to guide when and
where to conduct the subsequent therapy.
First, Len, ZnPc and ZnPc-C8-Len can be formulated in PEG2000-PLA2000 to form nanoparticles
of a uniform diameter around 124, 167 and 151 nm for Len NPs, ZnPc NPs and ZnPc-C8-Len
NPs respectively (Figure S8 and S9). ZnPc-C8-Len NPs still had the ability to produce ROS
generation (Figure S10). The antitumor efficacy of ZnPc-C8-Len NPs was also investigated in
4T1 cells (Figure S11).
Then in vivo imaging experiment was performed using the fluorescence properties of ZnPc-C8-
Len NPs. As shown in Figure 7A, for ZnPc-C8-Len NPs, fluorescence was clearly observed at
tumor site after 3 h post-injection. More importantly, 24 hours after the injection, clear
fluorescence signals could still be observed at the tumor site. These results suggested that the
tumor accumulation of ZnPc-C8-Len NPs could be real-time monitored by the fluorescence
changes. Moreover, according to the fluorescence intensity, the distribution of ZnPc-C8-Len NPs
in different tissues was compared. As shown in Figure 7B, after 24 h of injection, ZnPc-C8-Len
NPs accumulated mainly in tumor tissue, while the accumulation in other tissues was tolerable.
Figure 7C represents the mean fluorescence intensity of ZnPc-C8-Len in the tumor and other
organs, and the result was consistent with that found in Figure 7B. In all, after the encapsulation
of ZnPc-C8-Len into PEG2000-PLGA2000, ZnPc-C8-Len could efficiently accumulate in tumor
tissue and stay in tumor for long time, which will favor the enhanced antitumor effect in vivo.
Thus, the antitumor efficacy of ZnPc-C8-Len NPs in vivo was tested via intravenous injection.
As shown in Figure 7D, without irradiation, the normal saline groups and ZnPc NPs groups had
almost no effect on tumor growth. ZnPc-C8-Len NPs could slightly inhibit tumor growth, and
moderate therapeutic efficacy of Len NPs was observed in the mice administrated. This result
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was consistent with the conclusion that the modification of a bulky group on the amino group of
Len could reduce slight the anti-tumor activity of Len in the absence of light in vitro cell
experiments. And light irradiation had little effect on the anti-tumor effect of Len NPs, but light
had substantial effect on ZnPc NPs due to PDT. After light irradiation with 671 nm, the
antitumor activity of ZnPc NPs was similar to that of Len NPs. However, ZnPc-C8-Len NPs
display the best therapeutic efficacy and almost completely depress the tumor progression after
light irradiation. After PDT treatment, tumors were excised for photographing (Figure 7E) and
weighing (Figure 7F). During the treatment, body weight of mice was record and no significantly
changes were observed (Figure 7G).
To further evaluate the therapeutic effects, standard H&E staining analysis of tumor tissues and
organs was performed. From H&E staining images (Figure 8A), some tumor cells were damaged
in the Len NPs group and ZnPc-C8-Len NPs group without light irradiation, which should be the
contribution of Len. ZnPc NPs group with light irradiation also had some tumor cell damage,
which should be the contribution of PDT of ZnPc. There was a large area of cell damage in
ZnPc-C8-Len NPs group with laser irradiation, which should be the synergistic effect of
photodynamic and targeted therapy. Moreover, major organs (liver, spleen, kidney, heart and
lung) were collected and sliced for H&E staining (Figure 8B). No obvious sign of organ damage
or inflammatory lesion was observed. All these results suggested ZnPc-C8-Len NPs would be a
relatively safe agent with noninvasive in situ drug tracking and fluorescence imaging guided
efficient antitumor therapy in vivo.
3. Conclusions
In summary, a photosensitive multifunctional conjugate ZnPc-C8-Len was designed and
synthesized to realize the photodynamic therapy to reverse multidrug resistance and enhanced
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antitumor therapy. After the irradiation, the conjugate ZnPc-C8-Len could generate ROS to
deplete intracellular GSH. The decreased GSH would enhance apoptotic cell death by Bcl-
2/caspase 3 pathway and reduce expression of P-gp to reverse lenvatinib resistance. In vitro
ZnPc-C8-Len could accumulate effectively inside MCF7/ADR cells and possessed an excellent
synergistic effect. Moreover, through PEG2000-PLA2000 encapsulation, ZnPc-C8-Len
nanoparticles can be accumulated in tumor tissues. At the same time, the fluorescence
characteristics of ZnPc-C8-Len can monitor the changes of nanoparticles in vivo in real time so
as to guide when and where to conduct the subsequent therapy. With the guidance, ZnPc-C8-Len
NPs showed excellent in vivo antitumor activity. As a result, conjugate ZnPc-C8-Len had an
outstanding capability to enhance synergistic therapy of multidrug-resistant cancer by
glutathione depletion. And the approach reported here provide a promising strategy in
development of conjugate integrated targeted therapy with photodynamic therapy to reverse
targeted drug multidrug resistance and enhance synergistic therapy.
4. Experimental Section
4.1 Materials
1-(2-chloro-4-hydroxyphenyl)-3-cyclopropylurea, Len, Methyl 4-chloro-7-Methoxyquinoline-
6-carboxylate,
2’,7’-Dichlorofluorescin
diacetate
(DCFH-DA),
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride, 1,8-Diaminooctane, 1,12-Dodecanediamine,
Fetal Bovine Serum (FBS), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT), Dulbecco's modified Eagle's medium (DMEM), phosphate buffered saline (PBS), l- were
purchased from Sigma-Aldrich (USA). Phenyl chloroformate, 3-Bromo-1-propanol, 4',6-
Diamidino-2-phenylindole Dihydrochloride (DAPI),
9
10-anthracenediyl-bis(methylene)
(ABDA), buthionine-sulfoximine (BSO), reduced glutathione (GSH) 1,3-Diphenylisobenzofuran
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(DBPF), was obtained from Aladdin (China). PEG2000 -PLA2000 was purchased from Beijing
HWRK Chem Co., LTD. GSH Assay Kit, P glycoprotein rabbit monoclonal antibody, β-actin
mouse monoclonal antibody, HRP-labeled Goat Anti-Mouse IgG, HRP-labeled Goat Anti-Rabbit
IgG was obtained from Beyotime Biotechnology Co., LTD. Other chemicals were of analytical
grade and used without further purification. Preparative column chromatography was used with
1
200-300 mesh silica gel (Qingdao Haiyang Chemical, China). H and 13C NMR spectra were
recorded on a Bruker ARX 600 MHz spectrometer. The purity of ZnPc-Cn-Len (≥ 95%) were
determined by analytical HPLC. Analytical HPLC was performed at Hitachi L-2000 instrument
equipped with a UV-DAD detector. The chromatographic separation was achieved on an C18
column, (150 mm × 3.9 mm, Nova-Pak-C18, Waters) at a flow rate of 1.0 mL/min in two mobile
phases (THF-Distilled water=15:85).
4.2 Synthesis of ZnPc-Cn-Len
4-(3-chloro-4-(3-cyclopropylureido)phenoxy)-7-methoxyquinoline-6-carboxylate Methyl (S1)
10g (44.12 mmol) 1-(2-chloro-4-hydroxyphenyl)-3-cyclopropylurea and 8.88g (35.30 mmol)
Methyl 4-chloro-7-Methoxyquinoline-6-carboxylate was dissolved in 150 mL DMF. Then 18.29
g (132.36 mmol) K2CO3 was added and the suspension was stirred at 75 for 30 min. After that,
the suspension was stirred at room temperature for 2 h and 500 mL distilled water was added.
The precipitate was filtered and washed thoroughly with distilled water. After drying at vacuum,
the precipitate was re-crystallize in methanol and dichloromethane (2:1, v/v) to get white crystal
1
pure product with a yield of 89 %. H NMR (400 MHz, DMSO-d6) δ 8.70 (d, J = 5.3 Hz, 1H),
8.58 (s, 1H), 8.29 (d, J = 9.1 Hz, 1H), 7.99 (s, 1H), 7.57 – 7.47 (m, 2H), 7.26 (dd, J = 9.1, 2.8
Hz, 1H), 7.21 (d, J = 3.0 Hz, 1H), 6.54 (d, J = 5.3 Hz, 1H), 3.98 (s, 3H), 3.87 (s, 3H), 2.58 (dd, J
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= 7.0, 3.5 Hz, 1H), 0.67 (dt, J = 6.8, 3.3 Hz, 2H), 0.43 (dt, J = 7.0, 3.5 Hz, 2H). ESI-MS (m/z):
442.1 [M+H]+.
4-(3-chloro-4-(3-cyclopropylureido)phenoxy)-7-methoxyquinoline-6-carboxylic acid (S2) 10
g (22.63mmol) S1 was dissolved in 100 mL methanol, 50 mL THF and 1 M NaOH solution.
After stirred at 70 for 2.5 h, the solution was cooled to room temperature and concentrated
using rotary evaporator. Then, pH value of the mixture was buffered close to 4.5 using
hydrochloric acid. The precipitate was filtered and washed thoroughly with distilled water.
Finally, the white product S2 was dried at vacuum and recrystallized in methanol and
dichloromethane (2:1, v/v) with a yield of 92%. 1H NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H),
8.82 (d, J = 5.8 Hz, 1H), 8.61 (s, 1H), 8.32 (d, J = 9.1 Hz, 1H), 8.03 (s, 1H), 7.57 (d, J = 3.2 Hz,
2H), 7.31 (dd, J = 9.1, 2.8 Hz, 1H), 7.25 (d, J = 2.9 Hz, 1H), 6.73 (d, J = 5.8 Hz, 1H), 4.02 (s,
3H), 2.61 – 2.55 (m, 1H), 0.67 (dt, J = 6.7, 3.3 Hz, 2H), 0.48 – 0.38 (m, 2H). ESI-MS
(m/z):426.1[M-H]-.
N-(8-aminooctyl)-4-(3-chloro-4-(3-cyclopropylureido)phenoxy)-7-methoxy
quinoline-6-
carboxamide (S3). 500 mg (1.17 mmol) S2, 336.05 mg (1.75 mmol) EDCI and 337.18 mg (2.34
mmol) 1-Hydroxybenzotriazole (BtOH) was dissolved in DMF. The mixture was stirred at room
temperature for 2 h. After that, the mixture was added to DMF solution of 1,8-Diaminooctane
(337.18 mg, 2.34 mmol) and stirred at room temperature for 24 h. Then the mixture was added to
distilled water and the precipitate was filtered and washed thoroughly with distilled water. The
1
pure product S3 was purified by column chromatography with a yield of 48%. H NMR (400
MHz, DMSO-d6) δ 8.67 (d, J = 5.2 Hz, 1H), 8.51 (s, 1H), 8.40 (t, J = 5.7 Hz, 1H), 8.27 (d, J =
9.1 Hz, 1H), 8.14 (s, 1H), 7.64 (s, 1H), 7.51 (s, 1H), 7.47 (d, J = 2.8 Hz, 1H), 7.23 (dd, J = 9.1,
2.8 Hz, 1H), 6.54 (d, J = 5.2 Hz, 1H), 4.02 (s, 3H), 3.30 (q, J = 6.7 Hz, 2H), 2.78 – 2.69 (m, 2H),
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2.58 (dq, J = 7.0, 3.4 Hz, 1H), 1.54 (q, J = 7.2 Hz, 4H), 1.39 – 1.21 (m, 10H), 0.66 (dt, J = 6.8,
3.3 Hz, 2H), 0.49 – 0.39 (m, 2H). ESI-MS (m/z): 554.3 [M+H]+.
N-(12-aminododecyl)-4-(3-chloro-4-(3-cyclopropylureido)phenoxy)-7-methoxy quinoline-6-
carboxamide (S4). 500 mg (1.17 mmol) S2, 336.05 mg (1.75 mmol) EDCI and 337.18 mg (2.34
mmol) HoBt was dissolved in DMF. The mixture was stirred at room temperature for 2 h. After
that, the mixture was added to DMF solution of 1,12-Dodecanediamine (468.31 mg, 2.34 mmol)
and stirred at room temperature for 24 h. Then the mixture was added to distilled water and the
precipitate was filtered and washed thoroughly with distilled water. The pure product S4 was
purified by column chromatography with a yield of 34%. 1H NMR (400 MHz, DMSO-d6) δ 8.66
(d, J = 5.3 Hz, 1H), 8.49 (s, 1H), 8.42 – 8.32 (m, 2H), 8.25 (d, J = 9.1 Hz, 1H), 7.79 (s, 1H), 7.51
(s, 1H), 7.45 (d, J = 2.8 Hz, 1H), 7.22 (dd, J = 9.1, 2.8 Hz, 1H), 6.53 (d, J = 5.2 Hz, 1H), 4.01 (s,
3H), 2.64 (t, J = 7.5 Hz, 2H), 2.60 – 2.54 (m, 1H), 1.72 (s, 4H), 1.51 (dt, J = 25.3, 6.7 Hz, 4H),
1.31 – 1.21 (m, 16H), 0.64 (dt, J = 6.8, 3.3 Hz, 2H), 0.43 (p, J = 4.6 Hz, 2H). ESI-MS (m/z):
610.3 [M+H]+.
ZnPc-C0-Len. 500 mg (0.45 mmol) ZnPc, 228.26 mg (0.54 mmol) Len, 128.14 mg (0.67 mmol)
EDCI, 120.42 mg (0.89 mmol) HoBt and 90 µL N, N-Diisopropylethylamine was dissolved in
DMF. The mixture was stirred at room temperature for 24 h and evaporated to yield a blue oil
when reaction completed. Finally, the pure product was purified by column chromatography
(CH2Cl2/CH3OH/TEA 15:1:0.1) as a dark green solid with a yield of 15%. MALDI-TOF MS
(m/z):1529.751 (Calcd for [M+H]+: 1529.250). Chemical purity assessed by HPLC: 96.53%.
ZnPc-C8-Len. 500 mg (0.45 mmol) ZnPc, 296.26 mg (0.54 mmol) S3, 128.14 mg (0.67 mmol)
EDCI, 120.42 mg (0.89 mmol) HoBt and 90 µL N, N-Diisopropylethylamine was dissolved in
DMF. The mixture was stirred at room temperature for 24 h and evaporated to yield a blue oil
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when reaction completed. Finally, the pure product was purified by column chromatography
(CH2Cl2/CH3OH/TEA 15:1:0.1) as a dark green solid with a yield of 26%. MALDI-TOF MS
(m/z):1656.602 (Calcd for [M+H]+: 1656.386), 1678.611 (Calcd for [M+Na]+: 1678.368).
Chemical purity assessed by HPLC: 97.47%.
ZnPc-C12-Len. 500 mg (0.45 mmol) ZnPc, 326.31 mg (0.54 mmol) S4, 128.14 mg (0.67
mmol) EDCI, 120.42 mg (0.89 mmol) HoBt and 90 µL N, N-Diisopropylethylamine was
dissolved in DMF. The mixture was stirred at room temperature for 24 h and evaporated to yield
a blue oil when reaction completed. Finally, the pure product was purified by column
chromatography (CH2Cl2/CH3OH/TEA 15:1:0.1) as a dark green solid with a yield of 19%.
MALDI-TOF MS (m/z):1712.584 (Calcd for [M+H]+: 1712.449), 1734.593 (Calcd for [M+Na]+:
1734.431). Chemical purity assessed by HPLC: 96.02%.
4.3 Characterization of ZnPc-Len
UV−vis Spectroscopy. Len, ZnPc, ZnPc-C0-Len, ZnPc-C8-Len, ZnPc-C12-Len were dissolved
in DMSO at the same concentration of 5 µM. Then their UV-visible absorption spectra were
recorded using an UV-vis spectrophotometer (TECHCOMP, UV2600 spectrophotometer).
Fluorescence Spectroscopy. ZnPc, ZnPc-C0-Len, ZnPc-C8-Len, ZnPc-C12-Len were dissolved
in DMSO at the same concentration. Then their fluorescence spectra were recorded using a
spectrofluorophotometer (Fluoromax-4, Horiba, Japan). The fluorescence excitation spectra and
emission spectra were obtained through monitoring emission wavelength at 780 nm and exciting
at 560 nm, respectively.
Detection of ROS. Blank DMSO (control), ZnPc, ZnPc-C0-Len, ZnPc-C8-Len and ZnPc-C12-
Len were mixed with DBPF solution (60 µM) at a final concentration at 1 µM. After that, the
mixture was exposed to laser irradiation (671 nm, 20 mW cm-2) and the absorbance of the
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mixture at 416 nm was recorded. The generation capacity of ROS of ZnPc, ZnPc-C8-Len
nanoparticles in aqueous solution was acquired using the similar method and ABDA was used as
water soluble indicator. In brief, blank DMSO (control), ZnPc or ZnPc-C8-Len nanoparticles
were mixed with ABDA water solution (100 µM) at a final concentration of 5 µM (ZnPc
equivalent dose). After that, the mixture was exposed to laser irradiation (671nm, 100 mW cm-2)
and the absorbance of mixture at 401 nm was recoreded. The DBPF and ABDA degradation rate
was calculated as (A0-At)/A0 × 100% (A0: the absorbance of the mixture solution before
irradiation, At: the absorbance after irradiation;)
Photostability. The photostability of ZnPc, ZnPc-C0-Len, ZnPc-C8-Len and ZnPc-C12-Len
during PDT was measured by their degradation reflected by the decrease of their absorbance.
Photosensitizers were dissolved in DMSO at same concentration and exposed to laser irradiation
(671 nm,100 mW cm-2). And the photobleaching capacity was calculated as At/A0. (A0: the
absorbance of mixture before irradiation, At: the absorbance after irradiation;)
4.4 Cellular assay
Cell culture. 4T1 murine breast carcinoma cells, MCF7 and MCF7/ADR (adriamycin drug-
resistant) human breast carcinoma cells were obtained from Laboratory Animal Center of Sun
Yat-sen University (Guangzhou, China). The culture medium was RPMI 1640 or DMEM
containing 10% FBS and antibiotics (penicillin 100 U mL-1 and streptomycin 100 µg mL-1).
In vitro Cytotoxicity Study. The in vitro cytotoxicity of Len, ZnPc, Len/ZnPc (equimolar
mixture), ZnPc-C0-Len, ZnPc-C8-Len and ZnPc-C12-Len against MCF7, MCF7/ADR and 4T1
for 72 h was determined by MTT assay. Cells were seeded at 96-well plates (5×104 cells per
well) with 100 µL DMEM or RPMI 1640 contain 10% FBS. Then the culture medium was
removed and 100 µL of drug solution (20 µM for MCF7 and 4T1 cells and 100 µM for
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MCF7/ADR cells) or blank culture medium containing 0.5% DMSO (control) was added and
incubated for 24 h. After that, drug solution was removed and cells were rinsed twice with PBS
and continuously cultured in fresh culture medium. Then cells were exposed to irradiation (660-
670 nm, 50 mW cm-2, 10 min) and continuously incubated. The same irradiation was
implemented to cells at 48 h and MTT solution was added at 72 h. The absorbance at 570 nm
was determined using a microplate reader (ELX800, Bio-Tek, USA). Cell viability was
determined by the percentage of the optical density value of the study group over the control
group. The in vitro cytotoxicity of Len affect by GSH or BSO was measured by simple
pretreatment. Cells were incubated with 2.5 mM GSH or 500 µM BSO for 24 h. Then cells were
rinsed twice with PBS and Len in various concentration was added for co-incubation for 24 h.
Then cells were rinsed twice with PBS again and fresh culture medium was added for another 48
h. Cell viability was determined by MTT assay. The in vitro cytotoxicity of various
concentration of Len, ZnPc and ZnPc-C8-Len against MCF7, MCF7/ADR and 4T1 cells for 72 h
was determined by MTT assay. Drug solution was added and firstly incubated for 24 h. Then
drug solution was removed and cells were continuously cultured in fresh culture medium. Cells
were exposed to light irradiation (660-670 nm, 50 mW cm-2, 10 min) and then continuously
incubated in incubator. The same irradiation was implemented to cells at 48 h and MTT solution
was added at 72 h.
Combination index (CI) analysis. The following equation was used to calculate the dose-
effect profiles of Len and ZnPc combined in ZnPc-C8-Len: CI = D/Da + D/Db, where D is the
IC50 values of ZnPc-C8-Len. Da and Db represent the IC50 values of Len and ZnPc, respectively.
CI values less than 1 indicate synergism, CI values equal to 1 indicate an additivity effect, and CI
values more than 1 correspond to an antagonism effect.
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Glutathione determination. Intracellular GSH was measured as described by instruction of the
GSH and GSSG Assay Kit. First, 1× 106 MCF7 or MCF7/ADR cells were plated in cell culture
dish and incubated overnight. Then cells were incubated with 4 µM Len, ZnPc, ZnPc-C8-Len or
500 µM BSO for 24 h and exposed to irradiation (660-670 nm, 50 mW cm-2, 10 min) after
removing drug solution and rinsed with PBS. These cells were treated with trysin and collected
by centrifuge, then treated with proper volume of protein removal reagent S. Samples were
frozen by liquid nitrogen and thawed at 37 °C for at least three times. After placed at 4 °C for 5
min, the extract was collected by centrifuging at 4 °C at 10000 rpm for 5 min. Proper volume of
extract and Proper volume detection solution was mixed in 96-well plates and incubated at 25 °C
for 30 min. The intracellular GSH was determined by the absorbance at 405 nm using a
microplate reader.
Apoptosis assay. Flow cytometry analysis was used for apoptosis assay. First, MCF7 or
MCF7/ADR cells were seeded on 6-well plates (2 × 105 cells per well) and incubated overnight,
then the cells were treated with Len, ZnPc and ZnPc-C8-Len for 24 h. After replacing with the
fresh culture medium, cells were exposed to irradiation (50 mW cm-2, 660-670 nm, 10min) at 24
h and 48 h. At 72 h, the cells were digested by trypsin without EDTA, collected in centrifuge
tubes, washed with PBS and binding buffer and stained in PI and Annexin-V-FITC containing
binding buffer for 15 min, and finally analyzed by Flow Cytometry (Guava EasyCyte 6-2L,
Merck Millpore).
Western blot. The change of P-gp, caspase 3 and Bcl-2 was investigated by Western blotting
method. Briefly, MCF7/ADR and MCF7 cells were seeded in 6-well plates at a density of 1 ×
106 per well. Then the cells were treated with 500 µM BSO, 2.5 mM GSH, 20 µM Len, ZnPc-C8-
Len with or without irradiation. The irradiation (50 mW cm-2,660-670 nm) was given at 24 h and
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48 h for 10 min once time. Untreated cells were used as the negative control. After 72 h
incubation, cells were harvested and the cellular proteins were extracted. The protein
concentration was determined using the BCA protein assay. Equal amounts of protein in each
treatment were separated by SDS-PAGE and electrotransferred to 0.22 µM PVDF membranes
for western blot. And the normalized gray value was analyzed by image analysis software
(Image J 1.50i).
Internalization and efflux assay. MCF7 or MCF7/ADR cells were seeded on the sheet glass
and incubated overnight. Then the culture medium was replaced with fresh culture medium
containing ZnPc-C8-Len or verapamil was added. The cells of light group was exposed to
irradiation (50 mW cm-2,660-670 nm) at 6 h and 12 h during 24 h incubation. After that, drug
solution was removed and cells were rinsed twice with PBS. The cells were incubated with fresh
culture medium for another 24 h. Then the cells were incubated with DCFH-DA for 30 min and
exposed to irradiation (50 mW cm-2, 660-670 nm) for 5 min. Finally, the cells were fixed with
4% paraformaldehyde and observed by confocal laser scanning microscope (Olympus FV3000).
Preparation and characterization of PLA2000-PEG2000 nanoparticles. The mixed THF
solution of ZnPc-C8-Len and PEG2000-PLA2000, ZnPc and PEG2000-PLA2000 or Len and PEG2000
-
PLA2000 (drug: 1 mM, polymer: 8 mM, 100 µL) was slowly added into the distilled water (1 mL)
while ultrasound. Then the mixtures were vigorous stirred overnight at room temperature to
evaporate the organic solvent. To determine the drug concentration in micelles, micelles were
collected by centrifugation and THF was added for demulsification. Then, DMSO was added to
dissolve and disperse the ZnPc and ZnPc-C8-Len. The concentration of Len in micelles was
determined by the UV–vis absorbance and the concentration of ZnPc and ZnPc-C8-Len in
micelles was determined by fluorescence assay. The micelles were characterized by Zetasizer
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Nano instruments (Zetasizer Nano ZS90) and transmission electron microscopy (JEOL
JEM1400).
4.5 In vivo assay
Female BALB/c mice (20 ± 2 g, 5–6 weeks) were obtained from the Laboratory Animal Center
of Sun Yat-sen University (Guangzhou, China). All experimental procedures were approved and
supervised by the Institutional Animal Care and Use Committee of Sun Yat-sen University. To
develop the xenograft tumor model, 4T1 cell suspension was subcutaneously injected into the
right flank of each mouse. Tumor volumes were calculated as (L×W2)/2, (L and W represent the
longest and shortest tumor diameter (mm)).
In vivo biodistribution. Mice bearing xenograft tumor were randomly divided into two groups
(n=3) and received a tail intravenous injection of free ZnPc-C8-Len and ZnPc-C8-Len NPs at a
dosage of 2 µmol kg-1. After narcotized, mice were imaged on the detersile area directly via in-
vivo imaging Systems (NightOWL LB983, Berthold, Germany). After that, mice were sacrificed
and major organs and tumors were excised for imaging. Mean fluorescence intensity (MFI) of
excised hearts, livers, spleens, lungs, kidneys and tumor was calculated as fluorescence counts
per square millimeter.
In vivo antitumor efficacy. Once the xenograft tumor reached about 100 mm3, mice were
divided randomly into 8 groups (n = 6) and tail intravenous injection with saline, Len NPs, ZnPc
NPs, ZnPc-C8-Len NPs at a dosage of 1 µmol kg-1 every four days. And the group with light
irradiation was exposed to a laser irradiation (671 nm, 500 mW cm-2, 10 min) at 24 h after drug
administration. Tumor volume and body weight were recorded every two days. When the PDT
treatment was finished, mice were sacrificed tumors were excised for imaging and recording the
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weight. The major organs and tumors were excised for histological examination by standard
H&E staining.
4.6 Statistical analysis
Quantitative data were demonstrated by a format as mean ± standard deviation (SD). Anova
analysis was used for statistical comparisons. Statistical Package for the Social Sciences version
22 (SPSS 22, SPSS Inc., USA) was used for statistical analyses. p < 0.05 was considered as
statistically significant, p < 0.01 and p < 0.001 were considered as highly significant.
Supporting Information
Electronic supplementary information (ESI) available: Chemical synthesis and characterization,
Fluorescence Spectroscopy, Photobleaching, Cytotoxicity of GSH and BSO, Cellular drug
absorption, TEM images, ROS generation of Len NPs, Intro cytotoxicity of Len NPs.
Conflicts of interest
There are no conficts to declare.
Acknowledgment
We gratefully acknowledge the financial support from National Natural Science Foundation of
China (81673369, 81703335), Natural Science Foundation of Guangdong (2017A030313069),
Guangdong Medical Research Foundation (A2017114) and the Fundamental Research Funds for
the Central Universities (18ykpy25).
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