Journal of Colloid and Interface Science 598 (2021) 213–228

Contents lists available at ScienceDirect

Journal of Colloid and Interface Science

journal homepage: www.elsevier.com/locate/jcis

Regular Article

Redox-responsive hyaluronic acid-based nanoparticles for targeted

photodynamic therapy/chemotherapy against breast cancer
Rujuan Wang 1, Haotong Yang 1, Abdur Rauf Khan, Xiaoye Yang, Jiangkang Xu, Jianbo Ji, Guangxi Zhai ⇑
Department of Pharmaceutics, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan 250012, PR China

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o a b s t r a c t

Article history: Specific cellular uptake and sufficient drug release in tumor tissues are important for effective cancer
Received 2 February 2021 therapy. Hyaluronic acid (HA), a skeleton material, could specifically bind to cluster determinant 44
Revised 10 April 2021 (CD44) receptors highly expressed on the surface of tumor cells to realize active targeting. Cystamine
Accepted 12 April 2021
(cys) is sensitive highly reductive environment inside tumor cells and was used as a connecting arm to
Available online 16 April 2021
connect docosahexaenoic acid (DHA) and chlorin e6 (Ce6) to the HA skeleton to obtain redox-sensitive
polymer HA-cys-DHA/Ce6 (CHD). Nanoparticles were fabricated and loaded with chemotherapeutic drug
Keywords:
docetaxel (DTX) by physical encapsulation. The prepared nanoparticles had significantly increased
Chemo-photodynamic therapy
Redox-responsive
uptake by MCF-7 cells that overexpressed CD44 receptors, and DTX was effectively released at high
Tumor-targeting reducing condition. Compared with mono-photodynamic therapy (PDT) or mono-chemotherapy, the

Abbreviations: CD44, cluster determinant 44; Ce6, chlorin e6; CHD, HA-cys-DHA/Ce6; CLSM, confocal laser scanning microscope; cys, cystamine; DAPI, 40 ,6-diamidino-2-
phenylindole; DCFH-DA, dichlorofluoresceindiacetate; DHA, docosahexaenoic acid; DLS, dynamic light scattering; DMA, 9, 10-dimethyl anthracene; DMEM, Dulbecco’s
modified eagle medium; DMF, N, N-dimethylformamide; DMSO, dimethyl sulfoxide; DS, degree of substitution; DTT, dithiothreitol; DTX, docetaxel; EDCI, 1-(3-
Dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride; EDTA, ethylene diamine tetraacetic acid; EPR, enhanced permeability and retention; FCM, flow cytometry; FITC,
fluorescein isothiocyanate; FRET, fluorescence resonance energy transfer; FT-IR, Fourier transform infrared spectroscopy; GSH, glutathione; HA, hyaluronic acid; HER-2,
human epidermal growth factor receptor-2; HOBt, hydroxybenzotriazole; HPLC, high performance liquid chromatography; MFI, mean fluorescence intensity; NAC, n-
acetylcysteine; NDDSs, nano drug delivery systems; NHS, N-Hydroxysuccinimide; NIR, near-infrared; PBS, phosphate buffer saline; PDI, polydispersity index; PDT,
photodynamic therapy; PI, propidium iodide; PLA, poly lactic acid; ROS, reactive oxygen species; PS, photosensitizer; TEM, transmission electron microscope; TNBC, triple-
negative breast cancer; UV Vis, ultraviolet-visible; VEGF, vascular endothelial growth factor.
⇑ Corresponding author at: Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua Xilu, Jinan 250012, PR China.
E-mail address: professorgxzhai@126.com (G. Zhai).
1
These two authors contributed equally to this study.

https://doi.org/10.1016/j.jcis.2021.04.056
0021-9797/Ó 2021 Elsevier Inc. All rights reserved.
R. Wang, H. Yang, Abdur Rauf Khan et al.
Docosahexaenoic acid prepared nanoparticles exhibited superior anti-tumor effect by inhibiting microtubule depolymerization,
Chlorin e6 blocking cell cycle and generating reactive oxygen species (ROS). In vivo anti-tumor experiments proved
Hyaluronic acid that DTX/CHD nanoparticles had the best antitumor response versus DTX and CHD nanoparticles under
Docetaxel
near-infrared (NIR) irradiation. These studies revealed that redox-responsive DTX-loaded CHD nanopar-
ticles held great potential for the treatment of breast cancer.
1. Introduction
Breast cancer has a highest morbidity and mortality in females,
seriously threating to women’s health [1]. There are several obsta-
cles limiting the efficacy of chemotherapy. First, breast cancer has
inter- and intra-tumoral heterogeneity [2]. So, conventional treat-
ment does not work well, especially for triple-negative breast can-
cer (TNBC) which lacks endocrine and targeted therapy receptors
against human epidermal growth factor receptor-2 (HER-2) [3].
Second, platinum, paclitaxel, adriamycin, cyclophosphamide, fluo-
rouracil and other cytotoxic drugs commonly used in the treat-
ment of breast cancer cannot get ideal result and may even cause
systemic toxicity like immunosuppression, bone marrow suppres-
sion, cardiotoxicity and gastrointestinal reactions due to the rapid
clearance, non-selective distribution and inactivation of free drugs
in the body [4]. Third, chemotherapy may result in drug resistance
at cellular or tumor microenvironment level. Fourth, it is difficult
to eradicate cancer stem cells [5].
In recent years, nano drug delivery systems (NDDSs) have
attracted wide attention and the delivery of anti-breast cancer
drugs by NDDS has become a research hotspot. They can increase
the solubility of insoluble drugs, realize passive targeting to tumor
sites through enhanced permeability and retention effect (EPR) of
solid tumors, and reduce the accumulation of drugs at other sites.
In addition, carriers can be modified to allow them the ability of
active targeting. Therefore, drugs loaded in the NDDSs are consid-
ered to have a better chemotherapeutic index. To date, a number of
anti-tumor NDDSs have been approved by FDA for clinical applica-
tions, such as DoxilÒ, albumin-bound paclitaxel nanoparticles
(AbraxaneÒ), and poly lactic acid (PLA)-based micellar formulation
of paclitaxel (GenexolÒ) [6]. Currently, extensively studied recep-
tors exploited for active targeting in breast cancer include folate-
folate receptor that are highly expressed in 50–86% of patients
with metastatic TNBC [7] HA-HA receptor [8] transferrin-
transferrin receptor [9] RGD peptide (Arg-Gly-Asp)-integrin [10]
vascular endothelial growth factor (VEGF)-VEGF receptor.
With in-depth understanding of tumor and tumor microenvi-
ronment, researchers found that tumor proliferation, invasion
and metastasis are highly correlated with the abnormal character-
istics of tumor microenvironment (including hypoxia, increased
acidity, abnormal expression of enzymes, high tissue pressure,
abnormal expression of inflammation and tumor-related factors,
etc. [11]). Taking advantage of tumor microenvironment, drug
delivery system responsive to specific stimuli could be designed
to release drugs at specific sites, increase carrier delivery efficiency,
improve bioavailability of drugs, and reduce toxic and side effects
[12]. Glutathione (GSH) is a potent antioxidant that is widely found
in mammalian tissues. Intracellular GSH concentration (3–10 mM)
is about three times higher than extracellular GSH concentration
(about 2.8 lM) in tumor cells [13] and GSH concentration in tumor
tissues is higher than that in normal tissues. Therefore, the redox
potential difference can be used as a potential stimulus for drug
release. Currently, encapsulating drugs inside redox-sensitive
polymer nanocarriers has been the most studied method [14].
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Journal of Colloid and Interface Science 598 (2021) 213–228
Ó 2021 Elsevier Inc. All rights reserved.
PDT as a minimally invasive or non-invasive therapy has been
widely used in various types of cancer and other diseases. It leads
to more apoptosis and cell death at low and high light intensity,
respectively [15]. PDT can be used repeatedly with low cumulative
toxicity and no drug resistance. The combination of chemotherapy
and PDT is very promising in the treatment of tumors, which is
usually realized by co-loading chemotherapeutic drugs and photo-
sensitizers (PS) into nanocarriers.
HA is a natural linear mucopolysaccharide found in all verte-
brates. CD44 receptor, being a member of the transmembrane gly-
coprotein family, is the most widely studied HA receptor. Standard
CD44 (CD44s) is widespread in normal cells, while variant CD44
(CD44v) is overexpressed on the surface of various epithelial tumor
cells and is associated with tumor progression. CD44v shows
higher affinity for HA than CD44s receptor [16]. Because of its good
biocompatibility, biodegradability, non-immunogenicity and tar-
geting potential, HA can be modified to fabricate a good drug deliv-
ery carrier.
DHA is listed by FDA as a nutritional supplement, and many
studies have shown that it has anti-tumor effects [17–19]. Up to
now, the use of DHA in conjunction with PS has not been reported.
In this study, hydrophilic HA molecules were modified with
hydrophobic docosahexaenoic acid (DHA) and Ce6 via cys as a con-
necting arm to obtain amphiphilic HA derivatives to encapsulate
DTX (Scheme 1B). The hydrophilic skeleton HA conferred CD44
receptor targeting property to the amphiphilic polymer, which
could realize active targeting of tumor with high expression of
CD44 receptor. The polymers could self-assemble into nanoparti-
cles with core–shell structure in water. DHA acted as a hydropho-
bic core to load hydrophobic chemotherapy drug DTX. Ce6 was
used to achieve PDT by producing ROS to kill tumor cells. The
cys linking arm could give the nanoparticles reduction-response
characteristics to trigger drug release in tumor microenvironment.
In addition, the fluorescence characteristic of Ce6 was used to real-
ize fluorescence imaging in vivo to assist the diagnosis and treat-
ment of tumors. This study provided experimental and
theoretical basis for exploring the application of environment-
sensitive nanoparticles for combined chemotherapy and PDT of
tumor.
2. Materials and methods
2.1. Materials
Sodium hyaluronate (Mw = 9000 Da) (medicinal grade) was
obtained from Huaxi Biological Technology Co., Ltd. (Shandong,
China). DHA (98%) was purchased from Innochem (Beijing,
China). Ce6 (93–98%) was purchased from Frontier (Simi Valley,
CA). DTX (99.0%) was purchased from Sian Biochemical Technology
Co., Ltd. Cys (97.0%), N-(3-Dimethylaminopropyl)-N0 -ethylcarbodii
mide (EDCI) (98.0%), N-Hydroxysuccinimide (NHS) (98.0%) and 1-
Hydroxybenzotriazole (HOBt) (97.0%) were purchased from Alad-
din (Shanghai, China). Formamide (analytical reagent), N, N-
dimethylformamide (DMF) (analytical reagent) and dimethylsul-
R. Wang, H. Yang, Abdur Rauf Khan et al.
Scheme 1. (A) Synthesis routes of CHD. (B) Self-assembly of DTX/CHD NP (a) and selective accumulation in tumor tissue (b), and illustration of the synergetic chemotherapy/
PDT of DTX/CHD NPs (c).
foxide (DMSO) (analytical reagent) were purchased from Sino-
pharm. Methanol (HPLC grade) and Acetonitrile (HPLC grade) were
purchased from Oceanpak (Sweden). Dialysis bag (MWCO = 3500
Da) was purchased from Biosharp (Shanghai, China). The synthesis
processes of HA-cys, HA-cys-DHA and CHD were described in the
supporting information.
MCF-7 cells and 4T1 cells were provided by School of Pharmacy,
Shandong University. Animals were purchased from Beijing Viton
Lihua Experimental Animal Technology Co., Ltd.
2.2. Characterization of CHD conjugates
Fourier transform infrared spectroscopy (FT-IR) (Nicolet 67,000/
NXR, ThermoFisher, USA) and 1H NMR spectroscopy (600 MHz,
AV600, BRUKER, Germany) were used to evaluate the structure of
CHD conjugates. 1H NMR spectroscopy and ultraviolet–visible
(UV Vis) spectrophotometer were used for calculating the degree
of substitution (DS) of DHA and Ce6, respectively.
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Journal of Colloid and Interface Science 598 (2021) 213–228
2.3. Preparation and characterization of polymeric nanoparticles
2.3.1. Preparation of nanoparticles
Blank nanoparticles were prepared by probe ultrasound
method. Briefly, 3 mg CHD was dissolved in 1.8 mL phosphate buf-
fer saline (PBS) (pH = 7.4, 10 mM), and then treated with probe
sonicator (YM-150, Shanghai Yuming Instrument Co., Ltd, China)
for 5 min at 50 W (2 s on, 4 s off). The ultrasonic sample was fil-
tered through 0.8 lm microporous membrane filter to get CHD
nanoparticles solution.
DTX/CHD nanoparticles were prepared by the same method.
7.5 mg CHD was dissolved in 2.5 mL PBS, and DTX dissolved in
methyl alcohol was added drop-wise under agitation. Then the
solution was treated with ultrasound for 10 min at 90 W. After
that, solution was sonicated with probe sonicator (40 W, 4 s on,
2 s off, 5 min). The resulting solution was put into a dialysis bag
and dialyzed in distilled water overnight. The sample was then
centrifuged at 4000 rpm for 10 min and filtered through 0.8 lm
microporous membrane filters to get DTX/CHD nanoparticles.
R. Wang, H. Yang, Abdur Rauf Khan et al.
2.3.2. Characterization of nanoparticles
The prepared solution of nanoparticles was diluted with dis-
tilled water, and then the dynamic light scattering (DLS) (Delsa
Nano, Beckman Coulter, Inc., USA) was applied to measure the par-
ticle size and corresponding zeta potential of the nanoparticles.
The morphology of nanoparticles was observed under transmission
electron microscope (TEM) (JEM-100CX II, Hitachi, Japan).
2.4. In vitro study of the redox responsive behaviors
2.4.1. Particle size observation
DTX/CHD nanoparticles were put into PBS (pH = 7.4, 10 mM) or
PBS (pH = 7.4, 10 mM) containing 20 mM dithiothreitol (DTT).
Then, they were placed in a thermostatic oscillator (37 °C,
100 rpm) for 12 h. Afterwards, the particle size and size distribu-
tion were measured by DLS.
2.4.2. Fluorescence recovery
Ce6 was connected to the CHD polymer. Due to the hydrophobic
effect, Ce6 would accumulate in the hydrophobic core of nanopar-
ticles in large quantities, resulting in concentration quenching and
fluorescence attenuation. In the reduction environment, the frac-
ture of disulfide bond in the connecting arm led to dissociation
of Ce6 from the polymer and decrease concentration quenching,
which was manifested as the recovery of fluorescence and also
reflected the reduction responsiveness of CHD polymer [20].
The polymer CHD was placed in a reducing environment, and
the fluorescence intensity was compared with that of free Ce6
and CHD without redox-treatment. Briefly, an appropriate amount
of Ce6 was dissolved in DMSO, with a concentration of 1 mg/mL.
The blank CHD nanoparticle solution was prepared according to
the method above mentioned. The Ce6 solution was diluted with
PBS to 5 lg/mL. In addition, a portion of diluted CHD nanoparticle
solution was taken, and an appropriate amount of DTT solution
was added to achieve final the DTT concentration of 20 mM. The
nanoparticle solution was placed in a constant temperature oscilla-
tor and incubated for 2 h and 4 h (37 °C, 100 rpm). Fluorescence
emission spectrum of each sample were recorded (excitation
wavelength was 405 nm and emission wavelength was 500–
700 nm).
2.4.3. Production of singlet oxygen under reduction conditions
Solutions of free Ce6 and blank CHD nanoparticle were pre-
pared with Ce6 equivalent concentration of 5 lg/mL by the
above-mentioned method. An appropriate amount of DTT solution
was added to the blank nanoparticle solution to obtain CHD
nanoparticle solution to obtain final DTT concentration of
20 mM. In addition, an appropriate amount of 9, 10-dimethyl
anthracene (DMA) was added to each solution to get DMA concen-
tration of 20 lM. Then, each solution was irradiated with a 660 nm
laser (1 W, 10 min), and the fluorescence emission spectrum (exci-
tation wavelength: 360 nm, emission wavelength: 380–550 nm) of
fluorescence DMA was recorded after 1 h in dark condition
2.4.4. Drug release study
PBS (pH 7.4) containing 0.5% tween-80 was used as the releas-
ing medium [21]. The release media with DTT concentration of
20 mM and 20 lM were used to simulate the high-reduction tumor
microenvironment and the low-reduction environment of normal
tissues, respectively. The release behavior of DTX/CHD in different
media was studied by dialysis method [22]. Briefly, 1 mL moder-
ately diluted DTX/CHD nanoparticles solution was taken into a
dialysis bag, and the dialysis bag was immersed in a conical tube
containing 40 mL releasing medium and placed in an incubator
(37 °C, 100 rpm). Release medium (3 mL) was taken at each time
point and the same amount of release medium was added. The
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Journal of Colloid and Interface Science 598 (2021) 213–228
sample was injected to HPLC after filtered through 0.22 lm micro-
porous membrane filters, and the content of DTX was calculated
according to the established method, and then the time-release
curve was drawn.
2.5. In vitro cell studies
2.5.1. Cell culture
Human breast cancer MCF-7 cells were cultured in Dulbecco’s
modified eagle medium (DMEM) containing 1% triple antibody
and 10% fetal bovine serum in a cell incubator at 37 °C and 5% CO2.
2.5.2. Cell uptake study
MCF-7 cells were digested, centrifuged and collected to prepare
a cell suspension of 7.5  104 cell/mL, and 2 mL of cell suspension
was inoculated into a sterile 12-well plate (1.5  105 cell/well).
Well plate was cultured overnight in an incubator. Then, the cells
were washed twice with PBS and 2 mL free Ce6 or CHD with Ce6
concentration of 5 lg/mL were added and cultured in an incubator
(in the HA blocking group, culture medium containing HA 10 mg/
mL was used to saturate the CD44 receptor on the cell surface
before the addition of CHD medium). Cells were fixed with 4%
paraformaldehyde for 15 min at room temperature. After removing
paraformaldehyde, cells were washed with PBS for three times.
40 ,6-diamidino-2-phenylindole (DAPI) was added and kept at room
temperature for 5 min. The cells were washed with PBS and 200 lL
sterile PBS was added. The cells were observed under confocal laser
scanning microscope (CLSM) (LSM780, Carl Zeiss, Inc., Jena,
Germany).
Flow cytometry (FCM) (CytoFLEXS,Beckman Instruments, Inc.
USA) was used to quantitatively investigate the uptake of each
preparation by using Ce6 as a fluorescent probe. MCF-7 cells were
prepared into a cell suspension (8  104 cell/mL), which was inoc-
ulated into a 24-well plate (1 mL/well). After incubation overnight,
the cells were washed twice with sterile PBS. Then, free Ce6 solu-
tion or CHD with Ce6 concentration of 5 lg/mL was added, and the
cells were cultured in a cell incubator for 1 h or 4 h. The medium
was washed with PBS for three times, digested with trypsin with-
out ethylene diamine tetraacetic acid (EDTA), and then re-
suspended in 200 lL PBS for analysis with FCM
2.5.3. Cytotoxicity study
The phototoxicity and dark toxicity of the preparations were
investigated in this section. Free Ce6 and CHD nanoparticle solu-
tion were diluted with culture medium to get the concentration
of Ce6 of 0.1, 0.5, 2.5, 5, 10, 20 and 40 lg/ml. MCF-7 cells were
seeded into 96-well plates (6.0  103 cells/well) and incubated
for 24 h. Then the cells were washed twice with PBS and 100 lL
medium containing preparations was added and cells were further
incubated for 12 h. After that, for NIR irradiation group, the cells
were washed with PBS and 100 lL fresh medium was added, then
the cells were irradiated with a 660 nm laser for 1 min at 50 mW/
cm2. For non-irradiation group, cells were washed with PBS and
100 lL fresh medium was added without exposure to NIR. After
12 h further incubation, 10 lL cck-8 reagent was added and the
cells were incubated for 40 min to 1 h and then optical density
was measured at 450 nm with the help of microplate reader
(ELx800, Bio Tek Instruments, Inc., Highland Park, IL), and the cell
survival rate was calculated according to the following formula:
ODsampleODblank
Cell viabilityð%Þ ¼  100%
ODcontrol  ODblank
ODsample was the optical density value of the experimental
group; ODblank was the optical density value of the blank group;
ODcontrol was the optical density value of the control group.
R. Wang, H. Yang, Abdur Rauf Khan et al.
The cytotoxicity of DTX/CHD nanoparticles were evaluated by
the same method. The concentrations of DTX were 0.1, 0.1, 0.5,
1.0, 2.0, 4.0 and 6.0 lg/ml. The corresponding concentrations of
Ce6 were 0.015, 015, 0.75, 1.5, 3.0, 6.0 and 9.0 lg/ml.
2.5.4. Cell apoptosis assay
MCF-7 cells were seeded into 12-well plate (5  105 cell/mL)
and incubated for 24 h. Cells were washed with PBS twice and
medium containing free Ce6, CHD nanoparticles, free DTX or
DTX/CHD nanoparticles with Ce6 and DTX concentration of
1.5 lg/mL and 1 lg/mL, respectively, were added. After incubation
for 12 h, the cells were washed twice with PBS and 1 mL fresh med-
ium was added. NIR irradiation groups were irradiated with a
660 nm laser for 1 min at a power of 50 mW/cm2 and non-NIR
groups were not processed. After incubation of further 6 h, the cells
were washed twice with PBS and digested with trypsin. The cells
were collected and centrifuged. Finally, the cells were treated
according the procedure suggested in the Annexin V-FITC/PI Dou-
ble Dye Kit (Dojindo, Japan). The result was recorded using FCM
within 1 h (n = 3).
2.5.5. Co-localization of lysosome
The lysosomal escape of CHD nanoparticles under light condi-
tions was studied as follows. MCF-7 cells were digested and cen-
trifuged to prepare cell suspension of 5  104 cell/mL. Then they
were inoculated to black laser confocal culture 12-well plates
(1 mL/well). After incubation for 12 h, the cells were washed twice
with PBS. Then the medium containing free Ce6 or CHD (Ce6 = 4
lg/mL) was added and cells were incubated for further 4 h. After
that, the cells were washed twice with PBS and NIR irradiation
groups were irradiated by a 660 nm laser for 1 min at a power of
50 mW/cm2 and non-NIR groups were not processed. Thereafter,
lysosome green fluorescent probe of LysoTracker Green DND-26
prepared according to the supplier’s specification was added
(100 lL/well) and the cells were further incubated for 1 h. Then
the cells were washed twice with PBS and observed under high
intensity confocal microscope (Opera Phenix, PerkinElmer, USA)
after nuclei staining with DAPI.
2.5.6. Detection of intracellular ROS
PDT could induce cell damage or apoptosis through ROS gener-
ated by PS, and the amount of ROS generated determined the effect
of PDT. In this study, the ability of preparation to produce ROS in
cells after irradiation was investigated through ROS detection kit.
MCF-7 cells were digested, and a cell suspension of 5  105 cell/
mL was prepared. Cell suspension was inoculated into a 12-well
plate and incubated for 12 h in the cell incubator for adherent
growth. Then the original medium was discarded, and fresh med-
ium, medium containing free Ce6 or CHD (Ce6 = 4 lg/ml) was
added, then the cells were further cultured for 4 h. For the negative
control group, the cells were pre-incubated with n-acetylcysteine
(NAC) for 1 h before the drug containing medium was added to
remove the ROS generated by the subsequent operation. There-
after, the culture medium was discarded, and medium containing
10 lM dichlorofluorescein diacetate (DCFH-DA) probe was added
(1 mL/well). Then NIR irradiation groups were irradiated with a
660 nm laser for 1 min at a power of 50 mW, whereas, non-NIR
groups and negative control groups were not processed. The cells
were washed three times with serum free medium after incubated
for 30 min in dark. Finally, the cells were observed with fluores-
cence microscope (Olympus, Japan).
2.5.7. Cell cycle study
MCF-7 cells were digested and centrifuged to prepare a cell sus-
pension of 7.5  104 cell/mL. The cells were seeded into 6-well
plate (1.5  105 cells/well) and inoculated for 12 h. After that,
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Journal of Colloid and Interface Science 598 (2021) 213–228
the culture medium was discarded and blank medium, medium
containing CHD nanoparticles, free DTX or DTX/CHD nanoparticles
were added (Ce6 = 1.5 lg/mL, DTX = 1 lg/mL]. After incubation for
12 h, fresh medium was added to replace the original medium. The
NIR irradiation groups were irradiated with a 660 nm laser for
1 min at a power of 50 mW/cm2, and non-NIR groups were not pro-
cessed. The cells were washed twice with PBS after incubation for
6 h. After that, the cells were digested and centrifuged, and 0.5 mL
of precooled 70% ethanol was added for fixation at 4 °C overnight.
After that, an appropriate amount of PBS was added, and the fixa-
tion solution was removed by centrifugation. Then, 50 lL RNase A
solution was added to the cell precipitation. The cells were resus-
citated and bathed in water at 37 °C for 30 min. Then the cells were
mixed with 200 lL PI staining solution and incubated at 4 °C for
30 min under dark condition. Thereafter, the cells were detected
by FCM and the results were analyzed using Modfit software.
2.5.8. Microtubules detection test
The cytoskeleton, the subsurface struts of cells containing actin
and tubulin, is a dynamic structure essential for a wide range of
normal cellular processes, including maintenance of cell morphol-
ogy, membrane dynamics, and signal transduction [23,24]. DTX
can interfere with the microtubule system in mitosis and block
the proliferative cells in G2/M phase. Apoptosis, or necrosis, is usu-
ally associated with morphological features, including nuclear
shrinkage, plaque rupture, plasma membrane rupture, etc [25]. In
order to explore the influence of DTX and PDT on microtubules
in cell division, and further explore the antitumor effect of prepa-
ration, the microtubules were detected with a red fluorescent
probe. Briefly, MCF-7 cells were digested and centrifuged to pre-
pare a cell suspension of 5  104 cell/mL. Then the cells were
seeded into black laser confocal culture 12-well plates (1 mL/well)
and the culture medium was abandoned after incubation for 12 h.
Thereafter, fresh medium containing free DTX, CHD NPs or DTX/
CHD NPs (Ce6 = 1.5 lg/mL, DTX = 1 lg/ml] were added and the
cells were further incubated for 12 h. Then, fresh medium was
added to replace the original medium and NIR irradiation groups
were irradiated with a 660 nm laser for 1 min at a power of
50 mW/cm2, and non-NIR groups were not processed. After incu-
bation for 4 h, the cells were washed twice with PBS. Next, the cells
were fixed with 4% paraformaldehyde for 15 min and washed
twice with PBS containing 0.1% Triton, 5 min each time. The work-
ing solution of the microtubule red fluorescent probe was prepared
according to the supplier’s instructions. The prepared staining
solution was added to the 12-well plate and the cells were incu-
bated at room temperature for 45 min under dark condition. Then
the cells were washed twice with PBS containing 0.1% Triton, 5 min
each time. After DAPI staining, 200 lL serum-free medium was
added to each well and the cells were observed with high intensity
confocal microscope.
2.6. In vivo biological evaluation of nanoparticles
2.6.1. Animal and tumor models
Female BALB/C mice (6–7 weeks old) were purchased from Bei-
jing Charles River Co. Ltd., and all animals were handled in compli-
ance with the guidelines outlined in the Guide for the Care and Use
of Laboratory Animals (SYXK 2019 0005). 4T1 cells (1  107) were
injected into the armpit to establish a mouse breast tumor model.
2.6.2. Biodistribution study of nanoparticles
When the tumor volumes reached 150 mm3, mice bearing 4T1
cells were randomly divided into two groups (n = 3 per group) and
then intravenously injected with free Ce6 or CHD NPs via tail vein
at a Ce6 dose of 4 mg/kg. At predetermined time points (4 h, 8 h,
12 h, 24 h), in vivo fluorescence in tumor bearing mice was
R. Wang, H. Yang, Abdur Rauf Khan et al.
detected using a Xenogen IVIS Lumina system (Caliper Life
Sciences, USA). At 24 h post-injection, the mice were sacrificed,
and the solid tumor tissues and main organs including heart, liver,
spleen, lung and kidney were collected, washed with saline and
imaged using the Xenogen IVIS Lumina system for semi-
quantitative analysis.
2.6.3. In vivo anti-tumor efficacy
When the tumor volumes of the mice reached 100 mm3, the
mice bearing 4T1 tumors were randomly divided into six groups
(n = 5 per group) of normal saline group (NS group), Ce6 + NIR
group, CHD + NIR group, DTX group, DTX/CHD group, DTX/
CHD + NIR group and intravenously injected at DTX dose of
10 mg/kg. In the NIR radiation groups, the tumor sites of the mice
were illuminated by a 660 nm laser at 1 h and 24 h after adminis-
tration (200 mW/cm3, 15 min). The tumor volume of mice was
measured by Vernier Caliper at a fixed time every other day after
administration, and the tumor volume was calculated by the fol-
lowing formula:
2
ab
Tumor volume ¼
2
where a representing the tumor length diameter (mm), and b was
the tumor short diameter (mm).
Tumor growth curve of each group was obtained by plotting by
the ratio of tumor volume to initial tumor volume. The mice were
sacrificed at 14th day, and the tumors were stripped out, washed
with normal saline. The tumor was weighed and photographed.
The exfoliated tumor tissues were fixed with 4% paraformalde-
hyde, and H&E staining and histological analysis were performed.
2.6.4. Safety analysis
Weight changes of mice were used to evaluate the safety of
each preparation. The mice were weighed by electronic balance
every other day. At 14th day of tumor suppression experiment,
mice in NS group and DTX/CHD + NIR group were sacrificed, and
the heart, liver, spleen, lung and kidney were separated and fixed
with 4% polyformaldehyde for H&E staining analysis.
2.7. Statistical analysis
Statistical analysis was performed using SPSS 19.0. The results
were reported as mean ± the standard deviation (SD). *p values <
0.05 and **p values < 0.01 were considered statistically significant
in all analysis.
3. Results and discussion
3.1. Characterization of CHD conjugate
The characteristic peaks of HA mainly included hydroxyl
stretching vibration m(OH) around 3300 cm1, and carboxylic acid
(R-COO-) asymmetric and symmetric stretching vibration at
1605.53 cm1 and 1404.93 cm1 respectively (Fig. S1). Compared
with HA, there was no obvious absorption peak of amide bond in
HA-cys, it was assumed that the number of synthesized amide
bonds was small, and was covered by the absorption peak of ion-
ized carboxyl group (R-COO-). HA-cys-DHA showed obvious amide
I band at 1639.25 cm1. With the appearance of peaks at
1544.96 cm1 (amide II band) and 1318.95 cm1 (amide III band),
the successful formation of amide bond was proved.
In 1HNMR spectrum, the acetyl group proton signal of HA at the
chemical shift of 1.94 ppm, and a sugar ring proton peak at 3.2–
4.5 ppm were used to calculate the graft ratio of HA (Fig. 1). In
the 1HNMR spectrum of HA-cys, two methylene proton peaks of
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Journal of Colloid and Interface Science 598 (2021) 213–228
cys appeared at 3.28 ppm and 2.86 ppm, proving that cys was suc-
cessfully connected to HA [26,27]. The DS (number of cys mole-
cules grafted per 100 HA monomers) of cys in HA-Cys was 30.8
according to the ratio of peak area of acetyl methyl peak
(1.94 ppm) to cys imide peak (2.86 ppm) [28]. In the 1HNMR spec-
trum of HA-cys-DHA, characteristic peaks of DHA appeared at
chemical shifts of 5.3–5.5 ppm (m, 10H, 5-CH = CH-) and 2.75 to
2.9 ppm (m, 8H, 4CH2) and there was no peak of carboxyl proton
(chemical shift was 12.09 ppm), which proved the successful con-
nection of DHA and HA. The DS of DHA was calculated according to
the ratio of characteristic peak area of peak at 5.3–5.5 ppm (m,
10H, 5-CH = CH-) to the peak area at 1.94 ppm (acetyl methyl peak
of HA) [18]. DS of DHA in HA-cys-DHA was 3.4, 4.4 and 8.8, respec-
tively, at the feed ratios of 10:1, 5:1 and 2.5:1 (HA: DHA). DHA was
used to form hydrophobic core loading DTX. In order to increase
the drug loading, HA-cys-DHA with the highest degree of DHA sub-
stitution (8.8%) was selected as a substrate for the subsequent syn-
thesis reaction. In the 1HNMR spectrum of the final product CHD, in
addition to the characteristic peak of HA-cys-DHA, the characteris-
tic peak of Ce6 appeared in the range of 6.0–10 ppm, which proved
the successful connection of Ce6.
3.2. Characterization of nanoparticles
The nanoparticles were analyzed by DLS to obtain the particle
size and Zeta potential values (Fig. 2Aa, Fig. 2Ba). The average
diameter of CHD nanoparticles was 214.1 ± 3.3 nm, PDI was 0.25
5 ± 0.016, and Zeta potential was 29.4 ± 0.6 mV. The particle size
of the drug-loaded nanoparticles (DTX/CHD nanoparticles) was
181.4 ± 1.8 nm, the PDI was 0.241 ± 0.039, and the zeta potential
was 22.9 ± 0.7 mV. Compared with blank nanoparticles, the par-
ticle size of DTX/CHD nanoparticles reduced from 214.1 ± 3.3 nm to
181.4 ± 1.8 nm after loading DTX, which may be due to the
enhanced hydrophobic effect of the core of the nanoparticles after
DTX loading [29]. According to the TEM images of CHD and DTX/
CHD nanoparticles (Fig. 2Ab, Fig. 2Bb), both CHD and DTX/CHD
nanoparticles were spherical in shape and finely dispersed, the size
of which distributed in the range of 100 nm-350 nm, generally in
accordance with the size distribution profiles shown in Fig. 2Aa
and Ba.
3.3. In vitro study of redox responsive behavior
3.3.1. Particle size observation
After DTT incubation, the particle size of DTX/CHD nanoparti-
cles increased from 181.4 nm to 212.9 nm (Fig. 2C), polydispersity
index (PDI) changed from 0.241 to 0.517 and zeta potential chan-
ged from 22.9 mV to 25.6 mV. The unimodal size distribution
peak was changed to bimodal as nanoparticles larger than
1000 nm and smaller than 100 nm were observed. The breaking
of disulfide bond under the reducing environment resulted in sep-
aration of hydrophobic and hydrophilic segments of the polymer,
thereby nanoparticles had a large particle size and a wide particle
size distribution.
3.3.2. Fluorescence recovery
As Ce6 was connected to CHD polymer, it could aggregate in the
hydrophobic core of nanoparticles due to hydrophobic effect,
resulting in concentration quenching, which in turn reduced fluo-
rescence [30]. The fluorescence intensity of free Ce6 was the stron-
gest, while that of untreated CHD nanoparticles was the weakest
(Fig. 2D). With the extension of incubation time in the presence
of DTT, the fluorescence intensity of CHD nanoparticles was closer
to that of free Ce6. This was due to the fracture of disulfide bond in
the connecting arm in the reduction environment, which made Ce6
free from the polymer and reduced the concentration quenching
R. Wang, H. Yang, Abdur Rauf Khan et al.
Fig. 1. 1H NMR spectra of HA,HA-cys,DHA,HA-cys-DHA,Ce6 and CHD.
[31]. The results showed fluorescence recovery, which reflected the
reduction sensitivity of CHD polymer. The results of this experi-
ment proved that under the irradiation of laser, nanoparticles
could produce strong fluorescence in the reduced microenviron-
ment of tumor, which was more conducive to the fluorescence
imaging of nanoparticles and had the potential for tumor imaging
diagnosis.
3.3.3. Production of singlet oxygen under reduction conditions
The ability of nanoparticles to generate reactive oxygen under
NIR irradiation could be used to evaluate whether nanoparticles
could meet the requirements of PDT. DMA was a commonly used
organic fluorescent probe for the detection of singlet oxygen. It
could selectively capture singlet oxygen to form inner ring oxide,
resulting in fluorescence disappearance. In this experiment, DMA
was used to detect the reactive oxygen species. As shown in
Fig. 2E, the fluorescence intensity of free Ce6 group was the weak-
est, while that of CHD group was the strongest, indicating that free
Ce6 had the strongest ability to produce reactive oxygen species
under NIR irradiation, while CHD nanoparticles were the weakest.
In the presence of DTT, the ability of CHD to produce reactive oxy-
gen species was improved. The possible reason was the occurrence
of fluorescence resonance energy transfer (FRET) [32]. FRET was
due to the short distance between the molecules, about 7–
10 nm. When the emission spectrum of one fluorescent molecule
overlapped with the excitation spectrum of another fluorescent
molecule, the energy was transferred from the fluorescent donor
molecule to the fluorescent acceptor molecule, thereby preventing
the change of Ce6 molecule from singlet state to triplet state,
which in turn affected the generation of ROS, leading to enhanced
DMA fluorescence intensity. In the reductive environment, the
disulfide bond was broken by DTT, and the structure of the
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Journal of Colloid and Interface Science 598 (2021) 213–228
nanoparticles was disturbed, so the distance between Ce6 mole-
cules became longer, which broke the FRET, resulting in an increase
in the amount of reactive oxygen produced [33]. This result indi-
cated that in the reduced environment of tumor, the structure of
nanoparticles was destroyed to release free Ce6, which was more
conducive to the production of ROS.
3.3.4. Drug release study
The release of DTX from DTX/CHD nanoparticles was measured
at physiological pH 7.4 for 48 h. To simulate the reducing tumor
microenvironment and physiological environment, 20 mM DTT
and 20 lM DTT were used respectively. The obtained release data
was fitted to Weibull release model (DDsolver software) [34,35]. b
values of DTX/CHD, DTX/CHD + 20 lM DTT and DTX/CHD + 20 mM
DTT were 0.492, 0.524 and 0.616, respectively. As shown in Fig. 2F,
in the absence of DTT, 20 lM DTT and 20 mM DTT, the cumulative
48 h release of DTX from DTX/CHD nanoparticles was 63.0 ± 2.5%,
65.8 ± 2.7% and 84.5 ± 1.7%, respectively. The release of DTX from
experimental group treated with 20 mM DTT was significantly dif-
ferent from the other two groups, indicating a fast drug release
behavior at the tumor site. While, the drug release rate did not
change significantly at low reducing condition. The initial burst
release of DTX from DTX/CHD nanoparticles in each release med-
ium might be due to the fact that part of DTX was adsorbed on
the surface of nanoparticles and rapidly dissolved in the release
medium, resulting in a rapid release.
3.4. In vitro cell studies
3.4.1. Cellular uptake studies in MCF-7 cells
The fluorescence intensity of Ce6 in each group was observed by
CLSM to investigate the uptake of nanoparticles by MCF-7 cells. As
R. Wang, H. Yang, Abdur Rauf Khan et al. Journal of Colloid and Interface Science 598 (2021) 213–228

Fig. 2. (A) Particle size distribution (a) and TEM images (b) of CHD. (B) Particle size distribution (a) and TEM images (b) of DTX/CHD. (C) Comparison of particle size
distribution of DTX/CHD NPs with or without DTT incubation (a. without DTT incubation, b. with DTT incubation). (D) Fluorescence recovery of CHD nanoparticles in reduced
environment. (E) The production of singlet oxygen by CHD nanoparticles in reduced environment. (F) Cumulative release curve of DTX/CHD nanoparticles in different media.
Data shown as mean ± SD, n = 3. (DTX/CHD) Rsqr_adj = 0.9973, b = 0.492; (DTX/CHD + 20 lM DTT) Rsqr_adj = 0.9978, b = 0.524; (DTX/CHD + 20 mM DTT) Rsqr_adj = 0.9929,
b = 0.616.

shown in Fig. 3A, in different treatment groups, the fluorescence
intensity at 4 h was higher than that at 1 h, indicating that the
uptake of free Ce6 and CHD nanoparticles by MCF-7 cells increased
with the passage of time, representing a time-dependent uptake.
The fluorescence intensity of CHD group was significantly higher
than that of Ce6 group at 1 h and 4 h, indicating that CHD nanopar-
ticles could increase cell uptake. For the cells pre-treated with HA,
the fluorescence indensity decreased significantly, indicating that
HA receptor played an important role in cell uptake of CHD
220
nanoparticles and CHD nanoparticles entered cells through CD44
receptor-mediated endocytosis. Due to the high hydrophobicity
of Ce6 and its binding with plasma protein [36] the uptake of free
Ce6 by MCF-7 cells after incubation for 1 h and 4 h was very low.
The uptake of each preparation by MCF-7 cells was quantified
by FCM (Fig. 3B). The results of FCM analysis were consistent with
those of CLSM. The mean fluorescence intensity (MFI) of CHD
nanoparticle group at 1 h and 4 h of incubation was 9.7 and 13.9
times higher than that of the free Ce6 group, respectively, which
R. Wang, H. Yang, Abdur Rauf Khan et al.
was similar to the results in relevant literatures [37,38]. In addition
to the time-related cell uptake, this result might be related to
disulfide bond breaking in the intracellular reduction environment,
which caused Ce6 to fall off from the polymer, leading to fluores-
cence recovery. The fluorescence intensity of CHD nanoparticle
group at 1 h and 4 h was 1.7 and 1.9 times higher than that of
the HA pre-treated group, respectively, showing that the competi-
tion between HA and CHD to bind to CD44 receptor of tumor cells
significantly reduced the uptake of HA-CHD nanoparticles by MCF-
7 cells, demonstrating that CHD nanoparticles might target tumor
cells via its affinity to CD44.
3.4.2. Cytotoxicity assays
Cell viability of MCF-7 cells treated with Ce6 and CHD was
shown in Fig. 3C (a). In the non-NIR irradiated group, the cell activ-
ity of free Ce6 and CHD nanoparticles was above 90% after 24 h of
incubation. Even at 40 lg/mL (Ce6 concentration), the cell viability
of the two groups was 93.9% and 92.7%, respectively, indicating
that the toxicity of free Ce6 and blank CHD nanoparticles in the
experimental range was very low. Under NIR irradiation, when
Ce6 concentration was 0.5 lg/ml or above, the cell viability of
Ce6 and CHD nanoparticle treatment groups decreased with the
increase of Ce6 concentration, showing a concentration-
dependent effect. Cell viability of MCF-7 cells treated with Ce6
was significantly higher than that of CHD under NIR irradiation.
Under NIR irradiation, compared with Ce6, CHD nanoparticles
exhibited less cell viability, indicating that cancer cells could
uptake more CHD nanoparticles through HA receptors than Ce6.
Cytotoxicity of DTX and DTX/CHD nanoparticles was shown in
Fig. 3C (b). When the concentration of DTX was less than 0.5 lg/
ml, the cytotoxicity was low and there was no significant differ-
ence between the groups. At DTX concentration of higher than
0.5 lg/ mL, the cell survival rate of DTX/CHD group was signifi-
cantly lower than that of free DTX group, which might be related
to more cellular uptake of DTX/CHD nanoparticles. When the con-
centration of DTX was 1.0 g/mL, the cell viability of DTX group and
DTX/CHD group was 58.5 ± 2.6% and 50.3 ± 2.9%, respectively, and
Fig. 3. (A) CLSM analysis of cellular uptake in MCF-7 cells at different time. (B) FCM analysis (a) and MFI analysis (b) of MCF-7 cells at different time. Data shown as
mean ± SD, n = 3. (C) (a) Cell viability of MCF-7 cells treated with Ce6 and CHD with or without NIR irradiation. (b) Cell viability of MCF-7 cells treated with DTX, DTX/CHD,
DTX/CHD + NIR. Data shown as mean ± SD, n = 4. *indicates p < 0.05. **indicates p < 0.01.
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Journal of Colloid and Interface Science 598 (2021) 213–228
the cell viability of DTX/CHD + NIR group was 21.3 ± 2.3%, signifi-
cantly different from that of the non-NIR group, showing a good
therapeutic effect of PDT.
3.4.3. Cell apoptosis studies
In FITC-Annexin V/PI double staining analysis, Q1, Q2, Q3, and
Q4 represent necrotic cells, late apoptosis, early apoptosis, and
normal cells, respectively, and Q2 + Q3 represents total apoptosis.
As shown in Fig. 4A and Fig. 4B, in the absence of light, the total
apoptosis rate of CHD and Ce6 groups was 7.4 ± 0.3% and
7.7 ± 0.5%, respectively, with no significant difference
(p = 0.3818). After light treatment, the total apoptosis rate of
CHD + NIR and Ce6 + NIR was 29.0 ± 1.1% and 11.8 ± 0.7%, respec-
tively, with significant difference (p = 0.0397). This result was
related to the increased uptake of CHD nanoparticles by cells,
and late apoptosis mainly contributed in total, which was
26.5 ± 1.0% and 8.2 ± 0.7%, respectively, indicating that PDT led
to more late apoptosis of cells. In DTX containing formulations
groups, total apoptosis rate of DTX/CHD group was 51.2 ± 0.6%,
higher than that of DTX group (44.9 ± 1.9% (p = 0.0155), and the
early apoptosis had the major proportion (32.8 ± 1.1% and
41.6 ± 1.2%, respectively), indicating that chemotherapy led to
more total apoptosis and early apoptosis. The total apoptosis rate
of DTX/CHD + NIR group was 57.8 ± 1.1%, higher than that of
DTX/CHD group (51.2 ± 0.6% (p = 0.0204)), indicating that the com-
bined treatment group had the best therapeutic effect.
3.4.4. Co-localization of lysosome
The phagosomes formed by nanoparticle endocytosis would
fuse with lysosomes, thus achieving lysosomal escape and releas-
ing the drug was the key to killing the tumor cells [39]. According
to the results in Fig. 5A, before NIR irradiation, the red fluorescence
of Ce6 overlapped with the green fluorescence of the lysosome,
indicating that Ce6 and CHD nanoparticles were in the lysosome.
However, after NIR irradiation, the red and green fluorescence
did not coincide well (as indicated by the white arrow in the fig-
ure). At experimental doses, NIR light itself had no damage to cells,
R. Wang, H. Yang, Abdur Rauf Khan et al. Journal of Colloid and Interface Science 598 (2021) 213–228

but PS Ce6 under NIR irradiation produced ROS, destroying lyso-
some structure [40] and led to the release of substances in lyso-
somes and lysosomal fluorescence did not overlap with Ce6
fluorescence. The results showed that under the NIR irradiation
CHD nanoparticles could successfully escape lysosome uptake,
and then release DTX to kill tumor cells.
3.4.5. Detection of intracellular ROS
PDT could induce cell damage or apoptosis through ROS gener-
ated by PS, and the amount of ROS production determined the
effect of PDT. In the ROS detection kit, the fluorescent probe
DCFH-DA itself had no fluorescence, which can freely pass through
the cell membrane. It could be hydrolyzed into DCFH by esterase in
the cell, and the ROS generated in the cell could oxidize DCFH into
DCF that can emit fluorescence. The higher the concentration of
ROS in the cell, the stronger the intensity of the emitted fluores-
cence, so it can be used to evaluate the ability of cells to generate
ROS [41]. As shown in Fig. 5B, the fluorescence intensity of blank
control group was weak with or without light exposure. While,
the fluorescence of free Ce6 group and CHD group was very weak
in the absence of light, but the fluorescence intensity increased sig-
nificantly after illumination. The reason was that in the absence of
NIR, no ROS was produced after cells ingested Ce6 and CHD
nanoparticles. ROS was produced by PS Ce6 after NIR irradiation,
and the amount of ROS produced was related to cell uptake of
Ce6. Due to increased cellular uptake, the fluorescence intensity
of CHD group was much higher than that of free Ce6 group. NAC
could remove ROS produced by PDT, and the fluorescence intensity

Fig. 4. (A) FITC-Annexin V/PI double staining method for the apoptosis of MCF-7 cells (a. FITC single staining; b. PI single staining; c. double- staining control group; d. Ce6; e.
Ce6 + NIR; f. CHD; g. CHD + NIR; h. DTX; i. DTX/CHD; j. DTX/CHD + NIR). (B) Apoptosis of MCF-7 cell. Data shown as mean ± SD, n = 3. *indicates p < 0.05. **indicates p < 0.01.

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R. Wang, H. Yang, Abdur Rauf Khan et al.
of cells preincubated by NAC decreased significantly. The experi-
mental results indicated that CHD nanoparticles could play a better
PDT treatment effect.
3.4.6. Cell cycle study
The effect of DTX on the cell cycle is that it binds specifically to
the subunits of tubule proteins and inhibits their depolymeriza-
tion, leading to cell cycle arrest at G2/M phase and further activa-
tion of a series of apoptotic pathways [42]. According to the FCM
detection results (Fig. 6A), the cells at G0/G1, S and G2/M phases
Fig. 5. (A) Fluorescence localization of lysosomes and Ce6. (B) Intracellular reactive oxygen generation in MCF-7 cell observed by fluorescence microscope.
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Journal of Colloid and Interface Science 598 (2021) 213–228
of the CHD nanoparticle treatment group were 58.82%, 26.23%
and 15.16%, respectively, without NIR irradiation, and the cycle
distribution was similar to that of the control group. While, the
proportion of cells in G2/M phase increased to 22.03% after NIR
treatment, and the proportion in G0/G1 phase decreased to
49.66%, suggesting that PDT induced a series of intracellular reac-
tions and influenced the cell cycle [43–45]. Most cells in the DTX
treatment group were in the G2/M phase, accounting for 85.40%
of the total number of cells, while the proportion of G2/M phase
cells in the DTX/CHD nanoparticle treatment group was further
R. Wang, H. Yang, Abdur Rauf Khan et al. Journal of Colloid and Interface Science 598 (2021) 213–228

Fig. 6. (A) Cell cycle distribution detected by FCM with PI staining (a. Control; b. CHD; c. CHD + NIR; d. DTX; e. DTX/CHD; f. DTX/CHD + NIR). (B) Cell microtubules and cell
morphology of MCF-7 cell observed by confocal high-connotative microscope.

increased to 90.21%, which may be related to the increased uptake cated that combined with PDT, DTX/CHD nanoparticles could block
of nanoparticles by cells. After NIR treatment, 97.33% of cells in the most cells at the G2/M stage, thereby activating the apoptotic
DTX/CHD group were blocked at G2/M stage. These results indi- pathway and inducing apoptosis.

224
R. Wang, H. Yang, Abdur Rauf Khan et al.
Fig. 7. (A) Fluorescence imaging of mice at different time after injection of Ce6 and CHD NPs (a) and ex-vitro fluorescence imaging of mouse heart, liver, spleen, lung, kidney
and tumor (b). Fluorescence intensity of tumors and major viscera ex-vivo (c). Data shown as mean ± SD, n = 3. (B) Tumor volume and tumor reanalysis of each treatment
group. (a) Tumor volume-time curve; (b) tumor images; (c) tumor weight. Data shown as mean ± SD, n = 5. *indicates p < 0.05. **indicates p < 0.01.
3.4.7. Microtubules detection test
The results in Fig. 6B showed that in the control group, MCF-7
cells were normally fusiform, the nuclei were spherical or ellip-
soidal, and the microtubules were normal. After NIR treatment,
the cell morphology of the CHD group changed from fusiform to
round. After DTX treatment, tubulin aggregated to form non-
functional microtubule bundles and cell morphology was
destroyed. After NIR treatment, the microtubules in the DTX/CHD
group gathered and closely adhered to the nucleus, and the
cytoskeletal morphology changed greatly. DNA fragmentation
and apoptotic bodies appeared in the nucleus, indicating that
apoptosis was in progress. The above results indicated that DTX
played a role in inhibiting microtubule depolymerization. From
the perspective of cell morphology, the combined treatment of
DTX/CHD drug-loaded nanoparticles exhibited the best therapeutic
effect.
3.5. In vivo biological evaluation of nanoparticles
3.5.1. In vivo and ex-vivo imaging of nanoparticles
The in vivo fluorescence images of Ce6 and CHD in BALB/C mice
at different time points after tail vein injection were shown in
Fig. 7A (a). At the measured time points, the fluorescence intensity
of free Ce6 was strongest 4 h after injection, showing a systemic
distribution and focused on the location of tumors and abdominal
organs. With the extension of time, the fluorescence intensity was
decreased. There was very little fluorescence in the tumor site and
abdomen at 24 h, indicating that free Ce6 could be quickly cleared
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Journal of Colloid and Interface Science 598 (2021) 213–228
in mice [46]. Compared with free Ce6, the fluorescence intensity of
CHD was much higher at all the time points, especially in tumor
and liver, and decreased with the extension of time, indicating that
CHD had more accumulation in tumor sites than free Ce6. Since
CHD nanoparticles could slow the drug clearance rate, the tumor
sites of mice still had strong fluorescence at 24 h. In order to study
the distribution of nanoparticles in tumors and major organs more
clearly, mice were sacrificed at 24 h after the injection of the drug,
and their heart, liver, spleen, lung, kidney and tumor tissues were
collected for semi-quantitative fluorescence analysis ex-vivo. As
shown in Fig. 7A (b) and Fig. 7A (c), free Ce6 was accumulated
more in liver, and evenly distributed in other organs and tumors,
demonstrating the lack of selective distribution of free Ce6 to the
tumor. In the liver and tumor site, the fluorescence intensity of
the CHD group was much higher than that of the Ce6 group, and
the fluorescence intensity of the CHD group at the tumor site
was 4.2 times that of the Ce6 group. From the above results, it
could be seen that CHD nanoparticles had long-term circulation
in vivo and were concentrated in tumor tissues. The accumulation
of nanoparticles in the liver was mainly related to the phagocytosis
of nanoparticles by Kupffer cells, a large number of reticuloen-
dothelial cells with phagocytosis in the liver [47]. Therefore, this
drug-loading system can be considered for the study of liver
targeting.
3.5.2. In vivo antitumor efficacy
Tumor volume was commonly used to reflect the antitumor
effect. As shown in Fig. 7B (a), tumor volume of the NS group,
R. Wang, H. Yang, Abdur Rauf Khan et al.
Fig. 8. (A) H&E staining (a) and Tunel apoptosis (b) of tumors in normal saline control group and DTX/CHD + NIR group. (B) Weight-time curve of mice in each treatment
group. Data shown as mean ± SD, n = 3. (C) H&E staining analysis of heart, liver, spleen, lung and kidney of NS group and DTX/CHD + NIR group.
Ce6 + NIR group, CHD + NIR group, DTX group, DTX/CHD group and
DTX/CHD + NIR group after 14 days of administration was 1479.3
± 282.4 mm3, 1319.8 ± 257.0 mm3, 1048.2 ± 167.6 mm3, 764.4 ±
181.9 mm3, 433.4 ± 147.3 mm3, and 179 ± 82.4 mm3, respectively.
There was no significant difference of tumor volume between
Ce6 + NIR treatment group and NS group, probably because of
the non-selective distribution of Ce6. As CHD could accumulate
at tumor sites, internalize by tumor cells, and could rapidly release
drugs in tumor cells, CHD + NIR group showed better cytoxicity
than Ce6 group. At the given dose, DTX group exhibited better anti-
tumor effect compared with CHD + NIR group, and the relative
tumor volume of DTX group relative to NS group was 51.7%. The
relative tumor volume of the DTX/CHD nanoparticle treatment
group relative to NS group was 29.3%. The tumor growth in DTX/
CHD + NIR group was very slow with the volume below
200 mm3. Compared with single chemotherapy or PDT treatment,
the combined treatment of DTX/CHD + NIR group showed superior
anti-tumor effect. The same conclusion could be obtained from the
photographs of isolated tumors and the analysis of tumor weight
(Fig. 7B b and c). In the H&E staining results of tumor sections
(Fig. 8A a), the cell structure of NS group was intact. While in the
DTX/CHD + NIR group, the structure of large number of cell was
destroyed. The tunnel staining results of the tumor (Fig. 8A b)
showed large brown staining areas in the DTX/CHD + NIR group,
indicating a large amount of apoptosis of the tumor cells. To sum
up, the DTX/CHD + NIR treatment group showed the best anti-
tumor therapeutic effect.
3.5.3. Safety evaluation
As shown in Fig. 8B, each group of mice showed an upward
trend of tumor growth in eight days after the treatment. After
the eighth day, the NS group, Ce6 + NIR group and DTX group
showed a weight loss trend. The depressed state of mice in the
DTX group might be related to the excessive consumption of
mouse energy by large tumor volume and the toxic and side effects
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Journal of Colloid and Interface Science 598 (2021) 213–228
of free DTX, and a similar phenomenon was also observed in rele-
vant literatures [46]. The body weight of mice in CHD + NIR group,
DTX/CHD group and DTX/CHD + NIR group was still maintained,
indicating that the combination of CHD and DTX/CHD nanoparti-
cles with PDT had obvious anti-tumor ability and could reduce
the systemic toxicity of DTX. The H&E staining analysis of each
major organ (Fig. 8C) showed that the heart, liver, spleen, lung
and kidney tissue sections in the DTX/CHD + NIR treatment group
showed no abnormal cell morphology, tissue lesions or degenera-
tion compared with the NS control group. Therefore, DTX/
CHD + NIR treatment group did not cause damage to the normal
tissues, showing good safety.
4. Conclusion
In summary, redox-responsive DTX/CHD nanoparticles combin-
ing PDT with chemotherapy were successfully designed and
explored for breast cancer therapy. DHA was introduced as a
hydrophobic core to load hydrophobic chemotherapy drug DTX.
The nanoparticles could accumulate at tumor site through passive
targeting and effective cellular uptake could be achieved through
HA and CD44 receptor interactions, thus minimizing the side
effects to normal tissues and cells. In response to the highly reduc-
tive environment of tumor cells, DTX and Ce6 could be released.
Under NIR irradiation, PS and DTX played a synergistic anti-
tumor effect and significantly inhibited the tumor growth. Taken
together, the prepared formulation had good biosafety and supe-
rior antitumor effect, which was very promising in the treatment
of breast cancer.
Our study was the first to deliver both Ce6 and DTX using DHA
and HA as the carrier material in order to achieve the significant
anti-tumor effect. Compared to other Ce6 loaded HA nanoparticles
[48–50], the carrier materials used in this study, HA and DHA, have
higher safety. They have good biocompatibility and can be
degraded under physiological conditions. On the premise of ensur-
R. Wang, H. Yang, Abdur Rauf Khan et al.
ing safety, the prepared nanoparticles have excellent anti-tumor
effect. Moreover, we conducted a more in-depth study of the
mechanism by which the DTX/CHD nanoparticles worked [51–
53], which can provide guidance for future preparations. In the
future, we will investigate the long-term stability and pharmacoki-
netics of DTX/CHD nanoparticles, so that we can have a more com-
prehensive understanding of the nanoparticles.
CRediT authorship contribution statement
Rujuan Wang: Conceptualization, Methodology, Software,
Investigation. Haotong Yang: Validation, Formal analysis, Visual-
ization, Software, Writing - original draft. Abdur Rauf Khan: Writ-
ing - review & editing, Supervision, Data curation. Xiaoye Yang:
Writing - review & editing, Supervision, Data curation. Jiangkang
Xu: Writing - review & editing. Jianbo Ji: Writing - review & edit-
ing. Guangxi Zhai: Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
This work is supported by the Major Research Project of Shan-
dong Province, P.R.China (No.2018GSF118004) and Shandong
Provincial Major Science &Technology Innovation Project, P.R.
China (2018CXGC1411) and Major Basic Research Projects of Shan-
dong Natural Science Foundation, P.R.China (No. ZR2018ZC0232).
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jcis.2021.04.056.
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