NIR-/pH-Responsive Nanocarriers Based on Mesoporous Hollow
Polydopamine for Codelivery of Hydrophilic/Hydrophobic Drugs
and Photothermal Synergetic Therapy
Shanshan Li,† Ying Gan,† Chen Lin, Kunpeng Lin, Peng Hu, Lei Liu, Shuling Yu, Shuang Zhao,*
and Jiahua Shi*
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ABSTRACT: Combined therapy system has become an efficient strategy to overcome
drug resistance and strengthen therapeutic effects. Herein, an efficient NIR-/pH-triggered
dual-drug-loaded nanoplatform was designed for combined chemo-photothermal therapy.
The hydrophobic anticancer drug bortezomib (BTZ) was first loaded in mesoporous
polydopamine nanospheres (MPDAs) through the acid-sensitive borate ester bond.
Afterward, pH-responsive carboxymethyl chitosan (CMCS) conjugated on the surface of
MPDA could capture another anticancer drug doxorubicin (DOX) and exhibited
controlled release behavior in an acidic tumor microenvironment. Meanwhile, under NIR
laser irradiation, hyperthermia produced by the photothermal conversion agent MPDA
could efficiently ablate cancer cells and further promote drug release. In vitro and in vivo
experiments emphasized that the synthesized MPDA-BTZ@CMCS-DOX nanostructure
exhibited efficient accumulation in the tumor site, resulting in sustained release of BTZ
and DOX and realizing NIR-/pH-triggered chemotherapy and photothermal synergistic
ablation of cancer.
KEYWORDS: mesoporous hollow polydopamine, dual-drug delivery, NIR-/pH-responsive, controlled release, photothermal therapy,
synergistic therapy
1. INTRODUCTION
Nowadays, various therapeutic strategies, such as surgery,
chemotherapy, photodynamic therapy (PDT), and photothermal therapy (PTT), have been developed to treat dreadful
cancer.1,2 Among them, chemotherapy is still one of the main
methods in clinic. Many kinds of anticancer drugs, such as
doxorubicin (DOX), bortezomib (BTZ), paclitaxel (PTX),
gemcitabine (GEM), curcumin (CUR), and docetaxel (DOC),
have been commonly used in clinical cancer therapy.3,4 DOX is
frequently used for solid tumors, and it exhibits admirable
effects on many kinds of tumors through its interference with
topoisomerase II or inhibition to the synthesis of DNA.5
However, it has high cytotoxicity, low selectivity, and easy
degradability.6 Serious side effects, especially myeloid and
cardiac toxicity, impeded its applications. Bortezomib (BTZ), a
unique proteasome inhibitor, could suppress the activity of the
26S proteasome and induce apoptosis of cancer cells. This
anticancer agent was generally used in hematologic tumors,
including multiple myeloma and lymphoma, whereas clinical
studies demonstrated that BTZ induced distinct inhibition on
normal tissues simultaneously and patients suffered from
severe side effects, such as cardiomyocytes necrosis, nausea,
diarrhea, certain liver/kidney damage, etc.7,8 To reduce the
side effects and drug resistance caused by single use of
chemotherapeutic drugs, a combination of multidrugs with
different antitumor mechanisms has been an increasing
research focus.9−11 The synergistic use of BTZ and DOX has
endured different phases of clinical tests toward several types
of cancers.12−14
Over the past few years, nanotechnology has gained
exceptional growth in the medical field. The emergence of
nanocarriers has provided the chance of delivering multiple
drugs and overcoming the deficiency of traditional chemotherapy including poor water solubility, low efficacy, high
toxicity, and drug resistance.15−17 To date, a variety of
nanocarriers, such as porous nanospheres, nanogels, liposomes,
and micelles, have been designed to deliver anticancer
drugs.18,19 Among them, mesoporous nanomaterials with
high loading capacities are highly desired for efficient drug
delivery. Especially, stimuli-responsive nanocarriers sensitive to
external/internal stimuli (light, magnetic field, acidic pH,
Received: November 9, 2020
Accepted: January 21, 2021
Published: February 2, 2021
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hypoxia, etc.) have attracted much research interest for their
capacity of controlled drug release.20−22 However, chemotherapy alone is usually insufficient to cure cancers completely,
and tumor metastasis or relapse occurs easily due to multiple
drug resistance and limited therapeutic ability.23,24 Therefore,
multifunctional nanoplatforms combining multistimuli-sensitive drug release and other therapeutic strategies for synergistic
tumor therapy may be an effective strategy for accurate and
efficient treatment of tumors.
In recent years, tremendous attention has been focused on
the design and synthesis of multifunctional porous nanostructures, which combined chemotherapy and noninvasive PTT
due to their inherent porosity and photothermal conversion
capability.25,26 On the one hand, premature release was
prevented by encapsulating cancer drugs inside nanocarriers,
thus minimizing the damage to normal tissues. On the other
hand, near-infrared (NIR)-induced PTT exhibits high
selectivity and excellent therapeutic capability; meanwhile,
the hyperthermia produced by NIR radiation can promote the
release of drugs, further enhancing the inhibition effect toward
tumors.27−29 Various PTT nanocarriers, such as porous
carbon-based nanomaterials, metal chalcogenides, and noble
metal nanostructures, have been widely explored, and ideal
treatment effect was demonstrated.30−32 However, the synthesis of these nanomaterials is usually complicated and most
of them are difficult to degrade, which causes waste of
resources and potential risks. Among the reported nanocarriers
with PTT ability, mesoporous polydopamine (MPDA) nanoparticles have attracted extensive attention for smart drug
delivery. Previous investigations demonstrated that MPDA
nanoparticles not only could realize effective drug encapsulation but also exhibite admirable photothermal conversion
ability.33−35 However, every coin has two sides: biodegradability relieves metabolic pressure of the nanoparticles, while it
also causes the problem of material instability. One of the main
challenges seems to be proper regulation of the two sides.
Besides, despite previous studies on the use of MPDA
nanoparticles as drug delivery systems, there is still a need to
explore the use of MPDA for simultaneous encapsulation of
hydrophobic and hydrophilic drugs as well as independently
controlling the release behavior of each drug.
Herein, we developed a facile approach and successfully
fabricated versatile MPDA-BTZ@CMCS-DOX dual-responsive drug delivery systems for synergetic chemo-photothermal
therapy. First, hydrophobic BTZ was encapsulated in MPDA
through the borate ester bond, which exhibits acid-responsive
behavior and pH-triggered BTZ release in the tumor
microenvironment. Meanwhile, DOX could be connected
with the carboxymethyl chitosan (CMCS) film on the surface
of BTZ-loaded MPDA through electrostatic adsorption, which
was weakened under acidic conditions, thus releasing DOX
first from the synthesized MPDA-BTZ@CMCS-DOX. Besides,
the CMCS was stable under neutral conditions and could be
depolymerized under acidic pH, which effectively solved the
instability of MPDA, avoiding preleaking of BTZ and realizing
pH-regulating drug release simultaneously. A series of in vitro/
in vivo experiments were conducted, and ideal tumor
inhibition was achieved, demonstrating great promise of the
MPDA-BTZ@CMCS-DOX nanoplatform for enhanced cancer
therapy.
2. MATERIALS AND METHODS
2.1. Synthesis of Mesoporous Polydopamine Nanoparticles
(MPDA NPs). MPDA NPs were synthesized by a one-pot synthesis
method. Briefly, 0.36 g of Pluronic F127 and 0.36 g of 1,3,5-
trimethylbenzene (TMB) were dissolved in a mixed solution of 65 mL
of H2O and 60 mL of C2H5OH. After stirring for 30 min, 90 mg of
tris(hydroxymethyl) aminomethane (TRIS) dissolved in 10 mL of
H2O was added, followed by adding dopamine hydrochloride aqueous
solution (60 mg, 1 mL). After 24 h, the product was centrifuged and
washed with ethanol and acetone several times. Then, the template
was removed by ultrasonic treatment (three times, 30 min each time)
in a mixture of ethanol and acetone (2:1 v/v). The final product was
washed and redispersed in water for further use.
2.2. Preparation of BTZ-Loaded MPDA (MPDA-BTZ). A total
of 10 mL of MPDA aqueous suspension (1 mg mL−1
) was sonicated
for 15 min, followed by adding 10 mL of BTZ solution in a mixture of
water and dimethyl sulfoxide (DMSO) (0.5, 1, 2, and 3 mg mL−1
The mixture was stirred at room temperature for 12 h, and the final
products were collected by centrifugation (12 000 rpm), washed, and
redispersed in 10 mL of water for further use. All supernatants were
collected, and the absorbance was recorded at 270 nm. The loading
capacity of BTZ was determined by the following equation:
loading capacity (wt %)
mass of total BTZ mass of BTZ in supernatant
mass of total MPDA BTZ = 100% −
2.3. Synthesis of Carboxymethyl Chitosan (CMCS)-Coated
MPDA-BTZ (MPDA-BTZ@CMCS). CMCS-modified MPDA-BTZ
was obtained via Michael addition/Schiff-base reactions between
catechol/quinone groups of MPDA and the amino groups of
CMCS.36,37 The reaction can be shown as follows: 10 mg of
MPDA-BTZ, 200 mg of CMCS, and 10 mL of Tris buffer were mixed
by ultrasonication and stirred at room temperature for 24 h. Finally,
the synthesized MPDA-BTZ@CMCS were isolated by centrifugation
and washed with water several times.
2.4. Preparation of MPDA-BTZ@CMCS-DOX. For DOX
loading, 10 mg of MPDA-BTZ@CMCS dispersed in phosphatebuffered saline (PBS) buffer (pH 7.4) was mixed with 10 mg of DOX,
which could be loaded by MPDA-BTZ@CMCS via electrostatic
adsorption. The nanoparticles were collected by centrifugation and
washed with distilled water several times. To quantify the DOX
loading efficiency, the supernatants were collected and the mass of
unloaded DOX was determined by a standard curve obtained through
the absorbance of DOX with various concentrations at 480 nm. The
DOX loading capacity was calculated as follows:
loading capacity (wt %)
mass of total DOX mass of DOX in supernatant
mass of total MPDA BTZ@CMCS DOX = 100% −
2.5. Photothermal Performance. The photothermal conversion
capability was investigated by monitoring the temperature of
MPDA@CMCS (0, 50, 100, 200, 500, 1000 μg mL−1
) under the
irradiation of 808 nm NIR laser (1.5 W cm−2
). Meanwhile, MPDA@
CMCS (200 μg mL−1
) was exposed to an 808 nm laser with different
power densities (0.75, 1.5, 2.25, 3 W cm−2
). The photothermal
stability of the as-synthesized MPDA@CMCS was evaluated by five
cycles of laser on/off irradiation experiments (10 min laser irradiation
and then natural cooling). The photothermal conversion efficiency
(η) was calculated as follows:
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where m and C are the mass (0.5 g) and heat capacity (4.2 J g−1
water, respectively. h and S are the heat transfer coefficient and the
surface area of the container, respectively. I represents the power
density (1.5 W cm−2
) of an 808 nm laser. A808 (0.694) is the
absorbance of MPDA@CMCS (200 μg mL−1
) at 808 nm. Tmax,M and
TS are the maximum temperature during irradiation and room
temperature, which are 54.7 and 28.0 °C, respectively. τs is the time
constant obtained in Figure S6D, which was calculated to be 206.3.
2.6. Drug Release Profiles. In vitro release of BTZ and DOX was
monitored as follows: several groups of MPDA-BTZ, MPDA-BTZ@
CMCS, and MPDA-BTZ@CMCS-DOX aqueous solutions (2 mL)
were encapsulated in dialysis bags (MWCO: 3500 Da). The endsealed dialysis bags were incubated in 20 mL of PBS buffer (pH = 7.4,
6.8, 5.0) and shaken at a speed of 150 rpm using a thermostatic
shaker, separately. At different time intervals, 2 mL of the PBS
medium was taken out and equivalent fresh PBS was added
immediately. The amount of released BTZ and DOX was quantified
from the absorbance at 270 and 480 nm, respectively.
Drug release study under NIR laser was performed by irradiating
the MPDA-BTZ, MPDA-BTZ@CMCS, and MPDA-BTZ@CMCSDOX solution with an 808 nm laser at the first 3 min of every
specified time point and shaken using a thermostatic shaker at 37 °C
in the rest of the time.
2.7. In Vitro Cytotoxicity. Human hepatocellular carcinoma
(HepG2) cells and human cervical cancer (HeLa) cells were chosen
to evaluate the cytotoxicity of the synthesized nanomaterials.
Appropriate HepG2 and HeLa cells were seeded in 96-well plates
overnight and then incubated with MPDA, MPDA@CMCS, MPDABTZ, MPDA-BTZ@CMCS, MPDA@CMCS-DOX, and MPDABTZ@CMCS-DOX, separately. After incubating for specific times
(24 and 48 h), the cells were washed with PBS buffer and cultured
with 100 μL of fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) (10 μL, 5 mg mL−1
Finally, 100 μL of DMSO was added to each well and the absorbance
at 490 nm was measured to calculate cell viabilities.
For in vitro chemo-photothermal therapeutic efficacy, HepG2 and
HeLa cells (8 × 103 cells per well) were seeded in 96-well plates and
incubated with MPDA@CMCS (0, 6.25, 12.5, 25, 50, 100 μg mL−1
and MPDA-BTZ@CMCS-DOX dispersions (BTZ: 0.625, 1.25, 2.5, 5,
10 μg mL−1
). The normally incubated cells were used as the control.
After 24 h, the plates were exposed to an 808 nm laser (1.5 W cm−2
and then washed with PBS buffer. The cell viabilities were detected by
the standard MTT assay.
2.8. In Vitro Cellular Uptake. First, the synthesized MPDABTZ@CMCS-DOX was labeled by fluorescein isothiocyanate (FITC)
through the integration between isothiocyanate groups of FITC with
dangling NH2 of MPDA,38−40 as illustrated in Figure S2. Briefly,
FITC (1 mg) was mixed with 1 mL of Na2CO3 solution (0.5 M) to
form solution A. Then, 200 μL of solution A was added to an MPDA
aqueous solution (10 mL, 1 mg mL−1
) and shake cultivation (4 °C)
for 12 h. Finally, FITC-labeled MPDA was obtained by dialyzing for
48 h in water. Afterward, FITC-labeled MPDA-BTZ@CMCS-DOX
was obtained according to the processes mentioned above for MPDABTZ@CMCS-DOX without FITC modification and then was used
for the following cellular uptake assay.
Take HepG2 cells as an example. HepG2 cells were seeded in 24-
well plates (4 × 104 cells per well) and then incubated with BTZ,
DOX, BTZ + DOX (m/m = 1.27/1), MPDA-BTZ, MPDA-BTZ@
CMCS, MPDA@CMCS-DOX, and MPDA-BTZ@CMCS-DOX. Half
of the group treated with MPDA-BTZ@CMCS-DOX was irradiated
by an 808 nm laser (1.5 W cm−2
). After incubation for specific times
(24 and 48 h), the cells were successively washed with PBS, fixed by
paraformaldehyde solution (4%), stained with 4′,6-diamidino-2-
phenylindole (DAPI) for 10 min, and finally observed by a confocal
laser scanning microscope.
2.9. In Vivo Fluorescence Imaging. To verify the efficient
accumulation of the synthesized MPDA@CMCS at the tumor site,
Figure 1. Schematic illustration of the synthesis and application of MPDA-BTZ@CMCS-DOX for pH-/NIR-responsive dual-drug chemophotothermal therapy.
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MPDA was labeled with NIR-797 isothiocyanate first. Briefly, NIR-
797 isothiocyanate (1 mg) dissolved in 1 mL of DMSO was mixed
with 1 mL of Na2CO3 solution (0.5 M) to form solution A. Then, 400
μL of solution A was added to MPDA aqueous solution (10 mL, 1 mg
mL−1
) and shake cultivation (4 °C) for 12 h. Finally, NIR-797
isothiocyanate-labeled MPDA was obtained by dialyzing in water for 2
days. Afterward, NIR-797-labeled MPDA was modified with CMCS
referring to the procedures for MPDA-BTZ@CMCS. Finally, NIR-
797-labeled MPDA@CMCS was injected intravenously into tumorbearing mice. At different time intervals, the fluorescence images were
monitored on a Maestro.
2.10. Hemolysis Assay. Red blood cells were separated with
plasma by centrifugation at 2000 rpm for 10 min, washed with 0.9%
NaCl solution until the supernatant was colorless, and then mixed
with 1 mL of water (negative control), saline (positive control), and
MPDA, MPDA@CMCS, and MPDA-BTZ@CMCS-DOX solution
(12.5, 25, 50, 100, and 200 μg mL−1
, respectively) separately. After
incubating at 37 °C for 3 h, the supernatants were collected by
centrifugation and the absorbance at 540 nm was recorded using a
UV−vis spectrophotometer. The percentage of hemolysis was
calculated by the following equation:
where Asample, Anegative, and Apositive are the absorbance of samples, the
negative control, and the positive control, respectively.
2.11. In Vivo Antitumor Efficiency and Histological
Analysis. To evaluate the antitumor effect of the synthesized
nanomaterials, female Kunming mice were subcutaneously implanted
with H22 cells (1.0 × 107 cells per mL, 200 μL). When the tumor size
reached around 100 mm3
, the mice were randomly divided into eight
groups (n = 10 for each group): (1) saline, (2) NIR, (3) MPDA@
CMCS, (4) free BTZ, (5) free DOX, (6) free BTZ + DOX, (7)
MPDA-BTZ@CMCS-DOX, and (8) MPDA-BTZ@CMCS-DOX +
NIR (808 nm laser, 1.5 W cm−2
, 5 min). The treatment was
conducted every 3 days with a drug dosage of 5 mg kg−1
. The tumor
sizes and mice body weights were recorded every 2 days. The tumor
volumes were calculated by the following formula: tumor volume =
tumor width2 × tumor length/2. After 16 days, the tumor-bearing
mice were sacrificed and the main tissues (heart, liver, spleen, lung,
and kidney) as well as tumors were collected, immobilized in 4%
paraformaldehyde, stained with hematoxylin & eosin (H&E), and
finally observed by a fluorescence microscope.
3. RESULTS AND DISCUSSION
3.1. Characterization of MPDA-BTZ@CMCS-DOX
Nanoparticles. The controlled synthesis and potential
applications of the MPDA-BTZ@CMCS-DOX nanoplatform
are illustrated in Figures 1 and S3. First, MPDA nanoparticles
were synthesized using an emulsion-templated route using
triblock copolymer F127 and TMB as the organic
templates.41,42 From Figure 2A,C, it could be seen that the
obtained MPDA nanoparticles exhibited a mesoporous hollow
spherical morphology with an average diameter of 150 nm and
the mean hydrodynamic diameter was about 270.3 nm (red
line in Figure 2E). Besides, nitrogen sorption measurements
were conducted to further identify the porosity of MPDA. As
shown in Figure S4, the N2 adsorption−desorption isotherms
showed a distinct type IV isotherm with obvious H1 hysteresis
loops, and the Brunauer−Emmett−Teller (BET) specific
surface area was measured to be 43.57 m2 g−1
, exhibiting the
porous structure of the synthesized MPDA. From the pore size
distribution curves, three peaks at about 4.98, 6.53, and 9.52
nm were found in the range of 3−15 nm, corresponding to the
mesopores and hollow cavities of MPDA. All of these results
demonstrated great promise of the synthesized MPDA for drug
encapsulation.
After treating with CMCS, a thin CMCS layer could be
observed on the surface of MPDA (Figure 2B,D). The
successful coating of CMCS was further revealed by the FTIR
Figure 2. SEM and TEM images of (A, C) MPDA and (B, D) MPDA-CMCS. (E) Hydrodynamic diameters of MPDA (red line) and MPDACMCS (black line). Inset: digital photo of MPDA@CMCS (1) and MPDA (2) aqueous solution after 48 h. (F) ζ-Potentials of MPDA, MPDABTZ, MPDA-BTZ@CMCS, and MPDA-BTZ@CMCS-DOX. (G) Fourier transform infrared (FTIR) spectra of MPDA (black line) and MPDACMCS (red line). (H) UV−vis spectra of MPDA, BTZ, MPDA-BTZ, CMCS, DOX, MPDA-BTZ@CMCS, and MPDA-BTZ@CMCS-DOX.
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spectra. As shown in Figure 2G (black line), the broad peak at
3432 cm−1 was attributed to the stretching vibrations of −OH
and −NH2 groups of MPDA, while the peaks centered at about
1641, 1464, and 1296 cm−1 were caused by stretching
vibrations of −CC−, −CN, and C−N−C of the indoline
structure of MPDA, respectively.43 Compared with the
spectrum of MPDA, new peaks corresponding to the stretching
appear for that of MPDA@CMCS, demonstrating the
successful modification of CMCS on the surface of
MPDA.44,45 After modifying with CMCS, the stability of
MPDA was effectively increased, while the hydrodynamic
diameter decreased slightly due to the better dispersivity of
MPDA@CMCS than MPDA (Figure 2E), as shown in the
inset of Figure 2E (1: MPDA@CMCS; 2: MPDA). Meanwhile, the hydrodynamic diameters in different solutions
(water, saline, PBS buffer, Dulbecco’s modified Eagle’s
medium (DMEM), and fetal bovine serum (FBS)) demonstrated excellent stability of MPDA in various physiological
conditions after modifying with CMCS (Figure S5).
Besides, the surface charges of MPDA, MPDA-BTZ, MPDABTZ@CMCS, and MPDA-BTZ@CMCS-DOX were investigated by ζ-potential measurements. The initial ζ-potential of
MPDA was −26.1 mV (Figure 2F), while the value decreased
to −27.8 mV for MPDA-BTZ. After modification with CMCS,
the result became −42.8 mV, further confirming the successful
package of CMCS on MPDA-BTZ nanomaterials. When
combined with DOX through electrostatic adsorption, a
positive ζ-potential of 32.4 mV was monitored, demonstrating
successful construction of MPDA-BTZ@CMCS-DOX nanomaterials.
To further demonstrate the successful synthesis of MPDABTZ@CMCS-DOX, UV−vis spectra of MPDA, BTZ, CMCS,
DOX, MPDA-BTZ, MPDA-BTZ@CMCS, and MPDA-BTZ@
CMCS-DOX were measured. As depicted in Figure 2H,
characteristic peaks belonging to free BTZ (270 nm) and DOX
(480 nm) were observed from the synthesized MPDA-BTZ@
CMCS-DOX, indicating that BTZ and DOX were efficiently
loaded in the nanomaterials.
3.2. Photothermal Performance. To test the photothermal conversion capacity of the synthesized MPDA@
CMCS, temperature variations of MPDA@CMCS (0, 50, 100,
200, 500, 1000 μg mL−1
) were recorded under an 808 nm laser
irradiation (1.5 W cm−2
). As shown in Figure 3A,B, the
temperature changes exhibited a distinct concentrationdependent behavior during laser irradiation. The solution
temperature increased by 15 °C after irradiating for 10 min at
the MPDA@CMCS concentration as low as 50 μg mL−1
, while
the value for pure water was only 2.9 °C, demonstrating that
MPDA@CMCS nanoparticles could efficiently convert NIR
light to heat. The photothermal conversion efficiency was
Figure 3. (A, C) Concentration- and power-density-dependent heating curves of MPDA@CMCS. (B, D) Maximum temperature elevations (ΔT)
of MPDA@CMCS with different concentrations (1.5 W cm−1
, 0, 50, 100, 200, 500, and 1000 μg mL−1
) and power intensities (200 μg mL−1
, 0.75,
1.5, 2.25, and 3 W cm−2
). (E, F) Thermal camera images of MPDA@CMCS with various concentrations (1.5 W cm−1
, 0, 50, 100, 200, and 500 μg
mL−1
) and power intensities (200 μg mL−1
, 0.75, 1.5, 2.25, and 3 W cm−2
). (G) Thermal camera images of mice at 7 h intravenous injection with
saline, MPDA, MPDA@CMCS (M@C), and MPDA-BTZ@CMCS-DOX (M-B@C-D).
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calculated to be 22.71% (Figure S6B−D). Meanwhile, using
MPDA@CMCS (200 μg mL−1
) solution as a model, the
temperature changes versus laser density were investigated. It
can be seen from Figure 3C,D that the temperature elevations
exhibited a linear dependence on laser energy from 0.75 to 3
W cm−2
. The corresponding thermal pictures of MPDA@
CMCS (808 nm, 1.5 W cm−2
; 0, 50, 100, 200, 500 μg mL−1
and MPDA@CMCS (200 μg mL−1
; 0.75, 1.5, 2.25, 3 W cm−2
also exhibited concentration- and power-density-dependent
temperature behavior, demonstrating the efficient photothermal conversion capacity of the synthesized MPDA@
CMCS (Figure 3E,F).
To assess the photothermal stability, five cycles of laser on/
off operation (laser irradiation for 10 min and then natural
cooling) were performed on MPDA@CMCS solution (200 μg
mL−1
) and no obvious temperature change was detected
during this period, demonstrating the excellent photothermal
stability of the obtained MPDA@CMCS (Figure S6A).
3.3. Drug Release Profiles. To prove the superiority of
MPDA as drug carriers, we primarily compared the BTZ
loading capacity for solid and mesoporous PDA structures. As
shown in Figure 4A, the loading capacity of BTZ increased
almost linearly with increasing concentration. For MPDA, the
loading capacity reached 63.5% when the BTZ concentration
was 3 mg mL−1
, while the value was only 21.5% for solid PDA
nanospheres.
Afterward, the controlled release of BTZ was studied in PBS
buffer (pH: 5.0, 6.8, and 7.4) at 37 °C. The accumulative
releases of BTZ in pH 7.4 were 13.5 and 23.0% at 48 h for
MPDA-BTZ@CMCS and MPDA-BTZ nanoparticles, demonstrating that CMCS on the surface of MPDA-BTZ sealed the
mesopores of MPDA, thus avoiding the leakage of cancer drugs
before arriving at the tumor site. At pH 6.8 and 5.0, the
amount of BTZ released from MPDA-BTZ@CMCS nanoparticles increased to 20.3 and 35.2%, while the results were 40
and 67.2% for MPDA-BTZ, respectively (Figure 4B,C). This
may be ascribed to the pH sensitivity of the borate ester bond
formed between MPDA and BTZ, which could break in an
acid environment, resulting in an increase of the BTZ release
rate. Meanwhile, CMCS on the surface could dissemble and
endow the materials with a pH-responsive drug release
behavior (Figure 4C). Under the irradiation of NIR laser,
about 37.4 and 69.3% of BTZ were, respectively, released from
MPDA-BTZ@CMCS and MPDA-BTZ within 48 h at pH 5.0,
which was greater than that without laser irradiation,
demonstrating that BTZ release could be further promoted
through NIR irradiation due to the rise in temperature of the
solution. Besides, as shown in Figure S7B,C, more drug release
was observed under a higher laser power density. This was due
to the higher elevated temperature obtained by increasing the
NIR power density, which ultimately resulted in an accelerated
drug release. All of these results exhibited the typical NIR-/pHresponsive drug release property of the synthesized MPDA@
CMCS nanocarrier.
The loading capacity of DOX under various concentrations
is shown in Figure S7A. It could be seen that the loading
capacity increased with higher DOX concentrations (0.5, 1, 2
mg mL−1
) and almost reached saturation at 2 mg mL−1
. The
loading value was calculated to be 17.8%. The ratio of BTZ/
DOX in MPDA-BTZ@CMCS-DOX was 1.27 at the BTZ and
DOX concentration of 2 mg mL−1
The release of DOX from MPDA-BTZ@CMCS-DOX was
then studied at pH 5.0, 6.8, and 7.4 (Figure 4D). It was found
that the release rate increased obviously at pH 5.0 compared
with that at pH 7.4 (55.3 versus 19.7), indicating that the
acidic tumor environment contributed to the release of DOX
from the MPDA-BTZ@CMCS-DOX structure. This phenomenon is due to the fact that DOX was loaded through the
electrostatic interaction with CMCS. In an acidic environment,
carboxyl groups of CMCS were protonated and the interaction
between DOX and CMCS was weakened, thus exhibiting the
pH-responsive release of DOX. Besides, the NIR irradiation
also enhanced the cumulative release of DOX and more DOX
was released under an NIR higher power density (Figure S7D)
similarly. Overall, a pH-/NIR-triggered release system was
successfully constructed, which efficiently prevented premature
release of cancer drugs.
3.4. In Vitro Cytotoxicity. HeLa and HepG2 cells were
chosen to investigate the cytotoxicity of the synthesized
MPDA and MPDA@CMCS nanomaterials. As shown in
Figures 5A1,B1 and S8A1,B1, the cell viability remained above
85% at the MPDA concentration of 100 μg mL−1 even when
the incubation time was extended to 48 h, indicating good
biocompatibility of the MPDA@CMCS nanocarriers. Under
NIR irradiation, the cell viability decreased due to the
photothermal conversion effect of MPDA@CMCS and
obvious cancer cell death was observed with a prolonged
irradiation time (6 min, 1.5 W cm−2
), as shown in Figures
5A2,B2 and S8A2,B2. Meanwhile, a series of orthogonal MTT
experiments were conducted to verify the ablation efficacy of
the synthesized drug-loaded systems toward cancer cells, where
free BTZ and DOX were used as the control.
Herein, HepG2 and HeLa cells were treated with BTZ,
DOX, BTZ + DOX (BTZ/DOX = 1.27:1), and drug-loaded
nanomaterials with various BTZ (0.625, 1.25, 2.5, 5, and 10 μg
mL−1
) and DOX concentrations (0.492, 0.984, 1.968, 3.936,
and 7.872 μg mL−1
). As shown in Figures 5A3 and S8A3,
Figure 4. (A) Concentration-dependent BTZ loading capacity of
solid PDA and mesoporous MPDA. Drug release profiles of BTZ
from (B) MPDA-BTZ, (C) MPDA-BTZ@CMCS, and (D) DOX
from MPDA-BTZ@CMCS-DOX in PBS at pH 5.0, 6.8, and 7.4,
respectively (NIR: 808 nm, 1.5 W cm−2
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obvious cancer cell death toward both HepG2 and HeLa cells
was observed for the group of MPDA-BTZ@CMCS-DOX
after incubation for 24 h. The cell viabilities were 48.21 and
39.54% at the BTZ concentration of 10 μg mL−1
. At the same
BTZ concentration, free BTZ + DOX showed a higher
cytotoxicity than MPDA-BTZ@CMCS-DOX, which was due
to the delayed release of BTZ and DOX from MPDA-BTZ@
CMCS-DOX. Besides, NIR irradiation further enhanced the
inhibition effect due to hyperthermia produced through the
PTT effect (Figures 5A4 and S8A4), which also promoted
drug release from the MPDA-BTZ@CMCS-DOX system.
On further extending the incubation time to 48 h (Figures
5B3 and S8B3), the survival rate of the two cells (9.87 and
7.47%) treated with MPDA-BTZ@CMCS-DOX apparently
decreased compared to that incubated for 24 h. As expected,
the cell viabilities decreased as the drug concentration and
incubation time increased, indicating a dose- and timedependent cytotoxic effect. Similarly, cell inhibition was also
enhanced by NIR irradiation and prolonged laser-treated time
(Figures 5B4 and S8B4). All of these results demonstrated
efficient cancer cell ablation of the synthesized MPDA-BTZ@
CMCS-DOX.
3.5. In Vitro Cellular Uptake. MPDA-BTZ@CMCSDOX was labeled with fluorescein isothiocyanate (FITC);
then, the cellular uptake of MPDA-BTZ@CMCS-DOX and
pure BTZ + DOX was studied by fluorescence microscopy.
After incubation for 24 h, we can see from Figure S9 that DOX
could accumulate in the nucleus and efficiently kill cancer cells.
At the same DOX concentration, strong green and red
fluorescence signals belonging to FITC and DOX were
simultaneously monitored in cancer cells when incubated
with MPDA-BTZ@CMCS-DOX nanoparticles, demonstrating
efficient internalization of the synthesized nanocarriers. Under
NIR irradiation, the fluorescence signals were relatively
stronger than those treated with BTZ + DOX or MPDABTZ@CMCS-DOX in the absence of the 808 nm laser and
enhanced red signals of DOX were observed by increasing the
irradiation time to 6 min (Figure S10), indicating more
endocytosed MPDA-BTZ@CMCS-DOX in HepG2 cells. This
phenomenon was mainly due to the hyperthermia by NIR
irradiation inducing minor disruptions in the cell membrane,
which improved the cellular uptake of NPs. The results were
consistent with the in vitro cytotoxicity assay that MPDABTZ@CMCS-DOX exhibited better ablation of cancer cells
with NIR laser treatment. All results considered, the
synthesized MPDA-BTZ@CMCS-DOX could be effectively
internalized by cancer cells and the therapeutic effect could be
enhanced by NIR light.
3.6. Hemolysis and In Vivo Fluorescence Imaging.
Good biocompatibility of drug carriers is the prerequisite for
their employment of medical treatment. Herein, hemolysis was
conducted preferentially to evaluate the biocompatibility of
MPDA, MPDA@CMCS, and MPDA-BTZ@CMCS-DOX,
respectively, in which deionized water and saline were used
as the positive negative and control, respectively. As shown in
Figure S11, negligible hemolysis was observed with the MPDA,
MPDA@CMCS, or MPDA-BTZ@CMCS-DOX concentration
of 200 μg mL−1
, demonstrating admirable blood compatibility
of the synthesized MPDA@CMCS.
To verify whether the synthesized MPDA-BTZ@CMCSDOX can preferentially accumulate at the tumor site, MPDA@
CMCS was labeled with NIR-797 and fluorescence images
Figure 5. Concentration-dependent HepG2 cell viabilities after incubating with MPDA and MPDA@CMCS for 24 h (A1) and 48 h (B1).
Concentration-dependent HepG2 cell viabilities after incubating with MPDA and MPDA@CMCS for 24 h (A2) and 48 h (B2) with NIR laser
irradiation (1.5 W cm−2
; 0, 3, and 6 min). Cytotoxicity study of HepG2 cells after different treatments at various BTZ or DOX concentrations for
24 h (A3) and 48 h (B3), respectively. Cytotoxicity study of HepG2 cells incubated with MPDA-BTZ@CMCS-DOX for 24 h (A4) and 48 h (B4)
with NIR laser irradiation (1.5 W cm−2
; 0, 3, and 6 min). (ns, no significance; *P < 0.05; **P < 0.01; and ***P < 0.001).
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were monitored at 1 h post intravenous injection. As shown in
Figure 6, the intensity of the fluorescence signal at the tumor
site increased over time, and the maximum value was observed
at 7 h, indicating efficient accumulation of MPDA@CMCS at
the tumor region.
3.7. In Vivo Antitumor Analysis. In vivo antitumor
efficacy was investigated on H22 tumor-bearing Kunming
mice, which were randomly divided into eight groups including
(1) saline, (2) NIR, (3) MPDA@CMCS, (4) BTZ, (5) DOX,
(6) BTZ + DOX, (7) MPDA-BTZ@CMCS-DOX, and (8)
Figure 6. (A) In vivo fluorescence imaging and (B) relative intensity of mouse after injecting with NIR-797-labeled MPDA@CMCS intravenously
at different time points.
Figure 7. (A) Images of tumors in each group taken out from the sacrificed H22 tumor-bearing mice at the end point of research. (B) Relative
tumor volumes and (C) weight changes in each group of mice during the treatments: (1) saline, (2) NIR, (3) MPDA@CMCS, (4) BTZ, (5) DOX,
(6) BTZ + DOX, (7) MPDA-BTZ@CMCS-DOX, and (8) MPDA-BTZ@CMCS-DOX + NIR. (D) H&E staining of tumors after different
treatments (equivalent drug concentration was 5 mg kg−1
; scale bars are 50 μm) (ns, no significance; *P < 0.05; **P < 0.01; and ***P < 0.001).
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MPDA-BTZ@CMCS-DOX +NIR. As shown in Figure 7A,B,
the final tumor sizes and mean relative tumor volumes were
obviously smaller for the groups of free BTZ, DOX, BTZ +
DOX, MPDA-BTZ@CMCS-DOX, and MPDA-BTZ@CMCSDOX + NIR than that of saline, NIR, and MPDA@CMCS
groups, suggesting efficient tumor inhibition effect of the
synthesized MPDA-BTZ@CMCS-DOX nanomaterial. Moreover, drug-loaded MPDA@CMCS (MPDA-BTZ@CMCSDOX) demonstrated a remarkable tumor growth inhibition
effect than free drugs, which demonstrated that the synthesized
MPDA@CMCS could be a promising drug delivery system.
Besides, MPDA-BTZ@CMS-DOX + NIR had a better effect
compared with MPDA-BTZ@CMS-DOX, exhibiting that
more effective tumor inhibition was realized by synergetic
therapy of chemotherapy and PTT.
Figure 7C shows the mean body weights of the groups with
various treatments. As can be seen from the body weight
profiles, the average body weight of groups treated with
MPDA-BTZ@CMCS-DOX (group 7) was not significantly
affected in comparison with that of saline treatment (group 1),
whereas the groups treated with free drugs (groups 4−6)
exhibited distinct weight loss, suggesting a relatively low
systematic toxicity by encapsulating cancer drugs in MPDA@
CMCS nanocarriers. In addition, hematoxylin & eosin (H&E)
staining of tumor slices was carried out to further evaluate the
tumor damage with different treatments. As shown in Figure
7D, obvious pathological changes, such as severe nuclear
shrinkage and apoptosis or necrosis of tumor cells, were
observed in the groups of BTZ, DOX, BTZ + DOX, MPDABTZ@CMCS-DOX, and MPDA-BTZ@CMCS-DOX + NIR.
Among them, the MPDA-BTZ@CMCS-DOX + NIR group
showed the best tumor cell ablation capacity, which was in
accordance with the results of final tumor sizes. Finally,
histological analysis toward normal tissues (liver, heart, lung,
spleen, and kidney) was implemented to study the side effects
of the as-synthesized nanomaterials, and negligible damage or
pathological changes were monitored after the treatments
(Figure S12). All of these results above suggested that the
design of the MPDA-BTZ@CMCS-DOX nanoplatform by
combining PTT and dual-drug therapy could be a promising
strategy to achieve enhanced therapeutic efficacy of the tumor.
4. CONCLUSIONS
In this study, a novel MPDA-BTZ@CMCS-DOX nanostructure was constructed for combined dual hydrophilic/hydrophobic drug delivery and PTT. In this structure, mesoporous
MPDA nanospheres with a hollow cavity exhibited a high
payload of hydrophobic BTZ loading and typical acid-induced
drug release behavior due to the pH sensitivity of the borate
ester bond. Meanwhile, CMCS was successfully decorated on
the surface of MPDA-BTZ, which could not only capture
hydrophilic DOX through electrostatic interaction but also
exhibit acid-responsive drug release in the acidic tumor
microenvironment. In vitro studies demonstrated that the
drug release behavior of MPDA-BTZ@CMCS-DOX was
controllable by pH values and NIR irradiation. Besides,
MPDA-BTZ@CMCS-DOX could be efficiently internalized
by cancer cells and distinct tumor inhibition was realized,
which was emphasized by a series of in vitro/vivo experiments.
Therefore, the MPDA-BTZ@CMCS-DOX nanoplatform
could be a promising host for synergistic chemo-photothermal
therapy.
■ ASSOCIATED CONTENT
*sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsabm.0c01451.
Materials, characterization, nitrogen absorption/desorption isotherms, hydrodynamic sizes, HeLa cell viabilities,
fluorescence images and confocal microscopic images,
hemolysis, and H&E staining assay (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
Shuang Zhao − Key Laboratory of Natural Medicine and
Immune-Engineering of Henan Province, Henan University,
Kaifeng 475004, Henan, P. R. China; Email: szhao@
henu.edu.cn
Jiahua Shi − Key Laboratory of Natural Medicine and
Immune-Engineering of Henan Province, Henan University,
Kaifeng 475004, Henan, P. R. China; orcid.org/0000-
0002-5051-0530; Email: [email protected]
Authors
Shanshan Li − Key Laboratory of Natural Medicine and
Immune-Engineering of Henan Province, Henan University,
Kaifeng 475004, Henan, P. R. China
Ying Gan − Key Laboratory of Natural Medicine and
Immune-Engineering of Henan Province, Henan University,
Kaifeng 475004, Henan, P. R. China
Chen Lin − Key Laboratory of Natural Medicine and
Immune-Engineering of Henan Province, Henan University,
Kaifeng 475004, Henan, P. R. China
Kunpeng Lin − Key Laboratory of Natural Medicine and
Immune-Engineering of Henan Province, Henan University,
Kaifeng 475004, Henan, P. R. China
Peng Hu − Key Laboratory of Natural Medicine and ImmuneEngineering of Henan Province, Henan University, Kaifeng
475004, Henan, P. R. China
Lei Liu − Key Laboratory of Natural Medicine and ImmuneEngineering of Henan Province, Henan University, Kaifeng
475004, Henan, P. R. China
Shuling Yu − Key Laboratory of Natural Medicine and
Immune-Engineering of Henan Province, Henan University,
Kaifeng 475004, Henan, P. R. China; orcid.org/0000-
0003-1919-8734
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsabm.0c01451
Author Contributions
S.L. and Y.G. are co-first authors.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was supported by grants from the Joint Fund of
National Natural Science Foundation of China and Henan
Province (U1404508), the Natural Science Foundation (Grant
No. 192102210021), Innovation Scientists and Technicians
Troop Construction Projects (C20150011), Key Scientific
Research Projects of Higher Education Institutions (Grant No.
20A430006) of Henan Province, and the Young Talents
Cultivation Program of the School of Medicine, Henan
University in 2019 (Grant No. 201902).
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