Acta Biomaterialia 135 (2021) 164–178

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actbio

Full length article

Intelligent and spatiotemporal drug release based on multifunctional

nanoparticle-integrated dissolving microneedle system for synergetic
chemo-photothermal therapy to eradicate melanoma
Yiting Zhao a,1, Yixian Zhou a,1, Dan Yang b, Xinyi Gao b, Ting Wen a, Jintao Fu a, Xinguo Wen c,
Guilan Quan b,∗, Xin Pan a,∗, Chuanbin Wu b
a
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
b
College of Pharmacy, Jinan University, Guangzhou 510632, China
c
Guangzhou Neworld Micnanobio Pharmatech Co., Ltd, Guangzhou 510670, China

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

Article history: Cutaneous melanoma is one of the most common malignant skin cancer with high lethality. Chemother-
Received 24 May 2021 apy and photothermal therapy are important and extensively studied treatment modalities for melanoma.
Revised 3 September 2021
However, these therapies still face some challenges, which severely restrict their further applications, such
Accepted 7 September 2021
as unsatisfactory efficacy of monotherapy, nonspecific uptake and release during drug delivery, and un-
Available online 13 September 2021
expected adverse effects from system administration. Recently, the strategies of collaboration, functional
Keywords: modification, stimuli-responsive design, and topical administration all show great prospect for solving
Cellular uptake above problems. In this research, a multifunctional nanoparticle-integrated dissolving microneedle drug
Specific distribution delivery system was constructed, in which the nanoparticles were prepared based on the framework with
Synergistic therapy the incorporation of photothermal agent (CuS) into Zeolitic imidazolate framework-8 and functionalized
Dissolving microneedles by hyaluronic acid. This system can co-load multi-modal drugs, improve specific uptake and distribu-
Superficial tumor
tion of targeted tumor, deliver drug locally, and release drug intelligently and spatiotemporally, thereby
promising a low-dose administration with high efficiency. The high inhibiting tumor performance and
excellent systematic safety were verified both in vitro and in vivo. Together, this smart design overcame
the drawbacks of monotherapy and conventional system administration. We believe the nanoparticle-
integrated dissolving microneedles will be in prospect of clinical application for more superficial tumors
with further delicate optimization.

Statement of significance

Melanoma is one of the most common skin cancers with high lethality. Extensively studied chemother-
apy and photothermal therapy still face some challenges, such as the limited therapeutic efficacy and the
severe system adverse effects. In order to overcome these drawbacks, the multifunctional nanoparticle-
integrated dissolving microneedles (DMNs) were designed. Especially, the nanoparticles could co-load
multi-modal drugs, improve specific uptake, and release drug intelligently and spatiotemporally. The mi-
croneedles could increase the drug accumulation in tumor, thus achieving excellent therapeutic efficacy
and reducing side effects. This system paved the way to a less invasive, more focused and efficient ther-
apeutic strategy for melanoma therapy.
© 2021 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

1. Introduction cer, resulting in nearly 12 thousand deaths yearly [1,2]. In the con-
text of skin cancer, melanoma is the most life-threatening type
Skin cancer has been one of the ruthless killers for human [2] and responsible for over 75% of deaths [3]. Chemotherapy and
health. In USA, over 10 0,0 0 0 patients are diagnosed with skin can- photothermal therapy (PTT) are important and extensively studied
treatment modalities for melanoma. Chemotherapy directly kills
∗
Corresponding authors. susceptible cancer cells by damaging the chromosome structural
E-mail addresses: quanguilan@jnu.edu.cn (G. Quan), panxin2@mail.sysu.edu.cn integrity and disrupting DNA synthesis [4], and PTT can destroy
(X. Pan). tumor cell membranes and denature proteins via hyperthermia to
1
These authors equally contributed to this work.

https://doi.org/10.1016/j.actbio.2021.09.009
1742-7061/© 2021 Published by Elsevier Ltd on behalf of Acta Materialia Inc.
Y. Zhao, Y. Zhou, D. Yang et al.
induce tumor cell apoptosis [5–8]. However, the above therapies
still face some challenges, which severely restrict their further ap-
plications. First, the therapeutic efficacy of monotherapy is unde-
sirable due to tumorous complexity and heterogeneity. The strat-
egy focused on combination therapy has attracted considerable at-
tention, which can achieve additive and/or synergistic antitumor
efficacy through pleiotropic and complex mechanisms. For exam-
ple, in combination therapy based on PTT and chemotherapy, PTT
can cause cellular damage by hyperthermia, simultaneously the el-
evated temperature can increase the blood flow to accelerate drug
delivery into deep-seated tumors, thus achieving synergistic effi-
cacy [9–12]. Second, nonspecific uptake and release during drug
delivery cause decreased damage for tumor cells. To tackle this
problem, intelligent nano-formulation exploitation [13–15], func-
tionally targeted modification [16,17], and spatiotemporally con-
trolled release design [18,19] are introduced. Third, system admin-
istration remains the most conventional mode for tumor therapy,
while it always results in severe system adverse effects and con-
siderable drug wastage in local tumor therapy. Therefore, topical
drug delivery may be an effective solution to concentrate drugs in
the tumor site and reduce drugs absorbed into blood circulation,
thereby minimizing side effects.
Metal organic frameworks (MOFs) are a type of porous mate-
rial with uniform and tunable porosity [20–23]. Among them, Ze-
olitic imidazolate framework-8 (ZIF-8) is one of the most represen-
tative MOFs built from zinc ions and 2-methylimidazolate (2-Mim),
and is regarded as a promising drug carrier in tumor treatment
due to its unique advantages [24–27]. For example, ZIF-8 can load
multi-modal drugs on account of its unique porous interior. In ad-
dition, owing to the pH-responsive degradation of ZIF-8, the ZIF-8
based nanoparticles can release drugs intelligently in the acid tu-
mor microenvironments (pH 5.5-6.0) [28,29]. Simultaneously, the
zinc ions and 2-Mim produced by decomposition are nontoxic and
easy to be metabolized and excreted under physiological condi-
tions, demonstrating its high biocompatibility. Apart from that, due
to the adjustable surface functionality, ZIF-8 can be modified to
improve active targeting, reduce rapid clearance, prolong blood cir-
culation, and so on. For instance, hyaluronic acid (HA) has been
widely used as a targeted “guider” in tumor cells with CD44 re-
ceptor overexpression [30,31], therefore, HA modification applied
to ZIF-8 based nanoparticles can specifically increase cellular up-
take [32–35] and thus improve therapeutic efficacy.
Microneedles (MNs) are a novel transdermal drug delivery sys-
tem (DDS), which can pierce the stratum corneum painlessly and
generate mechanical microchannels to remarkably facilitate drug
permeation in superficial tumor therapy. Dissolving microneedles
(DMNs) are the most extensively explored type of MNs during the
past years [36–38], because DMNs can encapsulate drug molecules
into the whole needle with high and precise drug dose, and re-
lease drug gradually after being dissolved in the tissue fluid. In
addition, compared with intravenous injection, DMNs can realize
uniform three-dimensional channel distribution, improve drug en-
richment in tumors, and minimize systematic side effects [37–40].
However, the direct loading of drug molecules into DMNs consists
of many problems, such as incompatible properties between drugs
and polymeric materials, uncontrollable drug release, and unsat-
isfactory efficacy of monotonic drug delivery [41]. Therefore, it is
urgent and necessary to develop a stable, intelligent, and multi-
modal DDS to overcome these drawbacks.
We are inspired to design a multifunctional nanoparticle-
integrated DMNs for multi-modal collaborative therapy of
melanoma. This system can co-load various drugs, improve
the specific uptake and distribution of tumor, and achieve intel-
ligent and spatiotemporal drug release, thereby promising a high
efficiency with low-dose administration. First, we simultaneously
encapsulated the photothermal agent (CuS nanoparticles) and hy-
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Acta Biomaterialia 135 (2021) 164–178
drophobic chemotherapeutic drug (Camptothecin, CPT) into ZIF-8
framework. Subsequently, the nanoparticles were coated with a
HA shell, denoted as CPT-CuS-ZIF-8@HA (Scheme 1A). Finally, CPT-
CuS-ZIF-8@HA nanoparticles were loaded into the tips of DMNs
through molding method [42] for local and synergetic therapy
(Scheme 1B). The successfully synthesized CPT-CuS-ZIF-8@HA
nanoparticles were characterized by scanning electron microscopy
(SEM), dynamic light scattering (DLS), Fourier transform infrared
ray (FT-IR), powder X-ray diffraction (PXRD), thermogravimetric
analysis (TGA), and ultra-violet and visible (UV-Vis) measurement.
As expected, CPT-CuS-ZIF-8@HA could achieve intelligent and spa-
tiotemporal control drug release under low pH and near infrared
ray (NIR) irradiation in vitro. In addition, the results of cellular
uptake suggested HA modification could significantly improve
the internalization of nanoparticles. Moreover, in vivo biodistribu-
tion and pharmacodynamics further confirmed the topical drug
enrichment and synergetic multi-modal therapy of the DDS. In
general, the strategic integration of DMNs and multifunctional
nanoparticles exhibited superior advantages in intelligent and
spatiotemporal drug delivery for superficial tumor therapy, and
we believe the future designs of diverse hierarchical targeting and
stimuli-responsivity could further broaden the therapeutic range.
2. Experimental section/methods
2.1. Materials
CPT and Copper(II) chloride dehydrate (CuCl2 ·2H2 O) were ac-
quired from Shanghai Macklin Biochemical Co., Ltd. (Shanghai,
China). HA, Zinc nitrate hexahydrate (Zn(NO3 )2 6·H2 O), 2-Mim, flu-
orescein (FL), polyvinyl alcohol (PVA 103), and sodium sulfide
(Na2 S) were supplied by Aladdin Bio-Chem Technology Co., Ltd.
(Shanghai, China). Polyvinyl Pyrrolidone (PVP-K30, 40 kDa) and 4 ,
6-Diamidino-2-phenylindole (DAPI) were purchased from Beijing
Solarbio Science & Technology Co., Ltd. (Beijing, China). Annexin
V-FITC/Propidium Iodide (PI) was obtained from MultiSciences
Biotech Co., Ltd. (Hangzhou, China). Calcein-AM/PI kit was supplied
by BestBio Biotechnology Co., Ltd. (Shanghai, China). Lyso Tracker
Red probe was acquired from G-clone Biotechnology Co., Ltd. (Bei-
jing, China).
2.2. Synthesis of CuS-ZIF-8 and CPT-CuS-ZIF-8
CuS nanoparticles were prepared by the hydrothermal method.
First, 112 mg of CuCl2 ·2H2 O was added into 80 mL of aqueous solu-
tion of PVP-K30 (4 mg/mL) and stirred for 30 min, and then 0.8 mL
of aqueous solution of Na2 S (60 mg/mL) was poured into the above
solution. After 5 min, the sample was heated at 90 °C for 15 min
to prepare CuS nanoparticles. Afterwards, 8 mL of as-prepared CuS
nanoparticle methanol dispersion (1.63 mg/mL) was mixed with 20
mL of Zn(NO3 )2 ·6H2 O methanol solution (293 mg, 0.98 mmol). Af-
ter stirring for 25 min, 12 mL of 2-Mim methanol solution (649
mg, 7.90 mmol) was added into the aforementioned mixture, and
the mixture was sequentially stirred for another 25 min. CuS-ZIF-8
nanoparticles were collected by centrifuging at 12,0 0 0 rpm for 5
min. Subsequently, the nanoparticles were washed with methanol
to remove redundant reagents for several times, and then were re-
dispersed in 3 mL of methanol for storage.
The only difference of the synthesis of CPT-CuS-ZIF-8 was that
CPT (20 mg, 0.06 mmol) was pre-dissolved into the Zn(NO3 )2 ·6H2 O
methanol solution. In the process, CuS nanoparticles were embed-
ded in ZIF-8 framework and CPT was in situ trapped into the pores
of the hybrid framework during crystal growth [43,44], thereby
forming CPT-CuS-ZIF-8 in one step. The supernatant during the
synthesis of CPT-CuS-ZIF-8 was collected for the measurement of
encapsulation efficiency (EE) and drug loading capacity (DLC) via
Y. Zhao, Y. Zhou, D. Yang et al.
Scheme 1. Schematic illustration of (A) preparation procedure of CPT-CuS-ZIF-8@HA and (B) multifunctional nanoparticle-integrated DMNs for local administration, targeted
delivery, intelligent drug release, and synergetic chemo-photothermal therapy in melanoma treatment.
high performance liquid chromatograph (HPLC). EE and DLC were
calculated according to the following equations:
 
EE(% ) = Minitial drug − Mdrug in supernatant /Minitial
 
DLC(% ) = Minitial drug − Mdrug in supernatant /Mnanoparticles × 100%
2.3. Preparation of CPT-CuS-ZIF-8@HA
CPT-CuS-ZIF-8@HA was prepared by HA surface modification on
CPT-CuS-ZIF-8 via coordination bond [45] and electrostatic interac-
tion [46]. First, 4.50 mg of HA was dissolved in 6 mL of water,
and 1.5 mL of as-prepared CPT-CuS-ZIF-8 nanoparticles (32 mg)
methanol dispersion was diluted with water to 3 mL. Afterwards,
the aforementioned HA water solution (0.50 mg/mL) and the CPT-
CuS-ZIF-8 nanoparticles methanol/water dispersion (3.56 mg/mL)
were mixed and stirred for 30 min. Subsequently, CPT-CuS-ZIF-
8@HA nanoparticles were collected by centrifuging at 15,0 0 0 rpm
for 3 min. Finally, the nanoparticles were washed with water for
several times, and then were redispersed in 1 mL of water for stor-
age.
The fluorescein labeled nanoparticles (FL-CuS-ZIF-8 and FL-CuS-
ZIF-8@HA) were also synthesized for cellular uptake, subcellular
location, and in vivo imaging and biodistribution studies using the
similar procedure mentioned above.
2.4. Characterization
Particle size and zeta potential were measured by DLS (Zeta-
sizer Nano, Malvern Instrument, Britain). Morphology was char-
acterized by transmission electron microscopy (TEM, FEI Tecnai
G2 Spirit, Netherlands) and Field-Emission-SEM (GeminiSEM500,
Zeiss/Bruker, Germany). The molecular interaction was indicated
by FT-IR (UATR Two, Perkin Elmer, America). The UV-Vis spec-
trum was measured by a UV-vis spectrophotometer (UV-2600, Shi-
madzu, Japan). Surface area and pore volume were evaluated by
Acta Biomaterialia 135 (2021) 164–178
nitrogen adsorption-desorption analysis (ASAP2460, Micromeritics,
America). Crystallography was analyzed by PXRD (D8 Advance,
Bruker, Karlsruhe, Germany) using Cu Kα radiation with 2θ in the
drug × 100%
range of 3o -40o at a scanning rate of 5o /min. Characteristic of mass
varying with temperature was monitored by TGA (TG209F1 libra,
NETZSCH-Gerätebau GmbH, Germany).
2.5. In vitro photothermal effect of nanoparticles
One milliliter of CuS, CuS-ZIF-8, and CPT-CuS-ZIF-8 samples
with the same CuS concentration (50 μg/mL), and 1 mL of CPT-
CuS-ZIF-8@HA samples with different concentrations (10 0, 30 0,
and 500 μg/mL) were put in 2 mL Eppendorf tube, and subse-
quently irradiated by an 808 nm laser (1.0 W/cm2 ) for 5 min. The
temperature was measured and photothermal images were taken
by an infrared thermal sensing and imaging equipment (TiS75,
Fluke, Everett, WA, America) every 30 s. ZIF-8 nanoparticles were
used as a contrast.
To evaluate the photostability, the CPT-CuS-ZIF-8@HA and pris-
tine CuS suspension were exposed to NIR for three cycles. Each cy-
cle was composed of 5 min irradiation followed by nearly 20 min
for cooling down.
2.6. In vitro drug release
To investigate the low pH and laser-responsive drug release of
CPT-CuS-ZIF-8@HA, the nanoparticles were dispersed in 6 mL of
PBS solution with various pH values (pH 5.0, 6.0 and 7.4), and then
the samples were incubated with continuous shaking (100 rpm) at
37 °C. At predetermined time points, 1.0 mL of supernatant was
collected and equal volume of corresponding fresh PBS solution
was added. Meanwhile, the samples were irradiated with an 808
nm laser (1.0 W/cm2 ) for 5 min, followed by similar sampling pro-
cess. Finally, the CPT concentration was measured by HPLC.
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2.7. Cell culture h. Finally, the cells were collected and handled according to the in-
struction of the kit, and subsequently were quantitatively analyzed
B16 cells (a cell line of mouse melanoma cells) were cul- via flow cytometry.
tured with the 1640 medium. A375 cells (a cell line of human
melanoma cells) were cultured with the DMEM medium. Both cell
2.10. Fabrication and characterization of CPT-CuS-ZIF-8@HA@MN
culture medium contained 10% FBS and 1% antibiotics (penicillin
and streptomycin). An incubator with 5% CO2 was used to culture
The nanoparticle-integrated microneedles were prepared by
cells at 37 °C.
multi-step centrifugation casting method referring to a previous
report [47]. First, the tip solution was made of PVP K30 and PVA
2.8. In vitro cellular uptake and subcellular location
103 with a ratio of 6:1 (w/w), and the base solution was 30% (w/v)
PVP K90 prepared by pre-swelling in ethanol overnight. And then,
B16 cells were pre-cultured in NUNCTM imaging 24-well plate
a quarter volume of tip solution was added to the CPT-CuS-ZIF-
for 24 h at 37 °C. The excessive amount of HA medium solution (10
8@HA nanoparticle suspension with a concentration of 16 μg/mL
mg/mL) was added into the plates of HA+FL-CuS-ZIF-8@HA group
to form tip mixture. The preparation procedure of CPT-CuS-ZIF-
for 2 h pre-incubation. After that, fresh FL-CuS-ZIF-8 and FL-CuS-
8@HA@MN was described below. Proper volume of tip mixture was
ZIF-8@HA medium suspension were added to replace the original
spread out on the poly (dimethylsiloxane) (PDMS) female mold and
medium in plates for 4 h incubation. Then, the cells were rinsed
centrifuged at 40 0 0 rpm for 5 min at 4 °C. Then, excess tip mix-
three times with cold PBS solution and fixed by 4% paraformalde-
ture on the surface of female mold was removed and the mold was
hyde at 4 °C for 10 min in dark. Later, the fixed liquid was re-
centrifuged at 40 0 0 rpm for 1 h to enrich the nanoparticles to tip.
moved, and the cells were rinsed with PBS again and stained by
Subsequently, the female mold was dried for 10 h in a dryer at
DAPI for 15 min. Finally, the cells were rinsed and fluorescence
room temperature, and the operation of centrifugation and drying
imaging was performed by FV30 0 0 Olympus confocal microscope
was repeated to improve drug loading. Afterwards, the tip solution
for cellular uptake observation. As for flow cytometry analysis, the
and base solution were poured on the female mold and then cen-
cells in plates were washed and collected to obtain quantitative re-
trifuged for 5 min in sequence. Finally, the microneedle patch was
sults of cellular internalization. Additionally, the lysosome imaging
collected after drying for 24 h in a dryer at room temperature. The
was performed with the aid of LysoTracker Red probe for subcel-
CPT loading amount of CPT-CuS-ZIF-8@HA@MN was calculated by
lular location study. After 2 h of incubation with nanoparticle sus-
HPLC after dissolving the whole excisional microneedle tips.
pension, the cells were rinsed three times with PBS solution, and
In addition, the morphology of DMNs was characterized un-
treated with pre-heated LysoTracker Red probe to stain lysosome
der a mobile microscope and Thermal Field Emission Enviromen-
at 37 °C for 30 min in dark. Later, the red probe solution was re-
tal SEM EDS EBSD (Quanta 400F, FEI/OXFORD/HKL, Netherlands).
moved and the cells were rinsed with PBS and stained by Hoechst
The nanoparticle distribution and 3D reconstruction were observed
for 15 min.
through FV30 0 0 Olympus confocal microscope with FL-CuS-ZIF-
8@HA@MN.
2.9. In vitro cytotoxicity, live/dead cell staining, and apoptosis assay

In vitro anti-tumor efficiency of CPT-CuS-ZIF-8@HA was eval-
uated by a standard CCK-8 method. B16 cells (12,0 0 0 cells/well)
and A375 cells (8,0 0 0 cells/well) were seeded in 96-well plate and
cultured for 24 h. After 4 h of incubation with nanoparticle sus-
pension of different concentrations and following 5 min NIR irra-
diation (1.0 W/cm2 ), the nanoparticle medium suspension was re-
moved and the cells were cultured in fresh medium for another 20
h. Subsequently, the cells were incubated with 110 μL/well of CCK-
8 solution (CCK-8: medium = 1:10) for 1 h at 37 °C. Finally, the
absorbance of each well at 450 nm was measured by a microplate
reader. The cell viability was calculated according to the following
Equation:
 
Cell Viability (% ) = Asample − Ablank /(Acon − Ablank ) × 100%
The live/dead cell staining assay was studied via a Cell Imager
(EVOS FL Auto, Life Technologies, America) after being stained by
the Calcein-AM/PI kit. B16 cells (10 0,0 0 0 cells/well) were seeded
in 24-cell plate and pre-cultured for 24 h. After 4 h of incubation
with nanoparticle suspension (CPT: 11.38 μg/mL, CuS: 9.85 μg/mL)
and following 5 min NIR irradiation (1.0 W/cm2 ), the nanoparticle
medium suspension was removed and the cells were rinsed with
fresh medium twice. Finally, cells in each well were stained accord-
ing to the instruction of the kit, and subsequently observed via a
Cell Imager.
The cell apoptosis assay was investigated using flow cytometry
after being stained by the Annexin V-FITC/PI apoptosis assay kit.
B16 cells (20 0,0 0 0 cells/well) were seeded in 6-well plate and pre-
cultured for 24 h. After 4 h of incubation with nanoparticle sus-
pension (CPT: 3.41 μg/mL, CuS: 2.96 μg/mL) and following 10 min
NIR irradiation (1.0 W/cm2 ), the nanoparticle suspension was re-
moved and the cells were cultured in fresh medium for another 20
2.11. Insertion capability evaluation
To evaluate the insertion capability, the CPT-CuS-ZIF-8@HA@MN
was pressed on the isolated depilated skin of rat and porket for 2
min. After removal, the insertion pores were imaged with a mo-
bile microscope. Furthermore, the inserted skin was also stained
by hematoxylin and eosin (H&E).
2.12. In vitro photothermal effect of DMNs
Blank DMN, CuS-ZIF-8@MN, and CPT-CuS-ZIF-8@HA@MN were
irradiated by an 808 nm laser with different power intensities (1.0,
0.5, 0.3 W/cm2 ) for 5 min. The temperature was measured and
photothermal images were taken every 30 s. The blank DMN was
applied as a contrast.
To investigate the photostability of nanoparticle-integrated
DMNs, the CPT-CuS-ZIF-8@HA@MN was exposed to NIR (1.0, 0.5
W/cm2 ) for three cycles. Each cycle was composed of 5 min irradi-
ation followed by 7 min or 4 min for cooling down.
2.13. Construction of animal melanoma models
C57BL/6 female mice were supplied by Guangdong Medical Ex-
perimental Animal Center, and all experiments were supervised by
the Animal Ethics Committee of Sun Yat-sen University (Approval
No. SYSU-IACUC-2021-0 0 0 029, Guangdong, China). After adaptive
feeding for 7 days in the Experimental Animal Center of Sun Yat-
sen University (Guangdong, China), the suspension of B16 cells (50
μL, 3 × 106 ) was inoculated subcutaneously into the depilated back
of the mice.

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Y. Zhao, Y. Zhou, D. Yang et al.
2.14. In vivo photothermal effect
The tumor-bearing C57BL/6 female mice were divided into
four groups randomly after the tumor volume being 100-120
mm3 : (1) control+NIR, (2) CuS-ZIF-8@MN+NIR, (3) CPT-CuS-ZIF-
8@HA@MN+NIR, and (4) CPT-CuS-ZIF-8@HA (I.T.)+NIR. The corre-
sponding DMN patches were pressed on the tumor site for 2 min
by thumb and subsequently secured on the back with medical
tape. Simultaneously equal amount of CPT-CuS-ZIF-8@HA nanopar-
ticles were intratumorally injected. After 4 h, the patches were
teared and the tumor site was irradiated by an 808 nm laser (1.0
W/cm2 ) for 5 min. The temperature was monitored and photother-
mal images were taken every 1 min.
2.15. In vivo imaging and biodistribution
The administration of FL-CuS-ZIF-8@HA@MN was almost simi-
lar to the aforementioned in vivo photothermal study. Live fluores-
cence imaging system (IVIS Lumina XRMS, PerkinElmer, America)
was used to detect the fluorescence signals at 0, 2, 4, 6, 8, and
12 h. Finally, the mice were sacrificed after the last time point,
and the fluorescence signal distribution in tumor and major organs
(heart, liver, spleen, lung, and kidney) was observed.
Additionally, the drug penetration capability was investigated.
After administration of FL-CuS-ZIF-8@HA@MNs for 4 h, the mice
were sacrificed and the excised tumor were quickly frozen in liquid
nitrogen. Afterwards, the frozen sections of tumor were stained by
DAPI and subsequently imaged by FV30 0 0 Olympus confocal mi-
croscope.
2.16. In vivo anti-tumor study
The tumor-bearing C57BL/6 female mice were divided into six
groups randomly after the tumor volume being 100-120 mm3 :
(1) untreated, (2) CuS-ZIF-8@MN, (3) CuS-ZIF-8@MN+NIR, (4) CPT-
CuS-ZIF-8@HA@MN, (5) CPT-CuS-ZIF-8@HA nanoparticles intratu-
moral injection (I.T.)+NIR, and (6) CPT-CuS-ZIF-8@HA@MN+NIR.
The administration of DMNs was according to the aforementioned
in vivo photothermal study. Meanwhile, equal amount of CPT-CuS-
ZIF-8@HA nanoparticles were intratumorally injected. After 4 h, the
tumor site was irradiated by an 808 nm laser (1.0 W/cm2 ) for 5
min for NIR groups. Subsequently, tumor volume and body weight
were measured and recorded every two days. Finally, the mice
were sacrificed, and the tumor and major organs were weighed af-
ter excising on day 12. During the experiment, the mice would be
sacrificed for kindness when tumor volume was over 20 0 0 mm3 .
The tumor volume was calculated according to the following equa-
tion:
Tumor volume = L(tumor length ) × W 2 (tumor width ) /2
In addition, the survival rates of the mice were monitored every
day in four weeks.
2.17. Safety evaluation
To evaluate the safety of DMNs administration, healthy
C57BL/6 female mice were divided into three groups randomly:
(1) untreated, (2) CPT-CuS-ZIF-8@HA@MN, and (3) CPT-CuS-ZIF-
8@HA@MN+NIR. After DMNs administration and following NIR ir-
radiation (1.0 W/cm2 ) for 5 min, the skin condition was observed
and imaged by a digital camera at predetermined date (day 0, 1, 3,
5, 7, and 10).
2.18. Histology and pathological investigation
The excised tumors and major organs were fixed in 4%
paraformaldehyde for 24 h before paraffin embedding, and then
168
Acta Biomaterialia 135 (2021) 164–178
the tissues were sliced for different tests. The tumor slices were
stained with H&E, cleaved caspase 3, terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP nick end labeling (TUNEL), and
Ki67 to assess the antitumor efficacy, and the major organs slices
were stained by H&E for safety evaluation.
2.19. Statistical analysis
Data are expressed as the means ± standard deviation (SD).
Statistical analysis was performed using a one-way ANOVA test
(Graphpad Prism 7.00). The post hoc comparisons of the means of
individual groups were performed using least significant difference
test. Differences were considered significant if P < 0.05.
3. Results and discussion
3.1. Preparation and characterization of CPT-CuS-ZIF-8@HA
The preparation procedure of CPT-CuS-ZIF-8@HA was illustrated
in Scheme 1A. First, one of the guests, CuS nanoparticles, were syn-
thesized with an extremely small size about 10 nm (Fig. S1) via
hydrothermal method. Then, CuS and CPT were co-encapsulated in
ZIF-8 and followed by HA coating to prepare CPT-CuS-ZIF-8@HA.
The elemental mapping confirmed CuS was distributed within the
framework of ZIF-8 (Fig. S2). As observed by SEM (Fig. 1A), the
morphology of CPT-CuS-ZIF-8@HA was roughly spherical, and their
diameters were approximately 50 nm, which was consistent with
the average hydrodynamic diameter determined by DLS (Fig. 1B).
For comparison, the morphology and hydrodynamic diameter of
ZIF-8, CuS-ZIF-8, and CPT-CuS-ZIF-8 were tested and exhibited in
Fig. S1 and Fig. S3A, respectively. The stepwise increment of par-
ticle size was attributed to the guest encapsulation and surface
modification during different synthetic stages. In addition, the zeta
potential was measured and exhibited in Fig. S3B. The potential of
ZIF-8 was +18.8 eV and slightly decreased to +16.4 eV for CuS-ZIF-
8. After CuS and CPT co-encapsulation, a significant change was
observed from +18.8 eV (ZIF-8) to +6.0 eV (CPT-CuS-ZIF-8). Fi-
nally, HA modification dramatically reversed the zeta potential to
-20.6 eV (CPT-CuS-ZIF-8@HA). This zeta potential variation initially
demonstrated the successful incorporation of negatively charged
CuS nanoparticles and CPT and the successful coating of negatively
charged HA. The negatively charged surface also endows CPT-CuS-
ZIF-8@HA with favorable stability for further biological tests.
Then, FTIR spectrum further qualitatively ascertained the mod-
ification processes of the nanoplatform. As presented in Fig. 1C,
the vibrations between CuS-ZIF-8 and ZIF-8 were nearly identical
except for the weak peak at 1701 cm−1 from the C=O stretch-
ing vibration of PVP existed in CuS nanoparticles, which implied
the existence of CuS nanoparticles. The stretching bands vibra-
tions in CPT-CuS-ZIF-8 corresponding to C-N (1097 cm−1 ) and C-
O (1235 cm−1 ) were indicative of CPT. In addition, the new broad
peak around 3310 cm−1 of CPT-CuS-ZIF-8@HA could be ascribed
to the stretching vibration of O-H of HA. The above representa-
tive signals further demonstrated the successful preparation of the
nanoparticles.
The UV-Vis spectra of various samples were displayed in
Fig. 1D. CPT and ZIF-8 did not present obvious absorption between
600 and 1000 nm, while samples containing CuS exhibited a broad
absorption band in this scope. In addition, compared with other
nanoparticles, CPT-CuS-ZIF-8 and CPT-CuS-ZIF-8@HA displayed an
obvious absorption peak of CPT at around 367 nm. These results
proved the successful encapsulation of guests. Simultaneously, the
concentrations of CPT were determined by an UV-detector in HPLC
for calculating EE and DLC. And the EE and DLC were determined
to be 74.18% and 11.38%, respectively.
Y. Zhao, Y. Zhou, D. Yang et al. Acta Biomaterialia 135 (2021) 164–178

Fig. 1. (A) SEM image and (B) size distribution of CPT-CuS-ZIF-8@HA. (C) FT-IR spectra of CPT-CuS-ZIF-8@HA, CPT-CuS-ZIF-8, CuS-ZIF-8, HA, CPT, CuS, and ZIF-8. (D) UV
absorption spectra of CPT-CuS-ZIF-8@HA, CPT-CuS-ZIF-8, CuS-ZIF-8, CPT, CuS, and ZIF-8. (E) Nitrogen adsorption-desorption isotherms and (F) pore size distribution of ZIF-8,
CuS-ZIF-8, and CPT-CuS-ZIF-8. (G) PXRD patterns and (H) thermogravimetric analysis of CPT-CuS-ZIF-8@HA, CPT-CuS-ZIF-8, CuS-ZIF-8, HA, CPT, CuS, and ZIF-8.

Nitrogen adsorption-desorption analysis was utilized to calcu-
late the surface area and pore volume of different samples. As ex-
hibited in Fig. 1E, the BET surface area of ZIF-8, CuS-ZIF-8, and
CPT-CuS-ZIF-8 were 1886, 1321, 1289 m²/g, respectively. The sur-
face area decreased with the encapsulation of CuS nanoparticles
and CPT, because the non-porous CPT and CuS nanoparticles con-
tributed to the mass of the composite and simultaneously ob-
structed the nitrogen flow [43]. Pore size distribution of the three
samples all exhibit a maximum at 1 nm (Fig. 1F), which indicated
the encapsulation could arise negligible influence on the pore
size.
PXRD can identify the crystallography of samples. As shown in
Fig. 1G, the diffraction peaks of CPT-CuS-ZIF-8 and CPT-CuS-ZIF-
8@HA were consistent with the characteristic peaks of ZIF-8, sug-
gesting that the incorporation of guests and HA modification did
not affect the crystal structure. However, the characteristic diffrac-
tion peaks related to CPT disappeared in the PXRD patterns of
CPT-CuS-ZIF-8, which indicated that CPT, as a guest molecule, was
loaded into ZIF-8 pores in amorphous or molecular state. Further-
more, the successful preparation process was further verified by
the weight loss observed in TGA (Fig. 1H).
3.2. In vitro photothermal effect and drug release properties of
CPT-CuS-ZIF-8@HA
Different samples with equal CuS concentration (50 μg/mL)
were irradiated to study the photothermal effect. After irradiation,
similar to the pure CuS, the temperature of CPT-CuS-ZIF-8@HA in-
creased from 28.2 °C to 57.4 °C, while the temperature increment
of ZIF-8 was negligible (Fig. 2A and B). The above results indicated
that the preparation procedure did not affect the photothermal
conversion efficacy of CuS, and the wonderful temperature-rising
performance of CPT-CuS-ZIF-8@HA made it potential to apply for
further PTT. Moreover, CPT-CuS-ZIF-8@HA displayed concentration-
dependent photothermal effects (Figs. 2C and S4). Therefore, the
intensity of PTT could be regulated on demand by dosing of CPT-
CuS-ZIF-8@HA. In addition, as shown in Fig. 2D, no obvious change
was observed in the temperature profiles among the three on/off
NIR irradiation cycles, which suggested that the nanoparticles pos-
sessed superior photothermal stability for repeated and pulsatile
therapy [42].
In addition, the drug release properties were investigated.
As photothermal agent, CuS nanoparticles were embedded in

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Fig. 2. (A) Temperature change curves of CPT-CuS-ZIF-8@HA, CPT-CuS-ZIF-8, CuS-ZIF-8, CuS, and ZIF-8 under laser irradiation (1.0 W/cm2 ) at different time points (equal CuS
concentration of 50 μg/mL). (B) The thermographic images of ZIF-8 and CPT-CuS-ZIF-8@HA (500 μg/mL) under NIR irradiation (1.0 W/cm2 ) at different time points (equal CuS
concentration of 50 μg/mL). (C) Temperature change curves of CPT-CuS-ZIF-8@HA at different concentrations under NIR irradiation (1.0 W/cm2 ). (D) Photothermal conversion
stability curves of CuS and CPT-CuS-ZIF-8@HA for three cycles (1.0 W/cm2 , equal CuS concentration of 50 μg/mL). (E) CPT release profiles in PBS with different pH values
under on/off NIR irradiation (1.0 W/cm2 ) cycles (mean±S.D., n=3).
the framework of ZIF-8. When CPT-CuS-ZIF-8@HA was irradi-
ated by NIR, the internal CuS could convert light energy to heat
[11,35,48,49]. However, the other components were not heated si-
multaneously for their low light absorption coefficient. The un-
even heating resulted in uneven volume change in different re-
gions on account of thermal expansion in the whole CPT-CuS-ZIF-
8@HA, which could cause the structure collapse, thereby facilitat-
ing the release of CPT. In Fig. S5, the results of TEM confirmed
that laser irradiation could cause the structure collapse of CPT-
CuS-ZIF-8@HA. Moreover, since the coordination bonds between
zinc ions and 2-Mim linkers would be broken for protonation of
2-Mim in acidic condition [50], the drug release of CPT-CuS-ZIF-
8@HA was speculated to be pH-responsive. To evaluate the drug
release behaviors, CPT-CuS-ZIF-8@HA was irradiated repeatedly by
an 808 nm laser for 5 min at predetermined time points in release
medium (PBS) with different pH. The PBS with the pH of 5.0, 6.0,
7.4 were designed to simulate lysosomes, tumorous microenviron-
ment, and normal tissue fluid, respectively. As presented in Fig. 2E,
a burst release was appeared with the laser irradiation, while the
release without laser was really gradual, confirming the laser ac-
celerated release effect. Furthermore, the release rate of CPT was
significantly enhanced in acid medium due to the disintegration of
ZIF-8 [25]. The cumulative drug release amount in PBS with the
pH of 5.0 was about 65% in 12 h, which was five times as much as
that in pH 7.4. Taken together, the release profiles confirmed the
pH-responsive and laser accelerated release properties of the DDS.
It could be predicted that the DDS would be further applied to var-
ious tumor therapy, because acid tumor microenvironment and ad-
justably external laser irradiation would control the drug release
intelligently and spatiotemporally at the lesion site on demand.
3.3. In vitro cellular uptake and subcellular location
The cellular uptake and intracellular distribution were evalu-
ated by fluorescein encapsulated nanoparticles (FL-CuS-ZIF-8 and
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Acta Biomaterialia 135 (2021) 164–178
FL-CuS-ZIF-8@HA) via fluorescence imaging and flow cytometry
analysis. The fluorescence images (Fig. 3A) under FITC channel and
quantitative analysis (Fig. 3B) indicated that the fluorescence in-
tensity from B16 cells incubated with FL-CuS-ZIF-8@HA was sig-
nificantly higher than that of FL-CuS-ZIF-8 group, and which de-
creased obviously when B16 cells were preincubated with an ex-
cessive amount of HA [51], confirming that HA coating notably
enhanced the cellular uptake of the nano-DDS. The quantitative
results of flow cytometry analysis (Fig. 3C) reconfirmed the en-
hanced cellular uptake through HA. To further investigate the cel-
lular uptake route of FL-CuS-ZIF-8@HA, B16 cells were stained
with two fluorescent tracers, LysoTracker Red probe and Hoechst,
to trace the intracellular lysosome and nucleic acid (nuclear), re-
spectively (Figs. 3D and S6). The green fluorescence of FL-CuS-ZIF-
8@HA matched well with the red fluorescence from lysosome af-
ter incubation for 2 h (Fig. 3D), and the intensity enhanced grad-
ually with the extension of incubation time (Fig. S6). These re-
sults demonstrated that FL-CuS-ZIF-8@HA was internalized to tu-
mor cell through lysosome-mediated endocytosis. Since the pH of
intracellular lysosomes ranges from 4.5 to 5.0 [28,50], we specu-
lated that the pH-responsive drug delivery system would disinte-
grate well after internalization, thereby releasing the encapsulated
drug molecules intelligently as designed.
3.4. In vitro cytotoxicity, live/dead cell staining, and apoptosis assay
The cytotoxicity was studied on B16 and A375 cells by CCK-8
assay. As displayed in Fig. 4A, the carrier (CuS-ZIF-8) exhibited no
obvious toxicity at concentrations ranging from 6.25 to 100 μg/mL
on B16 cells, indicating its superior biocompatibility. In contrast,
the groups of CPT-CuS-ZIF-8@HA and CPT-CuS-ZIF-8@HA+NIR dis-
played high toxicity in a dose-dependent manner. For instance,
with the concentration of CPT-CuS-ZIF-8@HA increasing from 6.25
μg/mL (CPT: 0.72 μg/mL) to 100 μg/mL (CPT: 11.38 μg/mL), the
cellular viability remarkably decreased from 68.8% to 23.7%, and
Y. Zhao, Y. Zhou, D. Yang et al. Acta Biomaterialia 135 (2021) 164–178

Fig. 3. (A) Cellular uptake of FL-CuS-ZIF-8, FL-CuS-ZIF-8@HA, and HA + FL-CuS-ZIF-8@HA by B16 cells after incubation for 4 h. (B) Corresponding quantitative green fluo-
rescence intensities of B16 cells after incubation with FL-CuS-ZIF-8, FL-CuS-ZIF-8@HA, and HA + FL-CuS-ZIF-8@HA for 4 h (∗ ∗ P < 0.01 vs. FL-CuS-ZIF-8@HA, ∗ ∗ ∗ P< 0.001 vs.
FL-CuS-ZIF-8@HA, n=3). (C) Flow cytometry analysis of B16 cells incubated with FL-CuS-ZIF-8, FL-CuS-ZIF-8@HA, and HA + FL-CuS-ZIF-8@HA for 4 h, respectively. (D) The
fluorescence images of the location of FL-CuS-ZIF-8@HA in B16 cells after incubation for 2 h. The cell nuclei and lysosomes were labeled by Hoechst and LysoTracker Red,
respectively. Scale bars for (A) and (D): 50 μm.

it was much lower after NIR irradiation. Unexpectedly, the group
of CuS-ZIF-8+NIR showed low toxicity even at the concentration
of 100 μg/mL. Previous studies verified that coagulative necrosis
would occur when the temperature was above 50°C through cell
membrane collapse, protein denaturation, and mitochondrial dys-
function [52–54]. While the heat-induced cellular injury under 45
°C could be reversed by tumor thermo-resistance at this concen-
tration. In addition, the results of cytotoxicity on A375 cells were
almost consistent with those on B16 cells (Fig. 4B). Taking all the
above results together, the CPT-CuS-ZIF-8@HA+NIR group demon-
strated the highest cytotoxicity among all groups in both cell lines,
which verified the nano-platform could achieve a synergistic effect
for cancer therapy in vitro as predicted. Meanwhile, the results of
live/dead cell staining assay (Fig. 4C) were almost in concert with
the synergistic cell inhibition effect of cytotoxicity study.
Besides, the apoptosis assay was performed to evaluate the
cell apoptosis. As exhibited in Fig. 4D, Q2 represents late apopto-
sis or necrosis cells, and Q4 represents viable cells. The apoptotic
ratios of cells treated by CuS-ZIF-8+NIR and CPT-CuS-ZIF-8@HA
were 8.99% and 18.9%, respectively, which were significantly lower
than that of CPT-CuS-ZIF-8@HA+NIR group (40.1%). The quantita-
tive analysis demonstrated the highest cellular apoptotic percent-
age of CPT-CuS-ZIF-8@HA+NIR group, which further manifested
the synergistic therapeutic effect consistent with the aforesaid cy-
totoxicity and live/dead cells staining assay.
3.5. Fabrication and characterization of CPT-CuS-ZIF-8@HA@MN
Systemic administration remains the most conventional mode
for tumor therapy, while the unspecific toxicity is still one of the
greatest problems [41]. Therefore, topical drug delivery was intro-
duced for regional and superficial tumor therapy. DMNs are a kind
of promising transdermal drug delivery (TDD) device in mediat-
ing a local delivery. In this research, the nanoparticle-integrated
microneedles were prepared by multi-step centrifugation casting
method (Fig. S7). The patch was composed of 12 × 12(144) mi-
croneedles with 1200 μm in height, and the CPT loading amount
was 74 μg per patch. The morphology of DMNs was observed by
SEM, optical microscope, and confocal laser scanning microscopy
(CLSM). As shown in Fig. 5A, the tips of both blank DMNs and
nanoparticle-integrated DMNs exhibited as the rectangular pyra-
mid, indicating that the encapsulation of nanoparticles did not
cause significant morphology change or deficiency on tips. In ad-
dition, the optical image (Fig. 5B), fluorescent image (Fig. 5C), and

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Fig. 4. Cell viability of (A) B16 and (B) A375 cells after incubation with various samples for 24 h (mean±S.D., n=6). (C) Fluorescence images of B16 cells with various
treatments co-stained with Calcein-AM/PI. Scale bar: 200 μm. (D) Flow cytometric analysis of apoptosis on B16 cells after incubation with various samples.

3D reconstruction (Fig. 5D) further confirmed the homogenous dis-
tribution of nanoparticles in the tips of FL-CuS-ZIF-8@HA@MN. The
tips enrichment of nanoparticles made it prospective to realize the
efficient and precise drug delivery after DMNs puncture and thus
enhance efficacy. Apart from that, the skin insertion capacity of
DMNs was a vital factor for effective TDD, which could be eval-
uated by isolated depilated skin of rat and porket. After puncture
of DMNs, the microchannels left in rat skin (Fig. 5E) and porcine
skin (Fig. S8A) were clearly observed via an optical microscope.
Additionally, H&E staining of the inserted skin (Figs. 5F and S8B)

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Fig. 5. (A) SEM images of blank DMN, CuS-ZIF-8@MN, and CPT-CuS-ZIF-8@HA@MN. Scale bar: 500 μm. (B) Photograph of CPT-CuS-ZIF-8@HA@MN. (C) CLSM images of
FL-CuS-ZIF-8@HA@MN. Scale bar: 300 μm. (D) 3D reconstruction image of FL-CuS-ZIF-8@HA@MN under CLSM. (E) Photograph of the excised rat skin after insertion of
CPT-CuS-ZIF-8@HA@MN. (F) H&E staining histological section of inserted rat skin. Scale bar: 200 μm.

further verified that the nanoparticle-integrated DMNs were strong
enough to penetrate cutaneous barrier for drug delivery.
To investigate the in vitro photothermal effect of CPT-CuS-
ZIF-8@HA@MN, the DMNs were irradiated by an 808 nm laser
with different power intensities (Figs. 6A, B, S9A, and S9B).
As displayed in Fig. 6A and B, compared with blank DMNs,
the temperature of CPT-CuS-ZIF-8@HA@MN rapidly elevated to
∼60 °C from room temperature only in 1 min irradiation (0.5
W/cm2 ) and subsequently remained steady, which demonstrated
the nanoparticle-integrated DMNs possessed satisfactory pho-
tothermal effect. Fig. 6C indicated their power intensity-dependent
photothermal property. Moreover, in photothermal stability anal-
ysis (Figs. 6D, S9C), the temperature variation curve of three laser
on/off cycles were nearly identical. The excellent photothermal sta-
bility of CPT-CuS-ZIF-8@HA@MN endowed the DDS with the char-
acteristics of repeated PTT and spatiotemporally controlled pul-
satile drug release for further application.
3.6. In vivo photothermal effect and biodistribution
In vivo photothermal effect was performed on B16 tumor-
bearing mice at 4 h post-administration. Compared with control
group, the localized temperatures in other three groups could dra-
matically elevate to around 50 °C in only 1 min irradiation fol-
lowed by gradual elevation (Fig. 7A and B). Previous studies have
proved that temperatures above 48 °C could induce a drastic and
nonreversible activation of cell apoptosis pathway to kill tumor
cells [17]. Therefore, CPT-CuS-ZIF-8@HA@MN was expected to be
an effective system for further in vivo PTT on account of its supe-
rior in vivo photothermal ability.
In order to investigate the duration of FL-CuS-ZIF-8@HA at tu-
mor site, the fluorescence signals of mice were detected at differ-
ent time points after administration of FL-CuS-ZIF-8@HA@MN. As
shown in Fig. 7C, the fluorescence signals were mainly concen-
trated at the tumor site. Quantitative analysis demonstrated that
the fluorescence intensity at the tumor site kept obvious during
12 h, indicating the formulation had a long duration at adminis-
tration site (Fig. 7D). The long duration might be attributed to the
formative drug reservoirs for continuous drug release in tumor af-
ter DMNs administration. To further investigate the biodistribution
of FL-CuS-ZIF-8@HA, the mice were sacrificed at 12 h, and the flu-
orescence signal distribution in the excised tumor and major or-
gans (heart, liver, spleen, lung, and kidney) was observed. Figs. 7E
and S10 showed that the fluorescence signal was nearly enriched
at the tumor site without appearing in major organs, suggesting
that the nanoparticles were difficult to exert adverse system effects

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Fig. 6. (A) The thermographic images of blank DMN, CuS-ZIF-8@MN, and CPT-CuS-ZIF-8@HA@MN under NIR irradiation (0.5 W/cm2 ) at different time points. (B) Temperature
change curves of blank DMN, CuS-ZIF-8@MN, and CPT-CuS-ZIF-8@HA @MN under NIR irradiation (0.5 W/cm2 ). (C) Temperature change curves of CPT-CuS-ZIF-8@HA@MN
under laser irradiation with different power intensities. (D) Temperature change curve of CPT-CuS-ZIF-8@HA@MN during three laser on/off cycles (0.5 W/cm2 ).
but dedicated to function at lesion site after DMNs administration.
In addition, after FL-CuS-ZIF-8@HA@MN administration, the corre-
sponding tumor was excised for frozen sections to inspect the drug
penetration capability. Fig. 7F displayed that the drug (green fluo-
rescence) could be delivered to deep tumor effectively. Taken to-
gether, these results suggested the topical DMNs delivery was a fa-
vorable and promising route for local tumor therapy.
3.7. In vivo anti-tumor study
Inspired by the in vitro pharmacodynamics, the in vivo anti-
tumor study was carried out, and the experimental process was
illustrated in Fig. 8A. The tumor-bearing C57BL/6 female mice
were randomly divided into six groups (n=6) after the tumor
volume being 100-120 mm3 : (1) untreated, (2) CuS-ZIF-8@MN,
(3) CuS-ZIF-8@MN+NIR, (4) CPT-CuS-ZIF-8@HA@MN, (5) CPT-CuS-
ZIF-8@HA nanoparticles intratumoral injection (I.T.)+NIR, and (6)
CPT-CuS-ZIF-8@HA@MN+NIR. The tumor volume curves (Fig. 8B)
showed that the volume of untreated group and CuS-ZIF-8@MN
group grew dramatically to nearly 20 0 0 mm3 on day 8 indicat-
ing that the CuS-ZIF-8@MN was incapable to inhibit tumor growth.
The mice in these two groups were sacrificed for kindness on day
10. By contrast, the tumor growth was suppressed to some extent
in CuS-ZIF-8@MN+NIR and CPT-CuS-ZIF-8@HA@MN groups. What
is more, the tumor volume of CPT-CuS-ZIF-8@HA@MN group was
always smaller than that of CuS-ZIF-8@MN+NIR group, which re-
vealed the chemotherapy was slightly better than PTT in tumor in-
hibition. Such result was also consistent with the aforementioned
in vitro cytotoxicity study. Moreover, the tumors in mice treated
with CPT-CuS-ZIF-8@HA@MN+NIR grew merely to 190 mm3 on
the terminal day (day 12), which were always the smallest in six
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Acta Biomaterialia 135 (2021) 164–178
groups, indicating that the combination of chemotherapy and PTT
realized a synergistic effect on melanoma inhibition in vivo. The
satisfactory synergistic effect might be attributed to the follow-
ing two reasons: First, the hyperthermia of PTT directly induced
thermal ablation of tumor cells; Second, ZIF-8 decomposition un-
der acid tumor microenvironment and external NIR irradiation in-
duced an intelligent and spatiotemporal drug release to improve
cell killing. Except that, the antitumor efficacy in different admin-
istration routes was also investigated. Compared with DMNs group,
the tumors of intratumoral injection group recurred on day 4 and
grew larger, revealing the superiority of DMNs administration. This
might because the nanoparticles with liquid formulation could eas-
ily diffuse into the surrounding tissues, thereby resulting in re-
duced drug accumulation at the tumor site after mono-point injec-
tion. While after CPT-CuS-ZIF-8@HA@MN administration, hundreds
of microchannels would deliver nanoparticles into tumor with a
three-dimensional uniform distribution, and the remained dissolv-
ing needles in skin could also act as drug reservoirs for continuous
drug release.
To further evaluate the anti-tumor effects, the tumors ex-
cised from each group were photographed (Fig. 8C) and weighed
(Fig. 8D) at the end of treatments. The tumor weight of CPT-CuS-
ZIF-8@HA@MN+NIR group was the lowest among all groups. In ad-
dition, the survival of CPT-CuS-ZIF-8@HA@MN+NIR group was sig-
nificant prolonged (Fig. 8E), further confirming the noticeable tu-
mor inhibiting ability of synergistic effect.
Histological sections were performed to verify the anti-tumor
effects at a microscopic level. H&E staining revealed the tumor tis-
sues of untreated and CuS-ZIF-8@MN groups were compact and
integrated, while varying degrees of apoptotic signs, including nu-
cleus pyknotic, cataclastic and dissolution of tumor cells, were ob-
Y. Zhao, Y. Zhou, D. Yang et al. Acta Biomaterialia 135 (2021) 164–178

Fig. 7. (A) The thermographic images and (B) corresponding temperature change curves of control, CuS-ZIF-8@MN, CPT-CuS-ZIF-8@HA@MN, and CPT-CuS-ZIF-8@HA intratu-
moral injection (I.T.) groups under NIR irradiation (1 W/cm2 ) for different time points (mean±S.D., n=3). (C) Fluorescent images of B16 tumor-bearing mice after adminis-
tration of FL-CuS-ZIF-8@HA@MN. (D) Total fluorescent intensity of tumor site at different time intervals after administration of FL-CuS-ZIF-8@HA@MN (mean±S.D., n=3). (E)
Fluorescent image of major organs at 12 h post administration. (F) Fluorescence images of histological section of the tumor after administration with FL-CuS-ZIF-8@HA@MN.
Scale bar: 200 μm.

served in other groups (Fig. 8F). Furthermore, cell apoptosis state
of tumor tissues was also evaluated. The positive brown signal
in cleaved caspase 3 sections and green signal in TUNEL images
represented early apoptosis cells and late apoptosis cells, respec-
tively. Fig. 8F showed that the positive signals of apoptosis in CPT-
CuS-ZIF-8@HA@MN+NIR group were obviously higher than other
groups, indicating the improved tumor apoptosis after synergistic
treatment. Moreover, we examined the state of tumor cell pro-
liferation. Ki67 is a kind of proliferating cell associated antigen
that can be used as a marker to evaluate tumor cell proliferation
[11,55,56]. The positive brown signal of Ki67 staining in CPT-CuS-
ZIF-8@HA@MN+NIR group was the least in the six groups (Fig. 8F),
which demonstrated the superior inhibiting effect on tumor expan-
sion. In general, histological assay confirmed that the great syner-
gistic efficacy in vivo was mainly attributed to promoting tumor
cells apoptosis and inhibiting their proliferation.
Apart from the antitumor efficacy, we also assessed the safety
of DMNs administration. The skin recovery photos of mice af-
ter CPT-CuS-ZIF-8@HA@MN administration were exhibited in Fig.
S11. The skin recovered to normal in only 1-3 days after dosing
DMNs alone. While with a laser irradiation, the penetration site
was slightly red and scabbed on the first day. Fortunately, the scab
faded away in 7-10 days. These results demonstrated the excel-
lent safety of DMN administration and the reversible skin dam-
age caused by irradiation. In addition, body weight variations were
monitored during the whole experimental process. No significant
weight change was observed among the six groups in Fig. S12A,
generally indicating the good biocompatibility and safety of the
formulation. Furthermore, the weight coefficient indexes and his-
tological analysis of major organs were also performed to eval-
uate the toxicity. The representative photos of major organs ex-
cised from tumor-bearing mice were exhibited in Fig. S12B. The
weight coefficient indexes of major organs (heart, liver, lung, and
kidney) among the six groups showed no noticeable difference ex-
cept spleen (Fig. S12C). The spleen weight indexes in untreated and
CuS-ZIF-8@MN groups were higher than other groups, probably be-
cause excessive tumor bearing may cause splenomegaly in mice.
Moreover, no obvious fibrosis, infiltration or inflammation was pre-

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Fig. 8. (A) Schematic illustration of the experimental process. (B) Tumor volume changes of mice after various treatments for 12 days (mean±S.D., n=6). ∗ P < 0.05 vs.
CuS-ZIF-8@MN+NIR; ∗ ∗ P < 0.01 vs. CuS-ZIF-8@MN+NIR. (C) Photographs and (D) average weight of the excised tumors in different groups at Day 12. (mean±S.D., n=6).
∗
P < 0.05 vs. CuS-ZIF-8@MN+NIR; ∗ ∗ P < 0.01 vs. CuS-ZIF-8@MN+NIR. (E) Survival rates of mice after various treatments for 28 days (n=6). ∗ P < 0.05 vs. Untreated;
∗∗
P < 0.01 vs. Untreated; ∗ ∗ ∗ P < 0.001 vs. Untreated. (F) Tumor section analysis by H&E, cleaved caspase 3, Ki67, and TUNEL staining. Scale bar: 400 μm.

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sented in H&E staining of the major organs (Fig. S12D), reflecting
the excellent histological safety of the DDS. Based on the results
of in vivo pharmacodynamics and safety, CPT-CuS-ZIF-8@HA@MN
demonstrated really high tumor inhibition rate and low toxicity,
which predicated our newly constructed DDS could be applied as
an efficient platform for synergistic therapy against various super-
ficial tumors.
4. Conclusion
In summary, we successfully constructed the multifunctional
nanoparticle-integrated DMNs (CPT-CuS-ZIF-8@HA@MN) for syn-
ergetic chemo-photothermal therapy against melanoma. Compre-
hensive studies revealed that this system possessed the following
unique advantages: local drug delivery, improved cellular internal-
ization, intelligent and spatiotemporal drug release, and synergistic
combination of chemotherapy and PTT. In addition, through in vivo
biodistribution and penetration studies, we found that the drug
could be enriched at the tumor site with long duration and deliv-
ered to deep tumor effectively after DMNs administration, which
indicated that this DDS promisingly realized a low-dose adminis-
tration with high efficiency in regional application. Furthermore,
the superior tumor inhibiting performance and excellent system-
atic safety of the DDS were confirmed in vitro and in vivo. To-
gether, this smart design overcame the drawbacks of monotherapy
and systematic administration. Nevertheless, there are some issues
need to be addressed before further utilization. For example, more
pharmacodynamics and long-term safety need to be further eval-
uated, scalable aseptic manufacturing and storage technology, and
standardized administration apparatus of the DMNs should be ex-
ploited for further practical applications. We believe that the mul-
tifunctional nanoparticle-integrated DMNs will be in prospect of
clinical application for more superficial tumor therapy with further
delicate optimization.
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.
Acknowledgments
This work was supported by the National Natural Science
Foundation (No. 81803466), the Key Areas Research and Devel-
opment Program of Guangdong Province (No. 2019B020204002),
the Natural Science Foundation of Guangdong Province (No.
2021A1515012525), and Guangdong Macao joint innovation fund-
ing project (No. 2020A050515009).
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.actbio.2021.09.009.
References
[1] M.R. Donaldson, B.M. Coldiron, No end in sight: the skin cancer epidemic con-
tinues, Semin. Cutan. Med. Surg. 30 (2011) 3–5.
[2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA Cancer J. Clin. 70 (2020)
7–30.
[3] P. Corrie, M. Hategan, K. Fife, C. Parkinson, Management of melanoma, Br. Med.
Bull. 111 (2014) 149–162.
[4] Y. Yan, S. Cao, X. Liu, S.M. Harrington, W.E. Bindeman, A.A. Adjei, J.S. Jang,
J. Jen, Y. Li, P. Chanana, A.S. Mansfield, S.S. Park, S.N. Markovic, R.S. Dronca,
H. Dong, CX3CR1 identifies PD-1 therapy-responsive CD8+ T cells that with-
stand chemotherapy during cancer chemoimmunotherapy, JCI Insight 3 (2018)
e97828.
177
Acta Biomaterialia 135 (2021) 164–178
[5] M.R. Ali, I.M. Ibrahim, H.R. Ali, S.A. Selim, M.A. El-Sayed, Treatment of natural
mammary gland tumors in canines and felines using gold nanorods-assisted
plasmonic photothermal therapy to induce tumor apoptosis, Int. J. Nanomed.
11 (2016) 4849–4863.
[6] H. Kim, K. Chung, S.J. Lee, D.H. Kim, H. Lee, Near-infrared light-respon-
sive nanomaterials for cancer theranostics, Wiley Interdiscip. Rev. Nanomed.
Nanobiotechnol. 8 (2016) 23–45.
[7] C. Yan, Q. Tian, S. Yang, Recent advances in the rational design of copper
chalcogenide to enhance the photothermal conversion efficiency for the pho-
tothermal ablation of cancer cells, RSC Adv. 7 (2017) 37887–37897.
[8] L. Li, L.H. Rashidi, M. Yao, L. Ma, L. Chen, J. Zhang, Y. Zhang, W. Chen, CuS
nanoagents for photodynamic and photothermal therapies: phenomena and
possible mechanisms, Photodiagn. Photodyn. Ther. 19 (2017) 5–14.
[9] L. Xu, J. Liu, J. Xi, Q. Li, B. Chang, X. Duan, G. Wang, S. Wang, Z. Wang, L. Wang,
Synergized multimodal therapy for safe and effective reversal of cancer mul-
tidrug resistance based on low-level photothermal and photodynamic effects,
Small (2018) 1800785.
[10] G. Gao, Y.W. Jiang, Y. Guo, H.R. Jia, X. Cheng, Y. Deng, X.W. Yu, Y.X. Zhu,
H.Y. Guo, W. Sun, X. Liu, J. Zhao, S. Yang, Z.W. Yu, F.M.S. Raya, G. Liang, F.G. Wu,
Enzyme-mediated tumor starvation and phototherapy enhance mild-tempera-
ture photothermal therapy, Adv. Funct. Mater. 30 (2020) 1909391.
[11] K. Poudel, A. Banstola, M. Gautam, Z. Soe, C.D. Phung, L.M. Pham, J.H. Jeong,
H.G. Choi, S.K. Ku, T.H. Tran, C.S. Yong, J.O. Kim, Macrophage-membrane-cam-
ouflaged disintegrable and excretable nanoconstruct for deep tumor penetra-
tion, ACS Appl. Mater. Interfaces 12 (2020) 56767–56781.
[12] K. Poudel, A. Banstola, M. Gautam, Z.C. Soe, L.M. Pham, J.H. Jeong, H.G. Choi,
S.K. Ku, C.S. Yong, T.H. Tran, J.O. Kim, Redox/photo dual-responsive, self-tar-
geted, and photosensitizer-laden bismuth sulfide nanourchins for combination
therapy in cancer, Nanoscale 13 (2021) 1231–1247.
[13] L. Liang, S.W. Peng, Z.W. Yuan, C. Wei, Y.Y. He, J.R. Zheng, Y.Q. Gu, H.Y. Chen,
Biocompatible tumor-targeting nanocomposites based on CuS for tumor imag-
ing and photothermal therapy, RSC Adv. 8 (2018) 6013–6026.
[14] R. van der Meel, E. Sulheim, Y. Shi, F. Kiessling, W.J.M. Mulder, T. Lammers,
Smart cancer nanomedicine, Nat. Nanotechnol. 14 (2019) 1007–1017.
[15] D.J. Irvine, E.L. Dane, Enhancing cancer immunotherapy with nanomedicine,
Nat. Rev. Immunol. 20 (2020) 321–334.
[16] R. Wang, Z. He, P. Cai, Y. Zhao, L. Gao, W. Yang, Y. Zhao, X. Gao, F. Gao, Sur-
face-functionalized modified copper sulfide nanoparticles enhance checkpoint
blockade tumor immunotherapy by photothermal therapy and antigen captur-
ing, ACS Appl. Mater. Interfaces 11 (2019) 13964–13972.
[17] W.H. Chen, G.F. Luo, X.Z. Zhang, Recent advances in subcellular targeted cancer
therapy based on functional materials, Adv. Mater. 31 (2019) 1802725.
[18] P. Majumder, U. Baxa, S.T.R. Walsh, J.P. Schneider, Design of a multicompart-
ment hydrogel that facilitates time-resolved delivery of combination therapy
and synergized killing of glioblastoma, Angew. Chem. Int. Ed. Engl. 57 (2018)
15040–15044.
[19] L. Yang, H. Sun, Y. Liu, W. Hou, Y. Yang, R. Cai, C. Cui, P. Zhang, X. Pan, X. Li,
L. Li, B.S. Sumerlin, W. Tan, Self-assembled aptamer-grafted hyperbranched
polymer nanocarrier for targeted and photoresponsive drug delivery, Angew.
Chem. Int. Ed. Engl. 57 (2018) 17048–17052.
[20] J. Jiang, Y. Zhao, O.M. Yaghi, Covalent chemistry beyond molecules, J. Am.
Chem. Soc. 138 (2016) 3255–3265.
[21] M. Bosch, S. Yuan, W. Rutledge, H.C. Zhou, Stepwise synthesis of metal-organic
frameworks, Acc. Chem. Res. 50 (2017) 857–865.
[22] C.Y. Sun, C. Qin, C.G. Wang, Z.M. Su, S. Wang, X.L. Wang, G.S. Yang, K.Z. Shao,
Y.Q. Lan, E.B. Wang, Chiral nanoporous metal-organic frameworks with high
porosity as materials for drug delivery, Adv. Mater. 23 (2011) 5629–5632.
[23] L. Gao, Q. Chen, T. Gong, J. Liu, C. Li, Recent advancement of imidazolate frame-
work (ZIF-8) based nanoformulations for synergistic tumor therapy, Nanoscale
11 (2019) 21030–21045.
[24] J.C. Yang, Y. Chen, Y.H. Li, X.B. Yin, Magnetic resonance imaging-guided multi–
drug chemotherapy and photothermal synergistic therapy with pH and NIR-s-
timulation release, ACS Appl. Mater. Interfaces 9 (2017) 22278–22288.
[25] C.Y. Sun, C. Qin, X.L. Wang, G.S. Yang, K.Z. Shao, Y.Q. Lan, Z.M. Su, P. Huang,
C.G. Wang, E.B. Wang, Zeolitic Imidazolate framework-8 as efficient pH-sensi-
tive drug delivery vehicle, Dalton Trans. 41 (2012) 6906–6909.
[26] J. Feng, W. Yu, Z. Xu, J. Hu, J. Liu, F. Wang, Multifunctional siRNA-laden
hybrid nanoplatform for noninvasive PA/IR dual-modal imaging-guided en-
hanced photogenetherapy, ACS Appl. Mater. Interfaces 12 (2020) 22613–
22623.
[27] L. He, G. Huang, H. Liu, C. Sang, X. Liu, T. Chen, Highly bioactive zeolitic imi-
dazolate framework-8-capped nanotherapeutics for efficient reversal of reper-
fusion-induced injury in ischemic stroke, Sci. Adv. 6 (2020) eaay9751.
[28] K. Dong, Y. Zhang, L. Zhang, Z. Wang, J. Ren, X. Qu, Facile preparation of met-
al-organic frameworks-based hydrophobic anticancer drug delivery nanoplat-
form for targeted and enhanced cancer treatment, Talanta 194 (2019) 703–708.
[29] B. Wu, J. Fu, Y. Zhou, S. Luo, Y. Zhao, G. Quan, X. Pan, C. Wu, Tailored coreshell
dual metal-organic frameworks as a versatile nanomotor for effective synergis-
tic antitumor therapy, Acta Pharm. Sin. B 10 (2020) 2198–2211.
[30] C. Underhill, CD44: the hyaluronan receptor, J. Cell Sci. 103 (1992) 293–298.
[31] Z. Luo, Y. Dai, H. Gao, Development and application of hyaluronic acid in tumor
targeting drug delivery, Acta Pharm. Sin. B 9 (2019) 1099–1112.
[32] H.Y. Seok, N. Sanoj Rejinold, K.M. Lekshmi, K. Cherukula, I.K. Park, Y.C. Kim,
CD44 targeting biocompatible and biodegradable hyaluronic acid cross-linked
zein nanogels for curcumin delivery to cancer cells: in vitro and in vivo evalu-
ation, J. Controll. Release 280 (2018) 20–30.
Y. Zhao, Y. Zhou, D. Yang et al.
[33] Z.X. Chen, M.D. Liu, M.K. Zhang, S.B. Wang, L. Xu, C.X. Li, F. Gao, B.R. Xie,
Z.L. Zhong, X.Z. Zhang, Interfering with lactate-fueled respiration for enhanced
photodynamic tumor therapy by a porphyrinic MOF nanoplatform, Adv. Funct.
Mater. 28 (2018) 1803498.
[34] O. Gotov, G. Battogtokh, Y.T. Ko, Docetaxel-loaded hyaluronic acid-cathepsin B–
cleavable-peptide-gold nanoparticles for the treatment of cancer, Mol. Pharm.
15 (2018) 4668–4676.
[35] K. Poudel, A. Banstola, T.H. Tran, R.K. Thapa, M. Gautam, W. Ou, L.M. Pham,
S. Maharjan, J.H. Jeong, S.K. Ku, H.G. Choi, C.S. Yong, J.O. Kim, Hyaluronic acid
wreathed, trio-stimuli receptive and on-demand triggerable nanoconstruct for
anchored combinatorial cancer therapy, Carbohydr. Polym. 249 (2020) 116815.
[36] C. Wang, Y. Ye, G.M. Hochu, H. Sadeghifar, Z. Gu, Enhanced cancer im-
munotherapy by microneedle patch-assisted delivery of anti-PD1 antibody,
Nano Lett. 16 (2016) 2334–2340.
[37] X. Lan, J. She, D.A. Lin, Y. Xu, X. Li, W.F. Yang, V.W.Y. Lui, L. Jin, X. Xie,
Y.X. Su, Microneedle-mediated delivery of lipid-coated cisplatin nanoparticles
for efficient and safe cancer therapy, ACS Appl. Mater. Interfaces 10 (2018)
33060–33069.
[38] A.F. Moreira, C.F. Rodrigues, T.A. Jacinto, S.P. Miguel, E.C. Costa, I.J. Correia, Mi-
croneedle-based delivery devices for cancer therapy: a review, Pharmacol. Res.
148 (2019) 104438.
[39] G. Mojeiko, M. de Brito, G.C. Salata, L.B. Lopes, Combination of microneedles
and microemulsions to increase celecoxib topical delivery for potential appli-
cation in chemoprevention of breast cancer, Int. J. Pharm. 560 (2019) 365–376.
[40] A.S. Rzhevskiy, T.R.R. Singh, R.F. Donnelly, Y.G. Anissimov, Microneedles as the
technique of drug delivery enhancement in diverse organs and tissues, J. Con-
troll. Release 270 (2018) 184–202.
[41] M. Chen, G. Quan, Y. Sun, D. Yang, X. Pan, C. Wu, Nanoparticles-encapsulated
polymeric microneedles for transdermal drug delivery, J. Controll. Release 325
(2020) 163–175.
[42] W. Qin, G. Quan, Y. Sun, M. Chen, P. Yang, D. Feng, T. Wen, X. Hu, X. Pan,
C. Wu, Dissolving microneedles with spatiotemporally controlled pulsatile re-
lease nanosystem for synergistic chemo-photothermal therapy of melanoma,
Theranostics 10 (2020) 8179–8196.
[43] Z. Wang, X. Tang, X. Wang, D. Yang, C. Yang, Y. Lou, J. Chen, N. He, Near-in-
frared light-induced dissociation of zeolitic imidazole framework-8 (ZIF-8)
with encapsulated CuS nanoparticles and their application as a therapeutic
nanoplatform, Chem. Commun. 52 (2016) 12210–12213.
[44] J. Zhuang, C.H. Kuo, L.Y. Chou, D.Y. Liu, E. Weerapana, C.K. Tsung, Optimized
metal-organic-framework nanospheres for drug delivery: evaluation of small–
molecule encapsulation, ACS Nano 8 (2014) 2812–2819.
178
Acta Biomaterialia 135 (2021) 164–178
[45] X. Zhang, L. Liu, L. Huang, W. Zhang, R. Wang, T. Yue, J. Sun, G. Li, J. Wang,
The highly efficient elimination of intracellular bacteria via a metal organic
framework (MOF)-based three-in-one delivery system, Nanoscale 11 (2019)
9468–9477.
[46] Q. Sun, H. Bi, Z. Wang, C. Li, X. Wang, J. Xu, H. Zhu, R. Zhao, F. He, S. Gai,
P. Yang, Hyaluronic acid-targeted and pH-responsive drug delivery system
based on metal-organic frameworks for efficient antitumor therapy, Biomate-
rials 223 (2019) 119473.
[47] A. Hou, G. Quan, B. Yang, C. Lu, M. Chen, D. Yang, L. Wang, H. Liu, X. Pan,
C. Wu, Rational design of rapidly separating dissolving microneedles for pre-
cise drug delivery by balancing the mechanical performance and disintegration
rate, Adv. Healthc. Mater. 8 (2019) 1900898.
[48] Y. Wang, X. Liu, G. Deng, J. Sun, H. Yuan, Q. Li, Q. Wang, J. Lu, Se@SiO2 -FA-CuS
nanocomposites for targeted delivery of DOX and nano selenium in synergis-
tic combination of chemo-photothermal therapy, Nanoscale 10 (2018) 2866–
2875.
[49] M. Zhang, X. Liu, Q. Luo, Q. Wang, L. Zhao, G. Deng, R. Ge, L. Zhang, J. Hu,
J. Lu, Tumor environment responsive degradable CuS@mSiO2 @MnO2 /DOX for
MRI guided synergistic chemo-photothermal therapy and chemodynamic ther-
apy, Chem. Eng. J. 389 (2020) 124450.
[50] H. Zhang, Q. Li, R. Liu, X. Zhang, Z. Li, Y. Luan, A versatile prodrug strategy
to in situ encapsulate drugs in MOF nanocarriers: A case of cytarabine-IR820
prodrug encapsulated ZIF-8 toward chemo-photothermal therapy, Adv. Funct.
Mater. 28 (2018) 1802830.
[51] M. Chen, D. Yang, Y. Sun, T. Liu, W. Wang, J. Fu, Q. Wang, X. Bai, G. Quan,
X. Pan, C. Wu, In situ self-assembly nanomicelle microneedles for enhanced
photoimmunotherapy via autophagy regulation strategy, ACS Nano 15 (2021)
3387–3401.
[52] K.F. Chu, D.E. Dupuy, Thermal ablation of tumours: biological mechanisms and
advances in therapy, Nat. Rev. Cancer 14 (2014) 199–208.
[53] M. Nikfarjam, V. Muralidharan, C. Christophi, Mechanisms of focal heat de-
struction of liver tumors, J. Surg. Res. 127 (2005) 208–223.
[54] L.F. Fajardo, B. Egbert, J. Marmor, G.M. Hahn, Effects of hyperthermia in a ma-
lignant tumor, Cancer 45 (1980) 613–623.
[55] L. Wang, T. Zhang, M. Huo, J. Guo, Y. Chen, H. Xu, Construction of nucleus-tar-
geting iridium nanocrystals for photonic hyperthermia-synergized cancer ra-
diotherapy, Small 15 (2019) e1903254.
[56] J. Wan, L. Huang, J. Cheng, H. Qi, J. Jin, H. Wang, Balancing the stability and
drug activation in adaptive nanoparticles potentiates chemotherapy in mul-
tidrug-resistant cancer, Theranostics 11 (2021) 4137–4154.