Responsive functionalized MoSe2 nanosystem for highly efficient synergistic therapy of breast cancer

Journal Pre-proof Responsive Functionalized MoSe2 Nanosystem for Highly Efficient Synergistic Therapy of Breast Cancer Yanan Liu (Conceptualization) (Methodology) (Writing - original draft) (Investigation) (Formal analysis) (Validation), ChunFang Wei (Conceptualization) (Methodology) (Investigation) (Formal analysis)Writing - original draft) (Validation), Ange Lin (Investigation) (Formal analysis), Jiali Pan (Conceptualization) (Methodology) (Writing - original draft) (Data curation), Xu Chen (Investigation), Xufeng Zhu (Investigation), Youcong Gong (Investigation), Guanglong Yuan (Investigation), Lanmei Chen (Resources) (Writing - review and editing) (Project administration), Jie Liu (Resources) (Writing - review and editing) (Project administration), Zhaohui Luo (Resources) (Writing - review and editing) (Project administration)

PII:

S0927-7765(20)30050-3

DOI:

https://doi.org/10.1016/j.colsurfb.2020.110820

Reference:

COLSUB 110820

To appear in:

Colloids and Surfaces B: Biointerfaces

Received Date:

5 August 2019

Revised Date:

12 January 2020

Accepted Date:

21 January 2020

Please cite this article as: Yanan L, ChunFang W, Ange L, Jiali P, Xu C, Xufeng Z, Youcong G, Guanglong Y, Lanmei C, Jie L, Zhaohui L, Responsive Functionalized MoSe2 Nanosystem for Highly Efficient Synergistic Therapy of Breast Cancer, Colloids and Surfaces B: Biointerfaces (2020), doi: https://doi.org/10.1016/j.colsurfb.2020.110820

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

Responsive Functionalized MoSe2 Nanosystem for Highly Efficient Synergistic Therapy of Breast Cancer Yanan Liu†a,b,c, ChunFang Wei†a,b,c, Ange Linb, Jiali Panb, Xu Chenb, Xufeng Zhub, Youcong Gongb, Guanglong Yuanb, Lanmei Chena,*, Jie Liua,b,*, Zhaohui Luob,* a

Guangdong Key Laboratory for Research and Development of Natural Drugs, School of Pharmacy,

Guangdong Medical University, Zhanjiang 524023, China b

College of Pharmacy, Guilin Medical University, Guangxi Guilin, 541004, China;

c

Guangzhou, 510632, China. *Corresponding

authors,

Jie

Liu,

E-mail:

[email protected];

These authors contributed equally to the work.

Chen,

E-mail:

Jo

ur

na

lP

re

Graphical Abstract

Lanmei

-p

[email protected]; Zhaohui Luo, E-mail: [email protected]. †

ro of

Department of Chemistry, College of Chemistry and Materials Science, Jinan University,

(A) Diagram of the synthesis of MoSe2@ICG-PDA-HA. (B) Schematic illustration of the targeted photothermal/photodynamic synergistic therapy in tumor.

Highlights



MoSe2@ICG-PDA-HA enables one-step simultaneous photothermal/photodynamic therapy.



Nanosystem can improve the optical stability of the loaded ICG.



Controlled release can be achieved via PDA.



Targeting tumors by HA reduces toxic side effects on normal tissues.

Abstract The photothermal/photodynamic synergistic therapy is a promising tumor treatment, but developing

ro of

nanosystems that achieve synchronous photothermal/photodynamic functions is still quite challenging. Here, we use a simple method to synthesize molybdenum selenide nanoparticles (MoSe2 NPs) with a photothermal effect as a carrier, and load a photosensitizer ICG to form a nanosystem (MoSe2@ICG-PDA-HA)with dual photothermal/photodynamic functions under near-infrared

-p

irradiation. In addition, the surface modification of the nanosystem with acid-responsive release polydopamine (PDA) and tumor-targeted hyaluronic acid (HA) enhanced the stability of the

re

photosensitizer ICG and the accumulation of ICG at tumor sites. The multicellular sphere assay simulated solid tumors and demonstrated that MoSe2@ICG-PDA-HA could significantly inhibit the

lP

4T1 cell growth. The anti-tumor experiments in tumor-bearing mice showed that MoSe2@ICG-PDAHA not only significantly inhibited the growth of 4T1 subcutaneous tumors, but also inhibited their metastasis. This study presented a nanosystem that could improve the photostability of optical

na

materials and enhance the photothermal/photodynamic synergy effect, providing a new idea for finding a way to effectively treat breast cancer. Keyword: Photothermal therapy; Photodynamic therapy; Multimodal imaging; Photostability;

ur

Molybdenum selenide; Breast cancer.

Jo

1. Introduction

In recent years, PTT and PDT, the new phototherapy method mediated by photosensitizers (PS),

have attracted much attention due to their excellent local therapeutic effect and minimal invasiveness. As we known, photothermal therapy uses photothermal agents to generate heat under laser irradiation, which can directly kill tumor cells under the local high heat (> 42℃). However, the local high heat can not distribute over all of the tumor area, especially in the vicinity of the main large blood vessels. The large blood flow to the large blood vessels causes the heat generated to dissipate rapidly, resulting in a sublethal heat dose in the area, which makes it impossible to completely eliminate all

tumor cells by photothermal therapy alone [1-3]. In addition, PDT is different to PTT, which uses photosensitizers to produce large amounts of reactive oxygen species (ROS) to kill tumor cells [4-7]. Nevertheless, the hypoxic problem of tumors can seriously affect the efficacy of PDT. We envisage that if PTT and PDT can be combined to play a synergistic effect, so as to compensate for the defects in the treatment process, which obtain better treatment effect [9, 10]. Because mild hyperthermia can increase membrane permeability to enhance the uptake of PS loaded nanocarriers in cancer cells, the intracellular PS concentration can be increased through mild PTT effect to improve intracellular ROS concentration, thereby enhancing the efficacy of PDT [11-14]. In addition, the mild hyperthermia can accelerate the blood flow and increase the concentration of saturated oxygen in

ro of

blood vessels, which is also conducive to increasing the production of 1O2 in oxygen-dependent type II PDT [15-19]. Therefore, compared to PTT or PDT alone, the photothermal/photodynamic synergistic therapy has a more obvious advantage which can effectively inhibit the growth and metastasis of tumor cells. But how combine the two treatment methods? A suitable carrier is required

-p

to deliver both the photothermal agent and the photosensitizer to the tumor lesion.

In recent years, the rapid development of nanotechnology and nanomedicine has brought dawn to cancer treatment. New design is carried out on the surface of nanomedicine, so as to add new

re

functions, such as making nano drug realize diagnosis and treatment at the same time, controlling the behavior of nanomedicine in vivo through near-infrared laser, combining the advantages of

lP

nanomedicine and photothermal-photodynamic therapy, etc. [20-25]. Inorganic nanomaterials are materials used earlier to study photothermal therapy. For example, carbon nanomaterials [26, 27], gold nanomaterials [20, 23] and copper sulfide nanoparticles [28-30], etc., have been extensively

na

studied for near-infrared mediated photothermal therapy. However, the disadvantages of gold nanomaterials are poor bio-metabolism and high cost; poor dispersion of carbon nanomaterials

ur

induces immune reactions; and the photothermal conversion efficiency of copper sulfide nanoparticles is relatively low. In addition, the latter organic photo-sensitizers generally have the

Jo

disadvantage of photo-bleaching. These all limit their use in the clinic. In recent years, new biological application materials two-dimensional (2D) layered transition

metal dichalcogenides MX2 (M = Mo, W; X = S, Se) have been widely studied in photocatalysis, sensors and biomedicine [31-33], owing to their wonderful electronic and optical properties, large surface area and controllable size [34-39]. It is noteworthy that two-dimensional layered transition metal dichalcogenide (TMDC) series materials have high load capacity, which benefits from their large specific surface area [40-43]. Once the photosensitizer is loaded on the surface of TMDC, not only its optical performance is more stable, but also its circulation time in blood is significantly

prolonged [44]. In addition, MoSe2 with a direct band gap of ∼ 1.5 eV [45] (compared with MoS2 ∼1.8 eV and WS2 ∼ 2.0 eV) [45-47], exhibits excellent long-wavelength NIR absorption, inducing a stronger penetrability in deep-tissue photothermal therapy [48]. Accordingly, MoSe2 is considered to be an innovative PTT agent. Therefore, the high loading capacity of TMDC and the synergistic absorption of TMDC materials and photosensitizers both give the photosensitizer high and stable optical absorption capacity, enhancing its PDT/PTT treatment effect. In this study, we innovatively designed and prepared a nano-system (MoSe2@ICG-PDA-HA) that achieved simultaneous photothermal/photodynamic action with one-step near-infrared illumination to improve the therapeutic effect of cancer. MoSe2 NPs have not only excellent

ro of

photothermal effects but also high load capacity, which were a good carrier to load ICG. In order to enhance the accumulation of ICG in the tumor area, we modified the surface of ICG-loaded MoSe2 NPs with PDA with acid-responsive release of tumor microenvironment [49], and simultaneously linked HA to target CD44 receptor on the surface of cancer cell membrane [50-51]. Finally, a

-p

nanosystem with photostability and enhanced photothermal/photodynamic synergistic effect was constructed. By studying its physical and chemical properties, biological distribution, biological toxicity and near-infrared mediated anti-tumor ability in vivo and in vitro, we found that the nano-

re

drug delivery system could not only significantly enhance the stability of ICG and tumor targeting ability, but also achieved excellent anti-tumor growth and migration ability through synchronous

lP

PDT/PPT. This study highlights nanosystems that could improve the photostability of optical materials and enhance the photothermal/photodynamic synergy effect, achieving significant

Jo

ur

na

anticancer therapeutic effects and providing a new strategy for cancer treatment.

ro of

Scheme 1. (A)Diagram of the synthesis of MoSe2@ICG-PDA-HA. (B) Schematic illustration of the

-p

targeted photothermal/photodynamic synergistic therapy in tumor.

re

2. Materials and methods 2.1. Chemicals and reagents

lP

Hydrazine hydrate solution, sodium hyaluronate, phthalocyanine green was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd; selenium powder was purchased from Tianjin Chemical Reagent Factory; dopamine hydrochloride was purchased from Guangzhou Hong Na

na

Chemical Co., Ltd.; lysosomal red fluorescent dye, SOSG singlet state Oxygen probe, DCFH-DA reactive oxygen probe, TUNEL apoptosis in situ detection reagent, DAPI/PI apoptotic double staining fluorescent dye cartridge purchased from Biyuntian Biotechnology Co., Ltd.; The AV-

ur

FITC/PI Apoptosis Detection Kit was purchased from Beijing Sizhengbai Biotechnology Co., Ltd. anhydrous ethanol purchased Tianjin Zhiyuan Chemical Reagent Co., Ltd.; Cell culture medium

Jo

DMEM and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies AG, Switzerland).; CCK-8 kit, DMSO, DAPI staining solution were purchased from Sigma Aldrich Chemical Co., Ltd.

2.2. Synthesis of MoSe2@ICG-PDA-HA 25 mL of hydrazine hydrate solution was poured into a beaker, and selenium powder (1.55 g) and molybdenum trioxide (0.85 g) were sequentially added while stirring. Then, 5 mL of deionized water and 5 mL of absolute ethanol were added during the stirring. The mixture was transferred to a

reaction kettle, and the reactor was placed in a 180 °C oven and heated for 6 hours. The black production was washed 3 times with absolute ethanol and deionized water, respectively. The black production was added to 50 mL of a 0.2 mol/L sodium hydroxide solution to incubatedat 80 °C for 2 h. MoSe2 NPs obtained by further filtration and purification The MoSe2 NPs were dissolved in a high concentration ICG solution and stirred for 24 h in the dark. The dopamine solution was diluted to 2.0 mg/mL with 10 mM Tris buffer to adjust the pH to 8.5.The mixture of MoSe2 NPs and ICG was added to the dopamine solution. The mixture was slowly stirred at room temperature for 24 h to deposit dopamine on the surface of MoSe2 NPs, and self-polymerized into a DPA layer by crosslinking. The product was collected by centrifugation,

ro of

washed three times with deionized water, and dried at 37 °C in a sample MoSe2@ICG-PDA. The collected MoSe2@ICG-PDA was dissolved in a (5.0 mg/mL) HA solution. The mixture was slowly stirred at room temperature for 24 h to adsorb HA on the PDA layer by electrostatic or hydrogen bonding. The product was collected by centrifugation, and washed 3 times with deionized

-p

water. Finally, the sample MoSe2@ICG-PDA-HA was dried in an oven at 37 °C. 2.3. Measurements and Characterizations.

re

The absorption spectra of MoSe2NFs and MoSe2@ICG-PDA-HA were measured by a UV-Vis spectrophotometer. The dynamic hydrodynamic diameter and zeta potential of MoSe2NFs,

lP

MoSe2@ICG-PDA, MoSe2@ICG-PDA-HA were analyzed by Nano-Zetasizer (Malvern Instruments Ltd.). The morphology of MoSe2 NPs and MoSe2@ICG-PDA-HA was observed on a transmission electron microscope. The crystallinity of MoSe2NFs was studied by Raman spectroscopy, and the

na

crystal structure of MoSe2 NPs was analyzed by a ray diffractometer. The 3H-2000PM1 high performance specific surface and pore size analyzer measures the pore size of MoSe2 NPs. The

ur

concentration of molybdenum ions was determined by ICP-AES, in which the sample was treated with nitric acid and 30% H2O2 (volume ratio of 4:1).

Jo

2.4. Cell uptake

4T1 mouse breast cancer cells were cultured in a high glucose medium in a 37 °C, 5% CO2

saturated humidity incubator. 100 μg/mL free ICG, MoSe2@ICG-PDA, MoSe2@ICG-PDA-HA were incubated with 4T1 cells for different time (2 h, 8 h, 12 h, 24 h, 36 h). Then the cells were washed with PBS three times, stained with DAPI for 5 minutes, washed with PBS three times, and finally fixed with 4% polyformaldehyde. Laser confocal microscopy and flow cytometry (FCM) were used to detect the uptake of the above drugs.

To observe the lysosomal escape process of nanomedicine, 100μg/mL MoSe2@ICG-PDA-HA was incubated with 4T1 cells for different time (4, 8, 12, 24, 36 h), then the cells were rinsed 3 times with PBS, then stained with Lyso Tracker for 30 min, with PBS. After washing 3 times, it was further stained with DAPI for 5 min, washed 3 times with PBS, and finally fixed with 4% of paraformaldehyde. The lysosomal escape process of MoSe2@ICG-PDA-HA was observed and photographed by laser confocal microscopy. 2.5. ROS detection SOSG fluorescent probes were added to different substances (MoSe2 NPs, free ICG, MoSe2@ICG, MoSe2@ICG-PDA-HA, or MoSe2@ICG-PDA-HA + tocopherol) in laser (0.5 W /cm2,

ro of

5 min) or no laser. The change in fluorescence intensity of SOSG was detected by fluorescence spectrometer.

After incubating 4T1 cells with ICG, MoSe2 NPs and MoSe2@ICG-PDA-HA) for 12 h, the cells were irradiated with a 0.5 W/cm2 808 nm laser for 1 min. The cells were then stained with a

-p

DCFH-DA probe and Rosup served as a positive control. Finally, the laser confocal microscope was used to observe and photograph the different substances to produce ROS.

re

2.6. Cell apoptosis assay

4T1 mouse breast cancer cells were incubated with different concentrations of ICG, MoSe 2NFs,

lP

MoSe2@ICG-PDA-HA for 24 h. The 808 nm laser was used to explore its photothermal effect. The therapeutic effects of the drug under different conditions of light, such as power and temperature,

na

were explored. The cells were then stained with Hoechst 33342 and PI for 15 minutes, washed 3 times with PBS, and then imaged by a laser confocal fluorescence microscope. Under the same conditions as above, the apoptosis of 4T1 cells was detected by flow cytometry by double staining

ur

with Annnexin V-FITI/PI.

2.7. Multicellular tumor sphere model

Jo

The low melting point agarose was placed in a sterilizing pot for high temperature sterilization

and stored in an incubator at 60 °C. A 2% low melting agarose medium was quickly configured before cooling. Agarose medium was added to a 96-well plate for laser confocal deposition, and stored in a CO2 incubator until the agarose medium was solidified. 4T1 cells (4 × 103/400 μL per well) were seeded in sterile agarose-coated 96-well plates. A multicellular sphere can be formed after about 1 week. The formed multicellular spheres were incubated with PBS, free ICG, MoSe2 NPs, and MoSe2@ICG-PDA-HA for 24 h, and then irradiated with 808 nm laser for 5 min for one week. At the same time, the size of the multicellular sphere was observed with a microscope and

photographed during this period. All cells were harvested after multi-cell pellet digestion one week later. Flow cytometry was used to detect the degree of apoptosis and necrosis of cells by FITCAnnexin V/PI double staining. 2.8. Mouse tumor model Female BALB/c nude mice aged 4 weeks were purchased from Guangdong Medical Laboratory Animal Center. All the animal procedures were performed under the protocols approved by the Institute of Laboratory Animal science, Jinan University. All animal experiments were in agreement with the guidelines of the Institute of Laboratory Animal science, Jinan University. Female BALB/c nude mice growing for 7-8 weeks and weighing about 18 g were used for the

ro of

experiment. The 4T1 cells in logarithmic growth phase were concentrated into cell suspension and subcutaneously injected into the left axilla of nude mice. The injection concentration of each nude mice was about 1×106 cells/200 μL. After a week of growth, the tumors grew to about 60 mm3 and

-p

began the experiment. 2.9. In vivo fluorescence imaging

re

Free ICG, MoSe2@ICG-PDA and MoSe2@ICG-PDA-HA (2 mg/kg per mouse) were intravenously injected into Female BALB/c nude mice for 48 hours. The distribution of nanoparticles in nude mice was detected by IVIS Lumina LT in vivo optical imaging system at predetermined time

lP

(0.5, 2, 4, 8, 12, 24, 48 h). Finally, the mice were killed and the contents of ICG in various organs (heart, liver, spleen, lung, and kidney) and tumors were quantitatively analyzed. MoSe2@ICG-PDA-

na

HA was injected intravenously into mice. Tumors and main organs of mice were collected and dissolved at different times. The distribution of Mo in mice was detected by ICP-AES. 2.10. In vivo photothermal imaging

ur

The mice were injected intravenously with PBS, free ICG, MoSe2 NPs, MoSe2@ICG-PDA-HA (1 mg/kg per mouse) every other day. After 24 hours of intravenous injection, the tumors of these

Jo

mice were continuously irradiated with 808 nm laser for 5 minutes. The temperature changes at the tumors of mice were recorded by infrared photothermal camera. The weight of mice was recorded before each administration, and the size of tumors was measured with the vernier calipers. 14 days later, all mice were euthanized, and the tumors were removed and weighed. The main organs, such as heart, liver, spleen, lung and kidney, were dissected and washed three times with PBS, then immersed in 4% polyformaldehyde for histopathological analysis. 2.11. MoSe2@ICG-PDA-HA induces apoptosis

4T1 cells were incubated (12 h, 37 °C) with borosilicate chambered cover glass (5 × 104 cells per well). Then, the medium was removed, and free ICG, MoSe2 NPs and MoSe2@ICG-PDA-HA 1640 medium solution with the same concentration of ICG was added. After incubation for 6 h, in order to evaluate nanosystem toxicity, cell viability after treatment with free ICG, MoSe 2NFs and MoSe2@ICG-PDA-HA was detected by CCK-8 kit in the absence of light. To explore the effects of different illumination times, power and temperature on the antitumor activity of nanomedicine systems, free ICG, MoSe2NFs and MoSe2@ICG-PDA-HA 1640 medium solution with a series of concentrations (0.5, 5, 25, 50, 100 μg/mL) of ICG were incubated 4T1 cells under different lighting times, power and temperature. The cell viability was detected by CCK-8 kit. Free ICG, MoSe2 NPs, MoSe2@ICG-PDA-HA were incubated with 4T1 cells for 24 h in the presence or absence of laser

ro of

irradiation (0.5 W/cm2, 2 min), and PBS was used as a control group. Finally, Hoechest 33342/PI double staining was performed, and the apoptosis of 4T1 cells was observed by laser confocal microscopy. Flow cytometry was used to detect apoptosis of 4T1 cells under the same conditions as

-p

above by Annexin V-FITC/PI double staining.

2.12. Anti-tumor activity of MoSe2@ICG-PDA-HA in combination therapy in vivo

re

When the tumors of 4T1 tumor-bearing mice reached 60 mm3, the mice were randomly divided into 7 groups (5 mice in each group). Nude mice injected intravenously with saline as control group

lP

(Group 1). The other groups were as follows: (Group 2) only near infrared irradiation (Group 3) MoSe2 NPs + Laser, (Group 4) free ICG + Laser, (Group 5) MoSe2 NPs + ICG mixed solution + Laser, (Group 6) MoSe2@ICG-PDA+Laser.(Group 7) MoSe2@ICG-PDA-HA+Laser. Tumor size

na

was measured with the vernier caliper every 2 days during treatment. In the above experimental group, the dose was 1 mg/kg, and the irradiation condition was 808 nm near infrared laser (0.5 W/cm2) for 5 minutes. At the end of the treatment, the mice were euthanized, then the tumors and

ur

major organs were dissected. Finally, the tumors and major organs were made into stained sections and stained by H&E, TUNEL and ki-67.

Jo

3. Results and discussion

3.1. Synthesis and characterization of MoSe2NFs and MoSe2@ICG-PDA-HA The synthesis process of MoSe2 @ICG-PDA-HA was presented in Scheme 1A. In this work, we

synthesized MoSe2 NPs by hydrothermal method to load the ICG. Based on the electronegativity of MoSe2 NPs, the electrically positive PDA biofilms were coated on MoSe2 NPs surface by simple chemical synthesis under alkaline conditions. HA is such a strongly negatively charged material that can be immobilized on a PDA biofilm by electrostatic adsorption or hydrogen bonding. The water-

soluble nanosystem MoSe2@ICG-PDA-HA was obtained after the PDA biomimetic membrane and the tumor targeting HA were modified on the MoSe2 NPs surface. MoSe2 NPs was a spherical structure with a particle size of ~ 200 nm characterized by TEM and dynamic light scattering (DLS). The irregular outer edge of MoSe2 NPs may have large number of vacancies to facilitate drug loading. The synthesized MoSe2 NPs was hexagonal crystal structure analyzed by X-ray diffraction (XRD) (Fig. 1C).The Raman spectrum indicated that MoSe2 NPs has good crystallinity (Fig. S3). MoSe2 NPs had a mesoporous structure with an average pore size of 4-20 nm by nitrogen adsorption desorption experiments (Fig. 1D). The specific surface area of MoSe2 NPs was calculated to be 17.47 m2/g by the BET multi-layer adsorption equation (Fig. S4). Therefore, MoSe2 NPs were good for

ro of

loading drugs. The structure of the MoSe2@ICG-PDA-HA becomes tight observed by the TEM (Fig.1B). Probably because of the cross-linking and polymerization of dopamine in the formation of the polydopamine layer, the loose structure of MoSe2 NPs became more compact and the surface becomes regular. The diameter of the MoSe2@ICG-PDA-HA was about 209 nm measured by DLS and TEM (Fig. S1B). The UV-vis spectrum indicated successful encapsulation of ICG (Fig.1E). It's

-p

worth noting that the absorption peak of ICG had a slight red shift, possibly due to the sulfonic acid groups in the ICG react with certain groups of dopamine. The potential changes of the synthesized

re

products in each process of zeta potential measurement indicated that the PDA modification layer

Jo

ur

na

lP

and HA were successfully wrapped on the surface of MoSe2 NPs (Fig.1F).

Fig. 1.Structural and compositional characterizations. TEM image of (A) MoSe2 NPs and (B)MoSe2@ICG-PDA-HA. (C) XRD spectra of MoSe2 NPs. (D) Nitrogen adsorption/desorption isotherms and pore-size distribution curves of MoSe2 NPs. (E) UV-Vis spectra of ICG, MoSe2 NPs, MoSe2@ICG and MoSe2@ICG-PDA-HA. (F) Zeta potentials of MoSe2 NPs, MoSe2@ICG-PDA and MoSe2@ICG-PDA-HA in water. 3.2. The Stability and simulated release in vitro of MoSe2@ICG-PDA-HA After laser irradiation, the absorption peaks of free ICG in both of them are gradually weakened and almost disappeared after 10 minutes, indicating the poor stability of ICG (Fig. 2A). On the contrary, the ICG characteristic absorption peak of MoSe2@ICG-PDA-HA showed only a slight

ro of

change, indicating that the optical stability of ICG can be significantly improved by encapsulating ICG in MoSe2 NPs (Fig. 2C). Compared to free ICG, ICG load on MoSe2@ICG-PDA-HA remained stable after 90 days of storage, also indicating that the nanosystem can significantly improve the stability of ICG (Fig. 2B&D).The drug loading efficiency of ICG was calculated by measuring the

-p

UV absorption intensity of ICG in the supernatant of the MoSe2@ICG-PDA-HA sample (Fig. 2E). The loading efficiency of ICG was 25.8% by calculated, exhibited a saturated load capacity of

re

MoSe2 NPs. Subsequently, the encapsulation ability of the PDA biomimetic layer was verified by in vitro drug release (Fig. 2F). The release efficiency of ICG in MoSe2@ICG-PDA-HA was only 12.8%

lP

within 24 h in simulated physiological conditions (pH 7.4), indicating that the PDA layer could stably encapsulate ICG in MoSe2 NPs. In contrast, the release efficiency of ICG in MoSe2@ICGPDA-HA was significantly increased within 24 h in the simulated tumor microenvironment (pH 5).

na

The release amount reached 68.3%, which is 5 times that under physiological conditions. More interestingly, the laser irradiation can also effectively increase the release efficiency of ICG (77.1%). It may be because ICG was adsorbed in MoSe2 NPs by physical adsorption, and the raising

ur

temperature would weaken the physical adsorption. The results above indicated that the MoSe2@ICG-PDA-HA nanosystem had an acid response and a near-infrared light response to

Jo

efficiently release ICG to maximize its anticancer effect.

ro of

Fig. 2.Uv-vis Spectra of (A) free ICG (10 μg/mL, in water) and (C) MoSe2@ICG-PDA-HA under

-p

laser (0.5 W/cm2) at different time. Uv-vis Spectra of (B) free ICG (10 μg/mL, in water) and (D) MoSe2@ICG-PDA-HA after a long-term storage for 90 days. (E) Loading efficiency in different

re

concentrations of ICG. (F) ICG released from MoSe2@ICG-PDA-HA overtime in phosphoric acid buffers at the different pH values (5.0 or 7.4) and under laser (0.5 W/cm2, 5 min every 4 h ).

lP

3.3. Photothermal/fluorescence of MoSe2@ICG-PDA-HA

The photothermal effect of MoSe2 NPs was positively correlated with its concentrations and the light power used (Fig. 3A&B&D&E).The temperature of MoSe2 NPs (10 μg/mL) increased rapidly

na

under 808 nm laser irradiation and the photothermal conversion efficiency was 61.31% by calculation (Fig. S5), which was higher than that of most reported photothermal nanomaterials, such

ur

as Au@Cu2@xS NCs (59%) and CuS-NCs (51.5%) [52-53]. More importantly, MoSe2 NPs also had the good photothermal stability (Fig. 3F). Under 808 nm laser irradiation, although ICG showed a

Jo

certain photothermal effect, MoSe2@ICG-PDA-HA exhibited more significant photothermal effect (~62 °C), probably due to the photothermal synergy between ICG and MoSe2 NPs. More interestingly, MoSe2@ICG-PDA-HA also has strong photothermal stability (Fig. 3G). Moreover, when the pH decreased from 7.4 to 5.5, the fluorescence intensity of ICG changed little. On the contrary, the fluorescence signal of MoSe2@ICG-PDA-HA was significantly enhanced, which proved that the encapsulation of ICG in MoSe2 NPs could achieve the control release of ICG and maximize its anti-cancer effect (Fig. S6).

ro of -p re lP

Fig. 3. IR thermal images of (A, D) different concentrations and (B, E) different power densities of

na

MoSe2 NPs under laser. (C) IR thermal images and (H) Photothermal heating curves and for PBS, MoSe2 NPs, ICG and MoSe2@ICG-PDA-HA under 808 nm laser. (F) Temperature records of MoSe2 NPs aqueous solution under five cycles of laser on/off at 0.5 W cm-2. (G) Temperature records of

ur

ICG and MoSe2@ICG-PDA-HA aqueous solution under five cycles of laser on/off at 0.5 W cm-2.

Jo

3.4. Uptake experiments of cells on MoSe2@ICG-PDA-HA Effective cellular uptake was particularly important to enhance the penetration and retention of

nano-anticancer drugs in the tumor area. 4T1 cells had the best absorption effect on MoSe2@ICGPDA-HA (94.52%) (Fig. 4A&B).To investigate the stability and targeting ability of MoSe2@ICGPDA-HA, 4T1 cells were co-incubated with ICG and MoSe2@ICG-PDA-HA with deferent time respectively. Compared with free ICG, the uptake of MoSe2@ICG-PDA-HA by 4T1 cells was obviously higher (Fig. 4C).The fluorescence of MoSe2@ICG-PDA-HA gradually increased with time, and became strongest at 24 h, indicating that loading ICG in MoSe2 NPs would significantly

enhance its cellular uptake and residence time in cells. Then the lysosome escape process of MoSe2@ICG-PDA-HA was observed and photographed by the laser confocal microscope. As shown in Fig. 4D, the fluorescence gradually increased with the incubation time, indicating that 4T1 cells had a time-dependent intake of MoSe2@ICG-PDA-HA. In addition, the MoSe2@ICG-PDA-HA began to adsorb on the cell membrane after 4 h of incubation. The nanoparticles began to enter the cytoplasm and were captured by lysosomes at 8h and escaped from the lysosome and began to enter

Jo

ur

na

lP

re

-p

ro of

the nucleus at 24 h.

Fig. 4. Cell uptake of ICG, MoSe2@ICG-PDA and MoSe2@ICG-PDA-HA by 4T1 cells analyzed by Confocal Laser Scanning Microscope (A) and the flow cytometry (B), scale bars: 50 μm. (C) Intracellular fluorescence intensity of free ICG and MoSe2@ICG-PDA-HA in 4T1 cells for incubation different time, scale bars: 50 μm. (D) Inctracellular localization of ICG (green) and

lysosome (red) in 4T1 cells was assessed by Confocal Laser Scanning Microscope at different time, scale bars: 50 μm.

3.5. Detection of ROS of MoSe2@ICG-PDA-HA In order to study the production of ROS in different nanometers in 4T1 cells, free ICG, MoSe2 NPs and MoSe2@ICG-PDA-HA were incubated with 4T1 cells for 12 h respectively. Then, the cells were stained with a DCFH-DA reactive oxygen probe after irradiated with an 808 nm laser (0.5 W/cm2) for 1 min. The free ICG and MoSe2@ICG-PDA-HA only produced active oxygen under laser irradiation, indicating that laser irradiation was a condition for generating active oxygen (Fig.

ro of

5A&B). The amount of ROS produced by MoSe2@ICG-PDA-HA was almost three times that of free ICG.

ROS produced by the photosensitizer can induce apoptosis and play a crucial role in photodynamic therapy. Compared with the control group, the fluorescence intensity of the singlet

-p

oxygen fluorescent probe Singlet Oxygen Sensor Green (SOSG) in MoSe2 NPs did not change much under laser irradiation, indicating that MoSe2 NPs could hardly produce ROS under illumination (Fig.

re

5C). The fluorescence intensity of SOSG in free ICG and MoSe2@ICG-PDA-HA was significantly enhanced under illumination, indicating that ICG the substance producing ROS. More importantly,

lP

MoSe2@ICG-PDA-HA showed a significantly enhanced SOSG fluorescence signal under laser irradiation. When a ROS-eliminating agent tocopherol was added, the SOSG fluorescence intensity was significantly reduced. It showed that MoSe2@ICG-PDA-HA can generate a large amount of

na

ROS under near-infrared illumination and had the better ability to produce ROS. The ROS production was mainly derived from ICG and was dependent on the illumination time and

Jo

ur

illumination power (Fig. 5D-G).

ro of -p re lP na ur Jo

Fig. 5. (A) Intracellular ROS detection of free ICG, MoSe2 NPs and MoSe2@ICG-PDA-HA with (0.5W/cm2, 1 min) or without laser. (−): without laser; (+): with laser. Scale bars: 20 μm. (B) Mean fluorescence intensity of DCFH-DA (ROS) inside the cells, n = 5. (C) SOSG Fluorescence intensity change with or without laser (0.5 W/cm2, 5 min) in MoSe2 NPs, ICG, MoSe2@ICG, MoSe2@ICGPDA-HA, and MoSe2@ICG-PDA-HA + tocopherol, n = 5. p values: *p < 0.05, **p < 0.01. (D&E) SOSG fluorescence intensity change with the increasing concentration of ICG or MoSe2 NPs (0.5 W/cm2, 5 min), n=5. The number in the brackets stands for drug concentration (μg/mL). (F&G)

SOSG fluorescence intensity change with the laser time (3~30 min) or enhanced laser power (power dose: 0.25 W~1W/cm2, 5 min), n=5. 3.6. MoSe2@ICG-PDA-HA induces apoptosis The antitumor activity of the nanosystem was demonstrated by the apoptosis. Free ICG, MoSe2 NPs and MoSe2@ICG-PDA-HA were respectively co-incubated with 4T1 cells. The cytotoxicity of free ICG, MoSe2 NPs and MoSe2@ICG-PDA-HA were relatively low under no light detected by CCK-8 kit (Fig. S7).Compared to free ICG and MoSe2 NPs, MoSe2 @ICG-PDA-HA have the best photocytotoxicity (~ 93%).The tumor cells-killing ability of MoSe2 @ ICG-PDA-HA has a positive correlation with its concentrations, power of near-infrared light, and illumination time((Fig. 6A-C&

ro of

Fig. S8). The experimental results above indicate that the anti-tumor activity of MoSe2@ICG-PDAHA was better than that of free ICG or MoSe2 NPs alone. Hoechest 33342/PI double staining was used to further evaluate treatment. The MoSe2@ICG-PDA-HA had the strongest effect on the apoptosis of 4T1 cells under the near infrared laser irradiation (Fig. 6D). Under the same conditions

-p

as above, apoptosis of 4T1 cells detected by flow cytometry by Annexin V-FITC/PI double staining were similar to those obtained by laser confocal microscopy (Fig. 6E). It was confirmed that

re

MoSe2@ICG-PDA-HA-mediated targeted photothermal/photodynamic synergistic therapy has the

Jo

ur

na

lP

strongest anti-cancer effect in vitro under the near-infrared laser mediated.

ro of -p re lP na ur Jo Fig. 6. Cytotoxicity in vitro. (A-B) Cytotoxicity of free ICG, MoSe2 NPs and MoSe2@ICG-PDAHA with different laser irradiation (0.5 W/cm2 or 1 W/cm2, 2 min), n = 5. (C) Cytotoxicity of free ICG, MoSe2 NPs and MoSe2@ICG-PDA-HA with laser irradiation (0.5 W/cm2, 5 min) at 37 ℃, n =

5. Representative fluorescence images (D) and the corresponding flow cytometry data (E) of 4T1 cells incubated with MoSe2 NPs, free ICG and MoSe2@ICG-PDA-HA with or without 808 nm laser irradiation (0.5 W/cm2, 5 min). Scale bars: 100 μm. 3.7. The experiment of multicellular spheroids study on anticancer effect of nanosystem In order to explore the inhibitory effect of MoSe2@ICG-PDA-HA on the growth of multicellular balls, multicellular spheres were incubated with PBS, ICG, MoSe2 NPs and MoSe2@ICG-PDA-HA about 24 h respectively, and then irradiated 5 min by laser (0.5W/cm2). The growth of multicellular spheres in each treatment group was observed under a microscope within 7 days (Fig. 7A&B). The MoSe2@ICG-PDA-HA+Laser group showed the best inhibitory effect on

ro of

multicellular spheres growth, and the cell sphere volume became the original 41.8%, which was better than the MoSe2 NPs + Laser group (66.7%) and the ICG+Laser group (62.2%). The results

Jo

ur

na

lP

re

-p

analyzed by the flow cytometry were the same (Fig. 7C).

Fig. 7. (A) Representative photomicrograph of 4T1 tumor spheroids after incubated with free ICG, MoSe2 NPs and MoSe2@ICG-PDA-HA followed by a laser exposure (0.5 W/cm2, 2 min) within 7 days, tumor spheroids only treated with laser were used as controls. Scale bars: 200 μm. (B)

Corresponding volume change curves of tumor spheroids, n = 5. (C) Flow cytometry results of FITC-Annexin V/PI double stained 4T1 tumor spheroids treated with MoSe2 NPs, free ICG and MoSe2@ICG-PDA-HA with laser irradiation (0.5 W/cm2, 2 min). 3.8. Fluorescence imaging and distribution of MoSe2@ICG-PDA-HA in vivo 4T1 tumor-bearing mouse were injected intravenously with free ICG, MoSe2@ICG-PDA and MoSe2@ICG-PDA-HA (2 mg/kg per mouse), respectively. The distribution of ICG in mice was observed by a living fluorescence imaging system at different time periods (Fig. 8A & Fig. S9). Finally, the mice were sacrificed and the content of ICG in various organs (heart, liver, spleen, lung, kidney) was quantitatively analyzed (Fig. S9 & S10).The free ICG would accumulate significantly in

ro of

the liver and kidney, due to the lack of targeting and rapid metabolism by organisms. The MoSe2@ICG-PDA-HA treatment group had the strongest fluorescence signal in the tumor and accumulated the least amount in liver and kidney. And the most significant fluorescence intensity occurred within 24 h with increasing time. Most of the Mo elements were distributed in the liver and

-p

kidney, which may be due to the strong phagocytosis of the reticuloendothelial system (RES) or the possibility that MoSe2@ICG-PDA-HA excreted through the kidneys (Fig. S11). The experimental

re

results above showed that MoSe2@ICG-PDA-HA could avoid the early release of ICG in the blood circulation and had excellent targeting effect, which enhanced the enrichment of ICG in tumor sites.

lP

The distribution of MoSe2@ICG-PDA-HA in the tumor was observed by CD31 fluorescence staining of tumor blood vessels (Fig. 8D). After 12 h of injection, most of the fluorescence (green) of MoSe2@ICG-PDA-HA did not completely coincide with the tumor blood vessels (red) and spread

na

throughout the tumor area. It showed that MoSe2@ICG-PDA-HA could exude from tumor blood vessels in a short time and exhibited good permeation retention effect.

ur

The photothermal efficacy of MoSe2@ICG-PDA-HA was evaluated in vivo. After 24 hours of intravenous injection, the MoSe2@ICG-PDA-HA with near-infrared strong absorption properties and effective tumor-targeted accumulation, the surface temperature of MoSe2@ICG-PDA-HA group

Jo

rapidly increased to nearly 56 °C within 5 min (Fig. 8B&Fig. S12). However, under the same experimental conditions, the treatment of the MoSe2 NPs and the free ICG only reach to 50 °C and 45 °C, respectively. MoSe2@ICG-PDA-HA has significant photothermal efficacy due to the synergistic heat production of MoSe2 and ICG. ROS produced in tumors was key indicators of PDT in vivo. MoSe2@ICG-PDA-HA was intravenously injected for 12 h to accumulate in the tumor. Then, after irradiated with near-infrared light, the tumor was dissected and subjected to immunofluorescence staining to evaluate the level of

generated ROS in vivo (Fig. 8C). MoSe2@ICG-PDA-HA could produce a large amount of ROS in the tumor after illumination, obviously higher than the free ICG and the non-targeted MoSe2@ICGPDA produced. It was indicated that MoSe2@ICG-PDA-HA could effectively accumulate in the tumor and produce a large amount of ROS under the condition of light, showing the best

ur

na

lP

re

-p

ro of

photodynamic therapeutic effect.

Jo

Fig. 8. (A) In vivo imaging of 4T1 tumor-bearing mice after administration of free ICG, MoSe2@ICG-PDA, MoSe2@ICG-PDA-HA at indicated time (0.5, 2, 4, 8, 12, 24, 48 h). (B) Infrared thermal images of mice bearing 4T1 tumor after 24 h i.v. injection with free ICG, MoSe2 NPs or MoSe2@ICG-PDA-HA (1 mg per kg per mouse) and with 808 nm laser irradiation (0.5 W/cm2, 5 min). (C) ROS generation level in tumor after 12 h i.v. injection with free ICG, MoSe 2@ICG-PDA or MoSe2@ICG-PDA-HA (1 mg per kg per mouse) and with 808 nm laser irradiation (0.5 W/cm2, 5 min). Scale bars, 50 μm. (D) Intratumoral distribution of MoSe2@ICG-PDA-HA at 2, 12 and 24 h

postinjection. The nuclei were stained with DAPI (blue) and the tumor blood vessels were stained with CD31 antibody (red). Scale bars, 50 μm. 3.9 Antitumor activity of MoSe2@ICG-PDA-HA-mediated synergistic therapy in vivo Subsequently, the 4T1 tumor bearing mice were used to evaluate nanomaterials anticancer effect in vivo. Fig. 9A showed the process of combination therapy with 4T1 tumor-bearing mice. we randomly divided the 4T1 tumor-bearing mice and treated them as follows: (Group 1)PBS , (Group 2) Laser, (Group 3)MoSe2 NPs + Laser, (Group 4) ICG + Laser, (Group 5) MoSe2 NPs + ICG mixed solution + Laser, (Group 6) MoSe2@ICG-PDA+Laser, (Group 7) MoSe2@ICG-PDA-HA+Laser. The inhibition effect of MoSe2 NPs or free ICG alone was not significant, indicating that it was

ro of

difficult to have better anticancer activity by single therapy. The tumors treated with MoSe2@ICGPDA-HA+Laser showed no obvious growth during treatment, indicating that MoSe2@ICG-PDAHA-mediated targeted photothermal/photodynamic synergistic therapy had the most significant anticancer effect (Fig. 9C).

-p

As shown in the H&E and TUNEL staining analysis, most of the tumor cells showed nuclear dissolution (green arrow) and nuclear fragmentation (black arrow) in the MoSe2@ICG-PDA-

re

HA+Laser treatment (Fig. 9E-F), which showed the best therapeutic effect. Moreover, the cells in the MoSe2@ICG-PDA-HA+Laser group exhibited low Ki-67 expression (positive cell ratio of 3.2%),

demonstrating

that

the

MoSe2@ICG-PDA-HA-mediated

targeted

lP

approximately

photothermal/photodynamic synergistic therapy significantly inhibited tumor cell proliferation. In general, the migratory 4T1 tumor model was capable of spontaneously forming lung

na

metastases. There were almost no metastatic lesions in the lung of the mice of MoSe2@ICG-PDAHA+Laser treatment, while the other groups had different degrees of pulmonary metastasis (Fig. S14

ur

and Fig. 9H). In addition, H&E staining analysis of lung revealed that there were significant tumor cell islands in the lungs of the other treatment groups except the MoSe2@ICG-PDA-HA+Laser treatment(Fig. 9I). Therefore, MoSe2@ICG-PDA-HA-mediated targeted photothermal/photodynamic

Jo

synergistic therapy could not only effectively inhibit tumor growth and proliferation, but also inhibited tumor metastasis. Moreover, the weight of mice had no significant changes during all the treatment (Fig. 9D). The H&E staining analysis of heart, liver, spleen and kidney of all treatment showed no obvious pathological changes, indicating that nanomedicine has good biocompatibility (Fig. S15). The index of mouse serum biochemical indicators and hematology studies indicated that MoSe2@ICG-PDA-HA had no significant side effects and had great clinical application value (Fig. S16).

ro of -p re lP na ur Jo

Fig. 9. In vivo antitumor efficacy of MoSe2@ICG-PDA-HA. (A) Therapy schedule for MoSe2@ICG-PDA-HA administration and targeted photothermal/photodynamic synergistic therapy in vivo. (B) The growth condition of the 4T1 tumors were measured in various treatments. The error bar is based on the standard error of mean (SEM). (C) The images and the mass of tumors collected

from mice at the end of treatments，*, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Body weight of mice from different groups during treatment. Error bars were based on the SEM of five mice per group. (E-G) Histological analysis of tumor sections stained with H&E, TUNEL (Scale bars, 20 μm) and Ki67 (Scale bars, 20 μm). (I) Corresponding photographs of the whole lungs from mice with different treatments and H&E staining of the lung tissue section (4× and 20×). Blue circles denote surface lung metastases.

4. Conclusion

ro of

In conclusion, the synthesized MoSe2 NPs carriers had the advantages of both photothermal therapy and loading photosensitizer ICG. We had successfully synthesized a nanosystem of diagnosis and treatment (MoSe2@ICG-PDA-HA) that can simultaneously achieve targeted photothermal/photodynamic synergistic therapy through one-step near-infrared light irradiation.

-p

Based on the excellent biocompatibility, light stability and targeting ability, MoSe2@ICG-PDA-HAmediated targeted photothermal/photodynamic synergistic therapy in vivo not only had achieved an

re

ideal healing effect, but also had significant anti-migration and anti-proliferative effects. This study highlights nanosystems that could improve the photostability of optical materials and enhance the photothermal/photodynamic synergy effect, achieving significant anticancer therapeutic effects and

lP

providing a new strategy for cancer treatment.

na

Credit author statement

Yanan Liu: Conceptualization, Methodology, Writing - Original Draft, Investigation, Formal

ur

analysis, Validation.

ChunFang Wei: Conceptualization, Methodology, Investigation, Formal analysis, Writing -

Jo

Original Draft, Validation.

Ange Lin: Investigation, Formal analysis. Jiali Pan: Conceptualization, Methodology, Writing - Original Draft, Data Curation. Xu Chen: Investigation. Xufeng Zhu: Investigation.

Youcong Gong: Investigation. Guanglong Yuan: Investigation. Lanmei Chen: Resources,Writing - Review & Editing, Project administration. Jie Liu: Resources,Writing - Review & Editing, Project administration. Zhaohui Luo: Resources, Writing - Review & Editing, Project administration.

ro of

All authors read and approved the manuscript.

Declaration of interests

-p

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

re

This work was supported by the National Natural Science Foundation of China (21877051, 81803027, 21701034), the Natural Science Foundation of Guangdong Province (2018A030310628)

lP

and Projects of Special Innovative of Department of Education of Guangdong Province (2017KTSCX078), Project of Young Innovative Talents in Universities and Colleges of Department of Education of Guangdong Province (2018KQNCX100) .

na

References

[1] Huang, W. C.; Shen, M. Y.; Chen, H. H.; Lin, S. C.; Chiang, W.H.; Wu, P. H.; Chang, C. W.;

ur

Chiang, C. S.; Chiu, H. C. Monocytic Delivery of Therapeutic Oxygen Bubbles for Dual-Modality Treatment of Tumor Hypoxia. J. Controlled Release, 2015, 220, 738−750.

Jo

[2] Zheng, X.; Tang, H.; Xie, C.; Zhang, J.; Wu, W.; Jiang, X. Tracking Cancer Metastasis in Vivo by Using an Iridium-based Hypoxia-activated Optical Oxygen Nanosensor. Angew. Chem., Int. Ed., 2015, 54, 8094−8099. [3] Liu, Y.; Liu, Y.; Bu, W.; Cheng, C.; Zuo, C.; Xiao, Q.; Sun, Y.; Ni, D.; Zhang, C.; Liu, J.; Shi, J. Hypoxia Induced by Upconversionbased Photodynamic Therapy: Towards Highly Effective Synergistic Bioreductive Therapy in Tumors. Angew.Chem.Int.Ed., 2015, 54, 8105−8109. [4] Vijayaraghavan P., Liu C. H., Vankayala R., Chiang C. S., Hwang K. C. Designing Multibranched Gold Nanoechinus for NIR Light Activated Dual Modal Photodynamic and Photothermal Therapy in the Second Biological Window. Advanced Materials 2014, 26, 6689−6695.

[5] Gollavelli G., Ling Y. C. Magnetic and Fluorescent Graphene for Dual Modal Imaging and Single Light Induced Photothermal and Photodynamic Therapy of Cancer Cells. Biomaterials 2014, 35, 4499−4507. [6] Chen R., Wang X., Yao X., Zheng X., Wang J., Jiang X. Near-IR-triggered Photothermal/photodynamic Dual-modality Therapy System via Chitosan Hybrid Nanospheres. Biomaterials 2013, 34, 8314−8322. [7] Wenpei Fan, Bryant Yung, Peng Huang, Xiaoyuan Chen. Nanotechnology for Multimodal Synergistic Cancer Therapy. Chemical Reviews 2017, 117, 13566-13638. [8] Liu JJ, Liang HN, Li MH, Luo Z, Zhang JX, Guo XM, Cai KY. Tumor acidity activating multifunctional nanoplatform for NIR-mediated multiple enhanced photodynamic and photothermal tumor therapy. Biomaterials 2018, 157, 107-124.

ro of

[9] Guo W., Guo C., Zheng N., Sun T., Liu S. CsxWO 3 Nanorods Coated with Polyelectrolyte Multilayers As A Multifunctional Nanomaterial for Bimodal Imaging-Guided Photothermal/Photodynamic Cancer Treatment. Advanced Materials 2017, 29, 1604157. [10] Bhana S., Lin G., Wang L., Starring H., Mishra S. R., Liu G., Huang X. Near-Infrared-Absorbing Gold Nanopopcorns with Iron Oxide Cluster Core for Magnetically Amplified Photothermal and Photodynamic Cancer Therapy. ACS Applied Materials & Interfaces 2015, 7, 11637−11647.

-p

[11] Chang G. Wang Y. Gong B. Xiao Y. Chen Y. Wang S. Li S. Huang F. Shen Y. Xie A. Reduced Graphene Oxide/Amaranth Extract/AuNPs Composite Hydrogel on Tumor Cells as Integrated Platform for Localized and Multiple Synergistic Therapy. ACS Applied Materials & Interfaces 2015, 7, 11246−11256.

re

[12] Huang Y., Qiu F., Shen L., Chen D., Su Y., Yang C., Li B., Yan D., Zhu X. Combining TwoPhoton-Activated Fluorescence Resonance Energy Transfer and Near-Infrared Photothermal Effect of Unimolecular Micelles for Enhanced Photodynamic Therapy. ACS Nano 2016, 10, 10489−10499.

lP

[13] Gong H., Dong Z., Liu Y., Yin S., Cheng L., Xi W., Xiang J., Liu K., Li Y., Liu Z. Engineering of Multifunctional Nano-Micelles for Combined Photothermal and Photodynamic Therapy Under the Guidance of Multimodal Imaging. Advanced Functional Materials 2014, 24, 6492−6502.

na

[14] Wang J., Zhu G., You M., Song E., Shukoor M. I., Zhang K., Altman M. B., Chen Y., Zhu Z., Huang, C. Z. Assembly of Aptamer Switch Probes and Photosensitizer on Gold Nanorods forTargeted Photothermal and Photodynamic Cancer Therapy. ACS Nano 2012, 6, 5070−5077.

ur

[15] Vijayaraghavan P., Liu C.-H., Vankayala R., Chiang C.-S., Hwang K. C. Designing MultiBranched Gold Nanoechinus for NIR Light Activated Dual Modal Photodynamic and Photothermal Therapy in the Second Biological Window. Advanced Materials 2014, 26, 6689−6695.

Jo

[16] Abbas M., Zou Q., Li S., Yan X. Self-Assembled Peptide- and Protein-Based Nanomaterials for Antitumor Photodynamic and Photothermal Therapy. Advanced Materials 2017, 29, 1605021. [17] Liu X., Yang G., Zhang L., Liu Z., Cheng Z., Zhu X. Photosensitizer Cross-Linked NanoMicelle Platform for Multimodal Imaging Guided Synergistic Photothermal/Photodynamic Therapy. Nanoscale 2016, 8, 15323−15339. [18] Kim Y.-K., Na H.-K., Kim S., Jang H., Chang S.-J., Min D.-H. One-Pot Synthesis of Multifunctional Au@Graphene Oxide Nanocolloid Core@Shell Nanoparticles for Raman Bioimaging, Photothermal, and Photodynamic Therapy. Small 2015, 11, 2527−2535. [19] Yan X., Hu H., Lin J., Jin A. J., Niu G., Zhang S., Huang P., Shen B., Chen X. Optical and Photoacoustic Dual-Modality Imaging Guided Synergistic Photodynamic/Photothermal Therapies. Nanoscale 2015, 7, 2520−2526.

[20] Vankayala, R.; Lin, C. C.; Kalluru, P.; Chiang, C. S.; Hwang, K. C. Gold Nanoshells-Mediated Bimodal Photodynamic and Photothermal Cancer Treatment Using Ultra-low Doses of Near Infrared Light. Biomaterials, 2014, 35, 5527−5538. [21] Zhao, Z.; Shi, S.; Huang, Y.; Tang, S.; Chen, X. Simultaneous Photodynamic and Photothermal Therapy Using Photosensitizer functionalized Pd Nanosheets by Single Continuous Wave Laser. ACS Appl. Mater. Interfaces,2014,6,8878−8885. [22] Li, Y.; Lin, T. Y.; Luo, Y.; Liu, Q.; Xiao, W.; Guo, W.; Lac, D.; Zhang, H.; Feng, C.; Wachsmann-Hogiu, S.; Walton, J. H.; Cherry, S. R.; Rowland, D. J.; Kukis, D.; Pan, C.; Lam, K. S. A Smart and Versatile Theranostic Nanomedicine Platform Based on Nanoporphyrin. Nat. Commun.,

ro of

2014, 5, 4712−4726.

[23] Jang. B., Park. J. Y, Tung. C. H, Kim, I. H.; Choi, Y. Gold Nanorod-photosensitizer Complex for Near-Infrared Fluorescence Imaging and Photodynamic/photothermal Therapy in Vivo. ACS Nano, 2011, 5, 1086−1094.

-p

[24]Xia Y., Zhong J., Zhao M., Tang Y., Han N., Hua L., Xu Tn., Wang C., Zhu B. Galactosemodified selenium nanoparticles for targeted delivery of doxorubicin to hepatocellular carcinoma.

re

Drug Delivery, 2019, 26(1), 1-1.

[25] Tian B., Wang C.,Zhang S., Feng L., Liu Z. Photothermally Enhanced Photodynamic Therapy

lP

Delivered by Nano-graphene Oxide. ACS Nano, 2011, 5, 7000−7009. [26] L Lin.,Y Xu., S Zhang., I. M. Ross., A. C. M. Ong.,D. A. Allwood. Fabrication of Luminescent

na

Monolayered Tungsten Dichalcogenides Quantum Dots with Giant Spin-Valley Coupling. ACS Nano 2013, 7, 8214–8223.

ur

[27] S. Xu., D. Li., P. Wu. One‐Pot, Facile, and Versatile Synthesis of Monolayer MoS 2/WS2 Quantum Dots as Bioimaging Probes and Efficient Electrocatalysts for Hydrogen Evolution Reaction. Advanced Functional Materials 2015, 25, 1127–1136.

Jo

[28] Mou J., Li P., Liu C., Xu H., Song L., Wang J., Zhang K., Chen Y., Shi J., Chen, H. Ultrasmall Cu2–xS Nanodots for Highly Efficient Photoacoustic Imaging-Guided Photothermal Therapy. Small 2015, 11, 2275– 2283. [29] Liang G., Jin X., Qin H., Xing D. Glutathione-capped, renal-clearable CuS nanodots for photoacoustic imaging and photothermal therapy. J. Mater. Chem. B 2017, 5, 6366– 6375. [30] Y Liu., M Ji., P Wang. Recent Advances in Small Copper Sulfide Nanoparticles for Molecular Imaging and Tumor Therapy. Mol. Pharmaceutics 2019, 16, 8, 3322-3332.

[31] Vipul Agarwal.; Kaushik Chatterjee, Recent Advances in the Field of Transition Metal Dichalcogenides for Biomedical Applications. Nanoscale 2018, 10, 16365–16397. [32] Zhao YY, Wei CF, Chen X, Liu JW, Yu QQ, Liu YA, Liu J. Drug Delivery System Based on Near-infrared Light-responsive Molybdenum Disulfide Nanosheets Controls the High-efficiency Release of Dexamethasone to Inhibit Inflammation and Treat Osteoarthritis. ACS Applied Materials & Interfaces 2109, 11(12), 11587-11601. [33] Jiali Pan., Xufeng Zhu., Xu Chen, Yingyu Zhaoa., Jie Liu. Gd 3+-Doped MoSe2 Nanosheets Used as a Theranostic Agent for Bimodal Imaging and Highly Efficient Photothermal Cancer Therapy. Biomaterials Science 2018, 6, 372–387, DOI: 10.1039/c7bm00894e.. [34] M. Chhowalla., H. S. Shin., G. Eda., L. J. Li., K. P. Loh., H. Zhang. The chemistry of twodimensional layered transition metal dichalcogenide nanosheets. Nature Chemistry 2013, 5, 263– 275.

ro of

[35] D. Gopalakrishnan.; D. Damien.; M. M. Shaijumon. MoS2 Quantum Dot-Interspersed Exfoliated MoS2 Nanosheets. ACS Nano 2014, 8, 5297–5303. [36]H. D. Ha., D. J. Han., J. S. Choi., M. Park.,T. S. Se. Dual Role of Blue Luminescent MoS 2 Quantum Dots in Fluorescence Resonance Energy Transfer Phenomenon. Small 2014, 10.

-p

[37] C. Zhu.; X. Mu.; P. A. van Aken.; Y. Yu.; J. Maier. Single-Layered Ultrasmall Nanoplates of MoS2 Embedded in Carbon Nanofibers with Excellent Electrochemical Performance for Lithium and Sodium Storage. Angewandte Chemie-International Edition 2014, 53, 2152–2156.

re

[38] L. Lin., Y. Xu.,S. Zhang., I. M. Ross., A. C. M. Ong.,D. A. Allwood. Fabrication of Luminescent Monolayered Tungsten Dichalcogenides Quantum Dots with Giant Spin-Valley Coupling. ACS Nano 2013, 7, 8214–8223.

lP

[39] S. Xu., D. Li., P. Wu. One‐Pot, Facile, and Versatile Synthesis of Monolayer MoS 2/WS2 Quantum Dots as Bioimaging Probes and Efficient Electrocatalysts for Hydrogen Evolution Reaction. Advanced Functional Materials 2015, 25, 1127–1136.

na

[40] L. Cheng., J. Liu., X. Gu., H. Gong., X. Shi., T. Liu., C. Wang., X. Wang., G. Liu., H. Xing., W. Bu., B. Sun., Z. Liu. PEGylated WS2 Nanosheets as a Multifunctional Theranostic Agent for in vivo Dual‐Modal CT/Photoacoustic Imaging Guided Photothermal Therapy. Advanced Materials 2014, 26, 1886–1893. [41]G. Guan., S. Zhang., S. Liu., Y. Cai., M. Low., C. P. Teng., I. Y. Phang., Y. Cheng., K. L. Duei., B. M. Srinivasan.,

ur

Y. Zheng., Y. W. Zhang., M. Y. Han. Protein Induces Layer-by-Layer Exfoliation of Transition Metal Dichalcogenides. Journal of the American Chemical Society 2015, 137, 6152–6155.

Jo

[42] X. Qian., S. Shen., T. Liu., L. Cheng., Z. Liu. Two-dimensional TiS2 nanosheets for in vivo photoacoustic imaging and photothermal cancer therapy. Nanoscale 2015, 7, 6380–6387. [43] S. Shen., Y. Chao., Z. Dong., G. Wang., X. Yi., G. Song., K. Yang., Z. Liu., L. Cheng. Bottom‐ Up Preparation of Uniform Ultrathin Rhenium Disulfide Nanosheets for Image ‐ Guided Photothermal Radiotherapy. Advanced Functional Materials 2017, 27,1700250. [44] J. Liu., M. Yu., C. Zhou., S. Yang., X. Ning., J. Zheng. Passive Tumor Targeting of RenalClearable Luminescent Gold Nanoparticles: Long Tumor Retention and Fast Normal Tissue Clearance. Journal of the American Chemical Society 2013, 135, 4978–4981. [45] Wang XL., Gong YJ., Shi G.., Chow WL., Keyshar K., Ye GL., Vajtai R., Lou J., Liu Z., Ringe E. Chemical Vapor Deposition Growth of Crystalline Mono layer MoSe 2. ACS Nano 2014, 8, 5,

5125-5131. [46] Li, ZW ., Liu, CX., Rong, X., Luo, Y., Cheng, HT., Zheng, LH., Lin, F., Shen, B., Gong, YJ ., Zhang, S. Tailoring MoS2 Valley-Polarized Photoluminescence with Super Chiral Near-Field. Advanced Materials 2018, 30, 34,1801908. [47] Velicky M., Donnelly GE., Hendren WR., McFarland S., Scullion D., DeBenedetti WJI., Correa GC., Han YM ., Wain AJ., Hines MA ., A. R. Beal.,W. Y. Liang. Mechanism of Gold-Assisted Exfoliation of Centimeter-Sized Transition-Metal Dichalcogenide Monolayers. ACS Nano 2018, 12(10), 10463-10472. [48] Z. Lei.,W. Zhu.,S. Xu.,J. Ding.,J. Wan., P. Wu. Hydrophilic MoSe2 Nanosheets as Effective Photothermal Therapy Agents and Their Application in Smart Devices. ACS Applied Materials & Interfaces 2016, 8, 20900–20908.

ro of

[49] Yanlan Liu., Kelong Ai., Lehui Lu. Polydopamine and Its Derivative Materials: Synthesis and Promising Applications in Energy, Environmental, and Biomedical Fields. Chemical Reviews 2014, 114(9), 5057-5115. [50]Xia Y., Xiao M., Zhao M., Xu T., Guo M., Wang C., Li Y., Zhu B., Liu H. Doxorubicin-loaded functionalized selenium nanoparticles for enhanced antitumor efficacy in cervical carcinoma therapy. Materials Science and Engineering: C, 2020, 106: 110100.

-p

[51] Zhong Yinan., Goltsche Katharina., Cheng Liang.Hyaluronic acid-shelled acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast tumor xenografts in vivo. Biomaterials 2016, 84, 250-261.

lP

re

[52] M. Ji, M. Xu, W. Zhang,Z. Yang, L. Huang, J. Liu, Y. Zhang, L. Gu, Y. Yu, W. Hao, P. An, L. Zheng, H. Zhu, J. Zhang Structurally Well ‐ Defined Au@Cu2-xS Core-Shell Nanocrystals for Improved Cancer Treatment Based on Enhanced Photothermal Efficiency. Advanced Materials, 2016, 28(16): 3094-3101.

Jo

ur

na

[53] T. Yang, Y. Wang, H. Ke, Q. Wang, X. Lv, H. Wu, Y. Tang, X.Yang, C. Chen, Y. Zhao, H. Chen. Protein-Nanoreactor-Assisted Synthesis of Semiconductor Nanocrystals for Efficient Cancer Theranostics. Advanced Materials, 2016, 28(28): 5923-5930.