Intrinsic, cancer cell-selective toxicity of organic photothermal nano

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Biological and Medical Applications of Materials and Interfaces

Intrinsic, cancer cell-selective toxicity of organic photothermal nano-agent: a simple formulation for combined photothermal-chemotherapy of cancer Zhuoxuan Lu, Feng-Ying Huang, Rong Cao, Guang-Hong Tan, Guohui Yi, Nongyue He, Lingfeng Xu, and Liming Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07801 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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ACS Applied Materials & Interfaces

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Intrinsic, cancer cell-selective toxicity of organic

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photothermal nano-agent: a simple formulation for

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combined photothermal-chemotherapy of cancer

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Zhuoxuan Lua‡, Feng-Ying Huanga‡, Rong Caoa‡, Guang-Hong Tana, Guohui Yi a, Nongyue Heb,c,

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Lingfeng Xua, Liming Zhanga*

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a

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Hainan Provincial Key Laboratory of Tropical Medicine, Hainan Medical College, Haikou

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571101, P. R. China

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b

Key Laboratory of Tropical Disease and Translational Medicine of the Ministry of Education &

Hunan Key Laboratory of Green Chemistry and Application of Biological Nanotechnology,

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Hunan University of Technology, Zhuzhou 412008, P. R. China

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c

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Southeast University, Nanjing 210096, P. R. China

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‡

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Email: [email protected].

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering,

These authors contributed equally to this work

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KEYWORDS: Nano-medicine, Photothermal therapy, Cancer cell-selective cytotoxicity, Cell

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cycle arrest, Biodegradability

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ACS Paragon Plus Environment

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ABSTRACT: Nano-agent-mediated photothermal therapy (PTT) combined with chemotherapy

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has been proposed as an effective strategy against cancer. However, chemotherapeutic agents

3

often cause serious side effects. Herein, a novel PTT nanoagent (Cy5.5-MSA-G250) with

4

unanticipated intrinsic tumor-selective cytotoxicity is developed. The Cy5.5-MSA-G250

5

nanoparticles (NPs) are created by mixing mouse serum albumin (MSA) and coomassie brilliant

6

blue (G250), and then conjugated with cyanine 5.5 (Cy5.5). As expected, Cy5.5-MSA-G250 NPs

7

can efficiently kill cancer cells in vitro and in vivo by PTT. Meanwhile, we accidentally discover

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that Cy5.5-MSA-G250 have intrinsic specific cytotoxicity against tumor cells but not against

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normal cells. Moreover, the tumor-specific cytotoxicity of Cy5.5-MSA-G250 is much stronger

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than that of cytarabine, an FDA-approved anticancer drug. In vivo experiments also prove that

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Cy5.5-MSA-G250 NPs can effectively eliminate residual tumor cells and prevent metastasis.

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Further study indicates that selective induction of G1 cell cycle arrest and inhibition of DNA

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duplication in tumor cells may be the possible mechanism of the tumor cell-selective cytotoxicity

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of Cy5.5-MSA-G250 NPs. In addition, direct visualization, low systematic toxicity, good

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biodegradation, and efficient body excretion further make Cy5.5-MSA-G250 NPs attractive for

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in vivo applications. Taken together, Cy5.5-MSA-G250 NPs are proven to be a promising

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platform for combined photothermal-chemotherapy.

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INTRODUCTION

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The ability to eradicate tumors, stop their metastasis, and prevent their recurrence is crucial for

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the development of effective therapeutic strategies against cancer.1-2 Conventional treatments,

4

although with strong tumoricidal capability, still have some limitations. Recently, the advent of

5

nanotherapeutics has changed the paradigm of cancer treatment by circumventing the

6

disadvantages of conventional treatment formulations in cancer therapy due to the unique

7

physical and chemical properties of nanomaterials.3-5

8

Nanomaterial-mediated photothermal therapy (PTT) has emerged as a potential means of

9

tumor ablation because of its high specificity, minimal invasiveness and limited side effects.4

10

Over the last decade, extensive efforts have been undertaken to develop a large number of light-

11

absorbing nanomaterials as photothermal agents for the treatment of cancer, including various

12

inorganic nanomaterials (e.g., various gold nanostructures,6-8 nanocarbons,9-10 and transition

13

metal sulphide/oxide nanomaterials11-13) and organic dye-based counterparts.14-15

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However, growing evidence suggests that photothermal nanotherapeutics remains sub-optimal,

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and is still far from clinical practice. One major obstacle facing nanomaterial-mediated PTT is

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cancer invasion and metastasis. In other words, it is of great importance to eliminate residual

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cancer cells left behind after tumor ablation. Therefore, some studies have been carried out to

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develop photothermal/chemo combination therapy to both kill the primary tumor and remove

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residual tumor cells.16-18 Despite the benefits of combination treatment, chemotherapy-associated

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toxicity cannot be ignored in the development of combined photothermal-chemotherapy.

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In this study, we develop an organic photothermal nano-agent (Cy5.5-MSA-G250) by co-

22

conjugating coomassie brilliant blue (G250) and cyanine 5.5 (Cy5.5) with mouse serum albumin

23

(MSA). As expected, the prepared Cy5.5-MSA-G250 nanoparticles (NPs) can efficiently kill


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cancer cells in vitro and in vivo by PPT. Most surprising, we accidentally discover that Cy5.5-

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MSA-G250 can specifically kill tumor cells alone but not damage normal cell. Compared with

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cytarabine, an FDA-approved anticancer drug, Cy5.5-MSA-G250 NPs demonstrated stronger

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cytotoxicity against cancer cells, but lower toxicity to normal cells. Owing to their characteristic

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dual functionality, the new designed Cy5.5-MSA-G250 is able to beat cancer like a boxer with

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two successive blows: (1) PTT to destroy tumors; (2) chemotherapy to kill residual cancer cells

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without damaging normal cells. In addition, direct visualization, high passive accumulation in

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tumors, good biodegradation, and efficient excretion through faeces and urine further make

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Cy5.5-MSA-G250 nanoparticles more attractive for in vivo applications. On the basis of these

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findings, we believe that this novel nano-agent has great potential in photothermal-

11

chemotherapy.

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EXPERIMENTAL SECTION

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2.1 Synthesis and characterization of Cy5.5-MSA-G250 NPs

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A synthesis procedure is described as follows: 100 mg of mouse serum albumin (MSA,

16

Equitech-Bio) solution (4 mg mL-1 in distilled water, 25 mL) was mixed with 62.5 mg of G250

17

(Sigma Aldrich) solution (2.5 mg mL-1 in DMSO, 25 mL), and kept stirring at room temperature

18

overnight. The consultant products were ultra-filtered (MWCO: 50 KD) and washed with 50 mL

19

of 30% DMSO solution twice to remove unbounded G250. Afterward, MSA-G250 were

20

dispersed with PBS buffer and filtered with a 0.22 µm membrane. To obtain Cy5.5-MSA-G250

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NPs, 50 mg of MSA-G250 solution were dispersed in 10 mL of 0.1 M sodium bicarbonate

22

solution, and 1.25 mg of Cyanine5.5 NHS ester (Cy5.5-NHS, Lumiprobe Corporation) solution

23

(5 mg mL-1 in DMSO, 250 µL) was added under stirring at room temperature. The mixture was


4

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kept stirring overnight, and then dialyzed for 48 h. The obtained Cy5.5-MSA-G250 solution

2

were filtered with a 0.22 µm membrane and stored at 4 ºC for future use. The size distribution of

3

NPs was measured using a Particle Sizer and Zeta Potential Analyzer NanoBrook Omni

4

(Brookhaven, USA), and the morphology of NPs was characterized by transmission electron

5

microscopy (TEM, JEM-2100, JOEL). UV-Vis-NIR spectra were recorded by a UV

6

spectrophotometer (UV2600, Shimadzu, Japan). A fluorescence spectrum was obtained using a

7

Lumina Fluorescence Spectrometer (Thermo Scientific, USA).

8 9

2.2 Photothermal property evaluation of Cy5.5-MSA-G250 NPs

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The photothermal properties of Cy5.5-MSA-G250 NPs were evaluated under different

11

conditions. The photothermal heating curves (660 nm, 1.27 W cm-2) were recorded by an IR

12

thermal camera (Fluke, USA). The photothermal conversion efficiencies (η) of Cy5.5-MSA-

13

G250 NPs, MSA-G250, and ICG were measured according to the previous literatures.19 The

14

detailed procedure is described as follows: the sample solutions with absorption value of 1 at 660

15

nm were first irradiated by a 660 nm with the same power density (1.27 W cm-2), and then cool

16

down to the room temperature. The temperature changes of Cy5.5-MSA-G250 NPs, MSA-G250,

17

and ICG solutions were recorded as a function of time under continuous irradiation of the 660

18

nm laser until the sample solutions reached a steady-state temperature. The photothermal

19

conversion efficiency was calculated according to the formula:

20 21 22

hS(Tmax﹣Tsur)﹣Qs η=

I(1﹣10-Aλ)


5


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where η is photothermal conversion efficiency, h is the heat transfer coefficient, S is the surface

2

area of the sample cuvette, Tmax is the steady-state temperature, Tsur is the temperature of the

3

surroundings, Qs is the baseline heat associated with the light absorbance of the solvent, I is the

4

incident laser power, and Aλ is the absorbance at a wavelength of 660 nm of the samples (in our

5

experiment, Aλ is 1).

6

To further evaluate the photostability of Cy5.5-MSA-G250 NPs, periodic laser on/off control

7

with 660 nm light at the power of 1.27 W cm-2 was employed. The temperature was recorded by

8

an IR thermal camera during six cycles of on/off laser irradiation. UV-Vis-NIR spectra were also

9

applied to study the photostability of Cy5.5-MSA-G250 NPs. The UV-Vis-NIR spectra of ICG,

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MSA-G250 and Cy5.5- MSA-G250 NPs were measured with or without six on/off cycles of

11

irradiation.

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2.3 In vitro photothermal ablation of tumor cells using Cy5.5-MSA-G250 NPs

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In vitro photothermal cytotoxicity of Cy5.5-MSA-G250 NPs was measured by performing

15

calcein-AM and propidium iodide (PI) fluorescent (live-dead) staining assay (Invitrogen,

16

Eugene, OR, USA) on 4T1 cells. Cells were seeded into a 48-well cell culture plate at a number

17

of 3×104 cells per well, and were cultured at 37 °C with 5% CO2 for 24 h; different

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concentrations of Cy5.5-MSA-G250 NPs were then added to the wells (the final concentrations

19

of G250 in different samples were 0, 3, 6 and 12 µg mL-1, respectively). The cells were

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subsequently incubated for 24 h. Afterward, the cells were exposed to an NIR laser (660 nm,

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1.27 W cm-2) for 0, 2, 5 and 8 min, respectively. Then calcein-AM and PI were added to the

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wells, and the final concentrations of calcein-AM and PI were 2 and 4 µM, respectively. After a

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45-minute incubation at 37 °C, cells were imaged by an Olympus confocal imaging system


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(Olympus FV100; Olympus Corporation) using excitation wavelengths of 488 nm (calcein AM)

2

and 533 nm (PI).

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2.4 Tumor cell-selective cytotoxicity assay of Cy5.5-MSA-G250 NPs and molecular

4

mechanism analysis

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The cytotoxicity was determined by Cell Counting Kit-8 (CCK-8, Beyotime) assay. The cells

6

were seeded into a 96-well plate and incubated at 37 °C for 24 h. Then, the various drug

7

formulations with G250 content ranging from 1.5 to 12 µg mL-1 were added. The cells were

8

incubated for another 24 h or 48 h. Afterward, the medium was replaced by CCK-8 solution. The

9

plate was incubated for 1 h at 37 °C in the incubator and then the absorbance at 450 nm was

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measured using a multiwell microplate reader (Biotek Instruments). The relative viability was

11

calculated

12

ODBlank)/(ODControl-ODBlank)×100%.

with the following formula: relative cell viability (%) = (ODTreatment-

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Cell apoptosis was tested using Cell Apoptosis Kit with Annexin-V FITC and PI (Thermo

14

Fisher Scientific). 4T1 and BALB/3T3 cells were seeded into six-well plates at a density of

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5×105 cells per well and incubated at 37 °C for 24 h. Then, the culture medium was replaced

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with fresh complete medium containing MSA, G250, MSA-G250 and Cy5.5-MSA-G250 NPs,

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respectively (MSA: 300 µg mL-1; others: 12 µg mL-1 of G250 content). After 24 h or 48 h, the

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cells were harvested and washed twice with cold PBS, and then resuspended in 500 µL of

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Binding Buffer. Then 5 µL of Annexin-V FITC and 10 µL of PI were added and incubated for 10

20

min

21

the CyFlow ® Cube 6 flow cytometry (Sysmex Partec, Germany). The data were evaluated using

22

FCS Express 5 Flow Cytometry Software.

at

room

temperature

in

the

dark.

Finally,


the

cells

were

analyzed

by

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Six types of tumor cells (4T1 mouse mammary carcinoma cells, LLC mouse lung carcinoma

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cells, Hepa1-6 mouse liver carcinoma cells, MFC mouse gastric carcinoma cells, HeLa human

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cervical cancer cells and MGC80-3 human gastric carcinoma cells) and four types of normal

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cells (BALB/3T3 mouse embryo fibroblast cells, L-929 mouse connective tissue cells, HFF-1

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human foreskin fibroblast cells and WI-38 human embryonic lung fibroblast cells) were selected

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to determine selective tumor cytotoxicity of Cy5.5-MSA-G250 NPs. All the cell lines were

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acquired from Cell Resource Centre of Shanghai Institutes for Biological Sciences of the

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Chinese Academy of Sciences. Tumor cells were maintained in RPMI 1640 or DMEM medium

9

with 10 % FBS (Gibco, Thermo Fisher Scientific), and normal cells were maintained according

10

to each instruction. All cells were cultured in tissue culture flasks in a humidified incubator at 37

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ºC in an atmosphere of 95% air and 5% carbon dioxide. The cells were seeded into a 96-well

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plate and incubated at 37 °C for 24 h; Cy5.5-MSA-G250 NPs were subsequently added with the

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final concentration of G250 ranging from 0 to 12 µg mL-1. After 48 h of incubation, the

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viabilities of the cells were tested by CCK-8 assay.

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Cell uptake of Cy5.5-MSA-G250 NPs was investigated. The cells were seeded into specified

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wells, incubated at 37 °C for 24 h, and then treated with NPs at different concentrations for 2

17

hours. Then the cells were collected and analyzed by the CyFlow ® Cube 6 flow cytometry.

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Intracellular reactive oxygen species (ROS) were detected using Cellular ROS/Superoxide

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Detection Assay (Abcam, UK). After being cultured with Cy5.5-MSA-G250 NPs, cells were

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incubated with 100 mM 2’,7’-dichlorofluorescein diacetate (DCFH-DA) (37 °C for 30 min).

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Oxidation of DCFH via intracellular ROS turns the molecule into fluorescent 2’,7’-

22

dichlorofluorescein (DCF). DCF was excited at 488 nm and emission was recorded with a


8

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525/30 bandpass filter using the CyFlow ® Cube 6 flow cytometry. Data were collected from

2

10 000 cells from at least three wells per condition.

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RNA sequencing was performed as follows: 4T1 cells were treated with or without Cy5.5-

4

MSA-G250 NPs treatment for 24 hours, and then RNA was extracted. Sequencing and analysis

5

were conducted by RiboBio (Guangzhou, China). Quantitative analysis of mRNA was performed

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by qPCR; the cDNA was synthesized from 2 mg of RNA using SuperScript III RT (Thermo

7

Fisher Scientific). Nine differentially regulated genes including CCND1, MCM3-7, Fen 1, Rpa

8

and PCNA, were validated by qPCR with SYBR™ Green PCR Master Mix (Applied

9

Biosystems™) on a ProFlex PCR System (Applied Biosystems™) using the primers listed in

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Supplementary Table 1. GAPDH and β-actin RNA were used as housekeeping loading controls

11

to normalize gene expression using the ∆∆Ct method.

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2.5 In vivo evaluation of photothermal therapeutic efficacy

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Female BALB/c mice with body weights of about 20 g were obtained from Tianqin

15

Biotechnology Co., Ltd, Changsha, China, and housed under protocols approved by Animal

16

Experimentation Ethics Committee of our institution. For in vivo photothermal therapy, the

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tumor-bearing mice were obtained by subcutaneous injection of 1 × 106 4T1 cells on the back of

18

each female BALB/c mouse. When the tumor sizes reach ~5 mm in diameter, the mice were

19

divided into three groups randomly (n = 7 per group): (1) Blank group (PBS only); (2) PBS +

20

laser group; and (3) Cy5.5-MSA-G250 + laser group. For groups 2 and 3, the 8-min irradiation

21

was carried out at 24th h after i.v. injection of PBS or Cy5.5-MSA-G250 (dose: 8 mg kg-1 of

22

G250; laser: 660 nm, 1.27 W cm-2). During irradiation, temperature variation of mice at different


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time points was monitored by an IR thermal camera. Tumor volumes were measured at 3-day

2

intervals by the Peira TM 900-a tumor measurement device (Peira, Belgium).

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2.6 In vivo evaluation chemotherapeutic efficacy

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For in vivo chemotherapy, tumor models were generated by i.v. injection of 1×105 luciferase-

6

expressed 4T1 (4T1-Luc) cells. The mice were randomly divided into four groups, including (1)

7

Blank group (PBS only), (2) Cy5.5-MSA-G250 group, (3) MSA-G250 group, (4) G250 group.

8

Then mice were treated by i.v. injection of 8 mg kg-1 of different medicines (dosage count by

9

G250) or PBS buffer. To in vivo detect the 4T1-Luc cells at the 10th, 14th and 21th day after

10

different treatments, mice were intraperitoneally injected 300 mg kg-1 of D-fluorescein

11

potassium, and the bioluminescence signals were detected using IVIS lumina II imaging system

12

(Caliper Life Sciences).

13 14

2.7 Distribution and metabolism of Cy5.5-MSA-G250 NPs

15

For biodistribution study, female BALB/c mice bearing 4T1 tumors were sacrificed at specified

16

time points after i.v. injection of Cy5.5-MSA-G250. Major organs and tissues were collected and

17

the fluorescence signals were recorded by IVIS lumina II imaging system. To determine the

18

blood circulation of Cy5.5-MSA-G250 NPs, 10 µL of blood was collected from the tail vein of

19

each mouse at different time intervals after i.v. injection of Cy5.5-MSA-G250 NPs. Fluorescent

20

images were captured by IVIS lumina II imaging system. The urine and faeces samples were

21

collected by metabolic cages at different time points after injection of Cy5.5-MSA-G250 NPs,

22

and the fluorescence signals were then recorded by IVIS lumina II imaging system.

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2.8 Histological analysis

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For histological analysis, organ samples (including heart, liver, spleen, lung and kidney) from

3

killed mice were harvested on the 14th day post-treatment of Cy5.5-MSA-G250 NPs (dose: 8 mg

4

kg-1), and fixed in 10% formalin. Then the samples were embedded with paraffin for standard

5

histological hematoxylin and eosin (H&E) staining and photographing. Age-matched healthy

6

untreated mice were used as the control.

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2.9 Data analysis

9

Data were presented as mean ± s.d. The in vitro experiments were performed in three

10

independent experiments with at least three repetitions for each condition. Statistical analysis of

11

the samples was performed using Student’s t-test, and P values ＜0.05 were considered to be

12

statistically significant (*P＜0.05, **P＜0.01 and ***P＜0.001).

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RESULTS AND DISCUSSION

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3.1 Synthesis and characterization of Cy5.5-MSA-G250 NPs

16

The procedure for the preparation of Cy5.5-MSA-G250 NPs is illustrated in Figure 1a. We first

17

prepared MSA-G250 conjugates by simply mixing MSA and G250 solutions, because G250 can

18

rapidly bind with proteins via hydrophobic interactions and van der Waals’ forces.20 After

19

incubation with Cy5.5-NHS ester, Cy5.5-MSA-G250 was then obtained through the covalent

20

conjugation of Cy5.5 to MSA-G250. The morphology of Cy5.5-MSA-G250 NPs was measured

21

by typical transmission electron microscopy (TEM). As shown in Figure 1b and 1c, self-

22

assembled Cy5.5-MSA-G250 has a nano-sized structure with an average diameter of 27.5 ± 5.7

23

nm, much larger than that of MSA and MSA-G250. Consistent with the results of TEM analysis,


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the average hydrodynamic diameter of Cy5.5-MSA-G250 NPs (analyzed by number) is

2

approximately 36.04 nm with a polydispersity index (PDI) of 0.259, which is larger than that of

3

MSA and MSA-G250 (6.48 and 12.92 nm, respectively, Figure S1). Both TEM analysis and size

4

distribution results indicated Cy5.5 is a critical agent to enable the formation of larger NPs, and

5

the possible reason is that the introduced Cy5.5 dye increases the hydrophobicity of Cy5.5-MSA-

6

G250 NPs. In addition, solutions of the prepared Cy5.5-MSA-G250 NPs showed quite stable,

7

and no precipitation was observed in PBS and serum-containing cell culture for 2 weeks (Figure

8

S2).

9

We next studied the optical behaviors of Cy5.5-MSA-G250 NPs. As shown in Figure 1d, the

10

absorbance peak of MSA-G250 conjugates is slightly red-shifted to 618 nm compared with

11

G250, with an absorption peak located at 598 nm, suggesting a successful binding between MSA

12

and G250. After conjugation with Cy5.5, Cy5.5-MSA-G250 NPs exhibit two characteristic

13

absorption peaks of Cy5.5 with a slight red shift (637 and 691 nm), indicating the successful

14

conjugation between MSA-G250 and Cy5.5. The mass percentages of G250 and Cy5.5

15

calculated by UV-Vis-NIR spectrophotometry are ~4% and 2.5%, respectively. In the

16

fluorescence emission spectrum (Figure 1e), Cy5.5-MSA-G250 NPs display the characteristic

17

fluorescence emission peak of Cy5.5 with a maximal wavelength at 700 nm, and this

18

fluorescence property enables visualization of Cy5.5-MSA-G250 NPs in vivo. On these bases,

19

we further explored the application of Cy5.5-MSA-G250 NPs in the following study.


12

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1 2

Figure 1. Synthesis and characterization of Cy5.5-MSA-G250 NPs. (a) Schematic illustration of

3

the synthesis of Cy5.5-MSA-G250 NPs. (b) TEM images of MSA, MSA-G250, and Cy5.5-

4

MSA-G250 NPs. (c) Statistical analysis of the diameter of 100 NPs determined by TEM. (d)

5

UV-Vis-NIR absorption spectra of Cy5.5, G250, MSA, MSA-G250 and Cy5.5-MSA-G250 NPs.

6

(e) Fluorescence emission spectrum of Cy5.5-MSA-G250 NPs under excitation at 660 nm.

7 8

3.2 Photothermal properties of Cy5.5-MSA-G250 NPs


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Page 14 of 37

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Since G250 has strong absorption in the red light region, and G250-based nanomaterials show

2

good performance in red-light-based photothermal therapy,21 we infer that MSA-G250 and

3

Cy5.5-MSA-G250 NPs have the capacity to convert red light energy into heat. To confirm our

4

speculation, temperatures of MSA-G250 and Cy5.5-MSA-G250 solutions under irradiation with

5

a red light laser (660 nm, 1.27 W cm-2) were monitored using an IR thermal camera (Fluke,

6

USA). As expected, the temperatures of MSA-G250 and Cy5.5-MSA-G250 solutions

7

respectively increased to 75 and 62 °C after 10-min laser irradiation, while the temperature of

8

indocyanine green (ICG, an FDA-approved NIR fluorescent agent) solution increased to only 54

9

°C (Figure 2a). In contrast, the temperature of pure water showed a negligible change under the

10

same conditions. To further assess the photothermal properties of these materials, the

11

photothermal conversion efficiency (η) was calculated according to the quantification method

12

developed by Roper et al.19 (the detailed calculations are presented in Figure 2b and Figure S3).

13

The η value of Cy5.5-MSA-G250 NPs was found to be 38.8%, which is lower than the value of

14

MSA-G250 (η = 49.7%). This can be attributed to the low photothermal conversion efficiency of

15

Cy5.5, which also has significant absorption at 660 nm. Nevertheless, the photothermal

16

performance of Cy5.5-MSA-G250 NPs is better than that of ICG (η = 30%) and many promising

17

inorganic PTT nano-agents (for example, 21% for Au nanorods,22 18.3% for PEGylated Bi2S3

18

nano-urchins,23 28.1% for Bi2S3 nanorods,13 and 28.5% for PEG-dBSA-RuS1.7 nanoclusters24).

19

High photothermal stability is also extremely important for PTT applications. Consequently,

20

the photothermal stability of MSA-G250 and Cy5.5-MSA-G250 NPs was evaluated via the

21

periodic laser on/off control with 660 nm red light. After six cycles of laser on/off irradiation, the

22

temperature elevation of both MSA-G250 and Cy5.5-MSA-G250 solutions showed negligible

23

change, whereas the photothermal effect of ICG deteriorated during the experiment (Figure 2c).


14

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1

Furthermore, the photostability of Cy5.5-MSA-G250 NPs was also evaluated by UV-Vis-NIR

2

spectroscopy. As shown in Figure S4a and S4b, the absorbance of MSA-G250 solution at 660

3

nm after laser irradiation has no obvious change, while that of ICG was greatly weakened. Note

4

that the absorbance of Cy5.5-MSA-G250 decreased by 50%, possibly as a result of the decline of

5

the absorbance of Cy5.5 under irradiation (Figure S4c). Since light in the region of 650-900 nm

6

has deep penetration in tissues and minimal absorption by hemoglobin and water in normal

7

tissues25 and Cy5.5-MSA-G250 NPs show favorable photothermal properties, we believe that the

8

prepared NPs have great potential as an effective photothermal agent. In addition, the

9

fluorescence intensity of Cy5.5-MSA-G250 NPs still decreases with the irradiation time.

10

However, compared with Cy5.5 dye, Cy5.5-MSA-G250 NPs display a relatively slow

11

photobleaching (Figure S5), possibly because of the protection of MSA and G250. Thus, even

12

under consecutive laser irradiation, Cy5.5-MSA-G250 NPs still retain the capacity for

13

fluorescence tracing.

14 15

3.3 In vitro PTT performance of Cy5.5-MSA-G250 NPs

16

To test the tumor-killing capacity of Cy5.5-MSA-G250 NPs as a photothermal agent in vitro,

17

4T1 cells were incubated with various concentrations of Cy5.5-MSA-G250 NPs for 24 h, and

18

then exposed to the laser for 0, 2, 5, and 8 min, respectively. The effective photothermal ablation

19

of 4T1 cells induced by 660 nm irradiation was detected through a live/dead assay using calcein-

20

AM and propidium iodide (PI). As shown in Figure 2d, an obvious decrease in cell viability was

21

observed, due to the prolonged irradiation time and the increased concentration of Cy5.5-MSA-

22

G250 NPs. In contrast, the cells without NP incubation were not killed by laser irradiation.

23

Interestingly, 4T1 cell death was found without irradiation after incubation with a high


15


Page 16 of 37

1

concentration of Cy5.5-MSA-G250 NPs (12 µg mL-1), which demonstrates the intrinsic toxicity

2

of the NPs to 4T1 cells. Motivated by that finding, we then investigated the cytotoxicity of

3

Cy5.5-MSA-G250 NPs in detail.

4 5

Figure 2. Photothermal performance of Cy5.5-MSA-G250 NPs. (a) Temperature elevation of

6

pure water, ICG, MSA-G250, and Cy5.5-MSA-G250 NPs as a function of irradiation time with a

7

power density of 1.27 W cm-2 under 660 nm laser irradiation (concentrations of all samples,

8

except distilled water, were adjusted to have absorbance value of 1 at 660 nm). (b) Plot of


16

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1

cooling time versus negative natural logarithm of the temperature driving force obtained from

2

the cooling stage as shown in Figure S2 (Supporting Information). The time constant for heat

3

transfer of the system (τs) is determined to be 260.3. (c) The temperature changes of ICG, MSA-

4

G250, and Cy5.5-MSA-G250 solutions for six laser on/off cycles. (d) Live/dead staining assays

5

of 4T1 cells treated with different concentrations of Cy5.5-MSA-G250 NPs for 24 h and then

6

irradiated by a 660 nm laser at 1.27 W cm-2 for 0, 2, 5, and 8 min, respectively. All the confocal

7

images share a same scale bar of 100 µm.

8 9

3.4 Tumor cell-selective cytotoxicity and molecular mechanism analysis of Cy5.5-MSA-

10

G250 NPs

11

To systematically investigate the cytotoxicity of Cy5.5-MSA-G250 NPs, we evaluated the

12

viability of the tumor cells (4T1 cells) and the normal cells (BALB/3T3 cells) after incubation

13

with the NPs using a cell counting kit-8 (CCK-8) assay. As shown in Figure 3a, Cy5.5-MSA-

14

G250 NPs (G250 content of 12 µg mL-1) induced ~55% and almost 100% cellular viability

15

inhibition in the 4T1 cells after 24 and 48 h of incubation, respectively, and exhibited much

16

stronger cytotoxicity than cytarabine, a clinically used anticancer drug. Meanwhile, The Cy5.5-

17

MSA-G250 NPs had a negligible impact on the proliferation of BALB/3T3 cells after 24 h and

18

48 h of incubation, even at a high G250 dose of 12 µg mL-1. By contrast, high cellular viability

19

inhibition was discovered after BALB/3T3 cells were incubated with cytarabine. Coherently, the

20

toxicity of Cy5.5-MSA-G250 NPs against cancer cells was also observed in the apoptosis

21

analysis. With the increase of Cy5.5-MSA-G250 NP concentration, more and more 4T1

22

cells undergoing apoptosis were scored (Figure S6). A significant apoptosis of 4T1 cells (~18%)

23

was induced by Cy5.5-MSA-G250 NPs after 24 h incubation, and nearly all the tumor cells (>

24

97%) were apoptotic after 48 h incubation with the NPs (Figure 3b and Figure S7, G250 content


17


Page 18 of 37

1

of 12 µg mL-1). In contrast, obvious apoptosis was not observed in NP-treated BALB/3T3 cells.

2

As illustrated in Figure 3b, more than 80% of BALB/3T3 cells were still alive, even after

3

incubation with Cy5.5-MSA-G250 NPs for 48 h. These observations indicate that Cy5.5-MSA-

4

G250 NPs have a strong cancer-cell-selective killing capacity. Moreover, just like Cy5.5-MSA-

5

G250 NPs, MSA-G250 also displays strong selective cytotoxicity to tumor cells based on the

6

CCK-8 assay and apoptosis analysis (Figure 3a and b). However, G250 and MSA show

7

significantly less cytotoxicity compared with MSA-G250 and Cy5.5-MSA-G250 NPs. Therefore,

8

we infer that the selective cytotoxic effects on tumor cells predominantly originate from the

9

binding of MSA and G250. To further determine whether the tumor cell-selective cytotoxicity

10

exists in other cell lines, we examined the effects of Cy5.5-MSA-G250 NPs on the viability of

11

various cultured cancer cells (HeLa, MGC80-3, LLC, 4T1, Hepa1-6, and MFC cells) and

12

normal cells (L-929, HFF-1, BALB/3T3, and WI-38 cells). As shown in Figure 3c, after 48 h of

13

incubation, the inhibitory effect on proliferation of all types of tumor cells was enhanced, with an

14

increase in the concentration of Cy5.5-MSA-G250 NPs, while normal cells maintained high

15

viability even after treatment with Cy5.5-MSA-G250 NPs at a maximum G250 dose of 12 µg

16

mL-1. These results confirm that the Cy5.5-MSA-G250 NPs can selectively kill cancer cells.

17

The above findings raise the question: what are the possible mechanisms of tumor cell-

18

selective cytotoxicity of Cy5.5-MSA-G250 NPs? According to the study by Dash et al.,26

19

different cellular uptake amounts of drugs could lead to tumor cell-selective cytotoxicity. To test

20

this hypothesis, we quantitatively examined the cellular uptake of the NPs using flow cytometry.

21

As shown in Figure S8, the 4T1 and BALB/3T3 cells have almost the same fluorescence

22

intensity after internalization of Cy5.5-MSA-G250 NPs, indicating that there is no difference in


18

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1

the cellular uptake between the 4T1 and BALB/3T3 cells. Therefore, the tumor cell-selective

2

cytotoxicity effect is irrelevant to cellular uptake.

3

In addition, there is emerging evidence that malignant cells are more vulnerable to oxidative

4

insults induced by reactive oxygen species (ROS)-generating agents,27 such as piperlongumine,28

5

since cancer cells are always under increased intrinsic oxidative stress. To investigate whether

6

Cy5.5-MSA-G250 NPs cause the rapid accumulation of ROS and then selectively kill tumor

7

cells, the intracellular concentration of ROS in 4T1 cells was measured using the 2’,7’-

8

dichlorofluorescence diacetate (DCFH-DA) fluorescence method. The results show that there is a

9

negligible difference in ROS level between 4T1 cells with or without NP treatment, while the

10

positive control (tert-butyl hydroperoxide, i.e. TBHP) greatly induced ROS generation (Figure

11

S9). The findings indicate that the selective cytoxicity is not caused by rapid accumulation of

12

ROS. Therefore, other mechanisms of tumor cell-selective cytotoxicity of Cy5.5-MSA-G250

13

NPs need to be explored.

14

To better understand how Cy5.5-MSA-G250 NPs selectively kill tumor cells, the differences

15

of mRNA expression between NP-treated and non-treated 4T1 cells were compared using the

16

RNA-Seq technique. Compared with the gene expression of non-treated 4T1 cells, the expression

17

level of 16 mRNAs (a total of 35) related with DNA replication and 20 mRNAs (a total of 125)

18

related with cell cycles were significantly down-regulated in NP-treated 4T1 cells. To confirm

19

the mRNA expression results obtained from RNA-Seq analysis, 9 mRNAs with high abundance

20

and more than 2-fold expression differences were selected from the total of 36 mRNAs, and were

21

validated by real-time quantitative PCR (qPCR). As shown in Figure 3d, the CCND1 mRNA

22

encoding the protein cyclin D1, is dramatically down-expressed by ~14 fold in Cy5.5-MSA-

23

G250 NP-treated 4T1 cells. It has been reported that cyclin D1 plays a key role in the G1-to-S


19


Page 20 of 37

1

transition of cells and the abrogation of its expression may lead to G1 cell cycle arrest.29-32 The

2

results of cell cycle analysis also clearly indicate that Cy5.5-MSA-G250 NP-treated 4T1 cells are

3

arrested in the G1 phase (evidenced by an increase of cells in the G0/G1 phase concomitant with

4

a decrease of cells in the S phase, as shown in Figure S10). Furthermore, the other 8 DNA

5

replication-related mRNAs, including MCM3-7, FEN1, Rpa and PCNA, were also significantly

6

down-regulated in Cy5.5-MSA-G250 NP-treated 4T1 cells, which can greatly influence DNA

7

replication.33-35 In addition, the expression profiles of the 9 mRNAs in the normal cells

8

(BALB/3T3) with or without NP-treatment were also analyzed in order to understand whether

9

this differential expression pattern of mRNAs occurs only in tumor cells. As illustrated in Figure

10

3d, the expression levels of most mRNAs display negligible differences between BALB/3T3

11

cells with and without treatment, and a small part of these mRNAs were even slightly up-

12

regulated after treatment. Based on the above analysis, we infer that selectively inducing cell

13

cycle arrest in G1 phase and inhibiting DNA duplication of tumor cells may be the main

14

mechanism of tumor cell-selective cytotoxicity of Cy5.5-MSA-G250 NPs (refer to the schematic

15

illustration of the main mechanism shown in Figure 3e).


20

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1 2

Figure 3. Tumor cell-selective cytotoxicity of Cy5.5-MSA-G250 NPs and mechanism analysis.

3

(a) In vitro viabilities of 4T1 cells and BALB/3T3 cells incubated for 24 h and 48 h with G250,


21


Page 22 of 37

1

MSA-G250, Cy5.5-MSA-G250 NPs, cytarabine, and MSA at varied concentrations (all the

2

sample concentrations expressed in the content of G250, except that of cytarabine and MSA). (b)

3

Apoptosis analysis of 4T1 cells and BALB/3T3 cells incubated for 24 h and 48 h with G250,

4

MSA-G250, Cy5.5-MSA-G250 NPs, and MSA (MSA: 300 µg mL-1; others: 12 µg mL-1 of G250

5

content). (c) Cellular viabilities of Cy5.5-MSA-G250 NP-treated normal cell lines (WI-38, L-

6

929, HFF-1, and BALB/3T3) and tumor cell lines (HeLa, LLC, Hepa1-6, MFC, 4T1, and

7

MGC80-3). (d) qPCR analysis of CCND1, MCM3-7, FEN1, Rpa, and PCNA mRNA expression

8

in 4T1 and BALB/3T3 cells (Cy5.5-MSA-G250 concentration: 12 µg mL-1 of G250 content). (e)

9

Schematic illustration of the main mechanism of selective antitumor activity.

10

3.5 In vivo PTT for tumor treatment

11

To evaluate the in vivo photothermal therapeutic effect of Cy5.5-MSA-G250 NPs, tumor-bearing

12

mice were prepared by subcutaneous injection of a suspension of 4T1 cells in PBS onto the back

13

of each mouse. When the tumors had grown to ~5 mm in diameter, the mice were divided into

14

three groups (n = 7 per group): (1) Blank (PBS only); (2) PBS + laser; and (3) Cy5.5-MSA-G250

15

+ laser. After being i.v. injected with PBS or Cy5.5-MSA-G250 NPs for 24 h, the mice were

16

exposed to a 660 nm laser at a power density of 1.27 W cm-2 for 8 min. As illustrated in Figure

17

4a, the tumor temperature of the mice injected with the Cy5.5-MSA-G250 NPs rapidly increased

18

to 67.4°C, which is sufficient to eradicate tumors immediately, while the temperature of the

19

blank group (PBS injection) exhibited only a slight increase. Complete tumor elimination was

20

achieved in group 3 (Cy5.5-MSA-G250 + laser) after irradiation, only leaving black scars at the

21

initial tumor sites, whereas the tumors in the other groups kept growing uncontrollably (Figure

22

4b,c). The Cy5.5-MSA-G250 NPs and laser irradiation greatly prolonged the survival of tumor-

23

bearing mice over 60 days (one death per 7 mice). In contrast, all the mice in the other groups


22

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1

died in two months (Figure 4d). These results show the Cy5.5-MSA-G250 NPs possess effective

2

PTT performance in vivo.

3 4

Figure 4. In vivo photothermal therapy. (a) Temperature variation and IR thermal images of

5

tumors with or without Cy5.5-MSA-G250 NP treatment during irradiation. (b) Photos of mice 14

6

days after different treatments. (c) Tumor volume growth curves of different groups of mice after


23


Page 24 of 37

1

various treatments. Error bars are based on standard error of the mean. (d) Survival curves of

2

mice after various treatments over a span of two months.

3

3.6 Preventing tumor metastasis by chemotherapy

4

Chemotherapy is mainly used to kill residual tumor cells and prevent post-operative recurrence

5

and metastasis.36-37 On account of the excellent tumor cell-selective cytotoxicity of Cy5.5-MSA-

6

G250 NPs, we used them as chemotherapeutic drugs to investigate their anti-metastatic abilities.

7

In this study, a total of 1× 105 4T1-luciferase cells were introduced by intravenous (i.v.) injection

8

into the lateral tail vein of each BALB/c mouse. Then the mice were randomly divided into four

9

groups (n = 6 per group); three groups of mice were injected with G250, MSA-G250, and Cy5.5-

10

MSA-G250 NPs respectively (the groups with the same G250 content of 8 mg kg-1) three times

11

at 3-day intervals, and the blank group of mice were injected with PBS (Figure 5a). The in vivo

12

localization of cancer cells was imaged via the injection of a solution of D-luciferin potassium

13

salt (a water-soluble chemiluminescent luciferase substrate). As shown in Figure 5b, Cy5.5-

14

MSA-G250 NP-treated mice showed significantly reduced tumor proliferation, as quantified by

15

the considerably lower photon flux values compared with untreated controls at day 10, 14, and

16

21. Even at day 21, tumor metastases were not detected in four NP-treated mice (a total of 6).

17

Since 4T1 cells have a high propensity to develop lung metastasis, we further calculated the

18

photon flux values in the lungs. The photon flux value of the NP-treated group was 150 times

19

lower than that of the untreated control group, suggesting that the prepared Cy5.5-MSA-G250

20

NPs exert a significant anti-metastatic effect (Figure 5c). These results further validate that the

21

Cy5.5-MSA-G250 NPs can effectively eliminate residual tumor cells and inhibit tumor

22

metastasis in vivo.

23


24

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1

2 3

Figure 5. In vivo inhibition of cancer cell invasion and metastasis by Cy5.5-MSA-G250 NPs. (a)

4

Schematic illustration of the experimental setup: mice were injected with G250, MSA-G250,

5

Cy5.5-MSA-G250 NPs, and PBS, respectively. (b) 4T1-luciferase cell growth was monitored by

6

bioluminescence imaging at day 10, 14, and 21 after tumor cell injection. (c) Quantitative lung

7

luminescence values of different groups of mice treated with G250, MSA-G250, Cy5.5-MSA-

8

G250 NPs and PBS, respectively.


25


Page 26 of 37

1

3.7 Biodistribution and metabolism of Cy5.5-MSA-G250 NPs

2

Before the NPs can be ready for clinical translation, a detailed survey of their in vivo behavior

3

should be carried out. Therefore, we investigated the biodistribution and metabolism of Cy5.5-

4

MSA-G250 NPs by near-infrared fluorescence (NIRF) imaging. First, we calculated the blood

5

circulation time of NPs by quantifying the fluorescence intensity of blood samples collected at

6

different time intervals after i.v. injection with Cy5.5-MSA-G250. As shown in Figure 6a,

7

Cy5.5-MSA-G250 NPs exhibit a relatively long blood circulation time (circulation half-life: ~8

8

h), which can be of great help for the tumor accumulation of Cy5.5-MSA-G250 NPs.38 The in

9

vivo imaging of tumors proved that Cy5.5-MSA-G250 NPs can be enriched in tumors. A strong

10

fluorescence signal was detected in tumors at 1 h post injection of Cy5.5-MSA-G250 NPs, and

11

the fluorescence intensity displayed a time-dependent increase within 24 h after injection (Figure

12

S11a). In addition, the tumor sites appeared as subcutaneous nodules with darker colors at 24 h

13

after injection, which was regarded as further evidence of effective tumor accumulation of NPs

14

(Figure S11b).

15

The metabolism of Cy5.5-MSA-G250 NPs was studied through the ex vivo fluorescence

16

measurement of organs, tissues, and excreta at different time points. In accordance with the in

17

vivo imaging data, strong fluorescence signals were observed in tumors, and the signals reached

18

peak levels at 24 h after i.v. injection with the NPs (Figure 6b), confirming the high tumor

19

accumulation of Cy5.5-MSA-G250 NPs in vivo. Notably, compared with other NPs, such as

20

RuS1.7 nanoclusters24 and VS2@lipid-PEG nanostructures,39 much fewer Cy5.5-MSA-G250 NPs

21

were accumulated in the spleen (Figure 6b), a main immune organ, suggesting that the NPs may

22

have the ability to escape macrophage clearance in vivo. The biodistribution of Cy5.5-MSA-

23

G250 NPs in excretory organs is also illustrated in Figure 6b. At 2 h post-i.v. injection, the


26

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1

fluorescence signals in the liver and intestine reached their peaks, and then decreased gradually

2

(Figure 6b). In the meantime strong fluorescence signals, peaking at 4 h, were detected in faeces

3

(Figure 6d). These results indicate that Cy5.5-MSA-G250 NPs can be metabolized in the liver

4

and then excreted through the digestive tract. In addition, high fluorescence signals were

5

observed in kidneys over a span of 3 days after NP-i.v. injection (Figure 6b). Correspondingly,

6

the urine displayed strong fluorescence. It should be mentioned that it took a long time (about 5

7

h) before a high fluorescence intensity could be detected in urine after i.v. injection (Figure 6c),

8

suggesting that the Cy5.5-MSA-G250 NPs may be degraded into small fragments before being

9

excreted through the urine.

10

To further understand the clearance mechanism of Cy5.5-MSA-G250 NPs, we explored their

11

biodegradable performance in vitro. The NPs were incubated with papain (a lysosomal cathepsin

12

B-like protease) for 6 h at 37 °C, pH 6.0 in order to simulate lysosomal degradation.40 As shown

13

in Figure 6e, a clear fluorescent band was observed in the electrophoretogram of Cy5.5-MSA-

14

G250 NPs, whereas no corresponding fluorescent band was observed in the electrophoretogram

15

of enzyme-digested Cy5.5-MSA-G250 NPs, indicating the degradation of Cy5.5-MSA-G250

16

NPs into small fragments. The biodegradable performance of Cy5.5-MSA-G250 NPs was further

17

verified by an ultrafiltration analysis using membranes with a cut-off of 50 KD. Without papain

18

treatment, Cy5.5-MSA-G250 NPs were cut off by the membrane, and weak fluorescence was

19

detected in the ultra-filtered fluid. However, Cy5.5-MSA-G250 NPs, treated by papain, were

20

degraded into fragments and passed through the membrane, and a strong fluorescence signal was

21

captured in the ultra-filtered fluid (Figure 6f). Based on the above results, we propose a possible

22

in vivo behavior of Cy5.5-MSA-G250 NPs (Figure 6g): (1) Cy5.5-MSA-G250 NPs can be

23

accumulated substantially in tumors via the enhanced permeability and retention (EPR) effect;


27


Page 28 of 37

1

(2) Cy5.5-MSA-G250 NPs can be degraded in liver, and excreted through faeces and urine; (3)

2

Cy5.5-MSA-G250 NPs may escape splenic macrophage engulfment, since few NPs remain in

3

spleen.

4 5

Figure 6. Distribution, metabolism and excretion of Cy5.5-MSA-G250 NPs. (a-d) The

6

fluorescent images of blood (a), organs and tissues (b), urine (c), and faeces (d) after i.v.

7

injection with Cy5.5-MSA-G250 NPs at different time points. (e) The electrophoretogram of

8

Cy5.5-MSA-G250 NPs with or without protease treatments. (f) The fluorescence intensity of the

9

ultra-filtered fluid of Cy5.5-MSA-G250 NP solution treated with or without papain (molecular


28

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1

weight cut-off (MWCO): 50 KD). (g) Schematic illustration of the in vivo degradation and

2

clearance processes of Cy5.5-MSA-G250 NPs.

3

3.8 In vivo toxicity of Cy5.5-MSA-G250 NPs

4

Since toxicity is an essential issue for clinical applications, we conducted a series of experiments

5

to assess the in vivo toxicity of Cy5.5-MSA-G250 NPs. First, a hemolytic assay of mouse red

6

blood cells (RBCs) was performed using deionized water and PBS as positive and negative

7

controls. Almost no hemolysis of RBCs was observed, even with a high concentration of Cy5.5-

8

MSA-G250 NPs (12 µg mL-1 of G250 content) after 3 h of incubation (Figure S12), suggesting

9

Cy5.5-MSA-G250 NPs have almost no side-effects on RBCs. Next, the growth and behavior of

10

normal mice after i.v. injection with Cy5.5-MSA-G250 NPs were carefully monitored. There

11

were no observed adverse effects on the mice, and they resumed normal activities (i.e., eating,

12

drinking, grooming) during the entire experimental period. In addition, normal mice injected

13

with Cy5.5-MSA-G250 NPs exhibited a similar body weight to that of mice without treatment,

14

indicative of unnoticeable toxic side effects. Finally, histology analysis was carried out on the

15

14th day after i.v. injection of Cy5.5-MSA-G250 NPs (8 mg kg-1 by G250 each time, 3 times at

16

3-day intervals) by hematoxylin and eosin (H&E) stain. As shown in Figure S13, no organ

17

damage or inflammatory lesion was evident compared to the controls. Based on the low residual

18

levels of NPs in the body after 7 days and the results of the above toxicity testing, it is reasonable

19

to believe that the prepared Cy5.5-MSA-G250 NPs will not cause severe in vivo toxicity.

20 21

CONCLUSIONS

22

In summary, we have developed a dye-based nano-agent for photothermal-chemo therapy.

23

Besides their excellent photothermal performance in vivo and in vitro, the as-prepared Cy5.5-


29


Page 30 of 37

1

MSA-G250 NPs per se showed cancer-specific chemotherapeutic effect. Compared with the

2

clinically used anticancer drugs, the NPs demonstrated stronger killing effect against cancer cells

3

but negligible cytotoxicity to normal cells. And the NPs can effectively eliminate residual tumor

4

cells and prevent metastasis in vivo. Selectively inducing G1 cell cycle arrest and inhibition of

5

DNA duplication may be the main mechanism of the tumor cell-selective cytotoxicity of Cy5.5-

6

MSA-G250 NPs. In addition, direct visualization, high passive accumulation in tumors, good

7

biodegradability, and efficient excretion through faeces and urine further make Cy5.5-MSA-

8

G250 NPs attractive for in vivo applications. On the basis of these findings, we believe that

9

Cy5.5-MSA-G250 is promising nano-drug for photothermal-chemo therapy.

10 11

ASSOCIATED CONTENT

12

Supporting Information

13

The hydrodynamic size distribution of MSA, MSA-G250, and Cy5.5-MSA-G250 NPs; The

14

photo of Cy5.5-MSA-G250 NPs dispersed in H2O, PBS, and medium (RPMI1640+10% FBS),

15

respectively; Temperature change in solutions of Cy5.5-MSA-G250, MSA-G250, and ICG when

16

irradiated with a 660 nm laser (1.27 W cm-2); UV-Vis-NIR absorbance spectra of ICG, MSA-

17

G250, and Cy5.5-MSA-G250 before and after 5 cycles of heating; Fluorescence intensity of

18

Cy5.5-MSA-G250 after laser irradiation for 0, 2, 5 10 min and Cy5.5 after laser irradiation for 0,

19

2 min; Apoptosis assays in 4T1 cells incubated for 24 h and 48 h with a series of concentrations

20

of Cy5.5-MSA-G250 NPs; Apoptosis assays in 4T1 cells and BALB/3T3 cells incubated for 24 h

21

and 48 h with G250, MSA-G250, Cy5.5-MSA-G250 NPs, and MSA; Cellular uptake analysis of

22

4T1 cells and BALB/3T3 cells incubated with Cy5.5-MSA-G250 NPs; Cellular ROS detection

23

assay of 4T1 cells incubated with or without Cy5.5-MSA-G250 NPs; Cell cycle analysis of 4T1


30

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1

cells and BALB/3T3 cells with or without Cy5.5-MSA-G250 treatment; In vivo Cy5.5 near-

2

infrared fluorescence optical images of 4T1 tumor-bearing BALB/c mice at different time

3

intervals after i.v. injection with Cy5.5-MSA-G250 NPs and the photos of 4T1 tumor-bearing

4

BALB/c mice 24 h post-i.v. injection with Cy5.5-MSA-G250 NPs; Hemolytic assay of mouse

5

RBCs incubated with Cy5.5-MSA-G250 NPs at various concentrations; Representative sections

6

of major organs collected from healthy mice with or without Cy5.5-MSA-G250 treatment were

7

stained with H&E and and observed under a light microscope; DNA primers for quantitative

8

PCR to measure the gene expression in 4T1 cells with or without Cy5.5-MSA-G250 NPs

9

treatment.

10 11

AUTHOR INFORMATION

12

Corresponding Author

13

* Email: [email protected].

14

Author Contributions

15

The manuscript was written through contributions of all authors. All authors have given approval

16

to the final version of the manuscript. ‡These authors contributed equally.

17

Notes

18

The authors declare no competing financial interest.

19

ACKNOWLEDGMENT

20

This work was financially supported by the National Science Foundations of China (No.

21

81660301, No. 81660592, No. 81673346, and No. 81760634).


31


Page 32 of 37

1

REFERENCES

2

1. Aceto, N.; Bardia, A.; Miyamoto, David T.; Donaldson, Maria C.; Wittner, Ben S.; Spencer,

3

Joel A.; Yu, M.; Pely, A.; Engstrom, A.; Zhu, H.; Brannigan, Brian W.; Kapur, R.; Stott,

4

Shannon L.; Shioda, T.; Ramaswamy, S.; Ting, David T.; Lin, Charles P.; Toner, M.; Haber,

5

Daniel A.; Maheswaran, S. Circulating Tumor Cell Clusters Are Oligoclonal Precursors of

6

Breast Cancer Metastasis. Cell 2014, 158, 1110-1122.

7

2. Fidler, I. J. Commentary on "Tumor Heterogeneity and the Biology of Cancer Invasion and

8

Metastasis". Cancer res. 2016, 76, 3441-3442.

9

3. Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Cancer Nanomedicine: Progress,

10

Challenges and Opportunities. Nat. Rev. Cancer 2017, 17, 20-37.

11

4. Chu, K. F.; Dupuy, D. E. Thermal Ablation of Tumours: Biological Mechanisms and

12

Advances in Therapy. Nat. Rev. Cancer 2014, 14, 199-208.

13

5. Huang, J.; Lin, L.; Sun, D.; Chen, H.; Yang, D.; Li, Q. Bio-Inspired Synthesis of Metal

14

Nanomaterials and Applications. Chem. Soc. Rev. 2015, 44, 6330-6374.

15

6. Kim, J.; Park, S.; Lee, J. E.; Jin, S. M.; Lee, J. H.; Lee, I. S.; Yang, I.; Kim, J.-S.; Kim, S. K.;

16

Cho, M.-H.; Hyeon, T. Designed Fabrication of Multifunctional Magnetic Gold Nanoshells and

17

Their Application to Magnetic Resonance Imaging and Photothermal Therapy. Angew.Chem.

18

2006, 118, 7918-7922.

19

7. Maltzahn, G. v.; Park, J.-H.; Agrawal, A.; Bandaru, N. K.; Das, S. K.; Sailor, M. J.; Bhatia, S.

20

N. Computationally Guided Photothermal Tumor Therapy Using Long-Circulating Gold

21

Nanorod Antennas. Cancer res. 2009, 69, 3892-3900.


32

Page 33 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

8. Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. Cancer Cell Imaging and Photothermal

2

Therapy in the Near-Infrared Region by Using Gold Nanorods. J. Am. Chem. Soc. 2006, 128,

3

2115-2120.

4

9. Li, Q.; Ruan, H.; Li, H. Nanocarbon Materials for Photodynamic Therapy and Photothermal

5

Therapy. Pharm. Nanotechnol. 2014, 2, 58-64.

6

10. Miyako, E.; Nagata, H.; Hirano, K.; Hirotsu, T. Micropatterned Carbon Nanotube–Gel

7

Composite as Photothermal Material. Adv.Mater. 2009, 21, 2819-2823.

8

11. Cheng, L.; Liu, J.; Gu, X.; Gong, H.; Shi, X.; Liu, T.; Wang, C.; Wang, X.; Liu, G.; Xing, H.;

9

Bu, W.; Sun, B.; Liu, Z. PEGylated WS2 Nanosheets as a Multifunctional Theranostic Agent for

10

in vivo Dual-Modal CT/Photoacoustic Imaging Guided Photothermal Therapy. Adv. Mater. 2014,

11

26, 1886-1893.

12

12. Liu, T.; Shi, S.; Liang, C.; Shen, S.; Cheng, L.; Wang, C.; Song, X.; Goel, S.; Barnhart, T. E.;

13

Cai, W.; Liu, Z. Iron Oxide Decorated MoS2 Nanosheets with Double PEGylation for Chelator-

14

Free Radiolabeling and Multimodal Imaging Guided Photothermal Therapy. ACS Nano 2015, 9,

15

950-960.

16

13. Liu, J.; Zheng, X.; Yan, L.; Zhou, L.; Tian, G.; Yin, W.; Wang, L.; Liu, Y.; Hu, Z.; Gu, Z.;

17

Chen, C.; Zhao, Y. Bismuth Sulfide Nanorods as a Precision Nanomedicine for in Vivo

18

Multimodal Imaging-Guided Photothermal Therapy of Tumor. ACS Nano 2015, 9, 696-707.

19

14.Chen, Q.; Xu, L.; Liang, C.; Wang, C.; Peng, R.; Liu, Z. Photothermal Therapy with Immune-

20

Adjuvant

21

Immunotherapy. Nat. Commun. 2016, 7, 13193

Nanoparticles

Together

with

Checkpoint

Blockade


for

Effective

Cancer

33


Page 34 of 37

1

15. Song, X.; Chen, Q.; Liu, Z. Recent Advances in the Development of Organic Photothermal

2

Nano-Agents. Nano Res. 2015, 8, 340-354.

3

16. Xu, X.; Huang, Z.; Huang, Z.; Zhang, X.; He, S.; Sun, X.; Shen, Y.; Yan, M.; Zhao, C.

4

Injectable, NIR/pH-Responsive Nanocomposite Hydrogel as Long-Acting Implant for

5

Chemophotothermal Synergistic Cancer Therapy. ACS Appl. Mater. & Interfaces 2017, 9,

6

20361-20375.

7

17. Zhang, R.; Su, S.; Hu, K.; Shao, L.; Deng, X.; Sheng, W.; Wu, Y. Smart

8

Micelle@Polydopamine Core-Shell Nanoparticles for Highly Effective Chemo-Photothermal

9

Combination Therapy. Nanoscale 2015, 7, 19722-19731.

10

18. Zhao, Y.; Alakhova, D. Y.; Kabanov, A. V. Can Nanomedicines Kill Cancer Stem Cells?

11

Adv. Drug Deliver. Rev. 2013, 65, 1763-1783.

12

19. Roper, D. K.; Ahn, W.; Hoepfner, M. Microscale Heat Transfer Transduced by Surface

13

Plasmon Resonant Gold Nanoparticles. J. Phys. Chem. C 2007, 111, 3636-3641.

14

20. Wei, Y.-j.; Li, K.-a.; Tong, S.-y. A Linear Regression Method for the Study of the Coomassie

15

Brilliant Blue Protein Assay. Talanta 1997, 44, 923-930.

16

21. Curry, T.; Epstein, T.; Smith, R.; Kopelman, R. Photothermal Therapy of Cancer Cells

17

Mediated by Blue Hydrogel Nanoparticles. Nanomedicine 2013, 8, 1577-1586.

18

22. Hessel, C. M.; Pattani, V. P.; Rasch, M.; Panthani, M. G.; Koo, B.; Tunnell, J. W.; Korgel, B.

19

A. Copper Selenide Nanocrystals for Photothermal Therapy. Nano Lett. 2011, 11, 2560-2566.


34

Page 35 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

23. Li, Z.; Hu, Y.; Chang, M.; Howard, K. A.; Fan, X.; Sun, Y.; Besenbacher, F.; Yu, M. Highly

2

Porous PEGylated Bi2S3 Nano-Urchins as a Versatile Platform for In Vivo Triple-Modal Imaging,

3

Photothermal Therapy and Drug Delivery. Nanoscale 2016, 8, 16005-16016.

4

24. Lu, Z.; Huang, F.-Y.; Cao, R.; Zhang, L.; Tan, G.-H.; He, N.; Huang, J.; Wang, G.; Zhang, Z.

5

Long Blood Residence and Large Tumor Uptake of Ruthenium Sulfide Nanoclusters for Highly

6

Efficient Cancer Photothermal Therapy. Sci. Rep. 2017, 7, 41571.

7

25. Weissleder, R. A Clearer Vision for in Vivo Imaging. Nat. biotechnol. 2001, 19, 316-317.

8

26. Dash, S. K.; Ghosh, T.; Roy, S.; Chattopadhyay, S.; Das, D. Zinc Sulfide Nanoparticles

9

Selectively Induce Cytotoxic and Genotoxic Effects on Leukemic Cells: Involvement of

10

Reactive Oxygen Species and Tumor Necrosis Factor Alpha. J. Appl. Toxicol. 2014, 34, 1130-

11

1144.

12

27. Trachootham, D.; Alexandre, J.; Huang, P. Targeting Cancer Cells by ROS-Mediated

13

Mechanisms: a Radical Therapeutic Approach? Nat. Rev. Drug Discov. 2009, 8, 579-591.

14

28. Raj, L.; Ide, T.; Gurkar, A. U.; Foley, M.; Schenone, M.; Li, X.; Tolliday, N. J.; Golub, T. R.;

15

Carr, S. A.; Shamji, A. F.; Stern, A. M.; Mandinova, A.; Schreiber, S. L.; Lee, S. W. Selective

16

Killing of Cancer Cells with a Small Molecule Targeting Stress Response to ROS. Nature 2011,

17

475, 231-234.

18

29. Fu, M.; Wang, C.; Li, Z.; Sakamaki, T.; Pestell, R. G. Minireview: Cyclin D1: Normal and

19

Abnormal Functions. Endocrinology 2004, 145, 5439-5447.

20

30. Musgrove, E. A.; Caldon, C. E.; Barraclough, J.; Stone, A.; Sutherland, R. L. Cyclin D as a

21

Therapeutic Target in Cancer. Nat. Rev. Cancer 2011, 11, 558-572.


35


Page 36 of 37

1

31. Otto, T.; Sicinski, P. Cell Cycle Proteins as Promising Targets in Cancer Therapy. Nat. rev.

2

Cancer 2017, 17, 93-115.

3

32. Hanai, J.-i; Dhanabal, M.; Karumanchi, S. A.; Albanese, C.; Waterman, M.; Chan, B.;

4

Ramchandran, R.; Pestell, R.; Sukhatme, V. P. Endostatin Causes G1 Arrest of Endothelial Cells

5

through Inhibition of Cyclin D1. J. Biol. Chem. 2002, 277, 16464-16469.

6

33. Tye, B. K. MCM Proteins in DNA Replication. Annu. Rev. Biochem. 1999, 68, 649-686.

7

34. Chapados, B. R.; Hosfield, D. J.; Han, S.; Qiu, J.; Yelent, B.; Shen, B.; Tainer, J. A.

8

Structural Basis for FEN-1 Substrate Specificity and PCNA-Mediated Activation in DNA

9

Replication and Repair. Cell 2004, 116, 39-50.

10

35. Dutta, A.; Ruppert, J. M.; Aster, J. C.; Winchester, E. Inhibition of DNA Replication Factor

11

RPA by P53. Nature 1993, 365, 79-82.

12

36. Chun, Y. S.; Laurent, A.; Maru, D.; Vauthey, J.-N. Management of Chemotherapy-

13

Associated Hepatotoxicity in Colorectal Liver Metastases. Lancet Oncol. 2009, 10, 278-286.

14

37. Chemotherapy in Adult High-Grade Glioma: a Systematic Review and Meta-Analysis of

15

Individual Patient Data from 12 Randomised Trials. Lancet 2002, 359, 1011-1018.

16

38. Barenholz, Y. Doxil® — The First FDA-Approved Nano-Drug: Lessons Learned. J. Control.

17

Release 2012, 160, 117-134.

18

39. Chen, Y.; Cheng, L.; Dong, Z.; Chao, Y.; Lei, H.; Zhao, H.; Wang, J.; Liu, Z. Degradable

19

Vanadium Disulfide Nanostructures with Unique Optical and Magnetic Functions for Cancer

20

Theranostics. Angew.Chem. 2017, 129, 13171-13176.


36

Page 37 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

40. Yang, Y.; Pan, D.; Luo, K.; Li, L.; Gu, Z. Biodegradable and Amphiphilic Block

2

Copolymer–Doxorubicin Conjugate as Polymeric Nanoscale Drug Delivery Vehicle for Breast

3

Cancer Therapy. Biomaterials 2013, 34, 8430-8443.

4 5

The TOC graphic of this manuscript

6


37

Intrinsic, cancer cell-selective toxicity of organic photothermal nano

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