Combined Phycocyanin and Hematoporphyrin Monomethyl Ether for

Nov 27, 2017 - †School of Life Science, ‡MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, and ...
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Combined PC and HMME for breast cancer treatment via photosensitizers modified Fe3O4 nanoparticles inhibiting the proliferation and migration of MCF-7 cells Shi-Wei Du, Ling-Kun Zhang, Kaibin Han, Shaoping Chen, Zhuoyan Hu, Wuya Chen, Kaikai Hu, Liang Yin, Baoyan Wu, and Yan-Qing Guan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01197 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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Biomacromolecules

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Revised manuscript submitted to Biomacromolecules (bm-2017-01197z.R1)

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Combined PC and HMME for breast cancer treatment via photosensitizers modified

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Fe3O4 nanoparticles inhibiting the proliferation and migration of MCF-7 cells

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Shi-Wei Du,†,# Ling-Kun Zhang,†,# Kaibin Han,† Shaoping Chen,† Zhuoyan Hu,† Wuya

8

Chen,† Kaikai Hu,‡ Liang Yin,† Baoyan Wu,‡ and Yan-Qing Guan*,†,‡,§

9 †

10 ‡

11

School of Life Science, South China Normal University, Guangzhou 510631, China

MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, 510631, China

12 §

13

Joint Laboratory of Laser Oncology with Cancer Center of Sun Yet-sen University, South China Normal University, Guangzhou 510631 (P. R. China)

14 15 16

1

17

*

Corresponding author at: MOE Key Laboratory of Laser Life Science & Institute of Laser Life

Science, College of Biophotonics, & Joint Laboratory of Laser Oncology with Cancer Center of Sun Yet-sen

University,

South

China

Normal

University,

Guangzhou

510631,

P.R.

Tel:(+86-20)85211241; E-mail address: [email protected] (Y. Q. Guan). #

These authors contributed equally to this work and should be considered co-first authors.

The authors declare no competing financial interest. 1

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[Abstract]

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Photodynamic therapy (PDT), combining the laser and photosensitizers to kill tumor

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cells, has the potential to address many currently medical requirements. In this study,

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magnetic Fe3O4 nanoparticles were firstly employed as cores, and modified with oleic acid

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(OA) and 3-triethoxysilyl-1-Propanamine. Then, the photosensitizers phycocyanin (PC) and

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hematoporphyrin monomethyl ether (HMME), which might be able to stimulate the cell

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release of ROS after the irradiation of near infrared (NIR) laser, were grafted on the surface of

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such nanoparticles. Our results revealed the high-efficiency inhibition of breast cancer MCF-7

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cells growing upon near-infrared irradiation both in vitro and in vivo. Furthermore, it was the

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synergy between the natural photosensitizers PC and the synthetic photosensitizers HMME

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that deeply influenced such inhibition, comparing to the groups that used either of these

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medicines alone. To utilize the combination of different photosensitive agents, our study thus

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provides a new strategy for breast cancer treatment.

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Keyword: Breast cancer, Photosensitive, Photodynamic therapy, Fe3O4 nanoparticle, Synergy

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1. Introduction

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Combination therapies are the future for the cancer clinical treatment, due to their

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enhanced therapeutic efficacy, reduced drug dosage, and avoided resistance.1-4 Combination

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regimens are complicated, however, since the administration dosage, time, and even sequence

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of different anticancer drugs must be appropriated to achieve a desired therapeutic effect with

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less toxic side effects.5-9

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Nanomedicine has been employed as a development treatment for the illness that is

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difficult to be conquered in clinical medicine, such as breast cancer. Nanoparticles bear

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identical properties, including the enhanced permeability and retention (EPR) effect for

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anti-cancer nanomedicine targeting, drug accurately delivery to the tumor tissues, and

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controlled drug release at the disease sites.10-13 Co-delivery of anticancer drugs using different

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nanoscales and functional nanocarriers have shown capabilities in improving the drug

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targeting delivery and therapeutic efficacies, while limiting the toxicity of drug by its

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targeting and controlled release only around cancer cells.7,14-20

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The strategies for combining PDT and chemotherapy have been widely studied for the

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treatment of tumors using nanomaterials.3,17-21 The PDT is an effective but noninvasive

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treatment modality for cancer therapy, and has enhanced the selectivity while fewer side

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effects comparing to the chemotherapy and radiotherapy.22,23 PDT kills tumor cells by singlet

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oxygen (1O2) produced by a photosensitizer (PS) after light illumination of the tumor. The

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reactive oxygen species (ROS) will lead to the apoptosis or necrosis of cancer cells, or

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through the destruction of tumor blood vessels. We have designed three nanoparticles by

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grafting the different photosensitizers on the surface of Fe3O4 nanoparticle. These strategies

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would effectively suppress breast cancer growth, significantly lower the cytotoxic drug

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dosage, and reduce the adverse effects. However, we wondered whether or not the

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combination of natural photosensitizer and synthetic photosensitizers would produce a better

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effect in cancer treatment.

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HMME is a promising second-generation porphyrin-related photosensitizer for PDT.

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Experimental studies and clinical trials have demonstrated that it can be selectively taken up

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tumor tissues. It has a strong photodynamic effect, with lower toxicity, shorter-term skin

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photosensitizations, and is less expensive than other photosensitizers. HMME has been tested

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in clinical trials as a treatment for skin diseases and it has been evaluated in clinical trials of

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port wine stains (PWS) in China.24-26 HMME-based PDT has proven to be an effective

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treatment for ovarian cancer, cutaneous malignancies and human glioblastoma cells.27-29

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Phycocyanin is separated from Spirulina sourced from Cyanophyta, Rhodophyta or

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Crytophyta.30 It is a pigment-protein complex with strong antioxidant, anti-free radical, and

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antitumor activities. Phycocyanin is a natural photosensitizer with minimum side effects.

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Although preclinical and clinical evaluations suggest that HMME and PC are promising PDT

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photosensitizers for the treatment of human disease, comparatively little has been published

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on the combined use of natural and synthetic photosensitizers.

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Iron oxide nanoparticles have been previously studied for their multitude of biological

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applications, and recent work suggests that the uptake of iron oxide nanoparticles can raise

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the concentration of loaded anticancer drugs, which may induced the death of cancer cells, 4

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especially combining with drug therapies.31-34 Moreover, magnetic nanoparticles would

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stimulate oxidative stress and greater ROS formation for the Fe3+ and Fe2+ ions being

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present.35 Meanwhile, we have provided several works focus on co-employing the Fe3O4

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nanoparticles and different drugs delivery for cancer treatment.36-38 Therefore, this study was

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focus on Fe3O4 nanoparticles modified with photosensitizers for a better PDT effect for breast

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cancer treatment.

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In our study, 3-triethoxysilyl-1-propanamine was used to link HMME and PC to Fe3O4

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nanoparticles covered with OA. This was followed by 633 nm laser irradiation, which

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activated the HMME and PC and generated 1O2 for PDT. We have successfully developed

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three

laser-sensitive

magnetic nanoparticles (LMN)

Fe3O4-OA-NH-PC

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Fe3O4-OA-NH-HMME (LMN-HMME) and Fe3O4-OA-NH-PC/HMME (LMN-PC/HMME).

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We demonstrate the photosensitivity of nanoparticles in both in vitro and in vivo assays.

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Firstly, we studied the effects of the nanoparticles and the 1O2 and Ca2+ release in the plasma.

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The effects of PDT nanoparticles on breast tumor cells (MCF-7) were then evaluated. Next,

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the tumor model of breast cancer mouse was constructed with subcutaneously implanted

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MCF-7 cells. Finally, we assessed the effects of the magnetic nanoparticles irradiated with

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laser in the treatment of BALB/c mice. This provides valuable information for the

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multi-functionalization of highly effective photosensitizers.

(LMN-PC),

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2. EXPERIMENTAL SECTION

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2.1. Synthesis of the nanoparticles LMN-PC, LMN-HMME, and LMN-PC/HMME 5

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According to the methods laid out in our previous study,38 the oleic acid-coated Fe3O4

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nanoparticles were successfully synthesized. Then, 20 mg 3-triethoxysilyl-1-propanamine and

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10 mg oleic acid-coated Fe3O4 nanoparticles were placed into the dispersant for 4 h, and

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washed four times with deionized water and left to rest for 12 h at 45℃. A total of 20 mg of

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1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 20 mg of

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N-Hydroxysuccinimide (NHS) were added as activators and 6 mg of the photosensitizer were

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added to the activator solution for 30 min. Six mg of the previously prepared LMN was added

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to the mixed solution at 25℃ for 12 h and the final reaction product was washed with ethanol

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and

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deionized

water

3

times.

The

purified

product

(LMN-PC,

LMN-HMME,

LMN-PC/HMME) was freeze-dried and stored at 4℃.

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2.2. Characterization of the nanoparticles

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It was critical to confirm whether the photosensitizers were immobilized on the surface

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of the LMN. The chemical structures of synthesized materials were characterized by

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ultraviolet absorption spectrum (Horiba, France) and fourier transform infrared spectroscopy

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(FTIR, TENSOR27, Bruker, Germany) spectra. The particle diameter and zeta potential of all

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nanoparticles were examined by the dynamic light scattering (DLS, Zetasizer Nano ZS90,

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Malvern Instruments, UK). The morphology of nanoparticles was evaluated by scanning

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electron microscopy (SEM, JEM-100 CXII). Thermal gravimetric analysis (TGA) was

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performed by thermal gravimetric analyzer (TG209F1, NETZSCH, Germany). The details of

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these characterization analysis were reference our recent paper in order to detect the 6

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characteristics of nanoparticles.[37-39]

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In order to estimate the grafting efficiency of PC and HMME on Fe3O4-OA-NH2. The

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powder of LMN-PC, LMN-HMME and LMN-PC/HMME were collected quantified by

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UV-visible spectrophotometer at 618 and 396 nm, respectively. Every experiment was

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repeated for three times. The drug grafting efficiency (DGE) was calculated by the following

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equations:

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The stability of nanoparticles was evaluated by DLS. Briefly, LMN-PC, LMN-HMME

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and LMN-PC/HMME nanoparticles (10 mg) were incubated with the PBS solution (pH = 7.4,

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10 mL) containing 20% fetal bovine serum (FBS) at 37℃. The average size of drug loaded

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nanoparticles in the mixtures were monitored and analyzed at certain time intervals (0, 3, 6,

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12, 18, 24, 36, 48, 60 and 72 h).

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2.3. Cell culture

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The MCF-7 cells were obtained from the Laboratory Animal Center Sun Yat-Sen

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University, China. The MCF-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium

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(DMEM, Gibco) containing 10% new-born calf serum (NBCS, Gibco), and 100 µg/mL

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penicillin, 100 µg/mL streptomycin and were incubated in a humidified atmosphere

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containing 5% CO2 at 37℃.

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2.4. MCF-7 cells treated by nanoparticles and laser

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The cells were treated with HMME, PC, PC/HMME, LMN, LMN-PC, LMN-HMME

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and LMN-PC/HMME (8 µmol/L), and the cell viability were detected by the cell counting

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kit-8 assay on MCF-7 cells after 12, 24 and 48 h. In order to evaluate the inhibition growth of

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MCF-7 cells by photodynamic therapy effects, the MCF-7 cells were treated with the

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nanoparticals for 6 h and irradiated with laser (633 nm and 628.5 nm, 1 W/cm2) for 5 min.

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2.5. Apoptosis Analysis of MCF-7 by PDT in vitro

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MCF-7 cells were stained with DAPI and PI and the nuclei were observed under a

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fluorescence microscope (Nikon TE2000-U, Japan). The samples were then analyzed by flow

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cytometry (FCM) using the FACS Calibur (BD, Franklin Lakes, NJ). Datas were analyzed

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with the CellQuest software (FACS Calibur, Becton-Dickinson). The monoclonal antibodies

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against P53, Bax and STAT3 were purchased from Boster, China. The details of fluorescence

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staining, flow cytometry analysis and western blot was reference our recent paper in order to

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detect the effect of inhibiting cell growth in vitro.[37-39]

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2.6. Measurement of ROS and Ca2+ generation

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The cells were irradiated with a laser (633 nm, 1 W/cm2) for 5 min, and then incubated

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for another 12, 24 and 48 h, respectively. Meanwhile, the MCF-7 cells incubated with 10

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µmol/L of 2,7-dichlorofluorescien diacetate (DCFH-DA, Beyotime) solution and 5 µmol/L of

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Fluo-3 AM (Beyotime) for 45 min at 37℃. The intracellular formation of ROS and Ca2+ were 8

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measured at excitation/emission wavelengths of 485/535 nm by using Wallac 1420

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Fluorescent Plate Reader.

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2.7. Wound healing assay

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A scratch wound was made across each well using a pipette tip. After washing with PBS

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3 times in order to remove any loosely held cells, the remaining cells were treated with LMN,

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LMN-PC, LMN-HMME and LMN-PC/HMME for 12, 24 and 48 h, respectively. Migrated

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cells were observed under an inverted microscope (Nikon ECLIPSE TS100, Japan).

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2.8. Subcutaneous Tumor Growth in BALB/c Mice and in vivo experiment

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All animal experiments were performed under a protocol approved by Sun Yat-sen

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Laboratory Animal Center and ensured the humane care of the animals. The Life Science

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Ethics Committee reviewed and approved the entire animal protocol prior to initiation of the

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experiments. The MCF-7 tumor models were generated by subcutaneous injection of 2 × 106

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cells in 0.1 mL saline into the right abdomen of female BALB/c mice (18-20 g). The mice

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were used for in vivo anti-tumor experiments when the tumor volume was >100mm3 (14 days

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after injection).

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The tumors-bearing mice were randomly grouped into 6 groups including saline, saline

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with laser, LMN + saline, LMN-PC + saline, LMN-HMME + saline, LMN-PC/HMME +

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saline, of which the LMN, LMN-PC, LMN-HMME, LMN-PC/HMME. In situ injected with

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100 µl containning drugs and the dosage was 3 µg/g/d. Four hours later, the tumors sites were 9

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locally irradiated by 633 nm laser (1 W/cm2) for 5 min. The injection treatment was

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performed once every 2 days and continued for 14 days. Prussian blue staining,

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immunohistochemistry, hematoxylin eosin (HE) staining and serological detection methods

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were reference our recent paper.[37,39]

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2.9. Tissue distribution

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Eight mice in each group were sacrificed at defined time periods (1, 6, 24 and 48 h), and

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the blood, kidney, liver, spleen, lung, heart and tumor tissues were excised. The same weight

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tissue mass decomposed on heating in nitric acid, evaporated to dryness, re-dissolved, and the

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Fe concentration was measured by ICP-MS (7500A, Agilent, USA).

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2.10. Statistical analysis

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Statistical results analyses were obtained using SPSS17.0. Analysis of variance was used

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to analyze statistical differences between groups under different conditions, and the Student’s

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t-test was also performed. P values of < 0.05 were considered statistically significant.

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3. Results

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3.1.

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LMN-PC/HMME

Construction

and

characterization

of

LMN-PC,

LMN-HMME

and

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According to our recent studies,38 the Fe3O4-OA-NH2 nanoparticles were synthesized

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with 3-triethoxysilyl-1-propanamine at 37℃. The co-polymer-assembled nanoparticles were 10

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co-encapsulated with PC and HMME (Figure 1). In order to produce oxidative damage to

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tumor cells, PC and HMME are photosensitizing drugs activated at the appropriate

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wavelength of excitation light.

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The chemical bondings between the functional groups of photosensitizer and LMN were

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detected with FTIR and ultraviolet-visible absorption spectrum. The FTIR and

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ultraviolet-visible absorption spectra data for LMN, LMN-PC, LMN-HMME, and

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LMN-PC/HMME were shown in Figure 2A,B. The wave trough representative of amide bond

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(~1070 cm-1) in LMN-PC, LMN-HMME, LMN-PC/HMME implies that the synthesis of three

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nanoparticles were successful. Meanwhile, the absence of a trough from the amino groups in

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LMN-PC, LMN-HMME and LMN-PC/HMME suggest that the photosensitizer PC and

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HMME were grafted on the surface of LMN successfully.

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The size of LMN, LMN-PC, LMN-HMME and LMN-PC/HMME were detected with

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DLS analysis. The results shown that the size of LMN was around 103 nm. However, the

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modification of HMME and PC increased the particle size to 217.3 ± 4.7 nm and 307.5 ± 3.7

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nm. Meanwhile, we obtained LMN-PC/HMME with the PC/HMME mass ratios of 4/6, and

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found that the nanoparticle size reached 503.7 ± 5.5 nm (Figure 2C), which indicates that the

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photosensitizer PC and HMME were successfully grafted in LMN.

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Subsequently, the morphology, size distributions, and thermal stability analyses of

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LMN-PC, LMN-HMME, and LMN-PC/HMME were assessed. The concentration of the

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HMME solution was 1 mg/mL and the residual concentration after grafting was 5.84 µg/mL.

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The concentration of the phycocyanin solution was 1 mg/mL and the residual concentration 11

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after grafting was 554 µg/mL. As shown in Figure 3A, the grafted rate of the amino

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group-modified magnetic nanoparticles was 98.83%, and the grafting ratios of HMME to PC

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were 98.83% to 44.6%.

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Meanwhile, the zeta potential of LMN was 9.8 ± 0.5 mV, and an increase in PC and

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HMME coating gradually enhanced the zeta potential (Figure 3B). When LMN-PC,

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LMN-PC/HMME and LMN-HMME were incubated in PBS (pH 7.4) at 37℃ for 3 days, the

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size and zeta potential showed no overt change (Figure 3B,D), indicating the stability of the

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nanoformulation. Together, these results demonstrate that LMN-PC, LMN-HMME and

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LMN-PC/HMME exhibit favorable characteristics for further antitumor development,

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including uniform morphology and suitable size for tumor accumulation and high stability in

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circulatory system. The thermal stability of the nanoparticles was evaluated by measuring the

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percentage of weight loss during the thermal gravimetric analysis Fe3O4-OA-NH2 as shown in

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Figure 3C. TGA showed that LMN-PC had a weight loss of wt 39% at temperatures below

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800℃. LMN-HMME experienced a weight loss of wt 17% at temperatures below 800℃.

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However, the weight loss in the LMN-PC/HMME group dropped to wt 25% at temperatures

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below 800℃ after the PC was loaded, demonstrating that the thermal stability of

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LMN-PC/HMME was enhanced. The SEM images of LMN, LMN-PC, LMN-HMME and

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LMN-PC/HMME are presented in Figure 3E.

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3.2. Activities and toxicities in vivo In order to investigate whether the construction of MCF-7 induced breast cancer model 12

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was completed (Figure 4A), we observed morphological and fur changes in the mice.

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Significant symptoms were occurred in the MCF-7 administration group, such as tumor

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volume (Figure 4C). Results also showed that MCF-7 administration induced a slight change

4

in hair. Statistically significant differences were also observed between the pre-injection and

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MCF-7 induced group.

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The breast cancer model animals were divided into 6 groups for comparative efficacy

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studies. The LMN, LMN-PC, LMN-HMME and LMN-PC/HMME treatment resulted in the

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volume reduction of the saline-treated under conditions with the use of laserirradiation

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(Figure 4B,F). The mice survival rates and weight were observed in the groups (Figure 4D,E).

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The HE staining was used to assess the morphology as showed in Figure 4G. According to the

11

images, our nanoparticles could inhibit the growth of tumor and have low damage to body.

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Prussian blue staining was performed to detect the Fe3O4 distribution of in the tumor

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providing that our nanoparticles could target the breast tumors. The red color of the breast cell

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nucleus and the blue color of the nanoparticles were shown in Figure 4H. According to the

15

images, blue color can be found in the nanoparticles treated groups, confirming that the LMN,

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LMN-PC, LMN-HMME and LMN-PC/HMME had been effectively delivered to the breast

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tumor. Next, we compared the metabolism of LMN, LMN-PC, LMN-HMME and

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LMN-PC/HMME exhibit in tumor tissue in 48 h. In the initial 6 h, loaded drugs quickly

19

entered the tumor tissue. After that, the drug concentration quickly decreased. While for the

20

loaded drugs, obvious retention effect was seen (Figure 4I1-I4). These results demonstrated

21

that the drug-loaded nanoparticles would quickly diffuse into tumors after injection, and 13

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would keep high drug concentrations in tumor.

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Furthermore, we performed immunohistochemical staining of Ki67 and Bcl-2 in the 6

3

groups to evaluate the efficacy of LMN, LMN-PC, LMN-HMME and LMN-PC/HMME.

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Immunohistochemical staining in the tumor is shown in Figure 4J1,J2. A decrease in Ki67 and

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Bcl-2 levels were observed in the LMN, LMN-PC, LMN-HMME and LMN-PC/HMME

6

groups, revealing the induced down-regulation of Ki67 and Bcl-2 as the result of apoptosis.

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These results indicated that LMN-PC, LMN-HMME and LMN-PC/HMME all have the

8

potential to inhibit the migration of breast cancer and to promote apoptosis, as reflected by the

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down-regulation of Ki67 and Bcl-2.

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To quantify the toxicity of LMN, LMN-PC, LMN-HMME, LMN-PC/HMME, the next

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important step was the assessment of standard hematologic parameters. The assessment of the

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counts in the treated group on day 14 provided weak evidence for leukopenia or associated

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toxicities (Figure 5A). We further assessed the toxicity in vivo by HE staining of the heart,

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liver, spleen, lungs and kidney tissues, respectively. As shown in Figure 5B, the HE staining

15

revealed that LMN, LMN-PC, LMN-HMME and LMN-PC/HMME did not cause organ

16

damage, which indicates that they were safe for treatment.

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3.3. Molecular mechanism of cell death in vitro

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We then evaluated the biological effects of the nanoparticles. Figure 6A1-A4 showed the

20

survival rates of MCF-7 cells after treated with different concentration of HMME, PC,

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PC/HMME, LMN, LMN-PC, LMN-HMME and LMN-PC/HMME. The diagram revealed 14

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that the cells underwent their death after exposing to HMME, PC, PC/HMME, LMN,

2

LMN-PC, LMN-HMME and LMN-PC/HMME. We chose a death rate of 50% and the

3

concentration of 8 µmol/L LMN, LMN-PC, LMN-HMME or LMN-PC/HMME in 24 h per

4

well for the in vitro research.

5

As shown in Figure. 6B, in our results, a small amount of nuclear agglutination was

6

observed in the nuclei of LMN-PC, LMN-HMME and LMN-PC/HMME treated for 24 h. The

7

cells treated with the same concentration of LMN-PC/HMME treated cells also showed

8

nuclear agglutination, indicating apoptosis. PI staining, the whole cell was stained red,

9

indicating that the particles after treatment of the cell membrane caused damage.

10

Cell scratches are an indirect method of determining cell migration and repair capacity,

11

similar to in vitro wound healing models. As shown in Figure 6C1,C2, the cells were cultured

12

for 12h, 24h and 48h. The results showed that LMN-PC, LMN-HMME and LMN-PC/HMME

13

can inhibit the migration and proliferation of MCF-7 cells. Figure 6C1 shows that the effect of

14

LMN-HMME and LMN-PC/HMME on cell migration is more obvious in 12h. The particles

15

still had a good inhibitory effect on cell migration at 24 and 48h. We can initially show that

16

our particle synthesis was successful.

17

Concomitantly, at 24 h, the treated control group LMN-PC, LMN-HMME and

18

LMN-PC/HMME exhibited cell mortality rates of 63.2%, 39.4% and 27.6%, respectively

19

(Figure 6C2). These results demonstrated that the groups of LMN-PC, LMN-HMME and

20

LMN-PC/HMME had better effects in inducing cell apoptosis at 24 h. Moreover, 8 µmol/L of

21

LMN-PC/HMME could effectively inducing the apoptosis of MCF-7 cells compared to the 15

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1

other treatments.

2

In addition, the acridine orange staining of the MCF-7 cells, as characterized by

3

fluorescence microscopy (Nikon) and the cell apoptosis were detected by flow cytometry

4

(FCM) and are shown in Figure 7A,B. After binding double-stranded DNA, AO emits green

5

fluorescence. A representative fluorescent photomicrograph of MCF-7 cells exposure to LMN,

6

LMN-PC, LMN-HMME and LMN-PC/HMME for 24 and 48 h is shown in Figure 7A. The

7

cell nucleus showed yellow-green fluorescence, the nucleolus showed orange-red

8

fluorescence and the cell volume was large and spread out. As shown Figure 7B, there is no

9

better effect after the MCF-7 cells were treated with LMN-PC compared with the control

10

group, the laser irradiation and the LMN group. After treatment with LMN-HMME, the cells

11

had undergone more apoptosis and death. The LMN-PC/HMME treated cells showed a high

12

death rate, indicating that our particle synthesis was successful, but its specific role requires

13

elucidation and follow-up experiment to explore the phenomenon.

14

In order to investigated the production of ROS in the cell cultures. As shown in Figure

15

7C, the LMN-PC/HMME group displayed high ROS expression compared to the control

16

LMN, LMN-PC, and LMN-HMME groups at both 24 h and 48 h. With the prolongation of

17

the reaction time, the concentration of active oxygen increased markedly in the

18

LMN-PC/HMME treated group. The initial display of particles is activated by ROS to kill

19

cells. The concentration of Ca2+ increased in a certain subpopulation of the LMN-PC/HMME

20

treatment group and the other groups showed a significant increase with the prolongation of

21

the time (Figure 7D). The causes of this phenomenon are unclear, necessitating follow-up 16

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Biomacromolecules

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experiments. After undergoing treatment with LMN-PC, LMN-HMME and LMN-PC/HMME

2

for 24 h, the cell nucleus showed yellow-green fluorescence, there was cytoplasmic

3

uniformity, the nucleolus showed orange-red fluorescence, and the cell volume was large and

4

spread out.

5

We also analyzed the expression of Bax, STAT3 and P53 in response to different

6

treatments with the nanoparticles for 24 h, respectively (as shown in Figure 7E1,E2). The

7

expression levels of P53 (53 kDa) protein, the groups treated with LMN-PC, LMN-HMME

8

and LMN-PC/HMME, showed a significant up-regulation compared to the control, the laser

9

and the LMN groups. The results suggested that a greater activation of the control capability

10

on the outer membrane of the MCF-7 cells after treated with LMN-PC, LMN-HMME and

11

LMN-PC/HMME. Meanwhile, the treatments also induced a greater up-regulation of Bax and

12

STAT3 (22 kDa and 86 kDa, respectively) at 24 h. These results suggested that LMN-PC,

13

LMN-HMME and LMN-PC/HMME treatment may interfere with the expression of cell-death

14

related proteins and subsequently hinder programmed cell death by down-regulating Bcl-2

15

expression and up-regulating Bax and P53 expression.

16 17

4. Discussion

18

Breast cancer, as an invasive malignancy, is the most commonly leading cause of cancer

19

mortality in women worldwide.40 The main methods have been employed for the breast

20

cancer therapy, including surgical, drug or gene therapy. The treatments of breast cancer still

21

rely on surgical treatment, and adriamycin is one of the few chemotherapy drugs that inhibits 17

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1

the growth and migration of breast cancer effectively. However, chemotherapeutics and

2

surgical treatments take a severe toll on the body and can have very serious side effects.

3

PDT is a new method being applied in cancer therapy. Central challenges of PDT therapy

4

are involved how to efficiently deliver drugs, and how to accurately target breast cancer cells,

5

and how to significantly reduce toxicity and side effects. Most PDT photosensitizers are

6

synthetic, giving them a low extinction coefficient and enabling photo-bleaching. And tissue

7

penetration depths are very weak. The 1O2 was recently reported to be directly produced by

8

metal nanoparticles like iron ion with laser irradiation without any photosensitizers for

9

enhancing the penetration depth of bio-tissues.

10

Recently, nanocarriers have been employed for targeted drug delivery in cancer

11

therapy.41 Photosensitizers have been widely applied for tumors, virus infections, and other

12

diseases by PDT or photothermal therapy.42,43 In addition, the limitations of current breast

13

cancer treatments require the development of some novel and more effective therapeutic

14

methods. Of note, HMME and PC have been applied for effective treatment of breast

15

cancer,44,45 while many sorts of nano-drug delivery systems were employed to deliver

16

photosensitiveness drugs to the therapy of tumor by PDT or PTT.46,47 Nevertheless, just a few

17

studies demonstrated photosensitizers for treatment of breast cancer both in vivo and in vitro.

18

Meanwhile, the synergistic effect of photosensitizers has not been reported. Herein, we

19

highlight the Fe3O4 nanoparticles that were functionalized with immobilized photosensitizers.

20

Their nontoxic and great biocompatibility for nanoparticle synthesis combined with PDT thus

21

provide these nanoparticles an ideal sort of drug for breast cancer therapy. 18

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Photosensitizer can induced the ROS-response for targeting tumor therapy in vivo.48

2

While our present experiments revealed that the combination of photosensitizer can

3

effectively improve the treatment of breast cancer under NIR irradiation, demonstrating the

4

photosensitizers synergy is the preferred role of in developing new PDT drugs against cancers.

5

Meanwhile, no obvious weight loss can be detected in vivo treated by nanomedcine, implying

6

that the toxicity was lessness.49,50 Our results demonstrate that the morphologies of spleen,

7

kidney, liver, heart and lung are similar among all treatment groups. It reveals that the

8

nanoparticles have very low toxicity. What’s more, we detect the distribution of nanoparticles

9

in the tumor and the expression of Bcl-2 and Ki67 proteins. It revealed that nanoparticles can

10

effectively enter tumor cells and might induce apoptosis of breast cancer cells. The

11

LMN-PC/HMME nanoparticle is more effective than LMN-HMME and LMN-PC. That may

12

be because of the synergistic effect of the photosensitizers, which was unreported in the

13

previous researches.

14

In addition, there are many studies on cytotoxicity test and flow cytometry detection cell

15

death about photosensitizer.48-50 However, there was no report on the study of photosensitizers

16

inhibiting cell migration. Moreover, it was enhanced by exposure to LMN-PC/HMME for 48h,

17

which lead to the inhibition of migration rate (about 27.6%) compared with the control group.

18

It can obviously inhibit the cells migration and is significance for the PDT treatment of breast

19

cancer.

20

Furthermore, comparing with control, photosensitizers increase the expression of Bax

21

and decrease the expression of Bcl-2 in HepG2 cells.48 In tumor cells, STAT3 phosphorylation 19

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associates with apoptotic activity through the Nox/ROS/NF-κB/STAT3 signaling cascade.51,52

2

Whereas, whether the photosensitizer can activate the STAT3 signaling pathway is not clear.

3

In in vitro experiment, we find the up-regulation of STAT3 and Bax, as presented in Figure 8.

4

It reveals that the photosensitizer can induce apoptosis by STAT3 signaling pathways.

5 6

5. Conclusion

7

In summary, we have successfully designed three sorts of Fe3O4 nanoparticles for

8

photodynamic therapy. The photosensitizers, PC and HMME, were grafted on the surface of

9

such nanoparticles, which might be able to stimulate the release of ROS in cells after the

10

irradiation of near infrared (NIR) laser. Such ROS might inhibit the migration of breast cancer

11

cell significantly, and might kill these cells by apoptosis or necrosis directly. Furthermore, it

12

was the synergy between the natural photosensitizers PC and the synthetic photosensitizers

13

HMME that deeply influenced such inhibition, comparing to the groups treated with either of

14

these medicines alone. This work might be helpful for the synergy of the photosensitizers for

15

breast cancer treatment.

16 17

Acknowledgements

18

The expenses of this work were supported by the National Natural Science Foundation

19

of China (31370967, 31170919, 81371646), the Guangdong Province Universities and

20

Colleges Pearl River Scholar Fund Scheme (2014), China, the Science and Technology

21

Planning Project of Guangdong Province (No.2015A020212033), China.

22

20

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Figure and Figure Captions

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Figure1. Molecular structures and chemical processes for preparing LMN-PC, LMN-HMME and LMN-PC/HMME.

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Figure2. FTIR, Ultraviolet-visible absorption spectra and size of the nanoparticles. (A) FTIR spectra of HMME, PC, LMN, LMN-HMME, LMN-PC and LMN-PC/HMME nanoparticles. (B) Ultraviolet-visible absorption spectra of HMME, PC, LMN, LMN-HMME, LMN-PC and LMN-PC/HMME nanoparticles. (C) Size of Fe3O4, LMN, LMN-HMME, LMN-PC and LMN-PC/HMME nanoparticles.

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Figure 3. The stability, grafting rate and morphology analyzed of nanoparticles. (A) Photosensitive magnetic nanoparticles grafting rate of LMN-HMME, LMN-PC and LMN-PC/HMME nanoparticles. (B) Comparison of surface charges after HMME and PC modification. (C) Thermogravimetric analysis of LMN, LMN-HMME, LMN-PC and LMN-PC/HMME nanoparticles. (D) The average particles size of LMN-HMME, LMN-PC and LMN-PC/HMME nanoparticle after incubation with serum for 3 days. (E)Scanning electron microscope of LMN, LMN-HMME,LMN-PC and LMN-PC/HMME nanoparticles.

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Figure 4. Antitumor effects of nanoparticles-based PDT in vivo. (A) Images of tumor-bearing mice treated with the six types of substances: saline, laser + saline, LMN, laser +LMN-PC, laser + LMN-HMME, and laser + LMN-PC/HMME. (B) Representative images oftumors in each treatment group after treating 14 day. (C) Volume of tumors after injecting with MCF-7 cell. (D, E, F) Survival rate, body weight and tumors volume of the BALB/c mice a function of time (days) upon the treatment. (G and H) HE staining and Prussian blue staining of the tumor. (I1-I4) Tissue and tumor metabolism of LMN, LMN-PC, LMN-HMME and LMN-PC/HMME nanoparticles. (J1, J2) Immunohistochemical for Bcl-2 and Ki67 in tumor of 6 groups: saline, laser, LMN, LMN-PC, LMN-HMME, and LMN-PC/HMME groups, respectively. The black arrow means for Bcl-2 and Ki67 positive cells. Number of positive cells determined using Image pro-plus 6.0. Scale bars, 50 µm. The relative levels are plotted with the significance p