Combined Phycocyanin and Hematoporphyrin Monomethyl Ether for

Subscriber access provided by READING UNIV

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 45 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

Biomacromolecules

1

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

2 3 4

Combined PC and HMME for breast cancer treatment via photosensitizers modified

5

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

6 7

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

ACS Paragon Plus Environment

China.

Biomacromolecules 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

[Abstract]

2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

synergy between the natural photosensitizers PC and the synthetic photosensitizers HMME

11

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

12

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

13

provides a new strategy for breast cancer treatment.

14 15

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

16

2


Page 2 of 45

Page 3 of 45 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

Biomacromolecules

1

1. Introduction

2

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

3

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

4

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

5

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

6

less toxic side effects.5-9

7

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

8

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

9

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

10

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

11

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

12

nanoscales and functional nanocarriers have shown capabilities in improving the drug

13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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

3



1

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

2

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

3

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

4

combination of natural photosensitizer and synthetic photosensitizers would produce a better

5

effect in cancer treatment.

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

Phycocyanin is separated from Spirulina sourced from Cyanophyta, Rhodophyta or

14

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

15

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

16

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

17

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

18

on the combined use of natural and synthetic photosensitizers.

19

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

20

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

21

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


Page 4 of 45

Page 5 of 45 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

Biomacromolecules

1

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

2

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

3

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

4

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

5

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

6

cancer treatment.

7

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

8

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

9

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

10

three

laser-sensitive

magnetic nanoparticles (LMN)

Fe3O4-OA-NH-PC

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

multi-functionalization of highly effective photosensitizers.

(LMN-PC),

19 20

2. EXPERIMENTAL SECTION

21

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



Page 6 of 45

1

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

2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

and

10

deionized

water

3

times.

The

purified

product

(LMN-PC,

LMN-HMME,

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

11 12

2.2. Characterization of the nanoparticles

13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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


Page 7 of 45 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

Biomacromolecules

1

characteristics of nanoparticles.[37-39]

2

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

3

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

4

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

5

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

6

equations:

7 8 9

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

10

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

11

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

12

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

13

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

14 15

2.3. Cell culture

16

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

17

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

18

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

19

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

20

containing 5% CO2 at 37℃.

21 7



1

2.4. MCF-7 cells treated by nanoparticles and laser

2

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

3

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

4

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

5

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

6

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

7 8

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

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16 17

2.6. Measurement of ROS and Ca2+ generation

18

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

19

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

20

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

21

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


Page 8 of 45

Page 9 of 45 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

Biomacromolecules

1

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

2

Fluorescent Plate Reader.

3 4

2.7. Wound healing assay

5

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

6

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

7

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

8

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

9 10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

after injection).

18

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

19

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

20

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

21

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



Page 10 of 45

1

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

2

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

3

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

4

were reference our recent paper.[37,39]

5 6

2.9. Tissue distribution

7

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

8

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

9

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

10

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

11 12

2.10. Statistical analysis

13

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

14

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

15

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

16 17

3. Results

18

3.1.

19

LMN-PC/HMME

Construction

and

characterization

of

LMN-PC,

LMN-HMME

and

20

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

21

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


Page 11 of 45 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

Biomacromolecules

1

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

2

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

3

wavelength of excitation light.

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

HMME were grafted on the surface of LMN successfully.

12

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

13

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

14

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

15

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

16

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

17

photosensitizer PC and HMME were successfully grafted in LMN.

18

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

19

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

20

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

21

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



1

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

2

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

3

were 98.83% to 44.6%.

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19 20 21

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


Page 12 of 45

Page 13 of 45 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

Biomacromolecules

1

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

2

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

3

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

5

MCF-7 induced group.

6

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

7

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

8

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

9

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

10

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.

12

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

13

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

14

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,

16

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

17

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

18

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



1

would keep high drug concentrations in tumor.

2

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.

4

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

5

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.

7

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

9

down-regulation of Ki67 and Bcl-2.

10

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

11

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

12

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

13

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

14

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.

17 18

3.3. Molecular mechanism of cell death in vitro

19

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,

21

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


Page 14 of 45

Page 15 of 45 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

Biomacromolecules

1

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



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


Page 16 of 45

Page 17 of 45 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

Biomacromolecules

1

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



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


Page 18 of 45

Page 19 of 45 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

Biomacromolecules

1

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



1

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


Page 20 of 45

Page 21 of 45 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

Biomacromolecules

1

References

2

(1) Hu, C. M.; Zhang, L. Nanoparticle-based combination therapy toward overcoming drug

3

resistance in cancer. Biochem Pharmacol 2012, 83, 1104-1111.

4

(2) Li, X.; Takashima, M.; Yuba, E.; Harada, A.; Kono, K. PEGylated PAMAM

5

dendrimer-doxorubicin conjugate-hybridized gold nanorod for combined photothermal

6

chemotherapy. Biomaterials 2014, 35, 6576-6584.

7 8

(3) Kim, J.; Jeong, C.; Kim, W. J. Synergistic nanomedicine by combined gene and photothermal therapy. Adv. Drug Deliv. Rev. 2015, 98, 99-112.

9

(4) LoRusso, P. M.; Canetta, R.; Wagner, J. A.; Balogh, E. P.; Nass, S. J.; Boerner, S. A.;

10

Hohneker, J. Accelerating cancer therapy development: the importance of combination

11

strategies and collaboration. Summary of an institute of medicine workshop. Clin.

12

Cancer Res. 2012, 18, 6101-6109.

13

(5) Liu, J.; Wang, C.; Wang, X.; Cheng, L.; Li, Y.; Liu, Z. Mesoporous silica coated

14

single-walled carbon nanotube served as drug carrier used for cancer combination

15

therapy. Adv. Funct. Mater. 2015, 25, 384-392.

16

(6) Kim, K.; Park, D. Y.; Lee, J. Y.; Lee, B. S.; Kim, I. S.; Kim, K.; Kwon, I. C.; Sang, Y. K.;

17

Yuk, S. H. Doxorubicin/Gold-Loaded core/shell nanoparticles for combination therapy

18

to treat cancer through the enhanced tumor targeting. J. Control Release 2016, 228,

19

141-149.

20

(7) Eldar-Boock, A.; Polyak, D.; Scomparin, A.; Satchi-Fainaro, R. Nano-sized polymers and

21

liposomes designed to deliver combination therapy for cancer. Curr. Opin. Biotechnol 21



1 2 3

2013, 24, 682-689. (8) Ju, H. Nuclear-uptake nanodrug delivery system for drug-resistant cancer therapy. Sci. China Chem. 2015, 58, 438-438.

4

(9) Zhang, H. Z.; Lin, J. M.; Syed, R.; Zhou, C. H. Design, synthesis, and biological

5

evaluation of novel benzimidazole derivatives and their interaction with calf thymus

6

DNA and synergistic effects with clinical drugs. Sci. China Chem. 2014, 57, 807-822.

7

(10) Lee, G. Y.; Kim, J. H.; Choi, K. Y.; Yoon, H. Y.; Kim, K.; Kwon, I. C. Hyaluronic acid

8 9 10

nanoparticles for active targeting atherosclerosis. Biomaterials 2015, 53, 341-348. (11) Thambi, T.; Deepagan, V.; Yoon, H. Y.; Han, H. S. Hypoxia responsive polymeric nanoparticles for tumor-targeted drug delivery. Biomaterials 2014, 35, 1735-1743.

11

(12) Yoon, H. Y.; Koo, H.; Choi, K. Y.; Kwon, I. C.; Choi K.; Park J. H. Photo-crosslinked

12

hyaluronic acid nanoparticles with improved stability for in vivo tumor-targeted drug

13

delivery. Biomaterials 2013, 4, 5273-5280.

14

(13) Ju, E.; Liu, Z.; Du, Y.; Tao, Y.; Ren, J.; Qu, X. Heterogeneous assembled

15

nanocomplexes for ratiometric detection of highly reactive oxygen species in vitro and in

16

vivo. ACS Nano. 2014, 8, 6014-6023.

17

(14) Li, F.; Zhao, X.; Wang, H.; Zhao, R.; Ji, T.; Ren, H.; Anderson, G. J.; Nie, G.; Hao, J.

18

Multiple layer-by-layer lipid-polymer hybrid nanoparticles for improved folfirinox

19

chemotherapy in pancreatic tumor models. Adv. Funct. Mater. 2014, 25, 788-798.

20

(15) Zhang, J.; He, X.; Zhang, P.; Ma, Y.; Ding, Y.; Wang, Z.; Zhang, Z. Quantifying the

21

dissolution of nanomaterials at the nano-bio interface. Sci. China Chem. 2015, 8, 22


Page 22 of 45

Page 23 of 45 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

Biomacromolecules

1

761-767.

2

(16) Wu, C.; Shi, L.; Wu, C.; Guo, D.; Selke, M.; Wang, X. Enhanced in vitro anticancer

3

activity of quercetin mediated by functionalized CdTe QDs. Sci. China Chem. 2014, 57,

4

1579-1588.

5 6

(17) Wang, C. Bio-cluster nano-bomb for cancer drug delivery: efficacious fire at the target. Chinese Sci. Bull. 2015, 60, 403-404.

7

(18) Desale, S. S.; Soni, K. S.; Romanova, S.; Cohen, S. M.; Bronich, T. K. Targeted delivery

8

of platinum-taxane combination therapy in ovarian cancer. J. Control Release 2015, 220,

9

651-659.

10

(19) Zeynabad, F. B.; Salehi, R.; Alizadeh, E.; Kafil, H. S.; Hassanzadeh, A. M.; Mahkam, M.

11

pH-Controlled multiple-drug delivery by a novel antibacterial nanocomposite for

12

combination therapy. Rsc. Adv. 2015, 5, 105678-105691.

13

(20) Wang, W.; Song, H.; Zhang, J.; Li, P.; Li, C.; Wang, C.; Kong, D.; Zhao, Q. An

14

injectable,

15

encapsulation and independent release of a drug cocktail as an effective combination

16

therapy platform. J. Control Release 2015, 203, 57-66.

17 18

thermosensitive

and

multicompartment

hydrogel

for

simultaneous

(21) Shanmugam, V.; Selvakumar, S.; Yeh, C. S. Near-infrared light-responsive nanomaterials in cancer therapeutics, Chem. Soc. Rev. 2014, 43, 6254-6287.

19

(22) Fay, B. L.; Melamed, J. R.; Day, E. S. Nanoshell-mediated photothermal therapy can

20

enhance chemotherapy in inflammatory breast cancer cells. Int. J. Nanomed. 2015, 10,

21

6931-6941. 23



1

(23) Meng, Z.; Wei, F.; Wang, R.; Xia, M.; Chen, Z.; Wang, H.; Zhu, M. NIR-laser switched

2

in vivo smart nanocapsules for synergic photothermal and chemotherapy of tumors. Adv.

3

Mater. 2016, 28, 245-253.

4

(24) Shen, J. M.; Gao, F. Y.; Guan, L. P.; Su, W.; Yang, Y. J.; Li, Q. R.; Jin, Z. C. Graphene

5

oxide Fe3O4 nanocomposite for combination of dual-drug chemotherapy with

6

photothermal therapy. Rsc. Adv. 2014, 4, 18473-18484.

7

(25) Cheng, J.; Liang, H.; Li, Q.; Peng, C.; Li, Z.; Shi, S. Hemato-porphyrin monomethyl

8

ether-mediated photodynamic effects on THP-1 cell-derived macrophages. J. Photoch.

9

Photobio. B. 2010, 101, 9-15.

10 11 12 13

(26) Yuan, K.; Huang, Z. Evolving role of PDT in PWS. Photodiagn. Photodyn. 2007, 4, 149-150. (27) Huang, Z. Photodynamic therapy in China: over 25 years of unique clinical experience: Part two - clinical experience. Photodiagn. Photodyn. 2006, 3, 71-84.

14

(28) Song, K.; Kong, B.; Li, L.; Yang, Q.; Wei, Y.; Qu, X. Intraperitoneal photo-dynamic

15

therapy for an ovarian cancer as cite model in Fischer 344 rat using hematoporphyrin

16

monomethyl ether. Cancer Sci. 2007, 98, 1959-1964.

17

(29) Guyon, L.; Ascencio, M.; Collinet, P.; Mordon, S. Photodiagnosis and photodynamic

18

therapy of peritoneal metastasis of ovarian cancer. Photodiagn. Photodyn. 2012, 9,

19

16-31.

20

(30) Kuddus, M.; Singh, P.; Thomas, G.; Al-Hazimi A. Recent developments in production

21

and biotechnological applications of C-phycocyanin. Biomed. Res. Int. 2013, 24


Page 24 of 45

Page 25 of 45 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

Biomacromolecules

1

742859-742868.

2

(31) Klein, S.; Sommer, A.; Distel, L.V.R.; Neuhuber, W.; Kryschi, C. Superparamagnetic

3

iron oxide nanoparticles as radiosensitizer via enhanced reactive oxygen species

4

formation. Biochem. Biophys. Res. Commun. 2012, 425, 393-397.

5

(32) Weissleder, R.; Stark, D. D.; Engelstad, B. L.; Bacon, B. R.; Compton, C. C.; White, D.

6

L.; Jacobs, P.; Lewis, J. Superparamagnetic iron-oxide - pharmacokinetics and toxicity.

7

Am. J. Roentgenol. 1989, 152, 167-173.

8

(33) Alarifi, S.; Ali, D.; Alkahtani, S.; Alhader, M. S. Iron oxide nanoparticles induce

9

oxidative stress, DNA damage, and caspase activation in the human breast Cancer cell

10

line. Biol. Trace Elem. Res. 2014, 159, 416-424.

11

(34) Klein, S.; Sommer, A.; Distel, L. V. R.; Hazemann, J. L.; Kroner, W.; Neuhuber, W.;

12

Müller, P.; Proux, O.; Kryschi, C. Superparamagnetic iron oxide nanoparticles as novel

13

x-ray enhancer for low-dose radiation therapy. J. Phys. Chem. B 2014, 118, 6159-6166.

14

(35) Aranda, A.; Sequedo, L.; Tolosa, L.; Quintas, G.; Burello, E.; Castell, J. V., Gombau, L.

15

Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay: a quantitative method for

16

oxidative stress assessment of nanoparticle-treated cells. Toxicol. In vitro 2013, 27,

17

954-963.

18

(36) Li. Z.; Guan, Y. Q.; Liu, J. M. The role of STAT-6 as a key transcription regulator in

19

HeLa cell death induced by IFN-γ/TNF-α co-immobilized on nanoparticles. Biomaterials

20

2014, 35, 5016-5027.

21

(37) Niu, S.; Zhang, L. K.; Zhang, Li.; Zhuang, S.; Zhan, X.; Chen, W. Y.; Du, S.; Yin, L.; 25



1

You, R.; Li, C. H.; Guan, Y. Q. Inhibition by Multifunctional Magnetic Nanoparticles

2

Loaded with Alpha-Synuclein RNAi Plasmid in a Parkinson's Disease Model.

3

Theranostics 2017, 7, 344-356

4

(38) Zhan X. Y.; Guan Y. Q. Design of magnetic nanoparticles for hepatocellular carcinoma

5

treatment using the control mechanisms of cell internal nucleus and external membrane.

6

J. Mater. Chem. B. 2015, 3, 4191-4204.

7

(39) Guan, Y. Q.; Zheng Z.; Huang Z.; Li Z.; Niu S.; Liu J. M. Powerful inner/outer

8

controlled multi-target magnetic nanoparticle drug carrier prepared by liquid

9

photo-immobilization, Sci. Rep. 2014, 4, 4990-4998.

10

(40) Deng, X.; Cao, M.; Zhang, J.; Hu, K.; Yin, Z.; Zhou, Z. Hyaluronic acidchitosan

11

nanoparticles for co-delivery of MiR-34a and doxorubicin in therapy against triple

12

negative breast cancer. Biomaterials 2014, 35, 4333-4344.

13

(41) He, Q.; Zhang, J.; Shi, J. The effect of PEGylation of mesoporous silica nanoparticles on

14

nonspecific binding of serum proteins and cellular responses. Biomaterials 2010, 31,

15

1085-1092.

16

(42) Rosenholm, J. M.; Sahlgren, C.; Linden, M. Towards multifunctional targeted drug

17

delivery systems using mesoporous silica nanoparticles-opportunities & challenges.

18

Nanoscale 2010, 2, 1870-1883.

19

(43) Chen, A. M.; Zhang, M.; Wei, D. Codelivery of doxorubicin and Bcl-2 siRNA by

20

mesoporous silica nanoparticles enhances the efficacy of chemotherapy in multidrug

21

resistant cancer cells. Small 2009, 5, 2673-2677. 26


Page 26 of 45

Page 27 of 45 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

Biomacromolecules

1

(44) Lia, H. T.; Songa, X. Y.; Yanga, C.; Lia, Q. Effect of hematoporphyrin monomethyl

2

ether-mediated PDT on the mitochondria of canine breast cancer cells. Photodlagn.

3

Photodyn. 2013, 10, 414-421.

4

(45) Master, A. M.; Rodriguez, M. E.; Kenney, M. E. Delivery of the photosensitizer Pc 4 in

5

PEG-PCL micelles for in vitro PDT studies. J. Pharm. Sci-US. 2010, 5, 2386-2398.

6

(46) Hoven, J. M.; Tomme, S. R. V.; Metselaar, J. M.; Nuijen, B.; Beijnen, J. H.; Storm, G.

7

Liposomal drug formulations in the treatment of rheumatoid arthritis. Mol. Pharm. 2011,

8

8, 1002-1015.

9

(47) Ulmansky, R.; Turjeman, K.; Baru, M.; Katzavian, G.; Harel, M.; Sigal, A.

10

Glucocorticoids in nano-liposomes administered intravenously and subcutaneously to

11

adjuvant arthritis rats are superior to the free drugs in suppressing arthritis and

12

inflammatory cytokines. J. Control Release. 2012, 160, 299-305.

13

(48) Dai, L. L.; Yu, Y. L.; Luo, Z.; Li, M. H. Photosensitizer enhanced disassembly of

14

amphiphilic micelle for ROS-response targeted tumor therapy in vivo. Biomaterials 2016,

15

104, 1-17.

16

(49) Hao, Y.; Zhang, B.; Zheng, C.; Ji, R.; Ren, X.; Guo, F.; Sun, S.; Shi, J.; Zhang, H.;

17

Zhang, Z.; Wang, L.; Zhang, Y. The tumor-targeting core-shell structured DTX-loaded

18

PLGA@au nanoparticles for chemophotothermal therapy and X-ray imaging. J. Control

19

Release. 2015, 220, 545-555.

20

(50) Zhang, L.; Gao, S.; Zhang, F.; Yang, K.; Ma, Q.; Zhu, L. Activatable hyaluronic acid

21

nanoparticle as a theranostic agent for optical/photoacoustic image guided photothermal 27



1 2 3

therapy. ACS Nano 2014, 8, 12250-12258. (51) Jackson, N. M.; Ceresa, B. P. EGFR-mediated apoptosis via STAT3. Exp. Cell Res. 2017, 356, 93-103.

4

(52) Zhang, Z. L.; Duan, Q. K.; Zhao, H. Q. Gemcitabine treatment promotes pancreatic

5

cancer stemness through the Nox/ROS/NF-κB/STAT3 signaling cascade. Cancer Lett.

6

2016, 382, 53-63.

7

28


Page 28 of 45

Page 29 of 45 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

Biomacromolecules

1

Figure and Figure Captions

2 3 4 5 6

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

29



1

2 3 4 5 6 7

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.

8

30


Page 30 of 45

Page 31 of 45 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

Biomacromolecules

1

2 3 4 5 6 7 8 9 10

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.

31



1

2 3 4 5 6 7 8 9 10 11 12 13 14 15

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

Combined Phycocyanin and Hematoporphyrin Monomethyl Ether for

Recommend Documents